Cobalt-electrocatalytic HAT for functionalization of unsaturated C-C bonds.

The study and application of transition metal hydrides (TMHs) has been an active area of chemical research since the early 1960s1, for energy storage, through the reduction of protons to generate hydrogen2,3, and for organic synthesis, for the functionalization of unsaturated C-C, C-O and C-N bonds4,5. In the former instance, electrochemical means for driving such reactivity has been common place since the 1950s6 but the use of stoichiometric exogenous organic- and metal-based reductants to harness the power of TMHs in synthetic chemistry remains the norm. In particular, cobalt-based TMHs have found widespread use for the derivatization of olefins and alkynes in complex molecule construction, often by a net hydrogen atom transfer (HAT)7. Here we show how an electrocatalytic approach inspired by decades of energy storage research can be made use of in the context of modern organic synthesis. This strategy not only offers benefits in terms of sustainability and efficiency but also enables enhanced chemoselectivity and distinct, tunable reactivity. Ten different reaction manifolds across dozens of substrates are exemplified, along with detailed mechanistic insights into this scalable electrochemical entry into Co-H generation that takes place through a low-valent intermediate.

Modular Access to Diverse Chemiluminescent Dioxetane-Luminophores through Convergent Synthesis.

Adamantyl-dioxetane luminophores are an important class of chemiluminescent molecular probes for diagnostics and imaging. We have developed a new efficient synthetic route for preparation of adamantyl-enolether as precursors for dioxetane chemiluminescent luminophores. The synthesis is convergent, using an unusual Stille cross-coupling reaction employing a stannane-enolether, to directly afford adamantyl-enolether. In a following simple step, the dioxetane is obtained by oxidation of the enolether precursor with singlet-oxygen. The scope of this synthetic route is broad since a large number of haloaryl substrates are either commercially available or easily accessible. Such a late-stage derivatization strategy simplifies the rapid exploration of novel luminogenic molecular structures in a library format and simplifies the synthesis of known dioxetane luminophores. We expect that this new synthetic strategy will be particularly useful in the design and synthesis of yet unexplored dioxetane chemiluminescent luminophores.

Self-Immolative Polymers: An Emerging Class of Degradable Materials with Distinct Disassembly Profiles.

Self-immolative polymers are an emerging class of macromolecules with distinct disassembly profiles that set them apart from other general degradable materials. These polymers are programmed to disassemble spontaneously from head to tail, through a domino-like fragmentation, upon response to extremal stimuli. In the time since we first reported this unique type of molecule, several groups around the world have developed new, creative molecular structures that perform analogously to our pioneering polymers. Self-immolative polymers are now widely recognized as an important class of stimuli-responsive materials for a wide range of applications such as signal amplification, biosensing, drug delivery, and materials science. The quinone-methide elimination was shown to be an effective tool to achieve rapid domino-like fragmentation of polymeric molecules. Thus, numerous applications of self-immolative polymers are based on this disassembly chemistry. Although several other fragmentation reactions achieved the function requested for sequential disassembly, we predominantly focused in this Perspective on examples of self-immolative polymers that disassemble through the quinone-methide elimination. Selected examples of self-immolative polymers that disassembled through other chemistries are briefly described. The growing demand for stimuli-responsive degradable materials with novel molecular backbones and enhanced properties guarantees the future interest of the scientific community in this unique class of polymers.

Activity-Based Optical Sensing Enabled by Self-Immolative Scaffolds: Monitoring of Release Events by Fluorescence or Chemiluminescence Output.

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.

Chemiluminescence molecular probe with a linear chain reaction amplification mechanism.

A new signal amplification probe with a linear chain reaction amplification mechanism and distinct chemiluminescence output was developed. The probe is composed of a unique structural motif that combines a chemiexcitation mechanism with an intramolecular transesterification into a single molecular structure. As demonstrated with a probe designed to detect hydrogen peroxide, an auto-inductive chemiluminescence signal amplification was obtained through methanol release by an intramolecular transesterification of the generated 2-hydroxymethylbenzoate derivative. The methanol was then oxidized by alcohol oxidase to regenerate the analyte of interest, hydrogen peroxide. Our probe enabled direct measurement of light emission with a limit of detection of 2.5 µM, whilst the assay was rapidly completed within 14 to 150 minutes. Such molecular probes with chemiluminescence signal enhancement through analyte amplification could be used for the detection of other chemical and biological analytes.

Direct Real-Time Monitoring of Prodrug Activation by Chemiluminescence.

The majority of theranostic prodrugs reported so far relay information through a fluorogenic response generated upon release of the active chemotherapeutic agent. A chemiluminescence detection mode offers significant advantages over fluorescence, mainly due to the superior signal-to-noise ratio of chemiluminescence. Here we report the design and synthesis of the first theranostic prodrug monitored by a chemiluminescence diagnostic mode. As a representative model, we prepared a prodrug from the chemotherapeutic monomethyl auristatin E, which was modified for activation by ß-galactosidase. The activation of the prodrug in the presence of ß-galactosidase is accompanied by emission of a green photon. Light emission intensities, which increase with increasing concentration of the prodrug, were linearly correlated with a decrease in the viability of a human cell line that stably expresses ß-galactosidase. We obtained sharp intravital chemiluminescent images of endogenous enzymatic activity in ß-galactosidase-overexpressing tumor-bearing mice. The exceptional sensitivity achieved with the chemiluminescence diagnostic mode should allow the exploitation of theranostic prodrugs for personalized cancer treatment.

Self-Immolative Chemiluminescence Polymers: Innate Assimilation of Chemiexcitation in a Domino-like Depolymerization.

Self-immolative polymers are distinctive materials able to disassemble in a domino-like mechanism from head-to-tail upon a triggering event induced by an external stimulus. We have developed an effective molecular method to intrinsically assimilate a chemiluminescence turn-ON mechanism with a domino-like fragmentation mechanism. A unique molecular unit was synthesized, which could combine the abilities of executing the duel function of quinone-methide elimination and chemiexcitation. Incorporation of this unit as a monomer, results with the first class of stimuli-responsive self-immolative polymers with amplified chemiluminescence output. Responsive groups for various analytes were introduced as a head-trigger during the polymer synthesis. The polymers were demonstrated as chemiluminescence probes for detection of different chemical analytes. The obtained polymers were able to amplify the intensity and the duration of the light emission signal by factors correlated to their length. We anticipate that the chemiluminescence self-immolative polymers described here will find use for various research topics such as signal amplification, light-emitting new materials, and molecular probes with long-lasting light emission and imaging capabilities.

Near-Infrared Dioxetane Luminophores with Direct Chemiluminescence Emission Mode.

Chemiluminescent luminophores are considered as one of the most sensitive families of probes for detection and imaging applications. Due to their high signal-to-noise ratios, luminophores with near-infrared (NIR) emission are particularly important for in vivo use. In addition, light with such long wavelength has significantly greater capability for penetration through organic tissue. So far, only a few reports have described the use of chemiluminescence systems for in vivo imaging. Such systems are always based on an energy-transfer process from a chemiluminescent precursor to a nearby emissive fluorescent dye. Here, we describe the development of the first chemiluminescent luminophores with a direct mode of NIR light emission that are suitable for use under physiological conditions. Our strategy is based on incorporation of a substituent with an extended π-electron system on the excited species obtained during the chemiexcitation pathway of Schaap's adamantylidene-dioxetane probe. In this manner, we designed and synthesized two new luminophores with direct light emission wavelength in the NIR region. Masking of the luminophores with analyte-responsive groups has resulted in turn-ON probes for detection and imaging of ß-galactosidase and hydrogen peroxide. The probes' ability to image their corresponding analyte/enzyme was effectively demonstrated in vitro for ß-galactosidase activity and in vivo in a mouse model of inflammation. We anticipate that our strategy for obtaining NIR luminophores will open new doors for further exploration of complex biomolecular systems using non-invasive intravital chemiluminescence imaging techniques.

Tagging the Untaggable: A Difluoroalkyl-Sulfinate Ketone-Based Reagent for Direct C-H Functionalization of Bioactive Heteroarenes.

We have developed a new difluoroalkyl ketal sulfinate salt reagent suitable for direct derivatization of heteroarene C-H bonds. The reagent is capable of introducing a ketone functional group on heteroarene bioactive compounds via a one-pot reaction. Remarkably, in three examples the ketone analog and its parent drug had almost identical cytotoxicity. In a representative example, the ketone analog was bioconjugated with a delivery vehicle via an acid-labile semicarbazone linkage and with a photolabile protecting group to produce the corresponding prodrug. Controlled release of the drug-ketone analog was demonstrated in vitro for both systems. This study provides a general approach to obtain taggable ketone analogs directly from bioactive heteroarene compounds with limited options for conjugation. We anticipate that this sodium ketal-sulfinate reagent will be useful for derivatization of other heteroarene-based drugs to obtain ketone-taggable analogs with retained efficacy.

Quinone-methide species, a gateway to functional molecular systems: from self-immolative dendrimers to long-wavelength fluorescent dyes.

Over the last 30 years, the quinone-methide elimination has served as a valuable tool for achieving various important molecular functions. Molecular adaptors based on quinone-methide or aza-quinone-methide reactivity have been designed, synthesized, and used in diagnostic probes, molecular amplifiers, drug delivery systems, and self-immolative dendritic/polymeric molecular systems. These unique adaptors function as stable spacers between an enzyme- or reagent-responsive group and a reporter moiety and can undergo 1,4-, 1,6-, or 1,8-type elimination reactions upon cleavage of the triggering group. Such reactivity results in the release of the reporter group through formation of a quinone-methide species. This type of elimination was applied to design distinct molecular adaptors capable of multiple quinone-methide eliminations. Using this chemistry, we have developed unique molecular structures that are known today as self-immolative dendrimers. These dendrimers disassemble upon a single triggering event in a domino-like manner from the focal point to their periphery with the consequent release of multiple end-groups. Such molecular structures are used in self-immolative dendritic prodrugs and in diagnostic probes to obtain a significant amplification effect. To further enhance amplification, we have developed the dendritic chain reaction, which uses simple molecules to achieve functionality of high-generation virtual self-immolative dendrimers. In addition, we harnessed the quinone-methide elimination reactivity to design polymers that disassemble from head-to-tail initiated by an analyte-responsive event. Following this example, other chemical reactivities were demonstrated by scientists to design such polymeric molecules. In a manner analogous to the quinone-methide elimination, electron rearrangement can lead to formation of conjugated quinone-methide-type dyes with long-wavelength emission of fluorescence. We have recently applied an intramolecular charge transfer to form a unique kind of quinone-methide type derivative based on a donor-two-acceptors molecular structure. This intramolecular charge transfer produces a new fluorochrome with an extended conjugation of π-electron system that is used for the design of long-wavelength fluorogenic probes with a turn-ON option. The rapidly expanding use of quinone-methide species, reflected in the increased number of examples reported in the literature, indicates the importance of this tool in chemistry. These species provide a useful gateway to functional molecular structures with distinct reactivities and spectroscopic characteristics.

Potent antitumor activity of anti-HER2 antibody-topoisomerase I inhibitor conjugate based on self-immolative dendritic dimeric-linker.

Antibody-drug conjugates (ADCs) are a rapidly expanding class of anticancer therapeutics, with 14 ADCs already approved worldwide. We developed unique linker technologies for the bioconjugation of drug molecules with controlled-release applications. We synthesized cathepsin-cleavable ADCs using a dimeric prodrug system based on a self-immolative dendritic scaffold, resulting in a high drug-antibody ratio (DAR) with the potential to reach 16 payloads due to its dendritic structure, increased stability in the circulation and efficient release profile of a highly cytotoxic payload at the targeted site. Using our novel cleavable linker technologies, we conjugated the anti-human epidermal growth factor receptor 2 (anti-HER2) antibody, trastuzumab, with topoisomerase I inhibitors, exatecan or belotecan. The newly synthesized ADCs were tested in vitro on mammary carcinoma cells overexpressing human HER2, demonstrating a substantial inhibitory effect on the proliferation of HER2-positive cells. Importantly, a single dose of our trastuzumab-based ADCs administered in vivo to mice bearing HER2-positive tumors, showed a dose-dependent inhibition of tumor growth and survival benefit, with the most potent antitumor effects observed at 10 mg/kg, which resulted in complete tumor regression and survival of 100% of the mice. Overall, our novel dendritic technologies using the protease-cleavable Val-Cit linker present an opportunity for the development of highly selective and potent controlled-released therapeutic payloads. This strategy could potentially lead to the development of novel and effective ADC technologies for patients diagnosed with HER2-positive cancers. Moreover, our proposed ADC linker technology can be implemented in additional medical conditions such as other malignancies as well as autoimmune diseases that overexpress targets, other than HER2.

An electroaffinity labelling platform for chemoproteomic-based target identification.

Target identification involves deconvoluting the protein target of a pharmacologically active, small-molecule ligand, a process that is critical for early drug discovery yet technically challenging. Photoaffinity labelling strategies have become the benchmark for small-molecule target deconvolution, but covalent protein capture requires the use of high-energy ultraviolet light, which can complicate downstream target identification. Thus, there is a strong demand for alternative technologies that allow for controlled activation of chemical probes to covalently label their protein target. Here we introduce an electroaffinity labelling platform that leverages the use of a small, redox-active diazetidinone functional group to enable chemoproteomic-based target identification of pharmacophores within live cell environments. The underlying discovery to enable this platform is that the diazetidinone can be electrochemically oxidized to reveal a reactive intermediate useful for covalent modification of proteins. This work demonstrates the electrochemical platform to be a functional tool for drug-target identification.

Electrochemically driven desaturation of carbonyl compounds.

Electrochemical techniques have long been heralded for their innate sustainability as efficient methods to achieve redox reactions. Carbonyl desaturation, as a fundamental organic oxidation, is an oft-employed transformation to unlock adjacent reactivity through the formal removal of two hydrogen atoms. To date, the most reliable methods to achieve this seemingly trivial reaction rely on transition metals (Pd or Cu) or stoichiometric reagents based on I, Br, Se or S. Here we report an operationally simple pathway to access such structures from enol silanes and phosphates using electrons as the primary reagent. This electrochemically driven desaturation exhibits a broad scope across an array of carbonyl derivatives, is easily scalable (1-100 g) and can be predictably implemented into synthetic pathways using experimentally or computationally derived NMR shifts. Systematic comparisons to state-of-the-art techniques reveal that this method can uniquely desaturate a wide array of carbonyl groups. Mechanistic interrogation suggests a radical-based reaction pathway.

Light emission enhancement by supramolecular complexation of chemiluminescence probes designed for bioimaging.

Chemiluminescence offers advantages over fluorescence for bioimaging, since an external light source is unnecessary with chemiluminescent agents. This report demonstrates the first encapsulation of chemiluminescence phenoxy-adamantyl-1,2-dioxetane probes with trimethyl ß-cyclodextrin. Clear proof for the formation of a 1 : 1 host-guest complex between the adamantyl-1,2-dioxetane probe and trimethyl ß-cyclodextrin was provided by mass spectroscopy and NMR experiments. The calculated association constant of this host-guest system, 253 M-1, indicates the formation of a stable inclusion complex. The inclusion complex significantly amplified the light emission intensity relative to the noncomplexed probe under physiological conditions. Complexation of adamantyl-dioxetane with fluorogenic dye-tethered cyclodextrin resulted in light emission through energy transfer to a wavelength that corresponds to the fluorescent emission of the conjugated dye. Remarkably, the light emission intensity of this inclusion complex was approximately 1500-fold higher than that of the non-complexed adamantyl-dioxetane guest. We present the first demonstration of microscopic cell images obtained using a chemiluminescence supramolecular dioxetane probe and demonstrate the utility of these supramolecular complexes by imaging of enzymatic activity and bio-analytes in vitro and in vivo. We anticipate that the described chemiluminescence supramolecular dioxetane probes will find use in various biological applications.

Chemiluminescence molecular probe with intrinsic auto-inductive amplification: incorporation of chemiexcitation in a quinone-methide elimination.

A new signal amplification system with an auto-inductive mode of action and distinct chemiluminescence output was developed. The system is composed of a unique structural motif that combines a chemiexcitation mechanism with a quinone-methide elimination into a signal building block. As demonstrated with a probe designed to detect fluoride, an auto-inductive chemiluminescence signal amplification was obtained through self-immolative disassembly and light emission mechanisms.

The emergence of aqueous chemiluminescence: new promising class of phenoxy 1,2-dioxetane luminophores.

The majority of known chemiluminescent compounds produce light through oxidation-dependent mechanisms. The unique notion of triggering chemiluminescence by a chemical reaction other than oxidation was first introduced by Schaap in 1987 with the development of chemically and enzymatically activated phenoxy-dioxetanes. Such dioxetanes are distinctive among chemiluminescent molecules since the oxidized high-energy species, the dioxetane, is stable for years at room temperature. Light emission is selectively activated by deprotection of the phenol-protecting group. The chemiluminescence quantum yields of such dioxetanes are relatively high in organic solvents like DMSO. In aqueous solution, however, light emission efficiency drops by approximately 10 000-fold due to energy loss to water molecules. As we sought to understand the low light emission efficiency in water, we realized that the dioxetane chemiexcitation leads to the release of an excited state benzoate molecule, which is a very weak emitter under aqueous conditions. Thus, we reasoned that emission in aqueous solution could be enhanced, if the emissive nature of the excited benzoate formed in water is improved. Introduction of an electron-withdrawing acrylic group at the ortho position of the phenol donor resulted in an excited benzoate species that emits light with high efficiency in aqueous solutions. A striking 3000-fold increase in chemiluminescence emission was observed by simply using an acrylonitrile substituent on the dioxetane probe. For the first time, scientists now have an effective single-entity chemiluminescent probe that can be used to evaluate biological processes. This discovery promoted us to develop numerous highly efficient chemiluminescent probes for detection of different enzymes and analytes in aqueous solution. We anticipate that further studies in this direction will lead to even better chemiluminescence probes with quantum yield emissions that are even higher than that of the luciferin/luciferase system. In this Feature Article, we describe the insights that led us to develop these unprecedented luminophores and the historical perspective that led to the current generation of chemiluminescent phenoxy-dioxetane probes.