Light-controllable cell-membrane disturbance for intracellular delivery.

Highly polar and charged molecules, such as oligonucleotides, face significant barriers in crossing the cell membrane to access the cytoplasm. To address this problem, we developed a light-triggered twistable tetraphenylethene (TPE) derivative, TPE-C-N, to facilitate the intracellular delivery of charged molecules through an endocytosis-independent pathway. The central double bond of TPE in TPE-C-N is planar in the ground state but becomes twisted in the excited state. Under light irradiation, this planar-to-twisted structural change induces continuous cell membrane disturbances. Such disturbance does not lead to permanent damage to the cell membrane. TPE-C-N significantly enhanced the intracellular delivery of negatively charged molecules under light irradiation when endocytosis was inhibited through low-temperature treatment, confirming the endocytosis-independent nature of this delivery method. We have successfully demonstrated that the TPE-C-N-mediated light-controllable method can efficiently promote the intracellular delivery of charged molecules, such as peptides and oligonucleotides, with molecular weights ranging from 1000 to 5000 Da.

A reductively convertible nickel phthalocyanine precursor as a biological thiol-responsive turn-on photoacoustic contrast agent.

A nickel phthalocyanine precursor bearing poly(ethylene glycol) as a turn-on contrast agent for photoacoustic imaging was prepared. The water-soluble polymeric chains were smoothly eliminated through thiol-mediated reductive aromatization in cancer cells, enabling the detection of endogenous biological thiols in vitro and in vivo.

Steric Control in Activator-Induced Nucleophilic Quencher Detachment-Based Probes: High-Contrast Imaging of Aldehyde Dehydrogenase 1A1 in Cancer Stem Cells.

Turn-on fluorescence probes can visualize the enzyme activity with high contrast. We have established a new turn-on mechanism, activator-induced nucleophilic quencher detachment (AiQd), and developed AiQd-based turn-on fluorescence probes for the detection of enzymes. Herein, we demonstrate that the precise steric control efficiently quenches the fluorescence of AiQd-based turn-on probes before the enzymatic transformation. Theoretical calculation appropriately predicted the ratio of the fluorescence-quenched closed-ring form of probes. ßC5S-A, which has a sterically demanding methyl group at the ß-position of a fluorescence-quenching nucleophilic mercapto group, showed a low background signal. ßC5S-A responded to aldehyde dehydrogenase 1A1 (ALDH1A1) with high selectivity, thereby enabling high-contrast live imaging of cancer stem cells (signal-to-noise ratio >10). The ALDH1A1-responsiveness of ßC5S-A was not significantly affected by amino acids and biological thiols, such as cysteine and glutathione.

Identification of Breast Cancer Stem Cells Using a Newly Developed Long-acting Fluorescence Probe, C5S-A, Targeting ALDH1A1.

BACKGROUND/AIM: Aldehyde dehydrogenase (ALDH) 1A1 is a well-known marker for cancer stem cells (CSCs), characterized by self-renewal capacity and multidrug resistance in breast cancer. We developed a near-infrared turn-on fluorescence probe for ALDH1A1, C5S-A, which is suitable for observing and analyzing viable cells. Here, we demonstrated the utility of C5S-A in CSC research using breast cancer cell lines. MATERIALS AND METHODS: To evaluate concordance between C5S-A and conventional stem cell markers, breast cancer cells sorted for ALDEFLUOR-positive cells and for CD44+/CD24- cell populations were stained with C5S-A. Tumorigenicity of C5S-A-positive cells was examined by mammosphere formation assay and subcutaneous transplantation to immunodeficient mice. Additionally, to determine how long fluorescence from a single staining remained observable, we cultured breast cancer cells for 5 days after C5S-A staining. We then evaluated whether C5S-A-positive cells possessed resistance to cytotoxic drugs by chronological imaging. RESULTS: C5S-A staining showed good concordance with conventional breast CSC markers, and good utility for research into CSC characteristics in breast cancer cell lines, including tumorigenesis. Additionally, C5S-A was observable for more than 3 days with a single staining. Using this property, we then confirmed that C5S-A-positive cells possessed resistance to cytotoxic drugs, which is one of the characteristics of CSCs. CONCLUSION: We showed that C5S-A is suitable for CSC research using breast cancer cell lines, and confirmed its utility in observing cells over time.

An activator-induced quencher-detachment-based turn-on probe with a cationic substrate moiety for acetylcholinesterase.

We report a choline ester-grafted turn-on fluorescence probe to detect acetylcholinesterase (AChE) in living cells. The AChE-mediated hydrolysis of the choline ester moiety producing carboxylate initiates the activation of the Cy5 fluorophore quenched by an intramolecular nucleophilic mercapto group. The probe has the advantages of high AChE affinity and low cytotoxicity.

Deep-Red/Near-Infrared Turn-On Fluorescence Probes for Aldehyde Dehydrogenase 1A1 in Cancer Stem Cells.

Accumulating evidence supports that cancer stem cells (CSCs) are responsible for cancer proliferation, metastasis, and therapy resistance; therefore, an effective strategy to identify and isolate CSCs is required urgently. Because of their low invasiveness and high signal/noise ratio, "turn-on" fluorescence probes working in the deep-red/near-infrared (DR/NIR) region are one of the most attractive yet undeveloped tools for CSC detection. Herein, we report DR/NIR turn-on fluorescence probes, CS5-A and CS7-A, targeted to aldehyde dehydrogenase 1A1 as an intracellular CSC marker. In contrast to the conventional "always-on" green-fluorescent ALDEFLUOR, we succeeded in generating high-contrast (signal/noise ratio > 8.3) and wash-free in vitro CSC imaging with the DR probe C5S-A. This probe can facilitate CSC isolation with minimal contamination by autofluorescence from other tissues through fluorescence-activated cell sorting. Furthermore, the NIR absorbance/emission and turn-on properties of C7S-A allow simple and rapid CSC detection in vivo within 15 min.

Aluminum naphthalocyanine conjugate as an MMP-2-activatable photoacoustic probe for in vivo tumor imaging.

Matrix metalloproteinase-2 (MMP-2), which is one of MMPs family, is known as an extracellular gelatinase controlling cancer cell adhesion, growth, and metastasis. Because of the great interest in MMP-2 activity, the detailed protocols for evaluating MMP-2-responsive contrast agents, especially photoacoustic probes for in vivo use, are helpful for researchers in the field. We here describe the detailed synthetic procedure of MMP-2-activatable photoacoustic probe AlNc-pep-PEG consisting of aluminum naphthalocyanine, MMP-2-responsive peptide sequence, and poly(ethylene glycol), which has recently been developed in our research group. The detailed measurement protocol of photoacoustic signal intensity in vitro and in vivo by using in-house built photoacoustic signal measurement system and photoacoustic imaging apparatus are also summarized.

MMP-2-Activatable Photoacoustic Tumor Imaging Probes Based on Al- and Si-Naphthalocyanines.

Enzyme-activatable photoacoustic probes are powerful contrast agents to visualize diseases in which a specific enzyme is overexpressed. In this study, aluminum and silicon naphthalocyanines (AlNc and SiNc, respectively) conjugated with matrix metalloprotease-2 (MMP-2)-responsive PLGLAG peptide sequence and poly(ethylene glycol) (PEG) as an axial ligand were designed and synthesized. AlNc-peptide-PEG conjugates AlNc-pep-PEG formed dimeric species interacting with each other through face-to-face H-aggregation in water, while SiNc-based conjugates SiNc-pep-PEG hardly interacted with each other because of the two bulky hydrophilic axial ligands. Both conjugates formed spherical nanometer-sized self-assemblies in water, generating photoacoustic waves under near-infrared photoirradiation. The treatment of MNc-peptide-PEG conjugates (M = Al, Si) with MMP-2 smoothly induced the cleavage of the PLGLAG sequence to release the hydrophilic PEG moiety, resulting in the aggregation of MNcs. By comparing the PA signal intensity changes at 680 and 760 nm, the photoacoustic signal intensity ratios were shown to be enhanced by 3-5 times after incubation with MMP-2. We demonstrated that MNc-peptide-PEG conjugates (M = Al, Si) could work as activatable photoacoustic probes in the in vitro experiment of MMP-2-overexpressed cell line HT-1080 as well as the in vivo photoacoustic imaging of HT-1080-bearing mice.

Substituted meso-vinyl-BODIPY as thiol-selective fluorogenic probes for sensing unfolded proteins in the endoplasmic reticulum.

A new type of thiol probes based on the meso-vinyl-BODIPY (VB) scaffold were developed. The monochloro-substituted VB1Cl exhibited the largest fluorescence enhancement (>200-fold) as well as high selectivity upon biological thiol sensing. VB1Cl was successfully applied for reporting the protein unfolding process under ER stress in living cells.

pH-Activatable Cyanine Dyes for Selective Tumor Imaging Using Near-Infrared Fluorescence and Photoacoustic Modalities.

Photoacoustic (PA) imaging is an emerging molecular imaging modality that complements fluorescence imaging and enables high resolution within deep tissue. Fluorescence/PA multimodality imaging would be a powerful technique to extract more comprehensive information from targets than traditional single-modality imaging. In this paper, we developed a new pH-activatable sensor, CypHRGD, which is applicable to both fluorescence and PA detection. CypHRGD was derived from our previous near-infrared pH-sensing platform, in which substitution with a bulky phenyl group and functionalization with a cRGD peptide remarkably improved the sensor's biocompatibility with attenuated dye aggregation. The multimodality imaging applications of CypHRGD were demonstrated in cultured cells and cancer-xenografted mice with rapid kinetics and high sensitivity and specificity, which relies on cRGD-facilitated tumor targeting, probe accumulation and subsequent activation in the acidic organelles after endocytosis.

An enzyme-triggered turn-on fluorescent probe based on carboxylate-induced detachment of a fluorescence quencher.

We developed a new class of turn-on fluorescent probes for an esterase. After the esterase-mediated hydrolysis produced carboxylate (as a fluorescence activator), the fluorescence intensity was markedly increased through the detachment of a quencher moiety from the quenched Cy5 fluorophore. Because the probes based on this new activator-induced quencher-detachment (AiQd) adopt a non-immolative linker between the cleavable site and the fluorophore, the rate of the enzymatic reaction is greatly improved, without the generation of any by-products.

Bis-Metal Complexes of Doubly N-Confused Dioxohexaphyrins as Potential Near-Infrared-II Photoacoustic Dyes.

We investigated the detailed photophysical properties of a series of bis-metal (Zn and Cu) dioxohexaphyrin complexes as potential second near-infrared (NIR-II)-light responsive dyes. A cisoid-configured 28π-electron-conjugated dioxohexaphyrin analogue (c-3a) containing two peculiar "confused pyrrole" moieties in the framework is identified as a reduced isomer derivative of a transoid 26π-dioxohexaphyrin (t-2a). The symmetry-altered structure of c-3a affords a heteroleptic inner environment within the NNNN/NNOO donor core, which imparts its highly flexible electronic features and nonplanar geometry. The macrocycle c-3a can be transformed into the corresponding 26π-electron congener (c-2a) having a coplanar rectangular structure by unique solvent-mediated redox reactivity. Furthermore, upon metal complexation, saddle-distorted bis-metal complexes (c-M2-2a) were formed as the 26π-conjugated structural isomer of the trans-dioxohexaphyrin species (i.e., t-M2-2a). These isoelectronic dioxohexaphyrins demonstrate precise geometry-dependent photophysical properties. Broad tailing NIR-II absorption, weak emissive character, and rapid-decay of the S1 state are observed for c-Zn2-2a. In contrast, the coplanar t-M2-2a exhibits efficient photoacoustic response upon laser excitation with NIR-II light (λ > 1000 nm). To the best of our knowledge, this is the first example of an expanded porphyrin-based photoacoustic contrast agent responsive to NIR-II light.

π-Conjugated Macrocycles Bearing Angle-Strained Alkynes.

Angle-strained alkyne-containing π-conjugated macrocycles are attractive compounds both in functional materials chemistry and biochemistry. Their interesting reactivity as well as photophysical and supramolecular properties have been revealed in the past three decades. This review highlights the recent advances in angle-strained alkyne-containing π-conjugated macrocycles, especially their synthetic methods, the bond angles of alkynes (∠sp at C≡C-C), and their functions. The theoretical and experimental research on cyclo[n]carbons and para-cyclophynes consisting of ethynylenes and para-phenylenes are mainly summarized. Related macrocycles bearing other linkers, such as ortho-phenylenes, meta-phenylenes, heteroaromatics, biphenyls, extended aromatics, are also overviewed. Bond angles of strained alkynes in π-conjugated macrocycles, which are generable, detectable, and isolable, are summarized at the end of this review.

Near-Infrared Circularly Polarized Luminescence through Intramolecular Excimer Formation of Oligo(p-phenyleneethynylene)-Based Double Helicates.

Carbon-based double helicates consisting of two anthracene-containing oligo(p-phenyleneethynylene) units and two flexible chiral 1,1'-binaphthyl units or two rigid chiral 9,9'-spirobifluorene units were developed. The curved oligo(p-phenyleneethynylene) fragments in the double helicates were successfully constructed by tin-mediated reductive aromatization. Helical oligo(p-phenyleneethynylene) double strands fixed by two rigid spirobifluorene units showed little structural change under photoirradiation, thereby emitting circularly polarized luminescence (CPL) in the visible region with a high quantum yield (ΦPL =0.93). In contrast, flexible binaphthyl units induced dynamic structural change of the oligo(p-phenyleneethynylene) luminophores under photoirradiation, leading to strong CPL (|glum |=1.1×10-2 ) in the near-infrared (NIR) region. UV/Vis, circular dichroism (CD), CPL and NMR spectroscopic analyses of the binaphthyl-hinged double helicate suggested excimer formation between two π-conjugated strands in the excited state. Theoretical calculations highlight the importance of the tightly interlocked excimer structure of the carbon-based double helicate in controlling the angle between the electric and magnetic transition dipole moments for strong NIR CPL generation.

Recent Progress on Cyclic Nitrenoid Precursors in Transition-Metal-Catalyzed Nitrene-Transfer Reactions.

Nitrene-transfer reactions are powerful synthetic tools for the direct incorporation of nitrogen atoms into organic molecules. The discovery of novel nitrene-transfer reactions has been dominantly supported not only by improvements in transition-metal catalysts but also by the employment of novel precursors of nitrenoids. Since pioneering work involving the use of organic azides and iminoiodinanes as practical synthetic tools for nitrogen-containing compounds was reported, a new approach using various N-heterocycles containing strain energy or a weak bond has emerged. In this review, we briefly summarize the history of nitrene-transfer chemistry from the viewpoint of its precursors. In particular, the use of N-heterocycles such as 2H-azirines, 1,4,2-dioxazol-5-ones, 1,2,4-oxadiazol-5-ones, isoxazol-5(4H)-ones, and isoxazoles is comprehensively described, showing the recent remarkable progress in this chemistry.

Facile Construction of Tetrahydropyrrolizines by Iron-Catalyzed Double Cyclization of Alkene-Tethered Oxime Esters with 1,2-Disubstituted Alkenes.

The iron-catalyzed cycloaddition reaction of alkene-tethered oxime esters with 1,2-disubstituted alkenes afforded tetrahydropyrrolizines, the structural motif often seen in bicyclic alkaloids. The reaction proceeds through consecutive cycloaddition reactions. These include, first, intramolecular cyclization, followed by intermolecular cyclization with a 1,2-disubstituted alkene in a regioselective manner where an imine moiety first generated plays a pivotal role.

Unique Tube-Ring Interactions: Complexation of Single-Walled Carbon Nanotubes with Cycloparaphenyleneacetylenes.

Carbon nanotubes (CNTs) interlocked by cyclic compounds through supramolecular interaction are promising rotaxane-like materials applicable as 2D and 3D networks of nanowires and disease-specific theranostic agents having multifunctionalities. Supramolecular complexation of CNTs with cyclic compounds in a "ring toss'' manner is a straightforward method to prepare interlocked CNTs; however, to date, this has not been reported on. Here, the "ring toss" method to prepare interlocked CNTs by using π-conjugated carbon nanorings: [8]-, [9]-, and [10]cycloparaphenyleneacetylene (CPPA) is reported. CPPAs efficiently interact with CNTs to form CNT@CPPA complexes, while uncomplexed CPPAs can be recovered without decomposition. CNTs, which tightly fit in the cavities of CPPAs through convex-concave interaction, efficiently afford "tube-in-ring"-type CNT@CPPA complexes. "Tube-in-ring"-type and "ring-on-tube"-type complexation modes are successfully distinguished by spectroscopic, thermogravimetric, and microscopic analyses.

Iron-Catalyzed Aminative Cyclization/Intermolecular Homolytic Aromatic Substitution Using Oxime Esters and Simple Arenes.

Intermolecular C-H alkylation of simple arenes in the presence of an iron catalyst has been achieved in a cascade manner with an aminative cyclization triggered by N-O bond cleavage of an alkene-tethered oxime ester. Various arenes, including electron-rich and electron-poor arenes, and heteroarenes can be employed in the reaction system. Regioselectivity and radical trapping experiments support the involvement of alkyl radical species, which undergo a homolytic aromatic substitution (HAS) to afford the arylation products.

Asymmetric Synthesis of 2H-Azirines with a Tetrasubstituted Stereocenter by Enantioselective Ring Contraction of Isoxazoles.

Highly strained 2H-azirines with a tetrasubstituted stereocenter were synthesized by the enantioselective isomerization of isoxazoles with a chiral diene-rhodium catalyst system. The effect of ligands and the coordination behavior support the proposed catalytic cycle in which the coordination site is fixed in favor of efficient enantiodiscrimination by a bulky substituent of the ligand. In silico studies also support the existence of a rhodium-imido complex as a key intermediate for enantiodiscrimination.

Generation of Stable Ruthenium(IV) Ketimido Complexes by Oxidative Addition of Oxime Esters to Ruthenium(II): Reactivity Studies Based on Electronic Properties of the Ru-N Bond.

The reaction of an oxime ester with [Ru(PPh3 )3 X2 ] proceeded smoothly at room temperature to afford a stable RuIV ketimido complex as oxidative adduct. The structure of the complex was unambiguously determined by X-ray crystallographic analysis, which showed an almost linear Ru-N-C array. The electronic properties of the nitrogen atom were estimated by DFT calculations, and the results suggested double-bond character of the Ru-N bond. Kinetic studies and consideration of the substituent effect on the oxime ester led to the proposal of a reaction mechanism involving oxidative addition, which could proceed by N,O-chelating coordination to the Ru center prior to N-O bond cleavage. The obtained Ru ketimido complex could be transformed into a ruthenacycle by C-H activation by a concerted metalation-deprotonation mechanism in dichloromethane/methanol. Ru ketimido complexes with a tethered alkyne or alkene moiety underwent chloroamination of unsaturated C-C bonds followed by C-H activation, which resulted in the formation of a ruthenacycle. Considering the LUMO of an isolated Ru ketimido complex, the chloroamination should proceed by a synchronous 1,3-dipolar cycloaddition-type mechanism. Insight into the character and reactivity of Ru ketimido complexes will be helpful for developments in the catalytic transformation of oxime esters.