Simple biocatalytic access to enantiopure (S)-1-heteroarylethanols employing a microbial hydrogen transfer reaction

Article abstract of DOI:10.1002/adsc.200303210

Lyophilised cells of various Rhodococcus spp. were employed in an efficient hydrogen transfer-like process for the asymmetric bioreduction of heteroaryl methyl ketones using 2-propanol as hydrogen donor. Besides the genus Rhodococcus, only Mycoplana rubra R14 showed a comparable stability towards elevated concentrations of the co-substrate 2-propanol. Among the organisms tested, Rhodococcus ruber DSM 44541 and DSM 43338 showed best activity and selectivity. With these strains, the reaction proceeded with high stereoselectivity (ee >99%) and predictable stereochemical outcome regardless of the nature of the heteroaromatic ring system. The reaction could be performed at the exceptional substrate concentration of up to 0.4 mol L ^-1 in an environmentally friendly aqueous-organic solvent mixture at room temperature and is easy to handle, thus providing a very practical tool to access enantio-pure 1-heteroarylethanols.

Full text of DOI:10.1002/adsc.200303210

FULL PAPERS
Simple Biocatalytic Access to Enantiopure
S)-1-Heteroarylethanols Employing a Microbial
Hydrogen Transfer Reaction
(
Wolfgang Stampfer, Klaus Edegger, Birgit Kosjek, Kurt Faber, Wolfgang Kroutil*
Department of Chemistry, Organicand BioorganicChemistry, University of Graz, Heinri ch strasse 28, A-8010 Graz, Austria
Fax: ( ‡ 43)-316-380-9840, e-mail: wolfgang.kroutil@uni-graz.at
Received: August 28, 2003; Accepted: December 7, 2003
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de.
Abstract: Lyophilised cells of various Rhodococcus and predictable stereochemical outcome regardless of
spp. were employed in an efficient hydrogen transfer- the nature of the heteroaromaticring system. The
like process for the asymmetric bioreduction of reaction could be performed at the exceptional
À1
heteroaryl methyl ketones using 2-propanol as hydro- substrate concentration of up to 0.4 mol L in an
gen donor. Besides the genus Rhodococcus, only environmentally friendly aqueous-organicsolvent
Mycoplana rubra R14 showed a comparable stability mixture at room temperature and is easy to handle,
towards elevated concentrations of the co-substrate 2- thus providing a very practical tool to access enantio-
propanol. Among the organisms tested, Rhodococcus pure 1-heteroarylethanols.
ruber DSM 44541 and DSM 43338 showed best
activity and selectivity. With these strains, the reac- Keywords: biotransformation; enzyme catalysis; 1-
tion proceeded with high stereoselectivity (ee>99%) heteroarylethanol; hydrogen transfer; oxidoreductase
Introduction
We recently reported the synthesis of chiral m- and p-
substituted 1-phenylethanols via asymmetricbio ac ta-
Chiral 1-heteroaryl-1-alkanols are widely used as chiral lytichydrogen-transfer employing lyophilised ce lls of
ligands[ and as building blocks for the synthesis of Rhodococcus ruber DSM 44541.
1]
[14,15]
The high efficiency
[
2]
bioactive compounds. The chiral heteroaryl alcohols are of this system is a result of the exceptional tolerance of
generally prepared by chemical or biochemical methods. the sec-alcohol dehydrogenase involved towards elevat-
Examples for chemical methods are (i) the asymmetric ed concentrations of co-substrates used as hydrogen
alkylation of aldehydes, (ii) the asymmetricreduction of
the corresponding ketones via hydrogenation, transfer be used as hydrogen donor for the reduction of
[
3]
donor or acceptor. Thus, 2-propanol (up to 50% v/v) can
[
4]
[6]
[5]
[16]
hydrogenation, by using complex hydrides or via ketones
and acetone (up to 25% v/v) serves as
hydrogen acceptor for the oxidative kinetic resolution
hydrosilylation,[ (iii) the oxidative kineticresolution
7]
[
8]
[17]
employing Sharpless× reagent and (iv) the asymmetric of sec-alcohols. The high concentration of the water-
hydroboration of styrene derivatives. Examples for miscible co-substrate not only drives the reaction into
[
9]
enzymaticmethods are (i) the kineticresolution of the
rac-alcohol or the corresponding rac-esters by lipases,
the desired oxidation or reduction direction, but also
ensures sufficient reaction rates by acting as co-solvent
[
10]
(
(
ii) the oxidative kineticresolution of the rac-alcohol[11] or for the solubilisation of lipophilic± and thus poorly
iii) the asymmetricreduction employing whole (ferment-
water-soluble ± substrates.
[
11b,12]
ing) cells or isolated enzymes,
droxylation of the corresponding arylalkane and (v)
microbial stereoinversion.
Most of the biocatalytic methods show either high
(iv) benzylicbiohy-
[
12]
[
13]
Results and Discussion
activity and low enantioselectivity or they give high In order to extend this useful method to heteroaryl
enantiomeric excess but have low activity. All biocata- analogues, we initiated a screening for the biocatalytic
lyticapproa ch es are hampered by low substrate and
reduction of various heteroaromatic ketones in the
therefore low product concentration and in the case of presence of 2-propanol as hydrogen donor at elevated
kinetic resolutions, the theoretical yield of each enan- concentrations (Scheme 1). The aim was (i) to identify
tiomer is limited to 50%.
organisms which reduce heteroaryl ketones and (ii)
Adv. Synth. Catal. 2004, 346, 57 ±62
DOI:10.1002/adsc.200303210
¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
57

FULL PAPERS
Wolfgang Stampfer et al.
R. USA-AN-012 both showing low activity. For all active
strains the alcohol obtained possessed the (S)-config-
uration.
Scheme 1. Asymmetricredu ct ion of heteroaromaticketones
via biocatalytic hydrogen transfer.
which tolerate 2-propanol as hydrogen donor, which
would provide a simple and reliable access to chiral 1-
heteroaryl-1-ethanol derivatives in high enantiomeric
excess at high conversion. All screening experiments
À1
were performed at 10% v/v 2-propanol and 9 g L
substrate concentration employing lyophilised cells in
phosphate buffer (50 mM, pH 7.5).
In a first approach, 20 organisms, used as lyophilised
cell powder, were screened with 4-acetylpyridine 1a
using 2-propanol as hydrogen donor in aqueous buffer
From the group of Rhodococci, R. ruber DSM 44541
(
Table 1). Baker×s yeast and a red yeast (Rhodotorula) and R. ruber DSM 43338 were selected for the reduction
preparation showed insufficient activity (entries 1 ± 3) as of the positional isomers 3- and 2-acetylpyridine 2a and
did a variety of bacteria, such as Lactobacillus, Coryne- 3a, respectively (Table 2). For both isomeric substrates,
bacterium, Methylobacterium and Streptomyces sp. (en- the corresponding (S)-alcohols (S)-2b and (S)-3b were
tries 4 ± 8), although for a related Lactobacillus strain, obtained in excellent ee ( > 99%) with both strains, while
namely Lactobacillus kefir, positive results at 7% v/v 2- R. ruber DSM 43338 showed higher activity. A deriva-
propanol were reported in literature.
Mycoplana rubra (entry 9) showed moderate conver- drug candidate for treatment of AIDS.
[
12f]
In contrast, tive of 3b is needed for the synthesis of PNU-142721, a
[
2a,18]
Optically
sion (44% within 22 h) but good stereoselectivity (93% pure 1-(3-pyridyl)-ethanol 2b was used as starting
ee). Best results in terms of activity and stereoselectivity material for the synthesis of allo-heteroyohimbine
[
19]
in general were obtained with Rhodococcus spp. (entries alkaloids. For both strains, an increase in reaction
0 ± 20), except for Rhodococcus sp. NCIMB 11216 and rate was observed from the o- (3a) via the m- (2a) to the
1
Table 1. Screening of micro-organisms for the reduction of 4-acetylpyridine 1a in the presence of 2-propanol (10% v/v, 22
hours).
Entry
Micro-organism
C [%]
ee [%]
P
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Baker×s yeast (Astrˆ Co.)
Baker×s yeast (Hofer Co.)
Rhodotorula mucilaginosa DSM 70404
Lactobacillus sakei DSM 20017
Corynebacterium glutamicum ATCC 13032
Methylobacterium FCC 031
Mycobacterium paraffinicum NCIMB 10420
Streptomyces lavendulae ATCC 55209
Mycoplana rubra R14, FCC 009
Rhodococcus USA-AN-012, FCC 021
Rhodococcus ANT-AN-037, FCC 011
Rhodococcus SRB-AN-019, FCC 022
Rhodococcus SRB-AN-030, FCC 024
Rhodococcus ruber DSM 43338
Rhodococcus erythropolis DSM 312
Rhodococcus sp. NCIMB 11216
Rhodococcus equi IFO 3730
n. c.
3.2
3.4
n. c.
n. c.
0.2
±
±
±
±
±
±
±
±
0.1
n. c.
43.9
2.8
98.5
39.5
99.7
99.5
99.4
7.5
99.5
99.5
99.5
99.4
93 (S)
80 (S)
> 99 (S)
> 99 (S)
> 99 (S)
> 99 (S)
> 99 (S)
95 (S)
96 (S)
> 99 (S)
> 99 (S)
> 99 (S)
1
1
1
1
1
1
1
1
1
1
2
Rhodococcus ruber DSM 44540
Rhodococcus ruber DSM 44541
Rhodococcus erythropolis NCIMB 11539
58
¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2004, 346, 57 ±62

Biocatalytic Access to Enantiopure (S)-1-Heteroarylethanols
FULL PAPERS
Table 2. Microbial reduction of 3- and 2-acetylpyridine 2a and 3a in the presence of 2-propanol (10% v/v, 42 hours).
Substrate
Micro-organism
Product
C [%]
ee [%]
P
2a
2a
3a
3a
R. ruber DSM 43338
R. ruber DSM 44541
R. ruber DSM 43338
R. ruber DSM 44541
(S)-2b
(S)-2b
(S)-3b
(S)-3b
84
77
98
57
> 99
> 99
> 99
> 99
Table 3. Microbial reduction of 3-acetylthiophene 5a and 2-acetylfuran 7a in the presence of 2-propanol (10% v/v, 42 hours).
Entry
Micro-organism
3-Acetylthiophene 5a
2-Acetylfuran 7a
C [%]
ee [%]
P
C [%]
ee [%]
P
1
2
3
4
5
6
7
8
9
Mycoplana rubra R14, FCC 009
Rhodococcus ANT-AN-037, FCC 011
Rhodococcus SRB-AN-030, FCC 024
Rhodococcus SRB-AN-019, FCC 022
Rhodococcus equi IFO 3730
Rhodococcus ruber DSM 44540
Rhodococcus ruber DSM 44539
Rhodococcus sp. NCIMB 11216
Rhodococcus erythropolis NCIMB 11539
Rhodococcus erythropolis DSM 312
Rhodococcus ruber DSM 43338
Rhodococcus ruber DSM 44541
29.5
4.3
21.6
3.3
49.6
35.6
2.4
35 (S)
n. d.
94 (S)
n. d.
94 (S)
99 (S)
n. d.
17.9
n. c.
15.6
1.6
54.5
58.5
12.2
n. d.
26.1
19,7
56.8
58.7
12 (S)
n. d.
97 (S)
n. d.
92 (S)
99 (S)
> 99 (S)
n. d.
93 (S)
97 (S)
99 (S)
> 99 (S)
0.4
n. d.
51.9
47.9
58.5
67.1
99 (S)
99 (S)
99 (S)
99 (S)
1
1
1
0
1
2
n. d. not determined, due to too low conversion.
p-isomer (1a). It is presumed that this is due to steric biocatalytic hydrogen transfer revealed, that R. ruber
hindrance exerted by the proximity of the basic nitro- DSM 44540, DSM 44541 and R. ruber DSM 43338
gen, which is heavily hydrated in the aqueous medium.
(Table 3, entries 6, 12, 11) gave the highest conversion
Surprisingly, none of the 20 organisms listed above and best enantiomericpurity of al co hol ( S)-7b. Inter-
were able to reduce the N-methylpyrrole derivative 4a. estingly, some strains showed higher selectivity and
However, better results were obtained with thiophene activity for 3-acetylthiophene 5a as compared to 2-
analogues. Thus, 3-acetylthiophene 5a was reduced to acetylfuran 7a (entries 9 ± 11), and vice versa (entry 6,
the corresponding (S)-alcohol by various Rhodococcus activity; entry 3: selectivity), no clear trend was observ-
spp. and Mycoplana rubra R14 (Table 3), while the (red) able.
yeasts showed exceedingly low activity (data not
shown). As for the pyridine derivatives, Rhodococcus above, the substrate concentration was always 9 g L ,
sp. NCIMB 11216 showed low activity for 5a. Again, which corresponds to approx. 74 to 81 mmol L de-
In the small-scale biotransformations described
À1
À1
best results in terms of activity and stereoselectivity pending on the substrate. Since for preparative-scale
were obtained employing R. ruber DSM 43338 (en- transformations higher substrate concentrations are
try 11) and R. ruber DSM 44541 (99% ee, entry 12), desired, the conversion of pyridyl derivatives 1a ± 3a
whereby the latter showed better activities. The reduc- using R. ruber DSM 44541 and R. ruber DSM 43338 was
tion of 2-acetylthiophene (6a) to furnish (S)-6b pro- investigated at substrate concentrations between 100
À1
ceeded in a comparable manner as for the 3-acetyl and 700 mmol L after 17 h of reaction time (Figure 1).
derivative 5a with R. ruber DSM 43338 (98% ee, 40% As observed during the screening, 4-acetylpyridine 1a
conversion within 42 h).
was transformed fastest, while 2-acetylpyridine 3a was
It is noteworthy that the presence of the heterocyclic slowest. Both organisms tolerated a substrate concen-
À1
sulphur atom did not significantly interfere with the tration of up to 700 mmol L , which corresponds to
À1
catalytic activity nor the stereoselectivity.
1
84 g L . Depending on the substrate, the highest
À1
-(2-Furyl)ethanol 7b and its derivatives have been productivity was within a range of 150 ± 400 mmol L .
utilised as chiral building blocks for the synthesis of
In order to prove the applicability of the method on a
numerous natural products, such as carbohydrates, preparative scale, the bioreduction of 3-acetyl-pyridine
[
20]
À1
pheromones, alkaloids, and macrolides like macro- 2a was performed with 623 mg 2a (270 mmol L ,
spelides H, G[ and A, B. A screening to obtain this 32 g L ) in the presence of 10% v/v 2-propanol employ-
21]
[22]
À1
building block from the corresponding ketone 7a by ing R. ruber DSM 44541. After 48 hours, 412 mg of (S)-1-
Adv. Synth. Catal. 2004, 346, 57 ±62
asc.wiley-vch.de
¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
59

FULL PAPERS
Wolfgang Stampfer et al.
5
8C), spectroscopic data were consistent with those reported in
[10f]
[23]
[24]
[25]
[26]
the literature (1b ± 3b, 4b, 5b, 6b, 7b ).
Baker×s yeast was employed as obtained from the Hofer and
Astrˆ Co. (Graz), lyophilised cells of all other micro-organisms
were prepared as previously described.[ Rhodocccus spp.
Rhodococcus ANT-AN-037, FCC 011, Rhodococcus SRB-
AN-030, FCC 024 and Rhodococcus SRB-AN-019, FCC 022
were kindly donated by A. T. Bull (Canterbury, UK) and
Mycoplana rubra R14, FCC 009 was a gift of G. Braunegg
27]
(
Graz University of Technology). FCC numbers refer to our in-
house Fab-Crew-Culture-Collection. All other strains were
obtained from culture collections.
Figure 1. Conversion from the reduction of 2-, 3- and 4-
acetylpyridyl isomers with increasing substrate concentration
after 17 h. Open symbols: R. ruber DSM 44541, solid symbols: General Procedure for the Screening
R. ruber DSM 43338; ±D±: 4-acetylpyridine 1a, ±
acetylpyridine 2a, ±*±: 2-acetylpyridine 3a.
&
±: 3-
The heteroaryl methyl ketone (1a ± 7a, 5.0 mg) dissolved in 2-
propanol (55 mL) was mixed with a suspension of rehydrated
(
(
30 min) lyophilised cells (40 mg) in phosphate buffer
0.50 mL, pH 7.5, 50 mM) in 1.5 mL Eppendorf vials. The
(
4-pyridyl)-ethanol 2b (75% conversion, 65% isolated
mixtures were shaken horizontally for 42 h (308C, 130 rpm)
and afterwards extracted with ethyl acetate (0.7 mL). The
organicphase was dried (Na SO ) and analysed by GC (see
yield,>99% ee) were obtained.
2
4
Supporting Information).
Conclusions
Biocatalytic reduction of heteroaryl methyl ketones Conversion at Elevated Substrate Concentration
employing Rhodococcus spp. via hydrogen-transfer at
the expense of 2-propanol as hydrogen donor proved to
The screening-procedure was performed as described above
with varying amounts of o-, m- and p-acetylpyridine 1a ± 3a as
be a highly efficient method for the preparation of the
corresponding heteroaryl methylcarbinols in high
chemical and optical yields. The predictability of the
stereochemical outcome according to Prelog×s rule was
excellent and always furnished the corresponding (S)-
alcohols.
indicated in Figure 1.
Preparative Biocatalytic Reduction
Lyophilised cells (2.30 g) of R. ruber DSM 44541 were
Whereas Mycoplana rubra showed moderate activity rehydrated in phosphate buffer (15 mL, 50 mM, pH 7.5) for
3
0 minutes (308C, 130 rpm). 3-Acetylpyridine 2a (623 mg,
and stereoselectivity, a range of Rhodococcus spp. were
identified, which reduced heteroaryl methyl ketones
with excellent stereoselectivity ( > 98% ee). Among
them, Rhodococcus ruber DSM 44541 proved to be the
champion with respect to activity and selectivity. Strains
of the genus Rhodococcus erythropolis are also selective
5
.14 mmol) dissolved in 2-propanol (2 mL) was added and the
mixture was shaken at 308C and 130 rpm for 48 hours. Ethyl
acetate (15 mL) was added, the mixture was centrifuged, the
phases were separated and the aqueous layer was again
extracted with ethyl acetate (2 Â 15 mL). The combined
organiclayers were dried (Na SO ), the solvent evaporated
2
4
(
> 93% ee), however, they were less active on 2-
under reduced pressure and the crude product was purified by
acetylpyridine and 2-acetylfuran. The stereo-electronic column chromatography on silica gel to give (S)-1-(4-pyridyl)-
properties of the heteroaromaticsystem had some effect
on the reaction rate, but little influence on the stereo-
selectivity. Good reaction rates can be maintained at
ethanol (412 mg, yield: 65%, ee > 99%).
The absolute configurations of the heteroaryl methylcarbi-
nols 1b ± 3b and 5b ± 7b were determined by comparison of
optical rotation values with literature data (see Supporting
Information).
À1
substrate concentrations of up to 400 mmol L in an
environmentally friendly aqueous-organicsolvent mix-
ture at room temperature and thus the reaction provides
a practical and green tool for the synthesis of enantio-
pure 1-heteroarylethanols.
Acknowledgements
Rhodococcus spp. strains USA-AN-012, ANT-AN-037, SRB-
AN-019 and SRB-AN-030 were kindly donated by Alan T. Bull
(
University ofKent, Canterbury) and Mycoplana rubra R14 was
Experimental Section
Ketones 1a ± 7a are commercially available. For analytical
purposes, samples of the corresponding rac-alcohols 1b ± 7b
a kind gift of G. Braunegg (Graz). This study was performed
within the Spezialforschungsbereich −Biokatalyse× and the
Research Centre Applied Biocatalysis. Financial support by
Ciba SC (Basel), Fonds zur Fˆrderung der wissenschaftlichen
were synthesised by reduction of the ketones (NaBH , MeOH,
4
60
¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Adv. Synth. Catal. 2004, 346, 57 ±62

Biocatalytic Access to Enantiopure (S)-1-Heteroarylethanols
FULL PAPERS
Forschung (Vienna, project no. F115), TIG, SFG, Province of
Styria and City ofGraz is grate fu lly acknowledged.
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