Simultaneous determination of intracellular reduced and oxidized glutathiones by the König reaction.

The König reaction is commonly used for the detection of cyanide and its derivatives, including thiocyanate and selenocyanate. We found that this reaction can be used to quantify glutathione fluorometrically, and applied it to the simultaneous determination of reduced and oxidized glutathiones (GSH and GSSG) using a conventional LC system with isocratic elution. The limits of detection were 6.04 nM and 9.84 nM for GSH and GSSG, respectively, and the limits of quantification were 18.3 nM and 29.8 nM, respectively. We also determined GSH and GSSG levels in PC12 cells exposed to paraquat, an oxidative stressor, and observed a decrease in GSH/GSSG ratio, as expected. Total GSH levels quantified by this method and by the conventional colorimetric method with 5,5'-dithiobis(2-nitrobenzoic acid) were comparable. Our new application of the König reaction offers a reliable and useful method to simultaneously quantify intracellular GSH and GSSG.

Identification of a regiospecific S-oxygenase for the production of marasmin in traditional medicinal plant Tulbaghia violacea.

Marasmin [S-(methylthiomethyl)-L-cysteine-4-oxide] is a pharmaceutically valuable sulfur-containing compound produced by the traditional medicinal plant, Tulbaghia violacea. Here, we report the identification of an S-oxygenase, TvMAS1, that produces marasmin from its corresponding sulfide, S-(methylthiomethyl)-L-cysteine. The amino acid sequence of TvMAS1 showed high sequence similarity to known flavin-containing S-oxygenating monooxygenases in plants. Recombinant TvMAS1 catalyzed regiospecific S-oxygenation at S4 of S-(methylthiomethyl)-L-cysteine to yield marasmin, with an apparent K m value of 0.55 mM. TvMAS1 mRNA accumulated with S-(methylthiomethyl)-L-cysteine and marasmin in various organs of T. violacea. Our findings suggest that TvMAS1 catalyzes the S-oxygenation reaction during the last step of marasmin biosynthesis in T. violacea.

Asymmetric Total Synthesis and Structure Elucidation of Huperzine H.

A first asymmetric total synthesis of huperzine H has been achieved in a 12% overall yield from commercially available (+)-pulegone. The key steps of this synthesis include a highly diastereoselective Mukaiyama-Michael addition reaction of a pyrrole bearing a silyl enol ether and an intramolecular SN2 cyclization reaction with iodinated pyrrole acting as an effective nucleophile for the formation of the nine-membered ring. As a result, the relative and absolute stereochemistry of huperzine H is established.

Poly-ion complex (PIC) formation of heparin and polyamines: PIC with tetrakis (3-aminopropyl) ammonium allows sustained release of heparin.

Physical mixtures of cationic polymers and heparin have been developed to overcome the limitations of unfractionated heparin. In this study, we found that heparin associates with natural polyamines in water, resulting in the generation of a poly-ion complex (PIC). PIC formation (or stability) was influenced by the concentration and ratio of heparin and polyamines, molecular weight of heparin, nature of polyamines, and pH conditions. Interestingly, the PIC obtained when heparin and tetrakis (3-aminopropyl) ammonium (Taa) were mixed exhibited stability and was sticky in nature. PIC formation was due to an electrostatic interaction between heparin and Taa. Heparin-Taa PIC was administered subcutaneously to mice, and the time to maximum heparin concentration within the therapeutic range of heparin was markedly increased compared to that after a single dose of heparin. These results suggest that the quaternary ammonium structure of Taa is critical for the preparation of a stable PIC, thereby allowing the sustained release of heparin into the blood.

Design, synthesis, and pharmacological evaluation of 2-(4-sulfonylphenyl)-2-[(E)-pyrrolidin-1-ylimino]-N-thiazoleacetamides as glucokinase activators.

This paper presents the synthesis and glucokinase activity of novel hydrazone derivatives. The 2-(4-cyclopropylsulfonylphenyl)-2-[(E)-pyrrolidin-1-ylimino]-acetamide derivatives 5a-5h presented the in vitro glucokinase activities and in vivo blood glucose-lowering effects in mice. Particularly, 5h showed an oral hypoglycemic effect in rats at 1 mg/kg. These hydrazone derivatives are a potential new class of glucokinase activators for the treatment of type 2 diabetes.

Total Syntheses of Lycopodium and Monoterpenoid Indole Alkaloids Based on Biosynthesis-Inspired Strategies.

The merits of biogenetic considerations in the chemical syntheses of natural products have been emphasized by describing the total syntheses of Lycopodium alkaloids; lycodine, flabellidine, lycopodine, and flabelliformine, as well as monoterpenoid indole alkaloids; C-mavacurine, kopsiyunnanine K, koumine, and 11-methoxy-19R-hydroxygelselegine.

Asymmetric Total Synthesis of Biphenylquinolizidine Alkaloids 4″-O-Demethyllythridine and 14-epi-4″-O-Demethyllythridine.

The first asymmetric total synthesis of new biphenylquinolizidine alkaloids 4″-O-demethyllythridine and 14-epi-4″-O-demethyllythridine isolated from Heimia salicifolia was accomplished. The key steps in the synthesis were a copper(I)-catalyzed asymmetric intramolecular aza-Michael reaction to build a chiral 4-arylquinolizidine unit and an intramolecular Suzuki-Miyaura cross-coupling reaction to construct a macrolactone ring comprising a biphenyl moiety.

Total Synthesis of (-)-14-Hydroxygelsenicine and Six Biogenetically Related Gelsemium Alkaloids.

The first concise and collective asymmetric total synthesis of six 14-hydroxygelsenicine-related Gelsemium alkaloids, i.e., 14-hydroxygelsedilam, 14-acetoxygelsedilam, gelsefuranidine, gelsemolenine A, and gelselegandines B and C, was accomplished via the facile construction of a 7-azabicyclo[4.2.1]nonane skeleton by intramolecular aza-Michael addition, the preparation of an oxabicyclo[3.2.2]nonane ring core with a secondary hydroxy group at C14 by an intramolecular oxymercuration-hydroxylation strategy, and divergent transformations of 14-hydroxygelsenicine into biogenetically related alkaloids.

Total Syntheses of Pleiocarpamine, Normavacurine, and C-Mavacurine.

The total syntheses of C-mavacurine-type indole alkaloids, (±)-pleiocarpamine, (±)-normavacurine, and (±)- C-mavacurine, were accomplished. The key step in the syntheses was the cyclization between the metal carbenoid at C16 and the N1 position in a Corynanthe-type compound that was equipped with a diazo function. For this cyclization, the N4 modification of the substrate using an amine-borane complex was indispensable to fix the molecular conformation.

Recent Progress in the Chemistry of Pandanus Alkaloids.

The genus Pandanus (Pandanaceae) is widely distributed in the tropical and subtropical regions. With about 700 species worldwide, three Pandanus species (P. amaryllifolius, P. utilis, and P. dubius) have been investigated and found to contain new alkaloids possessing a pyrrolidinyl-α,ß-unsaturated γ-lactone, a γ-butylidene-α-methyl-α,ß-unsaturated γ-lactam, and/or indolizidine residues. Several total syntheses of Pandanus alkaloids have been accomplished. Several pharmacological studies on Pandanus species, including scientific validations of their antibacterial, antiinflammatory, antidiarrheal, and cytotoxic activities, have been conducted in relation to their traditional folk medicine uses.

Asymmetric Total Synthesis of Fawcettimine-Type Lycopodium Alkaloid, Lycopoclavamine-A.

An asymmetric total synthesis of lycopoclavamine-A (1), a structurally unique fawcettimine-type Lycopodium alkaloid, was achieved via a stereoselective Pauson-Khand reaction and a stereoselective conjugate addition to construct a quaternary carbon center at C-12.

New otonecine-type pyrrolizidine alkaloid from Petasites japonicus.

One new otonecine-type pyrrolizidine alkaloid secopetasitenine (1), along with petasitenine (fukinotoxine, 2), neopetasitenine (3), and senkirkine (4), was isolated from the whole plant of Petasites japonicus. The structure of 1 was determined by spectroscopic analyses and chemical conversion from the known alkaloid petasitenine (2).

Asymmetric Total Synthesis and Evaluation of Antitumor Activity of Ophiorrhisine A and Its Derivatives.

The first asymmetric total synthesis of ophiorrhisine A (1), a new cyclic tetrapeptide isolated from Ophiorrhiza nutans, was accomplished via an intramolecular aromatic nucleophilic substitution reaction (IMSNAr) of a linear tripeptide to construct a 14-membered paracyclophane ring, resulting in confirmation of its structure and absolute configuration. The structure-activity relationship study of 1 and its derivatives demonstrated that some derivatives possessed cytotoxicity toward human cancer cell lines A549, HT29, and HCT116.

Protective Effects of Brain Infarction by N-Acetylcysteine Derivatives.

BACKGROUND AND PURPOSE: We recently found that acrolein (CH2=CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. METHODS: Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N-acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. RESULTS: NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S-transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S-transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S-transferases. CONCLUSIONS: The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S-transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.

A Cyclopeptide and a Tetrahydroisoquinoline Alkaloid from Ophiorrhiza nutans.

A new cyclopeptide, ophiorrhisine A (1), a new tetrahydroisoquinoline alkaloid, 7',10-dide-O-methylcephaeline (2), two known ß-carboline alkaloids, and four known tetrahydroisoquinoline alkaloids were isolated from Ophiorrhiza nutans (Rubiaceae). Compound 1 is a tetrapeptide possessing a 14-membered paracyclophane ring and a novel N,N,N-trimethyltyrosine residue in the side chain. The stereochemistry at the aryl-alkyl ether bond was different from that of other known 14-membered paracyclophanes. The structure of 2 was established by spectroscopic analysis and semisynthesis.

Asymmetric Total Synthesis of Pentacyclic Indole Alkaloid Andranginine and Absolute Configuration of Natural Product Isolated from Kopsia arborea.

The first asymmetric total synthesis of andranginine (1) via an asymmetric Morita-Baylis-Hillman reaction and a diastereoselective intramolecular Diels-Alder reaction has revealed that natural andranginine (1) isolated from Kopsia arborea existed as a scalemic mixture and contained predominantly the (16R,21S) form rather than the (16S,21R) form.

Identification of novel glutathione adducts of benzbromarone in human liver microsomes.

Benzbromarone (BBR) is a potent uricosuric drug that can cause serious liver injury. Our recent study suggested that 1'-hydroxy BBR, one of major metabolites of BBR, is metabolized to a cytotoxic metabolite that could be detoxified by glutathione (GSH). The aim of this study was to clarify whether GSH adducts are formed from 1'-hydroxy BBR in human liver microsomes (HLM). Incubation of 1'-hydroxy BBR with GSH in HLM did not result in the formation of GSH adducts, but 1',6-dihydroxy BBR was formed. In addition, incubation of 1',6-dihydroxy BBR with GSH in HLM resulted in the formation of three novel GSH adducts (M1, M2 and M3). The structures of M1 and M2 were estimated to be GSH adducts in which the 1-hydroxyethyl group at the C-2 position and the hydroxyl group at the C-1' position of 1',6-dihydroxy BBR were substituted by GSH, respectively. We also found that the 6-hydroxylation of 1'-hydroxy BBR is mainly catalyzed by CYP2C9 and that several CYPs and/or non-enzymatic reaction are involved in the formation of GSH adducts from 1',6-dihydroxy BBR. The results indicate that 1'-hydroxy BBR is metabolized to reactive metabolites via 1',6-dihydroxy BBR formation, suggesting that these reactive metabolites are responsible for BBR-induced liver injury.

Characterization of a recently evolved flavonol-phenylacyltransferase gene provides signatures of natural light selection in Brassicaceae.

Incidence of natural light stress renders it important to enhance our understanding of the mechanisms by which plants protect themselves from harmful effects of UV-B irradiation, as this is critical for fitness of land plant species. Here we describe natural variation of a class of phenylacylated-flavonols (saiginols), which accumulate to high levels in floral tissues of Arabidopsis. They were identified in a subset of accessions, especially those deriving from latitudes between 16° and 43° North. Investigation of introgression line populations using metabolic and transcript profiling, combined with genomic sequence analysis, allowed the identification of flavonol-phenylacyltransferase 2 (FPT2) that is responsible for the production of saiginols and conferring greater UV light tolerance in planta. Furthermore, analysis of polymorphism within the FPT duplicated region provides an evolutionary framework of the natural history of this locus in the Brassicaceae.

Asymmetric Total Synthesis of Kopsiyunnanine K, a Monoterpenoid Indole Alkaloid with a Rearranged Skeleton.

A new monoterpenoid indole alkaloid, kopsiyunnanine K, was isolated from Kopsia arborea. Its intriguing rearranged structure and absolute configuration, which were inferred from spectral data and a possible biosynthetic pathway, were determined on the basis of a 13-step asymmetric total synthesis.

Asymmetric Total Synthesis of Sarpagine-Related Indole Alkaloids Hydroxygardnerine, Hydroxygardnutine, Gardnerine, (E)-16-epi-Normacusine B, and Koumine.

Sarpagine-related indole alkaloids (-)-hydroxygardnerine, (+)-hydroxygardnutine, (-)-gardnerine, (+)-(E)-16-epi-normacusine B, and (-)-koumine were divergently synthesized via a common intermediate possessing a piperidine ring with an exocyclic (E)-ethylidene side chain, which was constructed by a gold(I)-catalyzed 6-exo-dig cyclization strategy.