Epoxide Intermediates Research Articles

Sulfonated polydivinylbenzene bamboo-like nanotube stabilized Pickering emulsion for effective oxidation of olefins to 1,2-diol

Sulfonated polydivinylbenzene bamboo-like nanotube (SPDVB) with effective olefins oxidation activity is prepared by combining cationic polymerization and sulfonation. By merely adjusting sulfonation time, SPDVB with different sulfonic acid group (–SO3H) contents is achieved. SPDVB is used as both a solid emulsifier and catalyst to fabricate Pickering emulsion interface catalytic system for oxidizing olefins with 30% H2O2 acting as oxidant/water phase and olefins acting as reactants/oil phase. It is shown that Pickering emulsion interface catalytic system stabilized by SPDVB exhibits enhanced olefins oxidation efficiency than the conventional ones. At the optimum catalyst and reaction condition, the conversion of olefins by Pickering emulsion interface catalytic system stabilized by SPDVB for cyclohexene, 1-methylcyclohexene, cyclooctene, 2,3-dimethyl-2-butene oxidation is higher than 90.00% and the corresponding 1,2-diol selectivity exceeds 93.00% except the selectivity to 1-methyl-1,2-cyclohexanediol. The catalytic system also exhibits excellent cycling performance (>95.00% olefins conversion and >89.00% 1,2-diol selectivity for cyclohexene/2,3-dimethyl-2-butene oxidation after four cycles). A possible mechanism for oxidation of olefins to 1,2-diol by SPDVB stabilized Pickering emulsion is proposed: the high catalytic interface area between sulfonic acid group and H2O2 in water phase enhances the sulfonic acid group of SPDVB to convert into peroxysulfonic acid (catalytic activity centre) firstly; then the formed peroxysulfonic acid attacks the double bond of olefins to form epoxide intermediates, which will be hydrolyzed to 1,2-diol.

First insight on in vitro metabolism of three newly identified aryl organophosphate esters via a suspect coupled with nontarget screening approach

The ubiquity of organophosphate esters (OPEs) in the environment has triggered research into metabolic pathways of OPEs. Using liquid chromatography coupled with a hybrid quadrupole Orbitrap high-resolution mass spectrometer, a suspect and characteristic fragment ion-based nontarget screening strategy for the identification of unknown OPE metabolites was developed and evaluated. Then, this integrated approach was successfully used for investigation of three newly identified organophosphate esters (NOPEs), namely 2-biphenylol diphenyl phosphate (BPDPP), tris(2-biphenyl) phosphate (TBPHP), and naphthalen-2-yl diphenyl phosphate (NDPHP), in human liver microsomes (HLMs). The results demonstrated that BPDPP, TBPHP, and NDPHP were effectively metabolized by HLMs, with zero-order kinetics (R2 = 0.48−0.94) within the time frame of the assay. The suspected approach identified a considerable number of dearylated phosphate (DP), and hydroxylated metabolites for each of NOPEs after incubation with HLMs for 2 h. In addition, the nontarget approach further identified 9 novel metabolites including 2 epoxide intermediates and 7 oxidative ring-opening compounds, which were first reported in the Phase I metabolism of OPEs. Collectively, this study provided a novel suspect coupled with nontarget screening approach and was successfully used to screen metabolites of three NOPEs. For the first time, we observed direct evidence that oxidative ring-opening might serve as another primary metabolic pathway regarding the metabolism of aryl OPEs.

Unraveling the mechanism of latex clearing protein Lcp<sub>K30</sub> catalysed oxidative cleavage of poly(<italic>cis</italic>-1,4-isoprene)

<p indent="0mm">Natural rubber or synthetic rubber materials have been extensively used in our daily life and industrial production for more than <sc>100 years</sc> due to their unique physical properties. However, a amount of rubber waste has been released into the environment, posing great challenges to the disposal of these polymers. In contrast to the disposal strategies such as combustion or stockpiling, enzyme catalyzed rubber degradation constitutes an environmentally friendly strategy. Latex clearing protein (Lcp) is an iron-heme containing endotype dioxygenase that catalyses the oxidative cleavage of the chemically inert poly(<italic>cis</italic>-1,4-isoprene) which is the main component of rubber, affording the products of aldehyde and ketone. It was found that active site E148 residue is key to the reactivity of Lcp based on the mutation experiments. However, the role of E148 and the overall mechanism are still controversial. It is generally accepted that both of dioxygen atoms are incorporated into the substrate. In previous studies, two possible mechanisms of Lcp catalyzed cleavage of poly(<italic>cis</italic>-1,4-isoprene) are proposed. In the first one, the distal oxygen atom of Fe(III)−O₂⁻ complex attacks onto the C=C double bond, leading to a three-membered epoxide intermediate. This is followed by the attack of Fe=O complex on the epoxide intermediate and subsequent C−C bond cleavage, affording the final ketone and aldehyde products. While in the second one, the proximal oxygen atom of Fe(III)−O₂⁻ complex attacks onto the C=C double bond of the substrate, which is followed by the attack of the distal oxygen atom onto the substrate radical to generate a four-membered cyclic dioxetane intermediate. The final O−O as and C−C bond cleavage in the dioxetane intermediate would afford the final products. In this study, based on the crystal structure of Lcp_K30, the combined molecular dynamic (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations are performed to elucidate the catalytic mechanism of Lcp catalysed cleavage of central double bond of poly(<italic>cis</italic>-1,4-isoprene). Our study shows E148 is protonated during the reaction and forms stable H bond interactions with Fe(III)−O₂⁻ species. Thus, the plausible role of E148 is related to the positioning of both Fe(III)−O₂⁻ species and the substrate. Moreover, it can be ruled out that the E148 acts as a base to abstract the proton from the substrate. The proposed mechanism of Lcp works as follows. Firstly, the distal oxygen of Fe(III)−O₂⁻ attacks onto the carbon−carbon double bond to generate the peroxide alky radical intermediate. This is followed by the rebound of the proximal oxygen to the carbon radical to generate the dioxetane intermediate, with Fe(III) being reduced to Fe(II). The following O−O bond cleavage occurs via the Fe(II)-mediated reductive cleavage of O−O bond of the dioxetane intermediate, leading to the vicinal diol radical anion intermediate. Finally, a facile C−C cleavage is undergone to yield the ketone and aldehyde products. In particular, our calculations show that the direct O−O cleavage of the dioxetane intermediate in the absence of Fe(II) catalysis requires a remarkably high barrier of <sc>160 kJ/mol,</sc> indicating the Fe(II) catalysis is vital for the further transformation of the dioxetane intermediate. For comparison, we also investigated the previously proposed mechanism involving the three-membered epoxide intermediate. However, the attack of the epoxide by Fe^IV=O complex is found to be highly unfavourable thermodynamically and thus can be ruled out. As such, this study may not only expand our understanding of the catalytic mechanism of Lcp_K30, but also provide useful information for rational design or engineering of Lcp enzyme toward the degradation of synthetic rubber materials.

Analysis of 12/15-lipoxygenase metabolism of EPA and DHA with special attention to authentication of docosatrienes

A proposed beneficial impact of highly unsaturated “fish oil” fatty acids is their conversion by lipoxygenase (LOX) enzymes to specialized proresolving lipid mediators, including 12/15-LOX products from EPA and DHA. The transformations of DHA include formation of docosatrienes, named for the distinctive conjugated triene of the double bonds. To further the understanding of biosynthetic pathways and mechanisms, herein we meld together biosynthesis and NMR characterization of the unstable leukotriene A (LTA)-related epoxide intermediates formed by recombinant human 15-LOX-1, along with identification of the stable enzymatic products, and extend the findings into the 12/15-LOX metabolism in resident murine peritoneal macrophages. Oxygenation of EPA by 15-LOX-1 converts the initial 15S-hydroperoxide to 14S,15S-trans-epoxy-5Z,8Z,10E,12E,17Z-EPA (appearing as its 8,15-diol hydrolysis products) and mixtures of dihydroperoxy fatty acids, while mainly the epoxide hydrolysis products are evident in the murine cells. DHA also undergoes transformations to epoxides and dihydroperoxides by 15-LOX-1, resulting in a mixture of 10,17-dihydro(pero)xy derivatives (docosatrienes) and minor 7S,17S- and 14,17S-dihydroperoxides. The 10,17S-dihydroxy hydrolysis products of the LTA-related epoxide intermediate dominate the product profile in mouse macrophages, whereas (neuro)protectin D1, the leukotriene B4-related derivative with trans,trans,cis conjugated triene, was undetectable. In this study, we emphasize the utility of UV spectral characteristics for product identification, being diagnostic of the different double bond configurations and hydroxy fatty acid functionality versus hydroperoxide. LC-MS is not definitive for configurational isomers. Secure identification is based on chromatographic retention times, comparison with authentic standards, and the highly distinctive UV spectra.

Influence of Controlled Epoxidation of an Asymmetric Styrene/Butadiene Star Block Copolymer on Structural and Mechanical Properties.

The chemical modification (namely the epoxidation) of a star shaped block copolymer (BCP) based on polystyrene (PS) and polybutadiene (PB) and its effect on structural and mechanical properties of the polymer were investigated. Epoxidation degrees of 37 mol%, 58 mol%, and 82 mol% were achieved by the reaction of the copolymer with meta-chloroperoxy benzoic acid (m-CPBA) under controlled conditions. The BCP structure was found to change from lamellae-like to mixed-type morphologies for intermediate epoxidation level while leading to quite ordered cylindrical structures for the higher level of chemical modification. As a consequence, the glass transition temperature (Tg) of the soft PB component of the BCP shifted towards significantly higher temperature. A clear increase in tensile modulus and tensile strength with a moderate decrease in elongation at break was observed. The epoxidized BCPs are suitable as reactive templates for the fabrication of nanostructured thermosetting resins.

MCPBA-mediated dioxygenation of unactivated alkenes for the synthesis of 5-imino-2-tetrahydrofuranyl methanol derivatives

A mCPBA-mediated, metal-free, intramolecular dioxygenation reaction of unactivated alkenes is reported. In the presence of m-chlorobenzoic peracid, different unsaturated amide substrates could be cyclized via epoxide intermediates, producing the corresponding 5-imino-2-tetrahydrofuranyl methanol products in up to 94% yield at room temperature.

Epoxide Syntheses and Ring-Opening Reactions in Drug Development

This review concentrates on success stories from the synthesis of approved medicines and drug candidates using epoxide chemistry in the development of robust and efficient syntheses at large scale. The focus is on those parts of each synthesis related to the substrate-controlled/diastereoselective and catalytic asymmetric synthesis of epoxide intermediates and their subsequent ring-opening reactions with various nucleophiles. These are described in the form of case studies of high profile pharmaceuticals spanning a diverse range of indications and molecular scaffolds such as heterocycles, terpenes, steroids, peptidomimetics, alkaloids and main stream small molecules. Representative examples include, but are not limited to the antihypertensive diltiazem, the antidepressant reboxetine, the HIV protease inhibitors atazanavir and indinavir, efinaconazole and related triazole antifungals, tasimelteon for sleep disorders, the anticancer agent carfilzomib, the anticoagulant rivaroxaban the antibiotic linezolid and the antiviral oseltamivir. Emphasis is given on aspects of catalytic asymmetric epoxidation employing metals with chiral ligands particularly with the Sharpless and Jacobsen–Katsuki methods as well as organocatalysts such as the chiral ketones of Shi and Yang, Pages’s chiral iminium salts and typical chiral phase transfer agents.

Mechanical Insights into the Enzymatic Cleavage of Double C-C Bond in Poly(cis-1,4-isoprene) by the Latex Clearing Protein.

The b-type cytochrome LcpK30 is a latex clearing protein (Lcp), which acts as an endotype dioxygenase to catalyze the extracellular cleavage of the chemically inert aliphatic polymer poly(cis-1,4-isoprene), producing oligo-isoprenoids with different terminal carbonyl groups (aldehyde and ketone, -CH2-CHO and -CH2-COCH3). On the basis of the fact that the muteins of E148A, E148Q, and E148H have substantially reduced reactivity, and the E148-initiated reaction mechanism has been previously proposed, in which a cyclic dioxetane intermediate or an epoxide intermediate may be involved, however, open questions still remain. In this paper, on the basis of the crystal structure of LcpK30, the enzyme-substrate reactant model was constructed, and the cleavage mechanism of the central double bond of poly(cis-1,4-isoprene) was elucidated by performing quantum mechanics/molecular mechanics calculations. Our calculation results revealed that the oxidative cleavage reaction is triggered by the addition of the heme-bound dioxygen to the double bond of the polymer, and E148 does not act as the catalytic base to extract the allylic proton to assist the reaction as previously suggested. Of the two considered pathways, the pathway that involves the dioxetane intermediate was calculated to be more favorable. During the catalysis, the distal oxygen first adds to the double bond of the substrate to form a radical intermediate, and then the Fe-O1 (proximal oxygen) bond cleaves to generate the dioxetane intermediate, which can easily collapse affording the final ketone and aldehyde products. In general, the cleavage mechanism of double C-C bond catalyzed by LcpK30 is similar to those of indoleamine 2,3-dioxygenase, tryptophan 2,3-dioxygenase, and the nonheme stilbene cleavage oxygenase NOV1 that all depend on the iron-bound dioxygen to initiate the cleavage reaction.

Stereoselective total synthesis of (−)-galantinic acid and 1-deoxy-5-hydroxysphingolipids via prins cyclization

The stereoselective total synthesis of (−)-galantinic acid 1 and 1-deoxy-5-hydroxysphingolipids 4 is described via Prins cyclization protocol followed by reductive ring opening sequence of substituted pyrenol derivative 6. The target molecules were synthesized using a common synthetic intermediate epoxide 5. Besides, we also proposed synthetic pathways to achieve other structural analogues using common intermediates.

Formation of Tetrahydrofurano-, Aryltetralin, and Butyrolactone Norlignans through the Epoxidation of 9-Norlignans

Epoxidation of the C=C double bond in unsaturated norlignans derived from hydroxymatairesinol was studied. The intermediate epoxides were formed in up to quantitative conversions and were readily further transformed into tetrahydrofuran, aryltetralin, and butyrolactone products—in diastereomeric mixtures—through ring-closing reactions and intramolecular couplings. For epoxidation, the classical Prilezhaev reaction, using stoichiometric amounts of meta-chloroperbenzoic acid (mCPBA), was used. As an alternative method, a catalytic system using dimeric molybdenum-complexes [MoO2L]2 with ONO- or ONS-tridentate Schiff base ligands and aqueous tert-butyl hydroperoxide (TBHP) as oxidant was used on the same substrates. Although the epoxidation was quantitative when using the Mo-catalysts, the higher temperatures led to more side-products and lower yields. Kinetic studies were also performed on the Mo-catalyzed reactions.

Synthesis and evaluation of nevirapine analogs to study the metabolic activation of nevirapine

Nevirapine (NVP) is widely used as a non-nucleoside reverse transcriptase inhibitor of HIV-1, however, it is associated with severe skin and liver injury. The mechanisms of these adverse reactions are not yet clear, but the metabolic activation of NVP is thought to be related to the injury process. Until now, several metabolic activation pathways of NVP have been reported. In this study, in order to identify the reactive metabolite of NVP mainly responsible for CYP inhibition and liver injury, we synthesized five NVP analogs designed to avoid the proposed bioactivation pathway and evaluated their metabolic stabilities, CYP3A4 time-dependent inhibitory activities, and cytotoxicity. As a result, only a pyrimidine analog of NVP, which could avoid the formation of a reactive epoxide intermediate, did not inhibit CYP3A4. Outside of this compound, the other synthesized compounds, which could avoid the generation of a reactive quinone-methide intermediate, inhibited CYP3A4 equal to or stronger than NVP. The pyrimidine analog of NVP did not induce cytotoxicity in HepG2 and transchromosomic HepG2 cells, expressing major four CYP enzymes and CYP oxidoreductase. These results indicated that the epoxide intermediate of NVP might play an important role in NVP-induced liver injury.

Mechanistic Insight of the Catalytic Role of WOX/SiO2 Catalyst in 2,5-Dimethylfuran to Para-xylene Conversion by DFT Calculation

In this paper, a DFT calculation was used to demonstrate the catalytic role of a WOX/SiO2 catalyst and show the excellent performance in 2,5-dimethylfuran to para-xylene conversion. A model of highly dispersed tungsten oxide supported on the amorphous silica was firstly constructed. A periodic DFT calculation was thereafter carried out to determine the reaction pathway, transition state (TS) and important intermediate structures. For reference purpose, the calculation was also repeat on an alkali ion, representative of a Lewis acid catalyst. Unexpectedly and although tungsten oxide is generally considered and characterized to be a Lewis acid, its mechanism for catalyzing the conversion of 2,5-dimethylfuran and ethylene to PX is differs significantly from other Lewis acids like alkali metal. Typical epoxide intermediates observed in other Lewis acid catalyzed systems are not formed over a WOX/SiO2 catalyst. The calculated reaction barrier indicated that the WOX/SiO2 catalyst has no obvious catalytic effect on the Diels–Alder cycloaddition for 2,5-dimethylfuran and ethylene. It mainly accelerates the dehydration step. The reason for the unique catalytic mechanism of WOX/SiO2 catalyst relies on the coordination of 2,5-dimethylfuran and tungsten that leads to the Diels–Alder cycloaddition that follow the so-called inverse electron demand mechanism. A new catalyst for this reaction was proposed based on the obtained mechanistic understanding.

Synthesis of (-)-Conduramine A1, (-)-Conduramine A2 and (-)-Conduramine E2 in Six Steps from Cyclohexa-1,4-diene.

A method to enable the synthesis of conduramines and their N-substituted derivatives (enantiopure or racemic form) in six steps (five steps for N-substituted derivatives) from cyclohexa-1,4-diene is reported. Key features of this reaction sequence include a preparation of benzene oxide that is amenable to multigram scale, and its efficient ring-opening upon treatment with a primary amine. Epoxidation of the resultant amino alcohols (40% aq HBF4 then m-CPBA) is accompanied by hydrolytic ring-opening in situ to give the corresponding N-substituted conduramine derivatives directly. These may undergo subsequent N-deprotection to give the parent conduramines, as demonstrated by the preparation of enantiopure (-)-conduramine A1, (-)-conduramine A2, and (-)-conduramine E2 (the latter two for the first time). The selectivity of the epoxidation reaction is proposed to be the result of competitive ammonium-directed and hydroxyl-directed epoxidation processes, followed by either direct (SN2-type) or conjugate (SN2'-type) ring-openings of the intermediate epoxides.

Kinetics of in Vitro Guanine- N7-Alkylation in Calf Thymus DNA by (2 S,3 S)-1,2-Epoxybutane-3,4-diol 4-methanesulfonate and (2 S,3 S)-1,2:3,4-Diepoxybutane: Revision of the Mechanism of DNA Cross-Linking by the Prodrug Treosulfan.

Prodrug treosulfan, originally registered for treatment of ovarian cancer, has gained a use in conditioning prior to hematopoietic stem cell transplantation. Treosulfan converts nonenzymatically to the monoepoxide intermediate (EBDM), and then to (2 S,3 S)-1,2:3,4-diepoxybutane (DEB). The latter alkylates DNA forming mainly (2' S,3' S)- N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (THBG) and (2 S,3 S)-1,4-bis(guan-7'-yl)butane-2,3-diol cross-link (bis-N7G-BD) via the intermediate epoxide adduct (EHBG). It is believed that DNA cross-linking by DEB is a primary mechanism for the anticancer and myeloablative properties of treosulfan, but clear evidence is lacking. Recently, we have proved that EBDM alkylates DNA producing (2' S,3' S)- N-7-(2',3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)-guanine (HMSBG) and that free HMSBG converts to EHBG. In this paper, we investigated the kinetics of HMSBG, bis-N7G-BD, and THBG in DNA in vitro to elucidate the contribution of EBDM and DEB to treosulfan-dependent DNA-DNA cross-linking. Calf thymus DNA was exposed to ( A) 100 μM treosulfan, ( B) 200 μM treosulfan, and ( C) DEB at a concentration 100 μM, exceeding that produced by 200 μM treosulfan. Following mild acid thermal hydrolysis of DNA, ultrafiltration, and off-line HPLC purification, the guanine adducts were quantified by LC-MS/MS. Both bis-N7G-BD and THBG reached highest concentrations in the DNA in experiment B. Ratios of the maximal concentration of bis-N7G-BD and THBG to DEB (adduct Cmax/DEB Cmax) in experiments A and B were 1.7-3.0-times greater than in experiment C. EHBG converted to the bis-N7G-BD cross-link at a much higher rate constant (0.20 h-1) than EBDM and DEB initially alkylated the DNA (1.8-3.4 × 10-5 h-1), giving rise to HMSBG and EHBG, respectively. HMSBG decayed unexpectedly slowly (0.022 h-1) compared with the previously reported behavior of the free adduct (0.14 h-1), which revealed the inhibitory effect of the DNA environment on the adduct epoxidation to EHBG. A kinetic simulation based on the obtained results and the literature pharmacokinetic parameters of treosulfan, EBDM, and DEB suggested that in patients treated with the prodrug, EBDM could produce the vast majority of EHBG and bis-N7G-BD via HMSBG. In conclusion, EBDM can produce DNA-DNA lesions independently of DEB, and likely plays a greater role in DNA cross-linking after in vivo administration of treosulfan than DEB. These findings compel revision of the previously proposed mechanism of the pharmacological action of treosulfan and contribute to better understanding of the importance of EBDM for biological effects.

Mechanistic Insights into a Stibene Cleavage Oxygenase NOV1 from Quantum Mechanical/Molecular Mechanical Calculations.

NOV1, a stilbene cleavage oxygenase, catalyzes the cleavage of the central double bond of stilbenes to two phenolic aldehydes, using a 4‐His Fe(II) center and dioxygen. Herein, we use in‐protein quantum mechanical/molecular mechanical (QM/MM) calculations to elucidate the reaction mechanism of the central double bond cleavage of phytoalexin resveratrol by NOV1. Our results showed that the oxygen molecule prefers to bind to the iron center in a side‐on fashion, as suggested from the experiment. The quintet Fe−O2 complex with the side‐on superoxo antiferromagnetic coupled to the resveratrol radical is identified as the reactive oxygen species. The QM/MM results support the dioxygenase mechanism involving a dioxetane intermediate with a rate‐limiting barrier of 10.0 kcal mol−1. The alternative pathway through an epoxide intermediate is ruled out due to a larger rate‐limiting barrier (26.8 kcal mol−1). These findings provide important insight into the catalytic mechanism of carotenoid cleavage oxygenases and also the dioxygen activation of non‐heme enzymes.

Dictamnine-induced hepatotoxicity in mice: the role of metabolic activation of furan

Cortex Dictamni is extensively used as an herbal medicine worldwide, but is believed to induce hepatotoxicity and even causes mortality in many Asian and European countries. As the most abundant furoquinoline alkaloid ingredient of Cortex Dictamni, dictamnine (DIC) can be metabolically activated by CYP3A to an epoxide metabolite, which possesses the potential to induce hepatotoxicity by covalent binding with proteins. As yet, the hepatotoxicity of DIC and the role played by metabolic activation remain unknown. Here, we found that DIC caused acute liver injury in a time- and dose-dependent manner in mice. The hepatic and urinary DIC epoxide intermediates were observed in DIC-treated mice. Ketoconazole, a CYP3A inhibitor, significantly reduced the hepatotoxicity of DIC and inhibited the formation of reactive metabolites of DIC. Moreover, treatment with 2,3-dihydro-DIC, a DIC analog synthesized by selective reduction of the furan moiety, produced no hepatotoxicity in mice, and no reactive metabolite was formed, suggesting a structural necessity of furan moiety in DIC hepatotoxicity. A time course of gradual hepatic glutathione consumption was observed in DIC-treated mice, while depletion of hepatic glutathione by L-buthionine-S,R-sulfoximine enhanced the hepatotoxicity of DIC. Collectively, this study demonstrates that DIC induces acute hepatocellular injury in mice, and that metabolic activation of furan plays a crucial role in DIC-induced hepatotoxicity.

Synthesis of 2-Azaadamantan-6-one: A Missing Isomer.

2-Azaadamantan-6-one and its Boc and ethylene ketal derivatives were synthesized from 9-oxo endo-bicyclo[3.3.1]non-6-ene-3-carboxylic acid. Similarly, the Cbz, Boc, and ethylene ketal derivatives of 2-azaadamantan-4-one were synthesized from endo-bicyclo[3.3.1]non-6-ene-3-carboxylic acid. Key steps were Curtius rearrangements to form benzyl carbamates, followed by spontaneous intramolecular attack of the carbamate nitrogen on transient bromonium ion or epoxide intermediates to effect ring closure to azaadamantane intermediates. The reaction sequence leading to 2-azaadamantan-6-one is consistent with the formation of a transient tetracyclic keto aziridine intermediate.

A carbohydrate-derived trifunctional scaffold for medicinal chemistry library synthesis

For the generation of compound libraries for drug discovery a central scaffold containing three exit vectors with defined chirality was devised starting from commercially available tri-O-acetyl-glucal. Surprisingly, the reaction of a 4-O-mesylate with sodium azide did not lead to the expected 4-azido-4-deoxy derivative but to a 3-azido-3-deoxy regioisomer via intermediate epoxide formation. The absolute stereochemical configuration of the final tetrahydropyran building block was proven by X-ray crystallography. This scaffold endowed with a carboxylic acid, a secondary alcohol, and an azide functionality may be connected to a DNA tag at any of the three distinct exit vectors, thus providing ready access to several different compound libraries.Â

Investigations into the biosynthesis of the antifungal strobilurins.

The strobilurins are important antifungal metabolites isolated from a number of basidiomycetes and have been valuable leads for the development of commercially important fungicides. Isotopic labelling studies with early and advanced intermediates confirm for the first time that they are produced via a linear tetraketide, primed with the rare benzoate starter unit, itself derived from phenylalanine via cinnamate. Isolation of a novel biphenyl metabolite, pseudostrobilurin B, provides evidence for the involvement of an epoxide in the key rearrangement to form the β-methoxyacrylate moiety essential for biological activity. Formation of two bolineol related metabolites, strobilurins Y and Z, also probably involves epoxide intermediates. Time course studies indicate a likely biosynthetic pathway from strobilurin A, with the simplest non-subsubstituted benzoate ring, to strobilurin G with a complex dioxepin terpenoid-derived substituent. Precursor-directed biosynthetic studies allow production of a number of novel ring-halogenated analogues as well as a new pyridyl strobilurin. These studies also provide evidence for a non-linear biosynthetic relationship between strobilurin A and strobilurin B.

Evidence for a complex formation between CYP2J5 and mEH in living cells by FRET analysis of membrane protein interaction in the endoplasmic reticulum (FAMPIR)

The potential complex formation between microsomal epoxide hydrolase (mEH) and cytochrome P450-dependent monooxygenase (CYP) has been a subject of research for many decades. Such an association would enable efficient substrate channeling between CYP and mEH and as such represent an attractive strategy to prevent deleterious accumulation of harmful metabolic by-products such as CYP-generated epoxide intermediates. However, such complex formation is experimentally difficult to prove, because CYP and mEH are membrane-bound proteins that are prone to unspecific aggregation after solubilization. Here, we report the development of a FRET-based procedure to analyze the mEH–CYP interaction in living cells by fluorescence-activated cell sorting. With this non-invasive procedure, we demonstrate that CYP2J5 and mEH associate in the endoplasmic reticulum of recombinant HEK293 cells to the same extent as do CYP2J5 and its indispensible redox partner cytochrome P450 reductase. This presents final proof for a very close proximity of CYP and mEH in the endoplasmic reticulum, compatible with and indicative of their physical interaction. In addition, we provide with FAMPIR a robust and easy-to-implement general method for analyzing the interaction of ER membrane-resident proteins that share a type I topology.