A biocatalytic route towards rose oxide using chloroperoxidase

Article abstract of DOI:10.1016/j.foodchem.2011.05.068

The chiral monoterpene alcohol citronellol was converted to the corresponding bromohydrin by the haem-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago in the presence of hydrogen peroxide and bromide ions. A conversion rate of 51% could be achieved under adapted reaction conditions, which easily yield product in the gramme per litre range while only needing catalytic amounts of enzyme. The bromohydroxylation was shown to be highly regioselective yielding 6-bromo-3,7-dimethyloctane-1,7-diol as the sole product. Product identity was confirmed by GC-MS, ¹H- and ¹³C-NMR spectroscopy and the synthesis of reference compounds. However, the reaction was shown to be non-stereospecific because enantiopure (R)- and (S)-citronellol, respectively, gave 1:1-diasteromeric mixtures of the corresponding bromohydrins. A racemic mixture of (R/S)-citronellol was bromohydroxylated without any detectable enantiodiscrimination. The total lack of stereospecificity and enantiodiscrimination points to a reaction mechanism where the oxidised bromide intermediate is not a ligand to the Fe(III)-haem at the distal site but is released from the enzyme active site. The final bromide transfer occurs probably outside the active site via a diffusible oxidised bromide species and the demonstrated regioselectivity is purely chemically controlled. The generated bromohydrins can be straightforward converted via two reactions steps into rose oxide which is a highly valuable flavour and fragrance substance.

Full text of DOI:10.1016/j.foodchem.2011.05.068

Food Chemistry 129 (2011) 1025–1029
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
A biocatalytic route towards rose oxide using chloroperoxidase
a
b
c
c,
⇑
Umberto Piantini , Jens Schrader , Andrzej Wawrzun , Matthias Wüst
a
Institut Technologies du Vivant, Haute Ecole Spécialisée de Suisse occidentale, Route du Rawyl 47, CH-1950 Sion, Switzerland
DECHEMA e.V, Karl-Winnacker-Institut, Biochemical Engineering, Theodor-Heuss-Allee 25, D-60486 Frankfurt, Germany
Fachgebiet Lebensmittelchemie, Institut für Ernährungs- und Lebensmittelwissenschaften, Rheinische Friedrich-Wilhelms-Universität Bonn,
b
c
Endenicher Allee 11-13, D-53115 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The chiral monoterpene alcohol citronellol was converted to the corresponding bromohydrin by the
haem-thiolate enzyme chloroperoxidase (CPO) from Caldariomyces fumago in the presence of hydrogen
peroxide and bromide ions. A conversion rate of 51% could be achieved under adapted reaction condi-
tions, which easily yield product in the gramme per litre range while only needing catalytic amounts
of enzyme. The bromohydroxylation was shown to be highly regioselective yielding 6-bromo-3,7-
dimethyloctane-1,7-diol as the sole product. Product identity was confirmed by GC–MS, 1H- and C-
NMR spectroscopy and the synthesis of reference compounds. However, the reaction was shown to be
non-stereospecific because enantiopure (R)- and (S)-citronellol, respectively, gave 1:1-diasteromeric
mixtures of the corresponding bromohydrins. A racemic mixture of (R/S)-citronellol was bromohydroxy-
lated without any detectable enantiodiscrimination. The total lack of stereospecificity and enantiodis-
crimination points to a reaction mechanism where the oxidised bromide intermediate is not a ligand
to the Fe(III)-haem at the distal site but is released from the enzyme active site. The final bromide transfer
occurs probably outside the active site via a diffusible oxidised bromide species and the demonstrated
regioselectivity is purely chemically controlled. The generated bromohydrins can be straightforward con-
verted via two reactions steps into rose oxide which is a highly valuable flavour and fragrance substance.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 13 December 2010
Received in revised form 24 March 2011
Accepted 18 May 2011
Available online 25 May 2011
13
Keywords:
Halohydrin
Bioconversion
Haloperoxidase
Monoterpene
Enzymatic halogenation
1
. Introduction
Chloroperoxidase (CPO) is a 42-kDa haem-thiolate enzyme that
limonene yielded exclusively the 1,2-diols with high regioselectiv-
ity but very low stereoselectivity (Aguila et al., 2008). This work
describes the regioselective CPO catalysed bromohydroxylation of
the chiral monoterpene alcohol citronellol 1 to the corresponding
bromohydrins 2 (Fig. 1). This oxyfunctionalisation of citronellol is
of large interest for the flavour and fragrance industry because
the reaction is the key step of numerous synthetic routes to rose
oxide 6 which is a valuable flavour and fragrance compound and
is currently produced on >100 tons per year industrial scale by
photooxidation of citronellol (Bicas, Dionisio, & Pastore, 2009).
Although light can be regarded as a ‘‘green’’ reagent, the high
energy demand of artificial light sources must not be disregarded
(Bicas et al., 2009). The need of bulk amounts of organic solvents,
such as methanol or acetonitrile, and of pigments used as triplet
photosensitisers, which may interfere with product purification,
are additional drawbacks (Dincalp & Icli, 2001). However, biotech-
nological attempts to develop a straightforward and environmen-
tally friendly route from citronellol to rose oxide have been so
far less successful. One of the first publications in this area by
Onken and Berger (1999) reported the biotransformation of citro-
nellol by the basidiomycete Cystoderma carcharias in an aerated
membrane bioreactor yielding rose oxide in small amounts (up
to 10 mg per day) from the exhaust air of the bioreactor. Aspergillus
sp. and Penicillium sp. were screened for their ability to bioconvert
is secreted by the fungus Caldariomyces fumago. It catalyses the
hydrogen peroxide-dependent chlorination of the intermediate
cyclopentanedione during the biosynthesis of the antibiotic calda-
riomycin (Kühnel, Blankenfeldt, Terner, & Schlichting, 2006). Addi-
tionally, CPO catalyses the halogenations and oxidations of a wide
range of substances because it shows only low substrate specificity
(Blasiak & Drennan, 2009; Butler & Sandy, 2009). This feature
makes CPO an attractive catalyst for bio-oxidation reactions of a
wide range of applications using low cost oxidising agents like
hydrogen peroxide (Dembitsky, 2003). Surprisingly, despite the de-
tailed investigation of the catalytic potential of CPO, only until re-
cently monoterpenoids have been shown to be substrates for this
enzyme (Aguila et al., 2008; Kaup, Piantini, Wüst, & Schrader,
2
007). However, the chemistry of the catalysed reaction is highly
dependent on the chemical structure of the substrate and the reac-
tion conditions. In the presence of halide ions the bicyclic monoter-
pene carene yielded exclusively the corresponding halohydrins
with high stereoselectivity (Kaup et al., 2007) while monocyclic
⇑
Corresponding author. Tel.: +49 228 73 2361; fax: +49 228 73 3499.
E-mail address: matthias.wuest@uni-bonn.de (M. Wüst).
0
308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.05.068

1
026
U. Piantini et al. / Food Chemistry 129 (2011) 1025–1029
2.3. GC-FID
The bromohydrins were initially analysed with an Agilent
(
Böblingen, Germany) 6890 N gas chromatograph by split injection
at 250 °C of 1 l with a split ratio of 50:1. A DB-WAX capillary col-
umn (30 m, 0.25 mm i.d., 0.25 m) from J&W Scientific (Wald-
l
l
bronn, Germany) was used with He at 0.8 ml/min (22 cm/s) as
carrier gas. The oven was programmed from 50 to 240 °C at a rate
of 5 °C/min. The detector was a FID at 300 °C; hydrogen flow 40 ml/
min; air flow 450 ml/min.
2.4. GC–MS
Reaction products were analysed with a Varian (Darmstadt,
Germany) Chrompack CP 3800 gas chromatograph by split injec-
tion at 240 °C of 1 l with a split ratio of 50:1. A DB-225MS capil-
lary column (30 m, 0.25 mm i.d., 0.25 m) from J&W Scientific
Waldbronn, Germany) was used with He at 0.8 ml/min (22 cm/s)
l
l
(
as carrier gas. The oven was programmed from 50 to 240 °C at a
rate of 5 °C/min. The detector was a Varian Saturn 2000 ion trap.
Transfer line temperature 220 °C; ion trap temperature 150 °C;
scan range 40–650 amu; ionisation mode: EI with automatic gain
control (AGC); emission current 10
and multiplier delay 5 min.
lA; scan time 1.0 s; filament
Fig. 1. Chloroperoxidase-catalyzed formation of the diastereomeric bromohydrins
a/2b from (R)-citronellol (R)-1 and conversion of 2a/2b to the corresponding
2
epoxides 3a/3b or to rose oxide 6 via the diols 4 and 5; DMSO, dimethyl sulfoxide;
t-BuOK: potassium tert-butylate.
2.5. Enzymatic conversions
For monoterpene conversions a previously established protocol
was used (Kaup et al., 2007) and adapted (see Table 1). Briefly,
approximately 100 ll of CPO (4 U) was incubated in 100 mM citric
acid buffer, pH 3.6 with 30% (v/v) tert-butanol, 12 mM of the mon-
terpene substrate and 10 mM sodium bromide. Hydrogen peroxide
was added to a total concentration of 10 mM over a reaction time
of 60 min in 50 ll portions every minute. As negative control either
CPO or hydrogen peroxide was omitted from the assays described
above. Reaction was monitored by TLC (n-pentane/ethyl acetate 5/
citronellol to rose oxide (Demyttenaere, Vanoverschelde, & De
Kimpe, 2004) but the yields were rather low (1–3% of rose oxide).
More recently the group of Pastore was able to produce up to
1
00 mg/l of rose oxide using sporulated surface cultures of Penicil-
lium sp. (Marostica, Macedo, & Pastore, 2005). In this study a novel
biocatalytic approach for the synthesis of rose oxide is described by
combining the CPO catalysed oxyfunctionalisation of citronellol
with a chemical two step synthesis. The results are also discussed
in the context of the reaction mechanism of the CPO-catalysed oxy-
halogenation which remained up to now controversial, with two
different hypotheses (Libby, Beachy, & Phipps, 1996; Reddy et al.,
4
v/v) using anisaldehyde/sulphuric acid as spray reagent. Samples
were extracted with n-hexane, dried over sodium sulphate and
stored at ꢁ20 °C until GC–MS analysis. Enzymatic conversions
were conducted independently at least in triplicate. For NMR
analysis the samples were purified by flash chromatography (n-
pentane/ethyl acetate 5/4 v/v).
2
002).
2
. Materials and methods
2.1. Materials
Table 1
Conversion rates of CPO catalyzed oxidation of (R/S)-citronellol (12 mM) using
different reaction conditions.
(
R/S)-Citronellol and enantiomers were purchased from Aldrich
Hydrogen Bromide pH tert-butanol Temperature aConversion rate
(
(
Germany). Chloroperoxidase was purchased from Fluka
Germany). All other chemicals were analytical reagent grade.
peroxide
mM]
[mM]
[vol.%]
[°C]
[%]
[
Silica gel 60 (0.040–0.063 mm) for flash chromatography was
purchased from Merck (Germany). TLC plates Polygram Sil G/
UV254, 0.2 mm, 8 ꢀ 4 cm, were purchased from Macherey Nagel
10
10
10
10
10
10
10
10
10
10
–
2.1 10
25
25
25
25
25
25
25
45
25
25
25
0
30
36
26
51
48
19
11
0
1
1
1
1
1
1
1
–
0
0
0
0
0
0
0
2.7 10
3.6 10
4.6 10
3.6 30
3.6 40
3.6 46
3.6 10
3.6 10
3.6 10
3.6 10
(
Germany).
2
.2. NMR
1H- and 13C-NMR spectra were recorded with a XP-400 spec-
trometer (Bruker, Rheinstetten, Germany) at 400 ( H) and
00 MHz ( C) at ambient temperature. All chemical shifts are gi-
ven with respect to the solvent (CDCl
7.0 ppm for C). Hydrogen signals were assigned by H/ H-COSY
techniques using standard pulse programs as provided by Bruker
Rheinstetten, Germany).
1
10
10
0
0
1
3
b
1
10
1
3
= 7.26 ppm for H, and
a
Conversion rates were determined by GC–MS assuming equal response factors
1
3
1
1
7
for citronellol and its bromohydrine and expressed as% peak area of bromohydrine
with respect to the total peak area (bromohydrine + residual citronellol).
b
No enzyme added.
(

U. Piantini et al. / Food Chemistry 129 (2011) 1025–1029
1027
2
.6. Synthesis of reference compounds
spectra indicating the presence of isomers (Fig. 2). The products
could be identified as 1:1 diastereomeric mixture of
6-bromo-3,7-dimethyloctane-1,7-diol 2, the diastereomeric ,b-
a
All four stereoisomers of the citronellol bromohydrins (6-bro-
a
mo-3,7-dimethyloctane-1,7-diol 2) and the citronellol epoxides
5-(3,3-dimethyloxiran-2-yl)-3-methylpentan-1-ol 3) were syn-
thesised as previously published (Demuth, Xing, & Schaffner,
bromohydrins of citronellol (Fig. 1). The corresponding reference
compounds were synthesised as a 1:1 diasteromeric mixture by
regioselective formation of the bromohydrin using racemic
citronellol 1 and N-bromosuccinimide. The reference compounds
co-eluted with the CPO-generated products when co-injected in
GC. Products and reference compounds showed identical mass
(
2
(
001) using racemic citronellol as starting material. Spectral data
H/ C-NMR) were identical to those previously published (De-
1
13
muth et al., 2001). MS data (low resolution ion trap; EI 70 eV) for
both diastereomers of 6-bromo-3,7-dimethyloctane-1,7-diol 2:
m/e (relative intensity): Diastereomer 1: 178 (31), 176 (31), 137
1
13
spectra and identical H- and C-NMR spectra. The presence of a
1:1 diastereomeric mixture of the ,b-bromohydrins 2 is unequiv-
a
1
3
1
(
16), 121 (16), 97 (60), 81 (21), 70 (35), 59 (100), 55 (46). Diaste-
reomer 2: 178 (29), 176 (27), 137 (25), 121 (19), 97 (100), 81
37), 70 (49), 59 (64), 55 (65).
ocally proven by the splitting of the C- and H-NMR signals. An
illustrative example is the signal of the H-6 proton which is at-
tached to the chiral centre and shows two double doublets indicat-
ing the presence of a diastereomeric mixture (Fig. 3).
(
2
.7. Synthesis of rose oxide
Two minor peaks were detected as well by GC–MS analysis of
the generated CPO-catalysed reaction products (Fig. 2) and
identified as citronellol epoxide diastereomers (5-(3,3-dimethy-
loxiran-2-yl)-3-methylpentan-1-ol 3). The corresponding refer-
ence compounds were synthesised by epoxidation of racemic
citronellol by m-chloroperbenzoic acid (Demuth et al., 2001). These
epoxides were identified as artifacts and are generated in the cap-
illary column during the gas chromatographic analysis by an intra-
molecular SN2 reaction of the bromohydrin. In support of this
finding the epoxides are visible as well when pure, synthetically
derived, bromohydrin is injected. When a carbowax stationary
phase was used the bromohydrins were even completely con-
verted into the epoxides in the capillary column. Nevertheless,
the formation of the diastereomeric epoxides 3 when the CPO reac-
tion product is treated with sodium hydroxide is a second indepen-
dent proof of structure of the generated enzymatic products
Citronellol bromohydrins were converted into cis-/trans-rose
oxide 6 using a previously published procedure (Demuth et al.,
2
001).
3
. Results
Both enantiomers and a racemic mixture of citronellol 1 have
been tested as substrates for the CPO-catalysed bio-oxidation in
the presence of hydrogen peroxide and sodium bromide as halide
source. In all cases a single product was obtained as indicated by
thin layer chromatography on silica gel. The conversion was
adapted with respect to pH and volume of added tert-butanol
and conversion rates up to 51% could be achieved after 1 h reaction
time at pH 3.6 (see Table 1). Isolated yields were in the gramme
range after purification by flash chromatography. In a typical
reaction 1.87 g citronellol gave 1.84 g of pure product. Analysis
by GC–MS revealed the presence of two very closely eluting major
products peaks of equal peak height with almost identical mass
(
Fig. 1).
The fact that both enantiomers of citronellol 1 gave a 1:1
mixture of the corresponding bromohydrin diastereomers
demonstrates that the CPO-catalysed bromohydroxylation is
2
Fig. 2. GC–MS analysis of the reaction mixture after incubation of (R)-citronellol (R)-1 with chloroperoxidase. Enlargement of the peak which is attributed to the
bromohydrin reveals the presence of two closely eluting diastereomers 2a/2b (enlargement not shown). The presence of two diastereomers is visible for the epoxides 3a/3b
which are artifacts that are generated in the in the capillary column of the GC system.

1
028
U. Piantini et al. / Food Chemistry 129 (2011) 1025–1029
et al., 2002). Regardless of the exact species responsible for halo-
genations, our results corroborate reports on non-stereospecific
CPO-catalysed bromohydroxylation (Kollonitsch et al., 1970)
and show that large substrates probably are unable to access
haem intermediates in the active sites and likely react with dif-
fusible oxidised bromide species outside the active site. For effi-
cient CPO-catalysed epoxidation of short-chain alkenes with
double bonds close to the chain terminus the critical chain
9
length was C and demonstrates the rather small active site of
CPO (vanDeurzen, vanRantwijk, & Sheldon, 1997). The observed
regioselectivity in the CPO-catalysed formation of the bromohy-
drins is probably purely chemically controlled and in agreement
with a reaction that takes place via a distorted bromonium ion
that reacts with a hydroxyl ion via a trans addition at the
tertiary C atom.
The generated bromhydrins of citronellol 2 are interesting
intermediates for the synthesis of valuable flavour and fragrance
substances like rose oxide 6 which can be obtained after treatment
of the bromohydrins with potassium tert-butylate followed by acid
treatment (Demuth et al., 2001). This reaction sequence yields a
high percentage of cis-rose oxide which is the most valuable and
appreciated diastereomer in the flavour and fragrance industry.
Noteworthy, mimicking such monoterpene bio-halohydroxylation
in the presence of halide salts by chemical means would need a
sixfold stoichiometric concentration of fluoroboric acid to activate
the halide ion instead of a nanomolar enzyme preparation only,
as shown for the chemical conversion of 3-carene (Barluenga,
Marco-Arias, Gonzáles-Bobes, Ballesteros,
& Gonzáles, 2004).
Moreover, the CPO-catalysed oxyfunctionalisation of citronellol is
a green chemistry approach that avoids the high energy demand
of the artificial light driven, dye sensitised photooxidation of citro-
nellol which is currently performed industrially on >100 tons per
year scale for the synthesis of cis- and trans-rose oxide (Bicas
et al., 2009).
Fig. 3. Section of the 1H-NMR spectrum of the diastereomeric bromohydrins 2a/2b
generated from (R)-citronellol (R)-1 by chloroperoxidase. The section shows the
splitting of the H-6 proton of the respective diastereomers. The signal assignments
of the H-6 and H-6 protons of the different diastereomers were arbitrarily chosen
and are interchangeable.
0
Acknowledgement
We thank Stefan Kehraus for recording the NMR spectra.
non-stereospecific. The same result was obtained with racemic cit-
ronellol demonstrating the complete lack of enantiodiscrimination.
To illustrate the synthetic usefulness of the CPO-catalysed bro-
mohydroxylation of citronellol the bromohydrins were converted
into rose oxide 6 via the diols 4 and 5 in two reaction steps with
yields of 77 and 60%, respectively (Fig. 1).
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