Photoquenching: The dependence of the primary quantum yield of a monophotonic laser-induced photochemical process on the intensity and duration of the exciting pulse

Excited state absorption in large molecules leads to a decrease of the primary quantum yield of a photochemical or a photophysical process. Since then the quantum yield decreases with increasing light intensity this effect is called photoquenching.Kinetic analysis of the excitation in a general level scheme of a large molecule yields expressions for the quentum yield of a laser-induced photochemical process. Calculation of the quantum yield for various combinations of molecular parameters and laser pulse characteristics shows quenching of the photochemical process due to excited state absorption, at laser intensities for which bleaching effects and other nonlinear processes are negligeable. The applicability of the steady state approximation in analyzing laser-induced processes is discussed. Experiments are reported, which confirm the calculated intensity-dependent quantum yield function. Previous measurements of intensity-induced quenching can now be discussed quantitatively. Care should be taken in interpreting laser-induced photochemical yields, especially at mode locked laser intensities; correct values can only be obtained by extrapolation to zero laser intensity     

LASER CROSS-LINKING OF PROTEINS TO NUCLEIC ACIDS: PHOTODEGRADATION AND ALTERNATIVE PHOTOPRODUCTS OF THE BACTERIOPHAGE T4 GENE 32 PROTEIN

Pulsed laser cross-linking provides a means of introducing a covalent bond between proteins and the nucleic acids to which they are bound. This rapid cross-linking effectively traps the equilibrium that exists at the moment of irradiation and thus allows examination of the protein-nucleic acid interactions that existed. Laser irradiation may also induce photodestruction of protein and we have used the bacteriophage T4 gene 32 protein to investigate this phenomenon. Our results show that both nonspecific and specific photoproducts can occur, specifically at wavelengths where the peptide backbone of proteins is known to absorb. These results demonstrate that nonspecific photodegradation can be correlated with the formation of a specific photodegradation product. The formation of this product was monitored to show that product yield is nonlinearly dependent on laser power and wavelength. We have also investigated an unexpected photoproduct whose formation is dependent on the length of the polynucleotide to which the gene 32 protein binds and that further demonstrates the complexities of analyzing protein-nucleic acid interactions through the use of UV laser cross-linking. These data provide essential information for the establishment of appropriate conditions for future studies that use UV cross-linking of protein-nucleic acid complexes.     

UV-Induced Nucleic Acid–Protein Cross-Linking: Manual on Planning of Irradiation Experiments and Calculation of Absorbed Dose and Quantum Yield

We have developed methods of photochemical quantitation of photobiological studies on UV-induced nucleic acid–protein cross-linking. Cases relating to incoherent low-intensity UV sources, laser UV sources and high-intensity laser UV sources are considered. In the case of low-intensity UV radiation the most important point is the correct determination of absorbed dose. The laser UV pulse energy is easily measured and the short-pulse irradiation also has the advantage of "freezing" the conformation of complexes under study. However, the use of high-intensity laser UV irradiation leads to realizations of two-quantum processes both in nucleic acid chromophores–bases and in solvent–water, which complicates singificantly the processing of results. In this paper methods for calculating the absorbed dose and the quantum yield of cross-linking for all above-mentioned cases are given as well as practical advice.     

Control of photobleaching in photodynamic therapy using the photodecarbonylation reaction of ruthenium phthalocyanine complexes via stepwise two-photon excitation

In this study, we have investigated the photochemical properties and photodynamic effects of ruthenium phthalocyanine (RuPc(CO)(Py)) and naphthalocyanine (RuNc(CO)(Py)) complexes. When a nanosecond-pulsed laser is used, the photodecarbonylation of our Ru complexes efficiently proceeds via stepwise two-photon excitation, while the reaction yields are negligibly small when a continuous-wave (CW) laser is employed. The pulsed laser selective photodecarbonylation decreases the Q-band absorbance, which satisfies what the photodynamic therapy (PDT) requires of the photobleaching. For RuPc(CO)(Py), the photochemical reactions including both the photodecarbonylation and just photobleaching occur in HeLa cells in vitro. Toxicity and phototoxicity tests indicate that our RuPc(CO)(Py) and RuNc(CO)(Py) complexes in concentrations of 0.3-1 microM and 1-2 microM, respectively, are applicable as PDT agents. The phototoxicity is consistent with the photochemical properties of these complexes, namely, excited triplet lifetimes (10 and 4.8 micros for the Pc and Nc complexes, respectively) and singlet oxygen yields (0.48 and 0.35 for the Pc and Nc complexes, respectively). On the basis of these results, we propose a novel concept for achieving a greater depth of necrosis in PDT as follows: (1) PDT of upper cellular layers using CW-laser irradiation; (2) efficient photobleaching in upper cellular layers using pulsed dye-laser irradiation, which results in an increase in the therapeutic depth of red light; (3) PDT directed toward deeper tumor tissues using CW laser irradiation. In addition, these Ru complexes are promising as CO release agents for investigative biochemistry.     

Lasers used in analytical micropyrolysis

Laser micropyrolysis gas chromatography mass spectrometry (GC-MS) allows analytical pyrolysis to be conducted with micro-spatial resolution. Despite the large range of contemporary laser sources, most previous laser pyrolysis studies have been conducted with continuous wave (CW) infrared irradiation. Here, the laser micropyrolysis analysis of a Sydney torbanite was conducted with three different laser sources - 1. CW 532 nm; 2. Q-Switched (QSw) pulsed 1064 nm; and 3. QSw pulsed 266 nm - to compare the molecular analyses attributes of different laser types (λ: 266-1064 nm; CW or QSw). The CW 532 nm laser irradiation consistently produced high concentrations of n-hydrocarbons, with lesser amounts of cyclic and aromatic hydrocarbons, similar to previous analyses with both CW 1064 nm laser pyrolysis and conventional analytical pyrolysis [1]. In contrast, both the IR and UV QSw pulsed irradiation sources provided poor and varied data. Relatively low concentrations of n-hydrocarbons were occasionally produced, but most often no structurally significant products were detected. The poor maintenance of hydrocarbon structural units by the short pulse lasers can be attributed to the very high power density delivered, leading to excessive degradation of the irradiated macromolecule.     

Investigation of multiple electronic excited state relaxation pathways following 200 nm photolysis of gas-phase imidazole

Imidazole acts as a subunit in the DNA base adenine and the amino acid histidine-both important biomolecules which display low fluorescence quantum yields following UV excitation. The low fluorescence quantum yields are attributed to competing non-radiative excited state relaxation pathways that operate on ultrafast timescales. Imidazole is investigated here as a model compound due to its accessibility to high level ab initio calculations and time-resolved gas-phase spectroscopic techniques. Recent non-adiabatic dynamics simulations have identified three non-radiative relaxation mechanisms which are active following 6.0-6.2 eV excitation. Presented herein is a comprehensive investigation of each mechanism using a combination of femtosecond time-resolved ion yield and total kinetic energy release spectroscopies to monitor the formation of associated photoproducts. Relaxation along the (1)πσ state constitutes the predominant deactivation pathway. Timescales for NH-dissociation are extracted and distinguished from alternative H-atom sources based on their kinetic energy distributions. Larger photoproducts are observed to a lesser extent and attributed to ring fragmentation following NH-puckering and CN-stretching relaxation paths.     

Photoinitiated crosslinking of polyethylene

The main types of polyethylene (PE) crosslinking photoinitiators were examined. It was shown that the aromatic ketones and quinone are the best types of the photoinitiators for practical usage. The quantum yields of initiators' photoreduction and the crosslinking yields per one initiator molecule at the different stages of PE photochemical crosslinking were measured. The photoinitiators effectiveness dependence on their chemical structure was considered. The influences of the UV-irradiation intensity, temperature, photoinitiators' concentration, some copolymers, polyfunctional monomers, peroxides additives on the photocrosslinking and photooxidation processes in PE were investigated.     

新型水溶性硫杂蒽酮类光引发剂的光化学性能研究

本文对8种水溶性硫杂蒽酮类光引发剂进行了紫外 可见光谱、荧光光谱以及电子自旋共振光谱(ESR)等测试。测定了最大吸收波长,计算了摩尔消光系数,荧光量子产率,测定了ESR信号强度等。并对其结构与光化学性能之间的关系进行了讨论。     

Nanosecond photofragment imaging of adiabatic molecular alignment

Adiabatic alignment of CH(3)I, induced by the anisotropic interaction of this symmetric top molecule with the intense field of a nonresonant infrared laser pulse, has been studied using velocity map imaging. We are using photodissociation imaging with pulsed nanosecond lasers to probe the distribution of the molecular axis in the laboratory space. In contrast to the commonly used probing with femtosecond laser pulses, this technique directly yields the degree of alignment over an extended space-time volume. This will be relevant for future reactive scattering experiments with laser-aligned molecules. The obtained degree of alignment, (cos?(2)θ), measured as a function of the infrared laser intensity, agrees well with a quantum calculation for rotationally cold methyl iodide. The strong infrared laser is also found to modify the photofragmentation dynamics and open up pathways to CH(3)I(+) formation and subsequent fragmentation.     

Ultrafast UV-mid-IR investigation of the ring opening reaction of a photochromic spiropyran

We present a femtosecond UV-mid-IR pump-probe study of the photochemical ring-opening reaction of the spiropyran 1',3',3',-trimethylspiro-[-2H-1-benzopyran-2,2'-indoline] (also known as BIPS) in tetrachloroethene, using 70 fs UV excitation pulses and probing with 100 fs mid-IR pulses. The time evolution of the transient IR absorption spectrum was monitored over the first 100 ps after UV excitation. We conclude that the merocyanine product is formed with a 28 ps time constant, contrasting with a 0.9 ps time constant obtained in previous investigations where the rise of absorption bands at visible wavelengths were associated with product formation. We deduce from the observed strong recovery of the spiropyran IR absorption bleaches that, in tetrachloroethene, the main decay channel for the S(1) excited state of the spiropyran BIPS, is internal conversion to the spiropyran S(0) state with a quantum yield of > or = 0.9. This puts an upper limit of 0.1 to the quantum yield of the photochemical ring-opening reaction.     

First direct detection of 2,3-dimethyl-2,3-diphenylcyclopropanone

3,5-dihydro-3,5-dialkyl-3,5-diaryl-4H-pyrazol-4-ones stimulate interest as potential precursors for 2,3-diarylcyclopropanones. Photoreactions of trans-3,5-dihydro-3,5-dimethyl-3,5-diphenyl-4H-pyrazol-4-one were studied by continuous-wave (CW) and pulsed laser UV photolysis revealing an intermediate that undergoes rearrangement to form cis- and trans-1,3-dimethyl-1-phenyl-2-indanones with the yield of ca. 60%. Steady-state photolysis (254 and 350 nm excitation) in different solvents produced an intermediate cyclohexadiene as evidenced by UV/vis, IR, and 1H NMR spectra. In contrast, the nanosecond laser pulsed photolysis at 355 nm produced 2,3-dimethyl-2,3-diphenylcyclopropanone along with two products of retro-1,3-dipolar addition phenylmethylketene and 1-phenyldiazoethane. These can be observed by time-resolved IR (TRIR) spectroscopy as characteristic absorption bands at 1814, 2101, and 2038 cm-1, respectively. Similar retro-1,3-dipolar addition showed 1-phenyldiazoethane formed following flash photolysis of 1-pyrazoline (trans-4,5-dihydro-3,5-dimethyl-3,5-diphenyl-3H-pyrazol-4-ol). The formation of the corresponding cyclopropanone as well the products of retro-1,3-dipolar addition during photoreaction of starting pyrazol-4-one is directly confirmed by the nanosecond TRIR spectroscopy for the first time. On the basis of the CW and pulsed laser UV photolysis, a dynamic equilibrium between cyclopropanone and intermediate 2,4-diphenyl-3-pentanone-2,4-diyl (dimethyldiphenyloxyallyl) was proposed.     

Nitrate radical quantum yield from peroxyacetyl nitrate photolysis

Peroxyacetyl nitrate (PAN, CH3C(O)OONO2) is a ubiquitous pollutant that is primarily destroyed by either thermal or photochemical mechanisms. We have investigated the photochemical destruction of PAN using a combination of laser pulsed photolysis and cavity ring-down spectroscopic detection of the NO3 photoproduct. We find that the nitrate radical quantum yield from the 289 nm photolysis of PAN is Phi(NO3)PAN = 0.31 +/- 0.08 (+/-2 sigma). The quantum yield is determined relative to that of dinitrogen pentoxide, which is assumed to be unity, under identical experimental conditions. The instrument design and experimental procedure are discussed as well as auxiliary experiments performed to further characterize the performance of the optical cavity and photolysis system.     

Photochemical addition of amino acids and peptides to homopolyribonucleotides of the major DNA bases

Abstract— The photochemical quantum yields for addition of glycine and the L-amino acids commonly occurring in proteins (excluding proline) to polyadenylic acid, polycytidylic acid, polyguanylic acid and polyribothymidylic acid have been determined in deoxygenated phosphate buffer at Λ 254 nm and pH 7, using a fluorescamine assay technique. Polyadenylic acid was reactive with eleven of the twenty amino acids tested, with phenylalanine, tyrosine, glutamine, lysine and asparagine having the highest quantum yields. Polyguanylic acid reacted with sixteen amino acids; phenylalanine, arginine, cysteine, tyrosine, and lysine displayed the largest quantum yields. Polycytidylic acid showed reactivity with fifteen amino acids with lysine, phenylalanine, cysteine, tyrosine and arginine having the greatest quantum yields. Polyribothymidylic acid, reactive with fifteen of nineteen amino acids surveyed, showed the highest quantum yields for cysteine, phenylalanine, tyrosine, lysine and asparagine. None of the polynucleotides were reactive with aspartic acid or glutamic acid.
The quantum yields for photoaddition of eighteen dipeptides of the form glycyl X (X being one of the amino acids commonly occurring in proteins, including proline), and of L-alanyl-L-tryptophan, L-seryl-L-seryl-L-serine, L-threonyl-L-threonyl-L-threonine, L-cystine- bis -glycine, and N_α-acetyllysine to polyadenylic acid, polycytidylic acid and polyguanylic acid were measured. All of these were found to add photochemically to each of these polymers. Polyribothymidylic acid, tested with eleven of these peptides and with N_α-acetyllysine, was found to be reactive with all.     

Laser induced synthesis: Condensation of acrylonitrile on mesityl oxide

The photoaddition of acrylonitrile to mesityl oxide was accomplished at 355 nm with high quantum yield (15%) using a pulsed laser (intensity= 9 MW/cm², pulse duration 13 ns, repetition rate= 10Hz) and selectively led to three products in contrast to irradiation with a mercury lamp which principally gave an acrylonitrile polymer and with continuous wave laser (intensity= 5.7 W/cm²) which gave a much lower quantum yield (0.4%).     

Generation of DNA photolesions by two-photon absorption of a frequency-doubled Ti:sapphire laser

The formation of spatially localized regions of DNA damage by multiphoton absorption of light is an attractive tool for investigating DNA repair. Although this method has been applied in cells, little information is available about the formation of lesions by multiphoton absorption in the absence of exogenous or endogenous sensitizing agents. Therefore, we have investigated DNA damage induced in vitro by direct two-photon absorption of frequency-doubled femtosecond pulses from a Ti:sapphire laser. We first developed a quantitative polymerase chain reaction assay to measure DNA damage, and determined that the quantum yield of lesions formed by one-photon absorption of 254 nm light is 7.86×10(-4). We then measured the yield of lesions resulting from exposure to the visible femtosecond laser pulses, which exhibited a quadratic intensity dependence. The two-photon absorption cross section of DNA has a value (per nucleotide) of 2.6 GM at 425 nm, 2.4 GM at 450 nm, and 1.9 GM at 475 nm. A comparison of these in vitro results to several in vivo studies of multiphoton photodamage indicates that the onset of DNA damage occurs at lower intensities in vivo; we suggest possible explanations for this discrepancy.     

Improved Utility of Photolabile Solid Phase Synthesis Supports for the Synthesis of Oligonucleotides Containing 3'-Hydroxyl Termini

Oligonucleotides are synthesized on, and cleaved from, a solid phase support (6) using the o-nitrobenzyl intramolecular photochemical redox reaction. The yields of isolated oligonucleotides relative to yields obtained using conventional hydrolytic cleavage vary between 67% and 82.5%. Synthesis of oligonucleotides using phosphoramidites that do not contain N-benzoyl protecting groups enables one to photolytically cleave the biopolymers in good yields using a commonly available UV irradiation source. Tritium labeling indicates that less than 3% thymidine.thymidine photodimers are formed during photolytic cleavage of polythymidylates from 6 using a transilluminator. No UV-induced damage is detected via HPLC analysis of enzymatically digested oligonucleotides that were obtained following photolytic cleavage from 6.     

Radical quantum yields from formaldehyde photolysis in the 30,400-32,890 cm(-1) (304-329 nm) spectral region: detection of radical photoproducts using pulsed laser photolysis-pulsed laser induced fluorescence

The relative quantum yield for the production of radical products, H + HCO, from the UV photolysis of formaldehyde (HCHO) has been measured using a pulsed laser photolysis–pulsed laser induced fluorescence (PLP–PLIF) technique across the 30,400–32,890 cm(–1) (304–329 nm) spectral region of the ?(1)A2–X?(1)A1 electronic transition. The photolysis laser had a bandwidth of 0.09 cm(–1), which is slightly broader than the Doppler width of a rotational line of formaldehyde at 300 K (0.07 cm(–1)), and the yield spectrum shows detailed rotational structure. The H and HCO photofragments were monitored using LIF of the OH radical as a spectroscopic marker. The OH radicals were produced by rapid reaction of the H and HCO photofragments with NO2. This technique produced an “action” spectrum that at any photolysis wavelength is the product of the H + HCO radical quantum yield and HCHO absorption cross section at the photolysis wavelength and is a relative measurement. Using the HCHO absorption cross section previously obtained in this laboratory, the relative quantum yield was determined two different ways. One produced band specific yields, and the other produced yields averaged over each 100 cm(–1). Yields were normalized to a value of 0.69 at 31,750 cm(–1) based on the current recommendation of Sander et al. (Sander, S. P.; Abbatt, J.; Barker, J. R.; Burkholder, J. B.; Friedl, R. R.; Golden, D. M.; Huie, R. E.; Kolb, C. E.; Kurylo, M. J.; Moortgat, G. K.; et al. Chemical Kinetics and Photochemical Data for Use in Atmospheric Studies, Evaluation No. 17; Jet Propulsion Laboratory: Pasadena, CA, USA, 2011). The resulting radical quantum yields agree well with previous experimental studies and the current JPL recommendation but show greater wavelength dependent structure. A significant decrease in the quantum yield was observed for the 5(0)(1) + 1(0)(1)4(0)(1) combination band centered at 31,125 cm(–1). This band has a low absorption cross section and has little impact on the calculated atmospheric photodissociation rate but is a further indication of the complexity of HCHO photodissociation dynamics.     

Two-laser infrared and ultraviolet matrix-assisted laser desorption/ionization

Matrix-assisted laser desorption/ionization (MALDI) was performed using two pulsed lasers with wavelengths in the IR and UV regions. A 10.6 micro m pulsed CO(2) laser was used to irradiate a MALDI target, followed after an adjustable delay by a 337 nm pulsed nitrogen laser. The sample consisted of a 2,5-dihydroxybenzoic acid matrix and bovine insulin guest molecule. The pulse energy for both of the lasers was adjusted so that the ion of interest, either the matrix or guest ion, was not produced by either of the lasers alone. The delay time for maximum ion yield occurs at 1 micro s for matrix and guest ions and the signal decayed to zero in approximately 400 micro s. A mechanism is presented for enhanced UV MALDI ion yield following the IR laser pulse based on transient heating.     

Optomechanical Control of Quantum Yield in Trans–Cis Ultrafast Photoisomerization of a Retinal Chromophore Model

The quantum yield of a photochemical reaction is one of the most fundamental quantities in photochemistry, as it measures the efficiency of the transduction of light energy into chemical energy. Nature has evolved photoreceptors in which the reactivity of a chromophore is enhanced by its molecular environment to achieve high quantum yields. The retinal chromophore sterically constrained inside rhodopsin proteins represents an outstanding example of such a control. In a more general framework, mechanical forces acting on a molecular system can strongly modify its reactivity. Herein, we show that the exertion of tensile forces on a simplified retinal chromophore model provokes a substantial and regular increase in the trans ‐to‐cis photoisomerization quantum yield in a counterintuitive way, as these extension forces facilitate the formation of the more compressed cis photoisomer. A rationale for the mechanochemical effect on this photoisomerization mechanism is also proposed.