Biocatalytic asymmetric and enantioconvergent hydrolysis of trisubstituted oxiranes

Article abstract of DOI:10.1016/S0957-4166(01)00256-7

Asymmetric biohydrolysis of trialkyl oxiranes (±)-1a-3a using the epoxide hydrolase activity of whole bacterial cells proceeded in an enantioconvergent fashion and thus led to the corresponding (R)-configurated vicinal diols 1b-3b in up to 97% enantiomeric excess (e.e.) as the sole product. The mechanism of this enantioconvergence was investigated by ¹⁸O-labelling experiments and it was found that both enantiomers were hydrolysed with opposite regioselectivity.

Full text of DOI:10.1016/S0957-4166(01)00256-7

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 12 (2001) 1519–1528
Biocatalytic asymmetric and enantioconvergent hydrolysis of
trisubstituted oxiranes
Andreas Steinreiber,a Sandra F. Mayer,a Robert Safb and Kurt Fabera,*
aDepartment of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
bInstitute for Chemical Technology of Organic Materials, Graz University of Technology, Stremayrgasse 16,
A-8010 Graz, Austria
Received 21 May 2001; accepted 4 June 2001
Abstract—Asymmetric biohydrolysis of trialkyl oxiranes ( )-1a–3a using the epoxide hydrolase activity of whole bacterial cells
proceeded in an enantioconvergent fashion and thus led to the corresponding (R)-configurated vicinal diols 1b–3b in up to 97%
enantiomeric excess (e.e.) as the sole product. The mechanism of this enantioconvergence was investigated by 18O-labelling
experiments and it was found that both enantiomers were hydrolysed with opposite regioselectivity. © 2001 Elsevier Science Ltd.
All rights reserved.
1. Introduction
bial epoxide hydrolases.5,6 For instance, ( )-1-methyl-
cyclohexene oxide was hydrolysed by resting cells of
Corynebacterium C12,7 Rhodotorula glutinis CIMW
1478 and an isolated epoxide hydrolase from Rhodococ-
cus erythropolis DCL 14.9 In each case, the reaction
proceeded via kinetic resolution producing two enan-
tiomers (i.e. formed diol and unreacted epoxide) pos-
The asymmetric hydrolysis of epoxides using (bio)-
chemical catalysts provides a convenient access to non-
racemic vicinal diols. For monosubstituted oxiranes,
the Jacobsen CoII-salen catalyst1 gives the best selectiv-
ities, whereas microbial epoxide hydrolases are the bio-
catalysts of choice for more sterically demanding
disubstituted analogues.2 A particular feature of the
latter processes is the fact that (particularly for 2,3-di-
substituted oxiranes) the asymmetric biohydrolysis pro-
ceeds in an enantioconvergent fashion, which leads to
the formation of a single enantiomeric vicinal diol as
the sole product.3 The mechanistic reason for such a
‘deracemisation’ is based on the fact that both oxirane
enantiomers are attacked with opposite regioselectivity,
which is facilitated by the steric similarity of both
adjacent oxirane carbon atoms.
sessing
opposite
configuration.
In
contrast,
diastereoisomeric limonene oxides derived from either
(+)- or (−)-limonene were hydrolysed in a diastereocon-
vergent fashion to give a single enantiomeric diol.10,11
The only enantioconvergent transformation of a trisub-
stituted oxirane was reported for ( )-10,11-epoxyfar-
nesol using Helminthosporium sativum to give the
(S)-diol as the sole metabolite.12 In order to provide a
mechanistic explanation for the enantioconvergence, an
enzymatic cis-hydration was assumed, which (in view of
the nowadays generally accepted mechanism of action
for epoxide hydrolases13) is extremely unlikely and war-
rants a more detailed investigation.
For more sterically demanding trisubstituted epoxides,
however, enantioconvergent hydrolysis is rather
unlikely, since it would require a nucleophilic attack on
a sterically congested carbon atom. Although such a
pathway has been observed in mechanistic studies,4 no
preparative-scale reaction has been reported to date.
The reported data on the asymmetric biohydrolysis of
trisubstituted oxiranes show that these sterically
demanding substrates are not easily accepted by micro-
Herein, we present our results on the biocatalytic
hydrolysis of trisubstituted epoxides ( )-1a–3a using the
epoxide hydrolase activity of whole bacterial cells.
2. Results and discussion
Epoxides ( )-1a–3a were prepared via Wittig reaction
from commercially available phosphonium salts via the
corresponding alkenes 1–3 (Scheme 1). Due to their
volatility, the latter were not isolated and the crude
* Corresponding author. Present address: Department of Organic
Chemistry, Stockholm University, S-106 91 Stockholm, Sweden.
Fax: +46-8-154908; e-mail: kurt.faber@kfunigraz.ac.at
0957-4166/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(01)00256-7

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A. Steinreiber et al. / Tetrahedron: Asymmetry 12 (2001) 1519–1528
Scheme 1. Synthesis of substrates ( )-1a–3a.
either one of the oxirane carbon atoms which may be
different for both enantiomers (Scheme 4). As a conse-
quence, four stereochemical pathways (denoted via
their apparent first-order relative rate constants k1
through k4) are possible. For trisubstituted oxiranes
possessing only a single stereogenic centre, two pairs of
pathways are possible, which proceed either via inver-
sion (though attack at the stereogenic centre, k2, k3) or
via (apparent) retention of configuration (via attack at
the fully substituted oxirane carbon atom, k1, k4), and
their relative value determines the stereochemical out-
come of the reaction. The following parameters are
used to describe the characteristics of these reactions: (i)
The enantioselectivity (defined as the relative reaction
rate of enantiomers) is determined as E=(k3+k4)/
(k1+k2) or reciprocal value, and the (ii) regioselectivity
for each enantiomer is expressed as hS=k2/k1 and hR=
k4/k3. Relative rate constants were calculated from
progress curves based on datasets of e.e.S, e.e.P and
conversion versus time by employing the computer
program ‘EntCon’.14 The validity of the data obtained
was verified by 18O-labelling experiments (see below).
product was directly subjected to epoxidation using
m-chloroperbenzoic acid. In order to validate the out-
come of the bioreactions, spontaneous hydrolysis of
substrates ( )-1a–3a was proven to be negligible (<2%
within 48 h).
A screening for biocatalytic activity was performed by
using whole bacterial cells in Tris-buffer at pH 8.0 and
30°C for 48 h (Scheme 2). With one exception (Table 1,
entry 8) diols 1b–3b were isolated as the sole product of
the biotransformation. Their structural identity was
confirmed by co-injection on GLC with the racemic
diols ( )-1b–3b obtained via acid catalysed hydrolysis
of the corresponding epoxides. Enantiomeric purities of
products from the biohydrolysis were determined by
GLC on a chiral stationary phase. In line with previous
observations,3 diols 1b–3b were (R)-configured and
were obtained in moderate to excellent enantiomeric
excesses.
Absolute configurations of non-reacted epoxides and
formed diols were determined by co-injection on GLC
using independently synthesised reference material
(Scheme 3). Diol (S)-4 was obtained in 87% e.e. by
asymmetric dihydroxylation of 2-methylocta-2,7-diene
according to Sharpless.26 The latter material was hydro-
genated (Pd/C, H2) to furnish (S)-2b without loss of
enantiomeric purity. Products (S)-1b and (S)-3b were
obtained from alcohol (S)-5, which was synthesised
from (S)-4 via (i) protection of the diol as the ace-
tonide, followed by (ii) oxidative cleavage of the termi-
nal CꢀC bond (O3) and (iii) reductive work-up of the
ozonide (LiAlH4). Removal of the primary alcohol
moiety by mesylation and nucleophilic displacement by
hydride (LiAlH4) and deprotection of the vic-diol moi-
ety gave (S)-1b. C2-Chain extension of (S)-5 was
achieved by standard methodology (halogenation of the
primary alcohol moiety via the mesylate, followed by
nucleophilic displacement with EtMgBr and deprotec-
tion of the acetonide) to yield (S)-3b. Reference mate-
rial for (S)-1a–3a was obtained by treatment of diols
(R)-1b–3b (formed during the biotransformation) with
triflic anhydride in pyridine to give the corresponding
epoxides of opposite configuration.
The data depicted in Table 1 reveals the following
features of the process.
(i) In general, [for exceptions see (iii)], (R)-diols were
formed from the (faster reacting) corresponding (S)-
epoxides via attack at the less congested C(3) stereocen-
tre leading to inversion of configuration (k2>k1 and
k2>k4). As a consequence, it can be concluded that the
slower reacting epoxide possessed (R)-configuration.
(ii) Two Rhodococcus strains (previously denoted as
Nocardia,15 entries 3 and 4) were shown to prefer the
(R)-epoxide with opposite regioselectivity by attacking
the fully substituted oxirane C-atom, thus forming
(R)-diols with retention of configuration (k4>k3 and
k4>k2).16
In contrast to the majority of asymmetric biotransfor-
mations (where the absolute configuration of the stereo-
genic centre always remains unaffected throughout the
reaction), the enzymatic hydrolysis of epoxides may
take place with different regioselectivity via attack at
Scheme 2. Asymmetric biohydrolysis of epoxides ( )-1a–3a.

Table 1. Selectivities of asymmetric biohydrolysis of (9)-1a–3a
Entry
Microorganism
Substrate
Conversion (%)
Diol
Epoxide
Relative rate constantsa
Enantioselectivity
(Eb)
/
Abs. Config.
E.e. (%)
89
Abs. Config.
E.e. (%)
99
k1
k2
k3
k4
1
M. paraffinicum NCIMB
10420
(9)-1a
76
R
R
2
100
1
5
17
2
3
4
5
6
7
R. ruber DSM 43338
R. ruber SM 1789
R. ruber SM 1790
S. la6endulae ATCC 55209
S. la6endulae ATCC 55209
M. paraffinicum NCIMB
10420
(9)-1a
(9)-2a
(9)-2a
(9)-2a
(9)-3a
(9)-3a
49
85
80
49
60
90
R
R
R
R
R
R
86
85
83
91
97
83
R
S
S
R
R
R
9
92
91
51
23
93
1
1
1
1
1
1
25
15
18
61
126
723
1
9
12
3
1
24
19
27
80
49
99
1.3
2.3
4.7
1.2
1.3
4.2
1
151
(
8
R. ruber CBS 717.73
(9)-2a
46
R
55
R
99
1
−4.5
−2.2
−0.8
n.a.
159
a Calculated from progress curves of e.e.S, e.e.P and c versus time using computer program ‘EntCon’ (see Ref. 14).
b Defined as the ratio of relative rates of fast versus slow reacting enantiomer; E=(k3+k4)/(k1+k2) or the respective reciprocal value.
n.a., not applicable; due to enzymatic oxidation of diol (R)-2b leading to the formation of 2-methyl-2-hydroxyoctan-3-one (ca. 20%), the kinetics could not be applied, as indicated by negative k-values.
1

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A. Steinreiber et al. / Tetrahedron: Asymmetry 12 (2001) 1519–1528
Scheme 3. Synthesis of reference materials and determination of absolute configuration.
Scheme 4. Stereochemical pathways of biohydrolysis.
(iii) In all cases, both oxirane enantiomers were
hydrolysed with opposite regioselectivity (k2>k1 and
k4>k3), thus forming the same enantiomeric diol in an
enantioconvergent fashion.
enantioselectivity for (S)-2b, the kinetics of Scheme 4
could not be applied.
The preparative applicability of this reaction regarding
an optimal chemical and optical yield of product(s) can
only be assessed by kinetic analysis, the importance of
which is demonstrated along three selected examples.
(iv) Most remarkably, both enantiomers of substrates
were hydrolysed at comparable rates, leading to low
enantioselectivities [(k1+k2):(k3+k4)]. In contrast, the
regioselectivities were significantly higher, which
resulted in good optical purities of products (k2ꢀk1;
k4ꢀk3).
Case I (entry 5, Fig. 1): Hydrolysis of ( )-2a by Strep-
tomyces lavendulae ATCC 55209 proceeds without
noticeable enantioselectivity (E=1.2) and, as a conse-
quence, the time-course of conversion is completely
steady and does not show any drop near half-way of
the reaction. In accordance with that, the e.e.S remains
very low throughout the whole process and does not
exceed a value of ꢁ10%. It is impossible to perform
this reaction as a kinetic resolution. On the other hand,
the regioselectivities are high (hS=61, hR=16) and
show a matching opposite trend for both enantiomers.
Thus, the enantiomeric composition of (R)-2b remains
high throughout the whole reaction and complete de-
racemisation can be achieved.
(v) The calculation of the relative rate constants from
the hydrolysis of ( )-2a using Rhodococcus ruber CBS
717.73 gave negative k-values (entry 8). Thus, the
reaction cannot be explained by the four pathways
depicted in Scheme 4 and a more complex metabolism
has to be considered. Indeed, detailed investigation of
this reaction revealed that diol (R)-2b (formed during
hydrolysis) was further enzymatically oxidised at the
sec-alcohol moiety to furnish the corresponding
hydroxyketone (2-hydroxy-2-methyloctan-3-one) in
ꢁ20% yield. Since this latter pathway showed some

A. Steinreiber et al. / Tetrahedron: Asymmetry 12 (2001) 1519–1528
1523
Figure 1. Time-course of hydrolysis of ( )-2a using S. lavendulae ATCC 55209.
ence in reaction rates of enantiomers, a conversion of
ꢁ50% is already reached within 2.5 h, but the comple-
tion of the second half of the process takes considerably
longer (>100 h). Due to the high regioselectivity of the
faster reacting (S)-enantiomer (hS=50), the e.e.P is high
at the onset of the reaction but is gradually depleted by
the low regioselectivity of the slow (R)-enantiomer
(hR=5).
Case II (entry 3, Fig. 2): On the contrary, R. ruber SM
1789 shows some enantioselectivity for (R)-2a (E=2.3),
as indicated by a drop in the rate of the reaction at near
to 50% conversion. As a consequence, the remaining
non-reacted epoxide (S)-2a can be obtained in good e.e.
beyond 70% conversion. In this case, kinetic resolution
would be feasible, albeit at reduced chemical yields of
epoxide. Again, the regioselectivity for both substrate
enantiomers is opposite but rather low (hS=15, hR=3).
The e.e.P is rather low at the onset of the reaction
(caused by the low regioselectivity of the fast reacting
(R)-enantiomer) but it increases steadily towards the
end, when the hydrolysis of the slow reacting (S)-enan-
tiomer (occurring with better regioselectivity) takes
over. Overall, deracemisation is possible and the best
results are obtained at high conversion.
Typically, this process can conveniently be run in a
kinetic resolution mode leading to (R)-1a and (R)-1b in
>95% e.e. at about 50% conversion. Deracemisation,
however, is hampered by the long reaction times of
(R)-1a.
In order to elucidate the reason for the enantioconver-
gence and to prove the validity of the calculation of
relative rate constants, experiments were performed in
18O-labelled water using substrate ( )-2a. All four
diastereomers (with respect to the 18O-label) of diol 2b
were analysed by GC–MS on a chiral stationary phase
using electron-impact ionisation (Scheme 5).
Case III (entry 1, Fig. 3): As indicated by a sharp drop
in reaction rate at 50%, substrate ( )-1a is hydrolysed
by Mycobacterium paraffinicum NCIMB 10420 with
good enantioselectivity (E=17) and at this point the
e.e.S has already reached >95%. Due to the large differ-
Figure 2. Time-course of hydrolysis of ( )-2a using R. ruber SM 1789.

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A. Steinreiber et al. / Tetrahedron: Asymmetry 12 (2001) 1519–1528
Figure 3. Time-course of hydrolysis of ( )-1a using M. paraffinicum NCIMB 10420.
Scheme 5. 18O-Labelling experiments.
(ii) For the (R)-diol, opposite regioselectivities were
The number of oxygen atoms incorporated during
enzymatic hydrolysis was determined by using the [M−
CH3]+ signal, since the relative intensity of M+ was
rather small. Both R. ruber SM 1789 and CBS 717.73
gave exclusively a signal for 147 Da, indicating a clear
preference for the incorporation of a single oxygen
atom from 18OH2,4,17 no trace of double incorporation
(M=149 Da) was found.
observed with both strains: R. ruber SM 1789 predomi-
nantly attacked the C(2) atom (entry 3, k4>k2). Thus, it
can be concluded that the (R)-epoxide is hydrolysed
with retention to furnish the (R)-diol. Again, the mea-
sured ratio of regioselectivity corresponds nicely to the
calculated values, i.e. k2/k4 is 1.0:2.2 (exp.) and 1.0:1.8
(calcd). On the contrary, R. ruber CBS 717.73 exhibited
a preference for incorporation at C(3) (entry 8) and, as
a consequence, k4<k2. Thus, the (S)-epoxide is trans-
formed with inversion to the (R)-diol and k2:k4 equals
1.0:1.8.
The regioselectivity of the O-incorporation was deter-
mined by using the 16O/18O ratio of the smaller frag-
ment
resulting
from
glycol
cleavage,
i.e.
[(CH3)2-C-OH]+ (Table 2). The corresponding signal for
the matching heavy fragment [HO-CH-n-C5H11]+ was
very small. The data revealed the following.
In order to test the practical applicability of the pro-
cess, a preparative-scale experiment was performed:
When ( )-2a (597 mg) was subjected to lyophilised cells
of R. ruber SM 1789 (2.13 g) over a period of 140 h,
(R)-2b (547 mg) was obtained as the sole product with
79% e.e. in 92% yield.
(i) Both microorganisms exhibited the same predomi-
nance for 18O-incorporation at the C(3) atom in the
case of the (S)-diol (Table 1, entries 3 and 8). From the
experimentally determined enantiopreference and the
stereochemical outcome of the biotransformations, it
can be concluded that k3>k1. Thus, the (R)-epoxide is
predominantly transformed with inversion to the (S)-
diol. For R. ruber SM 1789, the measured ratios of
regioselectivity are in good agreement with the calcu-
lated values, i.e. k3/k1 is 12:1.0 (exp.) and 9:1 (calcd).
For R. ruber CBS 717.73, k3/k1 (exp.) is 4.6:1.0. Due to
further oxidative metabolism of (R)-2b, the correspond-
ing calculated values could not be obtained.
Table 2. Relative distribution of 16O/18O-label in the small
fragment [(CH3)2-C-OH]+
Strain
(R)-2ba
(S)-2bb
Rhodococus ruber SM 1789
Rhodococus ruber CBS 717.73
1.0:2.2
1.8:1.0
12:1.0
4.6:1.0
a Corresponding to the ratio of k2/k4.
b Corresponds to the ratio of k3/k1.

A. Steinreiber et al. / Tetrahedron: Asymmetry 12 (2001) 1519–1528
1525
3. Conclusion
bacterial cells; strains were obtained from culture col-
lections. SM numbers refer to the culture collection of
the Institute of Biotechnology, Graz University of
Technology. All strains were grown as previously
described.18–21
In summary, it was shown that the biohydrolysis of the
racemic trialkyl epoxides ( )-1a–3a using the bacterial
epoxide hydrolase activity from Rhodococcus and
Streptomyces spp. proceeds in an enantioconvergent
fashion. Due to its simplicity and high efficiency, this
process is feasible on a preparative scale and provides
an elegant method for the preparation of non-racemic
long-chain 2-methyl-2,3-diols, which constitute the chi-
ral moieties of numerous natural products derived from
isoprenoid pathways. The applicability of this method
for the asymmetric synthesis of bioactive terpenoids,
such as Juvenile hormone analogues, is currently under
investigation.
4.2. Synthesis of substrates and reference materials
4.2.1. General procedure for Wittig reaction and epoxi-
dation of alkenes to furnish ( )-1a, ( )-2a and ( )-3a.
Epoxides ( )-1a, ( )-2a and ( )-3a were synthesised via
a two-step sequence described in Method A. Due to the
high volatility of alkenes 1, 2 and 3, they were directly
converted into the corresponding epoxides 1a, 2a and
3a without isolation. Only alkene 2 was isolated to
prove the outcome of the first step.
4. Experimental
4.2.2. Method A. NaH (60%, 1.13 g) was washed with
pentane and suspended in DMSO (35 mL). The mixture
was heated at 80°C for 0.5 h and then cooled to rt.
n-Alkyltriphenylphosphonium bromide (46.1 mmol) in
DMSO (60 mL) was added during a period of 0.5 h.
After stirring the reaction mixture for a further 0.5 h,
acetone (2.57 g, 44.26 mmol) was added and stirring
was continued for 12 h. The reaction was quenched by
addition of water (700 mL) and pentane (100 mL). The
phases were separated and the aqueous phase was
extracted with pentane (3×100 mL). The combined
organic phase was dried (Na2SO4), evaporated and
cooled (0°C, 12 h) to precipitate residual phosphonium
salts, which were removed by filtration. The liquid
residue was dissolved in CH2Cl2 (300 mL), K2CO3 (9.5
g, 68.7 mmol) and m-chloroperbenzoic acid (10.0 g,
40.6 mmol, 70%) were added and the suspension was
stirred for 3 h. After the reaction had reached comple-
tion, the white suspension was filtered. The resulting
solution was treated with Na2S2O5 (200 mL, 10%).
After phase separation, the organic phase was washed
with sat. NaHCO3 (3×200 mL), dried and evaporated.
Kugelrohr distillation afforded ( )-1a, ( )-2a and ( )-
3a. The following compounds were thus obtained.
4.1. General remarks
NMR spectra were recorded in CDCl3 using a Bruker
AMX 360 at 360 (1H) and 90 (13C) MHz, a Bruker
DMX Avance 500 at 500 (1H) and 125 (13C) MHz or a
Varian XL-200 at 200 (1H) and 50 (13C) MHz. Chemi-
cal shifts are reported relative to TMS (l 0.00) with
CHCl3 as internal standard [l 7.23 (1H) and 76.90
(13C)], coupling constants (J) are given in Hz. 13C
NMR multiplicities were determined by using a DEPT
pulse sequence.
TLC plates were run on silica gel Merck 60 (F254) and
compounds were visualised by spraying with vanillin/
H2SO4 conc. (5 g/L) (detection I), or by dipping into a
KMnO4 reagent (2.5 g/L in H2O) (detection II). Com-
pounds were purified either by flash chromatography
on silica gel Merck 60 (230–400 mesh) or, for volatile
substances, by Kugelrohr distillation. Petroleum ether
had a boiling range of 60–90°C. GC analyses were
carried out on a Varian 3800 gas chromatograph
equipped with FID and either an HP1301 or an
HP1701 capillary column (both 30 m, 0.25 mm, 0.25
mm film, N2). Enantiomeric purities were analysed on a
Varian 3800 gas chromatograph equipped with FID,
using a CP-Chirasil-DEX CB column (25 m, 0.32 mm,
0.25 mm film, H2). High-resolution mass spectra were
recorded on a double-focussing Kratos profile mass
spectrometer with electron-impact ionisation (EI, 70
eV). A Shimadzu GC-14A directly coupled to the MS
was used for sample introduction.
4.2.3. 2-Methyl-2-octene 2. n-Hexyltriphenylphospho-
nium bromide (8.2 g, 19.2 mmol) and acetone (1.5 mL,
20.4 mmol) were used as described in method A. The
pentane extract was subjected to Kugelrohr distillation
to afford 0.71 g (28%) of 1 as a colourless liquid; Rf
(petroleum ether)=0.78 (detection II); bp (Kugelrohr)
95–100°C (400 mbar); 1H NMR (360.13 MHz,
CDCl3):22 l 0.89 (3H, t, J=7.0), 1.26–1.60 (6H, m),
1.60 (3H, s), 1.69 (3H, d, J=1.0), 1.97 (2H, m), 5.13
(1H, m); 13C NMR (90.56 MHz, CDCl3): l 14.06 (q),
17.56 (q), 22.69 (t), 25.67 (q), 28.08 (t), 29.67 (t), 31.66
(t), 125.02 (d), 131.04 (s).
Solvents were dried and freshly distilled by common
practice. For anhydrous reactions, flasks were dried at
150°C and flushed with dry argon just before use.
Organic extracts were dried over Na2SO4, and then the
solvent was evaporated under reduced pressure. H218O
(94.5% isotope content) was purchased from Pro-
mochem (Germany). n-Pentyl-, n-hexyl- and n-heptyl-
triphenylphosphonium bromide, as well as a- and
b-AD-mix, were purchased from Aldrich. m-Chloroper-
benzoic acid (m-CPBA, 70%, Fluka) and NaH sus-
pended in mineral oil (60%, Aldrich) were used.
Biotransformations were performed with lyophilised
4.2.4. 2-Methyl-2,3-epoxyheptane ( )-1a. Method A was
employed using n-pentyl-triphenylphosphonium bro-
mide (17.3 g, 41.8 mmol) and acetone (3.2 mL, 43.6
mmol). Work-up and Kugelrohr distillation afforded
( )-1a as
a colourless liquid (1.30 g, 40%); Rf
(petroleum ether/EtOAc, 5:1)=0.75 (detection I); bp
(Kugelrohr) 110–115°C (38 mbar); H and 13C NMR
1
data matched those previously reported.23

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A. Steinreiber et al. / Tetrahedron: Asymmetry 12 (2001) 1519–1528
4.2.5. 2-Methyl-2,3-epoxyoctane ( )-2a. Method A was
employed using n-hexyl-triphenylphosphonium bro-
mide (18.2 g, 42.5 mmol) and acetone (3.25 mL, 44.3
mmol). Work-up and Kugelrohr distillation afforded
2.02–2.18 (2H, br s), 3.36 (1H, d, J=6.3); 13C NMR
(90.56 MHz, CDCl3): l 14.26 (q), 22.82 (t), 23.30 (q),
26.70 (q), 26.96 (t), 29.53 (t), 31.90 (t), 32.02 (t), 73.36
(s), 78.84 (d).
( )-2a as
a colourless liquid (3.10 g, 49%); Rf
4.3.4. 2-Methyl-2-hydroxyoctan-3-one. Diol 2b (85 mg,
0.53 mmol) was dissolved in CH2Cl2 (3 mL) and Dess–
Martin periodinane oxidant (0.3 g, 0.71 mmol) was
added. After stirring the mixture for 3 h the solution
was diluted with Et2O (7 mL) and sat. aqueous
NaHCO3 (10 mL). The organic layer was dried
(Na2SO4) and evaporated to yield 2-methyl-2-hydroxy-
octan-3-one (45 mg, 54%) after flash chromatography
(petroleum ether/EtOAc, 5:1)=0.72 (detection I); bp
1
(Kugelrohr) 140°C, (32 mbar); H NMR (360.13 MHz,
CDCl3): l 0.89 (3H, t, J=7.1), 1.25 (3H, s), 1.30 (3H,
s), 1.31–1.55 (8H, m), 2.69 (1H, t, J=5.9); 13C NMR
(90.56 MHz, CDCl3): l 14.03 (q), 18.75 (q), 22.65 (t),
24.96 (q), 26.23 (t), 28.85 (t), 31.73 (t), 58.24 (s), 64.62
(d).
(pentane/Et2O, 3:1); Rf (petroleum ether/EtOAc, 1:1)=
4.2.6. 2-Methyl-2,3-epoxynonane ( )-3a. Method A was
employed by using n-heptyl-triphenylphosphonium
bromide (17.6 g, 39.8 mmol) and acetone (3.05 mL,
41.5 mmol). Work-up and Kugelrohr distillation
afforded ( )-3a as a colourless liquid (3.40 g, 43%); Rf
(petroleum ether/EtOAc, 5:1)=0.68 (detection I); bp
1
0.62 (detection I); H NMR (503.03 MHz, CDCl3):25
l
0.83 (3H, t, J=5.1), 1.30 (6H, s), 1.18–1.96 (4H, m),
2.46 (2H, t, J=7.3), 3.77 (1H, s); 13C NMR (125.76
MHz, CDCl3): l 13.80 (q), 22.35 (t), 23.33 (t), 26.38 (q),
31.27 (t), 35.34 (t), 68.05 (s), 214.53 (s). High-resolution
MS for C9H18O2: calcd 158.1291, found 158.1307.
1
(Kugelrohr) 145°C (20 mbar); H NMR (360.13 MHz,
CDCl3): l 0.84 (3H, t, J=6.8), 1.21 (3H, s), 1.26 (3H,
s), 1.28–1.50 (10H, m), 2.66 (1H, t, J=5.9); 13C NMR
(90.56 MHz, CDCl3): l 14.07 (q), 18.73 (q), 22.60 (t),
24.93 (q), 26.51 (t), 28.88 (t), 29.19 (t), 31.82 (t), 58.18
(s), 64.59 (d).
4.4. Determination of absolute configuration
Abolute configurations of the biotransformation prod-
ucts were determined by co-injection on chiral GC with
reference material (Table 3). This was independently
synthesised as follows: Sharpless dihydroxylation of
2-methyl-octada-2,7-diene with a-AD-mix was previ-
ously described to give (S)-4 (e.e. 87%).26 Hydrogena-
tion of (S)-4 using Pd/C (5% w/w) in dry EtOH under
a hydrogen atmosphere gave (S)-2b (e.e. 88%). Diol
(S)-4 was transformed into the corresponding acetonide
(2,2-dimethoxypropane, cat. IR 120 H+-form, rt) which
led after ozonolysis and reductive work-up (dry
CH2Cl2, O3, LiAlH4 in THF) to alcohol (S)-5 (spectro-
scopic data are given below). Mesylation of (S)-5
(MsCl, pyridine, CH2Cl2), reduction (LiAlH4, Et2O)
and deprotection (H2SO4 cat., H2O) led to (S)-1b (e.e.
90%). Bromination of (S)-5 (MsCl, pyridine, CH2Cl2
followed by LiBr in acetone) was followed by chain
extension via a Grignard reaction (EtMgBr, Et2O).
After deprotection, (S)-3b was obtained in 95% e.e.
Diols (R)-1b, (R)-2b and (R)-3b (e.e. >90%) were
4.3. Synthesis of 1,2-diols ( )-1b, ( )-2b and ( )-3b
Diols ( )-1b, ( )-2b and ( )-3b were obtained by acid
catalysed hydrolysis of the corresponding racemic oxi-
ranes 1a, 2a and 3a (0.2 M in water/THF 1/1, three
drops of 6N H2SO4). Extractive work-up and flash
chromatography (petroleum ether/EtOAc 5/1) gave
pure diols 1b, 2b and 3b. The following compounds
were thus obtained.
4.3.1. 2-Methylheptane-2,3-diol ( )-1b. Hydrolysis of 1a
(0.14 g, 1.09 mmol) afforded 1b as a colourless oil (0.12
g, 76%); Rf (petroleum ether/EtOAc, 1:1)=0.25 (detec-
1
tion I); H NMR (360.13 MHz, CDCl3): l 0.88 (3H, t,
J=6.9), 1.11 (3H, s), 1.16 (3H, s), 1.26–1.55 (6H, m),
2.31 (1H, s), 2.46 (1H, s), 3.32 (1H, d, J=8.2); 13C
NMR (90.56 MHz, CDCl3): l 14.08 (q), 22.75 (t), 23.10
(q), 26.53 (t), 29.02 (t), 31.42 (t), 73.25 (s), 78.65 (d).
The non-racemic reference material was obtained
according to the reported procedure.24
Table 3. GC retention times of compounds on a chiral
stationary phase
Compound
Conditions
Retention time [mm], (absolute
config.)
4.3.2. 2-Methyloctane-2,3-diol ( )-2b. Hydrolysis of 2a
(0.15 g, 1.05 mmol) afforded 2b as a colourless oil (0.11
g, 64%); Rf (petroleum ether/EtOAc, 5:1)=0.16 (detec-
1a
2a
3a
lb
12 psi, 60°C
(iso)
12 psi, 60°C
(iso)
12 psi, 80°C
(iso)
12 psi, 110°C
(iso)
12 psi, 120°C
(iso)
12 psi, 125°C
(iso)
12 psi, 125°C
(iso)
3.38 (S), 3.63 (R)
7.29 (S), 7.84 (R)
5.11(S), 5.43 (R)
4.56 (S), 5.03 (R)
3.45 (S), 3.81(R)
5.65 (S), 6.35 (R)
3.49 (S), 3.85 (R)
1
tion I); H NMR (360.13 MHz, CDCl3): l 0.86 (3H, t,
J=6.8), 1.11 (3H, s), 1.17 (3H, s), 1.36–1.55 (8H, m),
2.30–2.34 (2H, br s), 3.33 (1H, d, J=9.1); 13C NMR
(90.56 MHz, CDCl3): l 14.10 (q), 22.68 (t), 23.11 (q),
26.50 (t), 26.54 (q), 31.70 (t), 31.91 (t), 73.23 (s), 78.68
(d).
2b
3b
4
4.3.3. 2-Methylnonane-2,3-diol ( )-3b. Hydrolysis of 3a
(0.15 g, 1.05 mmol) afforded 3b as a colourless oil (0.15
g, 90%); Rf (petroleum ether/EtOAc, 1:1)=0.35 (detec-
1
tion I); H NMR (360.13 MHz, CDCl3): l 0.86 (3H, t,
J=6.0), 1.15 (3H, s), 1.20 (3H, s), 1.24–1.57 (10H, m),

A. Steinreiber et al. / Tetrahedron: Asymmetry 12 (2001) 1519–1528
1527
obtained from biotransformations. Treatment of these
References
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4.5. General procedure for the biocatalytic hydrolysis
of ( )-1a, ( )-2a and ( )-3a
Lyophilised cells (50 mg) were rehydrated in Tris-buffer
(1 mL, 0.05 M, pH 8.0) for 1 h, ( )-epoxides 1a, 2a and
3a (5 mL) were added and the mixture was shaken at
30°C at 130 rpm. At intervals of 3.5, 6.5, 22, 33.5, 49.5,
76.5 and 105 h, aliquots of 0.1 mL were withdrawn and
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at 30°C. After 140 h the suspension was extracted with
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numbers will be released immediately at <http://
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4.5.2. General procedure for the biocatalytic hydrolysis
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obtained by cell-disruption in a bead-mill (Vibrogen,
glass beads 0.3 mm diameter (10 g) in Tris-buffer (0.05
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17. The blank experiment performed in 16OH2 contained the
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18. Kroutil, W.; Osprian, I.; Mischitz, M.; Faber, K. Synthe-
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Acknowledgements
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This research was performed within the Spezial-
forschungsbereich Biokatalyse (Project No. F-104) and
financial support by the Fonds zur Fo¨rderung der
wissenschaflichen Forschung and the Austrian Ministry
of Science is gratefully acknowledged.

1528
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