Isolation,transposon mutagenesis,and characterization of the novel nicotine-degrading strain Shinella sp. HZN7

Nicotine is a significant toxic waste generated in tobacco manufacturing. Biological methods for the degradation of nicotine waste are in high demand. In this study, we report the identification and characterization of the novel nicotine-degrading strain Shinella sp. HZN7. This strain can degrade 500 mg/L nicotine completely within 3 h at 30 °C and pH values of 6.5?～?8.0. The biodegradation of nicotine by Shinella sp. HZN7 involves five intermediate metabolites: 6-hydroxy-nicotine (6HN), 6-hydroxy-N-methylmyosmine, 6-hydroxypseudooxynicotine (6HPON), 6-hydroxy-3-succinoyl-pyridine (HSP), and 2,5-dihydroxypyridine, as detected by ultraviolet spectrophotometry, HPLC, and LC-MS. We generated three mutants, N7-W18, N7-X5, and N7-M17, by transposon mutagenesis, in which the nicotine-degrading pathway terminated at 6HN, 6HPON, and HSP, respectively. The production of the five intermediate metabolites and their order in the degradation pathway were confirmed in the three mutants, indicating that strain HZN7 degrades nicotine via a variant of the pyridine and pyrrolidine pathways. The mutant gene from strain N7-X5, orf2, was cloned by self-formed adaptor PCR, but the nucleotide and amino acid sequence showed no similarity to any gene or gene product with defined function. However, orf2 disruption and complementation suggested that the orf2 gene is essential for the conversion of 6HPON to HSP in strain HZN7. This is the first study to provide genetic evidence for this variant nicotine degradation pathway.     

Molecular Mechanism of Nicotine Degradation by a Newly Isolated Strain,Ochrobactrum sp. Strain SJY1

A newly isolated strain, SJY1, identified as Ochrobactrum sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated vppB, vppD, and vppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The vppB gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. The vppD gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from Pseudomonas putida strain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.     

Conversion of nornicotine to 6-hydroxy-nornicotine and 6-hydroxy-myosmine by <Emphasis Type="Italic">Shinella</Emphasis> sp. strain HZN7

Nornicotine is a natural alkaloid produced by plants in the genus Nicotiana and is structurally related to nicotine. Importantly, nornicotine is the direct precursor of tobacco-specific nitrosamine N′-nitrosonornicotine, which is a highly potent human carcinogen. Microbial detoxification and degradation of nicotine have been well characterized; however, until now, there has been no information on the molecular mechanism of nornicotine degradation. In this study, we demonstrate the transformation of nornicotine by the nicotine-degrading strain Shinella sp. HZN7. Three transformation products were identified as 6-hydroxy-nornicotine, 6-hydroxy-myosmine, and 6-hydroxy-pseudooxy-nornicotine by UV spectroscopy, high-resolution mass spectrometry, nuclear magnetic resonance, and Fourier transform-infrared spectroscopy analyses. The two-component nicotine dehydrogenase genes nctA1 and nctA2 were cloned, and their product, NctA, was confirmed to be responsible for the conversion of nornicotine into 6-hydroxy-nornicotine as well as nicotine into 6-hydroxy-nicotine. The 6-hydroxy-nicotine oxidase, NctB, catalyzed the oxidation of 6-hydroxy-nornicotine to 6-hydroxy-myosmine, and it spontaneously hydrolyzed into 6-hydroxy-pseudooxy-nornicotine. However, 6-hydroxy-pseudooxy-nornicotine could not be further degraded by strain HZN7. This study demonstrated that nornicotine is partially transformed by strain HZN7 via nicotine degradation pathway.     

Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

Nicotine and its N-demethylation product nornicotine are two important alkaloids in Nicotiana tabacum L. (tobacco). Both nicotine and nornicotine have two stereoisomers that differ from each other at 2′-C position on the pyrrolidine ring. (S)-Nicotine is the predominant form in the tobacco leaf, whereas the (R)-enantiomer only accounts for ∼0.2% of the total nicotine pool. Despite considerable past efforts, a comprehensive understanding of the factors responsible for generating an elevated and variable enantiomer fraction of nornicotine (EF_nnic of 0.04 to 0.75) from the consistently low EF observed for nicotine has been lacking. The objective of this study was to determine potential roles of enantioselective demethylation in the formation of the nornicotine EF. Recombinant CYP82E4, CYP82E5v2, and CYP82E10, three known tobacco nicotine demethylases, were expressed in yeast and assayed for their enantioselectivities in vitro. Recombinant CYP82E4, CYP82E5v2, and CYP82E10 demethylated (R)-nicotine 3-, 10-, and 10-fold faster than (S)-nicotine, respectively. The combined enantioselective properties of the three nicotine demethylases can reasonably account for the nornicotine composition observed in tobacco leaves, which was confirmed in planta. Collectively, our studies suggest that an enantioselective mechanism facilitates the maintenance of a reduced (R)-nicotine pool and, depending on the relative abundances of the three nicotine demethylase enzymes, can confer a high (R)-enantiomer percentage within the nornicotine fraction of the leaf.     

3-hydroxyisobutyrate dehydrogenase-I from Pseudomonas denitrificans ATCC 13867 degrades 3-hydroxypropionic acid

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (k _cat/K _m) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD⁺ over NADP⁺ as a cofactor for its catalytic activity. The k _cat/K _m determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD⁺ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k _cat/K _m values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn²⁺ increased the activity of 3HIBDHI significantly, whereas the presence of Fe²⁺, Hg²⁺ and Ag⁺ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.     

Synthesis and characterization of pyridoxine,nicotine and nicotinamide salts of dithiophosphoric acids as antibacterial agents against resistant wound infection

The pyridine-derived biomolecules are of considerable interest in developing medicinal compounds with various specific activities. Novel ammonium salts of pyridoxine, (S)-(–)-nicotine and nicotinamide with O,O-diorganyl dithiophosphoric acids (DTPA) were synthesized and characterized. The complexation of chiral monoterpenyl DTPA, including (S)-(–)-menthyl, (R)-(+)-menthyl, (1R)-endo-(+)-fenchyl, (1S,2S,3S,5R)-(+)-isopinocampheolyl derivatives, with pyridoxine and nicotine provided effective antibacterial compounds 3a,b,e,f, and 5a,b,d,f with MIC values against Gram-positive bacteria as low as 10?µM (6?µg/mL). Two selected pyridoxine and nicotine salts based on menthyl DTPA 3a and 5a were similarly active against antibiotic-resistant bacteria from burn wounds including MRSA. The compounds had enhanced amphiphilic and hemolytic properties and effectively altered surface characteristics and matrix-secreting ability of P. aeroginosa and S. aureus. MBC/MIC ratios of 3a and 5a suggested the bactericidal mode of their action. Furthermore, the compounds exhibited moderate cytotoxicity towards human skin fibroblasts (IC₅₀?=?48.6 and 57.6?µM, respectively, 72?h), encouraging their further investigation as potential antimicrobials against skin and wound infections.     

Concerning the biosynthesis of prostaglandin I2

Two diastereoisomers, 5R,6R-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (7) and 5S,6S-5-hydroxy-6(9α)-oxido-11α,15S-dihydroxyprost-13-enoic acid (10) were synthesized for evaluation as possible biosynthetic intermediates in the enzymatic transformation of PGH₂ or PGG₂ into PGI₂. The synthetic sequence entails the stereospecific reduction of the 9-keto function in PGE₂ methyl ester after protecting the C-11 and C-15 hydroxyls as tbutyldimethylsilyl ethers. The resulting PGF_2α derivative was epoxidized exclusively at the C-5 (6) double bond to yield a mixture of epoxides, which underwent facile rearrangement with SiO₂ to yield the 5S,6S and 5R,6R-5-hydroxy-6(9α)-oxido cyclic ethers. It was found that dog aortic microsomes were unable to transform radioactive 9β-5S,6S[³H] or 9β-5R,6R[³H]-5-hydroxy-6(9α)-oxido cyclic ethers into PGI₂. Also, when either diastereoisomer was included in the incubation mixture, neither isomer diluted the conversion of [1-¹⁴C]arachidonic acid into [1-¹⁴C]PGI₂.     

Zur biogenese des aza-oxa-spiran-systems der steroidalkaloide vom spirosolan-typ in Solanaceen

5α,6-³H₂-Solacongestidine and 5α,6-³ H₂-(22S)-dihydrosolacongestidine administered to Solanum dulcamara as well as 16-³H₂-(22S: 25R)-22,26-epimino- cholest-5-en-3β-ol (25-isodihydroverazine) and 7α-³H-(22S: 25R)-22,26-epimino-cholest-5-en-3β,16β-diol administered to Solanum laciniatum were converted to coladulcidine and solasodine, respectively. These results are discussed in relation to spirosolane alkaloid biosynthesis.     

Neolignans from stem bark of ocotea veraguensis

The stem bark of Ocotea veraguensis has yielded nine neolignans of which five appear to be novel. The new neolignans, which were identified on the basis of spectral characteristics, are^* (7S,8R,1′S,2′S,3′R,4′S)-Δ^8′-2′,4′-dihydroxy-3,3′5′-trimethoxy-4,5-methylenedioxy-1′,2′,3′,4′-tetrahydro-7.3′,8.1′-neolignan, (7S,8R,1′S,3′S,4′S)-Δ^8′-4,4'-dihydroxy-3,3′,5′-trimethoxy-1′,2′,3′,4′-tetrahydro-2′-oxo-7.3′,8.1′-neolignan, (7S,8S,1′R)-Δ^8′-3′,5′-dimethoxy-3,4-methylenedioxy-1′,4′-dihydro-4′-oxo-7.0.2′,8.1′-neolignan, (7S,8S,1′R )-Δ^8′-1′-methoxy-3,4-methylenedioxy-1′,6′-dihydro-6′-oxo-7.0.4′,8.3′-neolignan and (7S,8S)-Δ^8′-2′,6′-dimethoxy-3,4-methylenedioxy-7.0.3′,8.4′,1′.0.7′-neolignan.     

22,23-Epoxides of Sitosterol and Related 7-Oxygenated Δ5-Sterols

(22S,23S)-22,23-Epoxysitosterol, (22R,23R)-22,23-epoxysitosterol, (22S, 23S)-22,23-epoxy-7-ketositosterol, (22R,23R)-22,23-epoxy-7-ketositosterol, (22S, 23S)-22,23-epoxy-7α-hydroxysitosterol, (22S,23S)-22,23-epoxy-7β-hydroxysitosterol, and (22R, 23R)-22,23-epoxy-7β-hydroxysitosterol were synthesized. Their ¹H and ¹³C NMR and the mass spectra of their trimethylsilyl derivatives were studied.     

Induction of d-6-hydroxynicotine oxidase in resting cells of Arthrobacter oxidans

Resting cell suspensions of Arthrobacter oxidans were shown to synthesize the inducible enantiozyme, d-6-hydroxynicotine oxidase, in the presence of d-nicotine or d-6-hydroxynicotine. The corresponding l-enantiomers, as well as -methylaminopropyl-(6-OH-pyridyl-3)-ketone, which is the product of the reaction catalyzed by the enzyme, were ineffective as inducers. l-6-Hydroxynicotine inhibited induction by d-nicotine and d-6-hydroxynicotine while l-nicotine inhibited induction by d-6-hydroxynicotine and had no effect on induction by d-nicotine. Enzyme induction was also found to be inhibited by glucose, 2-deoxy-d-glucose and by several intermediates of the tricarboxylic acid cycle. An absolute requirement for protein synthesis and for oxygen was also demonstrated to be necessary for the reactions involved in the covalent attachment of flavin adenine dinucleotide to pre-existing precursor protein to yield the catalytically active d-6-hydroxynicotine oxidase.H. C. R. completed these studies while on sabbatical leave from the Department of Botany and Microbiology, Arizona State University, Tempe, Arizona 85281, U.S.A.     

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K_m = 48 ± 5 μM, k_cat = 291 ± 30 s⁻¹), but turned over (2S)-ibuprofenoyl-CoA (K_m = 86 ± 6 μM, k_cat = 450 ± 14 s⁻¹) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His₆-tag had a catalytic efficiency (k_cat/K_m) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.     

Enantioselective Uptake and Degradation of the Chiral Herbicide Dichlorprop [(RS)-2-(2,4-Dichlorophenoxy)propanoic acid] by Sphingomonas herbicidovorans MH

Sphingomonas herbicidovorans MH was able to completely degrade both enantiomers of the chiral herbicide dichlorprop [(RS)-2-(2,4-dichlorophenoxy)propanoic acid], with preferential degradation of the (S) enantiomer over the (R) enantiomer. These results are in agreement with the recently reported enantioselective degradation of mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propanoic acid] by this bacterium (C. Zipper, K. Nickel, W. Angst, and H.-P. E. Kohler, Appl. Environ. Microbiol. 62:4318–4322, 1996). Uptake of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D (2,4-dichlorophenoxyacetic acid) was inducible. Initial uptake rates of cells grown on the respective substrate showed substrate saturation kinetics with apparent affinity constants (K_t) of 108, 93, and 117 μM and maximal velocities (V_max) of 19, 10, and 21 nmol min⁻¹ mg of protein⁻¹ for (R)-dichlorprop, (S)-dichlorprop, and 2,4-D, respectively. Transport of (R)-dichlorprop, (S)-dichlorprop, and 2,4-D was completely inhibited by various uncouplers and by nigericin but was only marginally inhibited by valinomycin and by the ATPase inhibitor N,N′-dicyclohexylcarbodiimine. Experiments on the substrate specificity of the putative transport systems revealed that (R)-dichlorprop uptake was inhibited by (R)-mecoprop but not by (S)-mecoprop, (S)-dichlorprop, or 2,4-D. On the other hand, the (S)-dichlorprop transport was inhibited by (S)-mecoprop but not by (R)-mecoprop, (R)-dichlorprop, or 2,4-D. These results provide evidence that the first step in the degradation of dichlorprop, mecoprop, and 2,4-D by S. herbicidovorans is active transport and that three inducible, proton gradient-driven uptake systems exist: one for (R)-dichlorprop and (R)-mecoprop, another for (S)-dichlorprop and (S)-mecoprop, and a third for 2,4-D.     

Nicotine Dehydrogenase Complexed with 6-Hydroxypseudooxynicotine Oxidase Involved in the Hybrid Nicotine-Degrading Pathway in Agrobacterium tumefaciens S33

Nicotine, a major toxic alkaloid in tobacco wastes, is degraded by bacteria, mainly via pyridine and pyrrolidine pathways. Previously, we discovered a new hybrid of the pyridine and pyrrolidine pathways in Agrobacterium tumefaciens S33 and characterized its key enzyme 6-hydroxy-3-succinoylpyridine (HSP) hydroxylase. Here, we purified the nicotine dehydrogenase initializing the nicotine degradation from the strain and found that it forms a complex with a novel 6-hydroxypseudooxynicotine oxidase. The purified complex is composed of three different subunits encoded by ndhAB and pno, where ndhA and ndhB overlap by 4 bp and are ∼26 kb away from pno. As predicted from the gene sequences and from chemical analyses, NdhA (82.4 kDa) and NdhB (17.1 kDa) harbor a molybdopterin cofactor and two [2Fe-2S] clusters, respectively, whereas Pno (73.3 kDa) harbors an flavin mononucleotide and a [4Fe-4S] cluster. Mutants with disrupted ndhA or ndhB genes did not grow on nicotine but grew well on 6-hydroxynicotine and HSP, whereas the pno mutant did not grow on nicotine or 6-hydroxynicotine but grew well on HSP, indicating that NdhA and NdhB are responsible for initialization of nicotine oxidation. We successfully expressed pno in Escherichia coli and found that the recombinant Pno presented 2,6-dichlorophenolindophenol reduction activity when it was coupled with 6-hydroxynicotine oxidation. The determination of reaction products catalyzed by the purified enzymes or mutants indicated that NdhAB catalyzed nicotine oxidation to 6-hydroxynicotine, whereas Pno oxidized 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde pyridine. These results provide new insights into this novel hybrid pathway of nicotine degradation in A. tumefaciens S33.     

Neolignans from an Aniba species

A benzene extract of the trunk of an Aniba species (Lauraceae) contained benzyl benzoate, benzyl salicylate, sitosterol and the neolignans (2S,3S,3aR)-3a-allyl-5-methoxy-3-methyl-2-piperonyl-2,3,3a,6-tetrahydro-6-oxobenzofuran (burchellin); (2S,3S,3aR)-3a-allyl-5-methoxy-3-methyl-2-veratryl-2,3,3a,6-tetrahydro-6-oxobenzofuran; (2S,3S,3aR)-3a-allyl-5,7-dimethoxy-3-methyl-2-veratryl-2,3,3a,6-tetrahydro-6-oxobenzofuran; (2S,3S,5S)-5-allyl-5-methoxy-3-methyl-2-veratryl-2,3,5,6-tetrahydro-6-oxo-benzofuran; (2R,3R)-7-methoxy-3-methyl-5-propenyl-2-veratryl-2,3-dihydrobenzofuran; rel-(1R,5R,6R,7R,8S)-1-allyl-8-hydroxy-3-methoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene (guianin); rel-(1S,5S,6S,7R,8R)-1-allyl-8-hydroxy-3,5-dimethoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene; rel-(1S,5S,6S,7R,8R)-8-acetoxy-1-allyl-3-hydroxy-5-methoxy-7-methyl-4-oxo-6-piperonyl-bicyclo[3,2,1]oct-2-ene; rel-1S,5S,6S,7R,8R)-8-acetoxy-3,5-dimethoxy-7-methyl-4-oxo-6-piperonylbicyclo[3,2,1]oct-2-ene; rel-(1R,5S,6R,7R)-1-allyl-3-methoxy-7-methyl-4,8-dioxo-6-piperonylbicyclo[3,2,1]oct-2-ene.     

Molybdenum-Containing Nicotine Hydroxylase Genes in a Nicotine Degradation Pathway That Is a Variant of the Pyridine and Pyrrolidine Pathways

Ochrobactrum sp. strain SJY1 utilizes nicotine as a sole source of carbon, nitrogen, and energy via a variant of the pyridine and pyrrolidine pathways (the VPP pathway). Several strains and genes involved in the VPP pathway have recently been reported; however, the first catalyzing step for enzymatic turnover of nicotine is still unclear. In this study, a nicotine hydroxylase for the initial hydroxylation step of nicotine degradation was identified and characterized. The nicotine hydroxylase (VppA), which converts nicotine to 6-hydroxynicotine in the strain SJY1, is encoded by two open reading frames (vppA_S and vppA_L [subunits S and L, respectively]). The vppA genes were heterologously expressed in the non-nicotine-degrading strains Escherichia coli DH5α and Pseudomonas putida KT2440; only the Pseudomonas strain acquired the ability to degrade nicotine. The small subunit of VppA contained a [2Fe-2S] cluster-binding domain, and the large subunit of VppA contained a molybdenum cofactor-binding domain; however, an FAD-binding domain was not found in VppA. Resting cells cultivated in a molybdenum-deficient medium had low nicotine transformation activity, and excess molybdenum was detected in the purified VppA by inductively coupled plasma-mass spectrometry analysis. Thus, it is demonstrated that VppA is a two-component molybdenum-containing hydroxylase.     

Neolignans from Nectandra miranda

Seven neolignans, isolated from a C₆H₆ extract of Nectandra miranda (Lauraceae) trunk wood, included the hitherto undescribed (2S, 3S, 3aS)- and (2S, 3S, 3aR)-5-allyl-3a-methoxy-2-(3′, 4′, 5′-trimethoxyphenyl)-3-methyl-2, 3, 3a, 6-tetrahydro-6-oxobenzofurans (respectively mirandin-A and mirandin -B), 7-allyl-6-hydroxy-5-methoxy-2-(3′, 4′, 5′-trimethoxyphenyl)-3-methylbenzofuran and (2R, 3R)-7-methoxy-2-(3′, 4′, 5′-trimethoxyphenyl)-3-methyl-5 -(E)-propenyl-2, 3-dihydrobenzofuran (licarin C).     

Structure–cytotoxic activity relationship of sesquilignan,morinol A

The cytotoxic activities of sesquilignans, (7S,8S,7′R,8′R)- and (7R,8R,7′S,8′S)-morinol A and (7S,8S,7′S,8′S)- and (7R,8R,7′R,8′R)-morinol B were compared, showing no significant difference between stereoisomers (IC₅₀ = 24–35 μM). As a next stage, the effect of substituents at 7, 7′, and 7″-aromatic ring on the activity was evaluated to find out the higher activity of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18 (IC₅₀ = 6–7 μM). In the research on the structure–activity relationship of 7″-position of (7S,8S,7′R,8′R)-7,7′,7″-phenyl derivative 18, the most potent compounds were 7,7′,7″-phenyl derivative 18 (IC₅₀ = 6 μM) against HeLa cells. Against HL-60 cells, 7″-(4-nitrophenyl)-7,7′-phenyl derivative 33 and 7″-hexyl-7,7′-phenyl derivative 37 (IC₅₀ = 5 μM) showed highest activity. We discovered the compounds showed four to sevenfold potent activity than that of natural (7S,8S,7′R,8′R)-morinol A. It was also confirmed that the 7′-benzylic hydroxy group have an important role for exhibiting activity, on the other hand, the resonance system of cinnamyl structure is not crucial for the potent activity.     

Afritoxinones A and B,dihydrofuropyran-2-ones produced by Diplodia africana the causal agent of branch dieback on Juniperus phoenicea

Two phytotoxic dihydrofuropyran-2-ones, named afritoxinones A and B, were isolated from liquid culture of Diplodia africana, a fungal pathogen responsible for branch dieback of Phoenicean juniper in Italy. Additionally, six others known metabolites were isolated and characterized: oxysporone, sphaeropsidin A, epi-sphaeropsidone, R-(−)-mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein. The structures of afritoxinones A and B were established by spectroscopic and optical methods and determined to be as (3aS¹,6R¹,7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one and (3aR¹,6R¹,7aS)-6-methoxy-3a,7a-dihydro-3H,6H-furo[2,3-b]pyran-2-one, respectively. The phytotoxic activity of afritoxinones A and B and oxysporone was evaluated on host (Phoenicean juniper) and non-host plant (holm oak, cork oak and tomato) by cutting and leaf puncture assay. Oxysporone proved to be the most phytotoxic compound. This study represents the first report of secondary metabolites produced by D. africana. In addition, the taxonomic implications of secondary metabolites in Botryosphaeriaceae family studies are discussed.