First synthesis of (1,2-C2)-monolignol glucosides

The monolignol glucosides (1,2-¹³C₂)-p-glucocoumaryl alcohol, (1,2-¹³C₂)-coniferin and (1,2-¹³C₂)-syringin, and the glucosides of (1,2-¹³C₂)-p-coumaric, (1,2-¹³C₂)-ferulic and (1,2-¹³C₂)-sinapic acids were synthesized by condensation of the corresponding aldehydes with (1,2,3-¹³C₃)-malonic acid. The free acids were converted to the acyl chlorides prior to their reduction to alcohols.     

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).     

Studies on the Biosynthesis of Spirostaphylotrichin A

Incorporation of ¹⁴C-labelled acetate and amino acids as well as of [1-¹³C]-, [2-¹³C]-, and [1,2-¹³C₂] acetate, L -[methyl¹³C] methionine, [2,3-¹³C₂] succinate, and L -[2,3-¹³C₂] aspartate into spirostaphylotrichin A ( 1 ) by Staphylotrichum coccosporum demonstrates that the building blocks of 1 are 5 units of acetate/malonate, 1 unit of methionine, and a C₄-dicarboxylic acid. The latter is most likely aspartate and derived from the citric-acid cycle. Using [2-¹³C, 2-²H₃] acetate as a precursor, the starter unit of the polyketide chain was identified.     

Studies on the Biosynthesis of Tabtoxin (Wildfire Toxin). Origin of the Carbonyl C-Atom of the β-Lactam Moiety from the C1-Pool

Re-isolation of Pseudomonas tabaci strain NCPPB 2730 from its host, the tobacco plant, led to an activation of the bacteria in order to produce the β-lactam dipeptide tabtoxin (Wildfire toxin, 1 ). Incorporation of several ¹⁴C-labelled amino acids as well as L -[methyl-¹³C]methionine, L -[1,2-¹³C₂]- and L -[3,4-¹³C₂]aspartate, rac -[1,2-¹³C₂]glycerol, and [1,2-¹³C₂]acetate into isotabtoxion ( 2 ) demonstrated that the building blocks of tabtoxin ( 1 ) are L -threonine, L -aspartate, the Me group of L -methionine and a C₂-unit derived from the C₃-pool (Fig. 3). The Me group of L -methionine provides the carbonyl C-atom of the β-lactam moiety. These findings represent a novel pathway in β-lactam biosynthesis. Mechanistic aspects with respect to the β-lactam ring formation are discussed. A biradical 16 is proposed as an intermediate during the cyclization of a N-formyl-α-amino ketone 15 .     

Biosynthesis of kinamycin D. incorporation of [1,2-13C] acetate and of [2-2H3,1-13C]acetate

Results from the incorporation of [1,2-¹³C₂]- and [2-²H₃,1-¹³C]acetates into kinamycin D. indicate the involvement of only unsymmetrical intermediates; the identity of some of these are suggested.     

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.     

Biosynthesis of the Cytochalasans. Biosynthetic studies on chaetoglobosin A and 19-O-acetylchaetoglobosin A

Incorporation of [1-¹³C]-, [2-¹³C]- and [1,2-¹³C₂]-acetate, [1-¹³C]-propionate, [¹³C-CH₃]-L -methionine and [3-¹⁴C]-DL -tryptophan into chaetoglobosin A ( 1 ) and 19-O-acetylchaetoglobosin A ( 2 ) by Chaetomium globosum demonstrated that the building blocks of 1 and 2 are 9 and 10 units of acetate/malonate respectively, 3 units of methionine and 1 unit of tryptophan. Propionate is incorporated indirectly after several biological transformations. Using [2-¹³C, 2-²H₃]-acetate as precursor, the starter unit of the polyketide-chain was identified. Experiments which [¹³C, ²H₃-CH₃-L -methionine demonstrated that the three C-methylations occur with retention of all three H-atoms of the methyl group. Incorporation experiments with various ¹⁴C- and ³H-labelled tryphtophan samples and with [2-²H]- and [2-¹⁵N]-L -tryptophan showed that the amino acid is incorporated intact with retention of both the α-H- and the α-N-atom. On the basis of these results a more detailed general scheme of the cytochalasan biogenesis is proposed.     

The biosynthesis of quadrone and terrecyclic acid

Feedings of [1-¹³C]- and [1,2-¹³C₂]acetate Aspergillus terreus gave quadrone and terrecyclic acid which were analyzed by ¹³C NMR. The pattern of ¹³C-enrichments and couplings is consistent with the formation of 1 and 2 by cyclization of farnesyl pyrophosphate.     

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.     

Studies on the biosynthesis of terrecyclic acid A II confirmation of the cyclization mechanism and hydrogen shifts using [2-2H3]acetate and [213C2H3]acetate

Feeding experiments with [2-³H₃]acetate and [2-¹³C²H₃]acetate in $\text{Aspergillus terreus}$ Thom No. 14 indicated that the hydrogen at C-2 in terrecyclic acid A is incorporated without migration from the precursor acetic acid; the results favour our group's earlier speculation for the cyclization to the tricyclic skeleton in the biosynthetic scheme.     

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.     

The biosynthesis of sorbicillinoids in Trichoderma sp. USF-2690: prospect for the existence of a common precursor to sorbicillinol and 5-epihydroxyvertinolide, a new sorbicillinoid member

Biosynthetic feeding studies of [1-¹³C], [2-¹³C], and [1,2-¹³C₂]-labeled sodium acetates into 5-epihydroxyvertinolide, a new sorbicillinoid, gives an incorporation pattern that proves the γ-lactone ring formation associated with a ring cleavage reaction of the precursor, a potential intermediate of sorbicillinol biosynthesis.     

A new approach to the synthesis of pyrrolo[1,2-a]indole derivatives

Reactions of 2-(3,3-diamino-2-cyanoprop-2-enylidene)indolin-3-ones with amide acetals afforded derivatives of 3-aminomethyleneamino-2-cyano-9-oxo-9H-pyrrolo[1,2-a]indole and 3-amino-2-cyano-9-oxo-9H-pyrrolo[1,2-a]indole. The rate and pathway of the reaction depend on the reagents and solvents. The formation schemes for the compounds obtained were proposed. Their structures were proven by IR and ¹H NMR spectroscopy and mass spectrometry and confirmed by independent syntheses. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 12, pp. 2186–2192, December, 2006.     

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.     

Biosynthesis of cytochalasans. Part 4. The mode of incorporation of common naturally-occurring carboxylic acids into cytochalasin D1.

Utilization of sodium [1-¹⁴C]-, [2–¹⁴C]-, and [1,2-¹³C]-acetates, [1-¹⁴C]-, [1-¹³C]-, or [2-¹⁴C]-propionates, [1-¹⁴C]-or [2-¹⁴C]-malonates, of [1-¹⁴C]- or of [1-¹⁴C]-myristic acid, or of [1-¹⁴C]- and [1-¹⁴C]-palmitic acid in the biosynthesis of cytochalasin D ( 1 ) by Zygosporium masonii was determined by degradation studies or by carbon magnetic resonance spectroscopy. The precursors were incorporated primarily via the acetate-malonate pathway to generate 1 from nine intact acetate units, eight of which are coupled in a head to tail fashion to form the C₁₆-polyketide moiety.     

Assignment of C NMR spectrum for blepharismin C based on biosynthetic studies

Blepharismins, toxic pigments of the ciliate Blepharisma japonicum, are polycyclic ring-condensed compounds. Assignment of ¹³C NMR signals for blepharismin C, a major constituent of blepharismins, was achieved by analyses of the HMQC, HMBC, and INADEQUATE spectra of ¹³C-enriched samples obtained by feeding experiments using sodium [1-¹³C], [2-¹³C], and [1,2-¹³C₂]acetates.     

Reactions of 3-(Polyfluoroalkyl)propane-1,2,3-trione-2-oximes with Diaminoarenes

Novel quinoxaline derivatives have been synthesized via the reaction of 3-trifhioromethyl-1,2,3-propanetrione-2-oximes with 1,2-diaminobenzene or 2,3-diaminonaphthalene: 2-trifluoromethyl-3-aroylquinoxaline and 2-trifluoromethyl-3-aroylbenzo[g]quinoxaline. Under similar conditions, 3-R^F-1,2,3-propanetrione-2-oximes [R^F = C₃F₇, H(CF₂)₄, C₄F₉, and C₆F₁₃] with the same diaminoarenes have given a mixture of the condensation and fragmentation products in different ratios. The structure of (4-methylphenyl)[3-(tri-fluoromethyl)benzo[g]quinoxalin-2-yl]methanone has been elucidated by means of X-ray diffraction analysis.

     

Preparation and protonation of 2-pyrimidyl- and 2-pyrazylpalladium(II) complexes

The oxidative addition of 2-chloropyrimidine or 2-chloropyrazine to [Pd(PPh₃)₄] yields a mixture of trans-[PdCl(C₄H₃N₂-C²)(PPh₃)₂] (I) and [PdCl(μ-C₄H₃N₂-C²,N¹)(PPh₃ (II) (C₄H₃N₂ = 2-pyrimidyl or 2-pyrazyl group). The mononuclear complexes I are quantitatively converted into the binuclear species II upon treatment with H₂O₂. The reaction of II with HCl gives the N-monoprotonated derivatives cis-[PdCl₂(C₄H₄N₂-C²)(PPh₃)] (III), from which the cationic complexes trans-[PdCl(C₄H₄N₂-C²)(L) (L = PPh₃, IV; PMe₂Ph, V; PEt₃, VI) can be prepared by ligand substitution reactions. Reversible proton dissociation occurs in solution for III–VI. The low-temperature ¹H NMR spectra of trans-[PdCl(C₄H₄N₂-C²)(PMe₂Ph)₂]ClO₄ show that the heterocyclic moiety undergoes restricted rotation around the PdC² bond and that the 2-pyrazyl group is protonated predominantly at the N¹ atom. These results and the ¹³C NMR data for the PEt₃ derivatives are interpreted on the basis of a significant d_π → π^★ back-bonding contribution to the palladium—carbon bond of the protonated ligands.     

K2S2O8-Mediated halogenation of 2-arylimidazo[1,2-a]pyridines using sodium halides as the halogen sources

A convenient halogenation of 2-arylimidazo[1,2-a]pyridines using sodium chloride/bromide/iodide as the halogen sources in the presence of K₂S₂O₈ as an easy-to-handle oxidizing agent was developed. The present work offers an efficient and rapid access to 3-chloro-, 3-bromo- and 3-iodo-2-arylimidazo[1,2-a]pyridines which can be readily converted to C3-substituted imidazo[1,2-a]pyridines by cross-coupling reactions.     

Application of biosynthetic 13C-enrichment using [1-13C]-, [2-13C]- and [1,2-13C2]-acetate as precursor for 13C NMR spectral assignment of higher plant metabolites. The assignments of some bryonolic acid derivatives

The ¹³C NMR spectra of some derivatives of bryonolic acid (1) (D:C-friedoolean-8-en-3β-ol-29-oic acid) were assigned by means of ¹³C-enrichment, lanthanide-induced shifts (LIS) and comparison of chemical shift data between derivatives. The ¹³C-enriched species of 1, i.e., 1a, 1b and 1c were biosynthesized by Luffa cylindrica (Cucurbitaceae) callus fed with [1-¹³C]-, [2-¹³C]- or [1,2-¹³C₂]-acetate, respectively. Methyl acetylbryonolates 2, 2a, 2b and 2c, methyl bryonolates 3, 3a, 3b and 3c, methyl bryononates 4 and 4a, diacetyl-3β,29-diols (3,29-diacetyl-D:C-friedoolean-8-en-β,29-diol) 5, 5a, 5b and 5c, and 3-acetyl-3β,29-diols 6, 6a and 6b were prepared from 1, 1a, 1b and 1c, and their ¹³C NMR spectra were recorded. The ¹³C concentration of the ¹³C-enriched species was high enough to exhibit the satellite peaks clearly, and the analysed data were very useful for this study. Thus, total assignments for 2, 3, 4, 5 and 6 were established. It was found that conversion of the methoxycarbonyl group at C-29 into an acetoxymethyl group caused complex changes in the chemical shifts of the C, D- and E-ring carbons and those of the methyl carbons linked to these rings.