Caged Molecules Research Articles

Sulfonated Hydroxyaryl-Tetrazines with Increased pKa for Accelerated Bioorthogonal Click-to-Release Reactions in Cells.

Bioorthogonal reactions that enable switching molecular functions by breaking chemical bonds have gained prominence, with the tetrazine-mediated cleavage of trans-cyclooctene caged compounds (click-to-release) being particularly noteworthy for its high versatility, biocompatibility, and fast reaction rates. Despite several recent advances, the development of highly reactive tetrazines enabling quantitative elimination from trans-cyclooctene linkers remains challenging. In this study, we present the synthesis and application of sulfo-tetrazines, a class of derivatives featuring phenolic hydroxyl groups with increased acidity constants (pKa). This unique property leads to accelerated elimination and complete release of the caged molecules within minutes. Moreover, the inclusion of sulfonate groups provides a valuable synthetic handle, enabling further derivatization into sulfonamides, modified with diverse substituents. Significantly, we demonstrate the utility of sulfo-tetrazines in efficiently activating fluorogenic compounds and prodrugs in living cells, offering exciting prospects for their application in bioorthogonal chemistry.

Sulfonated Hydroxyaryl‐Tetrazines with Increased pKa for Accelerated Bioorthogonal Click‐to‐Release Reactions in Cells

AbstractBioorthogonal reactions that enable switching molecular functions by breaking chemical bonds have gained prominence, with the tetrazine‐mediated cleavage of trans‐cyclooctene caged compounds (click‐to‐release) being particularly noteworthy for its high versatility, biocompatibility, and fast reaction rates. Despite several recent advances, the development of highly reactive tetrazines enabling quantitative elimination from trans‐cyclooctene linkers remains challenging. In this study, we present the synthesis and application of sulfo‐tetrazines, a class of derivatives featuring phenolic hydroxyl groups with increased acidity constants (pKa). This unique property leads to accelerated elimination and complete release of the caged molecules within minutes. Moreover, the inclusion of sulfonate groups provides a valuable synthetic handle, enabling further derivatization into sulfonamides, modified with diverse substituents. Significantly, we demonstrate the utility of sulfo‐tetrazines in efficiently activating fluorogenic compounds and prodrugs in living cells, offering exciting prospects for their application in bioorthogonal chemistry.

O-nitrobenzyl Caged Molecule Enables Photo-controlled Release of Thiabendazole.

Pesticides are essential in agricultural development. Controlled-release pesticides have attracted great attentions. Base on a principle of spatiotemporal selectivity, we extended the photoremovable protective group (PRPG) into agrochemical agents to achieve controllable release of active ingredients. Herein, we obtained NP-TBZ by covalently linking o-nitrobenzyl (NP) with thiabendazole (TBZ). Compound NP-TBZ can be controlled to release TBZ in dependent to light. The irradiated and unirradiated NP-TBZ showed significant differences on fungicidal activities both in vitro and in vivo. In addition, the irradiated NP-TBZ displayed similar antifungal activities to the directly-used TBZ, indicating a factual applicability in controllable release of TBZ. Furthermore, we explored the action mode and microcosmic variations by SEM analysis, and demonstrated that the irradiated NP-TBZ retained a same action mode with TBZ against mycelia growth.

Stabilization of Carbocation Intermediate by Cucurbit[7]uril Enables High Photolysis Efficiency.

A cucurbit[7]uril-based host-guest strategy is employed to enhance the efficiency of photolysis reactions that release caged molecules from photoremovable protecting groups. The photolysis of benzyl acetate follows a heterolytic bond cleavage mechanism, thereby leading to the formation of a contact ion pair as the key reactive intermediate. The Gibbs free energy of the contact ion pair is lowered by 3.06 kcal/mol through the stabilization of cucurbit[7]uril, as revealed by DFT calculations, which results in a 40-fold increase in the quantum yield of the photolysis reaction. This methodology is also applicable to the chloride leaving group and the diphenyl photoremovable protecting group. We anticipate that this research presents a novel strategy to improve reactions involving active cationics, thereby enriching the field of supramolecular catalysis.

Constructing pH-Responsive and Tunable Azo-Phenol-Based o-Nitrobenzyl Photocages.

Multiple triggered-release strategies are widely utilized to control the release of caged target molecules. Among them, photocages with conditional triggers provide extra layers of control in photorelease. In this work, a series of pH-responsive photocages was designed that could be triggered under irradiation and specific intracellular pH values. pH-sensitive phenolic groups were conjugated with o-nitrobenzyl (oNB) to form azo-phenolic NPX photocages with tunable pKa. These azo-phenol-based oNB photocages showed differentiable photoreleasing profiles at pH 5.0, 7.2 and 9.0. By attaching fluorogenic cargos, it was shown that one of the photocages, NPdiCl, could be used to differentiate between acidic pH 5.0 and neutral pH 7.2 in cells under artificial pH conditions. Finally, NPdiCl was identified as a promising pH-responsive photocage for photoreleasing cargo inside acidic tumor cells.

A Caged In‐Source Laser‐Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI‐MS Imaging

AbstractInsufficient vacuum stability of matrix chemicals is a major limitation in matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo‐cleavable caged molecule 4,5‐dimethoxy‐2‐nitrobenzyl‐2,5‐dihydroxyacetophenone (DMNB‐2,5‐DHAP) and employed it for lipid MALDI‐MSI of mouse brain tissue sections. DMNB‐2,5‐DHAP is vacuum‐stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built‐in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5‐DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum‐stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI‐2 laser‐induced postionization.

A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging.

Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.

Far-red BODIPY-based oxime esters: photo-uncaging and drug delivery.

Far-red BODIPY-based oxime esters for photo-uncaging were designed to release molecules of interest with carboxylic acids. The low power red LED light breaks the N-O oxime ester bond and frees the caged molecules. We studied the mechanism and kinetics of the uncaging procedure using a 1H NMR spectrometer. Moreover, the drug delivery strategy to release valproic acid (VPA) on demand was tested in vitro using this far-red BODIPY photo-uncaging strategy to induce apoptosis in tumor cells.

Surface-limited reactions for spatial control of kinesin-microtubule motility assays using indirect irradiation of an electron beam.

Gliding of microtubules (MTs) on kinesins has been applied to lab-on-a-chip devices, which enable autonomous transportation and detection of biomolecules in the field of bioengineering. For rapid fabrication and evaluation of the kinesin-MT based devices, optical control techniques have been developed for control of kinesin activity and density; however, use of caged molecules lacks spatial controllability for long-term experiments, and direct irradiations of UV light onto kinesin-coated surfaces are inherently damaging to MTs due to their depth limit since the heights of the kinesin-MT systems are at the tens of a nanometer scale. Considering surface electric fields in electrolytic solutions are shielded at the nanometer scale due to Debye shielding, in this study, we show that fine spatial control of kinesin density and activity is enabled using surface-limited electrochemical reactions induced by indirect irradiations of an electron beam (EB). An EB is indirectly irradiated onto the kinesins through a 100-nm-thick silicon nitride membrane, and the electrons scattered in the membrane can cause localized electrochemical effects to the kinesins. We show that these localized electrochemical effects cause both ablation of kinesins and motility control of kinesin activity by changing the EB acceleration voltage. In particular, the latter is achieved without complete ablation of MTs, though the MTs are indirectly irradiated by the EB. As a demonstration of on-demand control of gliding MTs, we show the accumulation of the MTs on a target area by scanning the EB. The proposed accumulation technique will lead to rapid prototyping of microdevices based on MT-kinesin motility assay systems.

Loose nanofiltration membrane constructed via interfacial polymerization using porous organic cage RCC3 for dye/salt separation

Fabricating loose membranes with well-tailored porosity for effective molecular separations remains challenging. Here, we introduce RCC3, a soluble porous organic cage (POC), as an amine monomer to fabricate polyamide (PA) membrane with loose structure via interfacial polymerization and evaluate the membrane performance in dye/salt separation. RCC3 is a [4 + 6] amine molecular cage with ideal stability and sub-nanometer macrocyclic cavity (∼7 Å). The intrinsic porosity and large interchain pores formed by its participation in interfacial polymerization provide extra transport channels in membrane and contribute to realizing precise aperture sieving with high permeation flux. Meanwhile, the unique structure of RCC3 (molecular-level size, organic framework and rich –NH– groups) helps to achieve molecular-level mixing between polymer chains and individual caged molecules in the membrane, thus overcoming the problem of interfacial defects. Under the condition of optimal RCC3/PIP mass ratio, the as-prepared membrane exhibited favorable water permeability of 52.55 LMH/bar (5.2 times of PIP/TMC membrane) with good dye rejection (98.6% for MEB) and salt permeability. Due to the extremely low surface roughness (Ra = 12.5 nm), the membrane exhibited outstanding anti-fouling capacity with high and stable flux recovery rate. Furthermore, the membrane possessed excellent long-term stability, which underlined the promising potential in practical application.

Isochronal superpositioning of the caged dynamics, the α, and the Johari-Goldstein β relaxations in metallic glasses.

The superposition of the frequency dispersions of the structural α relaxation determined at different combinations of temperature T and pressure P while maintaining its relaxation time τα(T, P) constant (i.e., isochronal superpositioning) has been well established in molecular and polymeric glass-formers. Not known is whether the frequency dispersion or time dependence of the faster processes including the caged molecule dynamics and the Johari-Goldstein (JG) β relaxation possesses the same property. Experimental investigation of this issue is hindered by the lack of an instrument that can cover all three processes. Herein, we report the results from the study of the problem utilizing molecular dynamics simulations of two different glass-forming metallic alloys. The mean square displacement 〈Δr2t〉, the non-Gaussian parameter α2t, and the self-intermediate scattering function Fsq,t at various combinations of T and P were obtained over broad time range covering the three processes. Isochronal superpositioning of 〈Δr2t〉, α2t, and Fsq,t was observed over the entire time range, verifying that the property holds not only for the α relaxation but also for the caged dynamics and the JG β relaxation. Moreover, we successfully performed density ρ scaling of the time τα2,maxT,P at the peak of α2t and the diffusion coefficient D(T, P) to show both are functions of ργ/T with the same γ. It follows that the JG β relaxation time τβ(T, P) is also a function of ργ/T since τα2,maxT,P corresponds to τβ(T, P).

Synthesis of Fullerenes from a Nonaromatic Chloroform through a Newly Developed Ultrahigh-Temperature Flash Vacuum Pyrolysis Apparatus.

The flash vacuum pyrolysis (FVP) technique is useful for preparing curved polycyclic aromatic compounds (PAHs) and caged nanocarbon molecules, such as the well-known corannulene and fullerene C60. However, the operating temperature of the traditional FVP apparatus is limited to ~1250 °C, which is not sufficient to overcome the high energy barriers of some reactions. Herein, we report an ultrahigh-temperature FVP (UT-FVP) apparatus with a controllable operating temperature of up to 2500 °C to synthesize fullerene C60 from a nonaromatic single carbon reactant, i.e., chloroform, at 1350 °C or above. Fullerene C60 cannot be obtained from CHCl3 using the traditional FVP apparatus because of the limitation of the reaction temperature. The significant improvements in the UT-FVP apparatus, compared to the traditional FVP apparatus, were the replacement of the quartz tube with a graphite tube and the direct heating of the graphite tube by impedance heating instead of indirect heating of the quartz tube using an electric furnace. Because of the higher temperature range, UT-FVP can not only synthesize fullerene C60 from single carbon nonaromatic reactants but sublimate some high-molecular-weight compounds to synthesize larger curved PAHs in the future.

An intramolecular coupling approach to alkyl bioisosteres for the synthesis of multisubstituted bicycloalkyl boronates.

Bicyclic hydrocarbons, and bicyclo[1.1.1]pentanes (BCPs) in particular, are playing an emerging role as saturated bioisosteres in pharmaceutical, agrochemical and materials chemistry. Taking advantage of strain-release strategies, prior synthetic studies have featured the synthesis of bridgehead-substituted (C1, C3) BCPs from [1.1.1]propellane. Here, we describe an approach to access multisubstituted BCPs via intramolecular cyclization. In addition to C1,C3-disubstituted BCPs, this method also enables the construction of underexplored multisubstituted (C1, C2 and C3) BCPs from readily accessible cyclobutanones. The broad generality of this method has also been examined through the synthesis of a variety of other caged bicyclic molecules, ranging from [2.1.1] to [3.2.1] scaffolds. The modularity afforded by the pendant bridgehead boron pinacol esters generated during the cyclization reaction has been demonstrated through several downstream functionalizations, highlighting the ability of this approach to enable the programmed and divergent synthesis of multisubstituted bicyclic hydrocarbons.

Disorder and dynamics of free and caged molecules in crystals

Disorder and dynamics of free and caged molecules in crystals

A Light‐Controllable Chemical Modulation of m6A RNA Methylation

AbstractBioactive small molecules with photo‐removable protecting groups have provided spatial and temporal control of corresponding biological effects. We present the design, synthesis, computational and experimental evaluation of the first photo‐activatable small‐molecule methyltransferase agonist. By blocking the functional N‐H group on MPCH with a photo‐removable ortho‐nitrobenzyl moiety, we have developed a promising photo‐caged compound that had completely concealed its biological activity. Short UV light exposure of cells treated with that caged molecule in a few minutes resulted in a considerable hypermethylation of m6A modification in transcriptome RNAs, implicating a rapid release of the parent active compound. This study validates for the first time the photo‐activatable small organic molecular concept in the field of RNA epigenetic research, which represents a novel tool in spatiotemporal and cellular modulation approaches.

A Light-Controllable Chemical Modulation of m6 A RNA Methylation.

Bioactive small molecules with photo-removable protecting groups have provided spatial and temporal control of corresponding biological effects. We present the design, synthesis, computational and experimental evaluation of the first photo-activatable small-molecule methyltransferase agonist. By blocking the functional N-H group on MPCH with a photo-removable ortho-nitrobenzyl moiety, we have developed a promising photo-caged compound that had completely concealed its biological activity. Short UV light exposure of cells treated with that caged molecule in a few minutes resulted in a considerable hypermethylation of m6 A modification in transcriptome RNAs, implicating a rapid release of the parent active compound. This study validates for the first time the photo-activatable small organic molecular concept in the field of RNA epigenetic research, which represents a novel tool in spatiotemporal and cellular modulation approaches.

Phase Diagrams for sII Clathrate Hydrates of CO2 from First-Principles Thermodynamics.

Clathrate hydrates are crystalline solid compounds consisting of a water caged framework and guest molecules such as CH4, C2H6, and CO2. Understanding the phase equilibrium conditions of hydrates is significantly important for the industrial exploitation and experimental synthesis of hydrates. Based on the correct description of the intermolecular noncovalent interactions of clathrate hydrates with vdW-DF2, we studied the crystal structures and the chemical potential phase diagrams of sII hydrates encapsulated with CO2 molecules to provide a deep understanding of the stability mechanism of hydrates. Under the given p-T conditions, the partially occupied hydrates (136H2O·1CO2 and 136H2O·16CO2) and fully occupied hydrates (136H2O·24CO2) are thermodynamically stable, and the equilibrium temperature decreases as the relative CO2 chemical potential increases at the same pressure. We expect that the present study may provide vital information on the stability conditions of CO2 hydrates and trigger new experiments to establish an effective replacement strategy for CO2/CH4.

Enamine N-Oxides: Synthesis and Application to Hypoxia-Responsive Prodrugs and Imaging Agents.

Tumor hypoxia induces the large-scale adaptive reprogramming of cancer cells, promoting their transformation into highly invasive and metastatic species that lead to highly negative prognoses for cancer patients. We describe the synthesis and application of a hypoxia-responsive trigger derived from previously inaccessible enamine N-oxide structures. Hypoxia-dependent reduction of this motif by hemeproteins results in the concomitant activation of a caged molecule and a latent electrophile. We exploit the former in a hypoxia-activated prodrug application using a caged staurosporine molecule as a proof-of-principle. We demonstrate the latter in in vivo tumor labeling applications with enamine-N-oxide-modified near-infrared probes. Hypoxia-activated prodrug development has long been complicated by the heterogeneity of tumor hypoxia in patients. The dual drug release and imaging modalities of the highly versatile enamine N-oxide motif present an attractive opportunity for theranostic development that can address the need not only for new therapeutics but paired methods for patient stratification.

In Situ Electrochemical Monitoring of Caged Compound Photochemistry: An Internal Actinometer for Substrate Release

Caged compounds are molecules that release a protective substrate to free a biologically active substrate upon treatment with light of sufficient energy and duration. A notable limitation of this approach is difficulty in determining the degree of photoactivation in tissues or opaque solutions because light reaching the desired location is obstructed. Here, we have addressed this issue by developing an in situ electrochemical method in which the amount of caged molecule photorelease is determined by fast-scan cyclic voltammetry (FSCV) at carbon-fiber microelectrodes. Using p-hydroxyphenyl glutamate (pHP-Glu) as our model system, we generated a linear calibration curve for oxidation of 4-hydroxyphenylacetic acid (4HPAA), the group from which the glutamate molecule leaves, up to a concentration of 1000 μM. Moreover, we are able to correct for the presence of residual pHP-Glu in solution as well as the light artifact that is produced. A corrected calibration curve was constructed by photoactivation of pHP-Glu in a 3 μL photoreaction vessel and subsequent analysis by high-performance liquid chromatography. This approach has yielded a linear relationship between 4HPAA concentration and oxidation current, allowing the determination of released glutamate independent of the amount of light reaching the chromophore. Moreover, we have successfully validated the newly developed method by in situ measurement in a whole, intact zebrafish brain. This work demonstrates for the first time the in situ electrochemical monitoring of caged compound photochemistry in brain tissue with FSCV, thus facilitating analyses of neuronal function.

General Strategy for Integrated Bioorthogonal Prodrugs: Pt(II)-Triggered Depropargylation Enables Controllable Drug Activation In Vivo.

Bioorthogonal decaging reactions for controllable drug activation within complex biological systems are highly desirable yet extremely challenging. Herein, we find a new class of Pt(II)-triggered bioorthogonal cleavage reactions in which Pt(II) but not Pt(IV) complexes effectively trigger the cleavage of O/N-propargyl in a variety of ranges of caged molecules under biocompatible conditions. Based on these findings, we propose a general strategy for integrated bioorthogonal prodrugs and accordingly design a prodrug 16, in which a Pt(IV) moiety is covalently connected with an O2-propargyl diazeniumdiolate moiety. It is found that 16 can be specifically reduced by cytoplasmic reductants in human ovarian cancer cells to liberate cisplatin, which subsequently stimulates the cleavage of O2-propargyl to release large amounts of NO in situ, thus generating synergistic and potent tumor suppression activity in vivo. Therefore, Pt(II)-triggered depropargylation and the integration concept might provide a general strategy for broad applicability of bioorthogonal cleavage chemistry in vivo.