Controlling Quantum Interference between Virtual and Dipole Two-Photon Optical Excitation Pathways Using Phase-Shaped Laser Pulses.

Two-photon excitation (TPE) proceeds via a "virtual" pathway, which depends on the accessibility of one or more intermediate states, and, in the case of non-centrosymmetric molecules, an additional "dipole" pathway involving the off-resonance dipole-allowed one-photon transitions and the change in the permanent dipole moment between the initial and final states. Here, we control the quantum interference between these two optical excitation pathways by using phase-shaped femtosecond laser pulses. We find enhancements by a factor of up to 1.75 in the two-photon-excited fluorescence of the photobase FR0-SB in methanol after taking into account the longer pulse duration of the shaped laser pulses. Simulations taking into account the different responses of the virtual and dipole pathways to external fields and the effect of pulse shaping on two-photon transitions are found to be in good agreement with our experimental measurements. The observed quantum control of TPE in the condensed phase may lead to an enhanced signal at a lower intensity in two-photon microscopy, multiphoton-excited photoreagents, and novel spectroscopic techniques that are sensitive to the magnitude of the contributions from the virtual and dipole pathways to multiphoton excitations.

XtalFluor-E® mediated proto-functionalization of N-vinyl amides: access to N-acetyl N,O-acetals.

XtalFluor-E® has been extensively used in a broad range of reactions in the past few years. Here we report its use with protic nucleophiles in a catalytic manner for the in situ generation of protons that lead to the proto-functionalization of activated olefins. Utilizing the latter protocol, proto etherification of enamides gives rise to N,O-acetals in nearly quantitative yields.

On the bioactive conformation of the rhodopsin chromophore: absolute sense of twist around the 6-s-cis bond.

Incubation of opsin with synthetic 6-s-locked retinoids 2a and 2b only led to pigment formation from the alpha-locked 2a, the CD spectrum of which was similar to that of native rhodopsin (Rh). This establishes that the 6-s-bond of the chromophore in rhodopsin is cis, and that its helicity is negative. Earlier cross-linking studies showed that the 11-cis to all-trans photoisomerization occurring in the batho-Rh to lumi-Rh conversion induces a flip over of the side carrying the ring moiety. The GTP-binding assay of pigment Rh-(2a), incorporating retinal analogue 2a, has shown that its activity is 80% that of the native pigment. That is, the overall conformation around the 6-s bond is retained in the steps leading to G-protein activation.

Regiochemical control in intramolecular cyclization of methylene-interrupted epoxydiols.

[reaction: see text] Methylene-interrupted epoxydiols have multiple regiochemical routes for cyclization. The 5-exo process is the most prevalent under acidic conditions. However, the regioselectivity can be controlled by the appropriate choice of acid promoter and pendant groups adjacent to the epoxide. The 5-exo product is obtained exclusively without the presence of a carbocation-stabilizing pendant group. Alkenyl and thiophenyl groups adjacent to the epoxide alter the regioselectivity and enable access to the 5-endo tetrahydrofuran and 6-endo tetrahydropyran products.

Movement of retinal along the visual transduction path.

Movement of the ligand/receptor complex in rhodopsin (Rh) has been traced. Bleaching of diazoketo rhodopsin (DK-Rh) containing 11-cis-3-diazo-4-oxo-retinal yields batho-, lumi-, meta-I-, and meta-II-Rh intermediates corresponding to those of native Rh but at lower temperatures. Photoaffinity labeling of DK-Rh and these bleaching intermediates shows that the ionone ring cross-links to tryptophan-265 on helix F in DK-Rh and batho-Rh, and to alanine-169 on helix D in lumi-, meta-I-, and meta-II-Rh intermediates. It is likely that these movements involving a flip-over of the chromophoric ring trigger changes in cytoplasmic membrane loops resulting in heterotrimeric guanine nucleotide-binding protein (G protein) activation.

Zinc porphyrin tweezer in host-guest complexation: determination of absolute configurations of primary monoamines by circular dichroism.

A nonempirical exciton chirality circular dichroic (CD) method for determining the absolute configurations of primary monoamines with amino group directly linked to the stereogenic center is described. Conventional exciton chirality CD method cannot be applied to these compounds since they lack the two sites for attaching the interacting chromophores. This was solved by covalently linking the monoamine to a trifunctional bidentate carrier moiety 1. Treatment of the carrier/monoamine conjugate with the porphyrin tweezer 4 consisting of two pentanediol-linked zinc porphyrins gives rise to 1:1 host-guest macrocyclic complexes that exhibit exciton-coupled CD spectra. The sign of the CD couplet can then be correlated with the absolute configuration of the monoamine as follows: a clockwise arrangement of the L, M, and S (large, medium, small) groups in the Newman projection of the monoamine with the amino group in the rear gives rise to a positive CD couplet, and vice versa; the assignments of L, M, S groups are based on conformational energies (A values). This method is applicable to cyclic and acyclic aliphatic amines, aromatic amines, amino esters, amides, and cyclic amino alcohols, and can be performed at the several microgram level.

Spectroscopic studies of PhTX facilitated cation movement across membranes.

Philanthotoxins, noncompetitive inhibitors of the nicotinic acetylcholine receptor and various glutamate receptors, were found to be capable of mediating cation transport across lipid bilayers. With respect to the relatively weak binding constants of these amphiphilic polyamines to neuronal receptor proteins, this finding implies that their interaction with cell membranes might have to be considered in addition to that with protein receptors to fully understand the molecular mechanism of these neurotoxins.

Structure-activity relationships for substrates and inhibitors of mammalian liver microsomal carboxylesterases.

PURPOSE: Carboxylesterases are important in the detoxification of drugs, pesticides and other xenobiotics. This study was to evaluate a series of substrates and inhibitors for characterizing these enzymes. METHODS: A series of novel aliphatic esters and thioesters were used in spectral assays to monitor human, murine and porcine esterases. A series of transition state mimics were evaluated as selective esterase inhibitors. RESULTS: Several alpha-alkyl thioacetothioates were found to be approximately 2 to 11-fold superior to commonly used substrates for monitoring carboxylesterase activity. Insertion of a heteroatom in the acid portion of these esters in the beta or gamma position relative to the carbonyl had a dramatic effect on enzyme activity with S or O substituents often improving the kCAT/K(M) ratio of the substrate and N decreasing it. Several alpha,alpha'-bis (2-oxo-3,3,3-trifluoropropylthio)alkanes proved to be potent selective transition state mimics of the esterase activity with IC50's from 10(-5) to 10(-9)M. CONCLUSIONS: This library of substrates and inhibitors are useful research tools for characterizing the numerous isozymes of carboxylesterases present in mammalian tissues.

Novel metabolic pathways for linoleic and arachidonic acid metabolism.

Mouse liver microsomes oxidized linoleic acid to form 9,10- or 12,13-epoxyoctadecenoate. These monoepoxides were subsequently hydrolyzed to their corresponding diols in the absence of the microsomal epoxide hydrolase inhibitor, 1,2-epoxy-3,3,3-trichloropropane. Furthermore, both 9,10- and 12,13-epoxyoctadecenoates were oxidized to diepoxyoctadecanoate at apparently identical rates by mouse liver microsomal P-450 epoxidation. Both epoxyoctadecanoates and diepoxyoctadecanoates were converted to tetrahydrofuran-diols by microsomes. Tetrahydroxides of linoleate were produced as minor metabolites. Arachidonic acid was metabolized to epoxyeicosatrienoates, dihydroxyeicosatrienoates, and monohydroxyeicosatetraenoates by the microsomes. Microsomes prepared from clofibrate (but not phenobarbital) -treated mice exhibited much higher production rates for epoxyeicosatrienoates and vic-dihydroxyeicosatrienoates. This indicated an induction of P-450 epoxygenase(s) and microsomal epoxide hydrolase in mice by clofibrate and not by phenobarbital. Incubation of synthetic epoxyeicosatrienoates with microsomes led to the production of diepoxyeicosadienoates. Among chemically generated diepoxyeicosadienoate isomers, three of them possessing adjacent diepoxides were hydrolyzed to their diol epoxides which cyclized to the corresponding tetrahydrofuran-diols by microsomes as well as soluble epoxide hydrolase at a much higher rate. Larger cyclic products from non-adjacent diepoxides were not observed. The results of our in vitro experiments suggest that linoleic and arachidonic acid can be metabolized to their tetrahydrofuran-diols by two consecutive microsomal cytochrome P-450 epoxidations followed by microsomal or soluble epoxide hydrolase catalyzed hydrolysis of the epoxides. Incubation experiments with the S-9 fractions indicate that the soluble epoxide hydrolase is more important in this conversion. This manuscript is the first report of techniques for the separation and identification of regio and geometrical isomer of an interesting class of oxylipins and their metabolism by liver microsomes and S-9 fractions to THF-diols.

Development of an in situ toxicity assay system using recombinant baculoviruses.

A new method for experimentally analyzing the role of enzymes involved in metabolizing mutagenic, carcinogenic, or cytotoxic chemicals is described. Spodoptera fugiperda (SF-21) cells infected with recombinant baculoviruses are used for high level expression of one or more cloned enzymes. The ability of these enzymes to prevent or enhance the toxicity of drugs and xenobiotics is then measured in situ. Initial parameters for the system were developed and optimized using baculoviruses engineered for expression of the mouse soluble epoxide hydrolase (msEH, EC 3.3.2.3) or the rat cytochrome P4501A1. SF-21 cells expressing msEH were resistant to trans-stilbene oxide toxicity as well as several other toxic epoxides including: cis-stilbene oxide, 1,2,7,8-diepoxyoctane, allylbenzene oxide, and estragole oxide. The msEH markedly reduced DNA and protein adduct formation in SF-21 cells exposed to [3H]allylbenzene oxide or [3H]estragole oxide. On the other hand, 9,10-epoxyoctadecanoic acid and methyl 9,10-epoxyoctadecanoate were toxic only to cells expressing sEH, suggesting that the corresponding fatty acid diols were cytotoxic. This was confirmed by showing that chemically synthesized diols of these fatty acid epoxides were toxic to control SF-21 cells at the same concentration as were the epoxides to cells expressing sEH. A recombinant baculovirus containing a chimeric cDNA formed between the rat P4501A1 and the yeast NADPH-P450 reductase was also constructed and expressed in this system. A model compound, naphthalene, was toxic to SF-21 infected with the rat P4501A1/reductase chimeric co-infecting SF-21 cells with either a human or a rat microsomal EH virus along with P4501A1/reductase virus. These results demonstrate the usefulness of this new system for experimentally analyzing the role of enzymes hypothesized to metabolize endogenous and exogenous chemicals of human health concern.

Methyl and phenyl esters and thioesters of carboxylic acids as surrogate substrates for microassay of proteinase K esterase activity.

The development of a microassay for proteinase K esterase activity with carboxylic acid esters is reported using novel substrates of the general formula R-C(O)-XR'. Highest rates of hydrolysis have been obtained with the O-phenyl esters CH(3)(CH(2))(n = 1 - 2)-S-(CH(2))(n = 1 - 2) C(O)-O-phenyl and their thioester analogs in studies where R, X and R' have been varied. The phenol release has been measured with 4-aminoantipyrine and potassium ferricyanide to determine the rates of O-phenyl ester hydrolyses. Thioester hydrolyses have been monitored continuously with 5,5'-dithio-bis (2-nitrobenzoic acid).

Mechanism of soluble epoxide hydrolase. Formation of an alpha-hydroxy ester-enzyme intermediate through Asp-333.

18O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to determine the regioselectivity of sEH. The nucleophilic nature of sEH catalysis was demonstrated by comparing the enzymatic and nonenzymatic hydrolysis products of tDPPO. The results from single turnover experiments with greater or equal molar equivalents of sEH:substrate were consistent with the existence of a stable intermediate formed by a nucleophilic amino acid attacking the epoxide group. Tryptic digestion of sEH previously subjected to multiple turnovers with tDPPO in H2 18O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of 180. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attack by Asp-333 initiates enzymatic activity, leading to the formation of an alpha-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl-enzyme occurs by the addition of an activated water to the carbonyl carbon of the ester bond, after which the resultant tetrahedral intermediate collapses, yielding the active enzyme and the diol product.

Improved radiolabeled substrates for soluble epoxide hydrolase.

Two rapid assays for the soluble epoxide hydrolase (sEH) are described. First, a sensitive radiometric assay based on thin-layer chromatography of [(14)C]-cis-9,10-epoxystearic acid and its corresponding diol ((14)C]-9,10-dihydroxystearic acid) is described. The cis fatty acid oxide exhibits higher specific activity of hydration with sEH from mouse, rat, human, and potato compared to trans-stilbene oxide (TSO). The K(m) and V(max) obtained for [(14)C]-cis-9,10-epoxystearic acid with mouse sEH are 11.0 microM and 3460 nmol/min/mg protein, respectively. [(14)C]-cis-9,10- Epoxystearic acid might more closely mimic the structures of natural substrates for sEH. Second, [2-(3)H]-trans-1,3-diphenyl-propene oxide ([(3)H]-tDPPO) and [2-(3)H]-cis-1,3-diphenylpropene oxide ([(3)H]-cDPPO) were synthesized and rapid radiometric assays for epoxide hydrolases (EHs) were developed by differential partitioning of the epoxide into iso-octane and its corresponding diol into aqueous phase containing methanol. It was shown that sEHs from mouse, rat, human, and potato rapidly hydrolyze [(3)H]-tDPPO and in comparison to TSO have 20-,49-,28-, and 7-fold higher rates, respectively. Mouse sEH hydrates [(3)H]-tDPPO at 26,200 nmol/min/mg protein, and a K(m)p4 of 2.80 microM is observed.

Development of surrogate substrates for neuropathy target esterase.

Seventeen substrates were synthesized and their activities as surrogate substrates for Neuropathy Target Esterase were tested. Substrates investigated are carbon analogs of phenylvalerate (1) with oxygen and sulfur substituted at the alpha, beta and gamma positions. Phenol and thiophenol esters of these analogs constitute two series of compounds tested. The ratio of catalytic hydrolysis to background hydrolysis increased at lower pH values with all substrates tested including phenylvalerate (1). There was more than a 2.5-fold increase in specific activity with phenylthiopropylethanoate (6) at pH of 6.75 compared to phenylvalerate (1). Furthermore, a 19-fold decrease in Km is reported with compound 6. This and related compounds can be used as the basis of more sensitive assays for neuropathy target esterase. Thiophenyl esters in this series are sufficiently good substrates to hold promise in continuous assays.

Molecular and biochemical evidence for the involvement of the Asp-333-His-523 pair in the catalytic mechanism of soluble epoxide hydrolase.

In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.

Characterization of the cytosolic epoxide hydrolase-catalyzed hydration products from 9,10:12,13-diepoxy stearic esters.

In a previous report we hypothesized that diepoxy fatty methylesters are metabolized to tetraols and/or tetrahydrofurandiols through an epoxydiol intermediate. In this study, p-nitrophenyldiepoxystearate was incubated with affinity-purified liver cytosolic epoxide hydrolase and product formation was monitored by reverse phase HPLC. The diepoxystearate was converted to the corresponding 9,10,12,13-tetraol using a concentrated enzyme (greater than or equal to 100 micrograms/ml). When lower concentration of the enzyme was used, simultaneous elevation of 9,10-epoxy-12,13-dihydroxy and 12,13-epoxy-9,10-dihydroxystearate along with disappearance of tetraol was observed. The epoxydiols were intermediates which could be isolated and cyclized quantitatively to form two chromatographically distinct tetrahydrofurandiols (A with a low Rf value and B with a high Rf value on TLC). Gas chromatographic analysis on a cyclodex-beta capillary column revealed that each compound was composed of two different isomers. The structure of these isomers was 9(12)-oxy-10,13-dihydroxystearate and 10(13)-oxy-9,12-dihydroxystearate using mass spectrometry. Stereochemistry of the aliphatic chain across the tetrahydrofuran moiety was determined by nuclear Overhauser effect spectroscopy. Chemically and enzymatically generated tetrahydrofurandiols had similar retention time on GC and HPLC, and identical mass spectra using the electron impact mode.