CONFIGURATIONAL PHOTOISOMERIZATION OF BILIRUBIN IN VITRO–II. A COMPARATIVE STUDY OF PHOTOTHERAPY FLUORESCENT LAMPS AND LASERS

Abstract— A comparative investigation of configurational photoisomerization of bilirubin (Z.Z-BR) bound to human serum albumin (HSA) in vitro produced by fluorescent lamps currently employed in phototherapy of neonatal jaundice has been carried out by using a fast absorbance technique. Photoequilibrium Z, E-BR concentrations and rise-times are reported. Similar measurements effected with monochromatic laser lines in the blue-green spectral region are presented for comparison purposes.
Narrow-spectrum fluorescent lamps (violet, special-blue, filtered-green) produce Z,E-BR concentrations at photoequilibrium almost equal to those obtained with monochromatic lines. The photoequilibrium rise-times, however, strongly depend on the spectral bandwidth of the excitation light. Special-blue, daylight and green lamps produce Z,E-BR concentrations = 34,31, and 12%, respectively. For green lamps, the intense UV-blue-Hg lines are responsible for more than 50% of Z,E-BR concentration. Green light is found to be quite ineffective in forming Z,E isomers and very efficient in causing Z,E-BR to revert to native Z.Z-BR. Moreover, simultaneous irradiation of BR solution with special-blue and green fluorescent lamps produces almost complete inhibition of the Z→E reaction at suitable green/blue intensity ratio.
On the basis of these results a possible mechanism is presented to explain the clinical success of fluorescent green lamp phototherapy and the greater production of structural BR isomers recently reported for green than that for white lamps.     

QUANTUM YIELD and EQUILIBRIUM POSITION OF THE CONFIGURATIONAL PHOTOISOMERIZATION OF BILIRUBIN BOUND TO HUMAN SERUM ALBUMIN*

Abstract— At low fluence rates it is possible to observe a photoequilibrium between Z,Z-bilirubin(BR) and its E isomers(collectively called PBR) bound to human serum albumin(HSA).For excitation centered at 465 nm, the fraction of PBR/HSA in the photoequilibrium mixture was observed to be 0.22 + 0.02. The quantum yield for conversion of BR/HSA to PBR/HSA was found to be 0.20 0.02. The equality of the quantum yield value and the fraction of PBR/HSA in the photoequilibrium mixture is consistent with a simple mechanism for the photoisomerization in which optically excited singlet states of BR and PBR convert rapidly and with virtually total efficiency to a common excited intermediate with twisted geometry that subsequently decays to BR and PBR in the ratio 4:1, respectively. Quantum yields for other photoprocesses of BR bound to HSA are much lower than that for configur-ational isomerization. The central role suggested for configurational isomerization in the phototherapy for neonatal hyperbilirubinemia is supported by these observations.     

QUANTUM YIELDS FOR LASER PHOTOCYCLIZATION OF BILIRUBIN IN THE PRESENCE OF HUMAN SERUM ALBUMIN. DEPENDENCE OF QUANTUM YIELD ON EXCITATION WAVELENGTH

The quantum yield for laser photocyclization of bilirubin to lumirubin in the presence of human serum albumin (phi LR) was measured at five monochromatic excitation wavelengths in the range 450-530 nm. Solutions used were optically thin throughout the wavelength range and precautions were taken to exclude contributions from photocyclization of bilirubin XIII alpha impurities. The values obtained (7.2-18 x 10(-4] were lower than those previously reported and showed the following wavelength dependence: 457.9 less than 488.0 less than 501.7 less than 514.5 approximately equal to 528.7. However, the rate of lumirubin formation, normalized to constant fluence, decreased with wavelength over the same wavelength range and no evidence was found that photoisomerization of bilirubin to lumirubin is faster with green (514.5 or 528.7 nm) than with blue (457.9 or 488.0 nm) light. The stereoselectivity of the configurational isomerization of bilirubin to 4Z,15E and 4E,15Z isomers also was studied. This reaction became less regioselective for the 4Z,15E isomer with increasing wavelength. The observed wavelength dependence of phi LR and of the [4E,15Z]: [4Z,15E] ratio at photoequilibrium are consistent with an exciton coupling model in which intramolecular energy transfer can occur between the two pyrromethenone chromophores of the bilirubin molecule in the excited state. Relative rates of lumirubin formation in vivo at different excitation wavelengths and constant fluence were estimated for different optical thicknesses and for different skin thicknesses. These estimates suggest that the recently reported clinical equivalence of blue and green phototherapy lights probably reflects the marked variation of skin transmittance with wavelength more than wavelength-dependent photochemistry. The calculations also indicated that the optimal wavelength for phototherapy is probably on the long wavelength side of the bilirubin absorption maximum.     

ANALYTICAL MODEL OF PHOTOTHERAPY FOR NEONATAL JAUNDICE—I. THE INITIAL Z-E TRANSIENT

A simple model of phototherapy (PT) for neonatal jaundice is presented. Two coupled systems are considered: the "skin" (upper skin layers and vascular network where phototherapy light may penetrate) and the "body" (i.e. the "dark" ensemble of blood vessels and organs where circulation and metabolization of biliribin (BR) and its photoisomers occur).
The mathematics necessary to analyze the PT process is presented together with an appropriate optical model of skin based on a multilayered system to which the theory of radiation transfer in isotropically scattering and absorbing media is applied. A simple matrix formalism is introduced to determine analytically the light distribution in the multilayer skin. The results are. then, used to analyze the initial transient of PT, i.e. configurational isomerization up to photoequilibrium in the superficial skin layers under blue (450 nm) and green (500 nm) light irradiation within a time interval sufficiently short to neglect diffusion of pigments and formation of structural isomers of BR.
Strong coupling among the various BR layers due to the time variation of diffuse transmittance and reflectance is found, resulting in complicated time patterns with initial and final single-exponential behavior. The initial decay constant of BR concentration depends sensibly on the depth of the layer, while the final one is independent of it. As expected the amount of BR conversion at photoequilibrium is larger with blue than with green light. However, at sufficiently larger depths green light is more efficient than blue light to produce the initial conversion of ZZ to ZE isomers of BR.
The present analysis has some relevance for the understanding of the different efficiencies of coloured fluorescent lamps in clinical PT. The application of the complete model ("skin"+"body") requires the knowledge of the diffusion constants and absorption coefficients of pigments and of the optical parameters of deep cutaneous tissues.     

EXCITED STATES OF BILIRUBIN

Abstract— The excited states of bilirubin (BR) in a variety of environments have been studied by 347 nm laser flash photolysis. Quantum yields of formation of triplet BR have been shown to be less than 0.005 in solution in water ( p H 9–11), methanolic ammonia, 10% aqueous mulgofen and in cetyl trimethyl-ammonium bromide. In benzene the quantum yield was 0.01 although this diminished to less than 0.005 on addition of triethylamine. Permanent products are formed with benzene and with 1% methanolic ammonia. With BR in HSA a transient decaying with k = 3.5 × 10⁵ s^-1 is formed by a monophotonic process together with a permanent product. Neither species is affected by oxygen or by iodide ion. Both originate from BR molecules in the strongest binding site in the HSA. The yields of both species are unaffected by salt but are temperature dependent. The decay of the transient is strongly temperature dependent corresponding to an activation energy of about 50–60 kj mol^-1. If this transient is a triplet it is formed with a quantum yield of 0.13 ± 0.01. The relevance of these results to an understanding of the photo therapeutic process is discussed.     

A [3]rotaxane with three stable states that responds to multiple-inputs and displays dual fluorescence addresses

A [3]rotaxane molecular shuttle containing two alpha-cyclodextrin (alpha-CD) macrocycles, an azobenzene unit, a stilbene unit, and two different fluorescent naphthalimide units has been investigated. The azobenzene unit and the stilbene unit can be E/Z-photoisomerized separately by light excited at different wavelengths. Irradiation at 380 nm resulted in the photoisomerization of the azobenzene unit, leading to the formation of one stable state of the [3]rotaxane (Z1-NNAS-2CD); irradiation at 313 nm resulted in the photoisomerization of the stilbene unit, leading to the formation of another stable state of the [3]rotaxane (Z2-NNAS-2CD). The reversible conversion of the Z1 and Z2 isomers back to the E isomer by irradiation at 450 nm and 280 nm, respectively, is accompanied by recovery of the absorption and fluorescence spectra of the [3]rotaxane. The E isomer and the two Z isomers have been characterized by 1H NMR spectroscopy and by two-dimensional NMR spectroscopy. The light stimuli can induce shuttling motions of the two alpha-CD macrocycles on the molecular thread; concomitantly, the absorption and fluorescence spectra of the [3]rotaxane change in a regular way. When the alpha-CD macrocycle stays close to the fluorescent moiety, the fluorescence of the moiety become stronger due to the rigidity of the alpha-CD ring. As the photoisomerization processes are fully reversible, the photo-induced shuttling motions of the alpha-CD rings can be repeated, accompanied by dual reversible fluorescence signal outputs. The potential application of such light-induced mechanical motions at the molecular level could provide some insight into the workings of a molecular machine with entirely optical signals, and could provide a cheap, convenient interface for communication between micro- and macroworlds.     

Syntheses, structures, and photoisomerization of (E)- and (Z)-2-tert-butyl-9-(2,2,2-triphenylethylidene)fluorene

"Sterically geared" 9-(2,2,2-triphenylethylidene)fluorene (1) is of potential interest as a photoactive moiety in molecular devices, and the 2-tert-butyl derivative (6) has been synthesized to investigate photoisomerization. E and Z stereoisomers of 6 were separated and identified by X-ray crystallography. The tert-butyl group does not introduce additional strain, and its close proximity to the trityl group in the Z isomer suggests an attractive van der Waals interaction. The UV spectra of (E)-6 and (Z)-6 are nearly identical, showing absorption bands that are similar to those of fluorene occurring at wavelengths longer than 240 nm. Photoisomerization of 6 was investigated at 266, 280 and 320 nm. Solutions initially containing only (E)-6 or (Z)-6 were irradiated with pulsed laser light, monitoring isomerization by 1H NMR spectroscopy. Negligible photodecomposition was observed when the solutions were agitated by N2 ebullition. Experimental data were fitted to theoretical curves to obtain photoisomerization quantum yields (phi(ZE) and phi(EZ)) ranging from 0.04 to 0.09. This first photoisomerization study of a dibenzofulvene reveals significant quantum yields, despite theoretical prediction of inefficient or negligible isomerization of the parent hydrocarbon, fulvene. Thermal isomerization of 6 at 270 degrees C (t(1/2) = 120 min) was also followed by 1H NMR spectroscopy, resulting in an estimated activation energy (deltaG(double dagger)) of 43 kcal/mol.     

A new photoisomerization of bilirubin

Photoirradiation of solutions of natural (4Z,15Z)-bilirubin-IX alpha in chloroform with approximately 366 nm UV light leads rapidly to a new photoisomer that has been characterized by NMR spectroscopy and by its (ground-state reaction) adducts with methanol and other protic nucleophiles. Unlike the previously described Z-->E geometric isomerization important in phototherapy of neonatal jaundice, the new photoisomerization involves regiospecific photoautomerization to afford 2-ethenyl-18-ethylidene- 1,10,19,22,24-pentahydro-2,7,13,17-tetramethyl-1,19- dioxo-21H-biline-8,12-dipropanoic acid.     

The photochemistry of a bis-crown ether based on benzobis(thiazole) and its alkaline earth metal cation complexes

Irradiation of acetonitrile solutions of the bis-crown ether E,E-2,7-bis[2-(6,7,9,10,12,13,15,16-octahydro-5,8,11,14,17-pentaoxabenzocyclopentadecen-2-yl)vinyl]-benzo[1,2-d;3,4-d']bisthiazole (hereafter, 1) gives efficient E --> Z photoisomerization (initial phi(trans --> cis) = 0.48), leading to lambda(exc)-dependent quasi-photostationary states composed primarily of E,Z and E,E isomer mixtures. Further irradiation gives [2 + 2]-cycloadducts of 1. In the presence of Ba2+ ions, essentially quantitative formation of 2:2 complexes, 1(2) x (Ba2+)2 controls the photochemical outcome. E --> Z photoisomerization of the ligand is entirely suppressed and efficient intramolecular [2 + 2]-photocycloaddition in the complexes leads to cyclobutane dimers of 1 (phiCB = 0.26). The reactivity of 1 in the presence of Mg2+ ions for which 1:2 complex formation dominates gives both cis-trans photoisomerization and enhanced photocycloaddition.     

WAVELENGTH DEPENDENCE OF QUANTUM YIELDS AND PRODUCT DISTRIBUTION IN THE ANAEROBIC PHOTOCHEMISTRY OF BILIRUBIN DIMETHYL ESTER

Abstract— ( Z,Z )-Bilirubin dimethyl ester exists in two or more forms in solution, and photorearranges to the ( E,Z )- and ( Z,E )-isomers. Deaerated solutions of bilirubin dimethyl ester in 2-methyltetrahydrofuran and chloroform/ethanol were irradiated with light of 10nm bandwidth in the range 380–470 nm at room temperature. Absorption difference spectroscopy showed that the solution forms of bilirubin dimethyl ester differ in their photoreactivity, and that the quantum yields of their disappearance and the distribution of the photorearrangement isomers are wavelength dependent.     

MODEL STUDIES ON THE PHOTOCHEMICAL PRODUCTION OF LENTICULAR FLUOROPHORES

Abstract With aging, human lens proteins accumulate fluorophores having blue and green emissions. Model studies were undertaken to determine the role of 3-hydroxykynurenine (3-HK) and its glucoside (3-HKG) in the photochemical production of those fluorophores. Experiments were carried out using 10⁻³ M 3-HK solutions in the presence or absence of glycine (1 M ), which was used to mimic the environment of the lens. The solutions were photolyzed (transmission above 295 nm) for various periods of time while the loss of starting material and the formation of fluorescent photoproducts (blue emission at 470 nm, and green emission at 520 nm) were monitored using fluorescence and UV-visible spectroscopy and thin-layer and high-pressure liquid chromatography analysis. Several parameters were varied such as oxygen tension and the addition of the free radical scavenger, penicillamine. The photolytic loss of 3-HK in the absence of glycine occurred approximately 5-10 times faster than in its presence. Conversely, blue and green fluorophores formed in irradiated solutions containing glycine but not with the photolysis of 3-HK alone. The blue fluorophore was formed first and appeared then to be photochemically converted to the green one, with the rate of formation of the latter increasing with an increase in UV dosage or oxidizing conditions. The addition of penicillamine drastically reduced the photochemical formation of both fluorophores.
Both the blue and green fluorophores appear to result from the photochemically induced covalent attachment of 3-HK to glycine. In the human lens, these reactions can explain the age-related loss of 3-HKG with the concomitant formation of fluorophores covalently attached to lens proteins, probably via the amino group of lysine.     

WAVELENGTH-DEPENDENT QUANTUM YIELD FOR Z →E ISOMERIZATION OF BILIRUBIN COMPLEXED WITH HUMAN SERUM ALBUMIN

The quantum yield, phi ZE, for configurational photoisomerization (4Z,15Z----4Z,15E) of bilirubin bound non-covalently to human serum albumin was determined (at 23 +/- 2 degrees C) by laser excitation and chromatographic analysis of products. Values obtained for photoexcitation at 465 nm were about one-half those previously reported. The quantum yield was dependent on excitation wavelength, decreasing from a value of 0.109 +/- 0.010 for excitation at 457.9 nm to a value of 0.054 +/- 0.005 for excitation at 514.5 nm. The wavelength dependence is consistent with rapid transfer of excitation energy between the two non-identical pyrromethenone chromophores of bilirubin in the singlet excited state. Since the quantum yields for photoisomerization and luminescence of bilirubin bound to serum albumin at room temperature are both low, internal conversion processes, rather than Z----E configurational isomerizations, are probably the major pathways for deactivation of photo-excited bilirubin.     

HAS-硅钨杂多酸缔合纳米微粒体系的荧光猝灭

人血清白蛋白;瑞利散射;HAS-硅钨杂多酸缔合纳米微粒体系的荧光猝灭     

trans–cis and trans–cis–trans Microstructure Evolution of Azobenzene Liquid‐Crystal Polymer Networks

The photomechanics of azobenzene LCNs is modeled using a nonlinear continuum mechanics approach that couples photoisomerization of liquid crystal domain structures with light absorption and deformation of a glassy polymer network. The effects during UV‐stimulated trans–cis photomechanical deformation versus blue‐green light (trans–cis–trans) photomechanical deformation are simulated. Different bending deformation is predicted by assuming liquid‐crystal order/disorder behavior during trans–cis photoisomerization in comparison to light‐polarization‐driven reorientation of the trans phase during potential trans‐cis‐trans photoisomerization. Light‐controlled deformation mechanisms offer support for improved control of photo‐responsive morphing structures with a single blue‐green polarized light source.

     

Determination of proteins at nanogram levels by their quenching effect on large particle scattering of colloidal silver chloride

A novel quantitative method for the determination of proteins in aqueous solutions has been based on the quenching of the resonance scattering light of colloidal silver chloride in the presence of proteins. The detection limits for eight kinds of proteins (BSA, HSA, egg albumin, human gamma-IgG,alpha-chymotrypsin, E. Coli. alpsase, myoglobin, alpha-casein) were at about 8 ng/mL; the linear ranges of the calibration curves were 10-400 ng/mL under optimal conditions,except for human gamma-IgG (20-400 ng/mL), myoglobin (10-300 ng/mL), and alpha-casein (10-300 ng/mL). Three wavelengths (398 nm, 475 nm, 499 nm) were all suitable for the determination and any acidity from pH 3.0 to pH 9.0 could be chosen. A few non-protein substances at high concentration levels interfered with this method, but this problem could simply be overcome by diluting the samples before the assay. Mechanism studies showed that the quenching effect of proteins on the scattering light of colloidal silver chloride was mainly due to the coagulation of AgCl particles retarded by protein. The method was employed for the determination of total protein in human serum with satisfactory results.     

Photochemical and photophysical properties of a poly(propylene amine) dendrimer functionalized with E-stilbene units

A second generation poly(propylene amine) dendrimer functionalized at the periphery with eight E-stilbene and eight 4-tert-butylbenzenesulfonyl units has been prepared. The absorption spectrum, fluorescence spectrum and decay, E<==>Z photoisomerization, and photocyclization of the Z-isomer of the stilbene units have been investigated in air equilibrated acetonitrile solutions. For comparison purposes, a reference compound of the peripheral dendrimer units, namely 4-tert-butyl-N-propyl-N-(4-styryl-benzyl)-benzenesulfonamide, has also been studied. The quantum yield of the E-->Z photoisomerization reaction (0.30) and the fluorescence quantum yield of the E isomer (0.014) are substantially smaller for the units appended to the dendrimer compared to those of the reference compound (0.50 and 0.046, respectively). The presence of a red tail and the biexponential decay of the emission band of the dendrimer indicate formation of excimers between the stilbene units appended at the poly(propylene amine) dendritic structure. Under the experimental conditions used (lambda(exc)= 313 nm), a Z/E photostationary state (around 9 : 1 for both reference compound and dendrimer ) is reached in the time scale of minutes. On continuing irradiation, other photoreactions take place in the time scale of hours: the stilbene moiety of compound undergoes photocyclization to phenanthrene (quantum yield 0.015), whereas in dendrimer photocyclization to phenanthrene is accompanied by other processes, including a photoreaction involving the internal amine groups.     

Z/E PHOTOISOMERIZATION OF UROCANIC ACID*

The E ? Z photoisomerization of the title compound (UA) (a naturally occurring sunscreen) has been studied in aqueous solution. At a UA concentration of 6mM and using 313nm excitation, φ_E→z= 0.52, φ_Z→E= 0.47 and the photostationary state is 34% E. Under these conditions, loss of UA is minimal. Low energy triplet quenchers fail to impede the isomerization, but the reaction can be induced by several triplet sensitizers. The E_T for UA is estimated to be approximately 55 kcal/mol.     

Probing the interaction of human serum albumin with norfloxacin in the presence of high-frequency electromagnetic fields: fluorescence spectroscopy and circular dichroism investigations

The present study describes an investigation by fluorescence quenching, circular dichroism and UV-visible spectroscopy of the interaction between norfloxacin (NRF) and human serum albumin (HSA) in the presence of electromagnetic fields (EMFs). The results obtained from this study indicated that NRF had a strong ability to quench HSA at λ(ex) = 280 nm. In addition, a slight blue shift occurred, which suggested that the microenvironment of the protein became more hydrophobic after addition of NRF. The interaction between the NRF and HSA, whether in the absence or presence of an EMF, was considered to be a static quenching mechanism. Moreover, synchronous fluorescence demonstrated that the microenvironment around Trp became modified. Data of HSA-NRF in the presence of EMFs between 1 Hz-1 MHz confirmed the results of quenching and blue shifts. Corresponding Stern-Volmer plots were also drawn and the resultant Ksv and kq values were compared. Moreover, the binding parameters, including the number of binding sites, the binding constant and the distance, r, between donor and acceptor, were calculated based on F?rster's non-radiative energy transfer theory. According to far and near UV-CD, the formation of the complex caused changes of the secondary and tertiary structures of HSA. The obtained results are significant for patients who are subjected to high-frequency radiation as this was found to reduce the affinity of NRF to HSA.     

Determination of proteins at nanogram levels by their quenching effect on large particle scattering of colloidal silver chloride

A novel quantitative method for the determination of proteins in aqueous solutions has been based on the quenching of the resonance scattering light of colloidal silver chloride in the presence of proteins. The detection limits for eight kinds of proteins (BSA, HSA, egg albumin, human γ-IgG,α-chymotrypsin, E. Coli. alpsase, myoglobin, α-casein) were at about 8 ng/mL; the linear ranges of the calibration curves were 10–400 ng/mL ¶under optimal conditions,except for human γ-IgG (20–¶400 ng/mL), myoglobin (10–300 ng/mL), and α-casein (10–300 ng/mL). Three wavelengths (398 nm, 475 nm, 499 nm) were all suitable for the determination and any acidity from pH 3.0 to pH 9.0 could be chosen. A few non-protein substances at high concentration levels interfered with this method, but this problem could simply be overcome by diluting the samples before the assay. Mechanism studies showed that the quenching effect of proteins on the scattering light of colloidal silver chloride was mainly due to the coagulation of AgCl particles retarded by protein. The method was employed for the determination of total protein in human serum with sactifactory results.     

1,2‐Dithienyldicyanoethene‐Based,Visible‐Light‐Driven,Chiral Fluorescent Molecular Switch: Rewritable Multimodal Photonic Devices

Reported here is the first example of a 1,2‐dithienyldicyanoethene‐based visible‐light‐driven chiral fluorescent molecular switch that exhibits reversible trans to cis photoisomerization. The trans form in solution almost completely transforms into the cis form, accompanied by a 10‐fold decrease in its fluorescence intensity within 60 seconds when exposed to green light (520 nm). The reverse isomerization proceeds upon irradiation with blue light (405 nm). When doped into commercially available achiral liquid crystal hosts, this molecular switch efficiently induces luminescent helical superstructures, that is, a cholesteric phase. The intensity of the circularly polarized fluorescence as well as the selective reflection wavelength of the induced cholesteric phases can be reversibly tuned using visible light of two different wavelengths. Optically rewritable photonic devices using cholesteric films containing this molecular switch are described.