One-pot multi-enzymatic synthesis of the four stereoisomers of 4-methylheptan-3-ol

Article abstract of DOI:10.3390/molecules22101591

The use of pheromones in the integrated pest management of insects is currently considered a sustainable and environmentally benign alternative to hazardous insecticides. 4-Methylheptan-3-ol is an interesting example of an insect pheromone, because its stereoisomers are active towards different species. All four possible stereoisomers of this compound were prepared from 4-methylhept-4-en-3-one by a one-pot procedure in which the two stereogenic centres were created during two sequential reductions catalysed by an ene-reductase (ER) and an alcohol dehydrogenase (ADH), respectively.

Full text of DOI:10.3390/molecules22101591

molecules
Article
One-Pot Multi-Enzymatic Synthesis of the Four
Stereoisomers of 4-Methylheptan-3-ol
Elisabetta Brenna *, Michele Crotti , Francesco G. Gatti 1 , Daniela Monti 2,
1
,
1,†
ID
Fabio Parmeggiani 1
ID
and Andrea Pugliese 1
ID
1
Politecnico di Milano, Dipartimento di Chimica, Materiali, Ingegneria Chimica, Via Mancinelli 7,
I-20131 Milano, Italy; michele.crotti@polimi.it (M.C.); francesco.gatti@polimi.it (F.G.G.);
fabio.parmeggiani@polimi.it (F.P.); andrea.pugliese@polimi.it (A.P.)
Istituto di Chimica del Riconoscimento Molecolare—CNR, Via M. Bianco 9, I-20131 Milano, Italy;
daniela.monti@icrm.cnr.it
2
*
†
Correspondence: mariaelisabetta.brenna@polimi.it; Tel.: +39-02-2399-3077
Authors are listed in alphabetical order. M. C. is the principal author of the publication.
Received: 24 July 2017; Accepted: 21 September 2017; Published: 22 September 2017
Abstract: The use of pheromones in the integrated pest management of insects is currently
considered a sustainable and environmentally benign alternative to hazardous insecticides.
4
-Methylheptan-3-ol is an interesting example of an insect pheromone, because its stereoisomers are
active towards different species. All four possible stereoisomers of this compound were prepared from
-methylhept-4-en-3-one by a one-pot procedure in which the two stereogenic centres were created
during two sequential reductions catalysed by an ene-reductase (ER) and an alcohol dehydrogenase
ADH), respectively.
4
(
Keywords: pheromone; stereoselectivity; biocatalysis; ene-reductase; alcohol dehydrogenase
1
. Introduction
The control of native and invasive insects represents an increasingly urgent need as a consequence
of the changing climate, the increasing of season temperatures, and the altered rainfall patterns [1].
The thrust to green and sustainable insect control is produced not only by the concern about
the health of rural workers, but also by the necessity to improve food security for a growing
population while reducing the loss of environmental resources and biodiversity. The idea of replacing
hazardous insecticides with environmentally benign and species-specific semiochemicals (i.e., chemical
compounds produced by one organism in order to cause a behavioural change in an individual of
the same or a different species) is a current research challenge [2]. Semiochemicals which are used in
intraspecies communication are called pheromones; when interspecies communication is produced,
they are known as allelochemicals. Most of them are nontoxic to vertebrates, and they are released in
the environment in low amounts.
The most important applications of this strategy are related to the use of pheromones in the
integrated pest management of insects according to the following techniques: (i) monitoring a
population of insects to determine if they are present in a certain area or to determine if enough insects
are present to require a treatment; (ii) removal of large numbers of insects from the breeding and
feeding population by mass trapping; (iii) disruption of mating in populations of insects by dispersing
synthetic pheromone into crops to attract males away from females that are waiting to mate [3].
Many pheromones are enantiomerically enriched chiral compounds, since living organisms
employ chirality to expand and diversify their communication systems. The absolute configuration
of many naturally occurring pheromones has been established, and the relationship between
stereochemistry and bioactivity has been extensively studied [
4
]. The use of chiral pheromones
Molecules 2017, 22, 1591; doi:10.3390/molecules22101591
www.mdpi.com/journal/molecules

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in effective pest management campaigns requires the optimization of scalable and cost-effective
synthetic procedures to prepare the necessary stereoisomers in high enantiomeric purity and chemical
yield, using readily available starting materials.
4-Methylheptan-3-ol (
1) represents an interesting example of an insect pheromone whose
stereoisomers are active for different species. (3S,4S)-
1 [5] is one of the components of the aggregation
pheromone of the smaller European elm bark beetle (Scolytus multistriatus Marsham): it is produced
when unmated female beetles bore into American elm. Male large European elm bark beetles
(
S. scolytus) were found to produce traces of (3S,4S)-
tiny quantities of the (3R,4S)- ]. (3S,4S)- was also identified as the main component of the
aggregation pheromone of the almond bark beetle, S. amygdali Geurin-Meneville, together with
3S,4S)-4-methylhexan-3-ol, which acts as a synergist [ ]. In field tests, (3S,4S)- attracted beetles in
combination with the synergist (3S,4S)-4-methylhexan-3-ol, whereas (3R,4S)- and (3R,4R)- were
inhibitory [ ]. Furthermore, (3R,4S)- was discovered to be the trail pheromone of a southeast Asian
1 and (3R,4S)-1, while females produced only
1
[
6
1
(
7
1
1
1
8
1
ant, Leptogenis diminuta of the subfamily Ponerinae [9].
Herein we report on a very effective enzymatic approach for the enantioselective synthesis of all
four stereoisomers of compound 1, starting from unsaturated ketone 2 (Scheme 1), easily obtained by
mixed aldol condensation. The synthetic approach is based on a two-step one-pot multi-enzymatic
procedure for the sequential creation of the stereogenic centres under the stereochemical control
of two enzymes: an ene-reductase (ER) for the reduction of the C=C double bond and an alcohol
dehydrogenase (ADH) for the creation of the alcohol moiety.
Scheme 1. Retrosynthetic approach to the stereoisomers of compound 1. ADH: alcohol dehydrogenase;
ER: ene-reductase.
2
. Results
2
.1. Known Synthetic Routes
The synthetic routes of pheromone
1 reported in the literature are directed mainly to the
(3S,4S)-stereoisomer, taking advantage of a wide range of techniques: (i) enzymatic resolution of
threo-2-amino-3-methylhexenoic acid, and subsequent manipulation [10]; (ii) asymmetric induction
with boronic esters [11]; (iii) chirality transfer by ester enolate Claisen rearrangement [12];
(
iv) asymmetric [
baker’s yeast [14 15], or chiral palladium-phosphine catalysts [
by regioselective epoxide opening with trimethylaluminum [16].
Five methods have been described for the preparation of all four stereoisomers of compound
i) HPLC separations of the corresponding Mosher esters on analytical scale (stereoisomeric
purity = 94–98%) [17]; (ii) sequential creation of the two stereocentres with diastereoselectivity
99% by using chiral boronic esters [18]; (iii) separation of (3RS,4RS)- and (3RS,4SR)- by fractional
2
,
3
]-Wittig rearrangement [13]; (v) stereoselective synthesis catalysed by either
,
6
]; (vi) Sharpless epoxidation followed
1
:
(
>
1
1
crystallization of the corresponding 3,5-dinitrobenzoate esters, followed by hydrolysis and subsequent
lipase-mediated transesterification of each racemic diastereoisomer with Candida antarctica lipase B,
using S-ethyl octanethioate as an acyl donor (stereoisomeric purity = 61–93%) [19]; (iv) reduction of
3
-propyl-4-thianone to yield easily separable cis- and trans-3-propyl-4-thianols, submitted to resolution
by chromatographic separation of the corresponding esters with (S)-chlorofluoroacetic acid, followed
by hydrolysis and desulfurization (stereoisomeric purity = 91–94%) [20]; (v) preparation of (R)- and
(S)-4-methylheptan-3-ones using SAMP and RAMP reagents, reduction to the corresponding alcohols,

Molecules 2017, 22, 1591
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and enantioselective transesterification with vinyl acetate catalysed by lipase AK (stereoisomeric
purity = 89–95%) [8].
We devised a new synthetic approach to the stereoisomers of
ketone (Scheme 1), easily obtained in 56% isolation yield by aldol condensation of propanal and
-pentanone in the presence of potassium hydroxide, followed by dehydration with anhydrous
oxalic acid in refluxing toluene. The conversion of into the four stereoisomers of requires the
optimization of the enantioselective reduction of both the alkene and the carbonyl group, with either
1 starting from unsaturated
2
3
2
1
(R)- or (S)-enantioselectivity.
Biocatalysis offers appealing solutions for this kind of reaction. The enantioselective reduction of
the alkene bond can be catalysed by an ER which promotes the stereospecific anti hydrogen addition
to the C=C double bond, with the delivery of a hydride ion from the reduced flavin mononucleotide
+
prosthetic group, which is restored at the expense of the oxidation of NADPH to NADP [21
,
22].
To avoid the use of NADPH in stoichiometric quantity, its regeneration can be promoted by combining
the ER reduction with the oxidation of glucose catalysed by a glucose dehydrogenase (e.g., GDH
from Bacillus megaterium). The reduction of the carbonyl group of ketone 3 can be performed in the
presence of an ADH, using NADH or NADPH as a cofactor, and the same enzymatic system for
cofactor regeneration.
2
.2. Selection of Ene-Reductases and Alcohol Dehydrogenases with the Desired Stereoselectivity
Recently, we investigated the ER-mediated reduction of unsaturated ketone within a work
2
devoted to studying the different stereochemical courses of the hydrogenation of certain activated
alkenes catalysed by Old Yellow Enzyme 1 from Saccharomyces pastorianus (OYE1) and Old Yellow
Enzyme 3 from S. cerevisiae (OYE3) [23]. Although the sequence of OYE3 is 80% identical to that
of OYE1, the reduction of the C=C double bond of ketone
2
afforded (S)-
3
(conversion = c > 98%,
enantiomeric excess = ee = 78%) in the presence of OYE3, whereas (R)-
3
was obtained (c > 98%,
ee = 44%) when OYE1 was employed as a catalyst (Table 1). The investigation was also extended to the
use of the complete sets of W116 mutants of OYE1 and OYE3, because the amino acid residue in this
position had been recognized to be strategic in influencing the substrate binding mode in the active
site of these enzymes [24]. In particular, our screening showed that the variant OYE1-W116V was able
to completely convert 2 into (S)-3 showing ee = 86%.
Table 1. Ene-reductase-mediated hydrogenation 1 of unsaturated ketone 2.
ER
c (%) 2
ee (%) 3
OYE1 4
99
99
99
99
99
99
99
99
44, R
78, S
86, S
40 R
99, R
70, R
60, S
22, R
4
OYE3
OYE1-W116V 4
OYE2
OYE2.6
LeOPR1
YqjM
PETN
1
+
Substrate (5 mM), glucose (20 mM), Old Yellow Enzyme (OYE), glucose dehydrogenase (GDH), NADP , DMSO
1%), phosphate buffer pH 7.0, 24 h; see Table S1 in the Supplementary Information for data concerning the ERs
(
2
employed in this screening; Conversion calculated on the basis of GC analysis of the crude mixture after 24 h;
3
Enantiomeric excess calculated on the basis of GC analysis (see Materials and Methods) on a chiral stationary
4
phase; ref. [23].

Molecules 2017, 22, 1591
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With the aim of selecting the best biocatalysts for the first step of the synthetic procedure for this
work, we also considered other ERs for the C=C double bond reduction of compound
are reported in Table 1.
2. The results
This new screening highlighted that compound
2 is well-accepted by ERs, but its reduction
generally occurs with low enantioselectivity, with the exception of OYE2.6 (from Pichia stipitis).
This enzyme could be chosen to perform the first step of the biocatalysed synthesis of the
(
4R)-stereoisomers of pheromone
selected. (S)- is itself an interesting compound, because it is the principal alarm pheromone of the ant
Atta texana [25].
For the second step of the synthetic sequence, a set of 18 commercial ADHs (from EVOXX) were
screened to identify the enzymes capable of reducing the carbonyl group of ketone , and creating
the new stereogenic centre with either (S) or (R) absolute configuration. The preliminary screening
was performed on racemic : five ADHs were able to promote the carbonyl reduction and the results
1. As for the (S)-selective reduction, the W116V mutant of OYE1 was
3
3
3
are collected in Scheme 2. ADH440 was the only catalyst showing (S)-selectivity, and among the
pro-(R)-enzymes ADH270 was selected for the optimization of the one-pot synthetic procedure.
+
+
Scheme 2. ADH screening on racemic
3
(substrate 5 mM, ADH, GDH, glucose, NAD , NADP
◦
potassium phosphate buffer 50 mM pH 7.0, 30 C, 24 h). The stereoisomeric composition of the products
were obtained by GC analysis of the corresponding acetyl derivatives on a chiral stationary phase.
2
.3. One-Pot Multi-Enzymatic Synthesis of the Four Stereoisomers of 4-Methylheptan-3-ol
The one-pot sequential enzymatic synthesis of the four stereoisomers of
scale in potassium phosphate buffer solution (pH 7.0) at 30 C, using DMSO as a cosolvent for unsaturated
1
was performed on 100 mg
◦
ketone 2. The suitable pair of enzymes (OYE2.6/ADH440, OYE2.6/ADH270, OYE1-W116V/ADH440,
OYE1-W116V/ADH270) were added sequentially to the reaction medium in the presence of the same
+
cofactor regeneration system for both ER and ADH (NADP , GDH, glucose) (Scheme 3).
Extraction of the reaction mixture with EtOAc and purification of the residue by column
chromatography afforded the four stereoisomers of compound
and excellent stereoselectivity: (3S,4R)- ee = 99%, de = 99%; (3R,4R)-
ee = 99%, de = 94%; (3R,4S)-1 ee = 99%, de = 92%.
1
in good isolation yields (72–83%)
1
1
ee = 99%, de = 99%; (3S,4S)-
1
The relative configuration of the four stereoisomers was established by comparison of their
H-NMR spectra with those already described in the literature (see Materials and Methods). Once the
1
relative configuration was defined, the absolute configuration was assigned by comparing the optical
rotation values of the samples with those reported in the literature (see Materials and Methods).

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Scheme 3. One-pot multi-enzymatic conversion of 2 into the four stereoisomers of 1.
3
. Discussion
This synthetic procedure shows remarkable improvements with respect to those already reported
in the literature, especially in the light of the so-called “twelve principles” of green chemistry [26].
Most of the reagents are incorporated into the final product, and a reduced quantity of non-hazardous
waste is generated: water is produced as a by-product of the aldolic condensation affording compound
2
, and the two enzymatic reductions occur at the expense of the oxidation of glucose to gluconic acid.
Biocatalysts from renewable feedstocks are employed instead of stoichiometric reagents.
The combination of suitable proteins enables the complete control of the absolute configuration
of the two stereocentres, in order to produce all the possible stereoisomers without the use of complex
derivatization strategies. The high selectivity of the enzymatic reductions makes the one-pot procedure
possible, thus avoiding the work-up of intermediate products and reducing the waste of organic
solvents. The final result is a three-step sequence, starting from diethylketone and propanal, to afford
high-value chiral products with the creation of two vicinal stereogenic centres under enzymatic control.
4
. Materials and Methods
GC-MS analyses were performed using a HP-5MS column (30 m
×
0.25 mm
×
0.25 µm,
◦
Agilent, Santa Clara, CA, USA). The following temperature program was employed: 60
C
◦
−1
◦
◦
−1 ◦
1
13
(
1 min)/6 C
·
min /150 C (1 min)/12 C min /280 C (5 min). H and C NMR spectra
·
were recorded on a 400 or 500 MHz spectrometer (Bruker, Billerica, MA, USA) and the chemical
shift scale was based on internal tetramethylsilane. All the chromatographic separations were
carried out on silica gel. Chiral GC analyses of compounds
by treatment with acetic anhydride in pyridine) and were performed on a Chirasil DEX CB
25 m 0.25 mm 0.25 m, Chrompack, Agilent, Santa Clara, CA, USA) column, installed on HP 6890
gas chromatograph (Agilent, Santa Clara, CA, USA); (a) compound
1 (as acetyl derivatives obtained
3
(
×
×
µ
◦
◦
−1
◦
1
45‘ C/1.0 C
·
min /65 C
◦
−1
◦
(
(
(
1 min)/50
3R,4R)- tR = 17.3 min, (3R,4S)-
1 min)/90 C min /180 C·2 min), (R)-3 t = 5.0 min, (S)-3 t = 5.5 min.
C
·
min /180 C (5 min): (3S,4S)-
1
tR = 15.5 min, (3S,4R)-
1
tR = 16.0 min,
◦
◦
−1
◦
1
1
tR = 17.7 min; (b) compound
3
: 55 C/0.8 C
·
min /67 C
◦
−1
◦
R
R
4
.1. Strains and Enzymes
A screening kit of 18 ADHs was purchased from EVOXX (Monheim am Rhein, Germany).
Ene-reductases (OYE1-3, OYE1-W116V, OYE2.6, YqjM, and LeOPR1) and glucose dehydrogenase

Molecules 2017, 22, 1591
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(GDH) were overproduced in E. coli BL21(DE3) strains harbouring a specific plasmid prepared
as previously reported: pET30a-OYE1 from the original plasmid provided by Neil C. Bruce [27],
pET30a-OYE2 and pET30a-OYE3 from S. cerevisiae BY4741 and pKTS-GDH from B. megaterium
DSM509 [28]; pDJBx-OYE2.6, pDJB5-OYE1-W116V, and pDJBx-LeOPR1 from the original plasmids
provided by Prof. Jon D. Stewart. For YqjM, the original plasmid provided by Prof. M. Hall was used
directly as provided [29]. The enzymes were produced and purified as described in Section 4.2.
4
.2. Overproduction of Enzymes in E. coli BL21(DE3)
LB medium (5 mL) containing the appropriate antibiotic (50
·
mL 1 kanamycin for pET30a,
−
µg
−
1
−1
1
00
µ
g
·
mL ampicillin for pKTS, pDJBx, 30
µ
g mL chloramphenicol for YqjM) 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 LB medium (TB medium in the case of YqjM), which was incubated for 8 h
◦
under the same conditions and used to inoculate 1.5 L medium. The latter culture was shaken at 37 C
and 220 rpm until OD6 reached 0.4–0.5, then enzyme expression was induced by the addition of
00
−
1
0
.1 mM IPTG (50 ng mL anhydrotetracycline was also added in the case of the pKTS-GDH plasmid).
·
In the case of OYE1-3, GDH, and YqjM, after 5–6 h the cells were harvested by centrifugation
◦
(5000
×
g, 20 min, 4 C), resuspended in 50 mL of lysis buffer (20 mM KP buffer pH 7.0, 300 mM
NaCl, 10 mM imidazole) and disrupted by sonication (Omni Ruptor 250 ultrasonic homogeniser, Omni
International Inc., Kennesaw, GE, USA, five sonication cycles, 15 s each, 50% duty). YqjM was used
i
as cell-free extract, whereas for OYE1-3 and GDH the cell-free extract after centrifugation (20,000
×
g,
◦
2
0 min, 4 C) was chromatographed on IMAC stationary phase (Ni-Sepharose Fast Flow, GE Healthcare,
Little Chalfont, UK) with a mobile phase composed of 20 mM KP buffer pH 7.0, 300 mM NaCl, and
i
a 10–300 mM imidazole gradient. Protein elution was monitored at 280 nm, and the fractions were
collected according to the chromatogram and dialysed twice against 1.0 L of 50 mM KP buffer pH 7.0
i
◦
◦
80 C.
(
12 h each, 4 C) to remove imidazole and salts. Purified protein aliquots were stored frozen at
In the case of OYE2.6 and LeOPR1—produced as Glutathione S-transferase (GST)-fusion
proteins—cell pellets were resuspended in cold sepharose binding buffer (PBS, 140 mM NaCl, 2.7 mM
KCl, 10 mM Na HPO , 1.8 mM KH PO , pH 7.3) and lysed by sonication (Omni Ruptor 250 ultrasonic
−
2
4
2
4
homogeniser, five sonication cycles, 15 s each, 50% duty). The cell-free extract was centrifuged
◦
(
12,000 rpm, 40 min, 4 C). The resulting supernatant was passed through Glutathione Sepharose
4
Fast Flow (GE Healthcare), with PBS buffer as the mobile phase. Once the absorbance (280 nm)
returned to a baseline reading, the desired protein was eluted by adding a reduced glutathione (GSH)
buffer solution (10 mM -L-glutamyl-L-cysteinylglycine, 50 mM Tris-HCl, pH 8.0). Protein elution was
γ
monitored at 280 nm and the fractions were collected according to the chromatogram. Purified protein
◦
aliquots were stored frozen at −80 C.
4
.3. One-Pot Conversion of 2 into the Four Stereoisomers of 1
A solution of unsaturated ketone (100 mg, 0.794 mmol) in DMSO (500
potassium phosphate buffer solution (100 mL, 50 mM, pH 7.0) containing the suitable OYE (10–15 mg)
2
µL) was added to a
(
two batches with OYE2.6 and two batches with OYE1-W116V), glucose (4 eq., 3.18 mmol, 572 mg),
+
GDH (125 U), and NADP (0.025 eq., 20
an orbital shaker at 30 C. The intermediates (R) and (S)-
µmol, 15 mg). The reactions were incubated for 24 h in
◦
3
(ee = 99% and 92%, respectively, in both
batches) were not isolated, and were submitted to carbonyl reduction. ADH440 or ADH270 (5 mg,
two batches each), glucose (1 eq., 0.794 mmol, 143 mg), and GDH (125 U) were added to the four
◦
reaction mixtures. After 24 h incubation at 30 C, the final products were recovered and purified by
column chromatography, eluting with hexane and increasing quantities of EtOAc.
4
.3.1. (3R,4R)-4-Methylheptan-3-ol ((3R,4R)-1)
From
2
(100 mg, 0.794 mmol), after reduction with OYE2.6 and ADH270, compound (3R,4R)-
1
1
(
85.7 mg, 83%) was obtained: ee = 99% (chiral GC), de = 99% ( H-NMR); [
α
]D = + 23.0 (c 1.2, hexane)

Molecules 2017, 22, 1591
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1
[
lit. [
9] [
α
]D = +22.7 (c 0.264, hexane, for (3R,4R)-
1
ee = 98%, de = 99%)]; H-NMR (CDCl , 400 MHz) [19
]
3
δ
0
7
8
3.41 (dt, J = 8.4 and 4.3 Hz, CHOH), 1.60–1.00 (8H, m, 3CH +CH+OH), 0.95 (3H, t, J = 7.4 Hz, CH CH ),
2 3 2
13
.90 (3H, t, J = 7.1 Hz, CH CH ), 0.86 (3H, d, J = 6.8 Hz, CH CH); C-NMR (CDCl , 125 MHz) [19
]
δ
3
2
3
3
+
6.9, 37.6, 35.8, 27.4, 20.6, 14.5, 13.6, 10.7; GC/MS (EI) m/z: t S= 4.98 min, 112 (M
−
18, 1), 101 (16),
R
3 (25), 59 (100).
4
.3.2. (3S,4R)-4-Methylheptan-3-ol ((3S,4R)-1)
From
2
(100 mg, 0.794 mmol), after reduction with OYE2.6 and ADH440, compound (3S,4R)-
1
1
(
[
78.4 mg, 76%) was obtained: ee = 99% (chiral GC), de = 99% ( H-NMR); [
α
]D = +11.5 (c 1.0, hexane)
1
lit. [16] [
α
]D = +10.9 (c 1.87, hexane, for (3S,4R)-
1
ee = 92%, de = 99%)]; H-NMR (CDCl , 400 MHz) [19
]
3
δ
3.35 (ddd, J = 8.7, 5.2, and 3.5 Hz, CHOH), 1.60–1.00 (8H, m, 3CH +CH+OH), 0.96 (3H, t, J = 7.4 Hz,
2
13
CH CH ), 0.90 (3H, t, J = 7.1 Hz, CH CH ), 0.88 (3H, d, J = 6.8 Hz, CH CH); C-NMR (CDCl3,
3
2
3
2
3
1
1
25 MHz) [19
13 (M − 17, 1), 101 (16), 83 (20), 59 (100).
]
δ
77.8, 38.4, 34.3, 26.4, 20.5, 15.5, 14.5, 10.5; GC/MS (EI) m/z: tR = 5.02 min,
+
4
.3.3. (3R,4S)-4-Methylheptan-3-ol ((3R,4S)-1)
From (100 mg, 0.794 mmol), after reduction with OYE1-W116V and ADH270, compound
3R,4S)- (83.6 mg, 81%) was obtained: ee = 99%(chiral GC), de = 92% with respect to (3R,4R)-
H-NMR); [ ] = 10.7 (c 1.4, hexane) [lit. [16] [ ]D = +10.9 (c 1.87, hexane, for (3S,4R)- ee = 92%,
2
(
(
1
1
1
α
−
α
1
D
de = 99%)]; spectroscopic data were in agreement with those of the enantiomer.
4
.3.4. (3S,4S)-4-Methylheptan-3-ol ((3S,4S)-1)
From
2
(100 mg, 0.794 mmol), after reduction with OYE1-W116V and ADH440, compound
1
(
[
3S,4S)-
1
(74.3 g, 72%) was obtained: ee = 99% (chiral GC), de = 94% with respect to (3S,4R)-
18.9 (c 1.1, hexane) [lit. [ ] [ ]D = +22.7 (c 0.264, hexane, for (3R,4R)-
1
( H-NMR);
α
] =
−
9
α
1
ee = 98%, de = 99%)];
D
the spectroscopic data were in agreement with those of the enantiomer.
Supplementary Materials: The following are available online: synthesis and characterization of compound
synthesis and characterization of compounds (S) and (R)- , general procedure for ER-mediated reduction of
(screening),
2,
3
unsaturated ketone
2 (screening), general procedure for ADH-mediated reduction of racemic 3
representative GC chromatograms on chiral stationary phases, Table S1.
Acknowledgments: This work was supported by Fondazione Cariplo (grant No. 2014-0568).
Author Contributions: E.B. conceived and designed the experiments; M.C. and A.P. performed the experiments;
E.B., F.G.G., D.M. and F.P. analysed the data and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
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.
Witzgall, P.; Kirsch, P.; Cork, A. Sex Pheromones and Their Impact on Pest Management. J. Chem. Ecol. 2010
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Sample Availability: Samples of the compounds are not available from the authors.
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