Chiral porphyrin imine manganese complex as catalyst for asymmetric epoxidation of styrene derivatives

New chiral porphyrin imine was synthesized from (S)‐3‐benzyl‐2‐methyl‐4‐phenylbutanal according to dipyrromethane method using trifluoroacetic acid, BF₃ etherate, and p‐chloranil. Manganese complex of this chiral porphyrin imine ligand was used as catalyst in the asymmetric epoxidation of styrene derivatives possessing different substituents. Styrene derivatives possessing electron withdrawing groups gave the corresponding chiral epoxides in high yield up to 98% and ee up to 99%. The mechanism for the catalytic asymmetric epoxidation was also discussed based on transfer of oxygen.     

Synthesis, potentiometric titration, electrochemical investigation and biological properties of trans-[RuCl2(dinic)4] (dinic = 3,5-pyridinecarboxylic acid)

This work discusses both the synthesis of trans-[RuCl2(dinic)4], dinic = 3,5-pyridinecarboxylic acid, and its main characteristics including potentiometric titration, spectroscopic and electrochemical properties, and some biological properties. The complex was synthesized using ruthenium blue solution as the precursor in a synthetic route. The complex was characterized using electronic spectroscopy, vibrational FT-IR spectroscopy, and Raman spectroscopy, as well as 1H and 13C NMR. The results indicated that the complex exhibits a trans-geometry. Cyclic voltammetry carried out in water:acetone 1:1 solution revealed a quasi-reversible process centered on the Ru(II) atom, as well as a dependence of the redox potential, E1/2, on pH. An analysis of the electronic spectra revealed that the MLCT (metal ligand charge transfer) band underwent a hypsochromic shift as the pH increased. Spectroelectrochemical analysis indicated that the visible region band progressively faded out upon oxidation. The equilibrium constants for the eight protons of the complex were determined by potentiometric titration. The complex neither inhibits the activity of nitrogen monoxide synthase nor acts as a scavenger for nitrogen monoxide. Nevertheless, the complex shows antinociceptive properties and acts as a scavenger for hydroxyl radicals.     

Studies on the reaction of D-amino acid oxidase with beta-cyano-D-alanine. Observation of an intermediary stable charge transfer complex

The reaction of D-amino acid oxidase [EC 1.4.3.3] (DAO) from porcine kidney with beta-cyano-D-alanine (D-BCNA) was studied. DAO was found to catalyze elimination of the cyano group as well as oxidation of D-BCNA. During the course of the reaction in the presence of excess oxygen, an intermediate was observed which exhibited a characteristic absorption spectrum with a broad charge transfer band in the longer wavelength region. The CD spectrum of this intermediate resembles that of DAO-anthranilate complex. The rate of oxygen consumption in the aerobic reaction decreased with time, suggesting product inhibition due to complex formation between the enzyme and the product. Anaerobic addition of D-BCNA reduced the enzyme to its fully reduced state, the CD spectrum of which closely resembles that of the enzyme reduced by excess D-alanine. When an appropriate amount of D-BCNA was added to the enzyme under air, the charge transfer complex was observed immediately, and underwent a change to the reduced state as the oxygen was consumed. The binding strength in the charge transfer complex was found to be comparable to that in DAO-benzoate complex. The accumulating product in the oxidation of D-BCNA had a strong absorption at 285 nm. The aerobic reaction of beta-cyano-L-alanine (L-BCNA) with snake venom L-amino acid oxidase (LAO) produced the same product with an absorption at 285 nm as the reaction of DAO with D-BCNA. The product obtained in the reaction with LAO was found to form the same charge transfer complex with DAO. We tentatively identified this product as alpha-amino-beta-cyanoacrylate and the charge transfer complex as the complex of alpha-amino-alpha-cyanoacrylate with the oxidized enzyme. A hypothetical reaction pathway based on the present finding is proposed. Addition of L-BCNA to the enzyme produced an absorption spectrum very similar to that of the DAO-benzoate complex without oxidation or elimination. L-BCNA was found to be a competitive inhibitor of the oxidation of D-alanine.     

Direct determination of the stoichiometry of charge transfer complexes.

The stoichiometry of the charge transfer complex between N-acetyl-L-tryptophan and 1-methylnicotinamide chloride has been determined to be precisely 1:1 by direct measurement of the molecular weight of the complex. The result is of interest both in terms of a general method for determining the stoichiometry of charge transfer complexes, and in terms of the probable stoichiometry of specific charge transfer complexes between 1-methylnicotinamide chloride and the exposed tryptophyl side chains of certain proteins. In the latter case, the result provides experimental proof for the assignment of extinction coefficients of specified magnitudes to the homomorphic model complexes which serve as the basis for the interpretation of results with proteins.     

Reaction of vitamin A with acceptors of electrons. Formation of radical anions from 7,7,8,8-tetracyanoquinodimethane and tetrachloro-1,4-benzoquinone

1. The interactions of retinol and retinoic acid with two electron acceptors, 7,7,8,8-tetracyanoquinodimethane (TCNQ) and tetrachloro-1,4-benzoquinone (chloranil), were studied in an investigation on the ability of vitamin A to behave as a donor of electrons. 2. Retinol reacts with TCNQ in polar organic solvents with the formation, as judged by spectral studies, of the radical anion of TCNQ. 3. Addition of the products of this reaction to water is accompanied by a rapid consumption of OH(-) ions. 4. Consumption of OH(-) ions is also a feature of the reactions between retinol and chloranil, but the spectrum of the radical anion of chloranil is observed only when retinol and chloranil are suspended in aqueous salt solutions. 5. Retinoic acid behaves similarly to retinol in its reactions with TCNQ and chloranil, but it appears to be a weaker electron donor than retinol. 6. The reaction products that may be formed from retinol in its reactions with TCNQ and chloranil are discussed. 7. It is suggested that the ability of vitamin A to behave as a donor of electrons may be an important aspect of its biochemical mode of action.     

Intermolecular complexes between N-methyl-1,4-dihydronicotinamide and flavines. The influence of steric and electronic factors on complex formation and the rate of flavine-dependent dihydronicotinamide dehydrogenation.

The reaction of N-methyldihydronicotinamide (NMNH) with flavine analogs saturates at high dihydronicotinamide concentrations. Complex formation between the reactants depends mainly on steric but not on electronic factors. Thus flavine analogs that differ up to 243 mV in their oxidation-reduction potential vary only between 0.09 and 0.17 M in Kd. When the flavine plane becomes blocked by bulky substituents, however, complex stability decreases by more than an order of magnitude. NMNH-flavine complexes show long wave optical absorption. The energy of the long wave transition decreases with increasing oxidation-reduction potential of the flavine as expected for charge transfer complexes. The first-order rate constants of flavine-dependent dihydronicotinamide dehydrogenation increase with increasing oxidation-reduction potential of the flavine but they are almost independent of Kd. The reaction is not subject to general acid-base catalysis. Thus flavine-dependent dihydronicotinamide dehydrogenation may be interpreted to proceed via a charge transfer complex between oxidized flavine and reduced nicotinamide. In the rate-limiting conversion of the charge transfer complex into products hydrogen is transferred directly, the rate being governed by the difference in oxidation-reduction potential between flavine and dihydronicotinamide. An alternative mechanism where the observed charge transfer complex is not on the reaction pathway appears to be improbable but cannot be eliminated.     

Utilization of Electron Spin Resonance Spectroscopy for Observation of In Vivo Response to Fungicides

Electron spin resonance studies of the in vivo response to fungicides (chloranil and thiram) by Aspergillus niger of different ages are described. The spores possessed a natural free radical signal which grew rapidly upon application of chloranil. Thiram, on the other hand, produced weak changes in the free radical region but easily detectable changes at lower fields, indicating involvement of copper and iron. In addition, flow experiments are described in which the production and decay of the chloranil semiquinone radical produced in the reaction of solutions of chloranil with extracts of bakers' yeast were monitored, both in the absence and in the presence of several buffers and respiratory chain inhibitors.     

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c(6)), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment-protein complex (from plastocyanin or cytochrome c(6) through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.     

The role of the charge transfer complex in the transketolase catalyzed reaction

Thiamine pyrophosphate (TPP), when bound with transketolase (TK) induces some changes in the absorption of the enzyme and coenzyme which can be registered by difference spectrophotometry. The binding of a donor substrate to the binary complex give rise to changes in the absorption region of the TPP thiazolium ring and in the charge transfer spectrum. With low concentrations of hydroxypyruvate, the kinetics of these changes may be revealed. The possibility is discussed of the charge transfer complex (CTC) being involved in the catalytic reaction.     

Structure and function in the isolated reaction center complex of Photosystem II: energy and charge transfer dynamics and mechanism

The dynamics of energy and charge transfer in the Photosystem II reaction center complex is an area of great interest today. These processes occur on a time scale ranging from femtoseconds to tens of picoseconds or longer. Steady-state and ultrafast spectroscopy techniques have provided a great deal of quantitative and qualitative data that have led to varied interpretations and phenomenological models. More recently, microscopic models that identify specific charge separated states have been introduced, and offer more insight into the charge transfer mechanism. The structure and energetics of PS II reaction centers are reviewed, emphasizing the effects on the dynamics of the initial charge transfer. This revised version was published online in June 2006 with corrections to the Cover Date.     

A mobile tryptophan is the intrinsic charge transfer donor in a flavoenzyme essential for nikkomycin antibiotic biosynthesis

The flavoenzyme nikD is required for the biosynthesis of nikkomycin antibiotics. NikD exhibits an unusual long wavelength absorption band attributed to a charge transfer complex of FAD with an unknown charge transfer donor. NikD crystals contain an endogenous active site ligand. At least four different compounds are detected in nikD extracts, including variable amounts of two ADP derivatives that bind to the enzyme's dinucleotide binding motif in competition with FAD, picolinate (0.07 mol/mol of nikD) and an unknown picolinate-like compound. Picolinate, the product of the physiological catalytic reaction, matches the properties deduced for the active site ligand in nikD crystals. The charge transfer band is eliminated upon mixing nikD with excess picolinate but not by a reversible unfolding procedure that removes the picolinate-like compound, ruling out both compounds as the intrinsic charge transfer donor. Mutation of Trp355 to Phe eliminates the charge transfer band, accompanied by a 30-fold decrease in substrate binding affinity. The results provide definitive evidence for Trp355 as the intrinsic charge transfer donor. The indole ring of Trp355 is coplanar with or perpendicular to the flavin ring in "open" or "closed" crystalline forms of nikD, respectively. Importantly, a coplanar configuration is required for charge transfer interaction. Absorption in the long wavelength region therefore constitutes a valuable probe for monitoring conformational changes in solution that are likely to be important in nikD catalysis.     

Spectral and kinetic characterization of the michaelis charge transfer complex in monomeric sarcosine oxidase

Monomeric sarcosine oxidase is a flavoenzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine). Rapid reaction kinetic studies under anaerobic conditions at pH 8.0 show that the enzyme forms a charge transfer Michaelis complex with sarcosine (E-FAD(ox).sarcosine) that exhibits an intense long-wavelength absorption band (lambda(max) = 516 nm, epsilon(516) = 4800 M(-)(1) cm(-)(1)). Since charge transfer interaction with sarcosine as donor is possible only with the anionic form of the amino acid, the results indicate that the pK(a) of enzyme-bound sarcosine must be considerably lower than the free amino acid (pK(a) = 10.0). No redox intermediate is detectable during sarcosine oxidation, as judged by the isosbestic spectral course observed for conversion of E-FAD(ox).sarcosine to reduced enzyme at 25 or 5 degrees C. The limiting rate of the reductive half-reaction at 25 degrees C (140 +/- 3 s(-)(1)) is slightly faster than turnover (117 +/- 3 s(-)(1)). The kinetics of formation of the Michaelis charge transfer complex can be directly monitored at 5 degrees C where the reduction rate is 4.5-fold slower and complex stability is increased 2-fold. The observed rate of complex formation exhibits a hyperbolic dependence on sarcosine concentration with a finite Y-intercept, consistent with a mechanism involving formation of an initial complex followed by isomerization to yield a more stable complex. Similar results are obtained for charge transfer complex formation with methylthioacetate. The observed kinetics are consistent with structural studies which show that a conformational change occurs upon binding of methylthioacetate and other competitive inhibitors.     

Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase

The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD⁺ at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD⁺. A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD⁺ and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductase_TOL with dioxygen and thus present a solution toward conflicting requirements.     

Interaction of 2,6-dimethyl-3,5-dicarbethoxy-1,4-dihydropyridine with enzymes of the NADP.H2-specific electron transport chain of rat liver microsomes]

2,6-Dimethyl-3,5-dicarboethoxy-1,4-dihydropyridine (DHP) interacts with NADPH-dependent electron transfer system of rat liver microsomes: it forms a complex with the terminal oxidase-cytochrome P-450, according to type I, and inhibits clearly the activity of NADPH-cytochromes c-reductase and mitindione dimethylesterase. DHP repeatedly administered in vivo rendered no inducing influence upon the microsomal enzymes.     

Resolving the excited state equilibrium of peridinin in solution

The carotenoid peridinin is abundant in the biosphere, as it is the main pigment bound by the light-harvesting complexes of dinoflagellates, where it collects blue and green sunlight and transfers energy to chlorophyll a with high efficiency. Its molecular structure is particularly complex, giving rise to an intricate excited state manifold, which includes a state with charge-transfer character. To disentangle the excited states of peridinin and understand their function in vivo, we applied dispersed pump-probe and pump-dump-probe spectroscopy. The preferential depletion of population from the intramolecular charge transfer state by the dump pulse demonstrates that the S(1) and this charge transfer state are distinct entities. The ensuing dump-induced dynamics illustrates the equilibration of the two states which occurs on the time scale of a few picoseconds. Additionally, the dump pulse populates a short-lived ground state intermediate, which is suggestive of a complex relaxation pathway, probably including structural reorientation or solvation of the ground state. These findings indicate that the unique intramolecular charge transfer state of peridinin is an efficient energy donor to chlorophyll a in the peridinin-chlorophyll-protein complex and thus plays a significant role in global light harvesting.     

Interaction energy decomposition in protein-protein association: a quantum mechanical study of barnase-barstar complex

Protein-protein interactions are very important in the function of a cell. Computational studies of these interactions have been of interest, but often they have utilized classical modelling techniques. In recent years, quantum mechanical (QM) treatment of entire proteins has emerged as a powerful approach to study biomolecular systems. Herein, we apply a semi-empirical divide and conquer (DC) methodology coupled with a dielectric continuum model for the solvent, to explore the contribution of electrostatics, polarization and charge transfer to the interaction energy between barnase and barstar in their complex form. Molecular dynamic (MD) simulation was performed to account for the dynamic behavior of the complex. The results show that electrostatics, charge transfer and polarization favor the formation of the complex. Our study shows that electrostatics dominates the interaction between barnase and barstar ( approximately 73%), while charge transfer and polarization are approximately 21% and approximately 6%, respectively. Close inspection of the polarization and charge-transfer effects on the charge distribution of the complex reveals the existence of two, well localized, regions in barstar. The first region includes the residues between P27 and Y47 and the second region is between N65 and D83. Since no such regions could be detected in barnase clearly suggests that barstar is well optimized for efficiently binding barnase. Furthermore, using our interaction energy decomposition scheme, we were able to identify all residues that have been experimentally determined to be important for the complex formation and to suggest other residues never have been investigated. This suggests that our approach will be useful as an aid in further understanding protein-protein contacts for the ultimate goal to produce successful inhibitors for protein complexes.     

A high throughput screen identifies chemical modulators of the laminin-induced clustering of dystroglycan and aquaporin-4 in primary astrocytes

Background

Aquaporin-4 (AQP4) constitutes the principal water channel in the brain and is clusteredat the perivascular astrocyte endfeet. This specific distribution of AQP4 plays a major role in maintaining water homeostasis in the brain. A growing body of evidence points to a role ofthe dystroglycan complex and its interaction with perivascular laminin in the clusteringof AQP4 atperivascular astrocyte endfeet. Indeed, mice lacking components of this complex or in which laminin-dystroglycan interaction is disrupted show a delayed onset of brain edema due to a redistribution of AQP4 away from astrocyte endfeet. It is therefore important to identify inhibitory drugs of laminin-dependent AQP4 clustering which may prevent or reduce brain edema.

Methodolgy/Principal Findings

In the present study we used primary rat astrocyte cultures toscreen a library of >3,500 chemicals and identified 6 drugs that inhibit the laminin-induced clustering of dystroglycan and AQP4. Detailed analysis of the inhibitory drug, chloranil, revealed that its inhibition of the clustering is due to the metalloproteinase-2-mediated ß-dystroglycan shedding and subsequent loss of laminin interaction with dystroglycan. Furthermore, chemical variants of chloranil induced a similar effect on ß-dystroglycan and this was prevented by the antioxidant N-acetylcysteine.

Conclusion/Significance

These findings reveal the mechanism of action of chloranil in preventing the laminin-induced clustering of dystroglycan and AQP4 and validate the use of high-throughput screening as a tool to identify drugs that modulate AQP4 clustering and that could be tested in models of brain edema.     

Ionization of zwitterionic amine substrates bound to monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) binds the L-proline zwitterion (pKa = 10.6). The reactive substrate anion is generated by ionization of the ES complex (pKa = 8.0). Tyr317 was mutated to Phe to determine whether this step might involve proton transfer to an active site base. The mutation does not eliminate the ionizable group in the ES complex (pKa = 8.9) but does cause a 20-fold decrease in the maximum rate of the reductive half-reaction. Kinetically determined Kd values for the ES complex formed with L-proline agree with results obtained in spectral titrations with the wild-type or mutant enzyme. Unlike the wild-type enzyme, Kd values with the mutant enzyme are pH-dependent, suggesting that the mutation has perturbed the pKa of a group that affects the Kd. As compared with the wild-type enzyme, an increase in charge transfer band energy is observed for mutant enzyme complexes with substrate analogues while a 10-fold decrease in the charge transfer band extinction coefficient is found for the complex with the L-proline anion. The results eliminate Tyr317 as a possible acceptor of the proton released upon substrate ionization. Since previous studies rule out the only other nearby base, we conclude that L-proline is the ionizable group in the ES complex and that amino acids are activated for oxidation upon binding to MSOX by stabilization of the reactive substrate anion. Tyr317 may play a role in substrate activation and optimizing binding, as judged by the effects of its mutation on the observed pKa, reaction rates, and charge transfer bands.     

Structure of the transition state analog of medium-chain acyl-CoA dehydrogenase. Crystallographic and molecular orbital studies on the charge-transfer complex of medium-chain acyl-CoA dehydrogenase with 3-thiaoctanoyl-CoA

The flavoenzyme medium-chain acyl-CoA dehydrogenase (MCAD) eliminates the alpha-proton of the substrate analog, 3-thiaoctanoyl-CoA (3S-C8-CoA), to form a charge-transfer complex with deprotonated 3S-C8-CoA. This complex can simulate the metastable reaction intermediate immediately after the alpha-proton elimination of a substrate and before the beta-hydrogen transfer as a hydride, and is therefore regarded as a transition-state analog. The crystalline complex was obtained by co-crystallizing MCAD in the oxidized form with 3S-C8-CoA. The three-dimensional structure of the complex was solved by X-ray crystallography. The deprotonated 3S-C8-CoA was clearly located within the active-site cleft of the enzyme. The arrangement between the flavin ring and deprotonated 3S-C8-CoA is consistent with a charge transfer interaction with the negatively charged acyl-chain of 3S-C8-CoA as an electron donor stacking on the pyrimidine moiety of the flavin ring as an electron acceptor. The structure of the model complex between lumiflavin and the deprotonated ethylthioester of 3-thiabutanoic acid was optimized by molecular orbital calculations. The obtained theoretical structure was essentially the same as that of the corresponding region of the X-ray structure. A considerable amount of negative charge is transferred to the flavin ring system to stabilize the complex by 9.2 kcal/mol. The large stabilization energy by charge transfer probably plays an important role in determining the alignment of the flavin ring with 3S-C8-CoA. The structure of the highest occupied molecular orbital of the complex revealed the electron flow pathway from a substrate to the flavin ring.     

The first catalytic step of the light-driven enzyme protochlorophyllide oxidoreductase proceeds via a charge transfer complex

In chlorophyll biosynthesis protochlorophyllide reductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide) to chlorophyllide, providing a rare opportunity to trap and characterize catalytic intermediates at low temperatures. Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR. Different models involving the formation of either radical species or charge transfer complexes have been proposed for the initial photochemical step, which forms a nonfluorescent intermediate absorbing at 696 nm (A696). Our EPR data show that the concentration of the radical species formed in the initial photochemical step is not stoichiometric with conversion of substrate. Instead, a large Stark effect, indicative of charge transfer character, is associated with A696. Two components were required to fit the Stark data, providing clear evidence that charge transfer complexes are formed during the initial photochemistry. The temperature dependences of both A696 formation and NADPH oxidation are identical, and we propose that formation of the A696 state involves hydride transfer from NADPH to form a charge transfer complex. A catalytic mechanism of POR is suggested in which Pchlide absorbs a photon, creating a transient charge separation across the C-17-C-18 double bond, which promotes ultrafast hydride transfer from the pro-S face of NADPH to the C-17 of Pchlide. The resulting A696 charge transfer intermediate facilitates transfer of a proton to the C-18 of Pchlide during the subsequent first "dark" reaction.