Uncaging Reaction Research Articles

Repurposing Copper(II)/THPTA as A Bioorthogonal Catalyst for Thiazolidine Bond Cleavage.

Metal-mediated chemical transformations are promising approaches to manipulate and regulate proteins in fundamental biological research and therapeutic development. Nevertheless, unlike bond-forming reactions, the exploration of selective bond cleavage reactions catalyzed by metals that are fully compatible with proteins and living systems remains relatively limited. Here, it is reported that Copper(II)/tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), commonly used in copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, can be repurposed as a new bioorthogonal catalyst for thiazolidine (Thz) bond cleavage. This process liberates an α-oxo-aldehyde group under physiological conditions, without requiring additional additives. To showcase the utility of this method, this simple catalyst system is coupled with genetic code expansion technology to achieve on-demand activation of genetically encoded Thz-caged α-oxo-aldehydes, enabling further functionalization of proteins. For the first time, this cell-compatible Thz uncaging reaction allows for the site-specific installation of α-oxo-aldehydes at the internal positions of proteins in phage and bacterial surface display systems, expanding the chemical space of proteins. Overall, this study expands the toolkit of bioorthogonal catalysts and paves the way for metal-promoted chemical reactions in living systems, potentially benefiting various applications in the future.

Study on factors affecting quantum yield for the design of improved ortho-nitrobenzyl photoremovable protecting groups

Abstract The use of photoremovable protecting groups (PPGs) for the protection and deprotection of biologically active substances has become instrumental in elucidating functional mechanisms in cells. However, the development of chromophores responsive to visible and near-infrared (NIR) light often results in reduced quantum yields (Φu) of uncaging reactions. Because a high quantum yield is essential for practical PPGs, it is imperative to understand the factors influencing it and devise strategies to enhance it. In this study, we systematically investigated nitrobiphenyl scaffolds featuring amino substituents as electron donors, potentially serving as visible and NIR excitable chromophores. Among the synthesized structures, PPGs incorporating carbazole and phenothiazine units as amino substituents exhibited the highest Φu values of 0.2 and 0.1, respectively, rendering them promising candidates for physiological studies. Conversely, diphenylamine substituents yielded considerably lower Φu values. Photophysical properties and quantum chemical calculations provided insights into the nature of the lowest excited states and their impact on Φu, highlighting the significance of achieving the balance between local n–π* excitation and charge-transfer excitation for manipulating the uncaging quantum yield in ortho-nitrobenzyl derivatives.

Development of Biocompatible Cu(I)-Microdevices for Bioorthogonal Uncaging and Click Reactions.

Transition-metal-catalyzed bioorthogonal reactions emerged a decade ago as a novel strategy to implement spatiotemporal control over enzymatic functions and pharmacological interventions. The use of this methodology in experimental therapy is driven by the ambition of improving the tolerability and PK properties of clinically-used therapeutic agents. The preclinical potential of bioorthogonal catalysis has been validated in vitro and in vivo with the in situ generation of a broad range of drugs, including cytotoxic agents, anti-inflammatory drugs and anxiolytics. In this article, we report our investigations towards the preparation of solid-supported Cu(I)-microdevices and their application in bioorthogonal uncaging and click reactions. A range of ligand-functionalized polymeric devices and off-on Cu(I)-sensitive sensors were developed and tested under conditions compatible with life. Last, we present a preliminary exploration of their use for the synthesis of PROTACs through CuAAC assembly of two heterofunctional mating units.

Interplay of chloride levels and palladium(ii)-catalyzed O-deallenylation bioorthogonal uncaging reactions.

The palladium-mediated uncaging reaction of allene substrates remains a promising yet often overlooked strategy in the realm of bioorthogonal chemistry. This method exhibits high kinetic rates, rivaling those of the widely employed allylic and propargylic protecting groups. In this study, we investigate into the mechanistic aspects of the C-O bond-cleavage deallenylation reaction, examining how chloride levels influence the kinetics when triggered by Pd(ii) complexes. Focusing on the deallenylation of 1,2-allenyl protected 4-methylumbelliferone promoted by Allyl2Pd2Cl2, our findings reveal that reaction rates are higher in environments with lower chloride concentrations, mirroring intracellular conditions, compared to elevated chloride concentrations typical of extracellular conditions. Through kinetic and spectroscopic experiments, combined with DFT calculations, we uncover a detailed mechanism that identifies AllylPd(H2O)2 as the predominant active species. These insights provide the basis for the design of π-allylpalladium catalysts suited for selective uncaging within specific cellular environments, potentially enhancing targeted therapeutic applications.

Enhancing Tsuji-Trost deallylation in living cells with an internal-nucleophile coumarin-based probe.

In recent years, bioorthogonal uncaging reactions have been developed to proceed efficiently under physiological conditions. However, limited progress has been made in the development of protecting groups combining stability under physiological settings with the ability to be quickly removed via bioorthogonal catalysis. Herein, we present a new water-soluble coumarin-derived probe bearing an internal nucleophilic group capable of promoting Tsuji-Trost deallylation under palladium catalysis. This probe can be cleaved by a bioorthogonal palladium complex at a faster rate than the traditional probe, namely N-Alloc-7-amino-4-methylcoumarin. As the deallylation process proved to be efficient in mammalian cells, we envision that this probe may find applications in chemical biology, bioengineering, and medicine.

Microscopic Observation of Membrane Fusion between Giant Liposomes and Baculovirus Budded Viruses Activated by the Release of a Caged Proton.

Baculovirus (Autographa californica multiple nucleopolyhedrovirus, AcMNPV) is an envelope virus possessing a fusogenic protein, GP64, which can be activated under weak acidic conditions close to those in endosomes. When the budded viruses (BVs) are bathed at pH 4.0 to 5.5, they can bind to liposome membranes with acidic phospholipids, and this results in membrane fusion. In the present study, using the caged-proton reagent 1-(2-nitrophenyl)ethyl sulfate, sodium salt (NPE-caged-proton), which can be uncaged by irradiation with ultraviolet light, we triggered the activation of GP64 by lowering the pH and observed membrane fusion on giant liposomes (giant unilamellar vesicles, GUVs) by visualizing the lateral diffusion of fluorescence emitted from a lipophilic fluorochrome (octadecyl rhodamine B chloride, R18) that stained viral envelopes of BVs. In this fusion, entrapped calcein did not leak from the target GUVs. The behavior of BVs prior to the triggering of membrane fusion by the uncaging reaction was closely monitored. BVs appeared to accumulate around a GUV with DOPS, implying that BVs preferred phosphatidylserine. The monitoring of viral fusion triggered by the uncaging reaction could be a valuable tool for revealing the delicate behavior of viruses affected by various chemical and biochemical environments.

Enhanced Design of Gold Catalysts for Bioorthogonal Polyzymes

Bioorthogonal chemistry introduces nonbiogenic reactions that can be performed in biological systems, allowing for the localized release of therapeutic agents. Bioorthogonal catalysts can amplify uncaging reactions for the in situ generation of therapeutics. Embedding these catalysts into a polymeric nanoscaffold can protect and modulate the catalytic activity, improving the performance of the resulting bioorthogonal “polyzymes”. Catalysts based on nontoxic metals such as gold(I) are particularly attractive for therapeutic applications. Herein, we optimized the structural components of a metal catalyst to develop an efficient gold(I)-based polyzyme. Tailoring the ligand structure of gold phosphine-based complexes, we improved the affinity between the metal complex and polymer scaffold, resulting in enhanced encapsulation efficiency and catalytic rate of the polyzyme. Our findings show the dependence of the overall polyzyme properties on the structural properties of the encapsulated metal complex.

Bioorthogonal catalytic nanozyme-mediated lysosomal membrane leakage for targeted drug delivery.

Rationale: Employing in situ bioorthogonal catalysis within subcellular organelles, such as lysosomes, remains a challenge. Lysosomal membranes pose an intracellular barrier for drug sequestration, thereby greatly limiting drug accumulation and concentrations at intended targets. Here, we provide a proof-of-concept report of a nanozyme-based strategy that mediates in situ bioorthogonal uncaging reactions within lysosomes, followed by lysosomal escape and the release of uncaged drugs into the cytoplasm.Methods: A model system composed of a protein-based nanozyme platform (based on the transition metals Co, Fe, Mn, Rh, Ir, Pt, Au, Ru and Pd) and caged compound fluorophores was designed to screen for nanozyme/protecting group pairings. The optimized nanozyme/protecting group pairing was then selected for utilization in the design of anti-cancer pro-drugs and drug delivery systems.Results: Our screening system identified Pd nanozymes that mimic mutant P450BM3 activity and specifically cleave propargylic ether groups. We found that the intrinsic peroxidase-like activity of Pd nanozymes induced the production of free radicals under acid conditions, resulting in lysosomal membrane leakage of uncaged molecules into the cytoplasm. Using a multienzyme synergistic approach, our Pd nanozymes achieved in situ bioorthogonal catalysis and nanozyme-mediated lysosomal membrane leakage, which were successfully applied to the design of model pro-drugs for anti-cancer therapy. The extension of our nanozyme system to the construction of a liposome-based “all-in-one” delivery system offers promise for realizing efficacious in vivo tumor-targeted therapies.Conclusions: This strategy shows a promising new direction by utilizing nanotechnology for drug development through in situ catalyzing bioorthogonal chemistry within specific subcellular organelles.

Transition Metal-mediated Uncaging Chemistry in Prodrug Design.

Uncaging chemistry catalyzed by transition metals is developed from deprotection reactions and metal-organic catalytic reactions. Also, it has the characteristics of high efficiency, simplicity and rapidity in the living biological system. In the past decade, metal encapsulation systems (such as nanoparticles) and metal complexes have been developed to reveal the reactivity of transition metals (including palladium, ruthenium, and gold) in biological systems. Metal nanostructures provide huge possibilities for targeted drug delivery, detection, diagnosis and imaging. So far, palladium, ruthenium and gold nano-architectures have dominated the field, but there are some problems that hinder their wide application in clinical practice. In this review, based on palladium, ruthenium, gold and their complexes, the application of prodrug design through uncaging reaction has been widely discussed.

P-Nitroterphenyl units for near-infrared two-photon uncaging of calcium ions

Near-infrared two-photon (NIR-2P) uncaging reactions are useful for precise investigations of the roles of biologically active compounds in various phenomena. In this study, we synthesized new NIR-2P-responsive chromophores with terphenyl backbones featuring an ethylene glycol tetraacetic acid (EGTA) unit as a Ca2+-selective chelator. The photolabile Ca2+ chelators were synthesized in high yields (83 % for 1a, 45 % for 1b) from commercially available starting materials. One- and two-photon photochemical excitations of the synthesized terphenyl derivatives featuring two different electron-donating groups (dimethylamino (NMe2) and methoxyl (OMe)), which were thermally stable at least 36 h at 37 C in HEPES buffer (pH 7.4), were conducted to clarify the substituent effect on the photophysical and photochemical properties. The NMe2 group, which is a stronger electron-donating group, has led to a higher two-photon cross-section (σ2 = 75 GM at 780 nm) compared to the OMe group (σ2 = 9 GM at 720 nm) in a pH = 7.4 HEPES buffer solution, although the molar extinction coefficient (ε) of the NMe2-substituted one (ε = 10,371 at λmax =363 nm) was lower than that of the OMe-derivative (ε = 14,351 at λmax = 344 nm). The quantum yield (Φ = 0.04) for the decomposition of the NMe2-substituted caged compound was lower than that for the OMe-substituted caged compound (Φ = 0.40). One and two-photon uncaging efficiency values of the OMe substituted compound, ε × Φ = 5740 and σ2 × Φ = 3.6, were higher than those of the NMe2-substituted compound, 415 and 3.0, respectively. Laser flash photolysis experiments clarified the decay rate constants (2.5 × 106 and 1.1 × 102 s−1) of aci-nitro intermediates formed during the uncaging process. A concentration jump of Ca2+ was confirmed in the photolysis of caged calcium compounds newly synthesized in this study, indicating the terphenyl derivatives can be also utilized in physiological experiments. The corresponding acetoxymethyl esters were also prepared for physiological studies.

Switchable Fluorescence of Doxorubicin for Label-Free Imaging of Bioorthogonal Drug Release.

Monitoring the release and activation of prodrug formulations provides essential information about the outcome of a therapy. While the prodrug delivery can be confirmed by using different imaging techniques, confirming the release of active payload by using imaging is a challenge. Here, we have discovered that the switchable fluorescence of doxorubicin can validate drug release upon its uncaging reaction with a highly specific chemical partner. We have observed that the conjugation of doxorubicin with a trans-cyclooctene (TCO) diminishes its fluorescence at 595 nm. This quenched fluorescence of the doxorubicin prodrug is recovered upon its bond-cleaving reaction with tetrazine. Clinically assessed iron oxide nanoparticles were used to formulate a doxorubicin nanodrug. The release of doxorubicin from the nanodrug was studied under various experimental conditions. A fivefold increase in doxorubicin fluorescence is observed after complete release. The studies were carried out in vitro in MDA-MB-231 breast cancer cells. An increase in Dox signal was observed upon tetrazine administration. This switchable fluorescence mechanism of Dox could be employed for fundamental studies, that is, the reactivity of various tetrazine and TCO linker types under different experimental conditions. In addition, the system could be instrumental for translational research where the release and activation of doxorubicin prodrug payloads can be monitored by using optical imaging systems.

Mechanistic insights into transition metal-mediated bioorthogonal uncaging reactions.

Cleavage of C-O and C-N bonds mediated by transition metals is a promising bioorthogonal approach to rescue the activity of caged molecules, such as proteins and cytotoxic drugs, under biological conditions. However, the precise mechanism of such uncaging reactions remains elusive. This review provides mechanistic insights into metal-mediated bond-cleavage reactions, with the goals of understanding the main factors that influence the reaction and aiding the rational development of new caging groups/catalysts for chemical biology and drug-delivery applications.

"Close-to-Release": Spontaneous Bioorthogonal Uncaging Resulting from Ring-Closing Metathesis.

Bioorthogonal uncaging reactions offer versatile tools in chemical biology. In recent years, reactions have been developed to proceed efficiently under physiological conditions. We present herein an uncaging reaction that results from ring-closing metathesis (RCM). A caged molecule, tethered to a diolefinic substrate, is released via spontaneous 1,4-elimination following RCM. Using this strategy, which we term “close-to-release”, we show that drugs and fluorescent probes are uncaged with fast rates, including in the presence of mammalian cells or in the periplasm of Escherichia coli. We envision that this tool may find applications in chemical biology, bioengineering and medicine.

Mechanism of Palladium(II)-Mediated Uncaging Reactions of Propargylic Substrates

The palladium(II)-mediated chemical uncaging reaction of propargylic substrates is a recent addition to the field of chemical biology and medicinal chemistry in the activation of bio and prodrug mo...

Photocatalysis Enables Visible‐Light Uncaging of Bioactive Molecules in Live Cells

AbstractThe photo‐manipulation of bioactive molecules provides unique advantages due to the high temporal and spatial precision of light. The first visible‐light uncaging reaction by photocatalytic deboronative hydroxylation in live cells is now demonstrated. Using Fluorescein and Rhodamine derivatives as photocatalysts and ascorbates as reductants, transient hydrogen peroxides were generated from molecular oxygen to uncage phenol, alcohol, and amine functional groups on bioactive molecules in bacteria and mammalian cells, including neurons. This effective visible‐light uncaging reaction enabled the light‐inducible protein expression, the photo‐manipulation of membrane potentials, and the subcellular‐specific photo‐release of small molecules.

Photocatalysis Enables Visible-Light Uncaging of Bioactive Molecules in Live Cells.

The photo-manipulation of bioactive molecules provides unique advantages due to the high temporal and spatial precision of light. The first visible-light uncaging reaction by photocatalytic deboronative hydroxylation in live cells is now demonstrated. Using Fluorescein and Rhodamine derivatives as photocatalysts and ascorbates as reductants, transient hydrogen peroxides were generated from molecular oxygen to uncage phenol, alcohol, and amine functional groups on bioactive molecules in bacteria and mammalian cells, including neurons. This effective visible-light uncaging reaction enabled the light-inducible protein expression, the photo-manipulation of membrane potentials, and the subcellular-specific photo-release of small molecules.

Harnessing Cyanine Reactivity for Optical Imaging and Drug Delivery.

Optical approaches that visualize and manipulate biological processes have transformed modern biomedical research. An enduring challenge is to translate these powerful methods into increasingly complex physiological settings. Longer wavelengths, typically in the near-infrared (NIR) range (∼650-900 nm), can enable advances in both fundamental and clinical settings; however, suitable probe molecules are needed. The pentamethine and heptamethine cyanines, led by prototypes Cy5 and Cy7, are among the most useful compounds for fluorescence-based applications, finding broad use in a range of contexts. The defining chemical feature of these molecules, and the key chromophoric element, is an odd-numbered polymethine that links two nitrogen atoms. Not only a light-harvesting functional group, the cyanine chromophore is subject to thermal and photochemical reactions that dramatically alter many properties of these molecules. This Account describes our recent studies to define and use intrinsic cyanine chromophore reactivity. The hypothesis driving this research is that novel chemistries that manipulate the cyanine chromophore can be used to address challenging problems in the areas of imaging and drug delivery. We first review reaction discovery efforts that seek to address two limitations of long-wavelength fluorophores: undesired thiol reactivity and modest fluorescence quantum yield. Heptamethine cyanines with an O-alkyl substituent at the central C4' carbon were prepared through a novel N- to O-transposition reaction. Unlike commonly used C4'-phenol variants, this new class of fluorophores is resistant to thiol modification and exhibits improved in vivo imaging properties when used as antibody tags. We have also developed a chemical strategy to enhance the quantum yield of far-red pentamethine cyanines. Using a synthetic strategy involving a cross metathesis/tetracyclization sequence, this approach conformationally restrains the pentamethine cyanine scaffold. The resulting molecules exhibit enhanced quantum yield (ΦF = 0.69 vs ΦF = 0.15). Furthermore, conformational restraint improves interconversion between reduced hydrocyanine and intact cyanine forms, which enables super resolution microscopy. This Account then highlights efforts to use cyanine photochemical reactivity for NIR photocaging. Our approach involves the deliberate use of cyanine photooxidation, a reaction previously only associated with photodegradation. The uncaging reaction sequence is initiated by photooxidative chromophore cleavage (using wavelengths of up to 780 nm), which prompts a C-N bond hydrolysis/cyclization sequence resulting in phenol liberation. This approach has been applied to generate the first NIR-activated antibody-drug conjugates. Tumor uptake can be monitored in vivo using NIR fluorescence, prior to uncaging with an external irradiation source. This NIR uncaging strategy can slow tumor progression and increase survival in a MDA-MB-468- luc mouse model. Broadly, the vantage point of cyanine reactivity is providing novel probe molecules with auspicious features for use in complex imaging and drug delivery settings.

Colloidal LaPO4:Gd3+ nanocrystals: X-ray induced single line UV emission.

Colloidal solutions of nearly monodisperse 5 nm LaPO4:Gd3+ nanocrystals are shown to strongly emit UV radiation upon excitation with tungsten Kα radiation (59.3 keV) or vacuum UV radiation (160 nm). The UV emission of the particles consists mainly of a single line at 311 nm corresponding to the 6P7/2-8S7/2 transition of Gd3+. The highest emission intensity is observed for LaPO4 nanocrystals with a Gd3+ concentration of 20%. Since the absorption cross section of biomaterials is low for X-rays but high for 311 nm radiation, the UV emission of particles embedded in the biological tissue can only affect the direct vicinity of the particles. Nanocrystals of LaPO4:Gd3+ could, therefore, be interesting for biomedical applications such as strongly localized drug release by X-ray triggered UV uncaging reactions.

Defining the conditional basis of silicon phthalocyanine near-IR ligand exchange.

Bond cleavage reactions initiated by long-wavelength light are needed to extend the scope of the caged-uncaged paradigm into complex physiological settings. Axially unsymmetrical silicon phthalocyanines (SiPcs) undergo efficient release of phenol ligands in a reaction contingent on three factors - near-IR light (690 nm), hypoxia, and a thiol reductant. These studies detail efforts to define the mechanistic basis for this unique conditionally-dependent bond cleavage reaction. Spectroscopic studies provide evidence for the formation of a key phthalocyanine radical anion intermediate formed from the triplet state in a reductant-dependent manner. Computational chemistry studies indicate that phenol ligand solvolysis proceeds through a heptacoordinate silicon transition state and that this solvolytic process is favored following SiPc radical anion formation. These results provide insight regarding the central role that radical anion intermediates formed through photoinduced electron transfer with biological reductants can play in long-wavelength uncaging reactions.

Design and Synthesis of Two-Photon Responsive Chromophores for Near-Infrared Light-Induced Uncaging Reactions

Near-infrared two-photon (TP)-induced photorelease (uncaging) of bioactive molecules such as drugs has attracted considerable attention because of its ability to elucidate mechanistic aspects of biological processes. This short review summarizes recent developments in the design and synthesis of TP-responsive chromophores.1 Introduction2 Molecular Design of TP-Responsive Organic Chromophores for ‘Caging & Uncaging’2.1 π-Conjugation2.2 A Dipolar System2.3 A Quadrupolar System2.4 An Octupolar System3 Recent Developments of TP Uncaging Reactions3.1 4-Methoxy-7-nitroindolinyl Caged Auxins3.2 Uncaging of GABA and Tryptophan Using TP-Induced Electron-Transfer Reactions3.3 Effect of Position Isomery in Aminoquinoline-Derived Photolabile Protecting Groups (PPGs)3.4 Cooperative Dyads for TP Uncaging3.5 Caged Calcium with a Bis-styrylthiophene Backbone3.6 Cloaked Caged Compounds3.7 Three-Dimensional Control of DNA Hybridization by Orthogonal Two-Color TP Uncaging3.8 TP-Induced Release of Diethyl Phosphate (DEP) and ATP4 Our Contribution to TP Uncaging Reactions5 Summary