First systematic chiral syntheses of two pairs of enantiomers with 3,5-dihydroxyheptenoic acid chain, associated with a potent synthetic statin NK-104

First systematic chiral syntheses of two pairs of enantiomers with 3,5-dihydroxyheptenoic acid chain, associated with a potent synthetic statin NK-104 are reported. A pair of syn diol isomers (NK-104 and its enantiomer) was obtained efficiently by diastereomeric resolution. The synthesis of a pair of anti diol isomers (3-epimer and 5-epimer) was accomplished effectively by the asymmetric aldol reaction followed by anti stereoselective reduction as key steps. Their purity determinations were effected by chiral HPLC analysis.     

Incorporation of 18O2 and absence of stereospecificity in primary product formation during fungal metabolism of a lignin model compound

The metabolism of a lignin substructure model compound, 1,2-bis(3-methoxy-4-ethoxyphenyl)propane-1,3-diol (Ia) in ligninolytic cultures of Phanerochaete chrysosporium was studied to help elucidate the biochemical mechanism of lignin degradation. The primary reaction was cleavage of the model compound between C₁ and C₂ of the propane moiety to produce 1-(3-methoxy-4-ethoxyphenyl)ethane-1,2-diol and a C₆-C₁ product (probably 3-methoxy-4-ethoxybenzaldehyde). Other identified products arose secondarily; all were further metabolized. Even though the model compound was a mixture of four stereoisomers, no stereoselectivity was observed in its metabolism. In cultures under ¹⁸O₂, the initial cleavage produced the diol product with ≈70% enrichment by ¹⁸O in the benzyl alcohol group. The diol was a mixture of the two possible enantiomers, and the O₂-derived hydroxyl was incorporated at the asymmetric (benzyl) carbon. (Limited optical activity in the diol was traced to selective further metabolism of the D form.) These results show that the primary cleavage reaction lacked stereospecificity and was primarily oxygenative, implicating a nonspecific oxygenase or a nonenzymatic reaction involving activated oxygen. Preliminary experiments demonstrated no cell homogenate activity against Ia.     

Stereoselective synthesis of novel antiproliferative steroidal (E,E) dienamides through a cascade aldol/cyclization process

The stereoselective and metal-free protocol involving a cascade aldol/cyclization process for the synthesis of steroidal (E, E) dienamides from steroidal α, α-dicyanoalkene was reported. This protocol efficiently achieved the construction of CC bond and selective conversion of cyano group into carboxamide in one-pot procedure under mild condition. Further biological evaluation showed that some of these compounds had moderate to excellent cytotoxic activities against all the tested cancer cell lines and were more potent than well-known drug 5-fluorouracil. Particularly, compound 3c represented excellent inhibitory effect against MCF-7 (IC₅₀ = 0.76 μM), which was about 10-fold more potent than 5-fluorouracil.     

Substrate specificity and kinetics for VP14, a carotenoid cleavage dioxygenase in the ABA biosynthetic pathway

The plant growth regulator, abscisic acid (ABA), is synthesized via the oxidative cleavage of an epoxy-carotenoid. Specifically, a double bond is cleaved by molecular oxygen and an aldehyde is formed at the site of cleavage in both products. The Vp14 gene from maize encodes an oxidative cleavage enzyme for ABA biosynthesis and the recombinant VP14 protein catalyzes the cleavage reaction in vitro. The enzyme has a strict requirement for a 9-cis double bond adjacent to the site of cleavage (the 11-12 bond), but shows some plasticity in other features of carotenoids that are cleaved. A kinetic analysis with the 9-cis isomer of five carotenoids displays several substrate activity relationships. One of the carotenoids was not readily cleaved, but inhibited the cleavage of another substrate in mixed assays. Of the remaining four carotenoids used in this study, three of the substrates have similar V(max) values. The V(max) for the cleavage of one carotenoid substrate was significantly higher. Molecular modeling and several three-dimensional quantitative substrate-activity relationship programs were used to analyze these results. In addition to a 9-cis double bond, the presence and orientation of the ring hydroxyl affects substrate binding or the subsequent cleavage. Additional variations that affect substrate cleavage are proposed.     

Substrate-selective mechanisms in biocatalysis demonstrated with a versatile and efficient aldolase antibody.

A structure-activity relationship study with a series of aldol substrates shows that the mechanism of the antibody 38C2-catalyzed retrograde aldol reaction depends on the nature of the substrate With electron-deficient substrates an early deprotonation precedes the C-C bond cleavage while with electron-rich substrates the catalytic mechanism involves an initial C-C bond cleavage leading to a positively charged intermediate.     

Tritium exchange reactions catalyzed by 2-oxo-4-hydroxyglutarate aldolase from Escherichia coli K-12.

Tritiated water and tritiated substrates have been used to study exchange reactions catalyzed by Escherichia coli 2-oxo-4-hydroxyglutarate aldolase (4-hydroxy-2-oxoglutarate glyoxylate-lyase, EC 4.1.3.16, 2-oxo-4-hydroxyglutarate in equilibrium pyruvate + glyoxylate). With pyruvate, the enzyme catalyzes a rapid first-order exchange of all three methyl hydrogens in the absence of added acceptor aldehyde (i.e. glyoxylate). This reaction is not rate limiting for aldol condensation or cleavage; quite different pH-activity profiles for the exchange reaction versus aldol cleavage and also comparative effects that pH changes have on Km and V values for the two processes favor this conclusion. The exchange reaction with 2-oxobutyrate, a substrate analog, is stereoselective; one methylene hydrogen is removed at a 6-fold faster rate than the other but eventually both are exchanged. No tritium exchange occurs with glyoxylate.     

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C₇ having a relatively weak carbon–hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen (¹ΔgO₂) and ozone (O₃) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5β-, 6α- and 6β-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ_5,6 –double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.     

Synthesis of methyl 16-trideuteriohexadecanoate

Methyl 16-trideuteriohexadecanoate has been prepared in high isotopic purity and in 29% overall yield, from methyl 7-oxo-16-heptadecenoate. The oxo group was reduced with sodium cyanoborohydride and the CD₃ group was introduced by reduction with lithium aluminum deuteride, first of the ester group to the alcohol and then of the derived mesylate. The carboxyl group was formed by oxidative cleavage of the double bond.     

Activation and cleavage of the carbon-cobalt bond of adeninylethylcobalamin by diol dehydrase

Adeninylethylcobalamin (AdeEtCbl) underwent cleavage of the C-Co bond by interaction with apoprotein of diol dehydrase from Klebsiella pneumoniae ATCC 8724, although this analog was quite inactive as coenzyme. Spectroscopic observation indicates that AdeEtCbl was converted to the enzyme-bound hydroxocobalamin without intermediates. The conversion was stoichiometric (1:1) and obeyed the second-order reaction kinetics (k = 0.027 min-1 microM-1 at 37 degrees C) depending upon concentrations of apoprotein and AdeEtCbl. This suggests that the complex formation is the rate-determining step and that AdeEtCbl undergoes rapid C-Co bond cleavage once it binds to the apoenzyme. Substrates and oxygen did apparently not affect the rate of the C-Co bond cleavage. The experiments using [adenine-U-14C]AdeEtCbl and [1(3)-3H]glycerol demonstrated that 9-ethyladenine was the only product formed from the adeninylethyl group of AdeEtCbl during the conversion and that an additional hydrogen atom in the 9-ethyladenine is not derived from the substrate. 1H NMR measurement of the 9-ethyladenine formed enzymatically from AdeEtCbl and DL-1,2-[1,1,2-2H3]propanediol also led to the same conclusion. All of these results indicate that the C-Co bond of AdeEtCbl is activated by diol dehydrase and undergoes heterolysis forming Co(III) and a carbanion or a carbanion-like species, in clear contrast to the homolysis of the C-Co bond of adenosylcobalamin in the normal catalytic process. 9-Ethyladenine formed remained tightly associated with the enzyme. Longer chain homologs, i.e. adeninylpropylcobalamin, adeninylbutylcobalamin, and adeninylpentylcobalamin did not undergo such cleavage of the C-Co bond by diol dehydrase.     

Asymmetric synthesis of chiral 2-hydroxy ketones by coupled biocatalytic alkene oxidation and CC bond formation

Two different biocatalytic reactions – a CC cleavage and a CC forming reaction – were evaluated concerning their application in a reaction sequence. In the overall reaction, an aromatic alkene was converted to a chiral 2-hydroxy ketone. In the first step, the olefin trans-anethole was converted to para-anisaldehyde and acetaldehyde by an aqueous extract of the white rot fungus Trametes hirsuta G FCC 047. The selective oxidative cleavage of the carbon–carbon double bond was achieved using molecular oxygen as a substrate. In a second step p-anisaldehyde was ligated to acetaldehyde to yield either (R)- or (S)-2-hydroxy-1-(4-methoxyphenyl)-propanone. The reaction was catalyzed by the enantiocomplementary CC bond forming enzymes benzaldehyde lyase and benzoylformate decarboxylase, respectively.     

Dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli: dihydrofolate monooxygenase activity

Dihydrofolate reductase from strain MB 1428 of Escherichia coli was shown to catalyze the oxidative cleavage of dihydrofolate at the C(9)N(10) bond. One of the products of the reaction was identified as 7,8-dihydropterin-6-carboxaldehyde through its proton magnetic resonance spectrum. The maximal enzymatic rate was 0.05 moles dihydrofolate cleaved per minute per mole enzyme at 25° and pH 7.2, and the K_M for dihydrofolate was 17.5 ± 2.5 μM. The enzymatic reaction was fully inhibitable with methotrexate. The mechanism of enzyme action was proposed to be an apparent “acidification” of dihydrofolate upon binding to the enzyme. Folate underwent an analogous oxidative cleavage by enzyme with a turnover number of 0.0014, which produced pterin-6-carboxaldehyde. Methotrexate was also slowly degraded by the enzyme.     

Stereoselective aldol addition reactions of Fischer carbene complexes via electronic tuning of the metal center for enolate reactivity

The aldol reactions of tetracarbonyl(phosphine)methyl(methoxy)methylene chromium complexes and pentacarbonylmethyl (dialkylamino)methylene chromium complexes with aldehydes and ketones were examined. The reactions of the phosphine complexes give only aldol condensation products, but the desired aldol addition products can be isolated from the reactions of amino carbene complexes. This was attributed to the greater reactivity of the enolates of amino carbene complexes which is supported by a determination of the thermodynamic acidity of the dimethylamino complex 13 (pK_a=20.4). The aldol reactions of amino complexes with -chiral aldehydes occur with very high facial selectivities rivaling the best methods that have been developed for facial selectivity in the aldol reaction. The aldol reactions of amino complexes can be considered as direct synthons for amides since amide functions can be obtained in the oxidative cleavage of the aldol adducts of these complexes. As illustrative of the versatility of carbene complexes, it is also demonstrated in a photo-induced carbon-homologative demetallation, that in combination with the aldol addition reaction the unique reactions of carbene complexes provide powerful and novel overall transformations.     

IMDA/aldol strategy for transforming carbohydrates into functionalized trans-decalins

L-Rhamnal is readily converted into an allyl 2, 3-unsaturated-C-glycopyranoside. The (S) configuration of the alphaL-anomer defines the stereochemical outcome of the future IMDA reaction, leading to the absolute stereochemistry for the trans-decalin moiety in naturally occurring terpenoids. Selective cleavage of the terminal double bond of the allyl group provides an aldehydo function which serves for an aldol/Claisen addition with ethyl sorbate. Of the four possible diastereomers, one is obtained in pure form and processed to give the IMDA precursor. Cyclocondensation is achieved by heating in xylene to give a tricyclic trans-decalin whose structure is established by NMR and X-ray analysis.     

Stereodivergent synthesis of diastereoisomeric carba analogs of glycal-derived vinyl epoxides: a new access to carbasugars

A convenient method for the stereoselective synthesis of diasteroisomeric vinyl epoxides (-)-2α and (-)-2β, the carba analogs of D-galactal and D-allal-derived vinyl epoxides 1α and 1β, has been elaborated starting from tri-O-acetyl-D-glucal. The key step of this synthesis is an application of the known Claisen thermal rearrangement of a glucal derivative, the vinyl allyl ether (+)-3b, which allows to switch the glycal structure into the corresponding carba analog scaffold. Epoxides (-)-2α and (-)-2β derive from the same synthetic intermediate, the trans diol (+)-5.     

A new mode of B12 binding and the direct participation of a potassium ion in enzyme catalysis: X-ray structure of diol dehydratase

Background: Diol dehydratase is an enzyme that catalyzes the adenosylcobalamin (coenzyme B₁₂) dependent conversion of 1,2-diols to the corresponding aldehydes. The reaction initiated by homolytic cleavage of the cobalt–carbon bond of the coenzyme proceeds by a radical mechanism. The enzyme is an α₂β₂γ₂ heterooligomer and has an absolute requirement for a potassium ion for catalytic activity. The crystal structure analysis of a diol dehydratase–cyanocobalamin complex was carried out in order to help understand the mechanism of action of this enzyme.Results: The three-dimensional structure of diol dehydratase in complex with cyanocobalamin was determined at 2.2 Å resolution. The enzyme exists as a dimer of heterotrimers (α β γ)₂. The cobalamin molecule is bound between the α and β subunits in the ‘base-on’ mode, that is, 5,6-dimethylbenzimidazole of the nucleotide moiety coordinates to the cobalt atom in the lower axial position. The α subunit includes a (β/α)₈ barrel. The substrate, 1,2-propanediol, and an essential potassium ion are deeply buried inside the barrel. The two hydroxyl groups of the substrate coordinate directly to the potassium ion.Conclusions: This is the first crystallographic indication of the ‘base-on’ mode of cobalamin binding. An unusually long cobalt–base bond seems to favor homolytic cleavage of the cobalt–carbon bond and therefore to favor radical enzyme catalysis. Reactive radical intermediates can be protected from side reactions by spatial isolation inside the barrel. On the basis of unique direct interactions between the potassium ion and the two hydroxyl groups of the substrate, direct participation of a potassium ion in enzyme catalysis is strongly suggested.     

Evidence for Deuterium Retention in the Products after Enzymatic C-C and Ether Bond Cleavages of Deuterated Lignin Model Compounds

The mechanism of C-C and ether bond cleavages of C_α-or C_β-deuterated β-O-4 and β-l lignin substructure models and the vicinal diol compounds catalyzed by the enzyme system from Phanerochaete chrysosporium culture was investigated. The enzymatic oxidation of β-O-4 lignin model compounds in the presence of H₂O₂ and O₂ yielded C₆-C_α-derived benzaldehyde, and C_β-C_γ-derived product together with the arylglycerol product. Likewise, the β-l models and the diol compounds also underwent the C-C bond cleavage, yielding C₆-C_β-derived benzaldehyde and the arylglycol product. The results demonstrated that the d-labels at C_α and C_β of the substrates were retained in the products after the C_α-C_β and ether bond cleavages.     

Classroom Enters the Courtroom: Stereochemistry of SN1 and SN2 Reactions in Enantiomer Patent Litigations of the Antidepressant Escitalopram

The role of elementary stereochemistry is illustrated in the patent litigations of the blockbuster antidepressant drug escitalopram oxalate. An undergraduate student of organic chemistry would recognize the stereochemical courses of the intramolecular S_N2 and S_N1 reactions of the single‐enantiomer (S)‐diol intermediate in the synthesis of the blockbuster antidepressant drug escitalopram oxalate: retention of configuration of the chiral carbon atom under basic conditions and racemization under acidic conditions, respectively. He/she, in searching for a stereoselective ring‐closure reaction of the enantiomeric diol, will think of an S_N2 reaction in a basic medium. From these points of view, the process claim in the enantiomer patents of escitalopram is obvious/lacks an inventive step. An organic chemistry examination problem based on this scenario is offered. Chirality 28:39–43, 2016. © 2015 Wiley Periodicals, Inc.     

The Outcome of the Oxidations of Unusual Enediamide Motifs Is Governed by the Stabilities of the Intermediate Iminium Ions

We compare the results from the oxidation of two unusual “enediamide” motifs (3,4-dihydropyrazin-2(1H)-ones), where a double bond is flanked by two amides. In one case the oxidation led to a ring-opened product arising from the cleavage of the double bond, and in the other a rare cis-dioxygenated compound was obtained. Both products have been characterized by X-ray crystallography. The outcomes of the key reactions are rationalized based on calculated free energies of intermediates.     

Interactions of diol dehydrase and 3',4'-anhydroadenosylcobalamin: suicide inactivation by electron transfer

3',4'-Anhydroadenosylcobalamin (anAdoCbl) is an analogue of the adenosylcobalamin (AdoCbl) coenzyme (Magnusson, O.Th., and Frey, P. A. (2000) J. Am. Chem. Soc. 122, 8807-8813). This compound supports activity for diol dehydrase at 0.02% of that observed with AdoCbl. In a side reaction, however, anAdoCbl induces suicide inactivation by an electron-transfer mechanism. Homolytic cleavage of the Co-C bond of anAdoCbl at the active site of diol dehydrase was observed by spectrophotometric detection of cob(II)alamin. Anaerobic conversion of enzyme bound cob(II)alamin to cob(III)alamin, both in the absence and presence of substrate, indicates that the coenzyme derived 5'-deoxy-3',4'-anhydroadenosine-5'-yl serves as the oxidizing agent. This hypothesis is supported by the stoichiometric formation of 3',5'-dideoxyadenosine-4',5'-ene as the nucleoside cleavage product, as determined by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. Experiments performed in deuterium oxide show that a single solvent exchangeable proton is incorporated into the product. These data are consistent with the intermediate formation of a transient allylic anion formed after one electron transfer from cob(II)alamin to the allylic 5'-deoxy-3',4'-anhydroadenosyl radical. Selective protonation at C3' was demonstrated by spectroscopic characterization of the purified product. This study provides an example of suicide inactivation of a radical enzyme brought about by a side reaction of an analogue of the radical intermediate.     

Thrombomodulin induction in cultured human endothelial cells by 9-cis-locked retinoic acid analogues

9-cis-Retinoic acid (RA) analogues devised to lock the 9-cis double bond by ring formation were synthesized using two stereoselective carbon-carbon bond formation reactions as key steps. The palladium-mediated Suzuki reaction was adopted to construct a 7E-double bond (RA numbering) and the Horner-Emmons olefination was employed for stereoselective 11E-double bond (RA numbering) formation. The synthesized 9-cis-RA analogues that are locked by five-membered ring systems (cyclopentene, dihydrofuran, and dihydrothiophene) were shown to have comparable thrombomodulin induction activities to that of 9-cis RA. Conformational analysis of these compounds showed their similarity to 9-cis RA in the spatial orientation of the side chain and the terminal carboxy group.