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.

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.

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.

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.

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.

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.

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 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 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.

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.

Syntheses and cellular investigations of di-aspartate and aspartate-lysine chlorin e(6) conjugates.

Chlorin e6 is a tricarboxylic acid degradation product of chlorophyll a. Four chlorin e6 bis(amino acid) conjugates were regioselectively synthesized bearing two aspartate conjugates in the 13(1),17(3)- and 15(2),17(3)-positions, or at the 13(1),15(2)via an ethylene diamine linker. One additional conjugate bearing two different amino acids, lysine at 13(1)via an ethylene diamine linker and an aspartate at 15(2)via a ß-alanine linker was also synthesized. The cytotoxicity and uptake of four di(amino acid) chlorin e6 conjugates were investigated in human HEp2 cells, and compared with chlorin e6. The most cytotoxic and most taken up conjugates were the zwitterionic 13(1),15(2)-disubstituted conjugates 28 and 33; these also localized in multiple organelles. In contrast, the anionic 13(1),17(3)- and 15(2),17(3)-di-aspartyl chlorin e6 conjugates 12 and 13 showed low dark cytoxicity and lower phototoxicity compared with chlorin e6.

Synthesis and regioselective functionalization of perhalogenated BODIPYs.

Three perhalogenated BODIPYs (1b-3b), bearing chloro and bromo groups at all carbon positions, were synthesized and characterized. The reactivity of BODIPY 3b was investigated under Stille cross-coupling reactions, and single crystal X-ray analysis was used to confirm the regioselectivity of the reactions. Further substitution at the boron atom produced nona-functionalized BODIPYs 7a,b, which show 676 and 739 nm emissions with 91 and 100 nm Stokes shifts, respectively.

Synthesis of 3,8-dichloro-6-ethyl-1,2,5,7-tetramethyl-BODIPY from an asymmetric dipyrroketone and reactivity studies at the 3,5,8-positions.

The asymmetric BODIPY 1 a (BODIPY=4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), containing two chloro substituents at the 3,8-positions and a reactive 5-methyl group, was synthesized from the asymmetric dipyrroketone 3, which was readily obtained from available pyrrole 2 a. The reactivity of 3,8-dichloro-6-ethyl-1,2,5,7-tetramethyl-BODIPY 1 a was investigated by using four types of reactions. This versatile BODIPY undergoes regioselective Pd(0) -catalyzed Stille coupling reactions and/or regioselective nucleophilic addition/elimination reactions, first at the 8-chloro and then at the 3-chloro group, using a variety of organostannanes and N-, O-, and S-centered nucleophiles. On the other hand, the more reactive 5-methyl group undergoes regioselective Knoevenagel condensation with an aryl aldehyde to produce a monostyryl-BODIPY, and oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) gives the corresponding 5-formyl-BODIPY. Investigation of the reactivity of asymmetric BODIPY 1 a led to the preparation of a variety of functionalized BODIPYs with λmax of absorption and emission in the ranges 487-587 and 521-617 nm, respectively. The longest absorbing/emitting compound was the monostyryl-BODIPY 16, and the largest Stokes shift (49 nm) and fluorescence quantum yield (0.94) were measured for 5-thienyl-8-phenoxy-BODIPY 15. The structural properties (including 16 X-ray structures) of the new series of BODIPYs were investigated.

Stepwise Polychlorination of 8-Chloro-BODIPY and Regioselective Functionalization of 2,3,5,6,8-Pentachloro-BODIPY.

An effective, stepwise methodology for polychlorination of BODIPY using trichloroisocyanuric acid (TCCA) in acetic acid was developed. In this way, selectively substituted di-, tri-, tetra-, and pentachloro-BODIPYs 2-5 were prepared. The pentachloro-BODIPY is shown to undergo regioselective Pd(0)-catalyzed Stille and Suzuki coupling reactions, first at the 8-position followed by the 3,5- and then the 2,6-positions; nucleophilic substitution reactions occur first at the 8- followed by the 3,5-positions, while the 2,6 are unreactive.

Synthesis and reactivity of 4,4-Dialkoxy-BODIPYs: an experimental and computational study.

A series of boron-disubstituted O-BODIPYs were synthesized, and their structures and spectroscopic properties were investigated using both computational and experimental methods. Three methods were investigated for the preparation of 4,4-dimethoxy-BODIPYs bearing electron-donating or electron-withdrawing 8-aryl groups: method A employs refluxing in the presence of NaOMe/MeOH, method B uses AlCl3 in refluxing dichloromethane followed by addition of methanol as nucleophile, and method C involves activation of the BODIPYs using TMSOTf in refluxing toluene followed by addition of methanol. The yields obtained depend on the method used and the structure of the starting BODIPYs; for example, 1a and 3a were most efficiently prepared using method C (98 and 70%, respectively), while 2a was best prepared by method A (50%). Methods B and C were employed for the synthesis of seven new 4,4-dialkoxy-BODIPYs. 4,4-Dipropargyloxy-BODIPY 1e reacted under Cu(I)-catalyzed alkyne-azide Huisgen cycloaddition conditions to produce 4,4-bis(1,2,3-triazole)-BODIPY 4 in 78% yield. The substitution of the fluorides for alkoxy groups on the BODIPYs had no significant effect on the absorption and emission wavelengths but altered their fluorescence quantum yields. Among this series of dialkoxy-BODIPYs, the 4,4-dipropargyloxy 1e and its corresponding bis(1,2,3-triazole) 4 show the largest quantum yields in toluene and THF, respectively.

Synthesis and properties of a series of carboranyl-BODIPYs.

A series of four BODIPYs containing one or two ortho- or para-carborane clusters were synthesized using palladium(0)-catalyzed Suzuki cross-coupling or nucleophilic substitution reactions, at the 2,6- or the 8-positions of halogenated boron dipyrromethenes (BODIPYs). The spectroscopic, structural (including one X-ray) and in vitro BBB permeability of the BODIPYs using hCMEC/D3 brain endothelial cells were investigated.

Synthesis and transformations of 5-chloro-2,2'-dipyrrins and their boron complexes, 8-chloro-BODIPYs.

Symmetric dipyrrylketones 1 a,b were synthesized in two steps from the corresponding α-free pyrroles, by reaction with thiophosgene followed by oxidative hydrolysis under basic conditions. The dipyrrylketones produced the corresponding 5-chloro-dipyrrinium salts or 5-ethoxy-dipyrrins on reaction with phosgene or Meerwein's salt, respectively. Boron complexation of the dipyrrins afforded the corresponding 8-functionalized BODIPYs (borondipyrromethenes) in high yields. The 5-chloro-dipyrrinium salts reacted with methoxide or ethoxide ions to produce monopyrrole esters, presumably via a 5,5-dialkoxy-dipyrromethane intermediate. In contrast, 8-chloro-BODIPYs underwent a variety of nucleophilic substitutions of the chloro group in the presence of alkoxide ions, Grignard reagents, and thiols. In the presence of excess alkoxide or Grignard reagent, at room temperature or above, substitution at the boron center also occurred. The 8-chloro-BODIPY was a particularly useful reagent for the preparation of 8-aryl-, 8-alkyl-, and 8-vinyl-substituted BODIPYs in very high yields, using Pd(0) -catalyzed Stille cross-coupling reactions. The X-ray structures of eleven BODIPYs and two pyrroles are presented, and the spectroscopic properties of the synthesized BODIPYs are discussed.