alcohol linalool cyp oxidative

8/13/2019 Alcohol Linalool Cyp Oxidative

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Xenobiotica, June 2007; 37(6): 604617

Study on the cytochrome P450-mediated oxidative

metabolism of the terpene alcohol linalool: Indication of

biological epoxidation

R. J. W. MEESTERS1, M. DUISKEN1,2, & J. HOLLENDER1,3

1Institute of Hygiene and Environmental Medicine, RWTH Aachen University, Aachen, Germany,2LECO Instrumente GmbH, Monchengladbach, Germany, and 3Swiss Federal Institute of Aquatic

Science and Technology, Eawag, Dubendorf, Switzerland

(Received 1 March 2007; revised 23 March 2007; accepted 11 April 2007)

AbstractThe cytochrome P450-mediated oxidative metabolism of the terpene alcohol linalool was

studied in vitro by enzymatic assays using recombinant human cytochrome P450 enzymes.Three different enzymatic products of allylic hydroxylation and epoxidation were identified bygas chromatography-mass spectrometry. Identified enzymatic products were 8-hydroxylinalool((R/S)-3,7-dimethyl-1,6-octadiene-3,8-diol) and the cyclic ethers pyranoid-linalool oxide

((R/S)-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol) and furanoid-linalool oxide (R/S)-2-(1,1-dimethylethyl)-5-methyl-5-vinyltetrahydrofuran. The cyclic ethers result most likely from the

epoxidation of the 6,7-carbon double carbon bond of (R/S)-linalool, followed by the intramolecularrearrangement of the 6,7-epoxy-linalool. Allylic-hydroxylation of the 8-methyl group of linalool was

catalyzed by CYP2C19 and CYP2D6 while the enzymatic epoxidation of linalool was only observedwith CYP2D6. The results indicate that the electrophilic oxidation products of linalool such as6,7-epoxy-linalool which may cause sensitization and irritational skin reactions are not only producedby auto-oxidation reactions in the presence of air-oxygen as published in the past, but also by

P450-mediated oxidative biological transformation.

Keywords: Linalool, cytochromes P450 (CYP), epoxidation, pyranoid-linalool oxide, furanoid-linalool

oxide, 8-hydroxylinalool

Correspondence: J. Hollender, Swiss Federal Institute of Aquatic Science and Technology, Eawag, U berlandstr. 133, CH-8600


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of electrophilic compounds similar to the ones identified previously as auto-oxidation

products. The CYP-mediated oxidation by CYP2D6- and CYP2C19 enzymes, which next

to other CYP are expressed in skin (Ahmad et al. 1996; Yengi et al. 2003) and belong to the

five CYP enzymes responsible for approximately 99% of P450-mediated drug metabolism

(Bertz and Granneman, 1997), were used for the study.

Materials and methods

Chemicals and enzymes

All chemicals used in this study were of analytical grade quality or for biochemical use,

unless specified otherwise. Ethanol (100%), linalool (97%, (R/S)-3,7-dimethyl-1,

6-octadien-3-ol; Figure 1 (1a, b)), disodium hydrogen phosphate dihydrate

(Na2HPO4 2H2O) and potassium dihydrogen phosphate trihydrate (KH2PO4 3H2O)

were purchased from Sigma Aldrich (Taufkirchen, Germany). Glucose-6-phosphatepotassium salt (G6P), the enzyme glucose-6-phosphate dehydrogenase (G6PDH; EC

1.1.1.49) and nicotinamide adenine dinucleotide phosphate sodium salt (NADP, 98%) were

purchased from Roche Diagnostics (Basel, Switzerland); ethyl acetate (EtOAc) was of

residue analysis quality and was purchased from LGC Promochem (Wesel, Germany).

Purified water produced by a Milli-Q water purification system (Millipore, Eschborn,

Germany) was used for the preparation of phosphate buffers in enzymatic assays. The

reference substance furanoid-linalool oxide (97%, (R/S)-2-(1,1-dimethylethyl)-5-methyl-5-

vinyltetrahydrofuran; Figure 1 (2ad)) was purchased from Fluka (Buchs, Switzerland) and

O

HO

O

HO

O

HO

O

HO

4c (3R,6S)4b (3S,6R)4a (3R,6R) 4d (3S,6S)

O O O O

2a (2R,5R) 2c (2R,5S) 2d (2S,5R )2b (2S,5R)

OH OH

1a (S) 1b (R)

OH OH

3a (S) 3b (R)

OH

OH

OHHO

5c (cis)5a (trans)

OH

OH

5b (trans)

OH

HO5d (cis)

Figure 1. Structural formula of (R/S)-linalool (1a, b), (R/S)-furanoid-linalool oxide (2ad),

(R/S)-dihydrolinalool (3a, b) and (R/S)-pyranoid-linalool oxide (4ad) and (cis/trans-8-hydroxylina-l l (5 d)

606 R. J. W. Meesters et al.


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pyranoid-linalool oxide (97%, (R/S)-2,2,6-trimethyl-6-vinyltetrahydro-2H-pyran-3-ol;

Figure 1 (4ad)) was purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan).

Recombinant human cytochrome P450 enzymes and NADPH generating system

Recombinant human CYP (EC 1.14.14.1), CYP2D6 and CYP2C19 (amount 1 nmol)

with co-expressed CYP-reductase in Escherichia coli (bactosomes) were obtained from

Cypex (Dundee, Scotland) and stored at a temperature of 80C until usage.

The combination of the enzyme G6PDH (1.5 U ml1), NADP (0.5 mM) and G6P

(4.7 mM) dissolved in phosphate buffer ( pH 7.2) functioned as NADPH-generation

system in all enzymatic assays. The NADPH-generation system solution was always freshly

prepared.

Identification of enzymatic products by GC/MS analysis

A Hewlett-Packard gas chromatograph (GC) model 5860 Series II (Waldbronn, Germany)

equipped with a programmable temperature vaporizer (PTV ) and an MPS large volume

sampler (CIS 3) all from Gerstel (Muhlheim a. d. Ruhr, Germany) were directly connected

by a heated transfer line to a Hewlett-Packard 5972 mass spectrometer (MS). Samples were

separated on an RTX-5SIL MS (Restek, Bad Homburg, Germany) fused silica capillary

column (0.28 mm30 m, 0.25mm film thickness) using helium (linear gas velocity of

0.7 ml/min) as the carrier gas. The GC temperature program conditions were: initial oven

temperature 37C, heating to 200C by a temperature ramp of 6C min1 followed by

another temperature ramp of 15C min1 heating up to a temperature of 330C. From the

sample extracts 40ml was injected (injection speed: 29 mlmin1

) into the liner (93 1 mmI.D., Gerstel, Muhlheim a. d. Ruhr, Germany) of the PTV stuffed with deactivated silanized

glass wool. The PTV operated in the solvent vent stop flow mode using a vent flow of

200 ml min1 helium gas.

Purging of the samples organic solvent started directly after sample injection (injector

temperature of 20C) and lasted 0.5 min while the GC temperature program was running.

The PTV injector temperature was held for 2 min at a temperature of 20C, followed by an

increase of the injector temperature (split less mode) up to 300C by a temperature ramp of

600Cmin1. The GC/MS transfer line was set at a temperature of 300C, this resulted in

an ion source and quadrupole temperature of 180C. The electron impact (EI) ionization

voltage was set to 70eV and positive charged ions were analyzed in full scan modeapplying a scan range of m/z 30300. Quantification of metabolite concentrations was

performed by external calibration for the metabolites furanoid-and-pyranoid-linalool oxide.

8-Hydroxylinalool was quantified using linalool as surrogate reference substance.

P450-mediated enzymatic assay

Phosphate buffer ( pH 7.2) was prepared by mixing 62.1 ml of a Na2HPO4(0.05 M) solution

with 39.8ml of a KH2PO4 (0.02 M) solution. CYP-mediated enzymatic assays of the

substrate linalool using CYP2D6 and CYP2C19 enzymes were carried out as follows.

A substrate stock solution was prepared by dissolving 10 ml of linalool with phosphate buffercontaining 10% ethanol (100%). The enzymatic biotransformation assays were carried out

i 1 5 l f l k i t b f E d f (H b G ) Th CYP

P450-mediated oxidative metabolism of linalool 607


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suspension was divided by diluting 100 ml of the homogenized enzyme suspension with

1000 ml of the phosphate buffer and stored at a temperature of80C until use, for every

enzymatic assay one tube with CYP was used.

The metabolism and formation of enzymatic products of linalool by selected CYP

was studied using time-, enzyme- and substrate-dependent enzymatic assays.

The time-dependent formation of the enzymatic products was studied by mixing 200 ml ofthe phosphate buffer solution with 200 ml of the NADP/G6P solution and 200 ml of the

G6PDH solution. Thereafter 20 ml of the linalool stock solution (0.215mmol ml1) were

added and 100mL of the recombinant human CYP2D6 (74 pmol ml1) or CYP2C19

(110 pmol ml1), respectively. The safe-lock micro-tubes were vortexed and incubated and

vigorously shaken at a temperature of 37C using an Eppendorf Thermomixer (Hamburg,

Germany). This procedure was carried out with various incubation times

(e.g. 5, 10, 15, 20, 30 and 40 min), each in a separate safe-lock micro-tube.

Formation of enzymatic products depending on CYP enzyme concentrations was

identically carried out with two different CYP concentrations, namely CYP2D6, 74 and

148 pmol ml1

and CYP2C19, 110 and 220 pmol ml1

, respectively.Substrate dependency of the formation of the enzymatic products was studied with

enzyme concentrations as applied in the time-dependent enzymatic assay with 4, 6, 8, 10,

12, 14 and 20ml of a diluted linalool stock solution (0.0215 mmol ml1) as substrate.

Incubation time of the substrate-dependent enzymatic assay was 40 min. With each type of

enzymatic assay two separate control incubations were carried out: one control consisted of

incubation with CYP in combination with the NADPH regeneration system but without

substrate addition; the second control consisted of incubation with substrate in combination

with the NADPH regeneration system but without addition of CYP. Enzymatic reactions

were stopped by denaturation of the CYP by the addition of 1 ml of EtOAc to the enzymatic

assay and mixing vigorously for 1 min using a vortex mixer. The metabolites were extractedby the previously added EtOAc by mixing the safe-lock micro-tubes for an additional

30 min. The EtOAc layer was separated by centrifugation of the safe-lock micro tubes

4000 rpm for 15 min (Eppendorf centrifuge, Hamburg, Germany). The EtOAc with

extracted enzymatic products was transferred into brown-colored glass GC septum vials and

stored at 4C until GC/MS analysis.

Results

Identification of enzymatic products of linalool catalyzed by P450s

A typical enzymatic conversion of the substrate linalool (4.3 nmol) by one of the selected

enzymes (CYP2D6, 74 pmol ml1) is illustrated by the GC-MS chromatogram in Figure 2.

GC-MS analysis of the two different control incubations showed that no additional

substances such as auto-oxidation products of linalool or impurities of the enzyme

suspension were formed during incubation experiments.

Identification of the enzymatic products was carried out in two different ways:

(i) comparison of mass spectra and retention times of the enzymatic products with reference

substances and, if reference substances were not available, (ii) the use of mass spectral match

factors (MFs) automatically calculated by the used library software (NIST 98 library;

Hennig et al. 1994), as well as comparison of Kovacs indices. A structural identification ofthe enzymatic product was considered to be accurate if the probability (MF) of the unknown

ti d t b i th d id tifi d t b th lib d t b



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was490% (Table I), main fragments could be explained and the calculated Kovacs indices

showed good agreement with literature values.

The enzymatic conversion of linalool catalyzed by both CYP resulted in the formation andidentification of three different enzymatic products (#2, 4, 5). The substance ( peak #3) was

t t ti l id tifi d dih d li l l ((R/S) 3 7 di th l 6 t 3 l Fi 1 (3 b))

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.000

0

50

150

250

350

450

550

650

TIC * 10E3

50

150

250

350

450

550

650

750(a)

(b) 750

TIC *10E3

Retention time [min]

4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Retention time [min]

14.00 16.00

1

1

2

3

3

4

5

{

{

{3

Figure 2. (a) GC/MS total ion current (TIC) chromatogram of the substrate (R/S)-linalool after

incubation for 40 min without CYP, (R/S)-linalool (1), (R/S)-dihydro-linalool (3); (b) GC/MS totalion current (TIC) chromatogram of the substrate (R/S)-linalool after incubation for 40 min withCYP2D6 (74 pmol ml1), (R/S)-linalool (1), (R/S)-furanoid-linalool oxide (2), (R/S)-dihydro-linalool

(3), (R/S)-pyranoid-linalool oxide (4) and (cis/trans)-8-hydroxylinalool (5); GC/MS conditions seeMaterials and methods.



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by the GC/MS mass spectra library with a low reliability (Table I). The substance

was also detected in the linalool control incubations and could be thus excluded as being a

P450-mediated enzymatic product of linalool, but was an impurity of the linalool. The two

small chromatographic peaks (#2) were identified by the GC/MS library as (R/S)-furanoid-

linalool oxide with an MP of470%, whereby the first peak (Rt11.5 min) of the couple was

identified as cis-isomers (Figure 1 (2a, b)) and the second peak (Rt 12.0 min) as being the

trans-isomers (Figure 1 (2c, d)). A commercially available (R/S)-furanoid-linalool oxide

reference substance confirmed the identity of both these enzymatic products by retention

times. According to this, the two peaks (#4) were identified by the GC-MS

library comparison also with a high MF (Table I) and with the reference substance as

(R/S)-pyranoid-linalool oxide. The first peak (Rt 14.8 min) of the couple was most likely

the cis-isomer (Figure 1 (4a, b)) while the trans-isomer (Figure 1 (4c, d)) had

a retention time of 15.0 min. The enzymatic products ( peak #5) were identified as

(cis/trans)-8-hydroxylinalool by the GC/MS library with match factors of 94 or 95%,

respectively. Owing to the fact that a (cis/trans)-8-hydroxylinalool-reference substance wasnot available, further confirmation was carried out by calculation of Kovacs

indices (KI) and interpretation of the EI fragmentation pattern. Calculated Kovacs index

of (cis)-8-hydroxylinalool (Figure 1 (5c, d), Rt 18.5 min) was KI 1302 and of the

isomer (trans)-8-hydroxylinalool (Figure 1 (5a, b), Rt19.0 min) was KI 1322.

Both calculated RI indices varied only approximately2.5% from the KI values reported

by Chassagne et al. (1999), indicating high consistence. The EI-mass spectrum of

(cis/trans)-8-hydroxylinalool is presented in Figure 3; and typical fragment ions resulting

from the EI-ionization process and their elucidation and fragmentation reactions are

presented in Table II. The intensity of the molecular peak at m/z170 (M) was very low

(abundance 2%), which is very common for secondary and primary alcohols.Some of the fragment ions are very common for EI-induced fragmentation of alcohols.

Loss of H2O (dehydration) is an example of such a typical fragmentation. Dehydration of

(cis/trans)-8-hydroxylinalool and of other main fragments was observed in the mass

spectrum several times.

Accordingly, the enzymatic conversion of (R/S)-linalool by CYP2C19 was studied

(chromatograms not shown). Only 8-hydroxylinalool was detected as metabolites, both

cyclic ethers were not found.

P450-mediated enzymatic assays

The relationship between enzymatic product formations catalyzed by both CYP was

t di d d ib d b f i b ti ti t ti d b t t

Table I. Calculated match factors (MFs) by the NIST GC/MS library.

Match factor (MF) (%)

(Enzymatic) product P450 2C19 P450 2D6

Furanoid-linalool oxidea (2) 82.8 86.8

Dihydrolinalool (3) 79.2 76.9

Pyranoid-linalool oxidea (4) 78.7 77.8

8-Hydroxylinalool (5) 95.5 94.1

aConfirmed also by reference substance.



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53

71

152

137

119

84

110

97

40 60 80 100 120 140 160 180 200

10

20

30

40

50

60

70

80

90

100

Abundance[1

0E3]

m/z

HO

OH

m/z=71

m/z=43

H

H

m/z=18 H

m/z=18

Figure 3. EI-mass spectrum and proposed fragmentation of (cis/trans)-8-hydroxylinalool.

Table II. Fragment ions used for identification of the enzymatic product (cis/trans)-8-hydroxylinalool.

Fragment ion m/z Description Postulated dissociation reaction/fragment ion

152 Neutral loss MH2O

137 Loss of m/z 15 from m/z152 CH3119 Neutral loss of m/z18 from m/z137 H2O

84 Dissociation of fragment ion m/z137 C4H

5

71 Dissociation of alkyl group MC6H11O

53 Neutral loss of m/z 18 from m/z 71 H2O



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concentration dependency. The time dependency of the enzymatic product formation by

enzymatic reactions catalyzed by both CYP was linear for all identified enzymatic products.

Using incubation times from 0 to 40 min, calculated regression coefficients showed a mean

r2 of 0.95 with SD0.04. Increase of enzyme concentrations by a factor of 2 resulting in

CYP concentrations of 148 pmol ml1 for CYP2D6 and 220 pmol ml1 for CYP2C19,

respectively, led also to an increase of the enzymatic activity by a factor of 2 (mean

r2 0.992, SD0.01).

Linalool concentration dependency of the enzymatic products formation was studied by

the use of LineweaverBurk analysis and was linear for all identified enzymatic products

(mean r2 0.92, SD0.09). Calculated specific enzymatic properties of the CYP enzymes

are presented in Table III.

Discussion

The knowledge that CYP-mediated metabolism occurs in skin tissue resulted in the

hypothesis that reactive oxidation products that cause skin sensitization may not only result

from air auto-oxidation but also from enzymatic origin. To prove this hypothesis we studied

the CYP-mediated oxidative metabolism of linalool and tried to identify biological

reactive enzymatic products equal or different to reactive auto-oxidation products as

reported in the past.

Results from enzymatic assays showed that enzymatic reactions catalyzed by CYP2C19

and CYP2D6, two CYP identified in skin tissue (Ahmad et al. 1996; Yengi et al. 2003),

resulted in the formation of several different enzymatic products. The enzymatic product,

cis- and trans-8-hydroxylinalool, could be identified by the GC-MS library and

fragmentation pattern and was confirmed by comparison of calculated Kovacs index with

literature data. The enzymatic products are probably the result of the allylic-hydroxylation

substitution reaction of one of the methyl group situated at the 8-carbon atom of the linalool

molecule. Similar to our results Letizia et al. (2003) identified 8-hydroxylinalool as an

enzymatic product of linalool using mammalian CYP prepared from rat livers and lung.

Comparing the hydroxylation activity of both CYP, CYP2C19 had a higher enzymatic

affinity to linalool than CYP2D6, but the catalytic efficiency (Kcat/Km) was 25% less than

for CYP2D6 (Table III).

Allylic-hydroxylation reaction of substrates containing (conjugated)--bonded carbon

atoms resulting in allylic alcohols has been reported as a common P450-mediated catalyzed

enzymatic reaction (Bylund et al. 1998; Wrighton et al. 1990). Accordingly, allylic alcohols

have been identified as enzymatic products from other monoterpenes such as limonene

(Miyazawa et al. 1998), 1,8-cineole (Duisken et al. 2005b; Miyazawa and Shindo, 2001;

Miyazawa et al. 2001) and 3-carene (Duisken et al. 2005a). Catalyzed hydroxylation

activities for the enzymatic conversion of limonene by CYP2C19 for the enzymatic product

carveol (Km 0.46 mM) and perillyl alcohol (Km 0.26 mM) were in approximately the same

range as for the catalyzed 80-allylic hydroxylation reaction of linalool (Table III).

The hydroxylation of 1,8-cineol by other CYP such as CYP3A4 and CYP3A5 showed

significantly lowerKmvalues (between 19 mM and 141 mM; Duisken et al. 2005b; Miyazawa

and Shindo, 2001).

The other enzymatic products of CYP2D6 were identified by the use of referencesubstances as (R/S)-furanoid-linalool oxide and (R/S)-pyranoid-linalool oxide. Both were

t d i l i t d i th t id ti f li l l d th ll i



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Table

III.

Michaelis-Mentenkineticvaluesofthe6,7

0-epoxidationand80allylic-hydroxylationshowninFigure

4(F

furanoid,

P

pyranoidenzymaticproduct).

Km

mM

maxmmolmin1mmol1CYP

Kcats1

Kcat/

Kms1mmol1

450

Epox.

Hydrox.

Epox.

Hyd

rox.

Epox.

Hydrox

Epox.

Hydrox

6,7

0-F

6,7

0-P

80

6,7

0-F

6,7

0-P

8

0

6,7

0-F

6,7

0-P

80

6,7

0-F

6,70-P

80

C19

0.1

4

11

.7

5.1

103

3.6

104

D6

3.9

11.7

1.3

0.1

3

0.3

5

144.9

5.6

101

1.5

102

6.3

104

1.4

101

1.3

101

4.8

104



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properties of identified auto-oxidation products (Skold et al. 2004). In this study the

formation of (R/S)-furanoid-linalool oxide and (R/S)-pyranoid-linalool was explained by

the formation of a tertiary hydroperoxide (7-hydroperoxy-3,7-dimethylocta-1,5-diene-3-ol),

which can rearrange to produce an epoxide as a secondary oxidation product. This epoxide

is then readily attacked by the hydroxyl group of linalool, on either one of the two epoxide-

carbons (60 and 70carbon), which then results in the formation of two different cyclic ethers.

Both cyclic ethers were also identified in our study, but as the result of an enzymatic reaction

catalyzed by CYP2D6. Therefore, we postulate that the CYP2D6 first catalyzes the

enzymatic epoxidation of the 6,70-carbon double bond in the linalool molecule, which is

then followed by the intramolecular rearrangement leading to the two cyclic ethers

(Figure 4). We speculate that the rearrangement reaction is not a CYP2D6-catalyzed

reaction since, like Skold et al. (2004), we found a higher amount for the furanoid derivative

(0.3 nmol nmol-1 CYP2D6) than for the pyranoid derivative (0.04 nmol nmol1 CYP2D6).

The precursor 6,7-epoxylinalool and the furanoid and pyranoid ethers were detected as

natural products in fruits by Winterhalter et al. (1986). Interestingly, biodegradation of

linalool by different Aspergillus niger fungus strains yielded also the furanoid andpyranoid linalool oxides (Demyttenaere et al. 2001) showing that this biological

t f ti f li l l t b l i diff t i

OH

O

HO

OH

OH

(cis/trans)- 8-Hydroxylinalool

OH

O

6,7-Epoxy-linalool

O

HO

(R/S)-Linalool

8-Allylic-hydroxylation(P450 2C19 and P450 2D6)

6,7-Epoxidation

(P450 2D6)

(R/S)-pyranoid-linalooloxide (R/S)-furanoid-linalooloxide

Figure 4. Scheme of postulated enzymatic reactions of the substrate (R/S)-linalool by CYP2C19 andCYP2D6 enzymes followed by intramolecular rearrangement.



12/15

Enzymatic epoxidation reactions of substrates catalyzed by CYP are common for

substrates with -bonded carbons in the molecule, for example aromatic terpenes and

olefins (Martinez and Stewart 2000). Recently, Nilsson et al. (2005) described the formation

of epoxides from the diene 5-isopropenyl-2-methyl-1-methylene-2-cyclohexene by human

liver microsomes; and Duisken et al. (2005a) identified 3-carene-epoxide as an enzymatic

product of3-carene by CYP1A2. Epoxidation of linalool by recombinant human CYP has,

to our knowledge, not been reported until now.

Kinetic analysis of the ether formation needs to be carefully interpreted because Km and

maxvalues include the epoxidation as well as the following cyclization reactions. The kinetic

values characterize the biological epoxidation only if epoxidation is the rate-limiting reaction

step. The enzymatic affinity of the CYP2D6 for the enzymatic epoxidation of the 6,70-carbon

double bound of linalool was approximately a factor 310 lower then the enzymatic affinity

for the catalyzed 80-carbon allylic hydroxylation (Table III). The catalytic efficiencies for the

formation of the furanoid and pyranoid enzymatic products were approximatley in the same

range but about a factor 30004000 lower than for the 80-allylic hydroxylation. Results

from the enzymatic assays indicate that there is no mechanism-based inactivation of the CYPby the one of the enzymatic products as reported for other olefins by Murray and

Reidy (1990). Regression analysis between enzyme product concentration in time

and enzyme concentration and substrate concentrations obtained for both CYP regression

lines with regression coefficients r240.90.

Conclusion

The results of our study confirm our hypothesis that reactive oxidation products of linaloolsuch as epoxides may also be formed by CYP catalyzed biological transformation. This is in

accordance with other recently published studies (Bergstrom et al. 2006; Duisken et al.

2005a; Nilsson et al. 2005). The formation of the identified cyclic ethers seems to be

possible by two different reactions: (i) an enzymatic conversion by oxidative CYP enzymes

followed by a rearrangement; and (ii) an auto-oxidation in the presence of air forming

a hydroperoxide, followed by the formation of an epoxide as a secondary oxidation product,

which is again rearranged to the cyclic ethers. The intermediary formed electrophilic epoxide

may cause sensitization and irritational skin reactions by linalool-containing

consumer products similar to other previously described auto-oxidation products such as

hydroperoxides (Skold et al. 2004). As a result, the use of preservatives against oxidation ofthe cosmetic ingredients may prohibit air oxidation to a certain extent, but the formation of

epoxides by CYP located in skin is still possible. Although in this in vitro system

the biological oxidation mainly proceeds via the non-epoxide route, a more pronounced

6,7-epoxide route in vivo cannot be excluded.

Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft. We thankour project partner Brunhilde Blomeke of the University of Trier for helpful discussions as

ll W lf D tt f th RWTH A h f hi ti t



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