Oxidation of carenes to chaminic acids by Mycobacterium smegmatis DSM 43061

Summary (+)-2-Carene is oxidized to (–)-isochaminic acid by Mycobacterium smegmatis DSM 43061. The side product (+)-2-carene-4-one is formed partially by autoxidation. (+)-3-Carene yields, under the same conditions, (+)-chaminic acid together with (+)-3-carene-5-one and a compound with a cleaved cyclopropane ring, 2-(3-methylcyclohexa-3,5-dienyl)propane-2-ol. Offprint requests to: K. Kieslich     

Biotransformation of biphenyl by Paecilomyces lilacinus and characterization of ring cleavage products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Novel ring cleavage products in the biotransformation of biphenyl by the yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4'-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

A practical route to partially protected pyrrolidines as precursors for the stereoselective synthesis of alexines

Either 3-O-benzoyl- (2a) or 3-O-benzyl-1,2-O-isopropylidene-beta-D-fructopyranose (2b) were regioselectively O-benzylated at C-4 to give 4a and 4b, respectively, which were transformed into 5-azido-3-O-benzoyl-4-O-benzyl- (6a) and 5-azido-3,4-di-O-benzyl-5-deoxy-1,2-O-isopropylidene-alpha-L-sorbopyranose (6b) by nucleophilic displacement of the corresponding 5-O-mesyl derivatives 5a and 5b by sodium azide in DMF, respectively. Compound 6b was also prepared from 4b in one step by the Mitsunobu methodology. Deacetonation of 6a and 6b gave the partially protected free azidouloses 8a and 8b, respectively, that were protected as their 1-O-TBDPS derivatives 9a and 9b. Hydrogenation of 9b over Raney nickel gave stereoselectively (2R,3R,4R,5S)-3,4-dibenzyloxy-2'-O-tert-butyldiphenylsilyl-2,5-bis(hydroxymethyl)pyrrolidine (12) which was identified by transformation into the well known (2R,3R,4R,5S)-3,4-dihydroxy-2,5-bis(hydroxymethyl)pyrrolidine (1, DGDP).     

Microbial transformation of precocene II: oxidative reactions by Streptomyces griseus.

Various species of "Streptomyces," "Aspergillus," "Rhodotorula," "Brevilegnia," "Syncephalastrum," and "Stysanus" were found to transform precocene II to three major metabolites. These major biotransformation products were isolated from a preparative-scale incubation of precocene II with Streptomyces griseus and were conclusively identified as (-)cis- and (+)trans-precocene II-3,4-dihydrodiols and (+)-3-chromenol. 18O2 incorporation studies indicated the involvement of a monooxygenase enzyme system in precocene II transformation by S. griseus. A mechanism is proposed for the formation of (+)-3-chromenol.     

Microbial transformation of precocene II: oxidative reactions by Streptomyces griseus

Various species of "Streptomyces," "Aspergillus," "Rhodotorula," "Brevilegnia," "Syncephalastrum," and "Stysanus" were found to transform precocene II to three major metabolites. These major biotransformation products were isolated from a preparative-scale incubation of precocene II with Streptomyces griseus and were conclusively identified as (-)cis- and (+)trans-precocene II-3,4-dihydrodiols and (+)-3-chromenol. 18O2 incorporation studies indicated the involvement of a monooxygenase enzyme system in precocene II transformation by S. griseus. A mechanism is proposed for the formation of (+)-3-chromenol.     

Biotransformation of Biphenyl by Paecilomyces lilacinus and Characterization of Ring Cleavage Products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4′-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Cometabolism of 3-methylbenzoate and methylcatechols by a 3-chlorobenzoate utilizingPseudomonas: Accumulation of (+)-2,5-dihydro-4-methyl-and (+)-2,5-dihydro-2-methyl-5-oxo-furan-2-acetic acid

Summary 3-Chlorobenzoate grown cells ofPseudomonas strain B 13 readily co-oxidize 3-methylbenzoate yielding 82% (+)-2,5-dihydro-4-methyl- and 9% (+)-2,5-dihydro-2-methyl-5-oxo-furan-2-acetic acid (compounds I and II, X=CH₃). The concentration of the products in the culture fluid exceed 11 g per liter without affecting the activity of the cells. The products were formed in 89% and 93% yield when 3- or 4-methylcatechol is cometabolized correspondingly. The lactonization of methyl- and halomuconic acids is discussed with regard to the mechanism of halide elimination during utilization of chlorosubstituted aromatic compounds.     

Novel Ring Cleavage Products in the Biotransformation of Biphenyl by the Yeast Trichosporon mucoides

The yeast Trichosporon mucoides, grown on either glucose or phenol, was able to transform biphenyl into a variety of mono-, di-, and trihydroxylated derivatives hydroxylated on one or both aromatic rings. While some of these products accumulated in the supernatant as dead end products, the ortho-substituted dihydroxylated biphenyls were substrates for further oxidation and ring fission. These ring fission products were identified by high-performance liquid chromatography, gas chromatography-mass spectrometry, and nuclear magnetic resonance analyses as phenyl derivatives of hydroxymuconic acids and the corresponding pyrones. Seven novel products out of eight resulted from the oxidation and ring fission of 3,4-dihydroxybiphenyl. Using this compound as a substrate, 2-hydroxy-4-phenylmuconic acid, (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)acetic acid, and 3-phenyl-2-pyrone-6-carboxylic acid were identified. Ring cleavage of 3,4,4′-trihydroxybiphenyl resulted in the formation of [5-oxo-3-(4′-hydroxyphenyl)-2,5-dihydrofuran-2-yl]acetic acid, 4-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid, and 3-(4′-hydroxyphenyl)-2-pyrone-6-carboxylic acid. 2,3,4-Trihydroxybiphenyl was oxidized to 2-hydroxy-5-phenylmuconic acid, and 4-phenyl-2-pyrone-6-carboxylic acid was the transformation product of 3,4,5-trihydroxybiphenyl. All these ring fission products were considerably less toxic than the hydroxylated derivatives.     

Role of host volatiles in mate location by the Japanese pine sawyer, Monochamus alternatus hope (Coleoptera: Cerambycidae)

We evaluated the responses of male and female Monochamus alternatus Hope (Coleoptera: Cerambycidae) to various terpenes commonly associated with host trees. Electroantennogram (EAG) tests were conducted with 12 plant volatile compounds and ethanol. Antennae of both sexes were highly sensitive to (R)-(+)-alpha-pinene, (+)-3-carene, (-)-beta-pinene, and terpinolene. Both sexes of M. alternatus were attracted by traps baited with (+)-alpha-pinene, (-)-beta-pinene, (+)-3-carene, or terpinolene. Our results support the first of the three-stage hypothesis posed by Ginzel and Hanks that suggests that location of stressed trees by cerambycids involves three stages: (1) both sexes locate larval hosts by using plant volatiles as kairomones; (2) males produces sex pheromones to attract females after both sexes land on the larval hosts; (3) males and female recognize each other by contract pheromones in their epicuticular wax layer. Males and females showed differences in their EAG responses to several compounds, including (R)-(+)-alpha-pinene, (-)-beta-pinene, myrcene, (+)-3-carene, (R)-(+)-limonene, terpinolene, and trans-caryophyllene. In all cases, males exhibited greater sensitivity than females. In laboratory assays, male M. alternatus showed strong preference for 1% (+)-alpha-pinene and 1% (-)-beta-pinene over other compounds. In field assays, traps baited with (+)-alpha-pinene, (-)-beta-pinene, (+)-3-carene, or terpinolene caught more beetles than control traps. We found strong male bias in beetle catches in baited traps and those captured on the stem of stressed trees despite a strong female bias in emerging beetles in 2004. We hypothesize that male M. alternatus are more responsive than females to plant volatiles and that males have more capacity than females in finding mating locations.     

Red turpentine beetle primary attraction to β-pinene or 3-carene (with and without ethanol) varies in western US pine forests

1. Lure attraction strength for red turpentine beetle, Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae) observed previously in US Pacific Northwest ponderosa pine forests is (−)-β-pinene+ethanol > (+)-3-carene+ethanol, but untested elsewhere in its western US range. Thus, both were tested with (−)-β-pinene, (+)-3-carene, ethanol, and a blank in Oregon and California sites burned by wildfire, whereas in Arizona the first four lures were tested in a thinned-unburned site.
2. The D. valens responses in burned Oregon and California sites were similar, (−)-β-pinene+ethanol > (−)-β-pinene > 3-carene = 3-carene+ethanol > ethanol > blank, whereas in the cut-unburned Arizona site it was 3-carene+ethanol > 3-carene = (−)-β-pinene+ethanol > (−)-β-pinene. Whether this variation was influenced by beetle genetic differences, or chemical and physical parameters in the different environments and remaining stressed host resources 1-year post disturbance warrants additional study.
3. Responses to (−)-β-pinene varied, from a stronger attractant than (+)-3-carene in Oregon and California, to a weaker lure than (+)-3-carene in Arizona. This (−)-β-pinene variability was minimized when released in combination with ethanol, making (−)-β-pinene+ethanol the most consistent attractant of those tested across the three states, and a reliable lure for detection, monitoring, and management projects for D. valens in western US pine forests.

     

Metabolism of camphors and related compounds

1. The metabolism of (+/-)-norcamphor, (+)-camphor, (-)-camphor, (+)-epicamphor, (+/-)-camphorquinone, (+/-)-camphane-2,5-dione and camphane was investigated in rabbits. All the compounds except camphane-2,5-dione increased the content of glucuronide in the urine. 2. (+/-)-Norcamphor was reduced to endo-norborneol; (+)-camphor, contrary to expectation, was reduced to (+)-borneol, as well as being hydroxylated to (+)-5-endo-hydroxycamphor and (+)-3-endo-hydroxycamphor, 5-endo-hydroxycamphor being the predominant product. (+)-Epicamphor was reduced mainly to (+)-epiborneol; (+/-)-camphorquinone gave 3-endo-hydroxycamphor and 2-endo-hydroxyepicamphor, the former being the major metabolite. (+/-)-Camphane-2,5-dione was reduced to 5-endo-hydroxycamphor. Camphane was hydroxylated to borneol and epiborneol, the latter predominating. 3. An explanation of these findings is given in terms of steric hindrance and thermodynamic stability. 4. The possibility was investigated that NADH was involved in the reductions.     

A cDNA clone for 3-carene synthase from Salvia stenophylla

The essential oil of Salvia stenophylla contains (+)-3-carene as the principal monoterpene component. Using an enriched cDNA library prepared from mRNA isolated from S. stenophylla peltate glandular trichomes, and a homology-based cloning strategy, a full-length cDNA was isolated that encoded a preprotein of 69.7 kDa which resembled a monoterpene synthase in sequence. Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of geranyl diphosphate to (+)-3-carene and to lesser amounts of limonene, myrcene, 4-carene and beta-phellandrene. This multiple-product synthase is responsible for the production of all of the essential oil monoterpenes of S. stenophylla.     

Formation of cysteine conjugates from dihydroxyphenylalanine and its S-cysteinyl derivatives by peroxidase-catalyzed oxidation

Peroxidase-catalyzed oxidation of 3-(3,4-dihydroxyphenyl)alanine (DOPA) and its S-cysteinyl derivatives(cysteinyldopas) in the presence of cysteine was studied by analyzing the products with chromatography on Dowex 50W. Products of the oxidation of DOPA were found to be 5-S- and 2-S-cysteinyldopa, 2,5-S,S-dicysteinyldopa, and three unknown compounds A1, B, and C. 5-S- and 2-S-cysteinyldopa were also oxidized as easily as DOPA to give 2,5-S,S-dicysteinyldopa and similar patterns of the unknown compounds. Further oxidation of 2,5-S,S-dicysteinyldopa in the presence of cysteine yielded compounds A1, B, and C, whereas in its absence compound B was not formed. From these results coupled with the spectral data, it is suggested that compounds A1 and C are the two isomeric dihydrobenzothiazine derivatives of 2,5-S,S-dicysteinyldopa, while compound B is 2,5,6-S,S-tricysteinyldopa. These date suggest a possibility that peroxidase may play some role in the formation of cysteinyldopa and related metabolites in vivo.     

Chronic Use of Intracerebral Dialysis for the In Vivo Measurement of 3,4-Dihydroxyphenylethylamine and Its Metabolite 3,4-Dihydroxyphenylacetic Acid

The intracerebral dialysis technique was studied with a method in which the rat was directly connected to the HPLC equipment. The effect of three pharmacological treatments [perfusion of 60 mmol K+ or 5 X 10(-5) M (+)-amphetamine or subcutaneous injection of 2 mg/kg (+)-amphetamine] on the release of 3,4-dihydroxyphenylethylamine (dopamine) and 3,4-dihydroxyphenylacetic acid was followed over a period of 7 days. The marked rise of dopamine output seen after infusion of K+ had almost disappeared on day 3. Tissue reactions around the membrane presumably formed a barrier preventing K+ from reaching dopaminergic terminals. In contrast, the pronounced rise in dopamine level after amphetamine (infused as well as systemically administered) was still present (although diminished) 8 days after implantation. It is concluded that, with certain restrictions, brain dialysis of dopamine is still useful several days after implantation of the membrane.     

Regio- and stereoselective cyclizations of dianhydro sugar alcohols catalyzed by a chiral (salen)Co(III) complex

The (salen)Co(III)OAc ((R,R)-1 and (S,S)-1) catalyzed cyclizations of the chiral dianhydro sugars, 1,2:5,6-dianhydro-3,4-di-O-methyl-D-glucitol (2), 1,2:5,6-dianhydro-3,4-di-O-methyl-D-mannitol (3), 1,2:5,6-dianhydro-3,4-di-O-methyl-L-iditol (4), and 1,2:4,5-dianhydro-3-O-methyl-L-arabinitol (5), is a facile method for the synthesis of anhydroalditol alcohols. Cyclization of 2 using (R,R)-1 and (S,S)-1 proceeded diastereoselectively to form 2,5-anhydro-3,4-di-O-methyl-D-mannitol (6) and 2,5-anhydro-3,4-di-O-methyl-L-iditol (7), respectively. The cyclization of 3 and 5 is a novel method for obtaining 1,6-anhydro-3,4-di-O-methyl-D-mannitol (11) and a stereoselective route to 1,5-anhydro-3-O-methyl-L-arabinitol (13). It is proposed that the reaction occurs via endo-selective cyclization of an epoxy alcohol produced by the endo-selective ring-opening of one of the two epoxide moieties in the starting material.     

Key structural features in cis-carane, (+)-3-carene,cis-pinane, (+)-α-pinene,and (−)-β-pinene influencing red turpentine beetle primary attraction when released with ethanol

1. In US Pacific Northwest ponderosa pine forests the primary attraction order shown previously for red turpentine beetle, Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae), is (−)-β-pinene+ethanol > (+)-3-carene+ethanol > (+)-α-pinene+ethanol. The monoterpenes are bicyclic C₁₀H₁₆ isomers containing one 6-carbon ring with one double bond. Both pinenes have a 4-carbon second ring and differ only by their endocyclic or exocyclic double bond. The (+)-3-carene second ring has 3-carbons; its double bond is endocyclic like (+)-α-pinene.
2. Ring system and double bond influences on primary attraction were evaluated by hydrogenating (+)-3-carene and (+)-α-pinene to cis-carane and cis-pinane, respectively. Field test primary attraction strengths were (−)-β-pinene+ethanol > cis-carane+ethanol > cis-pinane+ethanol > ethanol.
3. In combination with ethanol (i) a double bond is not required in either ring system to attract D. valens, (ii) the cis-carane bicyclic 3, 6-carbon ring system provides stronger beetle attraction than the cis-pinane 4, 6-carbon bicyclic ring system, and likely structural basis for stronger (+)-3-carene attraction over (+)-α-pinene, (iii) adding an exocyclic double bond to the 4, 6-carbon ring system elevates attraction above the 3, 6-carbon ring system with no double bond, and (iv) the 4, 6-carbon ring system is a much stronger attractant with an exocyclic rather than endocyclic double bond.

     

Microbial Transformation of beta-Ionone and beta-Methylionone

Aspergillus niger JTS 191 was selected from many microorganisms tested as capable of converting ionones to other compounds having aromas. The individual transformation products from beta-ionone were isolated and identified by comparison with synthetically derived compounds. The major products were (R)-4-hydroxy-beta-ionone and (S)-2-hydroxy-beta-ionone. 2-Oxo-, 4-oxo-, 3,4-dehydro-, 2,3-dehydro-4-oxo-, 3,4-dehydro-2-oxo-, (S)-2-acetoxy-, (R)-4-acetoxy-, and 5,6-epoxy-beta-ionone and 4-(2,3,6-trimethylphenyl)-but-3-en-2-one were also identified. Analogous transformation products of beta-methylionone also were identified. Based on gas-liquid chromatographic analysis during the fermentation, we propose two main oxidative pathways of beta-ionone. The results of this study suggest that these transformations of beta-ionones may be useful as tobacco-flavoring compounds.