Enzyme-Catalyzed Hydrolysis of Cellulose in Ionic Liquids: A Green Approach Toward the Production of Biofuels

We investigated the reactivity and stability of a commercial mixture of cellulases in eight ionic liquids by optical and calorimetric techniques. First, hydrolysis by cellulases from Tricoderma reesei in these ionic liquids was benchmarked against that in aqueous buffer. Only 1-methylimidazolium chloride (mim Cl) and tris-(2-hydroxyethyl)methylammonium methylsulfate (HEMA) provided a medium in which hydrolysis could occur. While hydrolysis at 65 °C is initially much faster in buffer than in these two liquids, it reaches a plateau after 2 h, whereas the reaction progresses monotonically in the two ionic liquids. This difference in the rate of hydrolysis is largely attributed to two factors: (1) the higher viscosity of the ionic liquids and (2) the enzymes are irreversibly denatured at 50 °C in buffer while they are stable to temperatures as high as 115 °C in HEMA. We explored whether fluorescence quenching of aromatic amino acids of the enzymes was indeed a signature of protein denaturation, as has been suggested in the literature, and concluded that quenching is not necessarily associated with denaturation. When it does occur, for example, in the presence of ionic liquids formed from imidazolium cations and chloride anions, it arises from the imidazolium rather than the chloride. Finally, we conclude that HEMA is a promising, novel, green medium for performing cellulose hydrolysis reactions to convert biomass into biofuels. Because of the thermal stability it imparts to enzymes, its ability to solubilize biomass, and the fact that it does not quench tryptophyl fluorescence (thus permitting monitoring of the enzymes by fluorescence spectroscopy), HEMA provides an ideal starting point for the design of ionic liquids, not only for the hydrolysis of biomass, but also for use with a wide spectrum of enzymatic reactions

Role of Solvent in Excited-State Proton Transfer in Hypericin

The excited-state proton transfer of hypericin is monitored by the rise time (-6-1 2 ps in the solvents investigated) of the component of stimulated emission corresponding to the formation of the long-lived (-5 ns) fluorescent tautomer. The assignment of this excited-state process to proton transfer has been verified by noting that a hypericin analog (mesonaphthobianthrone) lacking labile protons is not fluorescent unless its carbonyl groups are protonated. Recent experimental studies on other systems have suggested that three solvent properties play important roles in excited-state proton transfer: viscosity, hydrogen-bonding character, and dynamic solvation. We find that for hypericin, in a range of protic, aprotic, hydrogen-bonding, and non-hydrogen-bonding solvents in which the viscosity changes by a factor of 60 and the average solvation time changes by a factor of 100, the excited-state proton-transfer rate of hypericin is uncorrelated with these properties and varies not more than a factor of 2 (- 6-1 2 ps) at room temperature. The relative contribution of the bulk solvent polarity is considered, and the role of intramolecular vibrations of hypericin on the proton-transfer rate is discussed

Light-Induced Acidification by the Antiviral Agent Hypericin

The naturally occurring polycyclic quinone hypericin possesses light-induced antiviral activity against the human immunodeficiency virus (HIV) and other closely related enveloped lentiviruses such as equine infectious anemia virus (EIAV). We have previously argued that hypericin undergoes a fast proton transfer reaction in its singlet state (J. Phys. Chem. 1994, 98, 5784). We have also presented evidence that the light-induced antiviral activity of hypericin does not depend upon the formation of singlet oxygen (Bioorg. Med. Chem. Lett. 1994, 4, 1339). It is demonstrated here that steady-state illumination of a solution containing hypericin effects a pH drop. When hypericin and an indicator dye, 3-hexadecanoyl-7-hydroxycoumarin,a re both imbedded in vesicles, hypericin transfers a proton to the indicator within a time commensurate to its triplet lifetime. Proton transfer to the indicator is not observed when the indicator is protonated or when the system is oxygenated. Since hypericin is known to form triplets and to generate singlet oxygen with high efficiency, this latter result is taken to confirm triplet hypericin as a source, but not necessarily the only source, of proton

Solvation Dynamics of the Fluorescent Probe PRODAN in Heterogeneous Environments: Contributions from the Locally Excited and Charge-Transferred States

The coexistence of different excited states with different properties of the same chromophores could have significant consequences for the accurate characterization of solvation dynamics in a heterogeneous environment, such as a protein. The purpose of this work is to study the contributions of the locally excited (LE) and charge-transferred (CT) states of the fluorescent probe molecule 6-propionyl-2-(N,N-dimethylamino)naphthalene (PRODAN) to its solvation dynamics in the heterogeneous environment provided by reverse micelles formed by sodium 1,4-bis-(2-ethylhexyl) sulfosuccinate (AOT)/n-heptane/water. We have found that the LE and CT states of PRODAN solvate on different time scales in reverse micelles (2 and ∼0.4 ns, respectively), consistent with results suggested in the literature, and have concluded that PRODAN’s use as a probe of heterogeneous environments must be used with caution and that, more importantly, the same caution must be exercised with any chromophore capable of emitting from different excited states

Ultrafast excited-state processes in the antiviral agent hypericin

Hypericin (Figure 1) has been shown by Lavieet al. toinactivate mature and properly assembled retroviruses, notably human immunodeficiency virus (HIV).\u27 Recently Kraus, Carpenter, and co-workers demonstrated the antiretroviral activity of hypericin against equine infectious anemia virus (EIAV), a lentivirus closely related to HIV, and have determined that light is required for activity.2 The requirement of light leads to the fundamental questions, what is the mechanism of action of hypericin and what is the role of light

Solvation of 7-azaindole in alcohols and water: evidence for concerted, excited-state, double-proton transfer in alcohols

The proton inventory technique is used for the first time to investigate excited-state proton-transfer processes. The nonradiative pathways of the biological probe, 7-azaindole, in methanol, ethanol, and water are examined. Results in methanol and ethanol demonstrate the involvement of two protons in the transition state for the excited-state doubleproton transfer process. These data provide the first experimental evidence suggesting a concerted tautomerization reaction of 7-azaindole in alcohols. The data for 7-azaindole in water are interpreted in terms of a nonradiative pathway that is qualitatively different from that in alcohols. We propose abstraction of the N1 hydrogen by water as a possible nonradiative decay process

Nonradiative pathways of 7-azaindole in water

The 7-azaindole chromophore in water is studied by means of picosecond absorption and fluorescence spectroscopy in order to determine its nonradiative decay pathways. It is concluded that a small population of 7-azaindole molecules (-20%) undergo excited-state tautomerization in about 70 ps. Intersystem crossing and photoionization are also identified as nonradiative decay channels. Photoionization occurs largely from an electronic state lying slightly in energy above the fluorescent state. This new understanding of the photophysics of the 7-azaindole chromophore in water will be essential in interpreting its behavior when it is used as an optical probe of protein structure and dynamics

Monophotonic Ionization of 7-Azaindole, Indole, and Their Derivatives and the Role of Overlapping Excited States

7-Azaindole undergoes monophotonic ionization just as its counterpart, indole. This result suggests that 7-azaindole is qualitatively more similar to indole than has previously been recognized. The appearance of the solvated electron for zwitterionic and anionic 7-azatryptophan and for 7-azaindole in water and methanol is complete within 1 ps, which indicates that the fluorescent state whose lifetime is \u3elo0 ps cannot be the source of the electron. The origin of the electron is related to the presence of closely spaced or overlapping excited states in 7-azaindole, which is another similarity that this chromophore bears with respect to indole. The fluorescence quantum yield of 7-azaindole is shown to be excitation wavelength dependent. The excitation-wavelength dependence and the temperaturedependence of the fluorescence quantum yield of 7-azaindole are explored and related to the production of the solvated electron. The implications of these observations for the use of 7-azatryptophan as an alternative to tryptophan as a probe of protein structure and dynamics are discussed

Single-Exponential Fluorescence Decay of the Nonnatural Amino Acid 7-Azatryptophan and the Nonexponential Fluorescence Decay of Tryptophan in Water

The fluorescence decay of an optical probe, the nonnatural amino acid 7-azatryptophan, is measured as a function of pH, in varying mixtures of H20 and D2O and in various nonaqueous solvents. The spectroscopic distinguishability of 7-azatryptophan is demonstrated by the comparison of its fluorescence lifetime in mixtures of N-acetyltryptophanamide (NATA) with that of mixtures of 5-hydroxytryptophan in NATA. The observation of single-exponential fluorescence decay for 7-azatryptophan in water is discussed in terms of nonradiative processes that compete effectively with charge transfer from the excited-state 7-azaindole to the side chain groups and in terms of the dependence of the charge-transfer reaction on the excited-state energy of 7-azaindole. We propose that the absence of nonexponential fluorescence decay (owing to the relative insignificance of charge transfer to the side chain as a nonradiative process in 7-azatryptophan in water) arises from an unfavorable free energy of reaction. This free energy is determined largely by the energy of the fluorescent state, which lies 46 nm (9.8 kcal/mol) below that of tryptophan when the solvent is water