Multi-enzyme cascade synthesis of the most odorous stereoisomers of the commercial odorant Muguesia

Article abstract of DOI:10.1016/j.molcatb.2014.10.006

The most odorous stereoisomers of the chiral commercial fragrance Muguesia are prepared by a very effective linear biocatalysed cascade reaction, in which a suitable unsaturated ketone is submitted to the sequential action of two enzymes, an ene-reductase and an alcohol dehydrogenase, which are added together to the same reaction vessel with the cofactor regeneration system. Two stereogenic centres in 1,2 relative position are thus created under high stereochemical control by a two-step one-pot enzymatic procedure.

Full text of DOI:10.1016/j.molcatb.2014.10.006

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Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Multi-enzyme cascade synthesis of the most odorous stereoisomers of
the commercial odorant Muguesia®
Elisabetta Brennaa,∗, Michele Crottia, Francesco G. Gattia, Daniela Montib,
Fabio Parmeggiania, Andrea Pugliesea, Sara Santangeloa
a Politecnico di Milano, Dipartimento di Chimica, Materiali, Ingegneria Chimica, Via Mancinelli 7, I-20131 Milano, Italy
b Istituto di Chimica del Riconoscimento Molecolare–CNR, Via M. Bianco 9, I-20131 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
The most odorous stereoisomers of the chiral commercial fragrance Muguesia® are prepared by a very
effective linear biocatalysed cascade reaction, in which a suitable unsaturated ketone is submitted to
the sequential action of two enzymes, an ene-reductase and an alcohol dehydrogenase, which are added
together to the same reaction vessel with the cofactor regeneration system. Two stereogenic centres in
1,2 relative position are thus created under high stereochemical control by a two-step one-pot enzymatic
procedure.
Article history:
Available online xxx
Keywords:
Cascade reaction
Ene-reductase
Alcohol dehydrogenase
Chiral fragrance
Stereoselectivity
© 2014 Published by Elsevier B.V.
1. Introduction
impact their registration and use. The importance of the chiral
switch procedure in fragrance chemistry has been established,
The phenomenon of the enantioselectivity in odour perception
has been intensively investigated in the last years [1]. Human olfac-
tory receptors are chiral and can discriminate the two enantiomers
of a chiral odorous molecule [2]. The most common situation is
the one in which both enantiomers possess qualitatively similar
odours of quantitatively different intensities. It is also possible
that the two enantiomers are not differentiated by the receptors,
so both of them elicit a similar, weak odour. In the case of very
potent odorants, which are nearly totally complementary to the
binding pocket of the olfactory receptor proteins, the odour of
the molecule is most likely due to one enantiomer only, whereas
the other one is weak to odourless. The rarest case is when
both enantiomers of an odorant are mainly complementary to
different receptors, thus they possess completely different odour
profiles.
The evaluation of the olfactory properties of the enantiomers of
chiral odorants can lead to novel ways of assessing and enhanc-
ing product performance. As a matter of fact, the use of the
most potent stereoisomers allows the content of chemicals in per-
fumery products to be decreased, with a consequent beneficial
reduction of their environmental load. Furthermore, stereoiso-
mers may exhibit different biodegradation properties [3] that
and some commercial odorants are now being sold as single
enantiomers (e.g. Paradisone®, Dextro Norlimbanol®, Thesaron®,
Citronellyl Nitrile®, l-Laurinal®). However, the possibility to pro-
mote the use of enantiomerically enriched odorous ingredients
is strictly related to the viability of a high yield enantiose-
lective procedure to the desired enantiomer, showing advanta-
geous price-performance ratio in comparison with the racemic
material.
Nearly ten years ago we prepared [4] all the four stereoisomers
of the chiral odorant Muguesia® (1) (Scheme 1), according to a pro-
cedure based on the use of lipase-mediated kinetic resolution. The
synthetic sequence had been devised to obtain all the stereoisomers
in enantiopure form, in order to submit them to odour evaluation.
The configuration of the carbon atom in position 3 was found
to be important in establishing the odour properties of Muguesia®:
the (3R) stereoisomers 1c and 1d were described as weak and com-
pletely devoid of odour in the dry down note; the (3S) stereoisomers
1a and 1b were found to be the effective odour vectors of the com-
mercial odorant, with floral and lily of the valley notes.
We report now on an efficient biocatalysed procedure for the
stereoselective synthesis of the odorous (3S)-stereoisomers of
Muguesia®, based on a multi-enzyme cascade reaction which com-
bines the reduction of the activated carbon carbon double bond
of (E)-3-methyl-4-phenylbut-3-en-2-one (2), mediated by an ene-
reductase (ER), with the reduction of the carbonyl group catalyzed
by an alcohol dehydrogenase (ADH).
∗
Corresponding author. Tel.: +39 02 23993077; fax: +39 02 23993180.
E-mail address: mariaelisabetta.brenna@polimi.it (E. Brenna).
http://dx.doi.org/10.1016/j.molcatb.2014.10.006
1381-1177/© 2014 Published by Elsevier B.V.
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2
pKTS) was inoculated with a single colony from a fresh plate and
grown for 8 h at 37 ◦C and 220 rpm This starter culture was used to
inoculate 200 mL medium, which was incubated for 8 h at the same
conditions and used to inoculate 1.5 L medium. The latter culture
was shaken at 37 ◦C and 220 rpm until OD600 reached 0.4–0.5, then
enzyme expression was induced by the addition of 0.1 mM IPTG
(50 ng mL−1 anhydrotetracycline was also added in the case of the
pKTS-GDH plasmid). After 5–6 h the cells were harvested by cen-
trifugation (5000 × g, 20 min, 4 ◦C) resuspended in 50 mL of lysis
buffer (20 mM potassium phosphate buffer pH 7.0, 300 mM NaCl,
10 mM imidazole) and disrupted by sonication (Omni Ruptor 250
ultrasonic homogenizer, five sonication cycles, 15 s each, 50% duty).
The cell-free extract, after centrifugation (20,000 × g, 20 min, 4 ◦C),
was chromatographed on IMAC stationary phase (Ni-Sepharose
Fast Flow, GE Healthcare) with a mobile phase composed of 20 mM
potassium phosphate buffer pH 7.0, 300 mM NaCl and a 10–300 mM
imidazole gradient. Protein elution was monitored at 280 nm, the
fractions were collected according to the chromatogram and dial-
ysed twice against 1.0 L of 50 mM potassium phosphate buffer pH
7.0 (12 h each, 4 ◦C) to remove imidazole and salts. Purified protein
aliquots were stored frozen at −80 ◦C.
Scheme 1. The four stereoisomers of Muguesia®
.
2. Experimental
2.1. General methods
TLC analyses were performed on Merck Kieselgel 60 F254
plates. All the chromatographic separations were carried out on
silica gel columns. 1H and 13C NMR spectra were recorded on a 400
or 500 MHz spectrometer. The chemical shift scale was based on
internal tetramethylsilane. GC/MS (EI) analyses were performed
2.4. Synthesis of (E)-3-methyl-4-phenyl-3-buten-2-one (2)
Benzaldehyde (4.24 g, 40 mmol) and 2-butanone (2.88 g,
50 mmol) were dissolved in AcOH (80 mL) under magnetic stir-
ring and conc. H2SO4 (4 mL) was added dropwise to the solution.
The mixture was stirred at r.t. for 24 h, then it was poured in ice
water (100 mL), neutralized with aq. NaOH (20% w/v) and extracted
with EtOAc (3 × 100 mL). The combined organic phase was washed
with water (100 mL) and brine (100 mL) and dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure and the
crude residue was purified by column chromatography using n-
hexane/EtOAc (9:1) as eluent, affording the unsaturated ketone
2 (4.42 g, 69%): 1H NMR [6] (400 MHz, CDCl3): ı = 7.51 (q, 1 H,
J = 1.2 Hz, C = CH), 7.36–7.44 (m, 5 H, aromatic hydrogens), 2.44 (s,
3 H, COCH3), 2.04 (d, 3 H, J = 1.5 Hz, CH3) ppm; 13C NMR (100.6 MHz,
CDCl3): ı = 199.9, 139.4, 137.7, 135.8, 129.5, 128.4, 128.3, 25.6,
12.8 ppm; GC/MS: tR = 16.40 min, m/z (%) = 160 (M+, 70), 159 (100),
145 (35), 117 (80), 115 (85).
using
a
HP-5MS column (30 m × 0.25 mm × 0.25 ␮m, Agilent).
The following temperature programme was employed: 60 ◦C
(1 min)/6 ◦C min−1/150 ◦C (1 min)/12 ◦C min−1/280 ◦C (5 min).
Chiral GC analyses of Muguesia® isomers were performed
on
a
Chirasil DEX CB column (25 m × 0.25 mm, Chrompack).
The following temperature programme was employed: 50 ◦C
(3 min)/2 ◦C min−1/120 ◦C/30◦/min/180 ◦C; tR acetate of (2S,3S)-
1b = 24.11 min, tR acetate of (2R,3R)-1c = 26.20 min, tR acetate of
(2S,3R)-1d = 24.37 min, tR acetate of (2R,3S)-1a = 26.17 min
2.2. Enzymes
OYE3 from Saccharomyces cerevisiae BY4741 and GDH from
Bacillus megaterium DSM509 were overexpressed in E. coli
BL21(DE3) strains harbouring specific plasmids (pET30a-OYE3 and
pKTS-GDH, respectively) prepared according to standard molecular
biology techniques [5]. The enzymes were produced and purified
as described in Section 2.3.
The ADHs employed were obtained from commercial sources:
CPADH and READH were purchased from Jülich; BYADH,
HLADH, TBADH, KRED, PLADH and DRADH were purchased from
Sigma–Aldrich.
2.5. General procedure for ADH-mediated biotransformations of
an equimolar mixture of (E)-3-methyl-4-phenylbut-3-en-2-one
(2) and 3-methyl-4-phenylbutan-2-one (3) (ADH
chemoselectivity screening)
Specific activities were measured as follows:
A solution of the equimolar mixture of ketones 2 and 3
in DMSO (10 ␮L, total concentration 500 mM) was added to a
potassium phosphate buffer solution (1.0 mL, 50 mM, pH 7.0) con-
taining glucose (20 ␮mol), NADP+ (0.1 ␮mol) or NAD+ (0.1 ␮mol)
(according to the ADH preference), GDH (4 U) and the required
ADH (200 ␮g mL−1). The mixture was incubated for 24 h in an
orbital shaker (160 rpm, 30 ◦C). The solution was extracted with
EtOAc (2 × 250 ␮L), centrifuging after each extraction (15,000 × g,
1.5 min), and the combined organic solutions were dried over anhy-
drous Na2SO4.
OYE3: 1 U equals to the amount of enzyme that reduces 1 ␮mol
of ␣-methylcinnamaldehyde per minute, as measured by GC anal-
ysis (biotransformation conditions: 5 mM substrate, 1% v/v DMSO,
0.1 mM NADP+, an aliquot of OYE solution, 4 U GDH, 50 mM potas-
sium phosphate buffer pH 7.0, total volume 1 mL, 160 rpm, 30 ◦C,
30 min).
ADHs: according to the data provided by the manufacturers
(related to different substrates and conditions).
GDH: 1 U equals to the amount of enzyme that reduces 1 ␮mol of
NADP+ per minute, as measured spectrophotometrically at 340 nm
(reaction mixture: 20 mM D-glucose, 0.2 mM NADP+, an aliquot
of GDH solution, 50 mM potassium phosphate buffer pH 7.0, total
volume 1 mL).
2.6. General procedure for ADH-mediated biotransformations of
(S)-3-methyl-4-phenylbutan-2-one ((S)-3) (ADH stereoselectivity
screening)
Protein concentrations were determined according to Bradford,
using bovine serum albumin (BSA) as a standard.
A solution of ketone (S)-3 [7] in DMSO (10 ␮L, 500 mM) was
added to a potassium phosphate buffer solution (1.0 mL, 50 mM,
pH 7.0) containing glucose (20 ␮mol), NADP+ (0.1 ␮mol) or NAD+
(0.1 ␮mol) (according to the ADH preference), GDH (4 U) and the
required ADH (200 ␮g mL−1). The mixture was incubated for 24 h in
2.3. Overexpression of OYE3 and GDH in E. coli BL21(DE3)
LB medium (5 mL) containing the appropriate antibiotic
(50 ␮g mL−1 kanamycin for pET-30a, 100 ␮g mL−1 ampicillin for
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Scheme 2. Biocatalysed cascade reaction for the synthesis of the best stereoisomers of Muguesia®
.
an orbital shaker (160 rpm, 30 ◦C). The solution was extracted with
EtOAc (2 × 250 ␮L), centrifuging after each extraction (15,000 × g,
1.5 min), and the combined organic solutions were dried over anhy-
drous Na2SO4.
to prepare enantiopure building blocks [8]. We have also shown
the possibility to perform cascade reactions on ␣,␤-unsaturated-
␣-substituted aldehydes by combining a OYE-mediated reaction
with the reduction of the carbonyl group by means of an
alcohol dehydrogenase (ADH), in the aim of avoiding the for-
mation of the configurationally unstable ␣-substituted chiral
aldehydes [9].
In this work the concomitant action of the two types of enzymes
(ER and ADH) is exploited (Scheme 2) for the one-pot conversion of
the ␣,␤-unsaturated ketone 2 into the enantiopure stereoisomers
of the secondary alcohol 1, with the creation of two stereogenic
centres in 1,2 relative position under high stereochemical control.
All the enzymes are added to the reaction mixture from the begin-
ning and no isolation of the intermediate saturated ketone (S)-3 has
to be carried out. This is made possible by the use of ADHs showing
high preference for the reduction of the carbonyl group of the satu-
rated intermediate ketone 3, with no concomitant formation of the
allylic alcohol 4.
This is a linear biocatalysed cascade reaction [10], in which a
single substrate is converted into a single product in a two-step one-
pot fashion. The procedure is useful to save time and diminish waste
in a multi-step synthesis, because the telescoping process reduces
the number of intermediates to be submitted to purification
steps.
We had already investigated the OYE-mediated reduction of
substrate 2 [7], and we had established that OYE3 gave the best
results affording the reduced product (S)-3 showing an enan-
tiomeric excess (ee) value of 98% in quantitative yields. The
regeneration of the NADPH cofactor was performed using a glu-
cose dehydrogenase (GDH from Bacillus megaterium) with glucose
as a co-substrate.
As for the selection of the ADHs for the cascade procedures,
first we performed a screening of some commercial recombinant
and/or purified ADHs, in order to select those showing the highest
chemoselectivity towards the reduction of the saturated ketone,
and to avoid the formation of the allylic alcohol 4 which cannot be
transformed by OYEs.
The ADH-mediated reductions were carried out on an equimo-
lar mixture of the two ketone derivatives 2 and 3, using the
GDH/glucose system for cofactor regeneration. The results of this
screening are reported in Table 1.
For the choice of the most suitable ADHs, their stereoselectivity
was investigated by performing the carbonyl reduction on a sample
of (S)-3 (ee = 98%) obtained by OYE3-mediated reaction. The two
diastereoisomers of alcohol 1 that could be obtained from (S)-3,
i.e. anti-(2R,3S)-1a, and syn-(2S,3S)-1b, could be distinguished by
GC/MS analysis (see Experimental). The diastereoisomeric excess
(de) values obtained with the screened ADHs are reported in
Table 1, together with the corresponding conversion values.
READH and PLADH were selected for their chemoselectivity, and
their high conversion and stereoselectivity towards the two desired
diastereoisomers.
2.7. General procedure for OYE3-ADH-mediated synthesis of
(2R,3S)-3-methyl-4-phenylbutan-2-ol (anti-1a) and
(2S,3S)-3-methyl-4-phenylbutan-2-ol (syn-1b)
A
solution of unsaturated ketone
2
in DMSO (0.60 mL,
500 mM, 48 mg) was added to
a
potassium phosphate
buffer solution (9.40 mL, 50 mM, pH 7.0) containing OYE3
(0.6 U mL−1, 0.350 mg mL−1), the required ADH [PLADH 3 U mL−1
(0.170 mg mL−1) or READH 2.5 U mL−1 (0.750 mg mL−1)], GDH
(4 U mL−1), glucose (1.0 mmol, 180 mg) and NADP+ (2.5 ␮mol,
1.9 mg, for PLADH) or NAD+ (1.5 ␮mol, 1.0 mg, for READH). The
reaction mixture was shaken at 30 ◦C and 130 rpm for 48 h, and it
was monitored by GC/MS. The mixture was then extracted with
EtOAc (3 × 10 mL) and submitted to purification by bulb-to-bulb
distillation (135 ◦C, 20 mmHg).
2.7.1. Data of (2R,3S)-3-methyl-4-phenylbutan-2-ol (anti-1a)
From unsaturated ketone 2 by cascade reaction with OYE3 and
PLADH, after purification by distillation (38.4 mg, 78%): 98% purity
by GC/MS, de = 99% (GC/MS); ee = 99% (chiral GC of the correspond-
ing acetate derivative) [4]; [␣]D = + 29.0 (c 0.75, CHCl3); 1H NMR
(400 MHz, CDCl3) ı = 7.30–7.10 (m, 5H, aromatic hydrogens), 3.70
(apparent quintet, 1H, J = 6.1 Hz, CH3CHOH), 2.87 (dd, 1H, J = 13.6,
4.9 Hz, H-C(4)), 2.34 (dd, 1H, J = 13.6, 9.6 Hz, H-C(4)), 1.82 (m, 1H,
CHC(3)), 1.20 (d, 3H, J = 6.3 Hz, CH3CHOH), 0.83 (d, 3H, J = 7.0 Hz,
CH3C(3)) ppm; 13C NMR (100.6 MHz, CDCl3): ı 140.9, 128.9, 128.1,
125.4, 71.0, 42.2, 38.8, 19.7,14.6 ppm; GC/MS tR = 14.80 min, m/z (%)
164 (M+, 10), 146 (35), 131 (70), 91 (100).
2.7.2. Data of (2S,3S)-3-methyl-4-phenylbutan-2-ol (syn-1b)
From unsaturated ketone 2 by cascade reaction with OYE3 and
READH, after purification by distillation (36.9 mg, 75%): 95% purity
by GC/MS, de = 99% (GC/MS); ee = 99% (chiral GC of the correspond-
ing acetate derivative) [4]; [␣]D = –8.0 (c 0.75, CHCl3); 1H NMR
(400 MHz, CDCl3) ı 7.30–7.14 (m, 5H, aromatic hydrogens), 3.75
(dq, 1H, J = 3.9, 6.2 Hz, CH3CHOH), 2.83 (dd, 1H, J = 13.5, 5.9 Hz, H-
C(4)), 2.40 (dd, 1H, J = 13.5, 8.9 Hz, H-C(4)), 1.78 (m, 1H, CHC(3)),
1.20 (d, 3H, J = 6.2 Hz, CH3CHOH), 0.87 (d, 3H, J = 6.7 Hz, CH3C(3))
ppm; 13C NMR (100.6 MHz, CDCl3) ı 140.9, 129.1, 128.1, 125.6, 70.4,
41.8, 39.3, 20.6, 13.4 ppm; GC/MS tR = 14.61 min, m/z (%) 164 (M+,
5), 146 (35), 131 (43), 91 (100).
3. Results and discussion
We have recently investigated the use of ene-reductases (ERs)
belonging to the superfamily of Old Yellow Enzymes for the
enantioselective reduction of suitably substituted activated alkenes
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Table 1
Screening of a set of commercial ADHs for chemoa and stereoselectivityb evaluation.
Alcohol dehydrogenase
% Reduction of 2a,c
% Reduction of 3a,c
Chemoselectivity 3 vs 2
deb (%)
cb (%)
CPADH (from Candida parapsilosis)
1.5
42
n.r.
n.r.
n.r.
79
10
79
n.r.
n.r.
n.r.
99
6.7
1.9
–
–
–
1.3
3.2
1.2
33 (syn)
99 (syn)
–
–
–
99 (anti)
99 (anti)
78 (anti)
25
99
–
–
–
99
99
23
READH (from Rhodococcus erythropolis)
BYADH (from Saccharomyces cerevisiae)
HLADH (from horse liver)
TBADH (from Thermoanaerobium brockii)
KRED (ketoreductase, unspecified source)
PLADH (from Parvibaculum lavamentivorans)
DRADH (from Deinococcus radiodurans)
24
25
76
30
a
Experimental conditions: 0.8 g L−1 mixture of substrates 2 and rac-3, 200 ␮g mL−1 ADH, 4 U mL−1 GDH, 4 eq. glucose, 0.1 mM NAD+,0.1 mM NADP+,50 mMpotassium
phosphate buffer pH 7.0, 30 ◦C, reaction time 24 h.
b
Experimental conditions: 0.8 g L−1 (S)-3, 200 ␮g mL−1 ADH, 4 U mL−1 GDH, 4 eq. glucose, 0.1 mM NAD+,0.1 mM NADP+,50 mMpotassium phosphate buffer pH 7.0, 30 ◦C,
reaction time 24 h.
c
Calculated by using the percentage values of the GC analysis of the crude mixture after 24 h reaction time, respectively as (% compound 4)/(% compound 4 + % compound
2) and (% compound 1)/(% compound 1 + % compound 3).
(3 U mL−1), and READH (2.5 U mL−1), respectively. After 48 h reac-
100
(a)
tion time, the conversion to the corresponding alcohol was nearly
complete with productivity values of 4.8 and 4.4 g L−1 d−1, respec-
80
tively. Only in the reaction with PLADH traces of the allylic alcohol
were detected (2% by GC/MS).
3
60
1a
4
4. Conclusions
40
20
0
2
The high selectivity of biocatalysed reactions can be exploited to
develop effective and sustainable manufacturing processes to those
chiral fragrances for which the influence of absolute configuration
on odour properties could justify the commercialization in enan-
tiopure form. In the same reaction medium two enzymes, which are
able to catalyze two distinct and subsequent reductions without
substrate competition and with high stereoselectivity, are com-
bined for the one-pot conversion of the starting unsaturated ketone
into chiral compounds in enantiospecific and totally diastereose-
lective way.
This procedure represents a further demonstration of the syn-
thetic potential of enzyme-mediated reactions: the high chemo
and stereoselectivity that enzymes can achieve are key requisites
for the optimization of one-pot processes, which are now exten-
sively investigated for the synthesis of valuable compounds [11].
The diminished number of isolation steps and the high specificity
of biocatalysis, which minimizes undesired side-reactions, greatly
simplify existing manufacturing processes, with reduction of purifi-
cation costs and increase of sustainability.
0
10
20
30
40
50
Time [h]
100
80
60
40
20
0
(b)
3
1b
4
2
0
10
20
Time [h]
30
40
50
Fig. 1. Time course of (a) the OYE3-PLADH-mediated cascade reaction to (2R,3S)-1a
and (b) the OYE3-READH-mediated cascade reaction to (2S,3S)-1b.
Acknowledgement
Prof. Neil C. Bruce (Department of Biology, University of York) is
kindly acknowledged for the gift of plasmid pT7-OYE1. Prof. Sven
Panke and Christian Femmer (Department of Biosystems Science
and Engineering, ETH, Basel) are kindly acknowledged for the help
provided in the preparation of the plasmids and the overexpressing
strains.
The cascade reaction conditions were optimized by combining
the OYE3-mediated reduction of the carbon carbon double bond of
ketone 2, and the carbonyl reduction catalyzed either by READH or
PLADH, by using GDH and glucose to recycle the cofactors for both
reductions.
Surprisingly, the chemoselectivity of the two ADHs was found
to be higher in the cascade reactions, in spite of the presence of
the unsaturated ketone as a starting material. For example, when
substrate 2 (0.8 mg mL−1) was treated with OYE3 (0.3 U mL−1) and
either PLADH or READH (4 U mL−1), in the presence of the cofactor
regeneration system, stereoisomers (2R,3S)-1a and (2S,3S)-1b were
obtained in quantitative yields in 24 h reaction time without any
trace of the allylic alcohols.
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a preparative synthesis of the two stereoisomers 1a and 1b.
Fig. 1 shows the time course of the OYE3-PLADH- and OYE3-
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Please cite this article in press as: E. Brenna, et al., J. Mol. Catal. B: Enzym. (2014), http://dx.doi.org/10.1016/j.molcatb.2014.10.006