Photodynamic treatment of melanoma cells using aza-dipyrromethenes as photosensitizers.

In this study, we report for the first time the use of four aza-dipyrromethenes (ADPMs) as photosensitizers for cancer PDT. The synthesis and characterization of the ADPMs and their photodynamic action against B16F10 melanoma cells were assessed. ADPM 2 is the best singlet oxygen generator and the most phototoxic (at 2.5 µM) towards B16F10 cells.

A glutathione-responsive photosensitizer with fluorescence resonance energy transfer characteristics for imaging-guided targeting photodynamic therapy.

Here, we have synthesized and characterized a novel activatable photosensitizer (PS) 8a in which two well-designed boron dipyrromethene (BODIPY) derivatives are utilized as the photosensitizing fluorophore and quencher respectively, which are connected by a disulfide linker via two successive Cu (І) catalyzed click reactions. The fluorescence emission and singlet oxygen production of 8a are suppressed via intramolecular fluorescence resonance energy transfer (FRET) from the excited BODIPY-based PS part to quencher unit, but both of them can be simultaneously switched on by cancer-related biothiol glutathione (GSH) in phosphate buffered saline (PBS) solution with 0.05% Tween 80 as a result of cleavage of disulfide. Also, 8a exhibits a bright fluorescence image and a substantial ROS production in A549 human lung adenocarcinoma, HeLa human cervical carcinoma and H22 mouse hepatoma cells having a relatively high concentration of GSH, thereby leading to a significant photocytotoxicity, with IC50 values as low as 0.44 µM, 0.67 µM and 0.48 µM, respectively. In addition, the photosensitizer can be effectively activated and imaged in H22 transplanted hepatoma tumors of mice and shows a strong inhibition on tumor growth. All these results suggest that such a GSH-responsive photosensitizer based on FRET mechanism may provide a new strategy for tumor-targeted and fluorescence imaging-guided cancer therapy.

Crystal structure, DNA crosslinking and photo-induced cytotoxicity of oxovanadium(IV) conjugates of boron-dipyrromethene.

Cis-dichloro-oxovanadium(IV) complexes [VO(L1/L2)Cl2], where L1 is N-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4ʎ4,5ʎ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine in 1 and L2 is N-(4-(5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-5H-4ʎ4,5ʎ4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)benzyl)-1-(pyridin-2-yl)-N-(pyridin-2-ylmethyl)methanamine in 2) having 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene as boron-dipyrromethene (BODIPY) appended dipicolylamine bases were prepared, characterized and their photocytotoxicity studied. X-ray crystal structure of 1 showed distorted octahedral geometry with a VIVON3Cl2 core having Cl-V-Cl angle of 91.93(4)°. The complexes showed variable solution conductivity properties. They were non-electrolytes in dry DMF at 25 °C but showed 1:1 electrolytic behavior in an aqueous medium due to dissociation of one chloride ligand as evidenced from the mass spectral study. Complexes 1 and 2 showed absorption bands at 500 and 535 nm, respectively. The calf thymus DNA melting study revealed their interaction through DNA crosslinking on exposure to light which was further confirmed from the alkaline agarose gel electrophoresis using plasmid supercoiled pUC19 DNA. Complex 2 showed disruption of the mitochondrial membrane potential in the JC-1 (1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloroimidacarbocyanine iodide) assay. The complexes were photocytotoxic in visible light (400-700 nm, power: 10 J cm-2) in cervical cancer HeLa and breast cancer MCF-7 cells. Complex 2 having a photoactive diiodo­boron-dipyrromethene moiety gave a singlet oxygen quantum yield (ΦΔ) value of ~0.6. It showed singlet oxygen mediated apoptotic photodynamic therapy activity with remarkably low IC50 (half maximal inhibitory concentration) value of ~0.15 µM. The cis-disposition of chlorides gave a cis-divacant 4-coordinate intermediate structure from the density functional theory (DFT) study thus mimicking the DNA crosslinking property of cisplatin.

A novel distyryl boron dipyrromethene with two functional tags for site-specific bioorthogonal photosensitisation towards targeted photodynamic therapy.

A distyryl boron dipyrromethene based photosensitiser substituted with 1,2,4,5-tetrazine and alkyne moieties was prepared. Through site-specific bioorthogonal reactions with the complementary functional tags anchored on the membrane of A431 human epidermoid carcinoma cells, this versatile photosensitiser exhibited enhanced cellular uptake and photocytotoxicity. The bioorthogonal ligation was also demonstrated in tumour-bearing nude mice.

Effect of Substituents on the Photophysical Properties and Bioimaging Application of BODIPY Derivatives with Triphenylamine Substituents.

We investigated the intramolecular charge transfer characteristics in the S1 state of boron-dipyrromethene (BODIPY) derivatives with triphenylamine (TPA) substituents, depending on the substituted position and the number of substituents. Based on the spectroscopic and theoretical results, the ß-substitution of TPA on BODIPY hybridizes locally excited and intramolecular charge transfer characteristics in the S1 state because of strong coupling between the highest occupied molecular orbitals of BODIPY and TPA moieties, and consequently, the BODIPY derivatives with ß-substituted TPAs exhibit strong red-color fluorescence around 640 nm in nonpolar and moderately polar solvents. The TPA substituent with propeller-like nonplanar geometry could prevent H-type aggregation between neighboring BODIPY derivative units and induce aggregation-induced emission enhancement (AIEE) characteristics of the BODIPY derivatives with TPA substituents, which are helpful to maintain their emission efficiencies under highly concentrated and condensed conditions. Since the red-color emission and AIEE property of the BODIPY derivatives with ß-substituted TPAs are promising characteristics for a bioimaging application, we applied these derivatives to L-929 fibroblast cells for cellular imaging. The BODIPY derivative with a single ß-substituted TPA (compound 2) was effectively loaded into porous silica nanoparticles (SNPs). Consequently, we achieved good cellular uptake of 2-SNPs and good cellular imaging, which further confirmed the bioimaging ability of 2-SNPs.

A H2O2-Responsive Boron Dipyrromethene-Based Photosensitizer for Imaging-Guided Photodynamic Therapy.

In this study, we demonstrate a novel H2O2 activatable photosensitizer (compound 7) which contains a diiodo distyryl boron dipyrromethene (BODIPY) core and an arylboronate group that quenches the excited state of the BODIPY dye by photoinduced electron transfer (PET). The BODIPY-based photosensitizer is highly soluble and remains nonaggregated in dimethyl sulfoxide (DMSO) as shown by the intense and sharp Q-band absorption (707 nm). As expected, compound 7 exhibits negligible fluorescence emission and singlet oxygen generation efficiency. However, upon interaction with H2O2, both the fluorescence emission and singlet oxygen production of the photosensitizer can be restored in phosphate buffered saline (PBS) solution and PBS buffer solution containing 20% DMSO as a result of the cleavage of the arylboronate group. Due to the higher concentration of H2O2 in cancer cells, compound 7 even with low concentration is particularly sensitive to human cervical carcinoma (HeLa) cells (IC50 = 0.95 µM) but hardly damage human embryonic lung fibroblast (HELF) cells. The results above suggest that this novel BODIPY derivative is a promising candidate for fluorescence imaging-guided photodynamic cancer therapy.

Pyriplatin-Boron-Dipyrromethene Conjugates for Imaging and Mitochondria-Targeted Photodynamic Therapy.

Monofunctional pyriplatin analogues cis-[Pt(NH3)2(L)Cl](NO3) (1-3) having boron-dipyrromethene (BODIPY) pendants (L) with 1,3,5,7-tetramethyl-8-(4-pyridyl)-4,4'-difluoroboradiazaindacene moieties were designed and synthesized, and their photocytotoxic properties were studied. The Pt-BODIPY conjugates displayed an absorption band within 505-550 nm and a green emissive band near 535 nm in 1% DMSO/DMEM (Dulbecco's modified Eagle's medium) buffer. Complex cis-[Pt(NH3)2(4-Me-py)Cl](NO3) (4) was used as a control for determining the structural aspects by X-ray crystallography. The mono- and diiodinated BODIPY complexes 2 and 3 showed generation of singlet oxygen on light activation as evidenced from the 1,3-diphenylisobenzofuran (DPBF) titration experiments. The cytotoxicity of the BODIPY complexes was tested against A549 (human lung cancer), MCF-7 (human breast cancer), and HaCaT (human skin keratinocyte) cells in dark and visible light (400-700 nm, 10 J cm-2). While complexes 2 and 3 showed excellent photocytotoxicity (IC50 ≈ 0.05 µM), they remained essentially nontoxic in the dark (IC50 > 100 µM). The emissive bands of 1 and 2 were used for cellular imaging by confocal microscopy study, which showed their mitochondrial localization. This was further supported by platinum estimation from isolated mitochondria and mitochondrial depolarization through a JC-1 assay. The photomediated apoptotic cell death was evidenced from flow cytometric assays, annexin-V/FITC-PI (fluorescein isothiocyanate-propidium iodide) and cell cycle arrest in sub-G1 and G2/M phases. The complexes bind to 9-ethylguanine as a model nucleobase to form monoadducts. A mechanistic study on DNA photocleavage activity using pUC19 DNA showed singlet oxygen as the reactive oxygen species (ROS). The combination of photodynamic therapy with DNA cross-linking property enhanced the anticancer potential of the monofunctional BODIPY-conjugates of pyriplatins.

Intracellular modulation of excited-state dynamics in a chromophore dyad: differential enhancement of photocytotoxicity targeting cancer cells.

The photosensitized generation of reactive oxygen species, and particularly of singlet oxygen [O2 (a(1) Δg )], is the essence of photodynamic action exploited in photodynamic therapy. The ability to switch singlet oxygen generation on/off would be highly valuable, especially when it is linked to a cancer-related cellular parameter. Building on recent findings related to intersystem crossing efficiency, we designed a dimeric BODIPY dye with reduced symmetry, which is ineffective as a photosensitizer unless it is activated by a reaction with intracellular glutathione (GSH). The reaction alters the properties of both the ground and excited states, consequently enabling the efficient generation of singlet oxygen. Remarkably, the designed photosensitizer can discriminate between different concentrations of GSH in normal and cancer cells and thus remains inefficient as a photosensitizer inside a normal cell while being transformed into a lethal singlet oxygen source in cancer cells. This is the first demonstration of such a difference in the intracellular activity of a photosensitizer.

Functionalization of 3,5,8-trichlorinated BODIPY dyes.

Catalytic hydrogenation of dibenzyl 5-dipyrroketone-2,9-dicarboxylates followed by decarboxylative iodination affords a 2,9-diiododipyrroketone which gives a 2,5,9-trichlorodipyrromethene hydrochloride after nucleophilic addition/elimination, with adventitious chloride to replace the two iodide groups. Treatment with BF3·Et2O gives a 3,5,8-trichloro-BODIPY that readily undergoes regioselective Stille coupling at the 8-position, or homo/mixed couplings at the 3,8- or 3,5- and 8-positions. Stepwise and controlled replacement of the 3,5- and 8-chlorine atoms using Stille reagents results in formation of a completely unsymmetrical trisubstituted BODIPY. Several examples of unsymmetrical BODIPYs were synthesized and characterized using this methodology. Structure features of new BODIPYs are discussed within the context of 14 new X-ray structures, and photophysical parameters of all new BODIPY compounds are reported and discussed.

Photoinduced energy and charge transfer in a p-phenylene-linked dyad of boron dipyrromethene and monostyryl boron dipyrromethene.

Boron dipyrromethenes (BDPs) are excellent building blocks for design of artificial light harvesting and charge separation systems. In the present work, we report the results of photophysical studies of a novel dyad, in which a BDP and a mono-styryl BDP (MSBDP) are covalently linked to each other at the meso-position via a p-phenylene unit. It was found that the photophysical properties of the dyad dissolved in polar as well as nonpolar solvents are strongly affected by two different types of interactions between the BDP and MSBDP parts, namely excitation energy transfer and photoinduced electron transfer. The first process delivers the excitation energy to the first excited singlet state of the MSBDP-part upon excitation of the BDP unit. The direct or indirect (via excitation energy transfer) population of the first excited singlet state of the MSBDP moiety is followed by hole transfer to generate the charge-separated state. In non-polar toluene, the probability of charge separation is low, whereas in polar acetonitrile the charge separation quantum yield is close to unity, resulting in strong quenching of the MSBDP fluorescence.

DNA binding and anti-cancer activity of redox-active heteroleptic piano-stool Ru(II), Rh(III), and Ir(III) complexes containing 4-(2-methoxypyridyl)phenyldipyrromethene.

The synthesis of four novel heteroleptic dipyrrinato complexes [(η(6)-arene)RuCl(2-pcdpm)] (η(6)-arene = C6H6, 1; C10H14, 2) and [(η(5)-C5Me5)MCl(2-pcdpm)] (M = Rh, 3; Ir, 4) containing a new chelating ligand 4-(2-methoxypyridyl)-phenyldipyrromethene (2-pcdpm) have been described. The complexes 1-4 have been fully characterized by various physicochemical techniques, namely, elemental analyses, spectral (ESI-MS, IR, (1)H, (13)C NMR, UV/vis) and electrochemical studies (cyclic voltammetry (CV) and differential pulse voltammetry (DPV)). Structures of 3 and 4 have been determined crystallographically. In vitro antiproliferative and cytotoxic activity of these complexes has been evaluated by trypan blue exclusion assay, cell morphology, apoptosis, acridine orange/ethidium bromide (AO/EtBr) fluorescence staining, and DNA fragmentation assay in Dalton lymphoma (DL) cell lines. Interaction of 1-4 with calf thymus DNA (CT DNA) has also been supported by absorption titration and electrochemical studies. Our results suggest that in vitro antitumor activity of 1-4 lies in the order 2 > 1 > 4 > 3.

Mechanistic insight into the formation of tetraarylazadipyrromethenes.

The tetraarylazadipyrromethene chromophore class has gained increasing attention in the past decade for a diverse set of scientific interests and applications. The most direct synthetic route available for their generation is heating of 4-nitro-1,3-diarylbutan-1-ones with an ammonium source in an alcohol solvent. Despite the practical simplicity, the reaction pathway(s) for these conversions are lengthy and unclear. To gain insight into the steps involved, (15)N labeling experiments with MS and NMR analysis were utilized for conversion of 4-nitro-1,3-diphenylbutan-1-one 1 into tetraphenylazadipyrromethene 2 with (15)NH(4)OAc. To permit examination of later stages of the reaction sequence to 2, the (15)N-labeled potential intermediate 3,5-diphenyl-1H-pyrrol-2-amine 10 was synthesized. A study of the dimerization pathway utilizing (15)N-labeled 10 revealed an unprecedented nitrogen rearrangement in the final stages of the pathway involving a ring-opening/closing of a pyrrole ring. Utilizing (15)N labeling experiments we have shown that 2,4-diphenylpyrrole 8 can also react under the reaction conditions with 3,5-diphenyl-2H-pyrrol-2-imine 7 (from oxidation of 10) to produce 2. Overall in the conversion of 1 into 2, two related pathways are ongoing concurrently; the first involves a dimerization of 3,5-diphenyl-2H-pyrrol-2-imine 7, and the other a reaction of 7 with 2,4-diphenylpyrrole 8.

Highly photostable near-infrared fluorescent pH indicators and sensors based on BF2-chelated tetraarylazadipyrromethene dyes.

In this study, a series of new BF(2)-chelated tetraarylazadipyrromethane dyes are synthesized and are shown to be suitable for the preparation of on/off photoinduced electron transfer modulated fluorescent sensors. The new indicators are noncovalently entrapped in polyurethane hydrogel D4 and feature absorption maxima in the range 660-710 nm and fluorescence emission maxima at 680-740 nm. Indicators have high molar absorption coefficients of ~80,000 M(-1) cm(-1), good quantum yields (up to 20%), excellent photostability and low cross-sensitivity to the ionic strength. pK(a) values of indicators are determined from absorbance and fluorescence measurements and range from 7 to 11, depending on the substitution pattern of electron-donating and -withdrawing functionalities. Therefore, the new indicators are suitable for exploitation and adaptation in a diverse range of analytical applications. Apparent pK(a) values in sensor films derived from fluorescence data show 0.5-1 pH units lower values in comparison with those derived from the absorption data due to Förster resonance energy transfer from protonated to deprotonated form. A dual-lifetime referenced sensor is prepared, and application for monitoring of pH in corals is demonstrated.

Replacing phenyl ring with thiophene: an approach to longer wavelength aza-dipyrromethene boron difluoride (Aza-BODIPY) dyes.

In the orignial 1,3,5,7-tetraphenyl aza-BODIPY, replacing the phenyl rings with thiophene achieved significant bathochromic shifts. One of the target molecules, DPDTAB, emitting strong NIR fluorescence with a quantum yield of 0.46 in acetonitrile, is a very competitive NIR fluorophore.

Singlet oxygen generation by novel NIR BODIPY dyes.

Five novel near-infrared BODIPY dyes were prepared for improved singlet oxygen generation using thiophene and bromine. Theoretical, optical, photostable, and singlet oxygen generation characteristics of these dyes were assessed. Predicted excitation energies by TDDFT calculations were in good agreement (ΔE ≈ 0.06 eV) with experimental data. All five dyes showed both excitation and emission in the NIR range. In particular, two dyes having sulfur and bromine atoms showed efficient singlet oxygen generation with high photostability.

8-Hydroxyquinoline-substituted boron-dipyrromethene compounds: synthesis, structure, and OFF-ON-OFF type of pH-sensing properties.

A series of four novel 8-hydroxyquinoline-substituted boron-dipyrromethene derivatives, namely 4,4-difluoro-8-(5-(8-hydroxyquinoline))-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (1), 4,4-difluoro-8-(5-(8-hydroxyquinoline))-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (2), 4,4-difluoro-8-(5-azastyryl-(8-hydroxyquinoline))-3,5-dimethyl-4-bora-3a,4a-diaza-s-indacene (3), and 4,4-difluoro-8-(5-azastyryl-(8-hydroxyquinoline))-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (4), have been synthesized and characterized by a series of spectroscopic methods. The molecular structures of 1 and 2 have been determined by single-crystal X-ray diffraction analyses. The two methyl substituents attached at C-1 and C-7 positions of boron-dipyrromethene (Bodipy) in compound 2 was revealed to prevent the free rotation of the 8-hydroxyquinoline (8-HQ) moiety, resulting in an almost vertical 8-HQ-Bodipy configuration of this compound. This is obviously different from those for 1 with the dihedral angle between 8-hydroxyquinoline and Bodipy moieties of 65.44 and 66.79° due to the lack of methyl substituents in the latter compound. The intense fluorescence from the Bodipy subunit of these compounds was revealed to gradually get diminished along with either decreasing or increasing the pH value under acidic and basic conditions, respectively, in particular for 1, 2, and 4 because of the photoinduced intramolecular electron transfer from excited Bodipy moiety to 8-HQ unit and just an opposite process. This renders these compounds the first OFF-ON-OFF type of pH-dependent fluorescent sensors. Nevertheless, both the intrinsic fluorescence of these compounds and their fluorescent quenching properties along with the change in the pH value have been found to depend on the steric configuration as well as the linking group between 8-hydroxyquinoline and Bodipy moieties, revealing the effect of molecular structure on their fluorescence properties.

8-PropargylaminoBODIPY: unprecedented blue-emitting pyrromethene dye. Synthesis, photophysics and laser properties.

Highly emitting 8-propargylaminoBODIPY (8-PAB) 2 was prepared in 94% yield. Unlike any other BODIPY structure hitherto described in the literature, 2 displays efficient emission in the blue region of the visible spectrum with a fluorescence quantum yield up to 0.94 and high laser efficiency (35%) at 483 nm.

Cofacial boron dipyrromethene (Bodipy) dimers: synthesis, charge delocalization, and exciton coupling.

A series of compounds containing two boron dipyrromethene (Bodipy) units has been synthesized and fully characterized in which the spacer between the two Bodipy groups is varied from dibenzothiophene (BD1), to dibenzofuran (BD2), to 9,9-dimethylxanthene (BD3), and finally to diphenyl ether (BD4 and BD5). For BD1-BD4 the Bodipy units adopt, to varying degrees, cofacial conformations that allow for systematic variations of both the mutual orientation and the mean separation of the two Bodipy residues. In the remaining dimer, BD5, the Bodipy units are well-separated and cannot come into close proximity. Single-crystal X-ray structures have been determined for BD1-BD3 and reveal that the "bite angle" between the two Bodipy residues decreases progressively along the series with individual values of 41.33(5) degrees, 36.95(6) degrees, and 8.57(3) degrees. Detailed (1)H and (19)F NMR studies for BD3 and BD4 show the methylene protons to be diastereotopic due to restricted rotation of the two Bodipy groups. For BD4 conformational rocking is invoked to explain the variable-temperature NMR spectra, whereby the methyl and methylene groups become inequivalent. Cyclic voltammetry indicates reversible oxidation and reduction of the Bodipy groups. However, the close proximity of the Bodipy groups in BD3 and BD4 results in two well-resolved waves in the anodic region, and slight splitting of the cathodic wave. Peak splitting is attributed to charge delocalization. Spectroelectrochemical measurements at a fixed oxidative potential reveal an optical intervalence charge-transfer (IVCT) absorption band. This IVCT band is attributed to electron exchange between the cofacially arranged neutral and mono-oxidized Bodipy units. Various levels of exciton coupling are observed for BD1-BD4, but not BD5 since here the Bodipy groups remain isolated.

Water-solubilised BF(2)-chelated tetraarylazadipyrromethenes.

Strategic incorporation of sulfonic acid, carboxylic acid or ammonium salt motifs generate water soluble BF(2)-chelated tetraarylazadipyrromethenes which exhibit strong near infra-red (NIR) emissions above 720 nm and can be readily imaged in both eukaryotic and prokaryotic cells.