Enantioselective biocatalytic reduction of non-protected hydroxyacetophenones

Article abstract of DOI:10.1515/znb-2010-0317

Direct enantioselective reduction of -, - and -hydroxyacetophenone without protection of the hydroxy moiety was carried out in the presence of (R)- and (S)-alcohol dehydrogenases as bio-catalysts. Whereas reduction of -hydroxyacetophenone gave only low to

Full text of DOI:10.1515/znb-2010-0317

Enantioselective Biocatalytic Reduction of Non-protected
Hydroxyacetophenones
Adrian Neuperta, Tina Ressa, Ju¨rgen Wittmanna, Werner Hummelb, and Harald Gro¨gera
a Department of Chemistry and Pharmacy, University of Erlangen-Nu¨rnberg, Henkestraße 42,
91054 Erlangen, Germany
b Institute of Molecular Enzyme Technology at the Heinrich-Heine-University of Du¨sseldorf,
Research Centre Ju¨lich, Stetternicher Forst, 52426 Ju¨lich, Germany
Reprint requests to Prof. Dr. Harald Gro¨ger. Fax: +49 9131 8521165.
E-mail: harald.groeger@chemie.uni-erlangen.de
Z. Naturforsch. 2010, 65b, 337 – 340; received November 17, 2009
Dedicated to Professor Rolf W. Saalfrank on the occasion of his 70th birthday
Direct enantioselective reduction of 2ꢀ-, 3ꢀ- and 4ꢀ-hydroxyacetophenone without protection of
the hydroxy moiety was carried out in the presence of (R)- and (S)-alcohol dehydrogenases as bio-
catalysts. Whereas reduction of 2ꢀ-hydroxyacetophenone gave only low to medium conversions,
reduction of 3ꢀ- and 4ꢀ-hydroxyacetophenone proceeded efficiently leading to the resulting 1-(3-
hydroxyphenyl)ethan-1-ol and 1-(4-hydroxyphenyl)ethan-1-ol with high conversion (up to > 95 %)
and excellent enantioselectivity (up to > 99 % ee).
Key words: Alcohols, Asymmetric Synthesis, Biocatalysis, Enzymes, Reduction
Introduction
as a substituent (1a – c) by means of an alcohol de-
hydrogenase (ADH) as (bio-)catalyst. Such a biocat-
alytic one-step concept appears to be superior over a
typical “classic chemical” synthetic strategy compris-
ing the three steps of (i) protection of the hydroxy
group, followed by (ii) asymmetric reduction of the
ketone and (iii) subsequent removal of the protect-
ing group [5]. Although biocatalytic reduction is rec-
ognized as a highly efficient approach towards enan-
tiomerically pure alcohols [6], studies on enzymatic re-
duction of non-protected hydroxyacetophenones 1 un-
der enantioselective formation of hydroxy-substituted
1-phenylethan-1-ols [(R)- or (S)-2], which represent
substructures in a range of drugs [7], are so far sur-
prisingly rare [8].
In multi-step synthesis of complex molecules such
as, e. g., natural products and drugs, the use of pro-
tecting groups often is a prerequisite to achieve highly
efficient transformations in the presence of functional
group(s) which are not involved in the desired reac-
tion [1]. Such a protection of functional groups con-
tributes to a decrease of side-reactions of substrate and
product, but also helps to maintain the performance
of the catalyst when the catalyst would react with
a specific functional group. At the same time, how-
ever, the need for protecting groups requires additional
reagents and causes additional reaction steps, thus de-
creasing atom efficiency and economic attractiveness
of the overall process [2]. Accordingly, efficient syn-
thetic transformations in the presence of non-protected
sensitive functional groups such as, e. g., hydroxy and
amino groups, which avoid the requirement for protect-
Results and Discussion
For our studies, 2ꢀ-, 3ꢀ-, and 4ꢀ-hydroxyacetophen-
ing groups are desirable, and their development repre- one (1a – c) served as substrates. As biocatalysts
sents an important (and still challenging) task in or- we used (R)-enantioselective alcohol dehydrogenases
ganic process chemistry.
from Lactobacillus brevis [9] and Lactobacillus ke-
In continuation of our studies on enzymatic re- fir [10] as well as an (S)-enantioselective ADH from
dox processes [3, 4] herein we report the enantiose- Rhodococcus sp. [11], which are prepared in the advan-
lective reduction of prochiral acetophenones bearing tageous recombinant form. The enzymatic reductions
a non-protected hydroxy group at the aromatic ring of ketones of type 1 have been carried out by apply-
c
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338
A. Neupert et al. · Enantioselective Biocatalytic Reduction of Non-protected Hydroxyacetophenones
using the (S)-ADH from Rhodococcus sp. (Rsp.-
ADH) as a biocatalyst, leading to a somewhat in-
creased but still low conversion of 22 % (entry 2). At
a prolonged reaction time of 72 h and higher bio-
catalyst loading conversion increased to 49 %, and
enantioselectivity was excellent with > 99 % ee (en-
try 3). When starting from 3ꢀ-hydroxyacetophenone
(1b; meta-hydroxyacetophenone) the enantioselective
reduction proceeded successfully in the presence of
both (R)- and (S)-enantioselective enzymes (entries
4 – 6). The use of the (R)-enantioselective ADH from
L. brevis as a biocatalyst resulted in the formation
of (R)-1-(3-hydroxy)phenylethan-1-ol ((R)-2b) with
> 95 % conversion and > 99% ee after a reaction
time of 24 h (entry 4). The reduction of 1b by means
of an (R)-ADH from L. kefir (LK-ADH) as a biocat-
alyst also gave (R)-2b with excellent enantioselectiv-
ity of > 99 % ee, but with a decreased conversion of
76 % in spite of a prolonged reaction time of 120 h
and high amount of enzyme (entry 5). In the pres-
ence of the (S)-ADH from Rhodococcus sp. a high
conversion of 95 % was achieved accompanied with
an excellent enantioselectivity of > 99 % ee after
24 h reaction time (entry 6). In addition, we were
pleased to find that 4ꢀ-hydroxyacetophenone(1c; para-
hydroxyacetophenone) can also be transformed into
the corresponding alcohol (2c) with both high con-
version and enantioselectivity. The enantioselective re-
duction of 4ꢀ-hydroxyacetophenone (1c) catalyzed by
the (R)-ADH from L. brevis led to the formation of the
desired (R)-alcohol (R)-2c with > 95 % conversion and
> 95 % ee after 24 h reaction time (entry 7). The oppo-
site (S)-enantiomer (S)-2c has been formed with 58 %
conversion and an enantioselectivity of > 99 % ee after
a prolonged reaction time of 72 h when using the (S)-
ADH from Rhodococcus sp. as a biocatalyst (entry 8).
Scheme 1. Concepts for enzymatic reduction of hydroxyace-
tophenones 1a – c.
ing the concept of substrate-coupled cofactor regener-
ation or alternatively enzyme-coupled cofactor regen-
eration (Scheme 1) [6]. In the substrate-coupled co-
factor regeneration process (Scheme 1, method A), the
cofactor NAD(P)H is regenerated in situ by means of
an oxidation of isopropanol (i-PrOH) to acetone. This
enables the use of the expensive cofactor in catalytic
amount only, whereas the economically attractive iso-
propanol represents the reducing agent used in stoi-
chiometric amount. The enzyme-coupled cofactor re-
generation (Scheme 1, method B) is based on the use
of a glucose dehydrogenase for in situ-regeneration
of the cofactor NAD(P)H via consumption of a sto-
ichiometric amount of D-glucose under formation of
D-gluconolactone.
Conclusion
In conclusion, direct reduction of 2ꢀ-, 3ꢀ- and 4ꢀ-
hydroxyacetophenone (1a – c) without protection of
the hydroxy moiety was carried out by means of (R)-
enantioselective ADHs from L. kefir and L. brevis and
When using 2ꢀ-hydroxyacetophenone (1a; ortho- an (S)-enantioselective ADH from Rhodococcus sp. as
hydroxyacetophenone) as a substrate, reduction of 1a biocatalysts. Whereas reduction of 2ꢀ-hydroxyaceto-
in the presence of an ADH from L. brevis (LB- phenone (1a) only gave low to medium conversions,
ADH) gave the resulting (R)-alcohol (R)-2a with a direct reduction of 3ꢀ- and 4ꢀ-hydroxyacetophenone
excellent enantioselectivity of > 98 % ee but low (1b, c) proceeded efficiently without the need to pro-
conversion of 5 % after a reaction time of 24 h tect the hydroxy moiety. The resulting products 1-
(Table 1, entry 1). The difficulty in reducing 2ꢀ- (3-hydroxyphenyl)ethan-1-ol (2b) and 1-(4-hydroxy-
hydroxyacetophenonehas also been encountered when phenyl)ethan-1-ol (2c) were formed with high conver-
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A. Neupert et al. · Enantioselective Biocatalytic Reduction of Non-protected Hydroxyacetophenones
Table 1. Enzymatic reduction of 1a – c.
339
Entrya
Substrate
Enzymee
Method
Product
Conversion (%)
ee (%)
1
1a
1a
1a
1b
1b
1b
1c
1c
LB-ADH
Rsp.-ADH
Rsp.-ADH
LB-ADH
LK-ADH
Rsp.-ADH
LB-ADH
Rsp.-ADH
B
A
A
B
A
A
B
A
(R)-2a
(S)-2a
(S)-2a
(R)-2b
(R)-2b
(S)-2b
(R)-2c
(S)-2c
5
22
49
> 95
76
95
> 98
> 99
> 99
> 99
> 99
> 99
> 95
> 99
2
3b
4
5c
6
7
> 95
58
8d
a
For experimental protocols, see Experimental Section; the reactions were carried out at room temperature, and unless otherwise stated the
reaction time was 24 h; b prolonged reaction time of 72 h; c prolonged reaction time of 120 h; d prolonged reaction time of 72 h; e LB-ADH:
(R)-ADH from L. brevis; Rsp.-ADH: (S)-ADH from Rhodococcus sp.; LK-ADH: (R)-ADH from L. kefir.
R
ꢀ
sion (up to > 95 %) and excellent enantioselectiv- by means of chiral HPLC (2a: Chiralcel column OJ-H,
hexane : isopropanol 90 : 10, flow 0.8 mL min−1, 230 nm;
ity (up to > 99 % ee). Based on these encouraging
R
ꢀ
preliminary results for the reactions with 2ꢀ-, 3ꢀ- and
retention times tR = 14.0 min, 16.7 min, or Chiralpak
column IB, hexane : isopropanol 95 : 5, flow 1.0 mL min−1
,
4ꢀ-hydroxyacetophenone current work is in progress
to apply this enantioselective enzymatic reduction
method for the direct, protection group-free synthesis
of related pharmaceutically active compounds bearing
a hydroxy-substituted 1-phenylethan-1-ol scaffold.
230 nm; retention times tR = 11.6 min, 12.3 min; 2b:
R
ꢀ
Chiralcel column OJ-H, hexane : isopropanol 90 : 10, flow
0.8 mL min−1, 230 nm; retention times tR = 20.8 min,
R
ꢀ
23.5 min, or Chiralpak column IB, hexane : isopropanol
95 : 5, flow 0.8 mL min−1, 230 nm; retention times tR
=
R
ꢀ
30.3 min, 34.6 min; 2c: Chiralcel
column OJ-H, hex-
Experimental Section
General procedure 1: Reduction of 1a–c via substrate-
coupled cofactor recycling (method A)
ane : isopropanol 90 : 10, flow 0.8 mL min−1, 230 nm; reten-
R
ꢀ
tion times tR = 44.7 min, 49.7 min, or Chiralpak column
IB, hexane : isopropanol 95 : 5, flow 1.0 mL min−1, 230 nm;
retention times tR = 41.3 min, 42.3 min).
In a 25 mL flask the ketone of type 1 (entries 2, 3, 6, 8:
0.5 mmol, entry 5: 0.25 mmol) and NAD(P)+ (0.02 mmol;
NADP+ when using LK-ADH and NAD+ when using
Rsp.-ADH) are dissolved in a mixture of phosphate buffer
(7.5 mL, pH = 7, 0.05 M) and isopropanol (2.5 mL), fol-
General procedure 2: Reduction of 1a–c via enzyme-coupled
cofactor recycling (method B)
In a 100 mL Methrom Titrino reaction apparatus the ke-
lowed by addition of the alcohol dehydrogenase (entries 2, tone of type 1 (entries 1, 4, 7: 0.25 mmol) and NADP+
3, 6, 8: 72 U, entry 5: 155 U; U refers to acetophenone (0.02 mmol) are dissolved in a mixture of phosphate buffer
as standard substrate for LB-ADH and LK-ADH, and 4- (5 mL, pH = 7, 0.05 M) and distilled water (95 mL), followed
chloroacetophenone as standard substrate for Rsp.-ADH) un- by adjustment of the pH at pH = 7 using a NaOH solution
der stirring. After stirring the resulting reaction mixture for (0.2 M) and addition of the alcohol dehydrogenase LB-ADH
24 h (entries 2, 6), 72 h (entries 3, 8) or 120 h (entry 5) at r. t., (16 U; U refers to acetophenone as standard substrate) and
the aqueous phase was extracted three times with methylene glucose dehydrogenase from Bacillus sp. (from Amano En-
chloride (80 mL). The combined organic phases were dried zyme Inc., 8 U). After stirring the resulting reaction mixture
over magnesium sulfate, filtered and concentrated in vacuo. for 24 h at room temperature at constant pH (pH = 7, ad-
The conversion of this reaction was determined from the re- justed by automatical dosage of a NaOH solution (0.2 M)),
sulting crude product by means of 1H-NMR spectroscopy. the aqueous phase was extracted three times with methylene
The enantiomeric excess of the product 2 was determined chloride (100 mL). The combined organic phases were dried
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A. Neupert et al. · Enantioselective Biocatalytic Reduction of Non-protected Hydroxyacetophenones
over magnesium sulfate, filtered and concentrated in vacuo. Acknowledgement
The conversion of this reaction was determined from the re-
sulting crude product by means of 1H-NMR spectroscopy.
The authors thank Evonik Degussa GmbH, Amano En-
The enantiomeric excess of the product 2 was determined by
means of chiral HPLC (for chiral HPLC methods in detail,
see general procedure 1).
zymes Inc., and Oriental Yeast Company Ltd. Japan for gen-
erous support with chemicals.
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