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Presented is a methodology to oxidatively cleave α-hydroxyketones and α- or β-diones using the environmentally benign reagent KHSO5, prepared easily from Oxone, to diesters in one simple transformation. In addition, we undertook a mechanistic study to provide a plausible mechanistic interpretation. These reactions may prove to be valuable alternatives to other related metal-mediated processes.

The regioselectivity of cyclization in methylene-interrupted epoxydiols with a thiophenyl ether group adjacent to the epoxide can be controlled by the appropriate choice of reaction conditions. Thus, while the 5-exo mode of cyclization is observed under protic conditions with polar solvents, the intermediacy of an episulfonium ion generated in non-polar solvents lead to a regioisomeric THF product.

[reaction: see text] A highly efficient, mild, and simple protocol is presented for the oxidation of aldehydes to carboxylic acids utilizing Oxone as the sole oxidant. Direct conversion of aldehydes in alcoholic solvents to their corresponding ester products is also reported. These reactions may prove to be valuable alternatives to traditional metal-mediated oxidations.

The chemistry of various salt forms of Oxone, an environmentally friendly oxidant, has been investigated. In addition to advances in the preparation of analytically pure KHSO5·H2O and nBu4NHSO5, a soluble form of this oxidant, we have also studied some of the known oxidative chemistry that utilizes Oxone as the oxidant. Our results indicate that utilizing purified reagents makes these reactions easier to workup and amenable to large scale synthesis because the amount of salt in the reaction has been greatly reduced. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

The enzyme, 15,15′-β-carotene dioxygenase (BCDOX), facilitates the oxidation of β-carotene to yield retinal. This is a remarkable process in which one of 11 double bonds in β-carotene is selectively oxidized. To further probe the mechanistic aspects of BCDOX, the synthesis of all-trans-[10′-3H]-8′-apo-β-carotenoic acid is reported. This compound will be used as a photoaffinity labeling reagent to probe the β-carotene binding pocket within BCDOX. The synthesis outlines a simple and efficient route for the incorporation of tritium at the 10′ olefinic carbon of 8′-apo-β-carotenoic acid. Copyright © 2002 John Wiley & Sons, Ltd.

Chloramine-T·3H(2)O and other chlorosulfonamide salts can serve as readily available, stable, and inexpensive precursors of electrophilic chlorine in the organocatalytic asymmetric chlorofunctionalization of olefins. In conjunction with commercially available organocatalysts, they can be utilized in the asymmetric chlorocyclization of unsaturated amides to yield products with unprecedented levels of stereoselectivity even at ambient temperatures and high concentrations.

Kinetic resolution and chlorofunctionalization are mediated by the same catalyst and proceed in one step.

We report absolute and relative stereochemistry of addition in enantioselective chlorolactonizations of 4-phenyl-4-pentenoic acid and its related t-butyl ester, catalyzed by (DHQD)2PHAL. Predominant syn addition of the chlorenium and the nucleophile across the olefin is observed. As shown by isotopic labeling, NMR spectroscopy, and derivative studies, the two new stereocenters formed by addition across the double bond are set independently and influenced by different factors. These findings suggest a stepwise process via an intermediate capable of lactone closure with either stereochemistry, in contradistinction to the more familiar scenario in which anti addition is dictated by a bridging chloronium ion intermediate.

KMnO4 and OsO4 catalyze the oxidative cyclization of 1,4-dienes to provide 2,3,5-trisubstituted tetrahydrofuran-diols in 30% yield. This reaction proceeds stereoselectively via a proposed [3+2] cycloaddition. Competing oxidative pathways are the major non-productive processes that reduce the yield of the reaction; however, four stereogenic centers are established in one-step.

A reagent controlled organocatalytic enantioselective chlorolactonization reaction has been developed. Several 4-aryl pentenoic acids were cyclized in the presence of 0.1 equiv of (DHQD)(2)PHAL, employing various N-chlorinated hydantoins as the terminal chlorenium source. Ten examples are presented with selectivities ranging from 43 to 90% ee. This work represents the first example of an enantioselective reagent-controlled chlorolactonization that approaches synthetically useful enantioselectivities.

The evolution of a variety of important chromophore-dependent biological processes, including microbial light sensing and mammalian color vision, relies on protein modifications that alter the spectral characteristics of a bound chromophore. Three different color opsins share the same chromophore, but have three distinct absorptions that together cover the entire visible spectrum, giving rise to trichromatic vision. The influence of opsins on the absorbance of the chromophore has been studied through methods such as model compounds, opsin mutagenesis, and computational modeling. The recent development of rhodopsin mimic that uses small soluble proteins to recapitulate the binding and wavelength tuning of the native opsins provides a new platform for studying protein-regulated spectral tuning. The ability to achieve far-red shifted absorption in the rhodopsin mimic system was attributed to a combination of the lack of a counteranion proximal to the iminium, and a uniformly neutral electrostatic environment surrounding the chromophore.

The retinal chromophore (11Z)-3-diazo-4-oxoretinal (1) with two photo-labile moieties has been synthesized by semi-hydrogenation of an 11-yne precursor with activated Zn in aqueous media. Incorporation of 1 into opsin yielded diazoketo rhodopsin (DK-Rh), which, upon bleaching, gave rise to intermediates batho-Rh, lumi-Rh, meta-Rh, and meta-II-Rh corresponding to those of native Rh but at lower temperatures. Photoaffinity labeling of DK-Rh and these bleaching intermediates showed that the ionone ring cross-linked to Trp265 of helix F in DK-Rh and batho intermediate, and to Ala169 of helix D in lumi, meta-I, and meta-II intermediates. These results demonstrate the occurrence of large conformational changes along the visual transduction path, which, in turn, is responsible for activation of the G-protein.

Attempts were made to develop an exciton chirality method applicable to the unique cases represented by 3-hydroxycarotenoids. However, this approach has so far not been successful. The 3-hydroxy configurations were therefore determined by the extended Mosher 1H-NMR method. Nine carotenoids with seven different end groups and of known 3-hydroxy configurations were derivatized with (R)- and (S)-methoxyphenylacetic acid (MPA); they all gave the expected shift differences. Three other auxiliary reagents with naphthalene and anthracene nuclei gave larger and consistent NMR shift differences, but they are not yet commercially available.

The light sensitivity and unstable nature of 11-cis-retinoids makes them ideal visual chromophores in nature. The synthesis of 11-cis-retinal analogues is of paramount importance in bioorganic studies of rhodopsin, the photoreceptor of the visual transduction pathway, but the instability of 11-cis-retinoids complicates their synthesis and there is no general synthetic route. Common strategies to the cis geometry have failed in the case of 11-cis-retinoids, and most often low yields and complex isomeric mixtures are obtained. Herein we report an efficient, general, and mild preparation of 11-cis-retinoids by semi-hydrogenation of 11-yne-retinoid precursors with Cu/Ag-activated zinc dust.

Radical-mediated ring expansion methodology is presented wherein 7-membered carbocycles can be prepared from the corresponding xanthate derivatives of bicyclo[4.1.0]heptan-1-methanol. In certain systems, an intermediate cycloheptyl radical appears to be kinetically favored over the cyclohexyl radical, but the direction of cyclopropylcarbinyl radical fragmentation is subject to substitution about the bicyclo[4.1.0]heptan-1-methyl ring.

We report absolute and relative stereochemistry of addition in enantioselective chlorolactonizations of 4-phenyl-4-pentenoic acid and its related t-butyl ester, catalyzed by (DHQD)2PHAL. Predominant syn addition of the chlorenium and the nucleophile across the olefin is observed. As shown by isotopic labeling, NMR spectroscopy, and derivative studies, the two new stereocenters formed by addition across the double bond are set independently and influenced by different factors. These findings suggest a stepwise process via an intermediate capable of lactone closure with either stereochemistry, in contradistinction to the more familiar scenario in which anti addition is dictated by a bridging chloronium ion intermediate.

Recently, a rhodopsin protein mimic was constructed by combining mutants of the cellular retinoic acid binding protein II (CRABPII) with an all-trans retinal chromophore. Here, we present a combined computational quantum mechanics/molecular mechanics (QM/MM) and experimental ultrafast kinetic study of CRABPII. We employ the QM/MM models to study the absorption (λ(a)max), fluorescence (λ(f)max), and reactivity of a CRABPII triple mutant incorporating the all-trans protonated chromophore (PSB-KLE-CRABPII). We also study the spectroscopy of the same mutant incorporating the unprotonated chromophore and of another double mutant incorporating the neutral unbound retinal molecule held inside the pocket. Finally, for PSB-KLE-CRABPII, stationary fluorescence spectroscopy and ultrafast transient absorption spectroscopy resolved two different evolving excited state populations which were computationally assigned to distinct locally excited and charge-transfer species. This last species is shown to evolve along reaction paths describing a facile isomerization of the biologically relevant 11-cis and 13-cis double bonds. This work represents a first exploratory attempt to model and study these artificial protein systems. It also indicates directions for improving the QM/MM models so that they could be more effectively used to assist the bottom-up design of genetically encodable probes and actuators employing the retinal chromophore.

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 H218O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of 18O. 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 α-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.

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 α, β and γ 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 K m 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.

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.

Human Cellular Retinol Binding Protein II (hCRBP II), a member of the intracellular Lipid binding protein (iLBP) family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. HCRBP II and other members of this family have been shown to be remarkably stable to mutations. Previous studies on other proteins of this family have shown that they fold into monomeric species. However we report, for the first time, that wild-type (WT) hCRBP II gives rise to an extensive domain swapped dimer (involving almost 50% of the protein sequence) during bacterial expression. In addition, there is no evidence of interconversion between monomer and dimer at room temperature, even after weeks of incubation, suggesting that two non-interconverting folds can result from the same amino acid sequence. Though wild-type hCRBP II forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. Structural studies of wild-type and several mutant dimers suggest that an “open monomer” folding intermediate gives rise to both monomer and dimer, and their ratio depends on the relative orientation of the two halves of the protein in the open monomer intermediate. Therefore, Nature may build in an interaction (a Tyr60-Glu72 hydrogen bond) in a folding intermediate to prevent dimerization to yield the physiologically relevant monomeric protein, begging the question of whether other members of the iLBP family form physiologically relevant dimer species.

The carboxylation of aziridine derivatives bearing a silylated propargylic alkoxy group proceeds in the presence of Bu4NF and NaHCO3 to afford the target fused enamine carbonates in good yields.

Both enantiomers of the dihydropyrans are obtained applying alkaloid-derived catalysts (I) or (II), respectively.

Synthesis of C15,14′-ring locked all- trans -β-carotene is described. The symmetric nature of the β-carotene analog allowed for rapid construction of the carbon frame through bis-olefination of the central dialdehyde containing the ring functionality with a C15 ylide bearing the ionone moiety.

Good to excellent stereo-selectivity has been found in the addition reactions of Grignard and organo-zinc reagents to N-protected aziridine-2-carboxaldehydes. Specifically, high syn selectivity was obtained with benzyl-protected cis, tert-butyloxycar-bonyl-protected trans, and tosyl-pro-tected 2,3-disubstituted aziridine-2-car-boxaldehydes. Furthermore, rate and selectivity effects of ring substituents, temperature, solvent, and Lewis acid and base modifiers were studied. The diastereomeric preference of addition is dominated by the substrate aziri-dines’ substitution pattern and especially the electronic character and conformational preferences of the nitrogen protecting groups. To help rationalize the observed stereochemical outcomes, conformational and electronic structural analyses of a series of model systems representing the various substitution patterns have been explored by density functional calculations at the B3LYP/6–31G* level of theory with the SM8 solvation model to account for solvent effects.

Hydration of methyl diepoxystearate ( 2 ) at high cytosolic epoxide hydrolase (CEH) concentration produces the corresponding tetraol, while at physiological CEH concentration four tetrahydrofuran diol products ( 3–6 ) are produced. These same four products are obtained in the acid-catalyzed hydration of 2 . Spectroscopic studies, primarily EI mass spectrometry and difference spectrum NOE, are reported which establish the regio- and stereoselectivity of 2 → 3–6 (MS differentiating 3 4 from 5 6 and NOE differentiating 3 5 from 4 6 ). The observed syn arrangement of the two adjacent groups (i.e., substituents at C 2 and C 3 ) in the four THF-diols can only arise from an A 2 type opening of the first epoxide. The resulting epoxydiol intermediate then cyclizes by A 2 opening of the second cis -epoxide as established by synthesis of these hypothetical epoxydiol intermediates and their subsequent conversion to THF-diols 3–6 .

The 1,4-bis-13C-labeled isomeric compounds (Z,E)- and (E,E)-2-ethoxycarbonyl-1,4-diphenylbutadiene have been synthesized by two different methods. A double Horner–Wadsworth–Emmons (HWE) strategy was employed for the synthesis of the Z,E isomer. On the other hand, synthesis of the E,E isomer was achieved by Baylis–Hillman reaction of benzaldehyde with ethyl acrylate and subsequent rearrangement, followed by Wittig olefination with benzaldehyde. These routes provide stereoselective access to labeled compounds in good yields.

PKCδ has emerged as a novel regulatory molecule of oxidative phosphorylation by targeting the pyruvate dehydrogenase complex (PDHC). We showed that activation of PKCδ leads to the dephosphorylation of pyruvate dehydrogenase kinase 2 (PDK2), thereby decreasing PDK2 activity and increasing PDH activity, accelerating oxygen consumption, and augmenting ATP synthesis. However, the molecular components that mediate PKCδ signaling in mitochondria have remained elusive so far. Here, we identify for the first time a functional complex, which includes cytochrome c as the upstream driver of PKCδ, and uses the adapter protein p66Shc as a platform with vitamin A (retinol) as a fourth partner. All four components are necessary for the activation of the PKCδ signal chain. Genetic ablation of any one of the three proteins, or retinol depletion, silences signaling. Furthermore, mutations that disrupt the interaction of cytochrome c with p66Shc, of p66Shc with PKCδ, or the deletion of the retinol-binding pocket on PKCδ, attenuate signaling. In cytochrome c-deficient cells, reintroduction of cytochrome c Fe3+ protein restores PKCδ signaling. Taken together, these results indicate that oxidation of PKCδ is key to the activation of the pathway. The PKCδ/p66Shc/cytochrome c signalosome might have evolved to effect site-directed oxidation of zinc-finger structures of PKCδ, which harbor the activation centers and the vitamin A binding sites. Our findings define the molecular mechanisms underlying the signaling function of PKCδ in mitochondria.—Acin-Perez, R., Hoyos, B., Gong, J., Vinogradov, V., Fischman, D. A., Leitges, M., Borhan, B., Starkov, A., Manfredi, G., Hammerling, U. Regulation of intermediary metabolism by the PKCδ signalosome in mitochondria.

In an attempt to revise the structural assignment of mucoxin, and faced with 64 diastereomeric possibilities, we resorted to the synthesis of truncated structures that contained the core stereochemical sites. Twelve stereochemical analogues were synthesized, their (1)H and (13)C NMR spectra were analyzed and four recurring stereochemical trends were distilled from the data. Applying the observed trends to the diastereomeric population pared the possible choices for the correct structure of mucoxin from 64 to 4. Synthesis of these analogues led to the identification of the correct structure of mucoxin.

Hydration of methyl diepoxystearate ( 2 ) at high cytosolic epoxide hydrolase (CEH) concentration produces the corresponding tetraol, while at physiological CEH concentration four tetrahydrofuran diol products ( 3–6 ) are produced. These same four products are obtained in the acid-catalyzed hydration of 2 . Spectroscopic studies, primarily EI mass spectrometry and difference spectrum NOE, are reported which establish the regio- and stereoselectivity of 2 → 3–6 (MS differentiating 3 4 from 5 6 and NOE differentiating 3 5 from 4 6 ). The observed syn arrangement of the two adjacent groups (i.e., substituents at C 2 and C 3 ) in the four THF-diols can only arise from an A 2 type opening of the first epoxide. The resulting epoxydiol intermediate then cyclizes by A 2 opening of the second cis -epoxide as established by synthesis of these hypothetical epoxydiol intermediates and their subsequent conversion to THF-diols 3–6 .

A reagent controlled organocatalytic enantioselective chlorolactonization reaction has been developed. Several 4-aryl pentenoic acids were cyclized in the presence of 0.1 equiv of (DHQD)2PHAL, employing various N-chlorinated hydantoins as the terminal chlorenium source. Ten examples are presented with selectivities ranging from 43 to 90% ee. This work represents the first example of an enantioselective reagent-controlled chlorolactonization that approaches synthetically useful enantioselectivities.

Radical-mediated ring expansion methodology is presented wherein 7-membered carbocycles can be prepared from the corresponding xanthate derivatives of bicyclo[4.1.0]heptan-1-methanol. In certain systems, an intermediate cycloheptyl radical appears to be kinetically favored over the cyclohexyl radical, but the direction of cyclopropylcarbinyl radical fragmentation is subject to substitution about the bicyclo[4.1.0]heptan-1-methyl ring.

Reuterin is an antimicrobial compound produced by Lactobacillus reuteri, and has been proposed to mediate, in part, the probiotic health benefits ascribed to this micro-organism. Despite 20 years of investigation, the mechanism of action by which reuterin exerts its antimicrobial effects has remained elusive. Here we provide evidence that reuterin induces oxidative stress in cells, most likely by modifying thiol groups in proteins and small molecules. Escherichia coli cells subjected to sublethal levels of reuterin expressed a set of genes that overlapped with the set of genes composing the OxyR regulon, which senses and responds to various forms of oxidative stress. E. coli cells mutated for oxyR were more sensitive to reuterin compared with wild-type cells, further supporting a role for reuterin in exerting oxidative stress. The addition of cysteine to E. coli or Clostridium difficile growth media prior to exposure to reuterin suppressed the antimicrobial effect of reuterin on these bacteria. Interestingly, interaction with E. coli stimulated reuterin production or secretion by L. reuteri, indicating that contact with other microbes in the gut increases reuterin output. Thus, reuterin inhibits bacterial growth by modifying thiol groups, which indicates that reuterin negatively affects a large number of cellular targets.

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...

KMnO4 and OsO4 catalyze the oxidative cyclization of 1,4-dienes to provide 2,3,5-trisubstituted tetrahydrofuran-diols in 30% yield. This reaction proceeds stereoselectively via a proposed [3+2] cycloaddition. Competing oxidative pathways are the major non-productive processes that reduce the yield of the reaction; however, four stereogenic centers are established in one-step.

A mild, organometallic alternative to ozonolysis utilizing oxone and OsO4 is presented. This is a direct oxidation of olefins via the carbon−carbon cleavage of an osmate ester by the action of oxone. Twenty-four different olefins were converted to their corresponding ketones or carboxylic acids in high yields (>80%). Free alcohols, acetate- and benzyl-protected alcohols, and 1,2-diols were stable under these conditions. This method should be applicable for traditional organic synthesis.

Within cancer, there is a large wealth of diversity, complexity, and information that nature has engineered rendering it challenging to identify reliable detection methods. Therefore, the development of simple and effective techniques to delineate the fine characteristics of cancer cells can have great potential impacts on cancer diagnosis and treatment. Herein, we report a magnetic glyco-nanoparticle (MGNP) based nanosensor system bearing carbohydrates as the ligands, not only to detect and differentiate cancer cells but also to quantitatively profile their carbohydrate binding abilities by magnetic resonance imaging (MRI). Using an array of MGNPs, a range of cells including closely related isogenic tumor cells, cells with different metastatic potential and malignant vs normal cells can be readily distinguished based on their respective "MRI signatures". Furthermore, the information obtained from such studies helped guide the establishment of strongly binding MGNPs as antiadhesive agents against tumors. As the interactions between glyco-conjugates and endogenous lectins present on cancer cell surface are crucial for cancer development and metastasis, the ability to characterize and unlock the glyco-code of individual cell lines can facilitate both the understanding of the roles of carbohydrates as well as the expansion of diagnostic and therapeutic tools for cancer.

A simple and efficient methodology for the preparation of N-chlorinated hydantoins is presented. These versatile chlorenium sources were isolated in high yield after a simple recrystallization. Among the ten examples are the first chiral N-chlorohydantoins.

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.

Chemical cross-linking combined with proteolytic digestion and mass spectrometry (MS) is a promising approach to provide inter- and intramolecular distance constraints for the structural characterization of protein topologies and functional multiprotein complexes. Despite the relative straightforwardness of these methodologies, the identification and characterization of cross-linked proteins presents a significant analytical challenge, due to the complexity of the resultant peptide mixtures, as well as the array of inter-, intra-, or "dead-end"-cross-linked peptides that may be generated from a single cross-linking experiment. To address these issues, we describe here the synthesis, characterization, and initial evaluation of a novel "fixed charge" sulfonium ion-containing crosslinking reagent, S-methyl 5,5'-thiodipentanoylhydroxysuc-cinimide. The peptide products obtained by reaction with this reagent are all shown to fragment exclusively via facile cleavage of the C-S bond directly adjacent to the fixed charge during CID-MS/MS, resulting in the formation of characteristic product ions that enable the presence and type (i.e., inter, intra, or dead-end) of the cross-linked products to be readily determined, independently of the "proton mobility" of the precursor ion. Subsequent isolation and dissociation of these products by MS3 provides additional structural information required for identification of the peptide sequences involved in the cross-linking reactions, as well as for characterization of the specific site(s) at which cross-linking has occurred. The specificity of these gas-phase fragmentation reactions, as well as the solubility and stability of the cross-linking reagent under aqueous conditions, suggests that this strategy holds great promise for use in future studies aimed at the structural analysis of large proteins or multiprotein assemblies.

A simple and efficient methodology for the preparation of N-chlorinated hydantoins is presented. These versatile chlorenium sources were isolated in high yield after a simple recrystallization. Among the ten examples are the first chiral N-chlorohydantoins.

The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by providing a carboxylic acid dimer partner in the form of a Glu residue.

A microscale protocol for determination of absolute configurations of 2,3-epoxy alcohols is described. 2,3-Disubstituted (cis and trans), 2,2-disubstituted, 2,2,3-trisubstituted, and 2,3,3-trisubstituted epoxy alcohols rendered prominent ECCD signals upon complexing with a Lewis acidic porphyrin tweezer and consequently provide straightforward assignment of chirality for epoxy alcohols. This method proved to be rapid, simple, sensitive, and reliable for the class of molecules listed above.

Rational redesign of the binding pocket of Cellular Retinoic Acid Binding Protein II (CRABPII) has provided a mutant that can bind retinal as a protonated Schiff base, mimicking the binding observed in rhodopsin. The reengineering was accomplished through a series of choreographed manipulations to ultimately orient the reactive species (the epsilon-amino group of Lys132 and the carbonyl of retinal) in the proper geometry for imine formation. The guiding principle was to achieve the appropriate Bürgi-Dunitz trajectory for the reaction to ensue. Through crystallographic analysis of protein mutants incapable of forming the requisite Schiff base, a highly ordered water molecule was identified as a key culprit in orienting retinal in a nonconstructive manner. Removal of the ordered water, along with placing reinforcing mutations to favor the desired orientation of retinal, led to a triple mutant CRABPII protein capable of nanomolar binding of retinal as a protonated Schiff base. The high-resolution crystal structure of all-trans-retinal bound to the CRABPII triple mutant (1.2 A resolution) unequivocally illustrates the imine formed between retinal and the protein.