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Kungl Tekniska Högskolan, KTH, svarar för en tredjedel av Sveriges kapacitet av teknisk forskning och ingenjörsutbildning på högskolenivå. Utbildningen och forskningen är av hög internationell klass och omfattar förutom alla teknikgrenar även naturvetenskap, arkitektur, industriell ekonomi, samhällsplanering, arbetsvetenskap och miljöteknik. KTHs verksamhet är förlagd till flera campus och är förgrenad ut i ett antal olika forsknings- och kunskapscentra.
Biotransformations of Turpentine Constituents: Oxygenation and
Esterification
Marica Lindmark-Henriksson
Doctoral Thesis
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To my dear parents
To Emelie and Anton
with love
Lindmark-Henriksson Marica Biotransformations of Turpentine Constituents:
Oxygenation and Esterification
Chemistry, Department of Natural and Environmental Sciences Mid Sweden University, SE-851 70 Sundsvall, SWEDEN
Organic Chemistry, Department of Chemistry
Royal Institute of Technology (KTH), SE-100 44, Stockholm, SWEDEN
© Marica Lindmark-Henriksson
ISBN: 91-7283-476-5 ISRN: KTH/IOK/FR—03/76—SE
ISSN: 1100-7974 TRITA-IOK
Tryck: Ekonomi Print AB, 2003
v
Lindmark-Henriksson Marica
Biotransformations of Turpentine Constituents: Oxygenation and Esterification
Doctoral Thesis, 2003
Abstract
This thesis describes methods to obtain value–added compounds from TMP-turpentine obtained from the spruce, Picea abies. The methodology focuses on biotransformations using two approaches: an oxygenation approach (i.e. oxygenation of terpene hydrocarbons by cell cultures) and an esterification approach (i.e. lipase-catalysed transesterification of vinyl acetate with terpene alcohols, and a further fractionation of the TMP-turpentine).
The main constituents of the turpentine, α-pinene, β-pinene and limonene, were subjected to a P. abies suspension culture. Allylic oxidation formed the major products for α-pinene and β-pinene, which were further oxidised to their respective aldehyde or ketone. One of the minor products from α-pinene, cis-verbenol, was not only transformed into verbenone but also isomerised to trans-verbenol. Limonene gave limonene-(1,2)-epoxide as the major product.
Fractionation of monoterpenes is accomplished through physical separation methods, chromatography and distillation, and lipase-catalysed transesterification of vinyl acetate with terpene alcohols. The esters of myrtenol and trans-pinocarveol were separated from the more slowly reacting alcohols such as borneol and carveol by use of a combination of the Mucor miehei lipase and Candida antarctica lipase A as catalysts. Furthermore, the non-reacting tertiary terpene alcohols were separated from the reacting alcohols in a single step by Candida antarctica lipase A.
Lipase-catalysed (Candida antarctica lipase B and Pseudomonas cepacia lipase) transesterification of vinyl acetate with sterically hindered secondary alcohols unexpectedly yielded hemiacetals or hemiacetal esters. The reaction conditions required to obtain these side products have been studied.
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Keywords: Picea abies, Pinaceae, Essential oils composition; Terpene alcohol; Hemiacetal; Hemiacetal ester, TMP-turpentine; Monoterpene; α-Pinene; β-Pinene; Limonene; Verbenol; Pinocarveol; Borneol; Myrtenol; Suspension cell culture; Biotransformation; Lipase-catalysed; Οxidation; Allylic oxidation; Transesterification; Autoxidation; Separation.
vii
This thesis is mainly based on the following papers, manuscripts and appendices. In the thesis, these are referred to as I-V and Appendix 1 or 2.
I. Transformation of α-Pinene Using Picea abies Suspension Culture. Henriksson-Lindmark M., Isaksson D., Sjödin K., Högberg H.E., Vanek T., Valterová I., Journal of Natural Products 2003, 66, 337-343.
II. Transformation of Terpenes Using a Picea abies Suspension Culture. Henriksson-Lindmark M., Isaksson D., Sjödin K., Högberg H.E., Vanek T., Valterová I., submitted to Phytochemistry.
III. Formation of Hemiacetal Esters in Lipase-Catalysed Reactions of Vinyl Esters with Hindered Secondary Alcohols. Högberg H-E., Lindmark M., Isaksson D., Sjödin K., Franssen M.C.R., Jongejan H., Wijnberg J. B.P.A., de Groot A., Tetrahedron Letters 2000, 41, 3193-3196.
IV. Hemiacetals and their esters as side-products in lipase-catalysed transesterifications of vinyl esters with sterically hindered alcohols. Isaksson D., Henriksson-Lindmark M., Manoranjan, T., Sjödin K., Högberg H-E., Manuscript.
V. Lipase-Catalysed Acylation as a Method of Separating Monoterpene Alcohols in Picea abies Turpentine. Henriksson-Lindmark M., Isaksson D., Sjödin K., Högberg H-E., Manuscript.
Reprints of papers, I and III were made with kind permission from the publishers: American Chemical Society and Elsevier. Appendices:
1. Chemical analyses of turpentine from the thermomechanical pulping (TMP) process. Henriksson-Lindmark M.
2. Biotransformations of monoterpenes. Henriksson-Lindmark M.
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Abbreviations and equations
Abbreviations Ac acetyl Asp aspartic acid ATP adenosine triphosphate c conversion CAL Candidia antarctica lipase CoA Coenzyme A CRL Candida rugosa lipase DMAPP dimethylallyl diphosphate E enantiomeric ratio (equation 1) ee enantiomeric excess (equation 2) ent enantiomer FAD flavin adenine dinucleotide FPP farnesyl diphosphate GPP geranyl diphosphate His histidine HP Hewlett Packard IPP isopentenyl diphosphate LPP linalyl diphosphate MML Mucor miehei lipase MPLC medium pressure liquid chromatography NAA naphthalene acetic acid NADP nicotineamide adenine dinucleotide phosphate PCL Pseudomonas cepacia lipase SCA Svenska Cellulosa Aktiebolaget Ser serine TBME tert-butyl methyl ether TLL Thermomyces lanuginose lipase TMP thermomechanical pulping
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Equations The enantiomeric ratio (E) is defined as the initial reaction rate ratio between the two enantiomers in a racemic mixture. E can for example be defined by the conversion (c) of a reaction and the enantiomeric excess of the substrate (ees) as:
The percentage of enantiomeric purity, or enantiomeric excess (ee), of a chiral compound consisting of the enantiomeric R and S-forms, is for [S] > [R] defined as:
The enantiomeric excess (ee) could either be calculated for the substrate (ees) or for the product (eep).
(1)Equation )]1()1ln[()]1()1ln[(
Es
s
eeceec
−⋅−+⋅−
=
[ ] [ ][ ] [ ] (2)Equation 100 % ⋅
+−
=RSRS
ee
x
xi
Table of Contents
Abstract .................................................................................................................v List of papers, manuscripts and appendices ........................................................vii Abbreviations and equations ..............................................................................viii 1. Introduction.....................................................................................................1
1.1. Essential oils ..............................................................................................1 1.2. Commercial interests in natural products ...................................................1
2. Turpentine and terpenes .................................................................................2 2.1. Turpentine ..................................................................................................2 2.2. Terpenes .....................................................................................................3 2.3. Biosynthesis of monoterpenes....................................................................6 2.4. Constituents of the TMP-turpentine .........................................................11 2.4.1. Monoterpenes ........................................................................................12 2.4.2. Sesquiterpenes, diterpenes.....................................................................16 2.4.3. Non-terpenoid constituents ....................................................................17
3. Transformation of terpenes by a Picea abies suspension culture ..............18 3.1. Introduction..............................................................................................18 3.2. Biotransformation products......................................................................18 3.2.1. Biotransformation products of α-pinene ...............................................19 3.2.2. Biotransformation products of β-pinene................................................22 3.2.3. Biotransformation products of limonene ...............................................25 3.3. Monoterpenes as substrates for the P. abies culture .................................27 3.4. Biotransformation of monoterpene hydrocarbons....................................31 3.4.1. Autoxidation ..........................................................................................32
4. Biotransformation by a horseradish culture and chloroperoxidase .........36 4.1. Introduction..............................................................................................36 4.2. Horseradish hairy root culture..................................................................38 4.3. Chloroperoxidase .....................................................................................39
5. Transformation of terpenes by lipases.........................................................40 5.1. Introduction..............................................................................................40 5.2. Lipase-catalysed transesterification .........................................................43 5.2.1. Side-products .........................................................................................46
6. Separation of the terpenes from the TMP-turpentine................................52 6.1. Introduction..............................................................................................52 6.2. Chromatography.......................................................................................52 6.3. Distillation................................................................................................52 6.4. Lipase-catalysed transesterification as a separation method ....................53
7. Conclusions and future work .......................................................................55 Acknowledgements............................................................................................56 References ..........................................................................................................58
xii
1
1. Introduction
1.1. Essential oils
The utilisation of essential oils, as for example offerings or perfumes, did very early become a tradition of mankind. As early as 3200 BC the Egyptians had developed technologies for producing cedar wood oils (Ohloff, 1994).
Essential oils originate from plants, fruits and vegetables and hence are products from a natural resource. The essential oils can either be tapped directly from the source as for cedar or pine essential oil, or obtained by extraction with a solvent. Essential oils can also be obtained by pressing, as exemplified by orange peel essential oil.
The main constituents of several essential oils are terpenes (see below, 2.2), the principal subject of this thesis.
1.2. Commercial interests in natural products
The classification ‘Natural’ has an economical impact on commercial products because of the interest of the consumer in natural flavours and fragrances. This classification should not be confused with the terminology used by chemists for “Natural Product Chemistry”, which is defined as the formation, structure and properties of secondary metabolites (Torssell, 1997).
The definition of ‘Natural’ is stated by the Code of Federal Regulations (1990) in USA and by the previous council of the European communities. The USA regulations define a ‘Natural’ flavour as the “essential oil, oleoresin, essence
or extractive, distillate of any product of roasting, which containes the flavouring
constituents derived from a bark, root, leaf or similar plant material” (Cheetham, 1997). The European guidelines define ‘Natural’ aroma compounds as “isolated
by physical, enzymatic, or microbiological processes or traditional food preparation
processes” (Berger, 1995). If transformation of a ‘Natural’ raw material is needed, the reaction must thus be carried out using enzymes or microorganisms in order to retain its ‘Natural‘ classification. Isolation of the product from a ‘Natural’ source is similarly acceptable. If the product occurs in nature but is made using a non-natural process it is classified as ‘Nature-identical’ (Cheetham, 1997).
Because ‘Natural’ products are commercially more valuable than those of other origins, the flavour and fragrance industry is interested in producing them for economic profit. However, not all ‘Natural’ products have the desired
2
properties for commercial exploitation. For example, due to their generally uncharacteristic and undesirable odours, ‘Natural’ terpene hydrocarbons are of low interest for use in, for example, perfumes. Hence, these hydrocarbons are often physically removed from an essential oil to obtain a better quality. However, the terpene hydrocarbons are easily available at low cost and therefore they are ideal starting material for ‘Natural’ or ‘Nature-identical’ products (Ohloff, 1995). In contrast, the oxygenated terpenes have a more distinct and pleasant odour and hence, they are more attractive for the flavour and fragrance industry.
2. Turpentine and terpenes
2.1. Turpentine
In the thermomechanical pulping (TMP)-process (Tienvieri et al. 1999) pulp is produced mainly for manufacturing of wood-containing papers, such as newsprint and magazine papers. The conifer Norway spruce, Picea abies, is the main raw material for the TMP-process (see Figure 1 in Appendix 1). Hence, the SCA Ortviken paper mill in Sundsvall, Sweden, uses fresh non-barked spruce wood as the main raw material. Often, a small amount of saw chips from spruce is mixed with the wood.
The logs are barked and cut into chips. The chips are presteamed before they are introduced into the pressurised preheaters by plug screw feeders that reduce the water content. In the refining process the chips are heated to 140 °C at 4 bar and disintegrated to pulp. Then water is added until a proper pulp-concentration is reached. Thus, this process involves only mechanical treatment of the raw material. The production capacity of TMP-pulp at the Ortviken plant is about 780 000 tons / year (2002) and the annual production of thermomechanical pulp in Sweden is about 2 700 000 tons.
Turpentine is a mixture of volatile terpenoids originating from, for example, conifer wood and is always obtained when softwood is used as the raw material for the pulping process. When the heated wood chips pass through the refiner, the volatile extractives are distilled with the steam evolved. After cooling the steam from the refiner, the Ortviken plant yields a condensate (TMP-turpentine), which is collected and taken to a turpentine decanter. Because turpentine is
3
lighter than and poorly soluble in water, the turpentine will float on top of the water layer in the decanter. About 0.2 kg TMP-turpentine per ton pulp can be collected. Hence, it would be possible to obtain 540 tons of TMP-turpentine in Sweden every year. The TMP-turpentine is of a high quality compared to turpentine from other pulping processes such as the kraft pulp-turpentine, which due to the nature of the latter process contains odorous sulphur compounds.
Today, turpentine is used mainly as solvents and diluents for oil-based paints, varnishes, and pharmaceuticals as well as perfume additives. In the pulping industry, however, the turpentine produced is often used as fuel. Provided that the value of TMP-turpentine can be increased over the fuel price, the industry will be encouraged to exploit it. The goal of the work presented in this thesis, is to make it possible to produce value-added compounds or fractions from TMP-turpentine by using methods that can be classified as within the realm of ‘Natural’. I have chosen to work with biotransformations.
2.2. Terpenes
The common names for isoprenoids are terpenes (hydrocarbons) and terpenoids (oxygenated terpenes). The isoprenoids constitute the largest and most widely distributed class of secondary metabolites, i.e. compounds produced by plants and microbes, which are not essential to the growth of the organism. Terpenes are used for several tasks in nature, for example as pheromones and for chemical defence against microbes and insects (Torssell, 1997).
As early as 1887, von Wallach (1887) suggested that isoprene (2-methylbutadiene) is the building block of terpenes and he earned the Nobel Price in chemistry in 1910 for this concept. The isoprene units are ‘linked in a special way according to the isoprene rule (von Wallach, 1887, Ruzicka, 1953). Hence, the tail of one unit is linked to the head of the next one (Figure 1).
Figure 1. The isoprene rule: Head to tail coupling of two isoprene units giving limonene.
head
or
Limonene
head
tail tail
4
The terpenes are classified based on the number of isoprene-units linked (Table 1). More than 6 000 compounds with mono-, sesqui-, and diterpene structures have been detected and their structures determined (Connolly et al. 1991). The vast majority of these are hydrocarbons or oxygen containing terpenes such as alcohols. There are compounds with the isoprenoid skeleton, either lacking one, or with one extra carbon atom, named nor-terpenes and homo-terpenes respectively.
Table 1. Classification of terpenes based on Sjöström (1981).
Prefix Number of carbon
atoms Number of isoprene
(C5H8) units Hemi 5 1 Mono 10 2 Sesqui 15 3 Di 20 (e.g. resin acids) 4 Sester 25 5 Tri 30 (e.g. steroids) 6 Tetra 40 (e.g. carotenoids) 8 Poly >40 (e.g. rubber) >8
The monoterpenes are C10-terpenes. The monoterpenes can be further subdivided into acyclic, mono-, bi-, as well as various polycyclic compounds. These sub- classes can be even further divided into groups according to their carbon skeleton. For example, the menthanes, belong to the monocyclic monoterpenes, whereas the pinanes and fenchanes belong to the bicyclic monoterpenes (see 2.4.1, Figure 7).
5
Several terpenes are chiral. The two enantiomers of a chiral compound often show different biological activities, including odour (Ohloff 1990, Berger 1995). The fragrance of the menthane group member (4R)-(+)-limonene (citrus fruit) differs from the turpentine like fragrance of (4S)-(−)-limonene (Figure 2).
Figure 2. The two enantiomers of limonene have different fragrances.
(4R)-(+)-Limonene
citrus
(4S)-(-)-Limoneneturpentine
1
2
34
6
2.3. Biosynthesis of monoterpenes
The biosynthesis of terpenoids has been the subject of much research. The biosynthesis of monoterpenes, such as the rearrangement of GPP to linalyl diphosphate, cyclisation and dehydration of rearrangement products has been studied in, for example conifers (Croteau 1987, Bernard-Dagan 1988, Savage et al. 1995, Phillips and Croteau 1999, Sjödin et al., 2000). Enzymes catalysing the biosynthesis of monoterpenes have also been studied (Savage et al. 1994, Savage et al. 1995, Bohlmann and Croteau, 1999, Phillips et al. 1999, Fäldt et al., 2003).
It is still unclear where in the plant cell the biosynthesis of terpenoids takes place. One theory is that the monoterpenoids, diterpenoids and tetraterpenoids are synthesised in the plastids (chloro- or leucoplasts) of the cell, and that the sesquiterpenoids are synthesised in the cytosol of the cell (Rohmer, 1999, Rohmer et al. 2001). The diphosphates (i.e. IPP, GPP and FPP) are exchanged between the two pathways in the active transport between the cytoplasm and the plastid in the plant cell (Rohmer, 1999). The biosynthesis of monoterpenes from primary metabolites can schematically be divided into four steps: Step 1. Synthesis of the isoprene unit, isopentenyl diphosphate (IPP), which is the fundamental precursor of all isoprenoids (Figure 3). Two major biosynthetic pathways leading to IPP exist, i.e. the mevalonate pathway (Figure 3a, Dewick, 1999, McCaskill and Croteau, 1997) and the mevalonate independent pathway, ”the Rohmer pathway” (Figure 3b, Rohmer, 1999, Rohmer et al., 2001). Both pathways lead to IPP and enzymes catalyse the individual steps. Many of these enzymes have been isolated and are well characterised (Bach, 1995, McGarvey, 1995, Rohmer, 2001, Hoeffler et al., 2002). For instance, the isomerisation of IPP into DMAPP is catalysed by IPP isomerase (Figure 3).
7
Figure 3. The first step in the biosynthesis of terpenoids is shown. Formation of isopentenyl
diphosphate by: a. the mevalonate pathway (upper scheme). b. the mavalonate independent pathway
i.e. the “Rohmer pathway” (lower scheme). The diphosphate group is abbreviated as –OPP.
S-CoA
O
HO2COH
OH
OPP OPP
Mevalonic acid
Isopentenyldiphosphate
(IPP)
O
O
OPyruvate
+ O OP
Glyceraldehyde3-phosphate
OP
OH
OH
HO
O
HOOP
OH
OH
3 ATP
3 steps
3 steps
Under investigation
Thiamine-PP
EnzymeNADPH
Enzyme2 NADPH
Enzyme
a. Mevalonate pathway
b. Rohmer pathway
Acetyl-CoA
Dimethyl allyldiphosphate
(DMAPP)
IPPisomerase
Deoxyxylulose-5-phosphate
2-Methyl-erythritol-4-phosphate
8
Step 2. Formation of the allylic prenyl diphosphate, geranyl diphosphate (GPP) (Figure 4). GPP is the precursor of all monoterpenes.
Figure 4. The second step in the biosynthesis of monoterpenes. PP= diphosphate.
Step 3. The elaboration of the geranyl diphosphate, i.e. GPP into monoterpenes (Figure 5). These reactions are catalysed by specific monoterpene synthases (Schwab et al., 2002). Experimental evidence has shown that at least two enzymatic routes leading to each of the enantiomers of the monoterpenes exist (Wise and Croteau, 1999).
The formation of the pinanes begins with an isomerisation (Figure 5, step 1-2) and cyclisation (Figure 5, step 3) of GPP to the α-terpinyl cation via linalyl diphosphate (LPP) (Cane, 1999, Wise and Croteau, 1999). Although many efforts to observe LPP have been made, this postulated intermediate has not yet been found (Schwab et al., 2000).
OPP
GPP
Dimethylallyl diphosphate
(DMAPP)
OPP
Isopentenyl diphosphate
(IPP)
IPPisomerase OPP
OPP
Geranyl diphosphate (GPP)
=
OPP
9
Figure 5. The third step in the biosynthesis of the monoterpene class, pinanes. Formation of α-pinene
and β-pinene from the monoterpene precursor geranyl diphosphate (GPP) (Cane, 1999, Wise and
Croteau, 1999).
An intramolecular Markovnikov addition to the cyclohexenyl double bond of the α-terpinyl cation forms the pinyl cation (Figure 5, step 4). Further, the pinyl cation is deprotonated, either from the adjacent methylene (step 5a) or methyl group (step 5b) to form α-pinene or β-pinene respectively. Both enantiomers of α-pinene occur naturally, whereas β-pinene occurs almost always as the nearly enantiomerically pure (–)-(1S)-enantiomer.
terpinyl-4-cation
HHbH
HHa
α-pinene
β-pinene
cis-LPP
pinyl cation
Linalyl diphosphateLPP
GPP
OPP
OPP
5a
5b
OPP OPP1 2
4
3
10
Step 4. Secondary chemical modifications of the parent isoprenoid product. These secondary enzymatic reactions include hydroxylation by the monooxygenases, cytochrome P-450 (see below 3.3.1), redox transformations and isomerisations (McCascill and Croteau, 1997). Oxygenated pinane monoterpenes, such as verbenol and pinocarveol are formed from α- or β-pinene respectively (Figure 6, Wise and Croteau, 1999).
Figure 6. The transformation of α-pinene and β-pinene to verbenol and pinocarveol respectively is
shown as an example of the last step in the biosynthesis of oxygenated monoterpenes. This step
includes the secondary enzymatic reactions of the parent monoterpene to the oxygenated one.
Pinocarveol
VerbenolOH
α-Pinene
β-Pinene
OH
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2.4. Constituents of the TMP-turpentine
Softwood (e.g. spruce) oleoresin contains mono-, sesqui- and diterpenes while hardwood (e.g. birch) oleoresin mainly contains higher terpenes and their derivatives, such as sterols and other polyprenols. In spruce wood the oleoresin is deposited in resin ducts (canals), which can be found in all parts of the tree. Spruce oleoresin contains 50% resin acids and 20-30% monoterpenes (Sjöström, 1981). The remainder consists of other terpenoids and fatty acid esters.
Turpentine from the TMP-process at Ortviken was fractionated by distillation and medium pressure liquid chromatography (MPLC) and analysed by GC-MS (Appendix 1). The major constituents were identified using reference substances and retention indices (Davies, 1990, Appendix 1). The TMP-turpentine consisted of monoterpenes (74%, V/V), sesquiterpenes (13%, V/V) and diterpenes (1%, V/V). Ninety-two percent (V/V) of these terpenes were non polar and eight percent (V/V) were oxygenated, polar terpenes (Appendix 1).
Although 0.18% of the dry weight of wood from P. abies consists of monoterpenes and 0.04% consists of sesquiterpenes and diterpene hydrocarbons (Assarsson and Åkerlund, 1966), only 0.014% turpentine is obtained from one ton of dry wood at the Ortviken TMP-paper mill. In order to investigate the possibility to increase the terpene yield from the TMP-process, wood chips of P. abies were steamed (Appendix 1). This resulted in an increased yield (0.12% of the dry weight) and a similar composition of terpenes compared to that achieved in the TMP-process of Ortviken mill (Appendix 1). This result demonstrated that it would be possible to produce a sixteen-fold increase in the turpentine yield in the plant.
Leakage of highly volatile compounds at different points in the process or incomplete removal during the steam distillation of the raw material could explain the loss of terpenes in the process used today. In addition, the loss of turpentine during wood storage is dramatic if storage time exceeds four weeks (Gullichsen and Lindeberg, 1999). Thus, processing of fresh wood will result in higher turpentine yields.
Preliminary results showed that the content of chemicals, such as terpene derivatives in the press water from the plug screw feeders in the process at Ortviken was too low to be commercially exploitable, hence no further work was undertaken to utilise it. (Lindmark et al. 1998).
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2.4.1. Monoterpenes
The composition of monoterpene hydrocarbons in the xylem/phloem oleoresin of P. abies (Kimland and Norin, 1972, Persson et al., 1996) and in the oil obtained after steam distillation of spruce wood (Groth, 1958) has been investigated. This oil contains mainly (±)-α-pinene and (–)-β-pinene and minor quantities of other monoterpene hydrocarbons. This is in agreement with the results of our analysis of the composition of monoterpenes in the TMP-turpentine (Table 2). Thus, we have detected and identified about 30 major monoterpenes from the TMP-turpentine (Figure 7, 8, Appendix 1).
Figure 7. Some of the monoterpene hydrocarbon constituents of the TMP-turpentine. The structures
of the major constituents are framed.
α- and β-pinene are important intermediates in manufacturing of synthetic aroma compounds and are also used as flavouring and fragrance ingredients. Limonene is also an important monoterpene. It shows for example insecticidal properties. It is also used extensively in the perfume and flavour industries and in manufacturing of polymers and adhesives. It is also an important chiral starting material for organic synthesis (Connolly, 1991).
α-Pinene β-Pinene 3-Carene Tricyclene
Sabinene
α-FencheneCamphene
α-Terpinene Limonene p-CymeneTerpinolene
Myrcene
β-phellandrene
Acyclic: Bicyclic:Pinane skeleton
Camphane skeleton
Fenchaneskeleton
Carane skeleton
Tricyclic:
Bicyclic:Thujaneskeleton
Cylic:Menthaneskeleton
γ-Terpinene
13
Table 2. The relative composition of monoterpene hydrocarbons in the TMP-turpentine compared
with the monoterpene hydrocarbon content in kraft pulp-turpentine*.
TMP-turpentine Kraft pulp-turpentine Monoterpene
(%)* (+)/(–)** (%)* Tricyclene 0.7 0.2 Thujen - - 0.2 α-Pinene 42.0 62/38 54.3 Sabinene 0.7 0.4 α-Fenchene 0.2 0.2 β-Pinene 27.0 3/97 20.4 Camphene 1.7 1.1 3-Carene 5.4 10.0 α-Terpinene 0.2 0.2 Limonene 17.5 19/81 9.4 p-Cymene 0.7 0.4 γ-Terpinene 0.3 0.2 Terpinolene 1.2 1.3 Myrcene 2.2 1.5 β-Phellandrene 0.3 7/93 0.2
*The relative composition of the monoterpene hydrocarbon constituents analysed by GC-MS. The
differences in the response factors were neglected. **The ratios between the enantiomers were
analysed by GC-MS using the peak purity software for control of overlapping peaks.
Kraft pulp-turpentine is often based on wood from pine instead of spruce. The ratio of monoterpene hydrocarbons is lower in kraft pulp-turpentine (47%), from the Östrand paper mill in Sundsvall, than in the TMP-turpentine (60%). Furhtermore, the composition of monoterpenes in the kraft pulp-turpentine is slightly different from that in the TMP-turpentine (Table 2). For example, the relative amount of 3-carene is higher in the kraft pulp-turpentine compared to that in the TMP-turpentine.
The composition of the monoterpene hydrocarbons in the TMP-turpentine remained relatively constant during a sampling period of one year, which was also true for the relative amount of the fraction of terpenes of a higher molecular weight (Appendix 1). Thus, the TMP-mill of Ortviken was found to be a source of monoterpenes of even quality.
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The oleoresin from P. abies also contains minor amounts of volatile monoterpene alcohols, such as terpinene-4-ol, α-terpineol, myrtenol and borneol (Figure 8, Kimland and Norin, 1972). Our analyses show that these monoterpene alcohols are also the most abundant ones in the TMP-turpentine with α-terpineol as the dominating one (Table 3).
Figure 8. Some oxygenated monoterpenes in the TMP-turpentine. The structures of the major ones
are framed.
OH
OH
OH
Myrtenol Borneol
Terpinene-4-ol α-Terpineol
Bornyl acetate
Myrtenyl acetate
Fenchol
Pinocarveol
Linalool
O
VerbenoneO
Isopinocamphone/Pinocamphone
O
Pinocarvone
VerbenolOH
O
Camphor
OH
OH
OH OAc
OAcOH
Bicyclic:Pinane skeleton
Camphaneskeleton
Bicyclic:Fenchaneskeleton
Acyclic: Cyclic:Menthaneskeleton
15
Ketones and esters were also found in the oxygenated monoterpene fraction of the TMP-turpentine (Figure 8, Table 3).
Table 3. The relative amounts of some oxygenated monoterpenes in the TMP-turpentine from
P. abies.
Compound Relative amount
(%)1 Concentration
(mg/ml)
(+)/(–)2 Borneol 0.4 0.8 62/383 Bornyl acetate 0.5 0.7 - Myrtenol 0.2 0.6 3/974 Myrtenyl acetate 1.1 0.02 - trans-Pinocarveol 0.2 0.6 - Terpinene-4-ol 0.8 1.1 - α-Terpineol 2.7 7.1 19/814
1 The relative amount is the peak area of the compound divided by the sum of all peak areas from the
TMP-turpentine (GC-MS). 2 The ratios between the enantiomers were analysed by GC-MS using the
peak purity software (HP) for control of overlapping peaks. 3 The ee of borneol was determined by
integration of a single ion spectrum of m/z 110. 4 The ee of myrtenol and α-terpineol were
determined by integration of the full scan (TIC) spectrum.
α-Terpineol, borneol and their acetates, are of great importance in perfumery. Myrtenol, 1,8-cineol, fenchol, linalool, carvone, and terpinene-4-ol are also of importance as fragrance constituents (Ohloff, 1997). Pinocarvone is known as a sex attractant for the pine beetle and cis-verbenol is a sex attractant component of Ips typographus (Schlyter et al., 1987) and Ips confusus a pest on Pinus ponderosa (Connolly, 1991). trans- and cis-Verbenols have frequently been reported as insect semiochemicals and are therefore of use in the monitoring or mass trapping of bark beetles (Schlyter et. al., 1987, Lindgren and Borden, 1993, Miller, 2000).
16
2.4.2. Sesquiterpenes, diterpenes
Sesquiterpenes have also been found in the oleoresin of Norway spruce (Kimland and Norin 1972). Sesquiterpenes, both as hydrocarbons and oxygenated ones, constitute 13% (V/V) of the TMP-turpentine. Bicyclic sesquiterpenes with the cadinane skeleton are the main constituents of this fraction (Figure 9). Some diterpenes present in the TMP-turpentine have the cyclic cembrane skeleton (Figure 10).
Figure 9. Examples of sesquiterpenes with the cadinane skeleton that are found in the TMP-
turpentine.
Figure 10. The diterpene cembrene.
H
H H
H
H H
H
γ-Muurolene α-Cadinene δ-Cadinene α-Cadinol
OH
Cembrene
17
2.4.3. Non-terpenoid constituents
Our studies show that TMP-turpentine also contains non-terpenoids such as alcohols, aldehydes, and aromatic derivatives, as well as straight chain and branched alkanes (Figure 11). Among the aldehydes, the malodours smelling hexanal and heptanal were present along with decadienal. These aldehydes could be the result of either autoxidation or enzymatic degradation of fatty acids (Schieberie and Grosch, 1981, Back, 2001, Nordermeer et al., 2000).
Figure 11. Non-terpenoid constituents of the TMP-turpentine. 1) Hexanol, 2) hexanal, 3) heptanol, 4)
heptanal, 5) 2,4-decadienal, 6) palmitic acid, 7) 1-allyl-4-methoxybensene, 8) 1-isopropyl-2-
methoxy-4-methylbensene.
The spruce wood oleoresin also contained n-alkanes in homologous series ranging from C11 to C33, with C22 to C27 as main constituents (Assarsson and Åkerlund, 1966). Such compounds were also present in the TMP-turpentine. After distillation of the TMP-turpentine, the residue (12% V/V) was found to contain 17% palmitic acid (Figure 11), a small amount of some other fatty acids and diterperpenes as resin acids and aliphatic branched and unbranched hydrocarbons of high molecular weight. (Appendix 1).
OH
O
OH
OH
H
O
H
O
H
O
O
O
1 2
3 4
5
6
7 8
18
3. Transformation of terpenes by a Picea abies suspension culture
3.1. Introduction
Microbial transformations of terpenes, were carried out by Mayer and Neuberg (1915) as early as 1915. They transformed (+)-citronellal to (+)-citronellol by yeast. Biotransformation constitutes one approach to achieve the desirable classification of the products as ‘Natural’. Terpene flavour and fragrance compounds are synthesised by several microorganisms and higher plants and the conversion of low value monoterpenes to value-added ones has been investigated (Demyttenaere et al, 2001, van der Werf et al., 1997, Berger, 1995, Krasnobajew, 1984). However, these methods have been thwarted by the fact that the transformations lead to several products from a single substrate and in most cases, the yields are low.
The bacteria of the genus Pseudomonas, have the unusual ability to degrade and live on monoterpenes. They transform the monoterpenes before degrading them to carbon dioxide and water (Hungund et al., 1970). Most other bacteria and fungi can only perform the transformation of terpenes, when grown in energy-rich media. This has been called co-metabolism.
3.2. Biotransformation products
Preliminary results showed that oxygenated monoterpenes were formed when we used TMP-turpentine as substrate for a P. abies suspension culture. Thus, α- pinene, β-pinene and limonene - major constituents of P. abies turpentine - were chosen as substrates for further investigations of the oxygenation ability of these cultures. Because of the explorative nature of this investigation, we have focused on the qualitative product pattern observed rather than on the yields of the products. Hence, in our earlier work (I) no corrections for differences in response factors (GC-MS) were performed. For useful applications it is of course necessary to have control over yields and mass balances between products and substrates.
Cultivation of P. abies cultures has been discussed by Mårtensson (2002) and has been reported by for example von Arnold and Hakman (1986). The P. abies suspension culture was grown in liquid nutrient medium according to Gupta and Durzan (1986) with some exceptions (Appendix 1). No optimisation of the
19
composition of nutrient medium and its constituents was performed. As expected for undifferentiated cells (Corbier and Ehret, 1986), no terpenes
were produced by the P. abies suspension culture in the absence of a terpene substrate (I).
3.2.1. Biotransformation products of α-pinene
Biotransformations of α-pinene to verbenols and verbenone by a pure enzyme system of the monooxygenase P-450cam (Table 4, entry 2), by plant cells (Table 4, entry 3-5) and by the use of microorganisms (Table 4, entry 6-13) are reported.
Table 4. Biotransformations of α-pinene described in literature.
Entry Microorganism/ Main products Referenceplant/enzyme Allylic oxidation / and
further oxidation Epoxidation Other reactions1 Picea abies trans -verbenol, α-terpineol, I
verbenone, trans -pinocarveol, myrtenol trans- sobrerol
2 P-450cam cis-verbenol, α-pinene epoxide Bell et al ., 2003verbenone,
myrtenol3 Rosa centifolia trans, cis -verbenol Corbier and Ehret, 19864 Nicotiana tabacum verbenone, α-pinene epoxide verbanone Hirata et al ., 19945 Hyssop officinalis trans -verbenol Karp and Croteau, 19926 Aspergillus sp. trans -verbenol trans -pinocarveol, Agraval et al ., 1999
Penicillium sp. verbenone, sobrerol7 Bacidomycetes trans -verbenol, myrtenol trans -pinocarveol, Busmann and Berger,
verbenone, myrtenal sobrerol, α-terpineol, 1994acarveol, carvone
8 Pholiota squarrosa trans -verbenol, trans -pinocarveol, Busmann and Berger, verbenone, myrtenol p-menth-2-ene-1,8-diol 1994b
9 Aspergillus niger trans -verbenol trans -sobrerol Prema and Bhattacharyya, verbenon 1962
10 Aspergillus niger cis -verbenol sobrerol Rama Devi and verbenone Bhattarachyya, 1978
11 Serratia marcescens trans -verbenol, myrtenol trans -sobrerol Wright et al .,198612 Armillariella mellea trans -verbenol, sobrerol, Draczynska et al ., 1985
verbenone, myrtenol carveol, carvacrol13 Armillariella mellea verbenone trans -pinocarveol, Draczynska et al ., 1985
sobrerol, α-terpineol, carvacrol
14 Armillariella mellea α-terpineol,sobrerol Draczynska-Lusiak and Siewinski, 1989
15 Psuedomonas PL limonene, perillyl alcohol, Shukla et al ., 196816 P. putida α-pinene epoxide Gibbon and Pirth, 197117 Pseudomonas sp. myrtenol borneol, perrilic acid Shukla et al ., 196818 P. fluoroscens myrtenol α-pinene epoxide limonene, perillyl alcohol Best et al ., 1987
trans -limonene-epoxide19 P. maltophilia limonene, borneol, camphor, Narushima et al ., 1982
perillic acid, 2-(4-methyl-3-cyclohexenylidiene)-
propioninc acid20 Psudomonas sp. carveol Rhodes and Winskill, 198521 Candida tropicalis α-terpineol, Chatterjee et al ., 1999
p-menth-2-ene-1,2-diol
20
In our study α-pinene was transformed by the P. abies suspension culture to mainly trans-verbenol and verbenone (Figure 12, I). trans-Verbenol was further transformed to verbenone (Figure 13). trans-Pinocarveol, cis-verbenol, myrtenol and α-terpineol were minor products and sobrerol was present in only some samples (I). Hence, biotransformation of α-pinene by the P. abies suspension culture was characterised mainly by oxidation at the allylic position. Other observed reactions were oxidative cleavage of the cyclobutane ring and oxidation of the terminal carbons in the C6 positions, producing monocyclic terpene alcohols.
Figure 12. Biotransformation products of (1R)-α-pinene with a P. abies suspension culture. The
structures of the major products are framed.
OH
OH
O
OH
OH
HO
OH
(1R)-(+)-α-Pinene
(1R)-cis-Verbenol
(1R)-trans-Verbenol
(1R)-(+)-Verbenone
(1S)-(-)-trans-Pinocarveol
(1S)-(+)-Myrtenol
(4S,6R)-(-)-trans-Sobrerol
(4R)-(+)-α-Terpineol
OH
(4R,6S)-(+)-trans-Sobrerol
OH6
4
OH
16
5
4
3
2
7
21
Figure 13. Biotransformation of (1R)-α-pinene utilising a P. abies suspension culture. The main
product in short time (1-4 days) was trans-verbenol, which was further transformed into verbenone. *Integrated peak area (GC-MS) relative to total integrated area in percent. The differences in response
factors were neglected.
Enantioselectivity has previously been found in biotransformations of α-pinene by plant cell suspension cultures of Nicotania tabacum grown in the medium of Murashige and Skoog (1962) (Hirata et al., 1994). However, our transformation of rac-α-pinene by a P. abies suspension culture showed only a very low preference for (1R)-α-pinene (I).
3.2.1.1. Isomerisation of cis-verbenol cis-Verbenol has been transformed into verbenone using plant cells of Nicotiana tabacum and Cannabis sativa (Hamada, 1988, Takeya et al., 1977) and the formation of trans-verbenol from cis-verbenol has been observed after biotransformation using Solanum aviculare plant cells (Vanek et al., 1989). In the biotransformation of cis-verbenol by the P. abies suspension culture, the compound was isomerised to trans-verbenol (I).
Figure 14. Tentative mechanism of the biotransformation of (1S)-(42H)-cis-verbenol into (1S)-(42H)-
trans-verbenol, which is further transformed to (1S)-verbenone.
This isomerisation was studied by biotransformation of a deuterium labelled (1S)-(42H)-cis-verbenol, which formed trans-verbenol with retention of the 4-
(1S)-(+)-Verbenone
O
(1S)-(42H)-cis-Verbenol
DOH
D
allylic cation
P. abiessuspensionculture
OHD
(1S)-(42H)-trans-Verbenol
rel.
int.
area
(GC
-MS
) *
(1R)-trans-verbenol
(1R)-α-pinene(1R)-cis-verbenol
(1R)-verbenone
days0
20
40
60
80
100
0 5 10 15
Verbenone
trans-Verbenol
α-Pinene
rel.
int.
area
(GC
-MS
) *
(1R)-trans-verbenol
(1R)-α-pinene(1R)-cis-verbenol
(1R)-verbenone(1R)-trans-verbenol
(1R)-α-pinene(1R)-cis-verbenol
(1R)-verbenone
days0
20
40
60
80
100
0 5 10 150
20
40
60
80
100
0 5 10 15
Verbenone
trans-Verbenol
α-Pinene
22
deuterium (I). Only very small amounts of verbenols were formed when verbenone was used as a substrate. Thus, the isomerisation of cis-verbenol to trans-verbenol did not proceed via ketone. Instead an enzyme bound allylic cation was suggested to be involved in the reaction (Figure 14, I).
3.2.2. Biotransformation products of β-pinene
Several pathways and products have been reported for the biotransformation of β-pinene (Table 5, Figure 15).
Table 5. Biotransformations of β-pinene described in literature.
Dhavlikar et al. (1974) did not determine the structure of the compound, however the masspectra
(GC-MS) showed: M+ 150. Hence, it could have been pinocarvone.
The main biotransformation product from β-pinene by the P. abies suspension culture was trans-pinocarveol (Figure 15, 16, II). This is a common biotransformation product of β-pinene (Table 5, entry 1-8). The products myrtenol, α-terpineol, pinocarvone, pinocamphone, and some other minor products from β-pinene were also identified in the product mixture.
Entry Microorganism/ Main products Referenceplant Allylic oxidation / Other reactions
and further oxidation1 Picea abies trans- pinocarveol myrtenol, α-terpineol II2 Armillariella mellea trans -pinocarveol, myrtenol, Draczynska et al .,
pinocarvone, perillic alcohol 19855-hydroxy-pinocarveol hydroxy-α-terpineol
3 Aspergillus niger trans -pinocarveol Devi et al ., 1978NC1M 612
4 Aspergillus niger pinocarveol, myrtenol Bhattacharyya and pinocarvone Ganapathy, 1965
5 Pleurotus flabellatus trans -pinocarveol pinocamphone, eucalyptol, Busmann and Berger, myrtenol, myrtanol, myrtanal, 1994a
perillyl alcohol, 1,4-cineol, carveol, fenchone
6 Sewage bacterium α-terpineol, camphor Dhavlikar et al ., 1974(Seuberts medium) borneol, isoborneol,
7 Sewage bacterium trans -pinocarveol α-terpineol, myrtenol, Dhavlikar et al ., 1974(Czapek-Dox medium) pinocarvone?* myrtenic acid, fenchol
8 Hyssop officinalis trans -pinocarveol Karp and Croteau, 19929 Pseudomonas sp myrtenol, borneol Shukla et al ., 1968
10 Ganoderma applanatum eucalyptol, 1,4-cineol Busmann and Berger, myrtenol, myrtenal, 1994a
11 Bacidomycetes α-terpineol, eucalyptol, Busmann and Berger, perillyl alcohol, 1994a
1,4-cineol, carveol
23
0
20
40
60
80
100
0 5 10 15
rel.
int.
area
(G
C-M
S)*
(1S)-β-pinene
(1R)-pinocarvone
pinocamphone
(1R)-trans-pinocarveol
(4S)-α-terpineol(1R)-myrtenol
days
trans-pinocarveol
myrtenolpinocamphone
0
20
40
60
80
100
0 5 10 15
rel.
int.
area
(G
C-M
S)*
(1S)-β-pinene
(1R)-pinocarvone
pinocamphone
(1R)-trans-pinocarveol
(4S)-α-terpineol(1R)-myrtenol
days0
20
40
60
80
100
0 5 10 15
rel.
int.
area
(G
C-M
S)*
(1S)-β-pinene
(1R)-pinocarvone
pinocamphone
(1R)-trans-pinocarveol
(4S)-α-terpineol(1R)-myrtenol
days
trans-pinocarveol
myrtenolpinocamphone
Figure 15. Tentatively biotransformation pathways of β-pinene using a P. abies suspension culture.
The structure of the major biotransformation product are framed.
Figure 16. Biotransformation of (1S)-β-pinene using a P. abies suspension culture. The major
product was trans-pinocarveol. *Response factor corrected integrated peak area (GC-MS) relative to
the total corrected integrated area in percent.
OH O
OH O
O
β-Pinene
trans-Pinocarveol
Pinocarvon
Myrtenol
Pino-camphone
Myrtenal
12
3
45
O
Reduction
Oxidation
Oxidation
Reduction of double bound
OH
Myrtanol
Reduction of double bound
Ring cleavage
OHα-Terpineol
Oxidation of double bound
Migration of double bound, allylic reararrangement or ring closure leads to several products
Rearrange-ment
β-Pinene-epoxide
Formation of epoxide
24
The biotransformation of primary products from β−pinene was also investigated (Figure 17). Thus, (1R)-(–)-myrtenol, (1R)-(–)-trans-pinocarveol, (1S)-(–)-cis-pinocarvol, and (4S)-α-terpineol were used as substrates (II). Except for α-terpineol, all the substrates tested were transformed within one minute. The amounts of the products were low and the compositions did not change during the 15-days of observation period.
Figure 17. Biotransformation products of pinocarveols and myrtenol by means of a P. abies
suspension culture. The composition of the product mixtures was calculated from integrated areas (GC-
MS). The differences in response factors were neglected.
trans-Pinocarveol formed pinocarvone and myrtenol formed myrtenal and myrtanol. The reduction of the double bond in myrtenol leading to the saturated alcohol, myrtanol, was similar to the reduction observed during the formation of pinocamphone and isopinocamphone in the biotransformation of β-pinene. Biotransformation of cis-pinocarveol gave mainly pinocarvone and pinocamphone. Thus, no similar isomerisation to the one discussed above (3.2.1.1) involving a cis- to trans-verbenol epimerisation (I) was observed for
OH O
+
(1R)-Myrtenal
5%
(1R)-Myrtanol
2%
(1R)-Myrtenol
(93% remaining)
(1S)-Pinocarvone
12%
(1S)-trans-Pinocarveol
(88% remaining)
OH O OH
P.abiessusp. cult.
P.abies
susp. cult.
OH O
(1S)-Pinocarvone
(1S)-cis-Pinocarveol
P.abiessusp. cult.
O
(1S)-Pinocamphone
+
25
cis-pinocarveol. However, it may be possible that cis-pinocarveol isomerised to trans-verbenol, which thereafter rapidly formed pinocarvone.
3.2.3. Biotransformation products of limonene
Several biotransformation products of limonene have been reported in the literature (Figure 18, reviews Demyttenaere, 2001, Mikami, 1988). This interest for limonene as a starting material in biotransformations is likely due to the low cost of and wide availability of limonene in various essential oils such as oils of lemon, orange, caraway, dill and bergamot.
Several pathways for the biotransformation of limonene are reported (Figure 18).
Figure 18. Some biotransformation pathways for limonene found in the recent literature. (i) van der
Werf et al. (2000), (ii) van der Werf et al. (1999), (iii) Vanek et al. (1999), (iv) McCaskill and
Croteau (1997), (v) Onken and Berger (1999), (vi) Trytek and Fiedurek (2002), (vii) Karp et al.
(1990), (viii) Noma et al. (1992), (ix) Tan et al. (1998).
HO
HOLimonene
Limonene-(1,2)-epoxide
Carveol
Perillyl alcoholO
Carvone
OH
Isopiperitenol
O
α-TerpineolOH
Limonene-(8,9)-epoxide
O
Solanum aviculare (iii)Dioscorea deltoidea (iii)
Rhodococcus erythropolis
Pleurotus sapidus (v)Mentha spicata (iv)
61
2
3
4
5
7
8
Penicillium digitatum (ix)
Mentha piperita (vii)
Perilla frutescens (vii)Aspergillus cellulosae (viii)
910
Glucose oxidase and horseradish peroxidase (vi)
6 6
7
3
8
8 9
1 2
DCL14 (ii)(II)
Xanthobacter sp. C20 (i)
(II)
(II)
26
Most of the microorganism and plant cell cultures seem to use more than one biotransformation pathway, which gives rise to a complex mixture of products. However, some exceptions are the biotransformation of rac-limonene by Penicillium digitatum that formed optically pure (4R)-(+)-α-terpineol in a good yield (up to 0.46 g/l) (Kieslich et al., 1986) and the transformation of (+)-limonene by Corenyspora cassiicola, which resulted in (1S, 2S, 4R)-p-menth-8-ene-1,2-diol as the only product in an even better yield (900g / 1300g) (Abraham et al., 1986). An interesting result is that some microorganism and plant cell cultures have been shown to transform one pure enantiomer of limonene into a mixture of (6S)- and (6R)-carveol (Berger, 1995, Vanek et al., 1999).
When (4R)-(+)-limonene was subjected to transformation by the P. abies suspension culture, only a small amount of products was formed (Figure 19). Due to this low transformation rate compared to α- and β-pinene (Figure 20, II), limonene as a substrate was not further investigated.
Figure 19. Relative amounts of the biotransformation products of limonene in a P. abies suspension
culture after 24 hours. The amounts are the relative integrated GC-MS areas of the peaks. The
differences in response factors are neglected.
HO
HOO
(4R)-(+)-Limonene
Limonene-(1,2)-epoxide
Carveol Perillyl alcohol
96%(remaining)
2% 0,6%
O
1,8-Cineole
0,4% trace
+ + +
P.abies
susp.cult.
27
3.3. Monoterpenes as substrates for the P. abies culture
In comparison with β-pinene and limonene, α-pinene proved to be a rapidly reacting substrate for the P. abies suspension culture (Figure 20, II).
Figure 20. The biotransformation of a) α-pinene, b) β-pinene and c) limonene by a P. abies
suspension culture forming their main products, trans-verbenol, trans-pinocarveol and limonene-
(1,2)-epoxide, respectively. *Integrated peak area (GC-MS) relative to the total integrated area in
percent. The differences in response factors are neglected.
The main transformation products of α-pinene, β-pinene and limonene formed by a P. abies suspension culture are shown in Figure 21.
Allylic oxidation seems to be the most favourable pathway, although several other products also occurred. The enantioselectivity of the reactions leading to the major products were low. However, a small increase in the enantioselectivity in the further transformation of the alcohols into their respective ketones or aldehydes was observed.
Enzymes or enzyme systems present in the P. abies suspension culture mediate the formation of the products. No identification of enzymes involved in these reactions was performed.
Enzymes catalysing biotransformations (van der Werf, 1997) and degradation (Trudgill, 1994) of monoterpenes are described in reviews. Cytochrome P-450 dependent enzymes, as described below (3.3.1), catalyses the biosynthesis of monoterpenes (McCaskill and Croteau, 1997).
trans-verbenol
05
1015
202530354045
0 10 20 hours
rel.
int.
are
a (G
C-M
S)*
trans -pinocarveol
05
10152025
30354045
0 10 20 hours
rel.
int.
are
a (G
C-M
S)*
limonene-(1,2)-epoxide
05
10152025
30354045
0 10 20
rel.
int.
are
a (G
C-M
S)*
a) b) c)
trans-verbenol
05
1015
202530354045
0 10 20 hours
rel.
int.
are
a (G
C-M
S)*
trans -pinocarveol
05
10152025
30354045
0 10 20 hours
rel.
int.
are
a (G
C-M
S)*
limonene-(1,2)-epoxide
05
10152025
30354045
0 10 20
rel.
int.
are
a (G
C-M
S)*trans-verbenol
05
1015
202530354045
0 10 20 hours
rel.
int.
are
a (G
C-M
S)*
trans -pinocarveol
05
10152025
30354045
0 10 20 hours
rel.
int.
are
a (G
C-M
S)*
limonene-(1,2)-epoxide
05
10152025
30354045
0 10 20
rel.
int.
are
a (G
C-M
S)*
a) b) c)
28
Figure 21. The main transformation products of α-pinene, β-pinene and limonene in the
biotransformation by a P. abies suspension culture.
Substrate Reaction Major products
Allylic oxidation
trans -Verbenol cis -Verbenol Verbenone Myrtenol
Oxidative cleavage and oxidation
α-Pinene α-Terpineol (-)-trans -Sobrerol (+)-trans -Sobrerol
Other reactionstrans -Pinocarveol
Allylic oxidation
trans -Pinocarveol Pinocarvone Pinocamphone
Oxidative cleavage and oxidation at C7
β-Pinene α-Terpineol
Other reactions
Myrtenol
Allylic oxidation
Carveol Perillyl alcohol
Epoxidation
Limonene Limonene-1,2-epoxide
Oxidation with ring closure
1,8-Cineole
OH OH O
OH
OH OH
OH6
4
OH
HO
OH
OH
OH O O
OH
HO
HO
O
O
29
3.3.1. Monooxygenases Oxido-reductases, which catalyse oxidations and reductions, are divided into subclasses on the basis of the type of substrate and the type of hydrogen or electron donor (acceptor) that is utilised. Monooxygenases (subgroup of oxido-reductases) catalyses the introduction of one atom of dioxygen into the substrate using cofactors furnishing oxidised organic products and a molecule of water (Figure 22). Monooxygenases could also catalyse the formation of epoxides.
Several oxido-reductases require the presence of cofactors in order to carry out their reaction. Cofactors can either be covalently bound to the active site of the enzyme forming a prosthetic group or acting as a co-substrate, interacting with the enzyme. The most commonly utilised cofactor for biosynthetic processes with oxido-reductases tends to be the nicotineamide adenine dinucleotides, NADH or NADPH (Bugg, 1999).
Figure 22. Reactions catalysed by monooxygenases.
The monooxygenases, catalysing hydroxylation of monoterpenes usually consist of three components, which take part in transporting electrons and catalysing the reaction (Figure 23-24, van der Werf, 1995):
1. A FAD flavoprotein reductase component, catalysing the electron release from NAD(P)H;
2. An iron-sulphur protein acting as an electron carrier between the flavoprotein and the cytochrome P-450 component;
3. A cytochrome P-450 containing component, which catalyses the oxidation of the substrate.
A reaction of monooxygenases catalysing hydroxylation:
H2OO2RH NAD(P)H H+ ROH NAD(P)+.
A reaction of monooxygenases catalysing epoxide formation:
H2OO2 NAD(P)H H+ NAD(P)+.HC CH CH CH
O
30
Figure 23. An example of a monooxygenase enzyme catalysing the hydroxylation of hydrocarbons.
Figure 24. The process of electron transfer from NADPH to cytochrome P-450 based on Okita and
Masters (1992).
Cytochromes are protein cofactors that are essential as transporters of redox equivalents to the catalytically active sites of many oxidoreductases. Cytochrome P-450 is the terminal electron carrier of numerous monooxygenases of mammalian liver microsomes, adrenal mitochondria and bacterial systems. The cytochrome P-450 isolated from camphor-oxidising microorganisms has been studied most. Cytochrome P-450 dependent systems perform enzymatic hydroxylations in the biosynthesis of oxygenated isoprenoids (McCaskill and Croteau, 1997). One example is the allylic hydroxylation of (–)-β-pinene in hyssop (Hyssopus officinalis), forming pinocamphone and isopinocamphone via trans-pinocarveol (Karp and Croteau, 1992).
2 NADPH FAD2+ Fe2+ Fe3+ ROH + H2O
2 NADP+ FADH2 Fe3+ Fe2+ RH + O2
NADPH NADP+ Fe-S
Cyt P-450FADH2FAD2+2e-
2e-
2e-
1
23
RH + O2 ROH + H2O
FADreductase
component
31
3.4. Biotransformation of monoterpene hydrocarbons
There are some important facts to have in mind when working with biotransformation of monoterpenes:
• Monoterpenes are toxic substrates, even to whole cells. • Monoterpene hydrocarbons are poorly soluble in water. • The volatility of monoterpenes causes problems during
biotransformations, such as losses of both substrates and products. • Biotransformations of terpenes often proceed along several metabolic
pathways leading to a mixture of products. • Most monoterpene biotransformations result in low yields. • Monoterpenes are relatively unstable compounds. They can, for
example, undergo spontaneous autoxidation.
The autoxidation of monoterpenes is discussed below (3.4.1). Terpenoids in general have antimicrobial activities. This results in inhibition
of culture growth or lysis of cells if too great an amount of a substrate is used. Inhibition of growth of cell cultures normally starts at terpene concentrations higher than 0.05% V/V (van der Werf et al., 1997). According to Brown et al. (1987) α-pinene, for example, is fatal to the growth of the suspension culture of the plant Pelargonium fragrans, at a concentration of 0.5 g/l. The products resulting from the biotransformation can also be toxic. For example a bacteria of the Pseudomonas sp., obtained from soil, increases the cell lysis when exposed to borneol, which is a transformation product of α-pinene (Shukla et al., 1968). α-Pinene oxide, another biotransformation product of α-pinene, inhibits the growth of Pseudomonas putida at a concentration above 0.2% (v/v) (van Keulen et al., 1998).
In order to avoid the problems associated with antimicrobial substrates, repeated or continuous and slow additions of the substrate or an organic-liquid two-phase system can be useful when performing biotransformations (van der Werf et al., 1997). In order to avoid the toxic effects of monoterpenes, a low substrate concentration (0.16 - 0.32 g/l) was used in the biotransformations of monoterpenes by the suspension culture from P. abies (I-II).
32
The solubility of the monoterpene hydrocarbons in aqueous solutions is low. The contact area between the cell culture and the substrate could be increased either by using a two-phase system or by purging the substrate into the bottom of the suspension culture as a saturated vapour in an inert gas. Preliminary results of using heptane and the nutrition medium in a two-phase system in the biotransformation of α-pinene did not give any increased yields (unpublished results) and needs to be further optimised.
Due to the volatility of the monoterpenes the work-up procedure must be fast and efficient. The filtration procedure used for the reaction mixture from the P. abies biotransformation is one operation in which the monoterpenes can easily evaporate if the filtration does not proceed quickly enough.
However, the fact that many biotransformations result in low yields is mainly due to the mixture of products obtained from a single substrate. Choosing for example optimised growth conditions for the culture can solve this problem. Among others, Dhavlikar et al. (1974) have shown that the nutrition medium is an important factor in biotransformations of monoterpenes. Thus, the products can change depending on the medium used. When Seuberts medium is used in the biotransformation of β-pinene by a culture obtained from Pseudomonas sp., borneol is found as the main product. When, in contrast, Czapek-Dox medium is used as the nutrition medium, the main product is trans-pinocarveol (Dhavlikar et al., 1974).
It is also known, that an optimised amount of the hormone, naphthalene acetic acid (NAA), in the nutrient medium (Murishige and Skoog, 1962, supplemented with hormones) of callus cultures of Melissa officinalis sp., leads to the formation of an essential oil, that contains mainly oxygenated monoterpenes instead of sesquiterpene hydrocarbons (Binder and Abou-Mandour 2000).
So far, we have not performed any work on the optimisation of the conditions for the P. abies cell growth.
3.4.1. Autoxidation
The term autoxidation is used for oxidation of an organic compound with molecular oxygen, usually via a free radical chain process. An initiator begins the free-radical chain process. The initiator could for instance be a metal, which will form a peroxiradical bond to the metal (Figure 25).
33
Figure 25. Molecular oxygen forms a peroxiradical with a metal. This peroxiradical will react with
the olefin.
Autoxidation of olefins is mechanistically described as epoxidations and allylic oxidations (Sheldon and Kochi, 1981, Drago, 1991). The epoxidations start with an attack on the double bond, while allylic oxidations begin with abstraction of allylic hydrogen. These reactions involve free-radical intermediates (Figure 26). The allylic oxidation forms a hydroperoxide (Figure 26), which will, for example, decompose to an alcohol and oxygen.
Figure 26. Autoxidations of olefins could proceed either by attack on the double bond or by
abstraction of the allylic hydrogen via free-radical intermediates.
Metal(Ox) + O2 Metal(red)O-O.
H H H.+ Metal(red)OOH
Abstraction of allylic hydrogen:
Initiation
Metal(red)O2.
.
Attack on the double bond:
O+ Metal(red)O
.
Metal(red)O2.
H. O HO.
OMetal(Red)O
O HO.
H H+
O HHO
Hydroperoxide
O2 +
34
3.4.1.1. Autoxidation products of α-pinene, β-pinene and limonene Generally the autoxidation products of monoterpenes are the same as those formed by biotransformations (Moore et al., 1956, Bhattarchayya et al., 1960, Rothenberg et al., 1998). This makes it difficult to discriminate between autoxidation products and biotransformation products. For instance, the biotransformation products from α-pinene by the P. abies suspension culture - verbenone, verbenol and sobrerol - have also been found among the autoxidation products of α-pinene (Table 6, II). A small amount of autoxidation products was formed when α-pinene or β-pinene was added to our nutrient medium in the absence of suspension culture (Table 6, I, II). However, this amount was much smaller than the amounts of products formed from the same substrate by the P. abies suspension culture. The suspension culture and the nutrient medium treatments produced different compositions of the products, with a higher extent of the respective ketone in the nutrient medium (Table 6). Neither trans-pinocarveol - the main product by the P. abies suspension culture - nor pinocamphone was produced from β-pinene by autoxidation in the nutrient medium. The amount of the produced pinocarvone was also lower than the amount produced by the suspension culture.
We did not detect any autoxidation products from limonene at the conditions used. However, our biotransformation products limonene-(1,2)-epoxide, carveol and carvone have been detected among the autoxidation products of limonene (Anandraman and Reineccius, 1986).
35
Table 6. The major oxidation products of (1R)-α-pinene (24 hours) or (1S)-β-pinene (8 days) after
treatment with either a nutrient medium (autoxidation) or as biotransformation products after
treatment with a P. abies suspension culture in a nutrient medium.
Substrate Products
1 3 4 5 Autoxidation
0.024 mg 0.008 mg 0.003 mg
Biotransformation 0.009 mg 0.064 mg 0.021 mg
2 6 7 8 9 10 Autoxidation
0.018 mg 0.006 mg 0.006 mg - -
Biotransformation
0.066 mg 0.039 mg 0.008 mg 0.320 mg 0.303 mg
1) α-Pinene; 2) β-pinene; 3) verbenone; 4) trans-verbenol; 5) cis- verbenol; 6) pinocarvone; 7) α-
terpineol; 8) 1,8-cineole; 9) trans-pinocarveol; 10) pinocamphone.
OH
OH
OO
O OH
O
OH
36
4. Biotransformation by a horseradish culture and chloroperoxidase
4.1. Introduction
Peroxidases (EC 1.11.X.X) are a sub-group of oxido-reductases catalysing the oxidation of substrates using a peroxide, most often hydrogen peroxide as oxidant. Peroxidases catalyse the general reaction showed in Figure 27. To carry out their reactions peroxidases are either dependent or independent on a cofactor.
Figure 27. A peroxidase-catalysed reaction of a substrate (R2H).
Peroxidases can however also act as monooxygenases and hence molecular oxygen is consumed as the oxygen donor. The oxygenation of monoterpenes by peroxidases may be useful to yield value-added compounds. No biotransformation of α-pinene or β-pinene by a pure enzyme preparation of peroxidase appears to be described in the literature. However, limonene is transformed into carvone as the major product by means of a mixture of a peroxidase, horseradish peroxidase and an oxygenase, glucose oxidase (Trytek and Fiedurek, 2002).
In contrast to P-450 enzymes (3.3.1), peroxidases do require neither electron transport proteins nor cofactors. A characteristic feature of most peroxidases is their low substrate specificity.
The reaction cycle of most peroxidases is described in Figure 28. They react through a ping-pong mechanism, in which two radical intermediates are produced in each catalytic cycle (Dunford, 1991). The first step is an oxidation of the resting enzyme (ferriperoxidase Fe3+) by H2O2. The unstable intermediate compound I, consisting of ferryl iron (Fe4+) and a porphyrin π cation radical, is formed. A reducing substrate converts compound I back to the resting enzyme via compound II. Alternative reaction mechanisms for peroxidases do exist (Dunford, 1990, Campa, 1990, Griffin, 1990). Example of such is the mechanism for chloroperoxidase-catalysed reactions (4.3).
R1OOH + 2R2H R1OH + H2O + 2R2Peroxidase
37
Figure 28. The conventional cycle of peroxidase-catalysed reactions. RH=substrate: indicates a
free radical.
The reducing substrates are oxidised to free radical intermediates (Figure 28). These free radicals can thereafter react by several pathways and may for example dimerise, react with another substrate molecule, or attack various oxygen containing spieces. Finally alcohols, carbonyl compounds and epoxides are formed.
Peroxidases are divided into two superfamilies, the plant peroxidases and the animal peroxidases, each with different structural features. Plant peroxidases (POD) are widely distributed in plants and are important during plant growth and development and in stress responses. The plant peroxidases are further divided into three classes, I-III. Plants normally produce two classes of peroxidases, the intracellular Class I ascorbate peroxidases found in the cytosol and chloroplasts and the extracellular Class III peroxidases.
Horseradish peroxidase belong to the Class III peroxidases, which are heme containing (protoporphyrin IX), monomeric glycoproteins catalysing the oxidation of substrates with O2 or H2O2, forming water as a coproduct.
FeIII FeIV
OH2O2 H2O
Native enzyme(FeIII, resting)
FeIVO
RH
R+ RH
H2O + R
+ HH
Compound I(FeIV + porphyrin π-cation radical)
Compound II(FeIV)
38
4.2. Horseradish hairy root culture
Horseradish is known to contain large amounts of Class III peroxidases, which makes it enteresting to employ terpenes as substrates for a horseradish culture (Appendix 2). This culture may tentatively also host other enzyme classes, such as monooxygenases.
Biotransformation of α-pinene by the horseradish hairy root culture furnished a mixture of products (Figure 29) similar to that observed in the biotransformation of α-pinene by the P. abies suspension culture (as described in 3.2.1, I). Also the composition of the mixture changed with time similar to what was observed in the P. abies suspension culture. Hence, during prolonged reactions the verbenols were transformed to verbenone either via trans-verbenol (as described in 3.2.1.1) or directly by oxidation to verbenone.
Figure 29. Biotransformation products of (1R)-α-pinene by a horseradish hairy root culture. The
major products are enclosed with borders.
However, no enzyme assays were performed to asses the enzyme activities in the horseradish culture. In addition, no experiments were made in order to determine the extent of autoxidation in the enzyme-free reaction medium.
OHOH O
(1R)-(+)-α-Pinene
(1R)-cis-Verbenol
(1R)-trans-Verbenol
(1R)-(+)-Verbenone
trans-Sobrerol
OH
OHOH
(1S)-(+)-Myrtenol
39
4.3. Chloroperoxidase
The oxidative chlorination of certain substrates is catalysed by chloroperoxidase (CPO, EC 1.11.1.10). The catalytic mechanism of CPO differs somewhat from the conventional cycle of peroxidase-catalysed reactions (Figure 28). The enzyme compound III consisting of ferryl iron (Fe6+) is formed after the conventional compound II, with the last step leading to the resting enzyme under speculation. Furthermore, there are structural similarities of the heme group between chloroperoxidase and P-450 cytochromes (Griffin, 1990).
Several organic compounds can act as substrates for the CPO, forming epoxides and diols. Styrene and α-methyl styrene are used as substrates for CPO, furnishing styrene epoxide and 2-phenyl-1,2-propane diol, respectively (Zaks and Dodds, 1995).
The structural similarities of the above mentioned substrates to limonene led to the investigation of the catalytic effect of chloroperoxidase on limonene. α-Pinene and β-pinene were also used as substrates. However, no products were found in either of the experiments. This was due to the precipitation of the enzyme already after one hour of contact with the substrates (Appendix II).
40
5. Transformation of terpenes by lipases
5.1. Introduction
Lipases (EC 3.1.1.3) belong to a class of enzymes called hydrolases and are members of a family of enzymes, which in biological systems, mainly hydrolyse carboxylic esters in the form of triacyl glycerol esters (fats). In vivo, enzymes mostly perform their catalytic processes in aqueous media. Yet, as early as 1900 Kastle and Loevenhart (1900) showed that lipases are able to catalyse esterification reactions in nonaqueous media. It was not until the work of Klibanov and co-workers that enzyme-catalysis in organic media achieved attention (Zacks and Klibanov, 1984, Cambou and Klibanov, 1984). In water, hydrolysis is the dominating reaction. By changing to an organic medium, the reverse reaction - esterification - is favoured instead. Thus, in organic media, lipases catalyse acyl transfer reactions. When an ester is used as acyl donor a transesterification occurs (Figure 30).
Figure 30. General reaction of a transesterification catalysed by lipases.
Lipases react according to a mechanistic model named the bi-bi Ping-Pong model (Kraut, 1977, Straathof et al., 1992). Lipases follow the reaction mechanism used by all serine hydrolases, involving a catalytic triad of amino acid residues consisting of serine, histidine and aspartate or glutamate (Figure 31, Fersht, 1999).
R1 OR2
O+ R3 OH
R1 OR3
O+ R2 OH
Transesterification
41
Figure 31. Mechanism of serine hydrolases in the transesterification of vinyl acetate with a second
substrate, the alcohol.
Medium engineering can increase the efficiency of the lipase-mediated reactions for a specific substrate by changing the temperature, the nature of acyl donor, the enzyme to substrate/acyl donor ratio, the concentration, or by the influence of additives. Some recent reviews discussing lipase-catalysed reactions in organic solvents and their synthetic and industrial applications include those by Kazlauskas and Bornscheuer (1998), Berglund and Hult (2000), Carrea and Riva (2000), and Reetz (2002).
Hydrolase-catalysed resolution by acylation of alcohols is a useful method in organic chemistry (Faber, 1997, van der Werf et al., 1997). Examples of factors controlling the enantioselectivity of the reaction are the reaction medium (organic solvent, water activity, substrate and acyl donor concentration), the substrate structure and the lipase (chemical modification, DNA shuffling and directed evolution) as described in a review by Berglund (2001).
CH2
N N CH2
O
CH2
CO-O H
Histidine
Serine
Aspartate
H
OO
CH2
N N CH2
O
CH2
CO-O
H
O
O-
H
Tetrahedralintermediate
Acylation
CH2
N N CH2
O
CH2
CO-O
H
OH
Ser
His
Asp
O
Acyl enzyme
Deacylation
CH2
N N CH2
O
H2CC
O-O
H
Ser
His
Asp
OOR
H+
CH2
N N CH2
O
CH2
CO-O
H
RO
O-
H
Tetrahedralintermediate
CH2
N N CH2
O
H2CC
O-O H
H
ROO
H
Alcohol substrate
Vinylalcohol
Vinylacetate
His
Ser
Asp
42
For secondary alcohols Kazlauskas has suggested a rule for prediction of the expected enantioselectivity of the majority of lipases (Kazlauskas et al., 1991). The configuration of the fast reacting enantiomer is shown in Figure 32.
Figure 32. Model for predicting the enantioselectivity of lipases for a secondary alcohol based on
Kazlauskas et al. (1991). M and L indicate the medium- and the large-sized substituent, respectively
in the fast reacting enantiomer.
Vinyl acetate is known to undergo efficient lipase-catalysed irreversible transesterifications with alcohols as substrates. The irreversibility in such reactions is due to the liberation of vinyl alcohol, which is rapidly and irreversibly tautomerised to acetaldehyde (Figure 33, Faber, 1997, Santiello, 1993). The secondary monoterpene alcohol, menthol, has been successfully resolved via enantioselective acylation with vinyl acetate catalysed by commercially available lipases (Koshiro et al., 1985, Wu et al., 1996).
We have investigated the possibility to use transesterification of vinyl acetate catalysed by several lipases as a method for the resolution and separation of terpene alcohols (V).
Figure 33. Transesterification of an alcohol with vinyl acetate catalysed by a lipase. The liberated
vinyl alcohol is tautomerised to acetaldehyde.
HHO
M L
O
O
O
O Lipase
Organicsolvent
+ +R OH R HO
O
H
43
5.2. Lipase-catalysed transesterification
The lipases used in these experiments are listed in Table 7. They are all commercially available.
Table 7. Commercially available lipases used for the studies of resolution and separation of
monoterpene alcohols.
Lipase Short Supplier Trade name
Bacterial: Burkholderia cepacia other names:
Pseudomonas cepacia,
Pseudomonas fluorescens
PCL Amano Roche
lipase PS Chirazyme® L-6
Fungal: Candida antactica A CAL-A Novo Nordisk SP526 Candida antactica B CAL-B Novo Nordisk Novozyme 435 Mucor miehei other name:
Rhizomucor miehei
MML Novo Nordisk SP524
Thermomyces lanuginose other name:
Humicola lanuginosa
TLL Novo Nordisk SP523
In the first series of experiments, the major primary and secondary monoterpene alcohols in TMP-turpentine - borneol and myrtenol - were used as model substances, and were subjected to lipase-catalysed transesterification of vinyl acetate (Figure 34, V).
The primary alcohol, myrtenol reacted fast using PCL-L6 in TBME (c = 98%, 24 hours, room temperature) while the secondary alcohol, borneol, reacted slower. However, when CAL-A was used as catalyst, acceptable yields of the bornyl ester were obtained (c = 68%, 14 days, 30 °C). The enantioselectivities (E) of these reactions were low (E ≈ 2) in all experiments. When PCL-PS, PCL-L6 or CAL-B was used as a catalyst for the transesterification of vinyl acetate with borneol, the hemiacetal ester was detected as an unexpected side-product (Figure 34, entry 1, see 5.2.1, III, IV)
44
In the second series of experiments the polar fraction from the TMP-turpentine was subjected to transesterification catalysed by five lipases: TLL, MML, CAL-A, PCL-PS or PCL-L6 (Figure 34, entry 2, V). The primary alcohols were esterified in a short time, whereas the secondary alcohols needed a longer conversion time. Still, they did all act as substrates. As expected, due to steric hindrance the tertiary terpene alcohols were unreactive (cf. Ospiran et al., 1996). TLL did not show any activity.
Figure 34. Schematic picture of the experiments performed on the lipase-catalysed
transesterifications of vinyl acetate with terpene alcohols. 1) Borneol, 2) myrtenol, 3) trans-
pinocarveol.
Selected reaction with best yield or useSubstrate Rection parameters
1.
rac-Myrtenol (1) or rac-borneol (2) as model substrate
AcO
OAc
PCL-L6, TBME22 °C, vinyl acetate
CAL-A, TBME, 14 days22 °C, vinyl acetate,
CAL-B, PCL-PS,PCL-L6
Acyl donor: Vinyl acetate or vinyl propionateSolvent: Heptane or TBMETemperature (°C): 7, 22 or 40Lipase: CAL-A, CAL-B,PCL-PS, PCL-L6
Comment: Enantioselectivity low, E = 2
O OAc
Unexpected product
HO
OH
OH
Terpene alcohols from the TMP-turpentine
obtained by flash chromatography (V)
Acyl donor: Vinyl acetateSolvent: Heptane or TBMETemperature (°C): 22 or 30Lipase: TLL, MMLCAL-A, PCL-PS,PCL-L6
AcO
OAc
OAc
MML, TBME
CAL-A, TBMEc = 100%, 24 h
CAL-A, TBME, 24 h
Comment: The tertiary terpene alcohols did not react at all
2. HO
OH
OH
1
2
2
1
2
3
c = 98%, 24 hours
c = 14%, (c = 68%, 30 °C), (c = 4 5%, vinyl propionate)
c = 100%, 24 h
MML, TBMEc = 30%, 24 h
c = 30%, 22 °Cc = 50%, 30 °C
45
When MML or CAL-A was used as catalysts, the myrtenol in the TMP-alcohol mixture was fully converted to the corresponding ester within 24 h and a slower reaction rate was observed for PCL-L6 as a catalyst (Figure 35 a). In contrast, a very fast rate of conversion of myrtenol was observed when it was used as the single substrate for the PCL-L6 catalysed reaction (Figure 34, entry 1). Probably, compounds present in the terpene alcohol mixture could have acted as enzyme inhibitors or even as denaturating agents. Also, a slight change in the enzyme/substrate ratio might have been overlooked due to difficulties in determining the actual substrate concentration in the mixture of the terpene alcohols from the turpentine.
Figure 35. Formation of terpene esters in the transesterification of vinyl acetate with the mixture of
alcohols from the TMP-turpentine by means of lipases. a) Formation of myrtenyl acetate by MML,
CAL-A and PCL-L6-catalysed transesterifications. b) Formation of myrtenyl acetate and trans-
pinocarvyl acetate by MML-catalysed transesterification. *Integrated GC-MS area of peak in relation
to the integrated area of an internal standard.
However, MML only converted about 30% of trans-pinocarveol, in the mixture of alcohols obtained from the TMP-turpentine, in 14 days (Figure 35 b). This result indicated that it would be possible to separate myrtenol from trans-pinocarveol by MML-catalysed transesterification of vinyl acetate (see below 6.4). The highest yield of bornyl acetate from the terpene alcohol mixture was obtained when the catalyst was lipase CAL-A.
a
0
0.2
0.4
0.6
0.8
1
0 5 10 15 days
rel.
int.
area
(GC
-MS)
*
Formation of myrtenyl acetate
PCL-L6
MML or CAL-A
b
0.0
0.2
0.4
0.6
0.8
1.0
0 5 10 15 days
trans -pinocarvyl acetate
myrtenyl acetate
MML as catalyst
rel.
int.
area
(GC
-MS)
*
PCL-L6 MML CAL-A
a
0
0.2
0.4
0.6
0.8
1
0 5 10 15 days
rel.
int.
area
(GC
-MS)
*re
l.in
t. ar
ea (G
C-M
S)*
Formation of myrtenyl acetate
PCL-L6
MML or CAL-A
b
0.0
0.2
0.4
0.6
0.8
1.0
0 5 10 15 days
trans -pinocarvyl acetate
myrtenyl acetate
MML as catalyst
rel.
int.
area
(GC
-MS)
*
b
0.0
0.2
0.4
0.6
0.8
1.0
0 5 10 15 days
trans -pinocarvyl acetate
myrtenyl acetate
MML as catalyst
rel.
int.
area
(GC
-MS)
*re
l.in
t. ar
ea (G
C-M
S)*
PCL-L6PCL-L6 MMLMML CAL-ACAL-A
46
5.2.1. Side-products
When the transesterification of vinyl acetate with borneol was catalysed by either of the two Pseudomonas sp. lipases (PCL-PS, PCL-L6) or by the lipase from Candida antarctica B (CAL-B), the yield of bornyl acetate was reduced due to the formation of the hemiacetal or the acetylated hemiacetal of borneol (Figure 36, III, IV). It is known that side-reactions can occur when using vinyl esters as acyl donors (Bianchi et al., 1988, Xie, 1991), however the nature of these reactions has not been described.
Figure 36. Acetylated hemiacetal of borneol formed in the transesterification reaction of vinyl
acetate and borneol using the lipases PCL-PS, PCL-L6 or CAL-B as catalysts.
In order to determine whether the formation of hemiacetal esters is a general reaction for slow-reacting, sterically hindered alcohols, the alcohols 2-17 (Figure 37) were used as substrates in transesterifications of vinyl acetate using the above-mentioned lipases as catalysts (III, IV). Formation of hemiacetal ester side-products also occurred, when the alcohols 2-11 (Figure 37) were used as substrates in transesterifications catalysed by Pseudomonas cepacia lipases and Candida antarctica lipase B. Formation of hemiacetal side-products occurred with substrates 1, 7-11 (Figure 37).
O O
O
47
Figure 37. Transesterification of vinyl acetate with sterically hindered alcohols catalysed by
Pseudomonas cepacia or Candida antarctica lipase B. 1) (+)-Borneol, 2) Isoborneol, 3) Endo-
norborneol, 4) 3,3,8a-trimethyl-2,3,4,6,7,8,8a-octahydronaphthalen-1-ol, 5) 1,2,3,4-
tetrahydronaphthalen-1-ol, 6) decahydronaphthalen-1-ol, 7) 2-Adamantol, 8) 1-Phenyl-1-butanol, 9)
3-Methyl-1-phenyl-1-butanol, 10) 3,3-Dimethyl-1-phenyl-1-butanol, 11) 3-Chloro-1-phenyl-1-
propanol, 12) Exo-norborneol, 13) (+)-Isopinochampheol, 14) (–)-trans-pinocarveol, 15) 2-
Methylhexanol, 16) α-Terpineol, 17) Terpinene-4-ol.
From alcohols 12-15 (Figure 37) only esters were produced and the substrates 16-17 were found to be unreactive due to sterical hindrance. The identities of the hemiacetal esters from the substrates 1-11 (Figure 37) were determined by GC-MS analysis. The identity of the hemiacetal esters of the substrates 1, 4-6 was also confirmed by NMR analysis (III, IV). Based on our experiments, no general correlation could be established between the extent of formation of a hemiacetal or hemiacetal ester and the structure of the substrate, the lipase used or the reaction conditions.
OH
OH
OHH
H
OH
H
OH
2 3
12 13 14
OH OH OH
Cl
OHOH
1OH
Alcohols forming mixtures of esters, hemiacetals or/and hemiacetal esters
Alcohols forming esters only
OH OHOH
OH
4 5 6
7 8 9 10 11
15OH
OH
1716
Non-reactive alcohols
48
The hemiacetal ester was probably produced via esterification of the initially formed hemiacetal, from the acetaldehyde liberated in the acylation reaction (Figures 31, 33). Experiments excluding the enzyme did not result in any product while in experiments using a purified CAL-B, the hemiacetal ester was detected (IV). Hence, the lipase itself was the active catalyst in the formation of the hemiacetal ester side-products. Our results indicate that both steps yielding the hemiacetal ester are probably lipase-mediated (Figure 38, IV).
Figure 38. The formation of a hemiacetal ester from acetaldehyde and its further esterification.
Whereas 2-adamantol and vinyl acetate, either with or without added acetaldehyde in the presence of CAL-B or PCL-L6, furnished the hemiacetal, similar experiments in the absence of enzyme gave no trace of a hemiacetal or its ester (Table 8). Furthermore, experiments with added acetaldehyde but excluding the vinyl acetate produced no side product. Hence, both lipase and vinyl acetate seem to be prerequisites to obtain both the hemiacetal and the hemiacetal ester of the substrate (Figure 38).
Table 8. Formation of a hemiacetal of 2-adamantol.
Substrate Enzyme Product
Acyl donor
PCL-L6/ CAL-B
PCL-L6/ CAL-B
− No product
−
PCL-L6/ CAL-B
No product
2-adamantol
−
− No product
O
O
H
OOH
O
O
O
O
H
O
O OH
H3CH
ORO OH
H3C H
RO OAc
H3C H
Enz-Ac Enz-H
ROH
ROHEnz ?
H
O
H
O
49
Four diastereomers of the hemiacetal ester were formed from racemic borneol, i.e. two pair of enantiomers (Figures 39, 40). When one enantiomer of borneol was used as substrate, two hemiacetal esters were formed and one of them was formed in diastereomeric excess. We were unable to determine which one of the diastereomers, A or B that was the major one (Figure 39).
Figure 39. Formation of acetylated hemiacetal during lipase-catalysed transesterification of vinyl
acetate with borneol. Enz-H = PCL-PS, PCL-L-6 or CAL-B.
Figure 40. The GC-chromatogram (column: β-dex 120) of the lipase-catalysed (PCL-PS)
transesterification of vinyl acetate with racemic borneol in heptane after 14 days at roomtemperature.
The two enantiomers of bornyl acetate did not separate.1) A or B; 2) ent-A or ent-B; 3) ent-B or ent
A; 4) B or A (Figure 39).
Abundance
Time (min)46.2 51.0 51.9 56.1 57.4
(-)-
born
eol
(+)-
born
eol
born
yl a
ceta
te
1
42
3
Abundance
Time (min)46.2 51.0 51.9 56.1 57.4
(-)-
born
eol
(+)-
born
eol
born
yl a
ceta
te
1
42
3
OO
O H
OO
HO
O O
OH
O
H
O
O
O
O
O OH
H
O
HO O
+
(-)-borneol
(+)-borneol
+
Enz-H Enz-Ac
Enz-Ac Enz-H
Enz-Ac Enz-H
A B
ent-A ent-B
?
hemiacetal hemiacetal esters of borneol
Enz ?
Enz ?
50
However, assuming that Kazlauskas rule (Kazlauskas et al., 1991, described in 5.1) is valid for the acylation of hemiacetals, the major products from (–)- and (+)-borneol will be the hemiacetal esters B and ent-A respectively (Figure 41).
Figure 41. Tentatively the major hemiacetal achieved from (+)-borneol (Figure 39), docked into
Kazlauskas model pockets of the lipase.
Adding acetaldehyde to the reaction mixture with borneol as a substrate increased the diastereoselectivity (from dr = 2:1, to dr = 5:1) (Table 9, entry 1-2) (IV). The diasetereoselectivity could be even more improved by performing the same experiment on a larger scale (dr = 98: 2) (Table 9, entry 3). Products of aldol condensation reactions were detected in experiments with propanal as additive (IV). Lipase (CAL-B)-mediated aldol reactions have been reported in the literature (Branneby et al., 2003).
Table 9. Transesterification of vinyl acetate with (–)-borneol.
Entry Substrate
(–)-Borneol
Lipase/mg Additives/mg c (%) Products (%)
14 days
Ester Ent-A? Ent-B?
1
a PCL-L6/4.8 VinylAc/ 128 97-98 8-20 59-66 25-33
2 a PCL-L6/4.8 VinylAc/ 128
CH3CHO/ 11
74-86 0 83-85 15-17
3 b PCL-L6/55.5 VinylAc/ 282
CH3CHO/ 196
100 0.4 97.5 2.1
a. 0.65 ml solution of (–)-borneol 0.1 M in TBME. b. 123 mg (–)-borneol dissolved in 4 ml of TBME
(the conversion was determined after 20 days).
O
OHH
51
The hemiacetal esters are sensitive to acids, which catalyse the cleavage to acid, acetaldehyde and alcohol. Thus, borneol was recovered from its corresponding acetylated hemiacetal upon chromatography on silica gel. Therefore, these side products are easily overlooked in enzymatic resolutions and their presence could result in lower ee-values than expected due to decomposition of hemiacetal esters during work-up. Liu et al. (2000) report lower ee-values than expected after hydrolysis of their initial product. They did not report any side-product formation in their CAL-B-catalysed resolution of 3-chloro-2-(2-thienyl)-1-propanol. Formation of hemiacetal esters offers one explanation for the unexpectedly low ee observed for the remaining substrate.
Figure 42. Resolution of 1) 3-chloro-2-(2-thienyl)-1-propanol by CAL-B-catalysed
transesterification of vinylbutyrate gave a lower eep than expected from E=299, after hydrolyses of
the product (Lie et al, 2000). Could the formation of a hemiacetal or hemiacetal ester be the
explanation?
OO
CAL-B
Nearly resolved alcohol
+Hemiacetal ester of 1
?SCl
OHE=299
1
52
6. Separation of the terpenes from the TMP-turpentine
6.1. Introduction
Separation of the more valuable terpenes present in TMP-turpentine is one approach to value-added compounds. Such an approach will, of course, include physical methods such as chromatography or distillation. The processes for separation must be as simple as possible in order to facilitate future work on a larger scale. We have investigated whether enzymatic methods can be used to separate terpene alcohols from mixtures obtained by distillation and chromatography.
6.2. Chromatography
One fraction of oxygenated terpenes and one fraction of hydrocarbons were obtained from TMP-turpentine by the use of flash chromatography on silica gel (V). The terpene hydrocarbons were first filtered through the column and then eluted by pentane until almost no unpolar compounds were left on the column. Diethyl ether (15%) in pentane was used to elute the esters and the ketones from the column. The remaining terpene alcohols were eluted with diethyl ether.
6.3. Distillation
The crude TMP-turpentine was fractionated by distillation (Appendix 1). However, the monoterpene alcohols were mixed with some of the mono- and sesquiterpene hydrocarbons. To solve this problem, flash chromatography of the crude TMP-turpentine was performed prior to distillation (V). Distillation of the polar fraction gave a mixture of oxygenated compounds, which was now free from sesquiterpenes and other compounds of high molecular weight.
53
6.4. Lipase-catalysed transesterification as a separation method
Good yields of terpene acetates were obtained using lipase-catalysed transesterification of vinyl acetate with a polar terpene alcohol fraction, obtained by MPLC of the crude TMP-turpentine (Appendix 1, V). The content of acetates in was increased from 0.5% to 8.2% by CAL-A-catalysed transesterification, at room temperature. At 30 °C the acetate content was further increased to 13.6% in two weeks of treatment.
A second alcohol fraction was obtained by flash chromatography in combination with distillation (V). By starting with this latter alcohol fraction and by using a one step reaction with CAL-A as catalysts, an alcohol fraction containing α-terpineol as the major constituent was obtained in good yield (Figure 43, unpublished results).
Figure 43. Separation of oxygenated terpenes from TMP-turpentine utilising chromatography,
distillation and lipase-catalysed (CAL-A) transesterification of vinyl acetate with monoterpene
alcohols. 1) α-Pinene, 2) β-pinene, 3) limonene, 4) myrtenyl acetate, 5) trans-pinocarvyl acetate, 6)
bornyl acetate, 7) α-terpineol.
TMP-Turpentine
AlcoholsUnpolar terpenes
Monoterpene alcohols
Chromatography
Distillation
Chromatography
CAL-A, 30 °C, 15 days
AcO
OAcOAc
OH
1 2 3
4 5 6 7
2.0 g
1.1 g Purity (GC-MS): 79%0.4 g
54
If the lipases MML and CAL-A were used sequentially, each of the acetates of myrtenol, trans-pinocarveol, and borneol were obtained in good yields (Figure 44, V). These compounds are all attractive flavours and fragrances. The myrtenol acetete was however contaminated by the malodours esters of hexanol, heptanol, octanol, and nonanol (V).
Figure 44. Separation of oxygenated terpenes from TMP-turpentine using chromatography,
distillation and lipase-catalysed (MML and CAL-A) transesterification of vinyl acetate with
monoterpene alcohols. 4) Myrtenyl acetate, 5) trans-pinocarvyl acetate, 6) bornyl acetate, 7) α-
terpineol. Values in % refer to the purity of the isolated compound.
TMP-Turpentine
AlcoholsUnpolar terpenesas α-pinene and
β-pinene
Monoterpene alcohols
Remaining alcoholsMyrtenyl acetate
Remaining alcohols
Remaining alcoholstrans-
Pinocarvyl acetate
Bornyl acetate
Chromatography
Distillation
Chromatography
Chromatography
15 days, 30 °CCAL-A step 2
Chromatography
CAL-A step 1
MMLAcO
OAc
OAc
OH
4
5
6
7
2.2 gPurity (GC-MS): 86%
0.3 gPurity (GC-MS): 62%
4.0 g 72 hours
3.6 g11 days
0.3 gPurity (GC-MS): 33%
0.3 gPurity (GC-MS): 68%
3.3 g
55
7. Conclusions and future work The aim of this thesis was to demonstrate methods of achieving value-added compounds from the TMP-turpentine. The methods described are successful in this respect and the usefulness of biotransformations to reach the goals set has been demonstrated. Hence, by use of a P. abies suspension culture (I, II) and the horseradish hairy root culture (Appendix 2), the main constituents of the TMP-turpentine (Appendix 1) were oxygenated in a satisfactory way, however, in low yields. Additionally, the lipase-catalysed transesterifications of vinyl acetate made it possible to separate the TMP-turpentine into acetates or fractions of desirable terpene alcohols (V). When performing enzyme-catalysed resolutions involving vinyl acetate, the formation of hemiacetal esters in the lipase-catalysed reactions of sterically hindered alcohols (III, IV) is a discovery worth keeping in mind. Further experiments should be made with the aim to clarify the mechanism for the side-product formation during lipase-catalysed transesterification of vinyl acetate with sterically hindered secondary alcohols.
Other oxygenating enzymes should be screened for their ability to transform the monoterpene hydrocarbon constituents. For instance, hydratases, and a mixture of glucose peroxidase and horseradish peroxidase have been found to transform limonene into α-terpineol and carvone, respectively (Tan et al., 1998, Trytek and Fiedurek, 2002).
The yield of the transformation by P. abies suspension culture should be increased. The work-up procedure should also be further improved. The composition of the nutrition medium should be optimised in order to induce the cultures to perform biotransformations that result in a desired product pattern.
Also of interest is the possibility of the P. abies suspension culture in use for biotransformation of other substrates than terpenes, such as other cyclic unsaturated hydrocarbons. Is the isomerisation seen in the biotransformation of α-pinene (cis-verbenol into trans-verbenol) common for other types of non-terpene substrates? Such reactions may be useful in organic synthesis.
Further investigations using the horseradish hairy root culture should be performed. The P. abies suspension culture gave the same products as the horseradish culture. Work-up of the latter culture broth was much easier and therefore low product losses should be expected.
56
Acknowledgements I am most grateful to Kristina Sjödin and Hans-Erik Högberg for being my supervisors. I thank you for the scientific guidance and the valuable discussions, for your patience and for never giving up. I am also most grateful to Irena Valterová for sharing experiences of science and art of life with me, and your never-ending encouraging and enthusiasm.
I am also most grateful to Dan Isaksson for sharing almost everything with me, experiments, discussions in chemistry and life science, failure and success. You are a brilliant, skilled and warm-hearted friend.
Special thanks to Dr. Tomáš Vanek for sharing the Picea abies culture with me. Special thanks also to Ms H. Ernyeiova for excellent technical assistance and to Ms. Z. Hornycova for all the invaluable caring of the culture. I am also grateful to Per Berglund and Matt Richardson for valuable comments and to Siw Stenlund for all generous help.
My sincere thanks to Dag Nordvall and Nils Ekelund for the valuable guidance in the world of biochemistry and plant physiology.
Warm thanks also to Håkan Norberg and Torborg Jonsson for helping me with technical assistance, and to both of you for being my indispensable friends.
The time at the Mid Sweden University became a valuable part of my life. I deeply thank all former and present colleagues and friends for creating a warm and helpful atmosphere filled with friendship and guidance.
Warm thanks to my mother, father and sister for support, love and never-ending believe in me. Thanks to all friends for being there when needed and for giving me so much joy and pleasure. Thanks to all the people who have inspired me.
Helen, I deeply thank you.
Niklas, from the bottom of my heart I thank you for all support, encouragement and love.
/Marica
57
Grants from the Swedish KK-Foundation, the Royal Swedish Academy of Sciences (KVA), the Aulin-Erdtman Foundation, and the Bengt Lundqvists Minne Foundation and financial support from the Mid Sweden University are gratefully acknowledged.
58
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