Biosynthetic pathway of 24-membered macrolactam glycoside incednine

Incednine was isolated from Streptomyces sp. ML694-90F3 as an inhibitor of anti-apoptotic function of Bcl-2/Bcl-xL oncoproteins. The structure of incednine is quite unique with a characteristic 24-membered macrocyclic lactam aglycone and two unusual aminosugars. To understand its biosynthetic pathway, the incorporation studies were carried out with [1-¹³C]acetate, [1,2-¹³C₂]acetate, [1-¹³C]propionate, l-[¹³C₅,¹⁵N]glutamate, [1,2,3-¹³C₃]glycerol, d-[6,6-²H₂]glucose, and l-[CH₃-¹³C]methionine. As a result, acetate, propionate, and glycerol were well incorporated into the elongation units of the macrolactam moiety, which indicates that its basic skeleton could be constructed by standard polyketide synthase, whereas all atoms of the starter unit were labeled by [¹³C₅,¹⁵N]glutamate suggesting that glutamate is somehow decarboxylated and rearranged into 3-aminobutyrate as the unique starter unit. The origins of the sugar moieties and methyl groups were also clarified. Based on the incorporation pattern, a plausible biosynthetic pathway for incednine is proposed.     

Biosynthetic pathway of macrolactam polyketide antibiotic cremimycin

Biosynthetic origin of macrolactam polyketide antibiotic cremimycin was investigated by feeding experiments with [1-¹³C]acetate, [1,2-¹³C₂]acetate, [1-¹³C]propionate, succinate-d₄, and d-[6,6-²H₂]glucose. NMR analysis of the resultant isotope-enriched cremimycins showed distinctive incorporation patterns, which suggested that the aglycon of cremimycin was constructed from two propionates and eleven acetates. Thus, 3-oxononanoate was proposed as a potential polyketide intermediate, that is, aminated to be the unique nitrogen-containing moiety of cremymicin. Further, characteristic propionate biosynthetic pathway in the cremimycin-producing strain was also described.     

Biosynthetic incorporation of glucose [U-13C6] into the C7N2 unit of antibiotic A2387

The biosynthetic incorporation fo Glucose [U-¹³C₆] into the C₇N₂ unit of A23187 indicated that this functionality is formed by a divergence of the shikimate pathway. The possibility that 2,6-diaminobenzoic acid is a free intermediate inthe biosynthesis of this unit was eliminated.     

The biosynthesis of pentalenolactone

Feeding of [UL-¹³C₆]-glucose, an $\text{in}$$\text{vivo}$ precursor of [1,2-¹³C₂]-acetyl-CoA, gave pentalenolactone in which the pattern of ¹³C-enrichments and couplings supported a mevalonoid biosynthetic pathway.     

Glucose and glycerol concentrations and their tracer enrichment measurements using liquid chromatography tandem mass spectrometry

The present study describes a new liquid chromatography tandem mass spectrometry method for high‐throughput quantification of glucose and glycerol in human plasma using stable isotopically labeled internal standards and is suitable for simultaneous measurements of glucose and glycerol enrichments in connection to in vivo metabolic studies investigating glucose turnover and lipolytic rate. Moreover, in order to keep up with this new fast analysis, simple derivatization procedures have been developed. Prior to analysis, glucose and glycerol were derivatized using benzoyl chloride in order to form benzoylated derivatives via new simplified fast procedures. For glucose, two internal standards were evaluated, [U‐¹³C₆]glucose and [U‐¹³C₆, D₇]glucose, and for glycerol, [U‐¹³C₃, D₈]glycerol was used. The method was validated by means of calibration curves, quality control samples, and plasma samples spiked with [6,6‐D₂]glucose, [U‐¹³C₆]glucose, and [1,1,2,3,3‐D₅]glycerol in order to test accuracy, precision, and recovery of the method. Moreover, post preparative and freeze‐thaw sample stability were tested. The correlation of calibration curves for the glucose concentration were r² = 0.9998 for [U‐¹³C₆]glucose and r² = 0.9996 for [U‐¹³C₆, D₇]glucose, and r² = 0.9995 for the glycerol concentration. Interday accuracy for glucose using [U‐¹³C₆]glucose and glycerol determined in spiked plasma were respectively 103.5% and 106.0%, and the coefficients of variation were 2.0% and 9.7%, respectively. After derivatization, plasma samples were stable for at least 14 days. In conclusion, we have developed and validated a novel, accurate, and sensitive high‐throughput liquid chromatography tandem mass spectrometry method for simultaneous determination of glucose and glycerol concentrations and enrichment of infused tracers most commonly used in human metabolic kinetic studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Biosynthetic studies on the antibiotics PF1140: a novel pathway for a 2-pyridone framework

Incorporation of labeled acetate and l-serine into PF1140 in Eupenicillium sp. indicated that the skeleton of PF1140 is derived from five acetates and a l-serine. Upon administration of [1,3-¹³C₂]glycerol, a precursor of biotransformation into l-[1,3-¹³C₂]serine, the isotopic labels became contiguous in the resultant 2-pyridone of PF1140. Based on the feeding experiments, a novel and potentially general biosynthetic pathway for a 2-pyridone framework has been proposed, in which an acyl tetramic acid precursor could be converted via a ring expansion with loss of the hydroxyl group.     

The synthesis of novel hexa-C-labelled glucosinolates from [C6]-d-glucose

An isotopically labelled building block, 2,3,4,6-tetra-O-acetyl-1-thio-β-d-[¹³C₆]glucopyranose (4), is obtained from the commercially available [¹³C₆]-d-glucose. This hexa-¹³C-labelled thioglucose can be employed to make any glucosinolate (8) for use as an internal standard for isotopic dilution LCMS analysis. Herein three typical glucosinolates in their hexa-¹³C-labelled form: [glucose-¹³C₆]gluconasturtiin, [glucose-¹³C₆]sinigrin and [glucose-¹³C₆]glucoerucin are synthesised by coupling the isotopically labelled thioglucose (4) with the corresponding hydroximoyl chlorides followed by sulfation with pyridine sulfur trioxide and deacetylation with a catalytic amount of potassium methoxide, respectively.     

Reverse versus Normal Prenyl Transferases in Paraherquamide Biosynthesis Exhibit Distinct Facial Selectivities

Both a face-selective and a non-face-selective mode of formation of quaternary centers of isoprene-derived structural moieties of the natural alkaloid paraherquamide A ( 1 ) have been discovered by feeding experiments on Penicillium fellutanum with [U-¹³C₆]-glucose and [¹³C₂]-acetate. The labeling patterns suggest that the methyl groups (C22, C23) are introduced in a non-face-selective manner by a reverse prenyl transferase. The C₅ unit comprising the dioxepin moiety retains stereochemical integrity indicative of a single, face-selective addition of the phenolic group to the dimethylallyl group.     

Biosynthesis of highly modified meroterpenoids in aspergillus variecolor. Incorporation of 13C-labelled acetates and methionine into anditomin and andilesin C

Incorporations of [1-¹³C]?, [2-¹³C]?, [1,2-¹³C₂]-acetates and [¹³C]-methionine into anditomin, a metabolite of $\text{Aspergillus variecolor}$, indicate its formation by a mixed polyketide-terpenoid biosynthetic pathway similar to that elucidated for andibenin; observations are made on the possible biosynthetic relationship of the $\text{A. variecolor}$ metabolites with austin and terretonin, mycotoxins recently isolated from $\text{A. ustus}$ and $\text{A. terreus}$ respectively.     

Synthesis of [13C6]3,4-diaminobenzoic acid as a precursor for stable isotope labeled benzimidazoles

The synthesis of [¹³C₆]3,4-diaminobenzoic acid from commercially available [¹³C₆]aniline is described in six steps. Salient features of this route include the preparation of a differentially protected 3,4-diaminobenzonitrile, hydrogen gas free aromatic nitro group reduction with ammonium formate and facile benzimidazole ring closure of the ortho-arylenediamine with triethylorthoformate. This stable-labeled 3,4-diaminobenzoic acid is an ideal [M+6]isotopomer to synthesize complex benzimidazole fragments for mass spectrometry internal assays.     

Biosynthesis of plaunotol in Croton stellatopilosus proceeds via the deoxyxylulose phosphate pathway

Plaunotol, an acyclic diterpene alcohol that accumulates in the chloroplasts of Croton stellatopilosus leaves, was studied for its origin of isoprene units. Separate feedings of [U-¹³C]glucose and [1-¹³C]glucose into cut shoots of the plant showed that the labels from glucose were incorporated into plaunotol. NMR analysis of the purified plaunotol revealed that all four isoprene units of the molecule had the same labeling pattern which is consistent with exclusive acquisition via the deoxyxylulose phosphate pathway. The results of in vivo feeding experiments suggested that there was no significant metabolite exchange taking place between the mevalonate pathway and the deoxyxylulose pathway during the biosynthesis of plaunotol in this Croton stellatopilosus.     

Tetrahedron report : Concerning the biosynthesis of vitamin B12

The use of ¹³C-Fourier transform nuclear magnetic resonance (FT-NMR) has led to the observation that while 8 molecules of [2-¹³C]-ALA are incorporated into vitamin B₁₂ in P. shermanii, [5-¹³C]-ALA labels only seven of the carbon atoms of cyanocobalamin; i.e. one of the amino methyl groups of ALA is “lost” in the process. It has also been confirmed that seven of the methyl groups of vitamin B₁₂ are derived from [¹³CH₃]-enriched methionine and further that the chirality of the gem-dimethyl grouping at C₁₂ labeled with [¹³CH₃]-methionine is R. A soluble enzyme mixture from the 37,000 g or 100,000 g supernatant of disrupted cells of P. shermanii converts both ¹⁴C-labeled ALA and ¹⁴C-uro'gen III to cobyrinic acid, the simplest corrinoid material on the pathway to vitamin B₁₂ and the coenzyme, in presence of NADPH, Co²⁺, Mg²⁺, S-adenosylmethionine and glutathione. Multiply-labeled uro'gens (¹³C, ¹⁴C and ³H) have been used to show that incorporation takes place without randomization. A sequence for corrin synthesis from uro'gen III, involving as the first step decarboxylation of the ring-C acetic acid side chain, is proposed.     

The unimolecular and collisionally activated decompositions of fluorine-substituted tetraphenyltetrahedrane ions on the microsecond time-scale

Unimolecular and collision-induced decomposition products of [C₄(C₆H₅)₂(C₆H₄F)₂]⁺˙ generated from four unsymmetrical sources include [C₁₄H₁₀]⁺˙ and [C₁₄H₈F₂]⁺˙ and so provide evidence for a tetrahedral intermediate. Other decompositions show substantial influence of the position of the ρ-fluorophenyl ring on ion energy distributions. This influence may be related to the reported absence of peaks diagnostic for the tetrahedral intermediate from the spectrum of the equivalent ion from the appropriate ¹³C-labeled analog. Alternatively the difference in spectra can be correlated with lifetimes of ions.     

Studies of the intermediary metabolism in cultured cells of the insect <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> using <Superscript>13</Superscript>C- or <Superscript>15</Superscript>N-labelled tracers

Background  

Insect cells can serve as host systems for the recombinant expression of eukaryotic proteins. Using this platform, the controlled expression of ¹⁵N/¹³C labelled proteins requires the analysis of incorporation paths and rates of isotope-labelled precursors present in the medium into amino acids. For this purpose, Spodoptera frugiperda cells were grown in a complex medium containing [U-¹³C₆]glucose. In a second experiment, cultures of S. frugiperda were grown in the presence of ¹⁵N-phenylalanine.     

Study on the biosynthesis of the notoamides: Pinacol-type rearrangement of the isoprenyl unit in deoxybrevianamide E and 6-hydroxydeoxybrevianamide E

Two reverse-prenylated indole alkaloids, deoxybrevianamide E and 6-hydroxydeoxybrevianamide E, are proposed as biosynthetic precursors for advanced metabolites isolated from the marine-derived Aspergillus sp. In order to uncover the role of the alkaloids in the biosynthetic pathway, the feeding experiments of the [¹³C]₂-[¹⁵N]-labeled deoxybrevianamide E and 6-hydroxydeoxybrevianamide E were performed to afford the metabolites, which were produced by oxidation and successive pinacol-type rearrangement of the isoprenyl units.     

Theoretical study of interactions between 1-alkyl-3-methyimidazolium tetrafluoroborate and dibenzothiophene: DFT,NBO, and AIM analysis

Density functional theory is employed to study the interaction energies between dibenzothiophene (DBT) and 1-alkyl-3-methylimidazolium tetrafluoroborate ([C _n mim]⁺[BF₄]^?). The structures of DBT, 1-ethyl-3-methylimidazolium tetrafluoroborate ([C₂mim]⁺[BF₄]^?), 1-butyl-3-methylimidazolium tetrafluoroborate ([C4mim]+[BF₄]?), 1-hexyl-3-methylimidazolium tetrafluoroborate ([C₆mim]⁺[BF₄]^?), 1-octyl-3-methylimidazolium tetrafluoroborate ([C₈mim]⁺[BF₄]^?), [C₂mim]⁺[BF₄]^?–DBT, [C₄mim]⁺[BF₄]^?–DBT, [C₆mim]⁺[BF₄]^?–DBT and [C₈mim]⁺[BF₄]^?–DBT systems are optimized systematically at the B3LYP/6-31G(d,p) level, and the most stable geometries are obtained by NBO and AIM analyses. The results indicate that DBT and imidazolium rings of ionic liquids are parallel to each other. It is found that the [BF₄]^? anion prefers to be located close to a C1–H9 proton ring in the vicinity of the imidazolium ring and the most stable gas-phase structure of [C _n mim]⁺[BF₄]^? has four hydrogen bonds between [C _n mim]+ and [BF₄]?. There are hydrogen bonding interactions, π–π and C–H–π interactions between [C₈mim]⁺[BF₄]^? and DBT, which is confirmed by NBO and AIM analyses. The calculated interaction energies for the studied ionic liquids can be used to interpret a better extracting ability of [C₈mim]⁺[BF₄]^? to remove DBT, due to stronger interactions between [C₈mim]⁺[BF₄]^? and DBT, in agreement with the experimental results of dibenzothiophene extraction by [C _n mim]⁺[BF₄]^?.     

Synthesis of an Isotopically Isomeric Mixture of 1,4,6,8-Tetramethyl[13C2]azulene and Its Thermal Reaction with Dimethyl Acetylenedicarboxylate

Sodium [1,3-¹³C₂]cyclopentadienide in tetrahydrofuran (THF) has been prepared from the corresponding labelled [¹³C₂]cyclopentadiene which was synthesized from ¹³CO₂ and (chloromethyl)trimethylsilane (cf. Scheme 10) according to an established procedure. It could be shown that the acetate pyrolysis of cis-cyclopentane-1,2-diyl diacetate (cis- 22 ) at 550 ± 5° under reduced pressure (60 Torr) gives five times as much cyclopentadiene as trans- 22 . The reaction of sodium [1,3-¹³C₂]cyclopentadienide with 2,4,6-trimethylpyrylium tetrafluoroborate in THF leads to the formation of the statistically expected 2:2:1 mixture of 4,6,8-trimethyl[1,3a-¹³C₂], -[2,3a-¹³C₂]-, and -[1,3-¹³C₂]azulene ( 20 ; cf. Scheme 7 and Fig. 1). Formylation and reduction of the 2:2:1 mixture [¹³C₂]- 20 results in the formation of a 1:1:1:1:1 mixture of 1,4,6,8-tetramethyl[1,3-¹³C₂]-, -[1,3a-¹³C₂]-, -[2,3a-¹³C₂]-, -[2,8a-¹³C₂]-, and -[3,8a-¹³C₂]azulene ( 5 ; cf. Scheme 8 and Fig. 2). The measured ²J(¹³C, ¹³C) values of [¹³C₂]- 20 and [¹³C₂]- 5 are listed in Tables 1 and 2. Thermal reaction of the 1:1:1:1:1 mixture [¹³C₂]- 5 with the four-fold amount of dimethyl acetylenedicarboxylate (ADM) at 200° in tetralin (cf. Scheme 2) gave 5,6,8,10-tetramethyl-[¹³C₂]heptalene-1,2-dicarboxylate ([¹³C₂]- 6a ; 22%), its double-bond-shifted (DBS) isomer [¹³C₂]- 6b (19%), and the corresponding azulene-1,2-dicarboxylate 7 (18%). The isotopically isomeric mixture of [¹³C₂]- 6a showed no ¹J(¹³C,¹³C) at C(5) (cf. Fig. 3). This finding is in agreement with the fact that the expected primary tricyclic intermediate [7,11-¹³C₂]- 8 exhibits at 200° in tetralin only cleavage of the C(1)? C(10) bond and formation of a C(7)? C(10) bond (cf. Schemes 6 and 9), but no cleavage of the C(1)? C(11) bond and formation of a C(7)? C(11) bond. The limits of detection of the applied method is ≥96% for the observed process, i.e., [1,3a-¹³C₂]- 5 + ADM→ [7,11-¹³C₂]- 8 →[1,6-¹³C₂]- 9 →[5,10a-¹³C₂]- 6a (cf. Scheme 6).     

Biosynthesis of aspergiolide A, a novel antitumor compound by a marine-derived fungus Aspergillus glaucus via the polyketide pathway

Aspergiolide A, a novel antitumor compound, was produced by a marine-derived filamentous fungus Aspergillus glaucus. The biosynthesis of it was unambiguously determined by feeding experiments using [l-¹³C]sodium acetate, [2-¹³C]sodium acetate, and [1,2-¹³C₂]sodium acetate precursors followed by ¹³C NMR spectroscopic investigation of the isolated products. Analysis of the patterns of ¹³C-enrichment revealed that all 25 carbon atoms in skeleton of aspergiolide A were derived from labeled acetate. And among them, 12 carbon atoms were labeled from the carboxylic group of acetate, while the other 13 carbon atoms were labeled from the methylic group of acetate. Besides, the labeling pattern of [1,2-¹³C₂]sodium acetate feeding experiment demonstrated that 12 intact acetate units were incorporated in aspergiolide A by polyketide pathway.     

Synthesis of labelled dihydroartemisinic acid

[15-¹³C²H₃]-Dihydroartemisinic acid (2a), [15-C²H₃]-dihydroartemisinic acid (2b) and [15-¹³CH₃]-dihydroartemisinic acid (2c) have been obtained in good yield and high isotopic enrichment by a reconstructive synthesis from artemisinin. These labelled compounds were designed to be used in biosynthetic experiments to determine the origins of artemisinin and other sesquiterpene natural products from Artemisia annua.     

Quantitative evaluation of the biosynthetic pathways leading to δ-aminolevulinic acid from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus by analysis of 13C-labeled coproporphyrinogen III biosynthesized from [2-13C]glycine, [1-13C]acetate, and [2-13C]acetate using 13C NMR spectroscopy

The biosynthetic pathways leading to δ-aminolevulinic acid (ALA) from the Shemin precursor glycine via the C5 pathway in Arthrobacter hyalinus were quantitatively evaluated by means of feeding experiments with [2-¹³C]glycine, sodium [1-¹³C]acetate, and sodium [2-¹³C]acetate, followed by analysis of the labeling patterns of coproporphyrinogen III (Copro’gen III) (biosynthesized from ALA) using ¹³C NMR spectroscopy. Two biosynthetic pathways leading to ALA from glycine via the C5 pathway were identified: i.e., transformation of glycine to l-serine catalyzed by glycine hydroxymethyltransferase, and glycine synthase-catalyzed catabolism of glycine to N ⁵,N ¹⁰-methylene-tetrahydrofolic acid (THF), which reacts with another molecule of glycine to afford l-serine. l-Serine is transformed to acetyl-CoA via pyruvic acid. Acetyl-CoA enters the tricarboxylic acid cycle, affording 2-oxoglutaric acid, which in turn is transformed to l-glutamic acid. The l-glutamic acid enters the C5 pathway, affording ALA in A. hyalinus. A ¹³C NMR spectroscopic comparison of the labeling patterns of Copro’gen III obtained after feeding of [2-¹³C]glycine, sodium [1-¹³C]acetate, and sodium [2-¹³C]acetate showed that [2-¹³C]glycine transformation and [2-¹³C]glycine catabolism in A. hyalinus proceed in the ratio of 52 and 48 %. The reaction of [2-¹³C]glycine and N ⁵,N ¹⁰-methylene-THF, that of glycine and N ⁵,N ¹⁰-[methylene-¹³C]methylene-THF generated from the [2-¹³C]glycine catabolism, and that of [2-¹³C]glycine and N ⁵,N ¹⁰-[methylene-¹³C]methylene-THF transformed the fed [2-¹³C]glycine to [1-¹³C]acetyl-CoA, [2-¹³C]acetyl-CoA, and [1,2-¹³C₂]acetyl-CoA in the ratios of 42, 37, and 21 %, respectively. These labeled acetyl-CoAs were then incorporated into ALA. Our results provide a quantitative picture of the pathways of biosynthetic transformation to ALA from glycine in A. hyalinus.