Acyclic Monoterpene Research Articles

Enantioselective divergent syntheses of several polyhalogenated Plocamium monoterpenes and evaluation of their selectivity for solid tumors.

The family of polyhalogenated monoterpenes from Plocamium counts over a hundred known members. Using glyceraldehyde acetonide as a chiral-pool precursor, an enantioselective and divergent strategy was developed that provides a blueprint for the synthesis of many of the small yet complex acyclic members of this family. The broad applicability of this approach is demonstrated with the short, eight-step synthesis of four natural products and three analogues. These syntheses are the first of any members of the acyclic polyhalogenated Plocamium monoterpenes and permitted the evaluation of their selectivity against a range of tumor cell lines.

Enantioselective Divergent Syntheses of Several Polyhalogenated Plocamium Monoterpenes and Evaluation of Their Selectivity for Solid Tumors

AbstractThe family of polyhalogenated monoterpenes from Plocamium counts over a hundred known members. Using glyceraldehyde acetonide as a chiral‐pool precursor, an enantioselective and divergent strategy was developed that provides a blueprint for the synthesis of many of the small yet complex acyclic members of this family. The broad applicability of this approach is demonstrated with the short, eight‐step synthesis of four natural products and three analogues. These syntheses are the first of any members of the acyclic polyhalogenated Plocamium monoterpenes and permitted the evaluation of their selectivity against a range of tumor cell lines.

The oxygen-independent metabolism of cyclic monoterpenes in Castellaniella defragrans 65Phen.

BackgroundThe facultatively anaerobic betaproteobacterium Castellaniella defragrans 65Phen utilizes acyclic, monocyclic and bicyclic monoterpenes as sole carbon source under oxic as well as anoxic conditions. A biotransformation pathway of the acyclic β-myrcene required linalool dehydratase-isomerase as initial enzyme acting on the hydrocarbon. An in-frame deletion mutant did not use myrcene, but was able to grow on monocyclic monoterpenes. The genome sequence and a comparative proteome analysis together with a random transposon mutagenesis were conducted to identify genes involved in the monocyclic monoterpene metabolism. Metabolites accumulating in cultures of transposon and in-frame deletion mutants disclosed the degradation pathway.ResultsCastellaniella defragrans 65Phen oxidizes the monocyclic monoterpene limonene at the primary methyl group forming perillyl alcohol. The genome of 3.95 Mb contained a 70 kb genome island coding for over 50 proteins involved in the monoterpene metabolism. This island showed higher homology to genes of another monoterpene-mineralizing betaproteobacterium, Thauera terpenica 58EuT, than to genomes of the family Alcaligenaceae, which harbors the genus Castellaniella. A collection of 72 transposon mutants unable to grow on limonene contained 17 inactivated genes, with 46 mutants located in the two genes ctmAB (cyclic terpene metabolism). CtmA and ctmB were annotated as FAD-dependent oxidoreductases and clustered together with ctmE, a 2Fe-2S ferredoxin gene, and ctmF, coding for a NADH:ferredoxin oxidoreductase. Transposon mutants of ctmA, B or E did not grow aerobically or anaerobically on limonene, but on perillyl alcohol. The next steps in the pathway are catalyzed by the geraniol dehydrogenase GeoA and the geranial dehydrogenase GeoB, yielding perillic acid. Two transposon mutants had inactivated genes of the monoterpene ring cleavage (mrc) pathway. 2-Methylcitrate synthase and 2-methylcitrate dehydratase were also essential for the monoterpene metabolism but not for growth on acetate.ConclusionsThe genome of Castellaniella defragrans 65Phen is related to other genomes of Alcaligenaceae, but contains a genomic island with genes of the monoterpene metabolism. Castellaniella defragrans 65Phen degrades limonene via a limonene dehydrogenase and the oxidation of perillyl alcohol. The initial oxidation at the primary methyl group is independent of molecular oxygen.

A domain swapping approach to elucidate differential regiospecific hydroxylation by geraniol and linalool synthases from perilla

Geraniol and linalool are acyclic monoterpenes found in plant essential oils that have attracted much attention for their commercial use and in pharmaceutical studies. They are synthesized from geranyl diphosphate (GDP) by geraniol and linalool synthases, respectively. Both synthases are very similar at the amino acid level and share the same substrate; however, the position of the GDP to which they introduce hydroxyl groups is different. In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. Sequences of the synthases were divided into ten domains (domains I to IV-4), and each corresponding domain was exchanged between both enzymes. It was shown that different regions were important for the formation of geraniol and linalool, namely, domains IV-1 and -4 for geraniol, and domains III-b, III-d, and IV-4 for linalool. These results suggested that the conformation of carbocation intermediates and their electron localization were seemingly to be different between geraniol and linalool synthases. Further, five amino acids in domain IV-4 were apparently indispensable for the formation of geraniol and linalool. According to three-dimensional structural models of the synthases, these five residues seemed to be responsible for the different spatial arrangement of the amino acid at H524 in the case of geraniol synthase, while N526 is the corresponding residue in linalool synthase. These results suggested that the side-chains of these five amino acids, in combination with several relevant domains, localized the positive charge in the carbocation intermediate to determine the position of the introduced hydroxyl group.

Monoterpene Citral Derivatives as Potential Antimalarials

Conjugated acid derivatives containing nitrogen have been synthesized from the simple acyclic monoterpene citral, using various reactions, including Wittig, Baylis Hillman, amide and ester condensations. Similarly, amine peroxides were synthesized by subjecting citral to Mannich type reaction with amines and t-butyl hydroperoxide. Molecules 3-10 were evaluated for antimalarial activity against erythrocytic stages of chloroquine sensitive P. falciparum strain 3D7. Four derivatives displayed interesting activity with an IC50< 2.5 microM, and warrant further investigations.

Geraniol—a flavoring agent with multifunctional effects in protecting the gastric and duodenal mucosa

Geraniol is an acyclic monoterpene alcohol commonly used as a flavoring agent. The present study was undertaken to investigate antiulcerogenic effects of geraniol and to determine the possible mechanisms involved in this action. In the model of the ethanol-induced ulcer, treatment of rats with geraniol by oral route significantly inhibited gastric lesions by 70% (7.50mg/kg) to 99% (200mg/kg). Analysis of the gastric tissue of rats treated with geraniol (7.50mg/kg) revealed that total glutathione content levels (GSH) increased and levels of myeloperoxidase (MPO) decreased in the gastric mucosa. Oral treatment with geraniol significantly decreased the number of ulcerative lesions induced by ischemia/reperfusion injury by 71% and the duodenal ulcers induced by cysteamine by 68%. The action of geraniol was mediated by the activation of defensive mucosa-protective factors such as the nitric oxide (NO) pathway, endogenous prostaglandins, increased mucus production, increased sulfhydryl compounds, antioxidant properties and the stimulation of calcitonin gene-related peptide (CGRP) release through the activation of transient receptor potential vanilloid (TRPV). The multifaceted gastroprotective mechanisms of geraniol represent a promising option for the treatment of gastric and duodenal mucosa injury.

Synthesis, characterization, and thermal properties of new flavor compounds

Synthesis, characterization, and thermal properties of new, flavor, long chain esters were presented. The new compounds were obtained in the catalytic esterification process of a stoichiometric ratio of trans-3,7-dimethyl-2,6-octadien-1-ol, succinic anhydride, and aliphatic chain diol. As diols ethylene glycol, 1,4-buthylene glycol, 1,5-pentylene glycol, and 1,6-hexylene glycol were applied. The spectroscopic analyses completely confirmed that the applied synthesis conditions allowed obtaining the new compounds with high yield and purity. Their thermal properties were studied in inert and oxidative atmospheres. The esters were less thermally stable in inert (IDT 186–195 °C) than in oxidative (IDT 210–228 °C) atmosphere. Two, non-completely divided decomposition steps were visible during their pyrolysis. In contrast, the new, long chain compounds decompose in three major steps in air. The analyses of the volatile products emitted during their pyrolysis indicated on the asymmetrical disrupt of their bonds. The formation of acyclic and alicyclic monoterpene hydrocarbons, succinic anhydride, diols, alcohols, alkenes, and water was observed. It indicated mainly on the β-elimination reactions during their pyrolysis. Also, β-elimination reactions of esters are mainly expected in air. Initially, it resulted in the formation of acyclic and alicyclic monoterpene hydrocarbons, hydroxyl compounds (diols, alcohols), and its β-elimination products: aldehydes, alkenes, and water. However, the presence of oxygen in the medium causes the partial decarboxylation and oxygenation of aldehydes and thus the formation of alkenes and carbon dioxide. In addition, the beginning of evaporation of succinic anhydride was detected at T max1. At T max2 the evaporation of succinic anhydride, their partial decarboxylation to CO2, the small amounts of diols, alcohols, and aldehyde fragments were indicated. Finally, succinic anhydride, water, and carbon dioxide were only observed during decomposition of studied esters in air.

BIOACTIVE MONOTERPENES FROM ANTARCTIC PLOCAMIUM CARTILAGINEUM

In a chemical analysis of the red seaweed Plocamium cartilagineum collected in Antarctic Peninsula, four polyhalogenated monoterpenes were isolated. Their structures were established by spectroscopic techniques. Two of them are acyclic monoterpenes and the others are cyclic. We report the antimicrobial, insecticide and acaricide activities of compounds.

Chemical Composition and Antioxidant Activity of Essential Oils from Cinnamodendron dinisii Schwacke and Siparuna guianensis Aublet.

The objectives of this study were to chemically characterize and evaluate the antioxidant activity of essential oils Cinnamodendron dinisii Schwacke (pepper) and Siparuna guianensis Aublet (negramina). The essential oil was isolated by hydrodistillation using a Clevenger modified apparatus, and the identification and quantification of constituents, through GC/MS and GC-FID analysis. The antioxidant activity was evaluated using β-carotene/linoleic acid system and the DPPH radical sequestering method. In chromatographic analysis, the majority constituents found in the essential oil of C. dinisii were bicyclic monoterpenes, α-pinene (35.41%), β-pinene (17.81%), sabinene (12.01%) and sesquiterpene bicyclogermacrene (7.59%). In the essential oil of the fresh leaves of Siparuna guianensis Aublet, acyclic monoterpene, β-myrcene (13.14%), and sesquiterpenes, germacrene-D (8.68%) and bicyclogermacrene (16.71%) were identified. The antioxidant activity was low by the β-carotene/linoleic acid test and was not evidenced by the DPPH test, for both oils evaluated.

Induction of senescence and identification of differentially expressed genes in tomato in response to monoterpene.

Monoterpenes, which are among the major components of plant essential oils, are known for their ecological roles as well for pharmaceutical properties. Geraniol, an acyclic monoterpene induces cell cycle arrest and apoptosis/senescence in various cancer cells and plants; however, the genes involved in the process and the underlying molecular mechanisms are not well understood. In this study, we demonstrate that treatment of tomato plants with geraniol results in induction of senescence due to a substantial alteration in transcriptome. We have identified several geraniol-responsive protein encoding genes in tomato using suppression subtractive hybridization (SSH) approach. These genes comprise of various components of signal transduction, cellular metabolism, reactive oxygen species (ROS), ethylene signalling, apoptosis and DNA damage response. Upregulation of NADPH oxidase and antioxidant genes, and increase in ROS level after geraniol treatment point towards the involvement of ROS in geraniol-mediated senescence. The delayed onset of seedling death and induced expression of geraniol-responsive genes in geraniol-treated ethylene receptor mutant (Nr) suggest that geraniol-mediated senescence involves both ethylene dependent and independent pathways. Moreover, expression analysis during tomato ripening revealed that geraniol-responsive genes are also associated with the natural organ senescence process.

Synthesis and thermal behavior of linear neryl diesters in inert and oxidative atmosphere

The studies on the synthesis and thermal properties of linear neryl diesters were presented. The linear neryl diesters can be successfully obtained during butylstannoic catalyzed esterification process. The final conversion of nerol and carboxylic groups was higher than 95 % using a stoichiometric molar ratio of reagents in mild conditions. The high yield products were prepared after longer time than previously studied geranyl diesters. It was directly connected with the steric hindrance and lower susceptibility of nerol to esterification process than geraniol. The TG/FTIR/QMS studies proved that the thermal properties and decomposition mechanism of neryl diesters differ considerably in inert and oxidative atmosphere. The diesters were thermally stable up to 200 °C in inert atmosphere. Their decomposition was run as a one-step process. The analyses of the volatile products emitted during their pyrolysis indicated on the ester and O-neryl bonds cleavage. It resulted in the formation of monoterpene hydrocarbons, cyclic acid anhydrides, ketones, or aldehydes. However, the studied compounds were less thermally stable in air than in helium. Their decomposition happened in two steps. The first step ranges from 185–228 °C to almost 326–380 °C with mass loss above 88 %. The formation of acyclic or alicylic monoterpene hydrocarbons, cyclic acid anhydrides, ketones, alkenes, alkanes, carbon dioxide, and water was expected. It indicated on the asymmetrical distrupt of the bonds, partial oxygenation, and decarboxylation of emitted gaseous fragments. The second step of decomposition was observed in temperatures ranges from 380 to above 560 °C. In this step carbon dioxide and water were mainly emitted. It was the result of the oxidation of the residue formed during the fist step.

Bioefficacy of acyclic monoterpenes and their saturated derivatives against the West Nile vector Culex pipiens

Twenty acyclic monoterpenes with different functional groups (acetoxy, hydroxyl, carbonyl and carboxyl) bearing a variable number of carbon double bonds were assayed as repellent and larvicidal agents against the West Nile vector Culex pipiens. Seven of them were derivatives that were synthesized through either hydrogenation or oxidation procedures. All repellent compounds were tested at the dose of 1mgcm−2 and only neral and geranial were also tested at a 4-fold lower dose (0.25mgcm−2). Repellency results revealed that geranial, neral, nerol, citronellol, geranyl acetate and three more derivatives dihydrolinalool (3), dihydrocitronellol (5) and dihydrocitronellyl acetate (6) resulted in no landings. Based on the LC50 values the derivative dihydrocitronellyl acetate (6) was the most active of all, resulting in an LC50 value of 17.9mgL−1. Linalyl acetate, citronellyl acetate, neryl acetate, geranyl acetate, dihydrocitronellol (5), dihydrocitronellal (7), citronellol, dihydrolinalyl acetate (2), citronellic acid and tetrahydrolinalyl acetate (1) were also toxic with LC50 values ranging from 23 to 45mgL−1. Factors modulating toxicity have been identified, thus providing information on structural requirements for the selected acyclic monoterpenes. The acetoxy group enhanced toxicity, without being significantly affected by the unsaturation degree. Within esters, reduction of the vinyl group appears to decrease potency. Presence of a hydroxyl or carbonyl group resulted in increased activity but only in correlation to saturation degree. Branched alcohols proved ineffective compared to the corresponding linear isomers. Finally, as it concerns acids, data do not allow generalizations or correlations to be made.

Synthesis of fragrance compounds from acyclic monoterpenes: Rhodium catalyzed hydroformylation and tandem hydroformylation/acetalization of linalool and β-citronellene

Rhodium-catalyzed hydroformylation of acyclic monoterpenic compounds, i.e., linalool and β-citronellene, was studied in toluene and ethanol solutions in the presence of PPh3 or P(O-o-tBuPh)3 ligands. Although both substrates have a monosubstituted terminal double bond, they show different behavior under the hydroformylation conditions. In toluene, linalool gave almost quantitatively a cyclic hemiacetal; whereas the hydroformylation of β-citronellene resulted in two isomeric aldehydes also in a nearly quantitative combined yield. The reactions occurred approximately two times faster in ethanol than in toluene giving the corresponding acetals even in the absence of additional acid co-catalysts. In the absence of phosphorous ligands, linalool (differently from β-citronellene) was very resistant to hydroformylation probably due to the binding with rhodium through both the double bond and the hydroxyl group to form stable chelates. The P(O-o-tBuPh)3 ligand exerted a remarkable effect on the reactivity of both substrates accelerating the reactions by 5–20 times as compared to the system with PPh3. Several fragrance compounds were obtained in high yields through a simple one-pot procedure starting from the substrates easily available from natural bio-renewable resources.

Changes in the essential oil composition of leaves of Echinodorus macrophyllus exposed to γ-radiation

Leaves of Echinodorus macrophyllus (Kunth) Micheli, Alismataceae, were exposed to different doses of γ-radiation (0.00, 1.00, 3.00, 5.00, 10.00, and 20.00 kGy) and the chemical composition of their essential oils was investigated. The extractive process of the essential oil was more favored when the leaves were irradiated. The essential oil components were identified by correlation between GCFID data and retention parameters obtained from the Kováts method. Moreover, GCMS analyses of the essential oils were correlated with fragmentation profiles in the NIST standard mass fragmentation data bank. The essential oil of E. macrophyllus contains biologically active constituents of different chemical classes. Acyclic monoterpenes and sesquiterpenes showed increase in concentration when the leaves were exposed to γ-radiation. On the other hand, the component concentrations of some chemical classes were lightly decreased, i.e., for bicyclic monoterpenes, diterpenes, triterpenes, carboxylic esters, and carotenoid derivatives.

Structural evidence for the involvement of the residues Ser187 and Tyr422 in substrate recognition in the 3-methylcrotonyl-coenzyme A carboxylase from Pseudomonas aeruginosa

The enzyme 3-methylcrotonyl-CoA carboxylase from Pseudomonas aeruginosa (Pa-MCCase) is essential for the assimilation of leucine and acyclic monoterpenes. The structure of the Pa-MCCase was analysed by computational modelling to establish the molecular basis of substrate recognition. The active site is composed of two zones, which may play important roles in substrate recognition and catalysis. To further understand the interactions of the active site with the substrate, site-directed mutagenesis of the conserved residues S187 and R51 located in zone I, and F417, Y422 and G423 from zone II of the Pa-MCCase was carried out. The residue substitutions S187A and Y422D completely abolished the Pa-MCCase activity, whereas substitutions R51A, F417Y and G423A indicated that these residues are not essential. Interestingly, the residues R47, R51 and S187 form a well-defined pocket that may play important roles in substrate coupling to the Co-A motif. At zone one, mutation S187A was essential, but mutant R51A retained activity, suggesting that the R51 function could be relegated to neighbouring positive residues. Residue Y422 instead of contributing to substrate discrimination, it may participate in deprotonation of methyl group on MC-CoA, because it is located at adequate distances from the 3-methylcrotonyl-chain and carboxybiotin groups in the Pa-MCCase carboxylation site.

A touch of lavender: gas-phase structure and dynamics of the monoterpene linalool validated by microwave spectroscopy

The microwave spectrum of linalool, an acyclic monoterpene, was recorded for the first time in the range from 9 to 16 GHz. The only conformer observed under molecular beam conditions was assigned. Fitting the rotational spectrum with two different programs treating internal rotation yielded the rotational constants A = 1.64674020(46) GHz, B = 0.68219862(16) GHz, C = 0.61875100(20) GHz, and the centrifugal distortion constants. The standard deviation of the fit was close to experimental accuracy. A-E splittings due to the internal rotation of one methyl group could be resolved and the internal rotation barrier was determined to be 400.20(64) cm(-1). The results from microwave spectroscopy were used to validate the molecular geometry obtained from quantum chemical calculations.

AVALIAÇÃO DO POTENCIAL ANTIEPIMASTIGOTA E CITOTOXIDADE DO CITRONELOL

Chagas disease is a major health problems often found in the Americas, especially in Latin America. The process of drug development has not kept pace with the rapid development of microorganisms and science have gathered efforts to find new substances that can disrupt defenses developed as a function of drug exposure. Derivatives of nifurtimox and benznidazole are the drugs used to treat Chagas disease, thus there is an interest in the use of natural products for the well being of humanity given in ancient times. The citronelole a compound present in the essential oils of various aromatic plant species, is a natural acyclic monoterpene considered excellent flavoring environments, which besides being a powerful insect repellent, also has antimicrobial activity and presents neuroprotective activity. The aim of this study was to evaluate the activity of the essential oil component, citronellol, on the epimastigote form of this Trypanossomacruzi and cytotoxicity in mammalian

TG/DSC/FTIR characterization of linear geranyl diesters

The synthesis, thermal behavior, and characterization of the decomposition products of linear geranyl diesters: digeranyl succinate, digeranyl glutarate, digeranyl adipinate, and digeranyl sebacinate were presented. The linear geranyl diesters were prepared in direct esterification process of a molar stoichiometric ratio of geraniol and suitable acidic reagent in solvent-free medium at 130 °C using butylstannoic acid as a catalyst. Their structure was confirmed based on FTIR, 1H- and 13C-NMR spectra. It was proved that the use of tin catalyst allowed decreasing the reaction time and increasing the final conversion of substrates when compared to non-catalyzed process. It considerably simplifies the processing by reduction of the preparation cost and thus this new method of synthesis of aroma diesters may be attractive for practical applications. The thermal behavior of prepared compounds was studied by TG/DSC/FTIR coupled method. TG analysis showed that diesters are thermally stable up to temperatures above 200 °C. The DTG curves confirmed that these decomposition run as a single-stage process. The T max1 were in the range of 294.5–313.8 °C depending on the aliphatic chain length (–CH2–)n in the structure of aroma diesters, which was in accordance with DSC data. The analysis of the gases evolved during heating of diesters in inert atmosphere indicated on the asymmetrical disrupt of their bonds. The cleavage of ester bond and O-geranyl bond was expected. It resulted in production of the mixture of derivatives of geraniol (acyclic and alicyclic monoterpene hydrocarbons) like myrcene, ocimene, or limonene as main decomposition products. In addition, the formation of anhydride, lactone, or ketone functionalities among the degradation products clearly confirmed the proposed degradation path of studied diesters.

Chemical Composition of Leaf and Seed Oils of Dryobalanops aromatica Gaertn. (Dipterocarpaceae)

The essential oils of the leaves and seed of Dryobalanops aromatica Gaertn. obtained by hydrodistillation resulted in 0.07% and 1.89% yield, respectively. These oils were then examined by GC-MS. Eighty-three components (plus an unknown) were identified from the leaf oil, representing 92% of the oil. Oxygenated monocyclic monoterpenes (terpinen-4-ol 15%, α-terpineol 16%), bicyclic monoterpene (α-pinene 7%) and oxygenated bicyclic sesquiterpene (globulol 8%) were the major constituents. In the case of the seed oil, 31 components were identified, representing 100% of the oil, while acyclic monoterpene (myrcene 5%), monocyclic monoterpene (limonene 6%), bicyclic monoterpenes (α-pinene 41%, α-thujene and b-pinene 13% each, sabinene 6%), and bicyclic sesquiterpene (bicyclogermacrene 6%) made up the major components. The remaining constituents of each oil (54% and 10%, respectively) were found to be minor (≤4% each). The chemical compositions of both oils differed quantitatively but showed important qualitative similarities and differences. The results of this study serve as the first report of complete chemical profiles of both oils.

Physiology of deletion mutants in the anaerobic β-myrcene degradation pathway in Castellaniella defragrans.

BackgroundMonoterpenes present a large and versatile group of unsaturated hydrocarbons of plant origin with widespread use in the fragrance as well as food industry. The anaerobic β-myrcene degradation pathway in Castellaniella defragrans strain 65Phen differs from well known aerobic, monooxygenase-containing pathways. The initial enzyme linalool dehydratase-isomerase ldi/LDI catalyzes the hydration of β-myrcene to (S)-(+)-linalool and its isomerization to geraniol. A high-affinity geraniol dehydrogenase geoA/GeDH and a geranial dehydrogenase geoB/GaDH contribute to the formation of geranic acid.A genetic system was for the first time applied for the betaproteobacterium to prove in vivo the relevance of the linalool dehydratase-isomerase and the geraniol dehydrogenase. In-frame deletion cassettes were introduced by conjugation and two homologous recombination events.ResultsPolar effects were absent in the in-frame deletion mutants C. defragrans Δldi and C. defragrans ΔgeoA. The physiological characterization of the strains demonstrated a requirement of the linalool dehydratase-isomerase for growth on acyclic monoterpenes, but not on cyclic monoterpenes. The deletion of geoA resulted in a phenotype with hampered growth rate on monoterpenes as sole carbon and energy source as well as reduced biomass yields. Enzyme assays revealed the presence of a second geraniol dehydrogenase. The deletion mutants were in trans complemented with the broad-host range expression vector pBBR1MCS-4ldi and pBBR1MCS-2geoA, restoring in both cases the wild type phenotype.ConclusionsIn-frame deletion mutants of genes in the anaerobic β-myrcene degradation revealed novel insights in the in vivo function. The deletion of a high-affinity geraniol dehydrogenase hampered, but did not preclude growth on monoterpenes. A second geraniol dehydrogenase activity was present that contributes to the β-myrcene degradation pathway. Growth on cyclic monoterpenes independent of the initial enzyme LDI suggests the presence of a second enzyme system activating unsaturated hydrocarbons.