Synthesis and optical properties of tyramine-functionalized boron dipyrromethene dyes for cell bioimaging.

Tyramine signaling amplification (TSA) technology is generally applied in immunofluorescence, enzyme-linked immunoassays, in situ hybridization techniques, etc. Successful amplification of fluoresence signals cannot be achieved without excellent fluorescent dyes. BODIPY fluorophore is an ideal probe for cell fluorescence imaging, but pristine BODIPY cannot be direct used in the TSA system. In the paper, the new red-shifted tyramide-conjugated BODIPY (BDP-B/C/D) was synthesized via the Knoevenagel condensation reaction, which based on the tyramide-conjugated BODIPY (BDP-A). The synthesized dyes were combined with tyramine to obtain which could be used as a fluorescent substrate for enzymatic reaction of TSA. By using the selected substrate (BDP-C) in TSA, we found it to be more sensitive than the commercial dye 594 styramide for the detection of low-abundance antigen proteins.

Engineering Neuroglobin for Synthesis of Chiral Organoborons via Carbene B-H Insertion.

Organoborons have recently received much attention, while a biocatalytic platform for the synthesis of chiral organoborons is limited only to Rma cytochrome c. In this study, we exploited the other heme protein, neuroglobin (Ngb), and engineered a quadruple mutant, A15C/H64G/V68F/F28M Ngb, by redesigning the heme active site using the structural information on A15C Ngb and molecular docking studies. The enzyme was shown to be efficient in catalyzing carbene transfer B-H insertion reactions between pyridine/quinoline boranes and benzyl 2-diazopropanoates and their derivatives (29 examples). The designed cavity in the heme distal site favors the binding of large volume substrates such as those containing a quinoline, naphthyl, or biphenyl group. As further determined by the X-ray crystallography of 6c, the chiral products are in the R-configuration, with up to 98:2 e.r. Furthermore, both the whole cell and cell lysate containing the enzyme are reactive toward the B-H insertion reactions. This study presents a convenient biocatalytic platform that may be generally applicable for the synthesis of functional chiral organoborons.

WazaGaY: An Innovative Aza-BODIPY-Derived Near-Infrared Fluorescent Probe for Enhanced Tumor Imaging.

Aza-BODIPYs represent a class of fluorophores in which the π-conjugated system is rigidified and stabilized by a boron atom. A promising strategy to enhance their fluorescence properties involves replacing the boron atom with a metal ion. Here, we describe the synthesis and characterization of a water-soluble derivative where the metal is a gallium(III) ion, termed WazaGaY (water-soluble aza-GaDIPY). Water solubility is ensured by two ammonium substituents, inducing a bathochromic shift and a significant increase in quantum yield compared to that of the dimethylamino analog. The cellular behavior of WazaGaY-1 was observed across different tumor cells. In vivo, the distribution and safety profiles were determined, and tumor uptake was assessed in various tumor types. Following intravenous injection, WazaGaY-1 enabled clear discrimination of tumors engrafted subcutaneously in mice with high tumor-to-muscle ratios (ranging from 7 to 20), even in the absence of specific conjugation. Its potential as a contrast agent for fluorescence-guided surgery was confirmed.

Biotin-Pt(IV)-Ru(II)-Boron-Dipyrromethene Prodrug as "Platin Bullet" for Targeted Chemo- and Photodynamic Therapy.

Using the principle of "Magic Bullet", a cisplatin-derived platinum(IV) prodrug heterobimetallic Pt(IV)-Ru(II) complex, cis,cis,trans-[Pt(NH3)2Cl2{Ru(tpy-BODIPY)(tpy-COO)}(biotin)]Cl2 (Pt-Ru-B, 2), having two axial ligands, namely, biotin as water-soluble B-vitamin for enhanced cellular uptake and a BODIPY-ruthenium(II) (Ru-B, 1) photosensitizer having N,N,N-donor tpy (4'-phenyl-2,2':6',2″-terpyridine) bonded to boron-dipyrromethene (BODIPY), is developed as a "Platin Bullet" for targeted photodynamic therapy (PDT). Pt-Ru-B exhibited intense absorption near 500 nm and emission near 513 nm (λex = 488 nm) in a 10% dimethyl sulfoxide-Dulbecco's phosphate-buffered saline medium (pH 7.2). The BODIPY complex on light activation generates singlet oxygen as the reactive oxygen species (ROS) giving a quantum yield (ΦΔ) of ∼0.64 from 1,3-diphenylisobenzofuran experiments. Pt-Ru-B exhibited preferential cellular uptake in cancer cells over noncancerous cells. The dichlorodihydrofluorescein diacetate assay confirmed the generation of cellular ROS. Confocal images revealed its mitochondrial internalization. Pt-Ru-B showed submicromolar photocytotoxicity in visible light (400-700 nm) in A549 and multidrug-resistant MDA-MB-231 cancer cells. It remained nontoxic in the dark and less toxic in nontumorigenic cells. Cellular apoptosis and alteration of the mitochondrial membrane potential were evidenced from the respective Annexin V-FITC/propidium iodide assay and JC-1 dye assay. A wound healing assay using A549 cells and Pt-Ru-B revealed inhibition of cancer cell migration, highlighting its potential as an antimetastatic agent.

Potent BODIPY-based photosensitisers for selective mitochondrial dysfunction and effective photodynamic therapy.

The development of new and improved mitochondria-targeting photosensitisers (PSs) for photodynamic therapy (PDT) remains highly desirable, due to the critical role the mitochondria play in maintaining healthy cellular function. Here, we report the design, synthesis, photophysical properties and biological characterisation of a series of di-iodinated BODIPY-based PSs, BODIPY-Mito-I-n, for mitochondria-targeted PDT applications. Six BODIPY-Mito-I-n analogues were synthesised in good yields, with fast reaction times of between 30 and 60 min under mild conditions. The di-iodination of the BODIPY scaffold enabled highly efficient population of the triplet state, leading to high singlet oxygen (1O2) photosensitisation efficiencies (ΦΔ = 0.55-0.65). All BODIPY-Mito-I-n compounds exhibited very high photocytotoxic activity towards HeLa cells, with IC50,light values of between 1.30 and 6.93 nM, due to photoinduced 1O2 generation. Notably, the poly(ethylene glycol) (PEG)-modified BODIPY-Mito-I-6 showed remarkably lower dark cytotoxicity (IC50,dark = 6.68-7.25 µM) than the non-PEGylated analogues BODIPY-Mito-I-1 to BODIPY-Mito-I-5 (IC50,dark = 0.58-1.09 µM), resulting in photocytotoxicity indices up to 2120. Mechanistic studies revealed that BODIPY-Mito-I-6 induced reactive oxygen species overproduction and mitochondrial dysfunction in cells upon irradiation, leading to significant cell death through a combination of apoptosis and necrosis. It is anticipated that our design will contribute to the development of more effective mitochondria-targeting PSs for cancer therapy.

Synthesis and Photodynamic Activities of Pyridine- or Pyridinium-Substituted Aza-BODIPY Photosensitizers.

In this work, various novel pyridinyl- and pyridinium-modified Aza-BODIPY PSs were designed and constructed based on monoiodo Aza-BODIPY PSs (BDP-4 and BDP-15) in an attempt to construct "structure-inherent organelles-targeted" PSs to endow potential organelle-targeting ability. Pyridinyl PSs displayed potent photodynamic efficacy, and monorigidified PSs were very effective. The monorigidified PS 20 with meta-pyridinyl moiety displayed the most potent photoactivity and negligible dark toxicity with a favorable dark/phototoxicity ratio (>4800). To our surprise, monorigidified PS with meta-pyridinyl moiety (e.g., 20) was lipid droplet-targeted. 20 showed good cellular uptake and intracellular ROS generation compared with BDP-15. The preliminary cell death process exploration indicated that 20 resulted in lipid peroxidation and induced cell death through an iron-independent ferroptosis-like cell death pathway. In vivo antitumor efficacy experiments manifested that 20 significantly inhibited tumor growth and outperformed BDP-15 and Ce6 even under a single low-dose light irradiation (30 J/cm2).

BODIPYs α-appended with distyryl-linked aryl bisboronic acids: single-step cell staining and turn-on fluorescence binding with D-glucose.

Small-molecule sensors that are selective for particular sugars are rare. The synthesis of BODIPYs appended with two boronic acid units is reported, alongside cellular staining/labelling and turn-on fluorescence binding data for carbohydrates. The structural frameworks were designed using computational methods, leaning on the chelation characteristics of bis(boronic acids) and the photophysical properties of BODIPYs. Selective binding to glucose is demonstrated via emission and absorption methods, and the challenges of using NMR data for studying carbohydrate binding are discussed. Furthermore, crystal structures, cell permeability and imaging properties of the BODIPYs appended with two boronic acid units are described. This work presents boronic-acid-appended BODIPYs as a potential framework for tunable carbohydrate sensing and chemical biology staining.

Protein aggregation monitoring in cells under oxidative stress: a novel fluorescent probe based on a 7-azaindole-BODIPY derivative.

The development of new fluorescent probes as molecular sensors is a critical step for the understanding of molecular mechanisms. BODIPY-based probes offer versatility due to their high fluorescence quantum yields, photostability, and tunable absorption/emission wavelengths. Here, we report the synthesis and evaluation of a novel 7-azaindole-BODIPY derivative to probe hydrophobic proteins as well as protein misfolding and aggregation. In organic solvents, this compound shows two efficiently interconverting emissive excited states. In aqueous environments, it forms molecular aggregates with unique photophysical properties. The complex photophysics of the 7-azaindole-BODIPY derivative was explored for sensing applications. In the presence of albumin, the compound is stabilized in hydrophobic protein regions, significantly increasing its fluorescence emission intensity and lifetime. Similar effects occur in the presence of protein aggregates but not with other macromolecules like pepsin, DNA, Ficoll 40, and coconut oil. Fluorescence lifetime imaging microscopy (FLIM) and two-photon fluorescence microscopy on breast (MCF-7) and lung (A549) cancer cells incubated with this compound display longer fluorescence lifetimes and higher emission intensity under oxidative stress. Synchrotron FTIR micro spectroscopy confirmed that the photophysical changes observed were due to protein misfolding and aggregation caused by the oxidative stress. These findings demonstrate that this compound can serve as a fluorescent probe to monitor protein misfolding and aggregation triggered by oxidative stress.

A highly fluorescent and readily accessible all-organic photosensitizer model for advancing image-guided cancer PDT.

The potential of using image-guided photodynamic therapy (ig-PDT) for cancer, especially with highly biocompatible fluorescent agents free of heavy atoms, is well recognized. This is due to key advantages related to minimizing adverse side effects associated with standard cancer chemotherapy. However, this theragnostic approach is strongly limited by the lack of synthetically-accessible and easily-modulable chemical scaffolds, enabling the rapid design and construction of advanced agents for clinical ig-PDT. In fact, there are still very few ig-PDT agents clinically approved. Herein we report a readily accessible, easy-tunable and highly fluorescent all-organic small photosensitizer, as a model design for accelerating the development and translation of advanced ig-PDT agents for cancer. This scaffold is based on BODIPY, which assures high fluorescence, accessibility, and ease of performance adaptation by workable chemistry. The optimal PDT performance of this BODIPY dye, tested in highly resistant pancreatic cancer cells, despite its high fluorescent behavior, maintained even after fixation and cancer cell death, is based on its selective accumulation in mitochondria. This induces apoptosis upon illumination, as evidenced by proteomic studies and flow cytometry. All these characteristics make the reported BODIPY-based fluorescent photosensitizer a valuable model for the rapid development of ig-PDT agents for clinical use.

A golgi targeting viscosity rotor for cancer diagnosis in living cells and tissues.

Unveiling the intricate relationship between cancer and Golgi viscosity remains an arduous endeavor, primarily due to the lack of Golgi-specific fluorescent probes tailored for viscosity measurement. Considering this formidable obstacle, we have triumphed over the challenge by devising a bespoke Golgi-specific viscosity probe, aptly named GOL-V. This ingenious innovation comprises the viscosity rotor BODIPY intricately tethered to the Golgi-targeting moiety benzsulfamide. GOL-V exhibits remarkable sensitivity to fluctuations in viscosity, the fluorescence intensity of GOL-V increased 114-fold when the viscosity value was increased from 2.63 to 937.28 cP. Owing to its remarkable capacity to suppress the TICT state under conditions of heightened viscosity. Moreover, its efficacy in sensitively monitoring Golgi viscosity alterations within living cells is also very significant. Astonishingly, our endeavors have culminated in not only the visualization of Golgi viscosity at the cellular and tissue levels but also in the clinical tissue samples procured from cancer patients. Harnessing the prowess of GOL-V, we have successfully demonstrated that Golgi viscosity could serve as a discerning marker for detecting the presence of cancer. The convergence of these exceptional attributes firmly establishes GOL-V as an immensely potent instrument, holding immense potential in the realm of cancer diagnosis.

Hydrogen peroxide responsive theranostics for cancer-selective activation of DNA alkylators and real-time fluorescence monitoring in living cells.

Triple negative breast cancer (TNBC) is a notoriously difficult disease to treat, and many of the existing TNBC chemotherapeutics lack tumor selectivity and the capability for simultaneously visualizing and monitoring their own activity in the biological context. However, TNBC cells have been known to generate high levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). To this end, three novel small molecule theranostics 1a, 1c, and 2 consisting of both H2O2-responsive nitrogen mustard prodrug and profluorophore character have been designed, synthesized, and evaluated as targeted cancer therapeutics and bioimaging agents. The three theranostics comprise of boronate esters that deactivate nitrogen mustard functional groups and fluorophores but allow their selective activation through H2O2-specific oxidative deboronation for the release of the active drug and fluorophore. The three theranostics demonstrated H2O2-inducible DNA-alkylating capability and fluorescence turn-on properties in addition to selective anticancer activity. They are particularly effective in killing TNBC MDA-MB-468 cells with high H2O2 level while safe to normal epithelial MCF-10A cell. The conjugated boron-masked fluorophores in 1c and 2 are highly responsive towards H2O2, which enabled tracking of the theranostics in living cellular mitochondria and nucleus organelles. The three theranostics 1a, 1c, and 2 are capable of both selective release of the active drug to take effect in H2O2-rich cancer sites and simultaneously monitoring its activity. This single molecule system is of utmost importance to understand the function, efficacy, and mechanism of the H2O2-activated prodrugs and theranostics within the living recipient.

BODIPY in Alzheimer's disease diagnostics: A review.

Timely diagnosis and therapy of Alzheimer's disease remains one of the greatest questions in medicinal chemistry of neurodegenerative disease. The lack of low-cost sensors capable of reliable detection of structural changes in AD-related proteins is the driving factor for the development of novel molecules with affinity for AD hallmarks. The development of cheap, safe diagnostic methods is a highly sought-after area of research. Optical fluorescent probes are of great interest due to their non-radioactivity, low cost, and ability of the real-time visualization of AD hallmarks. Boron dipyrromethene (BODIPY)-based fluorophore is one promising fluorescent unit for in vivo labeling due to its high photostability, easy modification, low toxicity, and cell-permeability. In recent years, many fluorescent BODIPY-based probes capable of Aß plaque, Aß soluble oligomers, neurofibrillary tangles (NFT) optical detection, as well as probes with copper ion chelating units and viscosity sensors have been developed. In this review, we summarized BODIPY derivatives as fluorescent sensors capable of detecting pathological features of Alzheimer's disease, published from 2009 to 2023, as well as their design strategies, optical properties, and in vitro and in vivo activities.

Development of BODIPY-based fluorescent probes for imaging Aß aggregates and lipid droplet viscosity.

Alzheimer's disease (AD), gradually recognized as an untreatable neurodegenerative disorder, has been considered to be closely associated with Aß plaques, which consist of ß-amyloid protein (Aß) and is one of the crucial pathological features of AD. There are no obvious symptoms in the initial phase of AD, and thus the therapeutic interventions are important for early diagnosis of AD. Moreover, recent researches have indicated that lipid droplets might serve as a potential ancillary biomarker, and its viscosity changes are closely associated to the pathological process of AD. Herein, two newly fluorescent probes 5QSZ and BQSZ have been developed and synthesized for identifying Aß aggregates and detecting the viscosity of lipid droplet. After selectively binding to Aß aggregates, 5QSZ and BQSZ exhibited linear and obvious fluorescence enhancements (32.58 and 36.70 folds), moderate affinity (Kd = 268.0 and 148.6 nM) and low detection limits (30.11 and 65.37 nM) in aqueous solutions. Further fluorescence staining of 5QSZ on brain tissue sections of APP/PS1 transgenic mouse exhibited the higher selectivity of 5QSZ towards Aß aggregates locating at the core of the plaques. Furthermore, 5QSZ and BQSZ displayed strong linear fluorescence emission enhancements towards viscosity changes and would be utilized to monitor variation in cellular viscosity induced by LPS or monensin. These two probes were non-cytotoxic and showed good localization in lipid droplets. Therefore, 5QSZ and BQSZ could serve as potential bi-functional fluorescent probes to image Aß aggregates and monitor the viscosity of lipid droplets, which have significant implications for the early diagnosis and progression of AD.

Near-infrared BODIPY photosensitizers for two-photon excited singlet oxygen generation and tumor cell photodynamic therapy.

In this paper, two near-infrared BODIPY photosensitizers, Id-BDPI and Cz-BDPI, were obtained by modifying the indole and carbazole aromatic heterocycles in the core of BODIPY. The maximum absorption wavelengths of Id-BDPI and Cz-BDPI were 694 nm and 722 nm, and their singlet oxygen yields were 48% and 48.4%, respectively. In the simulated tumor cell photodynamic therapy, Id-BDPI and Cz-BDPI could effectively inhibit the growth of A549 tumor cells under near-infrared light. Meanwhile, the lysosomal co-localization coefficients of Id-BDPI and Cz-BDPI with A549 tumor cells were 0.94 and 0.89, respectively, showing high lysosomal targeting ability and biocompatibility. The two-photon absorption cross sections measured at 1050 nm by the Z-scanning method were 661.8 GM and 715.6 GM, respectively, and Cz-BDPI was further successfully applied to two-photon fluorescence imaging and two-photon excited singlet oxygen generation in zebrafish. The above results indicate that the introduction of aromatic heterocycles can effectively enhance the photodynamic efficacy of BODIPY photosensitizers, and the larger two-photon absorption cross section also brings potential for two-photon photodynamic therapy applications.

Borylation via iridium catalysed C-H activation: a new concise route to duocarmycin derivatives.

The synthesis of the ethyl ester analogue of the ultrapotent antitumour antibiotic seco-duocarmycin SA has been achieved in eleven linear steps from commercially available starting materials. The DSA alkylation subunit can be made in ten linear steps from the same precursor. The route involves C-H activation at the equivalent of the C7 position on indole leading to a borylated intermediate 9 that is stable enough for peptide coupling reactions but can be easily converted to the free hydroxyl analogue.

Heavy-atom-free BODIPY dendrimer: utilizing the spin-vibronic coupling mechanism for two-photon photodynamic therapy in zebrafish.

In this study, the heavy-atom-free BODIPY dendrimer TM4-BDP was synthesized for near-infrared photodynamic therapy, and was composed of a triphenylamine-BODIPY dimer and four 1-(2-morpholinoethyl)-1H-indole-3-ethenyl groups. The TM4-BDP could achieve near-infrared photodynamic therapy through two different photosensitive pathways, which include one-photon excitation at 660 nm and two-photon excitation at 1000 nm. In the one-photon excitation pathway, the TM4-BDP could generate singlet oxygen and superoxide radicals under 660 nm illumination. In addition, the one-photon PDT experiment in human nasopharyngeal carcinoma (CNE-2) cells also indicated that the TM4-BDP could specifically accumulate in lysosomes and show great cell phototoxicity with an IC50 of 22.1 µM. In the two-photon excitation pathway, the two-photon absorption cross-section at 1030 nm of TM4-BDP was determined to be 383 GM, which means that it could generate reactive oxygen species (ROS) under 1000 nm femtosecond laser excitation. Moreover, the two-photon PDT experiment in zebrafish also indicated the TM4-BDP could be used for two-photon fluorescence imaging and two-photon induced ROS generation in biological environments. Furthermore, in terms of the ROS generation mechanism, the TM4-BDP employed a novel spin-vibronic coupling intersystem crossing (SV-ISC) process for the mechanism of ROS generation and the femtosecond transient absorption spectra indicated that this novel SV-ISC mechanism was closely related to its charge transfer state lifetime. These above experiments of TM4-BDP demonstrate that the dendrimer design is an effective strategy for constructing heavy-atom-free BODIPY photosensitizers in the near-infrared region and lay the foundation for two-photon photodynamic therapy in future clinical trials.

Synthesis and characterization of organoselenium based BODIPY and its application in living cells.

Phenylselenide based BODIPY probe was successfully synthesized and characterized by NMR spectroscopic techniques (1H, 13C and 77Se NMR), mass spectrometry and single crystal XRD. Surprisingly, crystal packing diagram of the probe showed formation of 1-D strip through intermolecular F---H interaction. The probe was screened with various Reactive Oxygen Species (ROS) and found to be selective for superoxide ion over other ROS via "turn-on" fluorescence response. The probe selectively and sensitively detects superoxide with a lower detection limit (43.34 nM) without interfering with other ROS. The quantum yield of the probe was found to increase from 0.091 % to 30.4 % (334-fold) after oxidation. Theoretical calculations (DFT and TD-DFT) were also performed to understand the sensing mechanism of the probe. The probe was able to effectively detect superoxide inside living cells without any toxic effect.

Synthesis of New Bodipy Hydrazide Fluorescent Probes for the Detection of Carbonylated Proteins Generated by Oxidative Stress.

Oxidative stress is a cellular disorder implicated in various severe diseases and redox biology and represents an important field of research for the last decades. One of the major consequences of oxidative stress is the carbonylation of proteins, which is also a reliable marker to assess protein oxidative modifications. Accumulation of carbonylated proteins has been associated with aging and age-related diseases and can ultimately causes cell death. Detection of these oxidative modifications is essential to understand and discover new treatments against oxidative stress. We describe the design and the synthetic pathway of new BODIPY fluorescent probes functionalized with hydrazide function for protein carbonyl labeling to improve existing methodologies such as 2D-Oxi electrophoresis. Hydrazide BODIPY analogues show very good fluorescent properties such as NIR emission up to 633 nm and quantum yield up to 0.88. These new probes were validated for the detection and quantification of carbonylated proteins with 2D-Oxi electrophoresis using mouse muscle protein extracts, as well as both flow cytometry and microscopy using oxidant stressed C2 C12 cells.

Bora-Diaza-Indacene Based Fluorescent Probes for Simultaneous Visualisation of Lipid Droplets and Endoplasmic Reticulum.

Organelle selective fluorescent probes, especially those capable of concurrent detection of specific organelles, are of benefit to the research community in delineating the interplay between various organelles and the impact of such interaction in maintaining cellular homeostasis and its disruption in the diseased state. Although very useful, such probes are synthetically challenging to design due to the stringent lipophilicity requirement posed by different organelles, and hence, the lack of such probes being reported so far. This work details the synthesis, photophysical properties, and cellular imaging studies of two bora-diaza-indacene based fluorescent probes that can specifically and simultaneously visualise lipid droplets and endoplasmic reticulum; two organelles suggested having close interactions and implicated in stress-induced cellular dysfunction and disease progression.

Novel BODIPY-based nano-biomaterials with enhanced D-A-D structure for NIR-triggered photodynamic and photothermal therapy.

Near-infrared (NIR) responsive nanoparticles are an important platform for multimodal phototherapy. Importantly, the simultaneous NIR-triggered photodynamic (PDT) and photothermal (PTT) therapy is a powerful approach to increase the antitumor efficiency of phototherapic nanoparticles due to the synergistic effect. Herein, a boron dipyrromethene (BODIPY)-based amphiphilic dye with enhanced electron donor-acceptor-donor (D-A-D) structure (BDP-AP) was designed and synthesized, which could self-assemble into stable nanoparticles (BDP-AP NPs) for the synergistic NIR-triggered PDT/PTT therapy. BDP-AP NPs synchronously generated singlet oxygen (1O2) and achieved preeminent photothermal conversion efficiency (61.42%). The in vitro and in vivo experiments showed that BDP-AP NPs possessed negligible dark cytotoxicity and infusive anticancer performance. BDP-AP NPs provide valuable guidance for the construction of PDT/PTT-synergistic NIR nanoagents to improve the efficiency of photoinduced cancer therapy in the future.