Investigations on the Synthesis, Reactivity, and Properties of Perfluoro-α-Benzo-Fused BOPHY Fluorophores.

The synthesis and reactivity of 3,8-dibromo-dodecafluoro-benzo-fused BOPHY 2 are reported, via SN Ar with O-, N- S- and C-nucleophiles, and in Pd(0)-catalyzed cross-coupling reactions (Suzuki and Stille). The resulting perfluoro-BOPHY derivatives were investigated for their reactivity in the presence of various nucleophiles. BOPHY 3 displays reversible color change and fluorescence quenching in the presence of bases (Et3 N, DBU), whereas BOPHY 7 reacts preferentially at the α-pyrrolic positions, and BOPHY 8 undergoes regioselective fluorine substitution in the presence of thiols. The structural and electronic features of the fluorinated BOPHYs were studied by TD-DFT computations. In addition, their spectroscopic and cellular properties were investigated; BOPHY 10 shows the most red-shifted absorption/emission (λmax 659/699 nm) and 7 the highest fluorescence (Φf =0.95), while all compounds studied showed low cytotoxicity toward human HEp2 cells and were efficiently internalized.

Syntheses and Investigations of Conformationally Restricted, Linker-Free α-Amino Acid-BODIPYs via Boron Functionalization.

A series of α-amino acid-BODIPY derivatives were synthesized using commercially available N-Boc-l-amino acids, via boron functionalization under mild conditions. The mono-linear, mono-spiro, and di-amino acid-BODIPY derivatives were obtained using an excess of basic (histidine, lysine, and arginine), acidic (aspartic acid), polar (tyrosine, serine), and nonpolar (methionine) amino acid residues, in yields that ranged from 37 to 66%. The conformationally restricted mono-spiro- and di-amino acid-BODIPYs display strong absorptions in the visible spectral region with high molar extinction coefficients and significantly enhanced fluorescence quantum yields compared with the parent BF2-BODIPY. Cellular uptake and cytotoxicity studies using the human HEp2 cell line show that both the presence of an N,O-bidentate spiro-ring and basic amino acids (His and Arg) increase cytotoxicity and enhance cellular uptake. Among the series of BODIPYs tested, the spiro-Arg- and spiro-His-BODIPYs were found to be the most cytotoxic (IC50 ∼ 22 µM), while the spiro-His-BODIPY was the most efficiently internalized, localizing preferentially in the cell lysosomes, ER, and mitochondria.

Synthesis, characterization, and cellular investigations of porphyrin- and chlorin-indomethacin conjugates for photodynamic therapy of cancer.

Indomethacin is a potent non-steroidal anti-inflammatory drug (NSAID) with a strong selective inhibitor activity towards cyclooxygenase-2 (COX-2), an enzyme that is highly overexpressed in various tumour cells, being involved in tumourigenesis. Concomitantly, porphyrins have gained much attention as promising photosensitizers (PSs) for the non-invasive photodynamic therapy (PDT) of cancer. Herein, we report the design, and determine the singlet oxygen generation capacity and in vitro cellular toxicity of porphyrin- and chlorin-indomethacin conjugates (P2-Ind and C2-Ind). Both the conjugates were obtained in high yields and were characterized by 1H, 19F and 13C NMR as well as by high resolution mass spectrometry. The singlet oxygen generation properties were assessed by the 1,3-diphenylisobenzofuran singlet oxygen trap method, which showed that C2 and C2-Ind are the best singlet oxygen photosensitizers. In addition, it was found that the presence of indomethacin did not influence the singlet oxygen generation of porphyrin or chlorin. Cytotoxicity studies of the conjugate in human HEp2 cells revealed that the porphyrin- and chlorin-indomethacin conjugates have similar dark cytotoxicities, while chlorin C2 was shown to be the most phototoxic. Despite having lower cellular uptake than C2-Ind after 24 hours, chlorin C2 had a broad localization in HEp2 cells while the chlorin-indomethacin conjugate C2-Ind could be detected in the form of small aggregates. DFT calculations were performed to shed light on the reaction energy involved in the formation of the indomethacin conjugates and to compare the relative stability of selected isomers in solution. Moreover, the calculated energy of their first excited triplet state structures confirmed their use as suitable photosensitizers to generate singlet oxygen for PDT.

Synthesis and Investigation of Linker-Free BODIPY-Gly Conjugates Substituted at the Boron Atom.

An efficient synthesis of boron-functionalized cyclic BODIPY-Gly conjugates, using commercially available N-protected glycine amino acids and a BF2-BODIPY moiety as starting materials, is reported. The existence of two conformers (up and down) is revealed through comprehensive DFT calculations and 1H and 11B NMR analyses. The experimental and computational results indicate that all BODIPYs are stable in aqueous solutions at neutral pH and that Fmoc-BODIPY (4) is more stable than Ac-BODIPY (6) in the presence of trifluoroacetic acid (TFA). In part due to their enhanced rigidity, all BODIPY-Gly conjugates display increased fluorescence quantum yields (0.6 < Φ < 0.9) relative to the corresponding BF2-BODIPY, making them excellent candidates for fluorescence imaging applications.

Synthesis, Characterization, and Evaluation of Near-IR Boron Dipyrromethene Bioconjugates for Labeling of Adenocarcinomas by Selectively Targeting the Epidermal Growth Factor Receptor.

A series of five boron dipyrromethene (BODIPY) bioconjugates containing an epidermal growth factor receptor (EGFR)-targeted pegylated LARLLT peptide and/or a glucose or biotin ethylene diamine group were synthesized, and the binding capability of the new conjugates to the extracellular domain of EGFR was investigated using molecular modeling, surface plasmon resonance, fluorescence microscopy, competitive binding assays, and animal studies. The BODIPY conjugates with a LARLLT peptide were found to bind specifically to EGFR, whereas those lacking the peptide bound weakly and nonspecifically. All BODIPY conjugates showed low cytotoxicity (IC50 > 94 µM) in HT-29 cells, both in the dark and upon light activation (1.5 J/cm2). Studies of nude mice bearing subcutaneous human HT-29 xenografts revealed that only BODIPY conjugates bearing the LARLLT peptide showed tumor localization 24 h after intravenous administration. The results of our studies demonstrate that BODIPY bioconjugates bearing the EGFR-targeting peptide 3PEG-LARLLT show promise as near-IR fluorescent imaging agents for colon cancers overexpressing EGFR.

Synthesis and investigation of phthalocyanine-biotin conjugates.

An isothiocyanato-functionalized phthalocyanine (Pc) was synthesized in good yield from the corresponding amine-substituted Pc. This Pc reacted with ethanolamine, biotin hydrazine, and biotin diethylamine under mild conditions (room temperature in DMF or DMSO in the presence of TEA) to produce the corresponding thiourea products in 60-75% yields. All Pcs showed intense Q absorptions in DMF around 677 nm, emissions centered at 683 nm, and fluorescence quantum yields in the range 0.18-0.27. The Pcs were phototoxic to human carcinoma HEp2 cells (IC50 ~ 7 at 1.5 J/cm2) and localized in multiple organelles, including the lysosomes, Golgi and ER. One biotin-Pc conjugate was injected via tail vein into nude mice bearing HT-29 tumors and demonstrated selective localization in the tumor tissue.

Synthesis, photodynamic activities, and cytotoxicity of new water-soluble cationic gallium(III) and zinc(II) phthalocyanines.

The cationic Ga(III) and Zn(II) phthalocyanines carrying N-methyl-pyridinium groups at eight peripheral ß-positionshave been synthesized. These complexes are highly soluble in dimethyl sulfoxide (DMSO) and moderately soluble in water and phosphate buffered saline (PBS); both Ga(III)Cl and Zn(II) complexes have shown no aggregation in water up to 1.2 × 10-4 and 1.5 × 10-5 M, respectively. A higher water-solubility of Ga(III)Cl complex as compared to Zn(II) complex is ascribed to the presence of an axially coordinated chloride. The spectroscopic properties, photogeneration of singlet oxygen (1O2), and cytotoxicity of these complexes have been investigated. The absolute quantum yields (ΦΔabsolute) for the photogeneration of singlet oxygen using Ga(III)Cl and Zn(II) complexes have been determined to be 4.4 and 5.3%, respectively, in DMSO solution. The cytotoxicity and intracellular sites of localization of Ga(III)Cl and Zn(II) complexes have been evaluated in human HEp2 cells. Both complexes, localized intracellularly in multiple organelles, have shown no cytotoxicity in the dark. Upon exposure to a low light dose (1.5 J/cm2), however, Zn(II) complex has exhibited a high photocytotoxicity. The result suggests that Zn(II) complex can be considered as a potential photosensitizer for Photodynamic therapy (PDT).

Lewis-Acid-Catalyzed BODIPY Boron Functionalization Using Trimethylsilyl Nucleophiles.

A novel and straightforward strategy for boron functionalization in boron dipyrromethenes (BODIPYs) is developed. In particular, this synthetic strategy provides new possibilities for the synthesis of sp2 N-substituted (B-NCS and -NCO), benzotriazole- and trifluoroacetamide-substituted BODIPYs that were hitherto unknown. These new BODIPYs display an array of highly desirable photophysical properties (0.04 < Φf < 0.86), paving the road for further investigations in material applications.

Syntheses, Spectroscopic Properties, and Computational Study of ( E, Z)-Ethenyl and Ethynyl-Linked BODIPYs.

A series of ( E, Z)-ethenyl- and ethynyl-linked boron dipyrromethene (BODIPY) dimers were synthesized in 23-34% yields by condensation of pyrroles with the corresponding bis-benzaldehydes, followed by oxidation and boron complexation. The BODIPY dimers were characterized by 1H, 13C, and 11B NMR spectroscopy, high-resolution mass spectrometry, and, in the cases of 1b, 2, and 3, by X-ray crystallography. The spectroscopic properties for this series of dimers were investigated in tetrahydrofuran solutions, and very similar absorption and emission profiles were observed for all dimers. Density functional theory calculations show minimal conjugation between the two BODIPY units in the dimers, as a result of the large dihedral angle between the BODIPYs and the linker. The ( E)-ethenyl-linked dimer 1a showed the highest fluorescence quantum yield of all dimers investigated in this study.

Structure Based Modulation of Electron Dynamics in meso-(4-Pyridyl)-BODIPYs: A Computational and Synthetic Approach.

The effects of structural modification on the electronic structure and electron dynamics of cationic meso-(4-pyridyl)-BODIPYs were investigated. A library of 2,6-difunctionalized meso-(4-pyridyl)-BODIPYs bearing various electron-withdrawing substituents was designed, and DFT calculations were used to model the redox properties, while TDDFT was used to determine the effects of functionalization on the excited states. Structural modification was able to restructure the low-lying molecular orbitals to effectively inhibit d-PeT. A new meso-(4-pyridyl)-BODIPY bearing 2,6-dichloro groups was synthesized and shown to exhibit enhanced charge recombination fluorescence. The fluorescence enhancement was determined to be the result of functionalization modulating the kinetics of the excited state dynamics.

Stability of a Series of BODIPYs in Acidic Conditions: An Experimental and Computational Study into the Role of the Substituents at Boron.

Boron-dipyrromethene (BODIPY) dyes have been extensively investigated in recent years for a variety of bioanalytical and bioimaging applications. The success of these applications relies on the stability of BODIPYs, particularly under acidic conditions. In this work, the stability of a series of 4,4'-disubstituted BODIPYs (-F, -CN, -Ph, -Me, -OMe) toward addition of excess trifluoroacetic acid (TFA) was studied systematically and comprehensively through 11B and 1H NMR, UV-vis, fluorescence, thin layer chromatography, mass spectrometry, and infrared. The results indicate that 4,4'-dicyano-BODIPY 2 is the most stable among this series and remains unchanged even 3 days after addition of excess TFA. On the other hand, 4,4'-dimethyl-BODIPY 3 and 4,4'-dimethoxy-BODIPY 5 are the least stable, toward addition of TFA, and the 4,4'-diphenyl and 4,4'-difluoro-BODIPYs 1 and 4 were found to have intermediate stability. The experimental analysis and comparison with theoretical calculations indicate that the 4,4'-dicyano-BODIPY 2 has the greater aromaticity of the series, as evaluated by the BLA parameter, decreased charge on boron, and upon TFA addition it forms an unusually stable BODIPY 2···TFA complex. On the other hand, all other BODIPYs decompose within hours after TFA addition. Computational modeling demonstrates that 4,4'-dicyano substitution increases aromaticity and stabilizes the B-N bond, resulting in the most stable compound from the series studied.

Tetrafluorobenzo-Fused BODIPY: A Platform for Regioselective Synthesis of BODIPY Dye Derivatives.

A novel route for the synthesis of unsymmetrical benzo-fused BODIPYs is reported using 4,5,6,7-tetrafluoroisoindole as a precursor. The reactivity of the 3,5-dibromo tetrafluorobenzo-fused BODIPY was investigated under nucleophilic substitution and Pd(0)-catalyzed cross-coupling reaction conditions. In addition to the 3,5-bromines, one α-fluoro group on the benzo-fused ring can also be functionalized, and an unusual homocoupling with formation of a bisBODIPY was observed. This new class of fluorinated BODIPYs could find various applications in medicine and materials.

Syntheses and PDT activity of new mono- and di-conjugated derivatives of chlorin e6.

Syntheses of three new chlorin e6 conjugates for PDT of tumors are reported. One of the new compounds 17 is conjugated with lysine at the 131-position, but the others are mono-conjugated 14 or diconjugated 15 with the non-amino acid species ethanolamine. Cellular experiments with the three new compounds and previously synthesized non-amino acid 152-conjugates (7-10), 131-monoconjugates 14, 16, and a 131,152-diconjugate 12 are reported. In vitro cytotoxicity experiments show that the 131-conjugates are more toxic than the 152-conjugates, and the most toxic derivative (dark- and photo-toxicity) is the 131-ethylenediamine conjugate 11. The most useful PDT photosentitizers appear to be the ethanolamine derivatives, conjugated at the 152- and the 131,152-positions; these show high phototoxicity but relatively low dark toxicity compared with 11, and also the highest dark/photo cytotoxicity ratios.

BODIPY dyads from a,c-biladiene salts.

Asymmetric dimers of BODIPY dyes were synthesized from a,c-biladiene salts in good yields; this work constitutes a new versatile approach to the synthesis of BODIPY dyads, which display red-shifted absorptions and emissions in the visible spectral region, higher fluorescence quantum yields and larger Stokes shifts compared with monomeric BODIPYs. The X-ray structure of a 5,5'-dibromo-BODIPY dyad was obtained, and the reactivity of this compound under Suzuki cross-coupling reaction conditions was investigated.

Synthesis and Spectroscopic and Cellular Properties of Near-IR [a]Phenanthrene-Fused 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacenes.

A new synthetic method to build aryl-fused 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPYs) is reported. The intramolecular cyclization step was completed in a short time (1-2 h) and in high yields (>90%), due to the intrinsic rigid structural conformation of the precursor BODIPY and the high reactivity of its 1,7-bromo groups. The [a]phenanthrene-fused BODIPYs 4a-c were characterized by NMR spectroscopy, HRMS, DFT calculations, and, in the case of 4a, by X-ray crystallography. Spectroscopic studies show that 4a-c strongly absorb and emit in the NIR spectral region, in the range 642-701 nm. In addition, BODIPYs 4b and 4c exhibit no toxicity in the light or dark in HEp2 cells and accumulate intracellularly in a time-dependent manner, mainly in the cell endoplasmic reticulum. These results suggest the potential use of [a]phenanthrene-fused BODIPYs as NIR bioimaging probes.

Synthesis of BODIPY-Peptide Conjugates for Fluorescence Labeling of EGFR Overexpressing Cells.

Regioselective functionalization of 2,3,5,6,8-pentachloro-BODIPY 1 produced unsymmetric BODIPY 5, bearing an isothiocyanate group suitable for conjugation, in only four steps. The X-ray structure of 5 reveals a nearly planar BODIPY core with aryl dihedral angles in the range 47.4-62.9°. Conjugation of 5 to two EGFR-targeting pegylated peptides, 3PEG-LARLLT (6) and 3PEG-GYHWYGYTPQNVI (7), under mild conditions (30 min at room temperature), afforded BODIPY conjugates 8 and 9 in 50-80% isolated yields. These conjugates showed red-shifted absorption and emission spectra compared with 5, in the near-IR region, and were evaluated as potential fluorescence imaging agents for EGFR overexpressing cells. SPR and docking investigations suggested that conjugate 8 bearing the LARLLT sequence binds to EGFR more effectively than 9 bearing the GYHWYGYTPQNVI peptide, in part due to the lower solubility of 9, and its tendency for aggregation at concentrations above 10 µM. Studies in human carcinoma HEp2 cells overexpressing EGFR demonstrated low dark and photo cytotoxicities for BODIPY 5 and the two peptide conjugates, and remarkably high cellular uptake for both conjugates 8 and 9, up to 90-fold compared with BODIPY 5 after 1 h. Fluorescence imaging studies in HEp2 cells revealed subcellular localization of the BODIPY-peptide conjugates mainly in the Golgi apparatus and the cell lysosomes. The low cytotoxicity of the new conjugates and their remarkably high uptake into EGFR overexpressing cells renders them promising imaging agents for cancers overexpressing EGFR.

Synthesis and Spectroscopic Investigation of a Series of Push-Pull Boron Dipyrromethenes (BODIPYs).

A series of push-pull BODIPYs bearing multiple electron-donating and electron-acceptor groups were synthesized regioselectively from 2,3,5,6,8-pentachloro-BODIPY, and characterized by NMR spectroscopy, HRMS, and X-ray crystallography. The influence of the push-pull substituents on the spectroscopic and electrochemical properties of BODIPYs was investigated. Bathochromic shifts were observed for both absorbance (up to 37 nm) and emission (up to 60 nm) in different solvents upon introduction of the push-pull moieties. DFT calculations, consistent with the spectroscopic and cyclic voltammetry studies, show decreased HOMO-LUMO energy gaps upon the installation of the push-pull moieties. BODIPY 7 bearing thienyl groups on the 2 and 6 positions showed the largest λmax for both absorption (635-653 nm) and emission (706-707 nm), but also the lowest fluorescence quantum yields. All BODIPYs were nontoxic in the dark (IC50 > 200 µM) and showed low phototoxicity (IC50 > 100 µM, 1.5 J/cm2) toward human HEp2 cells. Despite the relatively low fluorescence quantum yields, the push-pull BODIPYS were effective for cell imaging, readily accumulating within cells and localizing mainly in the ER and Golgi. Our structure-property studies can guide future design of functionalized BODIPYs for various applications, including bioimaging and in dye-sensitized solar cells.

Enhanced Hypsochromic Shifts, Quantum Yield, and π-π Interactions in a meso,ß-Heteroaryl-Fused BODIPY.

We report the synthesis and investigation of an unprecedented 8-heteroaryl-fused BODIPY 4. This compound exhibits enhanced π-π stacking in the solid state, unusually large blue-shifts in the absorbance and emission spectra, and higher fluorescence quantum yield than its unfused precursor; DFT calculations suggest a small energy gap for 4 and strong electronic communication between the 8-OPh and the BODIPY core.

Synthesis and spectroscopic properties of ß,ß'-dibenzo-3,5,8-triaryl-BODIPYs.

A series of ß,ß'-bicyclo-3,5-diaryl-BODIPYs were synthesized from the corresponding ß,ß'-bicyclo-3,5-diiodo-BODIPYs (1a,b) via Pd(0)-mediated Suzuki cross-coupling reactions in 82-92% yields. Subsequent aromatization with DDQ afforded the corresponding ß,ß'-dibenzo-aryl-BODIPYs, which showed red-shifted absorptions and emissions in the near-IR range. The dibenzo-appended BODIPYs showed characteristic 1H-, 13C-, 11B- and 19F-NMR shifts, and nearly planar conformations by X-ray crystallography.

Targeting of the epidermal growth factor receptor with mesoporphyrin IX-peptide conjugates.

The synthesis and in vitro evaluation of four mesoporphyrin IX-peptide conjugates designed to target EGFR, over-expressed in colorectal and other cancers, are reported. Two peptides with known affinity for EGFR, LARLLT (1) and GYHWYGYTPQNVI (2), were conjugated to mesoporphyrin IX (MPIX, 3) via one or both the propionic side chains, directly (4, 5) or with a triethylene glycol spacer (7, 8). The conjugates were characterized using NMR, MS, CD, SPR, UV-vis and fluorescence spectroscopies. Energy minimization and molecular dynamics suggest different conformations for the conjugates. SPR studies show that conjugate 4, bearing two LARLLT with no PEG spacers, has the greatest affinity for binding to EGFR, followed by conjugate 7 with two PEG and two LARLLT sequences. Molecular modeling and docking studies suggest that both conjugates 4 and 7 can bind to monomer and dimer EGFR in open and closed conformations. The cytotoxicity and cellular targeting ability of the conjugates were investigated in human HEp2 cells over-expressing EGFR. All conjugates showed low dark- and photo-toxicities. The cellular uptake was highest for conjugates 4 and 8 and lowest for 7 bearing two LARLLT linked via PEG groups, likely due to decreased hydrophobicity. Among the conjugates investigated 4 is the most efficient EGFR-targeting agent, and therefore the most promising for the detection of cancers that over-express EGFR.