Fluorescence Quantum Distributions Research Articles

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor Archon2.

Archon2 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense using robotic multidimensional directed evolution approach. Here we report absorption and emission spectroscopic studies of Archon2 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of Archon2 was studied by long-time attenuation coefficient measurements at room temperature (21 ± 1 °C) and at refrigerator temperature (3 ± 1 °C). The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 63 ± 3 °C). In the protein melting process protonated retinal Schiff base (PRSB) with absorption maximum at 586 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Storage of Archon2 at room temperature and refrigerator temperature caused absorption coefficient decrease because of partial protein clustering to aggregates at condensation nuclei and sedimentation. At room temperature an onset of light scattering was observed after two days because of the beginning of protein unfolding. During the period of observation (18 days at 21 °C, 22 days at 3 °C) no change of retinal isomer composition was observed indicating a high potential energy barrier of S0 ground-state isomerization.

Absorption and Emission Spectroscopic Investigation of the Thermal Dynamics of the Archaerhodopsin 3 Based Fluorescent Voltage Sensor QuasAr1.

QuasAr1 is a fluorescent voltage sensor derived from Archaerhodopsin 3 (Arch) of Halorubrum sodomense by directed evolution. Here we report absorption and emission spectroscopic studies of QuasAr1 in Tris buffer at pH 8. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation spectra were determined. The thermal stability of QuasAr1 was studied by long-time attenuation coefficient measurements at room temperature (23 ± 2 °C) and at 2.5 ± 0.5 °C. The apparent melting temperature was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 65 ± 3 °C). In the protein melting process the originally present protonated retinal Schiff base (PRSB) with absorption maximum at 580 nm converted to de-protonated retinal Schiff base (RSB) with absorption maximum at 380 nm. Long-time storage of QuasAr1 at temperatures around 2.5 °C and around 23 °C caused gradual protonated retinal Schiff base isomer changes to other isomer conformations, de-protonation to retinal Schiff base isomers, and apoprotein structure changes showing up in ultraviolet absorption increase. Reaction coordinate schemes are presented for the thermal protonated retinal Schiff base isomerizations and deprotonations in parallel with the dynamic apoprotein restructurings.

Absorption and Emission Spectroscopic Investigation of Thermal Dynamics and Photo-Dynamics of the Rhodopsin Domain of the Rhodopsin-Guanylyl Cyclase from the Nematophagous Fungus Catenaria anguillulae.

The rhodopsin-guanylyl cyclase from the nematophagous fungus Catenaria anguillulae belongs to a recently discovered class of enzymerhodopsins and may find application as a tool in optogenetics. Here the rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase from Catenaria anguillulae was studied by absorption and emission spectroscopic methods. The absorption cross-section spectrum and excitation wavelength dependent fluorescence quantum distributions of CaRh samples were determined (first absorption band in the green spectral region). The thermal stability of CaRh was studied by long-time attenuation measurements at room temperature (20.5 °C) and refrigerator temperature of 3.5 °C. The apparent melting temperature of CaRh was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 62 ± 2 °C). The photocycle dynamics of CaRh was investigated by sample excitation to the first inhomogeneous absorption band of the CaRhda dark-adapted state around 590 nm (long-wavelength tail), 530 nm (central region) and 470 nm (short-wavelength tail) and following the absorption spectra development during exposure and after exposure (time resolution 0.0125 s). The original protonated retinal Schiff base PRSBall-trans in CaRhda photo-converted reversibly to protonated retinal Schiff base PRSBall-trans,la1 with restructured surroundings (CaRhla1 light-adapted state, slightly blue-shifted and broadened first absorption band, recovery to CaRhda with time constant of 0.8 s) and deprotonated retinal Schiff base RSB13-cis (CaRhla2 light-adapted state, first absorption band in violet to near ultraviolet spectral region, recovery to CaRhda with time constant of 0.35 s). Long-time light exposure of light-adapted CaRhla1 around 590, 530 and 470 nm caused low-efficient irreversible degradation to photoproducts CaRhprod. Schemes of the primary photocycle dynamics of CaRhda and the secondary photocycle dynamics of CaRhla1 are developed.

Photo-dynamics of photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva strain HT

The photoactivated adenylyl cyclase TpPAC from the spirochete bacterium Turneriella parva was synthesized and the purified recombinant protein was characterized by biochemical and optical spectroscopic methods. TpPAC consists of a BLUF domain (BLUF=Blue Light sensor Using Flavin) and an adenylyl cyclase homology domain (CHD). A light induced cAMP cyclase activity of≈53.3nmolmg−1min−1 was measured while in the dark the cyclase activity was approximately a factor of 240 lower. The photo-cycling dynamics of the BLUF domain of TpPAC was studied by absorption spectra, fluorescence quantum distribution, and fluorescence lifetime measurements. The quantum efficiency of BLUF domain signaling state formation was found to be ϕs≈0.59. A three-component exponential recovery of the signaling state to the receptor state was observed with the time constants τrec,1=4.8s, τrec,2=34.2s, and τrec,3=293s at 21.3°C. The protein thermal stability was studied by stepwise sample heating and cooling. An apparent TpPAC melting temperature of ϑm≈46°C was determined. The photo-degradation of TpPAC in the signaling state was studied by prolonged intense light exposure at 455nm. An irreversible flavin photo-degradation was observed with quantum yield ϕD≈8.7×10−6.

Photoluminescence behavior of riboflavin and lumiflavin in liquid solutions and solid films

The absorption and emission behavior of riboflavin and lumiflavin in water, tetrahydrofuran (THF), water–starch, THF–polystyrene, starch films, and polystyrene films was studied at room temperature. Absorption cross-section spectra, fluorescence quantum distributions, and fluorescence quantum yields were determined. For the starch films additionally phosphorescence and delayed fluorescence spectra as well as phosphorescence lifetimes and delayed fluorescence lifetimes were measured and their quantum yields of intersystem-crossing, intrinsic triplet-based phosphorescence quantum yields, T1–S0 radiative lifetimes, and S0–T1 absorption strengths were calculated. A method of absolute intrinsic luminescence quantum distribution and quantum yield determination for dye doped films on transparent plates with a fluorimeter is described.

Absorption and fluorescence characteristics of photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

The spectroscopic characteristics of BLUF (BLUF = sensor of blue light using flavin) domain containing soluble adenylate cyclase (nPAC = Naegleria photo-activated cyclase) samples from the amoeboflagellate Naegleria gruberi NEG-M strain is studied at room temperature. The absorption and fluorescence spectroscopic development in the dark was investigated over two weeks. Attenuation coefficient spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation distributions were measured. Thawing of frozen nPAC samples gave solutions with varying protein nano-cluster size and varying flavin, tyrosine, tryptophan, and protein color-center emission. Protein color-center emission was observed in the wavelength range of 360–900 nm with narrow emission bands of small Stokes shift and broad emission bands of large Stokes shift. The emission spectra evolved in time with protein nano-cluster aging.

Room temperature hydrolysis of lumiflavin in alkaline aqueous solution

The degradation of lumiflavin (7,8,10-trimethyl-isoalloxazine, LF) in aqueous 1–4M sodium hydroxide solutions (pH 14–14.6) at room temperature in the dark was studied by absorption spectroscopy, fluorescence spectroscopy, and mass spectrometry. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence lifetimes at certain times after sample preparation were determined. The degradation products were analyzed by combined liquid chromatography and mass spectrometry. Lumiflavin is present in anionic form (LF−). It degrades to anionic 7,8-dimethyl-isoalloxazine (DMIA−), anionic methyl-isoalloxazine (MIA−), and to the quinoxaline derivatives of 1,2-dihydro-2-keto-1,6,7-trimethyl-quinoxaline-3-carboxylic acid (QO1), 2-methoxy-6,7-dimethyl-quinoxaline-3-carboxylic acid (QO2), methyl-quinoxaline-2-ol (QO3), and 3-hydroxy-1,6,7-trimethyl-1H-quinoxaline-2-one (QO4). The rate constants of the NaOH catalyzed lumiflavin degradation are determined for the investigated samples.

Absorption and Emission Spectroscopic Characterization of Lumichrome in Aqueous Solutions†

The spectroscopic behavior of lumichrome (7,8-dimethyl-alloxazine, LC) in aqueous solutions in a pH range from -1.08 to 14.6 is studied. Absorption spectra, fluorescence quantum distributions, quantum yields, and lifetimes are determined. The ionization stage of ground-state LC changes with rising pH from the cationic form (LCH(2)(+)) to the neutral form (LCH) with a mid-point pH of pK(c) ≈ -0.53, and to the anionic form (LC(-)) with a mid-point pH of pK(a) ≈ 12.5. Above pH 7 a partial ground-state tautomerization of LCH to 7,8-dimethyl-isoalloxazine (IAH) occurs by N1-N10 intra-molecular proton transfer. For pH > pK(a) ≈ 12.5 LCH and IAH change to the anionic forms LC(-) and IA(-), and above pH 14 LC(-) tautomerizes completely to IA(-). In the excited state some neutral lumichrome (LCH*) converts to cationic lumichrome (LCH(2)(+)) at low pH by proton transfer from H(3)O(+) to LCH*. No photoinduced excited-state tautomerization of lumichrome was observed. LCH for pH > 3 and IAH are reasonably fluorescent. The fluorescence efficiencies of LC(-) and IA(-) are lower than those of LCH and IAH. The fluorescence of LCH(2)(+) is strongly quenched likely by intra-molecular diabatic charge transfer and excited-state relaxation by potential surface touching with the ground state.

PH dependence of the absorption and emission behaviour of lumiflavin in aqueous solution

The spectroscopic behaviour of lumiflavin (7,8,10-trimethyl-isoalloxazine, oxidized form LF ox) in aqueous solutions of pH range −1.08 to 14.6 is studied. Absorption spectra, fluorescence quantum distributions, quantum yields and lifetimes are determined. The ionization stage of ground-state LF ox changes from cationic (LF oxH 2 +) at low pH (p K c ≈ 0.38) via neutral (LF oxH) to anionic (LF ox −) at high pH (p K a ≈ 10.8). The cationic, neutral, and anionic forms are identified by their different absorption spectra. LF oxH in neutral aqueous solution is reasonably fluorescent (fluorescence quantum yield ϕ F ≈ 0.29, fluorescence lifetime τ F ≈ 5.2 ns), while LF ox − is weakly fluorescent ( ϕ F ≈ 0.0042, τ F ≈ 90 ps), and LF oxH 2 + is nearly non-fluorescent ( ϕ F ≈ 3.6 × 10 −5, τ F ≈ 0.4 ps). A theory of the pH dependent equilibration of cationic, neutral and anionic molecules in the ground state and their dynamics in the excited state is developed. For lumiflavin in aqueous solution in the excited state no equilibrium distributions are reached between the cationic, neutral, and anionic forms. Some neutral excited lumiflavin transforms to the cationic ground-state form at low pH by intermolecular photo-induced proton transfer from H 3O + to LF oxH*. At high pH no photo-induced intermolecular proton transfer takes place.

Photo-degradation behaviour of roseoflavin in some aqueous solutions

An absorption and emission spectroscopic characterization of roseoflavin (8-dimethylamino-8-demethyl-riboflavin, RoF) in aqueous solutions was carried out. The studies were concentrated on roseoflavin in pH 8 phosphate buffer. Absorption cross-section spectra, fluorescence excitation spectra, fluorescence quantum distributions, fluorescence quantum yields and fluorescence lifetimes were determined. The fluorescence of RoF is quenched by photo-induced intra-molecular charge-transfer at room temperature. The photo-degradation of RoF in un-buffered water, in Tris–HCl buffer, and in phosphate buffer was studied. Phosphate buffer and to a smaller extent Tris buffer catalyse the RoF photo-degradation. Photo-excitation of the primary photoproduct, 8-methylamino-riboflavin (8-MNH-RF), enhanced the RoF degradation by triplet 8-MNH-RF – singlet RoF excitation transfer with subsequent triplet-state RoF degradation.

Fluorescence spectroscopic behaviour of folic acid

Abstract The fluorescence spectroscopic behaviour of folic acid (FA) in 4 M HCl (dominant bi-cationic form), 0.1 M HCl (bi-cationic and cationic form), citric acid–NaOH pH 6 buffer (neutral form), 0.1 M and 4 M KOH (anionic form), and trifluoroacetic acid is studied. The thermal stability is investigated. Absolute absorption cross-section spectra are determined and compared with fluorescence excitation spectra. Intrinsic fluorescence quantum distributions and fluorescence quantum yields are extracted from fluorescence spectra measurements. The temporal fluorescence decay after picosecond pulse excitation is studied. The fluorescence quenching mechanisms for the different ionic forms of FA are discussed: excited-state proton release for bi-cationic FA, photo-physical non-radiative relaxation for cationic FA, and photo-induced intra-molecular electron transfer for neutral and anionic FA. Aerobic FA in 4 M KOH at elevated temperature dehydrated to 9,10-dehydro-folic acid. Its photo-dynamics was governed by twisted intra-molecular charge transfer and photo-isomerisation.

Fluorescence behaviour of 5,10-methenyltetrahydrofolate, 10-formyltetrahydrofolate, 10-formyldihydrofolate, and 10-formylfolate in aqueous solution at pH 8

The fluorescence spectroscopic behaviour of (6R,S)-5,10-methenyltetrahydrofolate (MTHF), (6R,S)-10-formyltetrahydrofolate (10-HCO-H4folate), 10-formyldihydrofolate (10-HCO-H2folate), and 10-formylfolate (10-HCO-folate) in aqueous Tris–HCl buffer at pH 8 is studied. MTHF and 10-HCO-folate were commercially available. 10-HCO-H4folate was prepared from MTHF by hydrolysis at room temperature under anaerobic conditions. 10-HCO-H2folate was prepared by oxidation of 10-HCO-H4folate under aerobic conditions. Fluorescence quantum distributions at room temperature and fluorescence signal decays at room temperature and liquid nitrogen temperature were measured. The fluorescence lifetimes determined at room temperature (liquid nitrogen temperature) are 10 ps (2.9 ns) for MTHF, 38 ps (3.7 ns) for 10-HCO-H4folate, 80 ps (10.5 ns) for 10-HCO-H2folate, and 7.1 ns (20 ns) for 10-HCO-folate. The results are discussed in terms of dyadic (pterin–benzoyl-glutamate) photo-induced electron transfer and dyadic fluorescent dynamics.

Spectroscopic and lasing characterisation of a luminescent phenylene/bipyridine polymer

The luminescent polymer, poly[2,2′-bipyridine-5,5′-diyl-(2,5-dihexyl-1,4-phenylene)] (abbreviated PPBpy) dissolved in tetrachloroethane (TCE) and as neat film is characterised by optical absorption and emission spectroscopy. The absorption and stimulated emission cross-section spectra, the fluorescence quantum distributions, fluorescence quantum yields, degrees of fluorescence polarisation, and fluorescence lifetimes are determined. A lasing characterisation is carried out by pumping with single second harmonic pulses of a mode-locked ruby laser (wavelength 347.15 nm, duration 35 ps). The excited-state absorption at the pump-laser wavelength is determined by saturable absorption measurements. Laser oscillation of the polymer in TCE in an uncoated rectangular cell was achieved by transverse pumping using the cell windows for light feedback. From the laser threshold pump pulse energy density the effective stimulated emission cross-section at the lasing wavelength is extracted. The travelling-wave lasing behaviour of the polymer as neat film is studied by analysis of the amplification of the transverse pumped spontaneous emission. Surface emitting distributed-feedback lasing was achieved with a neat film on corrugated second-order periodic gratings.

Spectroscopic and travelling-wave lasing characterisation of tetraphenylbenzidine and di-naphtalenyl-diphenylbenzidine

The organic light emitting diode (OLED) hole transport molecules N,N,N’,N’-tetraphenylbenzidine (TPB, triphenylamine dimer TAD or TPD) and N,N’-bis(2-naphtalenyl)-N,N’-bis(phenylbenzidine) (β-NPB, naphtyl-diphenylamine dimer β-NPD), dissolved in tetrahydrofuran (THF) and as neat film, are characterized by optical absorption and emission spectroscopy. The absorption and stimulated emission cross-section spectra, the fluorescence quantum distributions, fluorescence quantum yields, degrees of fluorescence polarization, and fluorescence lifetimes are determined. The lasing behaviour is studied by picosecond laser pulse excitation (excitation wavelength 347.15 nm, pulse duration 35 ps). The excited-state absorption at the pump laser wavelength is determined by saturable absorption measurement. Low-Q laser oscillation of TPB in THF is achieved by transverse pumping of the dye in a cell. The excited-state absorption of TPB in THF at the laser wavelength is extracted from the laser threshold. In TPB neat films, wave-guided travelling-wave lasing was obtained. No laser action was achieved for β-NPB because of small S1-S0 stimulated emission cross-section, and the presence of excited-state absorption in the fluorescence wavelength region. The TPB and β-NPB results are compared with the corresponding spectroscopic and lasing behaviour of the related methyl-substituted triphenylamine dimers, 3-methyl-TPD and 4-methyl-TPD, which are well established OLED hole transport materials.

Spectroscopic and lasing characterisation of a dicarbazovinylene-MEH-benzene dye

The dye 1,4-bis(9-ethyl-3-carbazovinylene)-2-methoxy-5-(2′-ethyl-hexyloxy)-benzene (abbreviated 2CzV-MEH-B) dissolved in tetrahydrofuran (THF) and as neat film is characterised by optical absorption and emission spectroscopy. The absorption and stimulated emission cross-section spectra, the fluorescence quantum distributions, fluorescence quantum yields, degrees of fluorescence polarisation, and fluorescence lifetimes are determined. A lasing characterisation is carried out by pumping with single second harmonic pulses of a mode-locked ruby laser (wavelength 347.15 nm, pulse duration 35 ps). The excited-state absorption at the pump laser wavelength is determined by saturable absorption measurements. Laser oscillation of the dye in THF in a rectangular cell is achieved by transverse pumping using the uncoated cell windows for light feedback. From the emission behaviour around threshold the excited-state absorption cross-section spectrum in the laser active spectral region is extracted. The wave-guided travelling-wave lasing behaviour of the dye as neat film is studied by analysis of the amplification of the transverse pumped spontaneous emission. Surface emitting distributed-feedback lasing was achieved with a neat film on corrugated second-order periodic gratings.

Absorption and emission behaviour of trans- p-coumaric acid in aqueous solutions and some organic solvents

The absorption and fluorescence behaviour of trans- p-coumaric acid ( trans-4-hydroxycinnamic acid) is investigated in buffered aqueous solution over a wide range from pH 1 to pH 12, in un-buffered water, and in some organic solvents. Absorption cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and degrees of fluorescence polarisation are measured. p-Coumaric acid exists in different ionic forms in aqueous solution depending on the pH. There is an equilibrium between the neutral form ( p-CAH 2) and the single anionic form ( p-CAH −) at low pH (p K na ≈ 4.9), and between the single anionic and the double anionic form ( p-CA 2−) at high pH (p K aa ≈ 9.35). In the organic solvents studied trans- p-coumaric acid is dissolved in its neutral form. The fluorescence quantum yield of trans- p-coumaric acid in aqueous solution is ϕ F ≈ 1.4 × 10 −4 for the neutral and the single anionic form, while it is ϕ F ≈ 1.3 × 10 −3 for the double anionic form. For trans- p-coumaric acid in organic solvents fluorescence quantum yields in the range from 4.8 × 10 −5 (acetonitrile) to 1.5 × 10 −4 (glycerol) were measured. The fluorescence spectra are 7700–10,000 cm −1 Stokes shifted in aqueous solution, and 5400–8200 cm −1 Stokes shifted in the studied organic solvents. Decay paths responsible for the low fluorescence quantum yields are discussed (photo-isomerisation and internal conversion for p-CA 2−, solvent-assisted intra-molecular charge-transfer or ππ ∗ to nπ ∗ transfer and internal conversion for p-CAH 2 and p-CAH −). The solvent dependence of the first ππ ∗ electronic transition frequency and of the fluorescence Stokes shift of p-CAH 2 is discussed in terms of polar solute–solvent interaction effects. Thereby the ground-state and excite-state molecular dipole moments are extracted.

Photophysical characterization of pyrromethene 597 laser dye in cross-linked silicon-containing organic copolymers

Samples of the dipyrromethene–BF 2 dye PM597 incorporated in copolymers of 3-trimethoxysilylpropyl 2-methylprop-2-enoate (TMSPMA, number of polymerizable CC double bonds: κ = 1) with 2-(2-methylprop-2-enoyloxy)ethyl 2-methylprop-2-enoate (EGDMA, κ = 2), [2-(hydroxymethyl)-3-prop-2-(prop-2-enoyloxymethyl)propyl] prop-2-enoate (PETA, κ = 3), and [3-prop-2-enoyloxy-2,2-bis(prop-2-2-enoyloxymethyl)propyl]prop-2-enoate (PETRA, κ = 4) are characterized. The fluorescence quantum distributions, fluorescence quantum yields, degrees of fluorescence polarization, and fluorescence lifetimes are measured. The radiative lifetimes are calculated from fluorescence lifetime and quantum yield. Absorption coefficient spectra are determined from transmission measurements. Absolute absorption cross-section spectra and dye concentrations are obtained by calibration to the radiative lifetimes and to saturable absorptions. Excited-state absorption cross-sections at 527 nm are determined by saturable absorption measurements. The photo-degradation is studied under cw laser excitation conditions and quantum yields of photo-degradation are extracted. The excited-state absorption cross-sections were found to be rather small, and the photo-stability turned out to be high (up to 3 million excitation cycles before degradation) making this class of dipyrromethene dye-doped polymers attractive active laser media. Structural and thermo-mechanical properties of the materials have been determined by Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetry, densitometry, and refractometry. They improve with increasing inter-crossing (copolymerization of TMSPMA with PETA and PETRA). The laser properties of the PM597 doped copolymers were evaluated by transverse pumping with 6 ns laser pulses at 532 nm. The best laser materials resulted to be the 7:3 and 9:1 TMSPMA-monomer copolymers.

2-Amino-7-nitro-fluorenes in Neat and Mixed SolventsOptical Band Shapes and Solvatochromism

Femtosecond transient absorption spectroscopy of amino-nitro-fluorenes in the UV-visible range shows that the dynamic Stokes shift of the emission band is sensitive to infrared-active modes of the solvent. Bandshapes for stationary absorption and emission are needed to quantify the observed spectral evolution. They are reported for 2-amino-7-nitro-fluorene (ANF), 2-dimethylamino-7-nitro-fluorene (dM-ANF), and 2-di(n-butyl)amino-7-nitro-9-di(n-propyl)-fluorene (dBdP-ANF) in a variety of solvents. Bands broaden systematically with increasing solvent polarity. This effect is taken into account in an improved location of band positions. The resulting solvatochromic plots differ significantly from those that use peak positions of absorption spectra and fluorescence quantum distributions. Absorption spectra were also measured in aqueous solvent mixtures, and shifts are described by binding curves for hydrogen bonding and stepwise solvent exchange.

Photo-induced dynamics in a pyrene–isoalloxazine(flavin)–phenothiazine triad

(α R)-α-[[[10-[4-(10-heptyl-10 H-phenothiazin-3-yl)phenyl]-4,10-dihydro-2,4-dioxobenzo[ g]pteridin-3(2 H)-yl]acetyl]amino]pyrenepropionic acid phenylmethyl ester, a pyrene–isoalloxazine(flavin)–phenothiazine triad (abbreviated as PYFPT), was designed to mimic the dye-based functions of cryptochromes. PYFPT dissolved in dichloromethane and acetonitrile is characterized by absorption and fluorescence spectroscopy. Absorption cross-section spectra, fluorescence quantum distributions and quantum yields, degrees of fluorescence polarisation, and fluorescence lifetimes are determined. The triad is highly photo-stable as has been shown by monitoring absorption spectra under prolonged blue-light exposure. The absorption spectrum of PYFPT resembles the superposition of the absorption of the isoalloxazine (flavin), pyrene, and phenothiazine subchromophores. Blue light ( λ exc = 428 nm) excites exclusively the isoalloxazine subunit causing reductive electron transfer from the phenothiazine subchromophore to the locally excited isoalloxazine with a time constant of approximately 600 fs under formation of a charge transfer state which subsequently undergoes charge recombination. Near ultraviolet radiation ( λ exc = 311 nm) causes simultaneous excitation of the isoalloxazine, pyrene, and phenothiazine chromophores. Both, the locally excited pyrene and phenothiazine moieties cause oxidative electron transfer to the flavin moiety with time constants of about 80 ps and 50 ps followed by subsequent charge recombination. Förster-type energy transfer processes have been found to be one to two orders of magnitude slower.

Absorption and fluorescence spectroscopic characterisation of a phenothiazine–flavin dyad

The phenothiazine–phenylene–isoalloxazine dyad, 3-methyl-10-[4-(10-heptyl-10 H-phenothiazin-3-yl)-phenyl]-10 H-benzo[ g]pteridine-2,4-dione, dissolved in either dichloromethane or acetonitrile is characterized by absorption and emission spectroscopy. Absorption cross-section spectra, stimulated emission cross-section spectra, fluorescence quantum distributions, fluorescence quantum yields, and degrees of fluorescence polarisation are determined. The fluorescence decay is determined by time-resolved measurements. The dye photo-stability is investigated by observation of absorption spectral changes due to prolonged blue-light excitation. The absorption spectrum of the dyad resembles the superposition of the absorption of the isoalloxazine (flavin) moiety and of the phenylphenothiazine moiety. Photo-excitation of the flavin moiety causes fluorescence quenching by ground-state reductive electron transfer from phenylphenothiazine to isoalloxazine followed by charge recombination. Photo-excitation of the phenothiazine moiety causes (i) efficient excited-state oxidative electron transfer from phenothiazine to isoalloxazine with successive recombination, and (ii) moderate energy transfer followed by ground-state phenothiazine electron transfer and recombination.