"Switched-on" flexible chalcogenopyrylium photosensitizers. Changes in photophysical properties upon binding to DNA

2,4-Bis(4-dimethylaminophenyl)-6-alkylthiopyrylium and selenopyrylium dyes are essentially nonfluorescent (phi F < 0.001) and are poor generators of singlet oxygen in aqueous solution. However, upon complexation to calf thymus DNA, quantum yields for both fluorescence and generation of singlet oxygen increased dramatically. Irradiation of the dye-DNA complexes produced strand breaks in the DNA. The photodamage is not observed in the absence of oxygen and is suppressed by the addition of the singlet oxygen quencher imidazole. The inactivation of the pseudo-rabies virus upon treatment of oxygenated leukodepleted 20% hematocrit red blood cell suspensions with the chalcogenopyrylium dyes and light followed the same trend observed with quantum yields for the generation of singlet oxygen in the dye-DNA complexes.     

Contribution of singlet oxygen to the accelerated decolorizing of dye mixtures

Binary mixtures of dispersed dyes, which during irradiation with polychromatic light give an effect of accelerated decolorization, were studied. Quantum yields of the formation of singlet oxygen by individual dyes were estimated during irradiation of solutions in ethyl acetate with light of 435 and 546 nm as well as quantum yields of the reaction of dyes with singlet oxygen. It was found that the contribution of the reaction of dyes with singlet oxygen to the effects of accelerated decolorization were vanishingly small. The quantum yield of the photodestruction of dyes in a mixture was 10^–3-10^–5 and the quantum yield of reaction with singlet oxygen was less than 10^–6. It was concluded that accelerated decolorization of the systems studied is not determined by reaction with singlet oxygen.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 9, pp. 2014–2017, September, 1991.     

Halogenated Squaraine Dyes as Potential Photochemotherapeutic Agents. Synthesis and Study of Photophysical Properties and Quantum Efficiencies of Singlet Oxygen Generation*

Abstract— We describe the synthesis and photophysical studies, including measurements of quantum yields of triplet excited states and singlet oxygen generation of bis(3,5-dibromo-2,4,6-trihydroxyphenyl)squaraine (2) and bis(3,5-diiodo-2,4,6-trihydroxyphenyl)squaraine (3). These dyes exist in solution in the protonated, neutral, single and double depro-tonated forms, depending on pH. The pK_a values of these dyes were found to be relatively lower than those of the parent bis(2,4,6-trihydroxyphenyl)squaraine (1). Only the single deprotonated forms (Sq) of 2 and 3 showed measurable fluorescence. In microheterogeneous media such as in the presence of β-cyclodextrin, cetyltrunethylammonium bromide and polyvinylpyrrolidone), bathochromic shifts in the absorption and emission spectra of Sq were observed with a substantial enhancement in their fluorescence yields. Triplet excited states are the main transient intermediates obtained upon 532 nm laser excitation of the various forms of 2 and 3 in methanol. These triplets have lifetimes in the range from 0.061 to 132 μs. The triplet quantum yields of double deprotonated forms are low (φT = <0.01), whereas the neutral and Sq^?forms of 2 (φr = 0.12 and 0.22) and 3 (φ_T= 0.24 and 0.5), respectively, exhibited significant triplet yields. Quantum yields of singlet oxygen generation by Sq^?forms of 2 and 3 were determined in methanol and were found to be 0.13 and 0.47, respectively, which are in good agreement with the triplet yields obtained in these systems.     

Synthesis and Characterization of Near‐Infrared Absorbing Water Soluble Squaraines and Study of their Photodynamic Effects in DLA Live Cells

Here, we report the synthesis, photophysical properties and photodynamic effects in DLA live cells of three water soluble squaraine dyes, viz. bisbenzothiazolium squaraine dyes SQMI and SQDI with iodine in one and both benzothiazolium units, respectively, and an unsymmetrical squaraine dye ASQI containing iodinated benzothiazolium and aniline substituents. The diiodinated SQDI showed an anomalous trend in both fluorescence and triplet quantum yields over the monoiodinated SQMI, with SQDI showing higher fluorescence and lower triplet quantum yields compared to SQMI. Nanosecond laser flash photolysis of SQDI and SQMI indicated the formation of triplet excited states with quantum yield of 0.19 and 0.26, respectively. On photoirradiation, both the SQDI and SQMI generate singlet oxygen and it was observed that both dyes undergoing oxidation reactions with the singlet oxygen generated. ASQI which exhibited a lower triplet quantum yield of 0.06 was, however, stable and did not react with the singlet oxygen generated. In vitro cytotoxicity studies of these dyes in DLA live cells were performed using Trypan blue dye exclusion method and it reflect an order of cytotoxicity of SQDI>SQMI>ASQI. Intracellular generation of the ROS was confirmed by dichlorofluorescein assay after the in vitro PDT.     

PHOTOSENSITIZING EFFICIENCIES, TUMOR- and CELLULAR UPTAKE OF DIFFERENT PHOTOSENSITIZING DRUGS RELEVANT FOR PHOTODYNAMIC THERAPY OF CANCER

Abstract Several parameters of the following dyes, all relevant as sensitizers for photochemotherapy of cancer, have been studied: Photofrin II (PII), hematoporphyrin (HP)-di-hexyl-ether, HP-di-ethyl-ether, tetra (3-hydroxyphenyl) porphyrin, (3THPP), tetraphenyl porphine tetrasulphonate (TPPS₄) aluminium phthalocyanine tetrasulfonate (A1PCTS), aluminium phthalocyanine (A1PC), chlorin e, (Chi e₆) and merocyanine 540 (MC 540). The following parameters and features of these dyes were studied: (1) Tumor uptake in C3H mouse mammary carcinomas. (2) Skin/tumor concentration ratio in the same animal system. (3) Triton X-114/H₂0 partition coefficients at different pH-values. (4) Uptake of the dyes by human cells of the line NHIK 3025. (5) Relative fluorescence quantum yields of the dyes bound to cells. (6) Absorption-, fluorescence-excitation- and fluorescence-emission spectra of the cell-bound dyes. (7) Relative quantum yields for photoinactivation of cells after 18 h incubation with the dyes. (8) Relative quantum yields of photodegradation of the singlet oxygen trap 1,3-diphenylisobenzofuran (DPBF) in cells after 18 h incubation with the dyes. The following main conclusions were drawn: (1) 3THPP was the best and most selective tumor localizer of the dyes tested, followed by AIPCTS, TPPS₄, PII and Chi e.,. (2) The Triton X-114/H₂0 partition coefficient of most of the dyes decreased with increasing pH. (3) The cellular uptake of the dyes (18 h incubation in medium with 3% serum) increased with increasing Triton X-114/H₂0 partition coefficient. (4) HP-di-hexyl-ether had the highest quantum yields both for photoinactivation of cells and degradation of cell-bound DPBF, followed by the other lipophilic porphyrins and Chi e₆. The water-soluble dyes TPPS₄ and AIPCTS had quantum yields of the order of ten times lower than those of the lipophilic porphyrins. (5) There was a clear correlation between the quantum yields for cell-inactivation and those for photodegradation of DPBF, suggesting that the same reactive photoinduced species is involved in both processes. This suggestion was strengthened by the observation that DPBF reduced the quantum yield of cell inactivation. Thus, all the tested dyes seem to act via a type II process. (6) All of the dyes, even the water-soluble TPPS₄ and AIPCTS, are aggregated in aqueous solutions, and the cells bind both monomers and aggregates. (7) A significant fraction of the cell-bound dyes was located close to tryptophan-containing proteins. (8) Cell-bound Chi e,₆ had the highest fluorescence quantum yield of the dyes.     

SINGLET OXYGEN GENERATION BY FUROCOUMARIN TRIPLET STATES—I. LINEAR FUROCOUMARINS (PSORALENS)

Abstract— Triplet extinction coefficients and hence singlet → triplet intersystem crossing quantum yields have been measured in benzene for a number of linear furocoumarins including pseudopsoralen, 5, 8-dimethoxypsoralen, 4, 5', 8-trimethylpsoralen and 3-carbethoxypseudopsoralen. These triplet yields were then used in conjunction with the corresponding quantum yields of singlet oxygen formation, measured in oxygenated solution, to estimate the fractions of furocoumarin triplets which when quenched by ground state oxygen produce singlet excited oxygen, similar data being obtained for psoralen, 5-methoxypsoralen, 8-methoxypsoralen and 3-carbethoxypsoralen. The superoxide anion radical was not detected from these oxygen quenching reactions, nor was a contribution to the singlet oxygen yield found from furocoumarin excited singlet state quenching by oxygen. The fraction of furocoumarin-oxygen quenching interactions leading to singlet oxygen varied between 0.13 (for 5, 8-dimethoxypsoralen) and unity (for 3-carbethoxypsoralen), and thus needs to be taken into account, as well as the triplet quantum yields, in assessing photobiological processes involving singlet oxygen.     

Aza‐BODIPY Derivatives: Enhanced Quantum Yields of Triplet Excited States and the Generation of Singlet Oxygen and their Role as Facile Sustainable Photooxygenation Catalysts

A new series of aza‐BODIPY derivatives ( 4 a – 4 c , 5 a , c , and 6 b , c ) were synthesized and their excited‐state properties, such as their triplet excited state and the yield of singlet‐oxygen generation, were tuned by substituting with heavy atoms, such as bromine and iodine. The effect of substitution has been studied in detail by varying the position of halogenation. The core‐substituted dyes showed high yields of the triplet excited state and high efficiencies of singlet‐oxygen generation when compared to the peripheral‐substituted systems. The dye 6 c , which was substituted with six iodine atoms on the core and peripheral phenyl ring, showed the highest quantum yields of the triplet excited state (Φ_T=0.86) and of the efficiency of singlet‐oxygen generation (Φ_Δ=0.80). Interestingly, these dyes were highly efficient as photooxygenation catalysts under artificial light, as well as under normal sunlight conditions. The uniqueness of these aza‐BODIPY systems is that they are stable under irradiation conditions, possess strong red‐light absorption (620–680 nm), exhibit high yields of singlet‐oxygen generation, and act as efficient and sustainable catalysts for photooxygenation reactions.     

Design,synthesis, and photophysical characterization of novel pentacyclic red shifted azine dyes

As part of our continuing efforts to develop second generation photodynamic therapeutic agents [1], we synthesized three pentacyclic azine dyes that were designed with the aid of MNDO calculations to absorb visible light having wavelengths longer than 600 nm. Photophysical measurements for the azine dyes 1,4,8,11-tetraethyl-1,2,3,4,8,9,10,11,13-nonahydrodipyrazino[2,3-b:2′, 3′-i] phenazinium acetate, 13 , 1,4,8,11-tetraethyl-2,3,4,8,9,10,11,12,13-octahydro-13-methyldipyrazino[2,3-b:2,3′-i]phenazinium iodide, 14 , and l,4,8,11,13-pentaethyl-2,3,4,8,9,10,11,12,13-octahydrodipyrazino[2,3-b:2′,3′-i]phenazinium iodide, 15 , are highlighted by a 35 nm red shift in their absorption spectra and a 5–7 fold increase in their singlet oxygen quantum yield relative to tricyclic model compounds 3,7-bis(diethylamino)phenazinium chloride, 20 , and 3,7-bis(diethylamino)-5-ethyl-phenazinium iodide, 21 , which were also synthesized for this study. Incorporation of rigid peripheral tetrahydropyrazino ring systems in the pentacyclic azines 13 , 14 , and 15 are responsible for the improved fluorescence and singlet oxygen quantum yields relative to the tricyclic azines 20 and 21 .     

Phototoxicity of Some Bromine-Substituted Rhodamine Dyes: Synthesis, Photophysical Properties and Application as Photosensitizers

Abstract— The synthesis of some bromine-substituted rhodamine derivatives viz. , 4,5-dibromorhodamine methyl ester (dye 2) and 4,5-dibromorhodamine n -butyl ester (dye 3) are reported. These dyes were synthesized to promote a more efficient cancer cell photosensitizer for potential use in in vitro bone marrow purging in preparation for autologous bone marrow transplantation. Spectroscopic and photo-physical characterization of these dyes together with rhodamine 123 (dye 1) are reported in water, methanol, eth-anol and also in a microheterogeneous system, sodium dodecyl sulfate. The possible mechanism of photosensi-tization is characterized in terms of singlet oxygen efficiency of these dyes. Singlet oxygen quantum yields for bromine-substituted dyes are in the range of 0.3-0.5 depending on the solvent. For dye 1 no singlet oxygen production is found. The photodynamic actions of these dyes in different cell lines are tested. It was found that dye 2 and dye 3 are efficient photosensitizers and mediate eradication of K562, EM2, myeloid cell lines (CML) and the SMF-AI rhabdomyosarcoma line.     

Diiodinated Mono- and Dipyridylvinyl BODIPY Dyes: Photophysicochemical Properties,in vitro Antibacterial Studies,Molecular Docking and Theoretical Calculations

In this study, novel mono- and dipyridylvinyl boron dipyrromethene dyes are prepared to compare their photodynamic antimicrobial chemotherapy (PACT) activities against Staphylococcus aureus to the corresponding core dyes. Pyridylvinyl substitution at the 3- or 3,5-positions of a meso-4-bromophenylBODIPY core dye via a Knoevenagel reaction with an aromatic 2-bromopyridinecarboxaldehyde shifts the major BODIPY spectral band to longer wavelength. The extended π-conjugation red shifts the main spectral band into the 602–618 nm region in CHCl₃, THF, ethanol and DMSO after monopyridylvinyl substitution and to 685–704 nm after dipyridylvinyl substitution. An enhancement of the population of the T₁ state through the incorporation of iodine atoms at the 2,6-positions results in moderately high singlet oxygen quantum yields in DMSO. The π-extended dyes were found to have significantly lower PACT activities than the diiodinated core dye.     

Synthesis and Photophysics of BF2‐Rigidified Partially Closed Chain Bromotetrapyrroles: Near Infrared Emitters and Photosensitizers

We report the synthesis, crystallographic, optical, and triplet and singlet oxygen generation properties of a series of BF₂‐rigidified partially closed chain bromotetrapyrroles as near infrared emitters and photosensitizers. These novel dyes were efficiently synthesized from a nucleophilic substitution reaction between pyrroles and the 3,5‐bromo‐substituents on the tetra‐ and hexabromoBODIPYs and absorb in the near‐infrared region (681–754 nm) with high molar extinction coefficients. Their fluorescent emission (708–818 nm) and singlet oxygen generation properties are significantly affected by alkyl substitutions on the two uncoordinated pyrrole units of these dyes and the polarity of solvents. Among them, dyes 4 ca and 4 da show good singlet oxygen generation efficiency and good NIR fluorescence emission (fluorescence quantum yields of 0.14–0.43 in different solvents studied).     

PHOTOPHYSICAL PROPERTIES OF 3,3'-DIALKYLTHIACARBOCYANINE DYES IN HOMOGENEOUS SOLUTION

The photophysical properties of 3,3′-dialkylthiacarbocyanine iodides and chlorides were measured in various solvents. It was found that photoisomerization and fluorescence are the major contributors to the deactivation of the excited singlet state; intersystem crossing occurs with only a very low efficiency. In ethanol, a triplet yield of 0.004 and a singlet oxygen quantum yield of 0.002 were determined. The photophysical parameters of these dyes are not substantially influenced by the length of the alkyl chain or the size of the halide counterion. The substitution of an ethyl with an octadecyl-chain only slightly hinders photoisomerization, and the replacement of the chloride with an iodide reduces only marginally the fluorescence lifetimes and fluorescence quantum yields in chloroform. A significant external heavy-atom effect is observed using dibromoethane as a solvent: triplet and singlet oxygen yields increase7–10-fold, and the triplet lifetime decreases from 55 μs to 15 mUs.     

Synthesis and photophysical properties of silicon phthalocyanines with axial siloxy ligands bearing alkylamine termini

Eleven silicon phthalocyanines which can be grouped into two homologous series [SiPc[OSi(CH3)2(CH2)(n)N(CH3)2]2, n = 1-6 (series 1), and SiPc[OSi(CH3)2(CH2)3N((CH2)(n)H)2]2, n = 1-6 (series 2)] as well as an analogous phthalocyanine, SiPc[OSi(CH3)2(CH2)3NH2]2, were synthesized. The ground state absorption spectra, the triplet state dynamics, and singlet oxygen quantum yields of 10 of these phthalocyanines were measured. All compounds displayed similar ground state absorption spectral properties in dimethylformamide solution with single Q band maxima at 668 +/- 2 nm and B band maxima at 352 +/- 1 nm. Photoexcitation of all compounds in the B bands generated the optical absorptions of the triplet states which decayed with lifetimes in the hundreds of microseconds region. Oxygen quenching bimolecular rate constants near 2 x 10(9) M(-1) s(-1) were measured, indicating that energy transfer to oxygen was exergonic. Singlet oxygen quantum yields, phi(delta), were measured, and those phthalocyanines in which the axial ligands are terminated by dimethylamine residues at the end of alkyl chains having four or more methylene links exhibited yields near > or = 0.35. Others gave singlet oxygen quantum yields near 0.2, and still others showed singlet oxygen yields of <0.1. The reduced singlet oxygen yields are probably caused by a charge transfer quenching of the 1pi,pi* state of the phthalocyanine by interaction with the lone pair electrons on the nitrogen atoms of the amine termini. In some cases, these can approach and interact with the electronically excited pi-framework, owing to diffusive motions of the flexible oligo-methylene tether.     

Synthesis,characterization and properties of some metallophthalocyanine complexes substituted by N -piperidineethanol

The preparation of eight metallophthalocyanine complexes substituted by N-piperidineethanol was achieved by tetramerization of 3-[2-(piperidin-1-yl)ethoxyl] phthalonitrile and 4-[2-(piperidin-1-yl)ethoxyl]phthalonitrile in the presence of a metal salt with n-pentanol as solvent and DBU as catalyst, respectively. These complexes were characterized by IR, elemental analysis, ¹H NMR and mass spectra. Some properties such as UV/visible absorption spectra, rate of singlet oxygen yields, fluorescence spectra and quantum yields were examined and discussed.     

Singlet oxygen generation ability of squarylium cyanine dyes

The quantum yields for singlet oxygen generation of several squarylium cyanine dyes derived from benzothiazole, benzoselenazole and quinoline, displaying absorption within the so-called “phototherapeutic window” (600–1000 nm), were determined, envisioning their potential usefulness for photodynamic therapy (PDT). The determination was performed by a direct method measuring the luminescence decay of the dyes in the near infrared. Considering the absorption and the quantum yields displayed by some of the dyes, these seemed to be potential candidates as sensitizers for PDT.     

Spectral and photophysical studies on cruciform oligothiophenes in solution and the solid state

The photophysical and spectroscopic properties of a new class of oligothiophene derivatives, designated as cruciform oligomers, have been investigated in solution (room and low temperature) and in the solid state (as thin films in Zeonex matrixes). The study comprises absorption, emission, and triplet-triplet absorption spectra, together with quantitative measurements of quantum yields (fluorescence, intersystem crossing, internal conversion, and singlet oxygen formation) and lifetimes. The overall data allow the determination of the rate constants for all decay processes. From these, several conclusions are drawn. First, in solution, the main deactivation channels for the compounds are the radiationless processes: S(1) --> S(0) internal conversion and S(1) --> T(1) intersystem crossing. Second, in general, in the solid state, the fluorescence quantum yields decrease relative to solution. A comparison is made with the analogous linear alpha-oligothiophenes, revealing a lower fluorescence quantum efficiency and, in contrast to the normal oligothiophenes, that internal conversion is an important channel for the deactivation of the singlet excited state. Replacement of thiophene by 1,4-phenylene units in the longer-sized cruciform oligomer increases the fluorescence efficiency. The highly efficient generation of singlet oxygen through energy transfer from the triplet state (S(Delta) approximately 1) provides support for the measured intersystem crossing quantum yields and suggests that reaction with this may be an important pathway to consider for degradation of devices produced with these compounds.     

Heavy-Atom-Free Bay-Substituted Perylene Diimide Donor-Acceptor Photosensitizers

Perylene diimide (PDI) dyes are extensively investigated because of their favorable photophysical characteristics for a wide range of organic material applications. Fine-tuning of the optoelectronic properties is readily achieved by functionalization of the electron-deficient PDI scaffold. Here, we present four new donor-acceptor type dyads, wherein the electron donor units – benzo[1,2-b : 4,5-b’]dithiophene, 9,9-dimethyl-9,10-dihydroacridine, dithieno[3,2-b : 2’,3’-d]pyrrole, and triphenylamine-are attached to the bay-positions of the PDI acceptor. Intersystem crossing occurs for these systems upon photoexcitation, without the aid of heavy atoms, resulting in singlet oxygen quantum yields up to 80 % in toluene solution. Furthermore, this feature is retained when the system is directly irradiated with energy corresponding to the intramolecular charge-transfer absorption band (at 639 nm). Geometrical optimization and (time-dependent) density functional theory calculations afford more insights into the requirements for intersystem crossing such as spin-orbit coupling, dihedral angles, the involvement of charge-transfer states, and energy level alignment.     

PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF BENZOPORPHYRIN DERIVATIVE MONOACID RING A (BPD-MA)*

The photophysical properties of benzoporphyrin derivative monoacid ring A (BPD-MA), a second-generation photosensitizer currently in phase II clinical trials, were investigated in homogeneous solution. Absorption, fluorescence, triplet-state, singlet oxygen (O₂(¹Δ_g)) sensitization studies and photobleaching experiments are reported. The ground state of this chlorin-type molecule shows a strong absorbance in the red (λ≈ 688 nm, ?≈ 33 000 M^?1 cm^?1 in organic solvents). For the singlet excited state the following data were determined in methanol: energy level, E_s= 42.1 kcal mol^?1, lifetime, Φ_f= 5.2 ns and fluorescence quantum yield, Φ_f= 0.05 in air-saturated solution. The triplet state of BPD-MA has a lifetime, τ_f >. 25 ns, an energy level, E_T= 26.9 kcal mol^?1 and the molar absorption coefficient is ?_T= 26 650 M^?1 cm^?1 at 720 nm. A dramatic effect of oxygen on the fluorescence (φ_f) and intersystem crossing (φ_T) quantum yields has been observed. The BPD-MA presents rather high triplet (φ_T= 0.68 under N₂-saturated conditions) and singlet oxygen (φ_Δ= 0.78) quantum yields. On the other hand, the presence of oxygen does not significantly modify the photobleaching of this photostable compound, the photodegradation quantum yield (φ_Pb) of which was found to be on the order of 5 × 10^?5 in organic solvents.     

Absorption and emission spectroscopic characterization of Ir(ppy)3

The absorption and emission spectroscopic behaviour of cyclometalated fac-tris(2-phenylpyridine) iridium(III) [Ir(ppy)₃] is studied at room temperature. Liquid solutions, doped films, and neat films are investigated. The absorption cross-section spectra including singlet–triplet absorption, the triplet–singlet stimulated emission cross-section spectra, the phosphorescence quantum distributions, the phosphorescence quantum yields and the phosphorescence signal decays are determined. In neat films fluorescence self-quenching occurs, in diluted solid solution (polystyrene and dicarbazole-biphenyl films) as well as deaerated liquid solution (toluene) high phosphorescence quantum yields are obtained, and in air-saturated liquid solutions (chloroform, toluene, tetrahydrofuran) the phosphorescence efficiency is reduced by triplet oxygen quenching. At intense short-pulse laser excitation the phosphorescence lifetime is shortened by triplet–triplet annihilation. No amplification of spontaneous emission in the phosphorescence spectral region was observed indicating higher excited-state absorption than stimulated emission.     

Determination of the quantum yield of singlet oxygen sensitized by halogenated boron difluoride dipyrromethenes

The spectral–luminescent, photophysical, and photochemical properties of dichloro-, dibromo-, and diiodo-derivatives of boron dipyrromethenate (BODIPY) have been studied, as well as the feasibility of generating singlet oxygen (¹O₂) via its photosensitization by the dihalogenated derivatives of BF₂ dipyrromethene in solutions. Quantum yields of singlet oxygen have been determined using 1,3-diphenylisobenzofuran as the ¹O₂ trap. The lowest fluorescence quantum yields have been shown to correspond to the maximum yields of singlet oxygen. It has been found that the best ¹O₂ photosensitizer among the three test dihalotetraphenylaza- BODIPY is dibromotetraphenylaza-BODIPY, which in addition possesses the highest photostability. Diiodotetramethyl-BODIPY results in the singlet oxygen yield close to unity, but it has significantly lower photostability. The yield of singlet oxygen is affected by the solvent. Dibromtetraphenylaza-BODIPY and diiodotetramethyl-BODIPY may find use as a medium in photodynamic therapy and photocatalysis of oxidation reactions.