Synthesis and resolution of a key building block for epothilones: A comparison of asymmetric synthesis, chemical and enzymatic resolution

Article abstract of DOI:10.1016/j.tetasy.2004.06.048

The asymmetric synthesis and kinetic resolution of a series of acyloins (α-hydroxy ketones) suitable as building blocks for the northern half of epothilones was studied. Three methods were applied to obtain nonracemic compounds at the eventual epothilone

Full text of DOI:10.1016/j.tetasy.2004.06.048

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2861–2869
Synthesis and resolution of a key building block for epothilones:
a comparison of asymmetric synthesis, chemical and
enzymatic resolutionq
Gunther Scheid,a Eelco Ruijter,a Monika Konarzycka-Bessler,b Uwe T.Bornscheuer b,* and
¨
a,
*
Ludger A.Wessjohann
aDepartment of Bioorganic Chemistry, Leibniz-Institute of Plant Biochemistry (IPB), Weinberg 3, D-06120 Halle (Saale), Germany
bInstitute of Chemistry and Biochemistry, Department of Technical Chemistry and Biotechnology, Greifswald University,
Soldmannstr. 16, D17487 Greifswald, Germany
Received 25 May 2004; accepted 23 June 2004
Available online 12 September 2004
Abstract—The asymmetric synthesis and kinetic resolution of a series of acyloins (a-hydroxy ketones) suitable as building blocks for
the northern half of epothilones was studied.Three methods were applied to obtain nonracemic compounds at the eventual epothi-
lone C15-position: asymmetric synthesis with Evansꢀ auxiliary, chemical resolution and enzymatic resolution.The success rate in
small scale applications increased in the order given, and the enzymatic resolution was studied in more detail.Out of a set of nine
lipases and esterases, lipases from Burkholderia cepacia, Pseudomonas sp., lipase B from Candida antarctica and recombinant ester-
ases from Streptomyces diastatochromogenes exhibited the highest enantioselectivities with E-values ranging from 60 to >200.Pig
liver esterase exhibited inverse enantiopreference and only with recombinant enzyme could a moderate selectivity (E = 50, commer-
cial PLE: E = 8) be observed.
ꢀ 2004 Elsevier Ltd. All rights reserved.
3
1. Introduction
petitive inhibitors of H-labelled paclitaxel binding to
tubulin, suggesting that the binding sites of both com-
pounds at least overlap partially.10 Moreover, the epo-
thilones are far more active than paclitaxel against
multidrug resistant cell lines.Several total syntheses of
epothilones A–E have been published,11,12 with
many derivatives being synthesized within a short period
of time after the first total syntheses.13,14 The chemistry
and biology of epothilones has been well described,14–18
and several members of the epothilone family, as well as
some synthetic derivatives, are currently in various
stages of clinical development.
Enantiomerically pure chiral acyloins are valuable inter-
mediates in the total synthesis of complex natural prod-
ucts.1 Acyloin derivatives such as 6 are useful fragments
for syntheses of epothilones B, D, and derivatives, which
use a northern/southern half approach (Scheme 1).2–5
However, installation of the remote C15 stereocentre
of the epothilones 1–5 proved to be a major challenge.
The epothilones are a family of closely related poly-
ketides (isolated from Sorangium cellulosum by Ho¨fle
et al.in 1994) that display a strong in vitro cytotoxicity
against mammalian cells.6,7 The mode of action of these
remarkable 16-membered macrolactones8 was soon
shown to be stabilization ꢁof microtubuli in a manner
similar to paclitaxel (Taxol ).9 The epothilones are com-
2. Results and discussion
In the course of our synthetic approach towards epothi-
lone B (D) and derivatives, we envisioned the C7-17
northern half precursor 6, in which the epothilone-C7–
C14 moiety is derived from the natural diprenol nerol
(cf.neryl bromide 8, Scheme 2).Three distinct strategies
to obtain 6 with the correct C3-stereocentre were inves-
tigated: (i) auxiliary-assisted asymmetric alkylation, (ii)
chemical resolution of a racemic intermediate, and
q Part of this work was also conducted at the Dept.of Organic and
Inorganic Chemistry, Vrije Universiteit Amsterdam, The
Netherlands.
*
Corresponding authors.Tel:. +49 3834 86 4367; fax: +49 3834 86
80066 (U.T.B.); tel.: +49 345 5582 1301; fax: +49 345 5582 1309
(L.A.W.); e-mail addresses: uwe.bornscheuer@uni-greifswald.de;
wessjohann@ipb-halle.de
0957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd.All rights reserved.
doi:10.1016/j.tetasy.2004.06.048

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G. Scheid et al. / Tetrahedron: Asymmetry 15 (2004) 2861–2869
R1
R
O
O
S
S
O
(15)
3
8
OTBS
15
O
15
O
OH
OH
R2
10
(8)
N
N
OR
O
OH
O
OH
O
1 Epothilone A: R1=R2=H
2 Epothilone B: R1=Me, R2=H
5 Epothilone E: R1=H, R2=OH
3 Epothilone C: R=H
4 Epothilone D: R=Me
6
Northern Half
Acyloin Building Block
Scheme 1. Epothilones A–E and northern half fragment 6 (in brackets: epothilone numbering).
(iii) enzymatic kinetic resolution of a racemic inter-
mediate (Scheme 2).
glycolic acid-derived Evansꢀ amide using an allylic
iodide and LiHMDS as base.22 However, at that
moment, our other two approaches towards 6 were
more promising and we decided not to pursue this
route further.
The first approach involved alkylation of chiral O-pro-
tected glycolic acid derivatives 7 (Scheme 3).Initially,
we considered using Evansꢀ oxazolidinones such as (S)-
5-benzyl-1,3-oxazolidin-2-one 11 as chiral auxiliaries
and TBS as the protecting group.Evans ꢀ amide 7a was
prepared by reaction of lithiated oxazolidinone 11 with
(tert-butyldimethylsilyloxy)acetyl chloride.19 However,
deprotonation of this Evansꢀ amide with LDA led to
extensive cleavage of the TBS ether.Alkylation of the
corresponding benzyloxy acetamide 7b with LDA fol-
lowed by neryl bromide 8 afforded the desired alkylation
product, albeit in poor yield (620%).
The second approach made use of our recently devel-
oped racemic acyloin synthesis1 to obtain C3-racemic
epothilone northern half precursors 9.2 Commercially
available tert-butyl acetoacetate was subjected to bro-
mination and subsequent displacement of the bromide
by acetate to give tert-butyl 2-acetoxyacetoacetate 17,
which was subsequently alkylated (NaH, neryl bromide
8, DMF) to give 18 (Scheme 4).1 A SeO2-oxidation can
be used to introduce the hydroxy group at C7.2 Catalytic
regioselective hydrogenation, for example, with Ru[(R)-
BINAP](OAc)2, gives C9/C10-reduced intermediates
with low (R)-preferential enantioselectivity at C10.23
Selective de-alkoxycarbonylation of the tert-butyl ester
gave C3-racemic, acetate mono-protected diol 20; TBS
protection afforded 21, and mild hydrolysis (K2CO3,
MeOH) resulted in the other mono-protected diol 22.
All the substrates for the enzymatic kinetic resolution
(v.i.). For the chemical resolution, C3-alcohol 22 was
reesterified with (R)-a-methoxyphenylacetic acid.The
resulting diastereomers 23a and 23b were easily separa-
ble by normal flash chromatography on silica.In con-
trast to the acyloin acetates, the mandelates require
much longer hydrolysis times.Under basic conditions,
this leads to increased or even complete racemization
of acyloins.Fortunately, mandelate can often serve as
Cardillo et al.reported that the more basic L-(ꢀ)-ephe-
drine-derived imidazolidinone 1220 is an efficient chiral
auxiliary in the alkylation of benzyloxyacetic acid deriv-
atives.21 Indeed, the alkylation of amide 7c resulted in a
significant increase in conversion with respect to the cor-
responding Evansꢀ amide 7b, but the isolated yield did
not exceed 49%.The absolute configuration of alkylated
imidazolinone 13 was assigned in analogy to the results
of Cardillo et al.21 Amide 13 was then treated with
AlMe3 and HN(Me)OMeÆHCl to give the corresponding
Weinreb amide 14, which was smoothly converted to
methyl ketone 15 (MeLiÆLiBr, CH2Cl2).
After completion of these initial studies, Danishefsky
et al.reported the alkylation of a TES-protected
O
(15)
3
OTBS
OR
6
O
O
+
O
O-PG
O-PG
Br
OMe
Ph
Aux*
O
O
O-PG
O
O
7
8
9
10
(i) auxiliary-assisted
asymmetric alkylation
(ii) chemical resolution
(separation of diastereomers)
(iii) enzymatic
kinetic resolution
Scheme 2. Strategies towards enantiomerically pure acyloin 6 derived from neryl derivatives as northern half precursor in the total synthesis of
epothilones B/D (PG = Protective Group or H, TBS = tert-butyldimethylsilyl, Aux* = chiral auxiliary).

G. Scheid et al. / Tetrahedron: Asymmetry 15 (2004) 2861–2869
2863
Aux* =
O
O
O
O
a
b
O
Aux*
R
Aux*
OBn
OBn
OPG
O
N
N
N
7a: Aux* = 11, PG = TBS
7b: Aux* = 11, PG = Bn
7c: Aux* = 12, PG = Bn
13: Aux* = 12
14: R = HN(Me)OMe
15: R = Me
c
Bn
Ph
12
11
Scheme 3. (a) LDA, neryl bromide 8, THF, ꢀ63ꢂC ! rt: no yield with 7a, 20% with 7b, 49% with 7c; (b) AlMe3, HN(Me)OMeÆHCl, CH2Cl2, 75%;
(c) MeLiÆLiBr, CH2Cl2, quant.
O
O
O
O
O
O
OR2
10
9
a
3
e,f
c
2
X
OtBu
OtBu
OR1
AcO CO2tBu
X
20: R1 = Ac, R2= H
16: X = Br
17: X = OAc
18: X = H
19: X = OH
g
h
i
b
d
21: R1 = Ac, R2 = TBS
22: R1 = H, R2 = TBS
23: R1 = (R)-C(O)CH(OMe)Ph,R2 = TBS
Scheme 4. (a) NBS, acetone, quant.; (b) NaOAc, DMF, 68%; (c) NaH, neryl bromide, DMF, 96%; (d) cat. SeO2, t-BuOOH, CH2Cl2, 45%; (e) 100bar
H2, cat.Ru[( R)-BINAP](OAc)2, MeOH/H2O, 89% (9R, ee = 21%); (f) TFA, CH2Cl2, 1h, 75%; (g) TBSCl, Et3N, cat.DMAP, CH 2Cl2, 80%; (h)
K2CO3, MeOH, 97%; or hydrolase (see Table 1) (i) (R)-a-methoxyphenylacetic acid, EDCI, cat.DMAP, CH 2Cl2, 80%.
a protecting group instead of acetate in further steps.
Substitution of the O-methyl-mandelic acid by THP-
protected mandelate is possible, whereas (1S)-camph-
anic acid did not allow diastereomeric separation on
silica.
able, that is, a preferential hydrolysis to (3R)-acyloins,
because the remaining (3S)-acyloin acetates are the ideal
protected building blocks for further synthetic steps
towards the epothilones.32
Acyloin acetate 20 could be resolved with high selectiv-
ity using lipase from Burkholderia cepacia (Amano PS),
E >300, lipase from Pseudomonas sp.( E >150) and a re-
combinant esterase from Streptomyces diastatochromog-
enes (E >100), affording the corresponding (3S)-diol in
excellent ee (>99%).Interestingly, commercial and re-
combinant pig liver esterase exhibited the desired inverse
enantiopreference compared to the other enzymes,
hydrolyzing (3R)-20 preferentially, thus leaving acetate
protected (3S)-epothilone building block (3S)-20 for fur-
ther synthesis.Furthermore, the E-value increased from
E = 8 (commercial PLE) to E = 50 for the recombinant
enzyme.This may be due to the presence of a single iso-
enzyme for the recombinant enzyme, which has already
been shown to have a strong effect on enantioselectivity
and preference.24,25 Similar enantioselectivities were
determined in the resolution of the TBS-protected deriv-
ative 21 (E > 200 for BCL and PSL).
The third approach was based on the enzyme-catalyzed
resolution of 21 and alcohol 20, protected and not pro-
tected at the primary hydroxy group, respectively.Such
an approach would not be confronted with additional
transesterification steps nor with basic racemization
1
upon hydrolysis.We have already demonstrated that
related simple acyloin acetates can be efficiently resolved
using lipase or esterase catalysis with high enantioselec-
tivity to give (S)-acyloins as hydrolysis products
(Schemes 3 and 4).
Here, we used a broader set of lipases and esterases and
investigated their use for the resolution of these two acy-
loin acetates using an aqueous biphasic system com-
posed of phosphate buffer/toluene (Table 1).Most
importantly, a biocatalytic system for the selective
hydrolysis with inverse stereopreference appeared desir-
Table 1. Results of the hydrolase screening for the resolution of 20 (left columns) and 21 (right columns) via hydrolysis in a phosphate buffer/toluene
biphasic system
Enzymea
Time (h)
EeS (%) 20
EeP (%)
cv (%)
Eb
Time (h)
EeS (%) 21
EeP (%) 22
cv (%)
Eb
BCL
PSL
24
2
>98
>99
>99
>99
55
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (R)
>99 (R)
48 (S)
50
50
50
50
35
25
24
75
50
ꢁ300
150
>100
50
24
24
24
37
87
3
>99 (S)
>99 (S)
>99 (S)
27
46
3
>200
>200
>20
PFL
4
CAL-B
PLEc
rPLEc
PFE I
PFE II
SDE
24
2
8
50
24
2
33
15
15
>1
24
4
<3
>99
<1
>99 (S)
>100
a For abbreviations of enzymes see Experimental section; cv=conversion.
b Calculated according to Chen et al.26
c Inversed stereopreference.

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G. Scheid et al. / Tetrahedron: Asymmetry 15 (2004) 2861–2869
3. Conclusions
Please note that in this section IUPAC-numbering and
not epothilone numbering is used.
We have demonstrated that the northern half of epothil-
ones can be best obtained in a chemoenzymatic synthe-
sis.Although the asymmetric synthesis using Evans ꢀ
auxiliary or separation of diastereomers also affords
the target compound, the lipase-catalyzed resolution
allows the fastest access and highest overall yields.
Asymmetric synthesis proved complicated, low yielding
and demanded many expensive steps.Chemical resolu-
tion was easy to scale-up, but required additional trans-
esterification, chromatographic separation, and if free
acyloin was required, a hydrolysis step, which is prone
to racemization.Enzymatic resolution emerged as the
most straightforward method, and scale-up using pH-
titration should be easy.Furthermore, enantioselectivi-
ties were outstanding, the biocatalysts are readily avail-
able, and each enantiomer can be obtained directly by
choosing either an (S)- or (R)-selective hydrolase.An
increase in the overall yield by a dynamic kinetic resolu-
tion with racemization of the nondesired enantiomer is
currently under study.
4.1.1. (4S)-4-Benzyl-3-(tert-butyldimethylsilyloxyacetyl)-
oxazolidin-2-one 7a. (tert-Butyldimethylsilyloxy)acetic
acid (1.05g, 5.0mmol) was dissolved in benzene
(20mL) and 5mL of benzene distilled off to remove
water.The mixture was allowed to cool to room temper-
ature and oxalyl chloride (0.73mL, 1.065g, 8.4mmol)
then added dropwise.After stirring for 30min at room
temperature, the mixture was heated to reflux for
30min.Subsequently, excess oxalyl chloride was dis-
tilled off with half of the benzene.05. 0mL of n-butylli-
thium (10M in hexane, 5.0mmol) was added to a
solution of (4S)-4-benzyloxazolidin-2-one (886mg,
5.0mmol) in THF (25mL) at ꢀ63ꢂC.After stirring for
5min at ꢀ63ꢂC, the benzene solution of (tert-butyldi-
methylsilyloxy)acetyl chloride was slowly added and
the mixture stirred for an additional 30min.After the
mixture was allowed to warm to room temperature,
H2O (10mL) was added and the organic solvent re-
moved in vacuo.The mixture was then extracted with
CH2Cl2 (2 · 25mL).The combined organic layers were
subsequently washed with 0.5M HCl (20mL), saturated
NaHCO3 solution (20mL) and brine (20mL) and dried
over Na2SO4.After filtration, the solvent was removed
in vacuo to yield (4S)-4-benzyl-3-(tert-butyldimethyl-
silyloxyacetyl)-oxazolidin-2-one 7a as a yellow oil.Yield
4. Experimental
4.1. General
All commercial reagents were purchased from Fluka,
Merck, Aldrich, Acros, Strem, Sigma and Lancaster,
and used without further purification, unless otherwise
stated.All oxygen- and water-sensitive reactions were
carried out in oven-dried glassware under argon.THF
was distilled from potassium/benzophenone ketyl,
Et2O was distilled from sodium/potassium/benzophe-
none ketyl, and CH2Cl2 was distilled from CaH2.Other
dry solvents were purchased from Fluka.Flash chroma-
tography was performed using silica gel 60 (230–400
mesh, Merck).Thin-layer chromatography (TLC) was
1
1.60g (4.58mmol, 92%). H NMR (200MHz, CDCl3,
TMS): d = 0.12 (s, 6H), 0.93 (s, 9H), 2.76 (dd, 1H),
3.31 (dd, 1H), 4.21 (s, 2H), 4.65 (m, 1H), 4.81 (s, 2H),
7.16–7.35 (m, 5H) ppm.
4.1.2. (4S)-4-Benzyl-3-(benzyloxyacetyl)oxazolidin-2-one
7b. n-Butyllithium (2.71mL, 10M in hexane,
27.1mmol) was added to a solution of (4S)-4-benzyl-
oxazolidin-2-one (4.78g, 27.1mmol) in THF (100mL)
at ꢀ63ꢂC.After stirring the mixture at ꢀ78ꢂC for
10min, benzyloxyacetyl chloride (3.0g, 16.0mmol) was
added and the mixture stirred at ꢀ63ꢂC for an addi-
tional 1.5h. The mixture was allowed to warm to room
temperature and then poured into brine (500mL) and
extracted with CH2Cl2 (4 · 100mL).The combined or-
ganic layers were dried over Na2SO4, filtered and con-
centrated in vacuo to yield a yellow oil.The crude
product was purified by flash chromatography (column
dimensions: 30 · 3cm, EtOAc/petroleum ether = 1:1) to
obtain (4S)-4-benzyl-3-(benzyloxyacetyl)oxazolidin-2-
one as a colourless solid. Yield 4.93g (15.1mmol,
95%). 1H NMR (200MHz, CDCl3, TMS): d = 2.80
(dd, 1H), 3.29 (dd, 1H), 4.19 (m, 2H), 4.64 (m, 1H),
4.68 (s, 2H), 4.70 (s, 2H), 7.17–7.43 (m, 10H) ppm.
13C NMR (50MHz, CDCl3, TMS): d = 37.46, 54.52,
67.14, 69.59, 73.28, 127.26–129.33 (10C), 134.96,
137.25, 153.29, 169.97ppm.
performed using silica plates (Merck, silica gel 60 F254
)
and developed using Cer-MOP reagent [molybdato-
phosphoric acid (5.0g), cerium (IV) sulfate (2.0g), and
concentrated H2SO4 (16mL) in water (200mL)].Optical
rotations were measured using a 1mL cell with 1dm
path length on a Perkin–Elmer 241MC polarimeter.
IR spectra were recorded as CHCl3 solutions or as thin
1
films between NaCl plates on a Bruker IFS 28. H and
13C NMR spectra were recorded in CDCl3 on either
Bruker ARX 200, ARX 250 and ARX 400 or Varian
Mercury VX 300 and VX 400 spectrometers using
TMS as the internal standard.Chemical shifts d are re-
ported in parts per million (ppm) and coupling con-
stants J given in hertz (Hz).Mass spectrometry was
performed on a Finnigan MAT-90 mass spectrometer
operating at an ionization potential of 70eV.High reso-
lution mass spectra were obtained from a Finnigan
MAT-90 mass spectrometer with isobutene as the ioni-
zation gas.
4.1.3. (4S,5R)-1-Benzyloxyacetyl-3,4-dimethyl-5-phenyl-
imidazolidin-2-one 7c. Compound 7c was synthesized
according to Refs. 20 and 21.
Derivatizations of alcohols for GC were done by expos-
ing an ethyl ether solution of the analyte for 10min with
an excess of trifluoracetic anhydride, followed by
removal of the reagent and solvent in a stream of dry
nitrogen.
4.1.4. Z-(4S,5R,20S)-3,4-Dimethyl-5-phenyl-1-(20-benzyl-
oxy-50,90-dimethyldeca-40,80-dienoyl)-imidazolidin-2-one
13. A solution of diisopropylamine (2.65mL, 1.90g,

G. Scheid et al. / Tetrahedron: Asymmetry 15 (2004) 2861–2869
2865
18.8mmol) in THF (25mL) was cooled to 0ꢂC and n-
butyl-lithium (1.88mL, 10M in hexane, 18.8mmol)
added.The resulting LDA solution was slowly added
at ꢀ63ꢂC to a solution of (4S,5R)-3,4-dimethyl-5-phen-
yl-1-(benzyloxyacetyl)-imidazolidin-2-one 7c (5.30g,
15.7mmol) in THF (25mL). After stirring the mixture
at ꢀ63ꢂC for 1h, neryl bromide (3.40g, 15.7mmol)
was added.After stirring for an additional 3h, the mix-
ture was allowed to warm to room temperature and stir-
red for 15min at room temperature.After addition of
saturated NH4Cl solution (25mL), the organic solvent
was removed in vacuo and H2O (25mL) added.The
mixture was extracted with Et2O (3 · 75mL) and the
combined organic layers dried over Na2SO4.After filtra-
tion, the solvent was removed in vacuo to give a yellow
oil.The crude product was purified by flash chromato-
graphy (column dimensions: 30 · 3cm, EtOAc/petroleum
ether = 1:2) to obtain (4S,5R,20S)-3,4-dimethyl-5-phen-
yl-1-(20-benzyloxy-50,90-dimethyldeca-40(Z),80-dienoyl)-
imidazolidin-2-one 13 as a yellow oil.Yield 36.4g
1.00mmol) in CH2Cl2 (15mL).After the mixture was
stirred for 40min at ꢀ63ꢂC, the reaction was quenched
by cannula transfer to a precooled (0ꢂC) and rapidly
stirred mixture of saturated NH4Cl solution (20mL)
and 3:1 hexane/CH2Cl2 (20mL).The mixture was al-
lowed to warm to room temperature and then diluted
with brine (50mL) and 3:1 hexane/CH2Cl2 (50mL).
After shaking the mixture vigorously, the organic layer
was separated and the aqueous layer was extracted with
CH2Cl2 (30mL).The combined organic layers were
washed with brine (150mL) and dried over Na2SO4.
After filtration, the solvent was removed in vacuo to
yield pure Z-(3S)-3-benzyloxy-6,10-dimethylundeca-
5,9-dien-2-one 15 as a yellow oil.Yield: 308mg
1
(1.03mmol, quantitative). H NMR (200MHz, CDCl3,
TMS): d = 1.59 (s, 3H), 1.67 (s, 3H), 1.70 (s, 3H), 2.02
(s, 4H), 2.17 (s, 3H), 2.42 (t, 2H), 3.77 (t, 1H), 4.52 (q,
2H), 4.78–4.98 (m, 2H), 7.29–7.34 (m, 5H). 13C NMR
(50MHz, CDCl3, TMS): d = 17.61, 23.44, 25.64, 25.70,
26.41, 30.55, 32.03, 72.21, 85.01, 119.03, 124.07,
127.75, 127.81, 128.38, 131.55, 137.60, 138.25. MS (CI,
1
(7.67mmol, 49%). H NMR (200MHz, CDCl3, TMS):
d = 0.79 (d, 3H), 1.56 (s, 3H), 1.65 (s, 6H), 1.98 (s,
4H), 2.45 (m, 2H), 2.80 (s, 3H), 3.84 (m, 1H), 4.54 (q,
2H), 5.08–5.35 (m, 4H), 7.09–7.37 (m, 10H) ppm.
13C NMR (50MHz, CDCl3, TMS): d = 14.88, 17.48,
23.34, 25.56, 26.26, 28.00, 31.61, 31.94, 53.99, 58.76,
72.00, 77.43, 119.72, 124.30, 126.88–128.21 (10C),
131.07, 136.22, 137.60, 138.22, 155.08, 171.85ppm.
ammonia), m/z (%): 301 (13), [M+H]+; 318 (100),
25
[M+NH4]+. ½aŠ ¼ ꢀ18:0 (c 1.0, CHCl3).
D
4.1.7. tert-Butyl-2-bromoacetoacetate 16 and tert-butyl-2-
acetoxyacetoacetate 17. Compounds 16 and 17 were
synthesized according to Ref. 1.
4.1.8.
Z-2-Acetoxy-2-acetyl-5,9-dimethyl-deca-4,8-di-
4.1.5. Z-(2S)-2-Benzyloxy-N-methoxy-N,5,9-trimethyl-
deca-4,8-dienoylamide
enoic-acid-tert-butylester 18. tert-Butyl-2-acetoxyaceto-
acetate 17 (19.5g, 90mmol) was added dropwise to a
stirred suspension of NaH (2.59g, 108mmol) in DMF
(180mL) at 0ꢂC.After liberation of the hydrogen gas
had stopped, neryl bromide (19.6g, 90mmol) was added
dropwise at 0ꢂC.Afterwards, the ice bath was removed
and the mixture stirred at room temperature for 16h.
The mixture was then diluted with ether (750mL) and
washed with water (3 · 200mL) as well as with brine
14. Trimethyl
aluminium
(4.50mL, 2M in toluene, 9.00mmol) was added at 0ꢂC
to a suspension of N,O-dimethyl hydroxylamine hydro-
chloride (878mg, 9.00mmol) in CH2Cl2 (12mL).The
mixture was stirred at room temperature for 15min, then
recooled to ꢀ10ꢂC and a solution of Z-(4S,5R,20S)-3,4-
dimethyl-5-phenyl-1-(20-benzyloxy-50,90-dimethyldeca-
40,80-dienoyl)imidazolidin-2-one 13 (1.42g, 3.00mmol)
in CH2Cl2 (12mL) added.After the mixture was stirred
at ꢀ10ꢂC for 1h, at 0ꢂC for 2h and at room temperature
for 0.5h, it was poured into a mixture of 0.5N HCl
(100mL) and CH2Cl2 (50mL).After shaking the mixture
vigorously for 5min, the organic layer was separated and
the aqueous layer extracted with CH2Cl2 (2 · 25mL).
The combined organic layers were subsequently washed
with 0.5M HCl (60mL) and 1M phosphate buffer
(60mL, pH = 7.0) and dried over Na2SO4.After filtra-
tion, the solvent was removed in vacuo.The crude prod-
uct was purified by flash chromatography (column
dimensions: 30 · 2cm, EtOAc/petroleum ether = 1:2) to
obtain Z-(2S)-2-benzyloxy-N-methoxy-N,5,9-trimethyl-
deca-4,8-dienoylamide 14 as a colourless oil.Yield:
(1 · 200mL).The solution was dried over Na SO4, fil-
2
tered and concentrated in vacuo, to give a slightly yellow
oil.Yield 303. g (86mmol, 96%). 1H NMR (200MHz,
CDCl3, TMS): d = 1.46 (s, 9H), 1.60 (s, 3H), 1.68 (s,
3H), 1.70 (s, 3H), 2.00–2.05 (m, 4H), 2.16 (s, 3H), 2.31
(s, 3H), 2.82–2.87 (m, 2H), 5.00–5.09 (m, 2H) ppm.13
C NMR (50MHz, CDCl3, TMS): d = 17.56, 20.60,
23.52, 25.61, 26.32, 26.95, 27.63, 31.90, 32.30, 83.08,
87.83, 116.22, 123.75, 131.84, 140.07, 165.96, 169.53,
201.06 ppm. IR (thin film): 845 (w), 1024 (w), 1072
(w), 1086 (w), 1115 (w), 1157 (s), 1236 (s), 1256 (s),
1314 (w), 1370 (s), 13895 (w), 1437 (w), 1452 (w), 1746
(s), 2861 (w), 2882 (w), 2932 (m), 2976 (m) cmꢀ1.MS
(CI, isobut.): m/z (%) = 353 (13) [M+H]+, 298 (21), 297
(100), 279 (14), 255 (10), 253 (13), 237 (27), 219 (20),
209 (65), 193 (10), 175 (6), 153 (7), 137 (16).HRMS: cal-
culated for C17H31O5 (MH+): 353.23282; found:
353.23245.
1
779mg (2.25mmol, 75%). H NMR (200MHz, CDCl3,
TMS): d = 1.58 (s, 3H), 1.66 (s, 3H), 1.71 (s, 3H), 2.03
(s, 4H), 2.46 (t, 2H), 3.20 (s, 3H), 3.55 (s, 3H), 4.27 (t,
1H), 4.55 (q, 2H), 5.08 (s, 1H), 5.23 (t, 1H), 7.16–7.36
(m, 5H) ppm.
4.1.9. (4Z,8E)-2-Acetoxy-2-acetyl-5,9-dimethyl-10-hydr-
oxy-deca-4,8-dienoic-acid-tert-butylester 19. Powdered
selenium dioxide (0.158g, 1.42mmol) was suspended in
CH2Cl2 (50mL), and a 70% aq tert-butyl hydroperoxide
solution (10.2g, 79.5mmol) was added and stirred at
room temperature for 30min. Then 10.0g (28.4mmol)
4.1.6. Z-(3S)-3-Benzyloxy-6,10-dimethylundeca-5,9-dien-
2-one 15. A methyllithium lithium bromide complex
(1.87mL, 1.5M in THF, 2.80mmol) was added at
ꢀ63ꢂC to a solution of Z-(2S)-2-benzyloxy-N-meth-
oxy-N,5,9-trimethyldeca-4,8-dienoylamide 14 (345mg,

2866
G. Scheid et al. / Tetrahedron: Asymmetry 15 (2004) 2861–2869
tert-butyl (4Z)-2-acetoxy-2-acetyl-5,9-dimethyldeca-4,8-
dienoate 18 were added and the reaction mixture stirred
at room temperature for 48h.After concentration in
vacuo, 50mL of toluene were added and removed in
vacuo (removal of excess tert-butyl hydroperoxide).This
was repeated three times and the obtained slightly
yellow oil separated by flash chromatography (column
dimensions: 5.0 · 19.5cm, ethyl acetate/petroleum
ether = 1:2). Yield: 4.65g (12.6mmol, 44%) colourless
oil. 1H NMR (250MHz, CDCl3, TMS): d = 1.45 (s,
9H), 1.66 (s, 3H), 1.71 (s, 3H), 1.90–2.15 (m, 4H), 2.15
(s, 3H), 2.31 (s, 3H), 2.85–2.88 (m, 2H), 3.99 (s, 2H),
5.02 (m, 1H), 5.38 (m, 1H) ppm.13C NMR (62.5MHz,
CDCl3, TMS): d = 13.62, 20.69, 23.47, 25.78, 27.03,
27.74, 31.62, 32.36, 68.71, 83.29, 87.97, 116.65, 124.95,
135.42, 139.69, 166.01, 169.60, 201.41 ppm. IR (thin
film): 755 (w), 845 (w), 1018 (w), 1049 (w), 1072 (w),
1157 (m), 1236 (m), 1258 (m), 1371 (m), 1395 (w),
1435 (w), 1456 (w), 1742 (s), 2854 (w), 2934 (w), 2978
(w) cmꢀ1.MS (CI, isobut).: m/z (%) = 369 (6) [M+H]+,
329 (6), 311 (26), 295 (100), 271 (11), 253 (24),
235 (14), 203 (10), 169 (9), 135 (10).HRMS: calcu-
lated for C20H33O6 (MH+): 369.22772; found:
369.22880.
4.1.11.
(10R)-3-Acetoxy-11-hydroxy-6,10-dimethyl-5-
undecen-2-one 20. 1.032g (2.79mmol) (9R)-(4Z)-2-
acetoxy-2-acetyl-5,9-dimethyl-10-hydroxydeca-4-enoic
acid tert-butyl ester 24 were dissolved in 28mL CH2Cl2
and 2.80mL TFA added. After stirring for 2h at room
temperature, all volatile matter was removed in vacuo
and the remaining oil dissolved in 28mL methanol.
Then 5.6mL saturated aqueous NaHCO3 solution was
added and the suspension stirred for 140min at ambient
temperature before dilution with 200mL ether was per-
formed.The organic layer was washed two times with
50mL water as well as once with 50mL brine.After dry-
ing over Na2SO4, filtration and removal of the solvents
in vacuo, a slightly yellow oil was obtained.Purification
was achieved by flash chromatography (column dimen-
sions: 2.0 · 20.0cm, ethyl acetate/petroleum ether =
2:3).Yield: 568mg (21.0mmol, 75%) colourless oil,
ee = 21%.The enantiomeric excess at C10 was deter-
mined by chiral HPLC on a cyclobond I 2000 column
(250 · 4.6mm, 5lL) with acetonitrile/0.2% triethylam-
monium acetate buffer.Diastereomers are not resolved
1
in NMR at the given resolution. H NMR (250MHz,
CDCl3, TMS): d = 0.92 (d, 3H, J = 6.6Hz), 1.00–1.20
(m, 1H), 1.30–1.50 (m, 3H), 1.60 (m, 1H), 1.70 (s, 3H),
1.73 (s, 1H, OH), 2.01 (m, 2H), 2.14 (s, 3H), 2.16 (s,
3H), 2.48 (m, 2H), 3.46 (m, 2H), 4.98 (m, 1H, CHOAc),
5.11 (m, 1H) ppm. 13C NMR (62.5MHz, CDCl3, TMS):
d = 16.57, 20.69, 23.42, 25.20, 26.28, 29.06, 32.04, 33.02,
35.71, 68.19, 78.56, 117.90, 139.08, 170.59, 205.47 ppm.
IR (thin film): 755 (w), 986 (w), 1047 (m), 1175 (w), 1242
4.1.10. Z-(9R)-2-Acetoxy-2-acetyl-5,9-dimethyl-10-hydr-
oxy-deca-4-enoic acid tert-butyl ester 24. 7.94g
(21.6mmol)
(4Z)-2-acetoxy-2-acetyl-5,9-dimethyl-10-
hydroxydeca-4,8-dienoic acid tert-butyl ester 19 was dis-
solved in 15.0mL absolute methanol and 750lL demin-
eralized water added.The solution was degassed with
three freeze-thaw-cycles before 185mg Ru[(R)-BINA-
P](OAc)2 were added and put in an autoclave under
nitrogen atmosphere together with a magnetic stirring
bar.After threefold purging with hydrogen (999.99%,
water 5vpm, oxygen 2vpm), the autoclave was set under
a pressure of 100bar hydrogen and stirred at room tem-
perature for 25h.The hydrogen pressure was released
and the solution concentrated in vacuo.The obtained
brown oil was purified by flash chromatography (col-
umn dimensions: 4.5 · 25.0cm, ethyl acetate/petroleum
ether = 1:2). Yield: 7.08g (19.1mmol, 88%) colourless
oil.The ee was determined after dealkoxycarbonylation
to compound 20 (cf.next procedure) to be 21%.The
racemic diastereomers behave like a single compound
and show only one set of signals in NMR, that is, the
stereocentres behave spectroscopically independent at
the given resolution. 1H NMR (250MHz, CDCl3,
TMS): d = 0.91 (d, 3H, J = 6.7Hz), 1.45 (s, 9H), 1.68
(s, 3H), 1.0–2.0 (m, 7H), 2.15 (s, 3H), 2.31 (s, 3H),
2.85 (m, 2H), 3.41 (dd, AB, J1 = 10.5Hz, J2=6.3Hz,
1H), 3.49 (dd, AB, J1 = 10.5Hz, J2 = 5.9Hz, 1H), 5.00
(m, 1H) ppm. 13C NMR (62.5MHz, CDCl3, TMS):
d = 16.59, 20.70, 23.53, 25.20, 27.03, 27.73, 32.04,
32.29, 32.98, 35.71, 68.15, 83.23, 88.03, 116.21, 140.32,
165.99, 169.92, 201.36ppm. IR (thin film): 756 (w), 845
(w), 1045 (m), 1065 (m), 1080 (m), 1157 (s), 1235 (s),
1258 (s), 1371 (s), 1395 (w), 1429 (w), 1456 (w), 1744
(s), 2874 (w), 2934 (m), 2972 (m) cm1.MS (CI, isobut).:
m/z (%) = 369 (6) [M+H]+.HRMS: calculated for
C20H33O6 (MH+): 371.2433; found: 371.242097. EA:
(s), 1375 (m), 1435 (w), 1456 (w), 1730 (s), 1744 (s),
22
D
2872 (m), 2932 (s) cmꢀ1. ½aŠ ¼ þ4:9 (c, 1); MS (ESI–
MS): m/z (%) = 563.3 (100) [2M+Na]+, 293.0 (54)
[M+Na]+, 271.1 (7) [M+H]+.EA: calculated, C 555.5,
H 7.46; found: C 55.39, H 7.54.
4.1.12.
oxy-6,10-dimethyl-5-undecen-2-one 21. 528mg (1.95
mmol) (10R)-3-Acetoxy-11-hydroxy-6,10-dimethyl-5-
Z-(10R)-3-Acetoxy-11-tert-butyldimethylsilyl-
undecen-2-one 20 were dissolved in 10.0mL absolute
CH2Cl2.After addition of 541 lL (395mg, 3.90mmol),
triethylamine and 12mg (0.10mmol) of DMAP, the
solution was cooled with an ice bath.After stirring for
5min 368mg (2.44mmol) of TBSCl were added at once
and the resulting solution stirred at 0ꢂC for 2h and
additional 14h at room temperature.The resulting col-
ourless suspension was again cooled to 0ꢂC before
460lL of methanol were added.After stirring for
30min at 0ꢂC, all solvents were removed in vacuo.Ether
(15mL) as well as 15mL saturated aqueous NH4Cl solu-
tion were then added and after intensive stirring and
separation, the aqueous phase extracted twice with
10mL ether.The combined organic layers were washed
with 15mL brine, dried over Na2SO4, filtered and con-
centrated in vacuo.The obtained oil was purified by
means of flash chromatography (column dimensions:
2.0 · 20.0cm, ethyl acetate/petroleum ether = 1:10).
Yield: 597mg (1.55mmol, 80%) colourless oil. The dia-
stereomers are not resolved in NMR at the given resolu-
1
tion. H NMR (250MHz, CDCl3, TMS): d = 0.04 (s,
6H), 0.87 (d, J = 6.7Hz, 3H), 0.89 (s, 9H), 1.00–1.20
(m, 1H), 1.30–1.50 (m, 3H), 1.52–1.60 (m, 1H), 1.70 (s,
3H), 1.97–2.03 (m, 2H), 2.13 (s, 3H), 2.15 (s, 3H),
calculated: C 64.84, H 9.25; found: C 64.46, H 9.36;
25
D
½aŠ ¼ þ5:2 (c 0.62, CHCl3) at ee ꢂ21%.

G. Scheid et al. / Tetrahedron: Asymmetry 15 (2004) 2861–2869
2867
2.44–2.50 (m, 2H), 3.37 (dd, J = 9.7Hz, J = 6.4Hz, 1H),
3.44 (dd, J = 9.7Hz, J = 6.0Hz, 1H), 4.98 (m, 1H), 5.09
(m, 1H) ppm. 13C NMR (62.5MHz, CDCl3, TMS):
d = ꢀ5.34, 16.72, 18.36, 20.68, 23.45, 25.30, 25.97,
26.59, 29.02, 32.16, 33.11, 35.74, 68.28, 78.57, 117.81,
138.89, 170.52, 205.16ppm. IR (thin film): 775 (m),
837 (m), 1053 (w), 1092 (m), 1248 (s), 1373 (w), 1458
(w), 1472 (w), 1734 (s), 1748 (s), 2857 (m), 2891 (w),
2905 (w), 2932 (m), 2955 (m) cmꢀ1.MS (CI, isobut).:
m/z (%) = 385 (13) [M+H]+, 327 (13), 267 (26), 253 (6),
193 (40), 175 (62), 117 (100).HRMS: calculated for
Na2SO4, filtrated and concentrated in vacuo.The
remaining yellow oil contained two diastereomeric
esters*, which were separated by flash chromatography
(column dimensions: 3.5 · 20.0cm, diethyl ether/petro-
leum ether = 2:9). Total yield 2.796g (5.697mmol,
81%).The absolute configuration at C1 was assigned
by NMR according to Raban and Mislow,30 and in
addition according to Riguera et al.,31 with identical
results.
Fraction one: (R)-a-methoxyphenylacetic acid 3Z-
(1R,8R*)-1-acetyl-9-(tert-butyldimethylsilyloxy)-4,8-di-
methyl-non-3-enyl ester 23a. Yield: 1.33g (2.710mmol,
C21H41O4Si (MH+): 385.27740; found: 385.278524.
25
D
½aŠ ¼ þ0:0 (c 0.33, CHCl3).
38%).R value: 0.31 (diethyl ether/petroleum ether =
f
4.1.13. Z-(10R)-11-(tert-Butyldimethylsilyloxy)-3-hydr-
oxy-6,10-dimethyl-undec-5-en-2-one 22. 1.943g (5.05
1:4). 1H NMR (250MHz, CDCl3, TMS): d = 0.05 (s,
6H), 0.89 (d, 3H, J = 6.6Hz), 0.91 (s, 9H), 1.00–1.65
(m, 5H), 1.68 (s, 3H), 1.82 (s, 3H), 1.99 (m, 2H), 2.50
(m, 2H), 3.35–3.46 (m, 2H), 3.47 (s, 3H), 4.84 (s, 1H),
5.00–5.09 (m, 2H), 7.37–7.51 (m, 5H) ppm. 13C NMR
(62.5MHz, CDCl3, TMS): d = ꢀ5.38, 16.68, 18.33,
23.42, 25.26, 25.93, 26.07, 29.11, 32.13, 33.07, 35.68,
57.42, 68.26, 78.98, 82.44, 117.64, 127.32, 128.63,
128.93, 135.82, 139.91, 170.08, 204.99ppm. IR (thin film):
665 (w), 697 (w), 732 (w), 757 (w), 776 (m), 814 (w), 837
(s), 916 (w), 939 (w), 1006 (w), 1033 (w), 1043 (w), 1097
(s), 1114 (s), 1170 (m), 1199 (m), 1254 (s), 1321 (w),
1360 (m), 1388 (w), 1459 (m), 1469 (m), 1729 (s), 1757
mmol)
(10R)-3-Acetoxy-11-(tert-butyl-dimethylsilyl-
oxy)-6,10-dimethyl-5-undecen-2-one 21, ee = 21%, were
dissolved in 20.0mL methanol and 400mL of a satu-
rated potassium carbonate solution then added.After
stirring at ambient temperature for 5–14min, 30mL
brine were added and fivefold extraction with 30mL
diethyl ether followed.The hydrolysis was already com-
pleted after 5min according to TLC.A timely work-up
is crucial, because the yield drops drastically if the reac-
tion is not stopped in time.For example, stirring for
90min resulted in dramatic drop of the yield to 43%.
The combined organic layers were washed with 50mL
brine and dried over Na2SO4.After filtration and re-
moval of the solvent in vacuo, the remaining oil was
purified by flash chromatography (column dimensions:
2.0 · 20.0cm, ethyl acetate/petroleum ether = 1:4).
Yield: 1.686g (4.92mmol, 97%). The diastereomers were
(s), 2855 (m), 2929 (s), 2952 (s) cmꢀ1
.
Fraction two: (R)-a-methoxyphenylacetic acid 3Z-
(1S,8R*)-1-acetyl-9-(tert-butyldimethylsilyloxy)-4,8-di-
methyl-non-3-enyl ester 23b. Yield: 1.466g (3.182mmol,
42%).R value: 0.22 (diethyl ether/petroleum ether =
f
1
1
not resolved in NMR at the given resolution. H NMR
1:4). H NMR (250MHz, CDCl3, TMS): d = 0.04 (s,
(250MHz, CDCl3, TMS): d = 0.03 (s, 6H, TBS), 0.83 (d,
J = 6.8Hz, 3H), 0.89 (s, 9H, TBS), 1.00–1.65 (m, 5H),
1.69 (s, 3H), 2.00 (m, 2H), 2.12 (s, 3H), 2.25–2.45 (m,
1H), 2.45–2.60 (m, 1H), 3.40 (m, 2H), 4.20 (m, 1H),
5.10 (m, 1H) ppm. 13C NMR (62.5MHz, CDCl3,
TMS): d = 5.43, 16.63, 18.27, 23.41, 25.21, 25.38,
25.88, 32.02, 32.22, 33.04, 35.61, 68.18, 76.73, 118.07,
139.59, 209.55 ppm. IR (thin film): 667 (w), 775 (m),
837 (s), 1006 (w), 1093 (s), 1147 (w), 1163 (w), 1249
(s), 1362 (m), 1373 (m), 1387 (w), 1419 (w), 1436 (w),
1457 (m), 1464 (m), 1472 (m), 1653 (w), 1684 (w), 1700
(w), 1733 (s), 1747 (s), 2855 (m), 2881 (w), 2902 (m),
6H), 0.87 (d, 3H, J = 6.7Hz), 0.91 (s, 9H), 0.95–1.75
(m, 5H), 1.57 (s, 3H), 1.75–2.05 (m, 2H), 2.11 (s, 3H),
2.30–2.50 (m, 2H), 3.37–3.51 (m, 2H), 3.47 (s, 3H),
4.84 (m, 1H), 4.90 (s, 1H), 5.00 (dd, 1H, J = 7.6Hz,
J = 5.3Hz), 7.35–7.49 (m, 5H) ppm. 13C NMR
(62.5MHz, CDCl3, TMS): d = ꢀ5.38, 16.67, 18.32,
23.31, 25.20, 25.93, 26.45, 28.83, 32.04, 33.05, 35.65,
57.47, 68.24, 79.04, 82.25, 117.42, 127.16, 128.58,
128.76, 135.98, 139.82, 170.31, 204.50ppm. IR (thin
film): 665 (w), 697 (w),733 (w), 756 (w), 776 (m), 814
(w), 837 (s), 916 (w), 939 (w), 1005 (w), 1032 (w), 1043
(w), 1098 (s), 1114 (s), 1171 (s), 1200 (m), 1254 (m),
2907 (m), 2929 (m), 2950 (m), 2956 (m) cmꢀ1
.
1388 (m), 1459 (m), 1470 (m), 1731 (s), 1757 (s), 2855
25
D
25
(m), 2897 (m), 2929 (s), 2952 (s) cmꢀ1. ½aŠ ¼ ꢀ23:9 (c
½aŠ ¼ þ0:0 (c 0.62, CHCl3).
D
0.91, CHCl3).
4.1.14. (R)-a-Methoxyphenylacetic acid Z-(1R,8R)-1-
acetyl-9-(tert-butyldimethylsilyloxy)-4,8-dimethylnon-3-
enyl ester 23a and (R)-a-methoxyphenylacetic acid
Z-(1S,8R)-1-acetyl-9-(tert-butyldimethylsilyloxy)-4,8-
dimethylnon-3-enyl ester 23b. To a solution of 2.413g
(7.04mmol) (10R)-11-(tert-butyldimethylsilyloxy)-3-hy-
droxy-6,10-dimethyl-undec-5-en-2-one 22 of ee = 21%,
1.287g (7.75mmol) (R)-a-methoxyphenylacetic acid
and 86mg (0.70mmol) of DMAP in 72.0mL CH2Cl2,
were added 2.701g (14.09mmol) of EDCI and the solu-
tion stirred 1h 40min at ambient temperature.Diethyl
ether (250mL) was then added and the resulting suspen-
sion extracted twice with 100mL water as well as twice
with 100mL brine.The organic layer was dried over
*C1/mandelic ester diastereomers.The additional ste-
reocentre at C8 (de = 21%) had no influence on the chro-
matographic separation and on NMR at the given
resolution.
4.2. Enzyme-catalyzed preparative scale resolutions of 21
and 20
All organic solvents were analytical grade or higher and
˚
dried overnight over activated molecular sieves (3A) be-
fore use.Commercial enzyme preparations were used as
delivered.Lipases from the following organisms were
used: Candida antarctica (Roche, Chirazyme L2, CAL-B),

2868
G. Scheid et al. / Tetrahedron: Asymmetry 15 (2004) 2861–2869
Burkholderia cepacia (Amano PS, BCL). Pseudo-
monas fluorescens (Amano AK, PFL), Pseudomonas sp.
(Chirazyme L6, PSL) and pig liver esterase (PLE, Sigma).
Production of the recombinant esterases from P. fluores-
cens (PFE-I,27 PFE-II28), Streptomyces diastatochromo-
genes (SDE29) by expression in E. coli has already been
described.Recombinant pig liver esterase (rPLE) was
produced by expression in the yeast Pichia pastoris.24,25
Amano, Nagoya, Japan; Novozymes, Bagsvaerd,
Danmark; Roche Diagnostics, Penzberg, Germany;
and Degussa, Hanau, Germany for their generous gift
of enzymes and amino acid derivatives.
References
1. Scheid, G. O.; Kuit, W.; Ruijter, E.; Orru, R. V. A.;
Henke, E.; Bornscheuer, U.; Wessjohann, L. A. Eur. J.
Org. Chem. 2004, 1063–1074.
4.2.1. Method for the screening of suitable enzymes.
.Five milligrams hydrolase (lipase or esterase) were dis-
solved in 500lL sodium phosphate buffer (pH7.5,
50mM) in 1.5mL Eppendorf tubes and thermostated
to 37ꢂC on a thermoshaker (Eppendorf, Hamburg, Ger-
many).Reactions were initiated by the addition of
200lL of a substrate solution (10mg/mL acyloin acetate
in toluene).Reaction mixtures were shaken at 900rpm.
For 24h, samples of 20lL from the organic phase were
taken from each reaction and transferred to a new vial
with 200lL dichloromethane.The mixture was vortexed
and centrifuged (2min, 13,000rpm) and the supernatant
transferred to a new vial and dried over a small amount
Na2SO4.After another centrifugation (2min,
13,000rpm), the organic phase was transferred to a
new vial.Samples were analyzed by GC with a chiral
column (Heptakis-(2,6-di-O-methyl-3-O-pentyl)-b-cyclo-
dextrin, 25m · 0.25mm). For baseline separation of the
enantiomers of alcohol 22, analytical derivatization with
trifluoro acetic acid anhydride was necessary (see gen-
eral part).
2.Gabriel, T;.Wessjohann, LA. .
Tetrahedron Lett. 1997, 38,
1363–1366; Gabriel, T.; Wessjohann, L. A. Tetrahedron
Lett. 1997, 38, 4387–4388; Scheid, G. O.; Eichelberger, U.;
Wessjohann, L.A.
publication.
Tetrahedron Lett. submitted for
3.Wessjohann, L. A;. Scheid, G. O. Deutsche Offen-
legungsschrift DE0010051136A1 (18.04.2002), Germany,
16.10.2000, CA 136: 325358.
4. Wessjohann, L. A.; Scheid, G. O.; Bornscheuer, U.;
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legungsschrift DE0010134172A1 (23.01.2003), Germany,
13.07.2001.
5. Wessjohann, L. A.; Scheid, G. O.; Bornscheuer, U.;
Henke, E.; Kuit, W.; Orru, R. V. A. Patent WO2002/
032844A2 (25.04.2002), EP 200111992, International,
16.10.2001, CA 136:340534.
6. Gerth, K.; Bedorf, N.; Ho¨fle, G.; Irschik, H.; Reichen-
bach, H. J. Antibiotics 1996, 49, 560–564.
7.Ho¨fle, G.; Bedorf, N.; Steinmetz, H.; Schomburg, D.;
Gerth, K.; Reichenbach, H. Angew. Chem., Int. Ed. 1996,
35, 1567–1569.
8. Wessjohann, L. A.; Ruijter, E.; Garcia-Rivera, D.; Brandt,
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4.2.2. Preparative enzymatic resolution of 20. Acyloin
acetate 20 (479mg, 1.248mmol) was added to a biphasic
mixture composed of 20mL distilled water and 2mL tolu-
ene at 37ꢂC.After addition of lipase CAL-B (98mg), the
reaction was stirred in a pH-stat (set to pH7.5) and
monitored by base consumption.The mixture was
extracted three times with diethyl ether and dried over
Na2SO4.After removal of excess solvent in vacuo, ester
and alcohol were separated by column chromatography
(hexane/diethyl ether = 10:1, 5:1).Yields: product (diol,
>99% ee): 179mg (42%), remaining substrate (acetate
20, 46% ee): 186mg (48%).Conv.31%, E >200.
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4.2.3. Preparative enzymatic resolution of 21. Acyloin
acetate 21 (253mg, 0.937mmol) was added to a biphasic
mixture composed of 30mL phosphate buffer (pH7.5,
50mM) and 20mL toluene at 37ꢂC.After addition of
lipase Amano PS (270mg), the reaction was stirred until
GC analysis revealed that the desired optical purity and
conversion was achieved.The enzyme was removed by
centrifugation, filtrated and extracted twice with toluene
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(20mL).The organic layer was dried over Na SO4 and
2
the excess solvent was removed in vacuum.Ester and
alcohol were separated by column chromatography
(hexane:ethyl acetate, 6:1).Yields: product (alcohol 22,
>99% ee): 59.7mg (28%), remaining substrate (acetate
21, 92% ee): 74.7mg (27%). Conv. 48%, E >200.
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