Biocatalytic racemization of α-hydroxycarboxylic acids using a stereo-complementary pair of α-hydroxycarboxylic acid dehydrogenases

Article abstract of DOI:10.1016/j.tet.2009.06.051

Biocatalytic racemization of aliphatic, (aryl)aliphatic and aromatic α-hydroxycarboxylic acids was achieved via a reversible oxidation-reduction sequence using a pair of stereo-complementary Prelog- and anti-Prelog d- and l-α-hydroxyisocaproate dehydrogenases from Lactobacillus confusus DSM 20196 and Lactobacillus paracasei DSM 20008, resp., overexpressed in Escherichia coli. The mild reaction conditions ensured essential 'clean' isomerization, undesired 'over-oxidation' of the substrate forming the α-ketoacid could be suppressed by exclusion of O₂ and adjustment of the NAD⁺/NADH-ratio.

Full text of DOI:10.1016/j.tet.2009.06.051

Tetrahedron 65 (2009) 7752–7755
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Biocatalytic racemization of
a-hydroxycarboxylic acids using a stereo-
complementary pair of -hydroxycarboxylic acid dehydrogenases
a
Anne Bodlenner a,b, Silvia M. Glueck a b, Bettina M. Nestl a , Christian C. Gruber ,
,
,
b
a
c
c
a
a,
*
Nina Baudendistel , Bernhard Hauer , Wolfgang Kroutil , Kurt Faber
a
Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria
Research Centre Applied Biocatalysis, Petersgasse 14, A-8010 Graz, Austria
b
c
BASF-AG, GVF/E-B9, D-67056 Ludwigshafen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 May 2009
Received in revised form 9 June 2009
Accepted 12 June 2009
Available online 18 June 2009
Biocatalytic racemization of aliphatic, (aryl)aliphatic and aromatic
ieved via a reversible oxidation-reduction sequence using a pair of stereo-complementary Prelog- and
anti-Prelog - and -hydroxyisocaproate dehydrogenases from Lactobacillus confusus DSM 20196 and
Lactobacillus paracasei DSM 20008, resp., overexpressed in Escherichia coli. The mild reaction conditions
a-hydroxycarboxylic acids was ach-
D
L-a
ensured essential ‘clean’ isomerization, undesired ‘over-oxidation’ of the substrate forming the
a-ke-
þ
toacid could be suppressed by exclusion of O
2
and adjustment of the NAD /NADH-ratio.
Keywords:
Biocatalytic racemization
Ó 2009 Elsevier Ltd. All rights reserved.
a
-Hydroxycarboxylic acids
Stereo-complementary
-hydroxyisocaproate dehydrogenases
Lactobacillus spp.
a
1. Introduction
intermediate catalyzed by NAD(P)H-dependent dehydrogenases
9
(Scheme 1). Kinetic analysis of a model system employing a set of
Racemization is an important (but often overlooked) tool for the
recycling of unwanted stereoisomers derived from kinetic resolu-
tions and the key to dynamic (kinetic) resolution processes (DKR).
stereo-complementary alcohol dehydrogenases possessing Prelog-
and anti-Prelog selectivity revealed that clean isomerization of sec-
1
10
alcohols was indeed feasible.
The majority of DKRs published so far either rely on (transition)
metal-catalysis,1
a–c,e,2
on free radicals oron (zeolite-type) acid-base
3
OH
catalyzed isomerization.4 Although enzymatic racemization plays
a somewhat minor role in this context, it shows great potential in
view of the mild reaction conditions and the excellent compatibility
–6
R
COOH
(S)-1a-e
L-HicDH
O
biocatalytic
NAD+ NADH
7
of enzymes with each other. In search for clean and efficient en-
racemization
R
COOH
2a-e
zymatic racemization of stereochemically stable a-hydroxycarbox-
OH
ylic acids we found that the desired activity was widespread among
D-HicDH
8
R
(
COOH
R)-1a-e
Lactobacillus spp., in particular Lactobacillus paracasei DSM 20008,
DSM 20207 and Lactobacillus delbrueckii DSM 20074, which allowed
the isomerization of awide range of aliphatic, (hetero)aryl- and aryl-
HicDH = α-Hydroxyisocaproate Dehydrogenase
aliphatic a-hydroxycarboxylic acids. Subsequent inhibitor-studies
using a cell-free extract of L. paracasei DSM 20207 proved that the
involvement of a putative racemase could be ruled out and sug-
gested that the racemization proceeded through a reversible oxi-
Substrate 1
a
b
c
d
e
R
3 2 2
4 9 6
(CH ) CH-CH - n-C H - n-C H13- Ph-CH2- Ph-
Scheme 1. Enzymatic racemization of
hydroxyisocaproate dehydrogenases (HicDH).
a-hydroxycarboxylic acids using D- and L-a-
dation–reduction sequence via the corresponding
a-ketoacid as
In order to extend this system to a-hydroxycarboxylic acids, we
conducted a search for a suitable set of stereo-complementary
a-
*
Corresponding author. Tel.: þ43 316 380 5332; fax: þ43 316 380 9840.
E-mail address: kurt.faber@uni-graz.at (K. Faber).
hydroxycarboxylic acid dehydrogenases ( -ketoacid reductases).
a
0
040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.06.051

A. Bodlenner et al. / Tetrahedron 65 (2009) 7752–7755
7753
Detailed analysis of the literature suggested two candidates:
-2-hydroxyisocaproate dehydrogenase12 (HicDH) from Lactobacil-
lus confusus DSM 20196 and L. paracasei DSM 20008, respectively,
which were investigated for the stereoselective reduction of
L
-11 and
The difference in racemization rates of enantiomers is a com-
mon phenomenon in enzymatic racemization and is a result of the
D
M
difference in the individual K - and kcat-values for enantiomers for
18
a
-
a given enzyme. Excellent racemization rates (50–96% relative to
ketoacids.1
0,11
Moreover, their substrate profile (in the reduction
the natural substrate 1a) were obtained for the aliphatic (entries 2–5,
mode) nicely matched that of the racemization-activity of Lactoba-
Table 1) and aryl-aliphatic a-hydroxycarboxylic acids (entries 6–7).
8
cillus spp. This unexpected observation prompted us to use
HicDH as stereo-complementary components for the design of
a redox-based racemization system for -hydroxycarboxylic acids
by mimicking a ‘racemase’. - and -HicDH from Lactobacillus DSM
0008 and DSM 20196 were cloned and overexpressed into E. coli
details will be published in a forthcoming paper).
D
- and
L-
Mandelate (entries 8–9) was racemized more slowly (average rel-
ative rate of both enantiomers ca. 25%), which illustrates the flex-
ibility of this system.
a
D
L
13
Table 1
2
Relative racemization rates of substrates 1a–e
(
Entry
Substrate
R
Relative ratea [%]
1
2
3
4
5
6
7
8
9
(S)-1a
(S)-1b
(R)-1b
(S)-1c
(R)-1c
(S)-1d
(R)-1d
(S)-1e
(R)-1e
(CH
3
)
2
CH–CH
2
–
100
74
2
. Results and discussion
n-C
n-C
n-C
n-C
4
H
H
H
H
9
–
–
4
6
6
9
65
81
50
82
96
32
15
13
13
–
–
–
The starting
a
-hydroxyacid enantiomer 1 was oxidized by the
þ
corresponding HicDH at the expense of NAD to furnish the cor-
responding prochiral
Ph–CH
Ph–CH2–
Ph–
2
a
-ketoacid 2 as transient species, which in
turn was stereoselectively reduced by the second stereo-comple-
mentary HicDH to produce the corresponding mirror-image en-
antiomer (Scheme 1). Since the cofactor is recycled internally, only
catalytic amounts are required and external recycling is not re-
quired. Due to the reversibility of both redox reactions, the overall
Ph–
a
Relative rates were measured from the slope of the decline of ee versus time at
the onset of the reaction (conversion <5%), values are expressed as % relative to the
natural substrate 1a, which was set as standard (100%).
result is an equilibrium between both
R)- and (S)-1 via -ketoacid 2. Moreover, the amount of
can be kept at a minimum by exclusion of O and by adjusting the
ratio of NADH/NAD in favor of NADH in order to shift the equi-
a
-hydroxyacid enantiomers
3
. Conclusion
(
2
a
a-ketoacid
2
In summary, efficient enzymatic racemization of a set of ali-
þ
phatic, aryl-aliphatic and aromatic
achieved via a reversible oxidation-reduction sequence using
a pair of stereo-complementary -hydroxycarboxylic acid de-
hydrogenases, i.e. - and -hydroxyisocaproate dehydrogenases
from L. confusus DSM 20196 and L. paracasei DSM 20008, resp.,
which were cloned and overexpressed in E. coli. Exclusion of mo-
lecular oxygen and careful adjustment of the NAD /NADH-ratio
prevented undesired ‘over-oxidation’ of the substrate and reduced
the formation of the non-chiral
a-hydroxycarboxylic acids was
10
libriums towards the
cemization proved to be essentially ‘clean’ and no by-products were
formed, except for traces of the intermediate
a-hydroxycarboxylic acid 1. Overall, the ra-
a
a
-ketoacid 2 (ꢀ3%).
D
L-a
In order to analyze the proper functioning of the system, race-
mization of (R)- and (S)-mandelic acid 1e was investigated using
both HicDH clones separately and in tandem using a 1:1 mixture of
þ
D- and L-HicDH (Fig. 1). As expected, only negligible isomerization
occurred in presence of a single HicDH, while good racemization
rates were obtained with the tandem-system (Fig. 1).
a
-ketoacid intermediate (ꢀ3%).
a
-Hydroxycarboxylic acids are widespread structural building
blocks of natural products and are versatile chiral synthons for the
synthesis of pharmaceuticals. 3-Phenyllactate 1d is used as chiral
4. Experimental
4.1. General
14
building block for the synthesis of bioactive compounds, such as
15
natural antibiotic agents. Mandelate 1e serves as building block
1
6
for the production of semisynthetic penicillins, cephalosporins
The following commercially available chemicals were used as
received: rac- and (S)-2-hydroxyisocaproic acid 1a, rac-2-hydroxy-
hexanoic acid 1b, rac-2-hydroxyoctanoic acid 1c (TCI), rac-, (R)- and
(S)-phenyllactic acid 1d (Sigma Aldrich), rac-, (R)- and (S)-mandelic
acid 1e (Fluka), Candida antarctica lipase immobilized on acrylic
resin was purchased from Sigma (L 4777), and C. antarctica lipase B
17
and anti-obesity agents. In order to prove the applicability of the
method, HicDH-catalyzed racemization of a set of structurally di-
verse substrates was investigated by using a series of (linear and
branched) aliphatic 1a–c, aryl-aliphatic 1d and aromatic 1e
a-
hydroxycarboxylic acids.
100
OH
CO2H
75
50
25
0
(S)-1e/L-HicDH
(
S)-1e
(S)-1e/D- and L-HicDH
-
-50
75
-100
25
(
R)-1e/D- and L-HicDH
OH
CO2H
R)-1e
-
(
R)-1e/D-HicDH
(
0
5
10
15
20
25
Time [h]
Figure 1. Time course of the racemization of (R)- and (S)-mandelic acid 1e in presence of a single (D- or L-) or tandem (D- and L-) HicDH system.

7754
A. Bodlenner et al. / Tetrahedron 65 (2009) 7752–7755
(
Novozym 435) was from Novozymes. The cloning and over-
expression of -hydroxyisocaproate dehydrogenase (HicDH) from L.
paracasei DSM 20008 and -HicDH from L. confusus DSM 20196 in E.
coli will be published in a forthcoming paper. NMR spectra were
4.4.1. General work up
D
The reaction mixture was acidified with 6 M HCl (1 drop) and
the cells were removed by centrifugation. The supernatant was
extracted with ethyl acetate and the organic phase was dried over
L
1
measured in CDCl
and 90 (13C) MHz. Chemical shifts are reported relative to TMS (
.00) and coupling constants (J) are given in Hz. TLC plates were run
on silica gel Merck 60 (F254) and compounds were visualized by
spraying with Mo-reagent [(NH Mo 24$4H (100 g/L),
Ce(SO $4H O (4 g/L) in H SO (10%)]. Conversion and enantio-
3
using a Bruker AMX spectrometer at 360 ( H)
2 4
Na SO . In case of the aryl-alkyl 1d and the aromatic substrate 1e
d
the determination of the conversion and enantiomeric excess was
carried out by HPLC on a chiral stationary phase. The alkyl sub-
strates 1a–c were derivatized to their corresponding 2-acetoxy-
acids derivatives or methyl carboxylates, resp.
0
4
)
6
7
O
2
O
4
)
2
2
2
4
meric excess were determined via GC or HPLC on a chiral stationary
phase. GC analyses were carried out on a Varian 3800 gas chro-
4.5. Synthesis of substrates
matograph equipped with a FID detector using H
2
as carrier gas
4.5.1. General procedure for lipase mediated kinetic resolution
(R)- and (S)-2-Hydroxyhexanoic acid 1b and (R)- and (S)-2-
hydroxyoctanoic acid 1c were synthesized by lipase kinetic resolution
(
14 psi) and an Astec Chiraldex B-TA column (column A, 40 m,
0
.25 mm, 0.12
m
m film). Temperature of the injector and detector
ꢁ
19
were 180 and 200 C, respectively, using a split ratio of 90:1. HPLC
analyses were carried out on a Shimadzu HPLC system equipped
with a Chiralpak AD column (column B, 25 cm, 0.46 cm). Optical
in analogy to a knownprocedure with the following modifications: To
a solution of rac-2-hydroxyacid in tert-butyl methyl ether, were added
vinyl acetate and lipase and the mixturewas vigorouslystirredat rt until
a conversion of 50% was reached. The enzymewas removed by filtration
and the solvent was evaporated under reduced pressure. Flash chro-
20
D
rotation values ([a] ) were measured on a Perkin–Elmer polari-
meter 341 at 589 nm (Na-line) in a 1 dm cuvette and are given in
units of [(degꢂmL)/(gꢂdm)].
2 2
matography on silica gel Merck 60 (230–400 mesh) using CH Cl /
MeOH/AcOH (gradient 95:5:0.1 to 70:30:0.1) resulted in optically pure
4
.2. HicDH-clone maintenance
E. coli clones ( -HicDH and
(S)-2-acetoxyacid and non-reacted (R)-2-hydroxy acid 1b,c.
L
D-HicDH) were maintained on
4.5.2. General procedure for the hydrolysis step
LB-plates (16 g/L agar) containing ampicillin (100 mg/L), spectino-
mycin (20 mg/L) and chloramphenicol (100 mg/L), resp. Sub-cul-
turing was performed every week, the plates were left in an
incubator for 7–9 h at 37 C, long-term storage was at þ4 C.
A mixture of (S)-2-acetoxy acid (1 mmol) and K
2
CO
3
(1 g) in
ꢁ
MeOH (8 mL) was stirred at 0 C until TLC analysis indicated com-
plete hydrolysis. After acidification with HCl (3 M) to pH 2–3, the
product was extracted three times with ethyl acetate, the organic
ꢁ
ꢁ
2 4
layer was dried over Na SO , the solvent was evaporated under
4
.3. Growth of microorganisms
reduced pressure and the residue was purified by flash chroma-
tography on silica gel Merck 60 (230–400 mesh) using CH
MeOH/AcOH (gradient 95/5/0.1 to 70/30/0.1).
2 2
Cl /
E. coli clones (
was prepared by sterilising a 1L solution of the following components in
baffled 1L-flasks: Trypton (10 g/L), NaCl (5 g/L), yeast extract (5 g/L). A
D- and L-HicDH) were grown in a LB-medium which
3
4.5.2.1. (R)-2-Hydroxyhexanoic acid 1b. Kinetic resolution of rac-2-
hydroxyhexanoic acid (1b, 2.22 g, 17 mmol) in tert-butyl methyl
ether (150 mL) using vinyl acetate (6.2 mL, 67 mmol, 4 equiv) and
immobilized C. antarctica lipase (Sigma L 4777) (2.33 g) gave (S)-2-
acetyoxyhexanoic acid (oil, 1.44 g, 48%) and (R)-2-hydroxyhexanoic
pre-culture was prepared by inoculating 125 mL of LB-medium con-
taining the sameconcentrations of antibiotics as described above for the
ꢁ
agar plates. The pre-culture was shaken at 120 rpm and 37 C until an
OD-value of 1 (600 nm) was reached. Afterwards the pre-culture was
transferred into three baffled 1 L-flasks containing a total amount of 1 L
of sterile LB-medium containing the antibiotics and inducers as follows:
ꢁ
20
acid 1b as white solid (mp 62 C, 844 mg, 38%, 85% ee). [
a
]
D
ꢃ0.62
H NMR (360 MHz, CDCl ):
¼0.93 (3H, t, J¼7.0 Hz),1.37–1.46 (4H, m), 1.71 (1H, m, CH –CHOH),
1.87 (1H, m, CH
-CHOH), 4.28 (1H, dd, CHOH, J¼7.4, 4.3 Hz);
NMR (90 MHz, CDCl ):
¼13.8, 22.3, 26.8, 33.8, 70.3, 179.7.
2
0
20 1
(c 1, CHCl
3
), [
a
]
D
ꢃ1.46 (c 7, EtOH);
3
100 mg/L ampicillin, 20 mg/L spectinomycin, 100 mg/L chlorampheni-
d
2
13
col, 0.5 g/L rhamnose and 100
m
M IPTG. The cultures were shaken at
2
C
ꢁ
120 rpm and 37 C overnight, then the cells were harvested by centri-
3
d
fugation (8000 rpm, 20 min), washed with potassium phosphate buffer
100 mM, pH 7), shock-frozen in liquid nitrogen, and lyophilized. Cells
(
4.5.2.2. (S)-2-Hydroxyhexanoic acid 1b. Hydrolysis of (S)-2-acetoxy-
hexanoic acid (1.44 g) with K CO (8 g) in MeOH (65 mL) gave (S)-2-
hydroxyhexanoic acid (1b, 1.04 g, 95%, 91% ee); [
ꢁ
were stored at 4 C and used as such for biotransformations.
2
3
2
0
a
]
D
þ0.90 (c 1,
¼0.92 (3H, t, J¼7.0 Hz), 1.26–
–CHOH), 1.81–1.88 (1H, m, CH
1
4
.4. General screening-procedure for the biocatalytic
CHCl
1.49 (4H, m), 1.66–1.74 (1H, m, CH
CHOH), 4.27 (1H, dd, CHOH, J¼7.3, 4.2 Hz); C NMR (90 MHz,
CDCl ):
¼13.8, 22.3, 26.8, 33.8, 70.3, 179.6.
3 3
); H NMR (360 MHz, CDCl ): d
racemization
2
2
–
13
All racemization experiments were performed in glass vials cap-
ped with septums. For degassing, potassium phosphate buffer
3
d
(
100 mM, pH7)wasfilteredusingaMilliporefilter(0.22
m
m)attached
4.5.2.3. (R)-2-Hydroxyoctanoic acid 1c. Kinetic resolution of rac-2-
hydroxyoctanoic acid (1c, 2.01 g, 12.5 mmol) in tert-butyl methyl
ether (75 mL) using vinyl acetate (5 mL, 54 mmol, 4 equiv) and C.
antarctica lipase B (Novozym 435,1.51 g) gave (S)-2-acetoxyoctanoic
acid (oil, 741 mg) and (R)-2-hydroxyoctanoic acid 1c as white solid
to a glass bottle by applying a vaccum using a water-jet pump. The
buffer solution was vigorously stirred for ca. 0.5 h during degassing,
until no more bubbles were visible. The system was ventilated and
maintained under an argon atmosphere. Lyophilized E. coli cells (
and -HicDH, 60 mg each; 120 mg - or -HicDH) were rehydrated in
degassed potassium phosphate buffer (2.3 mL,100 mM, pH 7) for 1 h
at 30 C on an orbit shaker at 120 rpm under an argon atmosphere.
Aliquots of cofactor solution (100
buffer) and substrate solution (15 mg 1a–e in 150
D-
ꢁ
20
20
L
D
L
(mp 57 C, 670 mg, 33%, 78% ee); [
a
]
D
ꢃ2.36 (c 1, CHCl
); H NMR (360 MHz, CDCl ):
¼0.88 (3H, m), 1.22–1.45
(8H, m),1,72 (1H, m, CH –CHOH),1.85 (1H, m, CH –CHOH), 4.28 (1H,
3
), [
a
]
D
ꢃ4.3
21 1
(c 1, CHCl
3
3
d
ꢁ
2
2
þ
13
m
L, NADH/NAD 5 mg/3 mg in 1 mL
m, CHOH), 6.2 (2H, s, OH, COOH); C NMR (90 MHz, CDCl
3
):
d¼14.0,
mL buffer) were
22.5, 24.7, 28.9, 31.6, 34.2, 70.3, 179.7.
ꢁ
added. Themixturewas shaken at 30 C and 120 rpmunderan argon
atmosphere. Samples of 850
m
L were withdrawn at intervals of 0.5–
4.5.2.4. (S)-2-Hydroxyoctanoic acid 1c. Hydrolysis of (S)-2-acetoxy-
octanoic acid (741 mg) using K CO (3.67 g) in MeOH (30 mL) gave
3
h, 4–6 h and 20–24 h depending on the substrate.
2
3

A. Bodlenner et al. / Tetrahedron 65 (2009) 7752–7755
7755
2
0
(
1
1
S)-2-hydroxyoctanoic acid (1c, 460 mg, 78%, 97% ee); [
a
]
D
þ2.23 (c
¼0.90 (3H, t, J¼6.9), 1.31–
–CHOH), 1.82–1.92 (1H, m, CH
Acknowledgements
1
, CHCl
.52 (8H, m), 1.66–1.78 (1H, m, CH
CHOH), 4.30 (1H, dd, CHOH, J¼7.5, 4.5 Hz); C NMR (90 MHz,
CDCl ):
¼14.0, 22.5, 24.7, 28.9, 31.6, 34.2, 70.3, 180.1.
3 3
); H NMR (300 MHz, CDCl ): d
2
2
–
This study was performed in cooperation within the Compe-
tence Center Applied Biocatalysis and the FWF project P 18537, fi-
nancial support from the FFG, the City of Graz, the province of Styria
and the FWF (Vienna) is gratefully acknowledged. Barbara Lar-
issegger-Schnell, Verena Resch and Axel Schlagenhauf (Graz) are
cordially thanked for their valuable assistance.
13
3
d
4
.6. General procedures for derivatization
4.6.1. Derivatization of substrate 1a to the corresponding O-acetyl
ester for GC analysis
To the solution of
acetate (700 mL) were added acetic anhydride (50 m
a
-hydroxycarboxylic acid 1a (ca. 5 mg) in ethyl
L) and a cata-
lytic amount of DMAP. The reaction mixture was shaken for 1 h at
rt. Water was added (500 L), the solution was centrifuged and the
organic phase was dried over Na SO
References and notes
1
. (a) Bel e´ n, M. M.; B a¨ ckvall, J.-E. Curr. Opin. Chem. Biol. 2007, 11, 226; (b) Kim, M.-J.;
m
Ahn, Y.; Park, J. Bull. Korean Chem. Soc. 2005, 26, 515; (c) Pamies, O.; B a¨ ckvall, J.-E.
Trends Biotechnol. 2004, 22, 130; (d) Turner, N. J. Curr. Opin. Chem. Biol. 2004, 8, 114;
2
4
.
(
e) Huerta, F. F.; Minidis, A. B. E.; B a¨ ckvall, J.-E. Chem. Soc. Rev. 2001, 30, 321.
. Berkessel, A.; Sebastian-Ibarz, M. L.; M u¨ ller, T. Angew. Chem., Int. Ed. 2006, 45,
567.
2
4
.6.2. Derivatization of substrates 1b,c to the corresponding methyl
esters for GC analysis
6
3. Gastaldi, S.; Escoubet, S.; Vanthuyne, N.; Gil, G.; Bertrand, M. P. Org. Lett. 2007,
9, 837.
4. (a) Zhu, Y.; Fow, K.-L.; Chuah, G.-K.; Jaenicke, S. Chem.dEur. J. 2007, 13, 541; (b)
Wuyts, S.; De Temmerman, K.; De Vos, D. E.; Jacobs, P. A. Chem.dEur. J. 2005, 11,
To 5 mg of dry
a
-hydroxycarboxylic acid 1b,c, was added
ꢁ
BF
h. The samples were cooled, water was added (500
were extracted with hexane (3ꢂ300 L) and the combined organic
phases were dried over Na SO
3
ꢂMeOH (300
mL). The vials were closed and heated at 100 C for
1
mL), products
3
86.
5. (a) O
¨
dman, P.; Wessjohann, L. A.; Bornscheuer, U. T. J. Org. Chem. 2005, 70, 9551;
m
(b) Gutierrez, M.-C.; Furstoss, R. D.; Alphand, V. Adv. Synth. Catal. 2005, 347,
2
4
.
1051.
6
. (a) Wang, Y.; Shen, Z.; Li, B.; Zhang, Y.; Zhang, Y. Chem. Commun. 2007, 1284; (b)
Crawford, J. B.; Skerlj, R.; Bridger, G. J. J. Org. Chem. 2007, 72, 669; (c) Szori, K.;
Szollosi, G.; Bartok, M. Adv. Synth. Catal. 2006, 348, 515; (d) Giacomini, D.;
Galleti, P.; Quintavalla, A.; Gucciardo, G.; Paradisi, F. Chem. Commun. 2007, 4038.
4
.7. Analytical procedures
See Tables 2 and 3.
7. Schnell, B.; Faber, K.; Kroutil, W. Adv. Synth. Catal. 2003, 345, 653.
8
. (a) Glueck, S. M.; Larissegger-Schnell, B.; Csar, K.; Kroutil, W.; Faber, K. Chem.
Commun. 2005, 1904; (b) Glueck, S. M.; Pirker, M.; Nestl, B. M.; Ueberbacher, B.
T.; Larissegger-Schnell, B.; Csar, K.; Hauer, B.; Stuermer, R.; Kroutil, W.; Faber, K.
J. Org. Chem. 2005, 70, 4028.
Table 2
GC-analyses using a chiral stationary phase
Compound
Columna
Conditionsb
t
R
[min]
9. Nestl, B. M.; Glueck, S. M.; Hall, M.; Kroutil, W.; Stuermer, R.; Hauer, B.; Faber, K.
Eur. J. Org. Chem. 2006, 4573.
0. Gruber, C. C.; Nestl, B. M.; Gross, J.; Hildebrandt, P.; Bornscheuer, U. T.; Faber, K.;
R
S
1
1
1
1
a
b
c
A
A
A
A
B
C
20.23
9.75
6.72
20.14
10.86
6.83
Kroutil, W. Chem.dEur. J. 2007, 13, 8271.
11. Sch u¨ tte, H.; Hummel, W.; Kula, M.-R. Appl. Microbiol. Biotechnol. 1984, 19, 167.
12. (a) Kallwass, H. K. W. Enzyme Microb. Technol. 1992, 14, 28; (b) Hummel, W.;
Sch u¨ tte, H.; Kula, M.-R. Appl. Microbiol. Biotechnol. 1985, 27, 7.
3. (a) Lerch, H.-P.; Bl o¨ cker, H.; Kallwass, H.; J u¨ rgen, H.; Tsai, H.; Collins, J. Gene
1989, 78, 47; (b) Lerch, H.-P.; Frank, R.; Collins, J. Gene 1989, 83, 263.
a
Column: (A) Astec Chiraldex B-TA column (40 m, 0.25 mm, 0.12 mm film).
Conditions: (A) 14 psi H
1
b
ꢁ
ꢁ
ꢁ
2
at 80 C, hold for 5 min, heat rate 2 C/min to 120 C,
ꢁ
ꢁ
ꢁ
ꢁ
heat rate 15 C/min to 170 C, hold for 10 min; (B) 14 psi H
heat rate 1 C/min to 90 C, heat rate 10 C/min to 110 C, heat rate 15 C/min to
2
at 85 C, hold for 5 min,
14. (a) Hanessian, S. Total Synthesis of Natural Products. The Chiron Approach; Per-
ꢁ
ꢁ
ꢁ
ꢁ
gamon: New York, NY, 1983; Chapter 2; (b) Coppola, G. M.; Schuster, H. F. a-
Hydroxy Acids in Enantioselective Syntheses; Wiley-VCH: Weinheim, 1997.
15. (a) For antifungal activity see: Str o¨ m, K.; Sj o¨ gren, J.; Broberg, A.; Schn u¨ rer, J.
Appl. Environ. Microbiol. 2002, 68, 4322; (b) For anti-Listeria activity see: Die-
uleveux, V.; van der Pyl, D.; Chataud, J.; Gueguen, M. Appl. Environ. Microbiol.
ꢁ ꢁ ꢁ ꢁ
ꢁ ꢁ ꢁ
C/min to 160 C, heat rate 15 C/min to 170 C, hold for 2 min.
170 C, hold for 2 min; (C) 14 psi H at 90 C, heat rate 10 C/min to 130 C, heat rate
2
ꢁ
5
Table 3
1
998, 64, 800.
6. Nishizawa, S.; Tamaki, S.; Kawamura, T.; Kakeya, N.; Kitao, K. WO Patent
8,501,046, 1985; Chem. Abstr. 1985, 103, 141736.
7. (a) Mill, J.; Schmiegel, K. K.; Shaw, W. N. U.S. Patent 4,391,826, 1983; Chem. Abstr.
981, 99, 128353; (b) Saravanan, P.; Singh, V. K. Tetrahedron Lett. 1998, 39, 167.
HPLC-analyses using a chiral stationary phase
1
Compound
Columna
Conditionsb
R
t [min]
1
R
S
1
1
1
d
e
B
B
B
A
26.2
26.3
31.0
21.4
18. Spies, M. A.; Woodward, J. J.; Watnik, M. R.; Toney, M. D. J. Am. Chem. Soc. 2004,
126, 7464.
1
9. Adam, W.; Lazarus, M.; Schmerder, A.; Humpf, H.-U.; Saha-M o¨ ller, C. R.;
a
Column: Chiralpak AD column (25 cm, 0.46 cm).
Conditions: (A) n-heptane/i-propanol/trifluoroacetic acid (90:10:0.1), flow 1 mL/
Schreier, P. Eur. J. Org. Chem. 1998, 2013.
20. Takeuchi, S.; Yonehara, H. Tetrahedron Lett. 1966, 7, 5197.
21. Schummer, A.; Yu, H.; Simon, H. Tetrahedron 1991, 47, 9019.
b
min; (B) n-heptane/i-propanol/trifluoroacetic acid (95:5:0.1), flow 0.8 mL/min.