Enzyme-catalysed regio- and enantioselective preparative scale synthesis of (S)-2-hydroxy alkanones

Article abstract of DOI:10.1039/c5ra02975a

α-Hydroxy alkanones were synthesised with high enantiomeric purity by stereoselective enzyme-catalysed diketone reduction. Both diketone reduction and cofactor regeneration were accomplished with purified carbonyl reductase from Candida parapsilosis (CPCR2). The reaction products were isolated by column chromatography and analysed by chiral GC measurements, ¹H-NMR spectroscopy and determination of optical rotations. Preparative-scale biotransformations yielded 350-600 mg of pure aliphatic α-hydroxy ketones including the difficult to obtain (S)-2-hydroxypentane-3-one. For all the products good enantiomeric excesses in the range of 89-93% were achieved.

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ARTICLE
Enzyme-catalysed regio- and enantioselective
preparative scale synthesis of (S)-2-hydroxy
alkanones
Cite this: DOI: 10.1039/x0xx00000x
C. Loderer, M. B. Ansorge-Schumacher*
Received 00th January 2012,
Accepted 00th January 2012
α-Hydroxy alkanones were synthesi
enzyme-cataly ed diketone reduction. Both diketone reduction and cofactor regeneration were
accomplished with purified carbonyl reductase from Candida parapsilosis (CPCR2). Reaction
products were isolated by column chromatography and analy ed by chiral GC measurements,
sed with high enantiomeric purity by stereoselective
DOI: 10.1039/x0xx00000x
s
www.rsc.org/
s
1H-NMR spectroscopy and determination of optical rotations. Preparative-scale
biotransformations yielded 350 - 600 mg of pure aliphatic α-hydroxy ketones including the
difficult to obtain (S)-2-hydroxypentane-3-one. For all products good enantiomeric excesses in
the range of 89 – 93 % were achieved.
In these enzymatic processes enantiomeric excesses between 90
and 99 % were achieved. If determined, isolated yields ranged
Introduction
between 23 and 70 %. However, especially the diketone
reduction has not been sufficiently exploited for efficient
preparation of α-hydroxy ketones so far.[1]
Chiral α-hydroxy ketones are important and valuable building
blocks for the synthesis of several different pharmaceuticals
such as anti-depressants and fungicides.[1] They contain a chiral
centre at the carbon atom of the hydroxy function, and in case
of unequal substituents at the carbonyl and the hydroxy moiety
not only enantiomers, but also structural isomers occur. Thus,
synthesis of this type of compounds requires regio- as well as
stereoselective approaches.
Presently, various organochemical methods for the synthesis of
α-hydroxy ketones are available. Examples are ranging from
peroxy acid catalysed oxidations[2] to metal catalysed diketone
reductions applying zinc[3] or titan iodide.[4] Some catalysts,
such as transition metal complexes, exhibit a notable regio- and
stereoselectivity, mostly, however, restricted to phenyl-
substituted hydroxy ketones.[5,6,7,8,9,10,11] Linear aliphatic α-
hydroxy ketones comprise particularly challenging targets due
to the structural similarity of the substituents.
Here, we demonstrate the application of a carbonyl reductase
from Candida parapsilosis CPCR2[24,25,26,27] for the highly
selective asymmetric reduction of prochiral linear aliphatic
diketones to a set of corresponding (S)-configured α-hydroxy
ketones in preparative scale. At the same time, the required
cofactor nicotinamide adenine dinucleotide (NADH) was
efficiently regenerated by oxidation of isopropyl alcohol with
CPCR2 (Figure 1). The reaction products were purified by
silica gel chromatography and analysed via gas
1
chromatography, H-NMR spectroscopy and determination of
optical rotation. The absolute configuration was determined via
1H-NMR analysis of Mosher´s esters obtained from the reaction
products.
In recent years, different biocatalytic approaches for the
production of α-hydroxy ketones have been introduced as
alternatives to chemical methods, including the use of isolated
enzymes such as lipases, lyases, and oxidoreductases.[1,12,13]
Lyases have been successfully applied in the synthesis of
symmetric α-hydroxy ketones such as acetoin[14] and propioin.
[15,16,17]
Utilizing a lyase and decarboxylase in cascade, some
non-symmetric compounds could also be synthesized.[18] With
lipases, kinetic resolutions of esters were conducted yielding
enantiopure aliphatic α-hydroxy ketones especially for larger
substituents.[19] The application of alcohol dehydrogenases for
the synthesis of aliphatic α-hydroxy ketones by means of diol
oxidation[20,21] or diketone reduction[21,22,23], was demonstrated.
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Figure 1. Reduction of 2,3-alkanedione substrates to the corresponding
(S)-2-hydroxy alkan-(3)-ones catalysed by CPCR2. Cofactor
regeneration is achieved by oxidation of the co-substrate isopropyl
alcohol to acetone, also catalysed by CPCR2.
Results and Discussion
For α-diketone substrates with varying structural determinants
and therefore varying potential for reduction by CPCR2, the
specific catalytic activity of the enzyme was investigated. A
selection of results is shown in Table 1. It was found that cyclic
diketones were not accepted at all, and substrates with
substituents larger than a methyl group next to both carbonyl
functions were converted with only low reaction rates.
Aliphatic diketones comprising one methyl group next to the
carbonyl functions, on the other hand, were converted with
reaction rates up to 10 U·mg-1, which is quite good.
Accordingly, 2,3-alkanedione substrates were investigated for
biotransformation in preparative scale.
Substrate
Spec. activity
[U mg-1]
2,3-Pentanedione (1a)
2,3-Hexanedione (2a)
2,3-Heptanedione (3a)
3,4-Hexanedione
10.0 ± 0.3
8.9 ± 0.2
9.1 ± 0.1
1.7 ± 0.1
0.0 ± 0.0
1,2-Cyclohexane-dione
Table 1. Specific catalytic activity of CPCR2 in the reduction of
diketones.
Preparative-scale biotransformations were successfully
conducted for 2,3-pentanedione, 2,3-hexanedione, and 2,3-
heptanedione (entries 1a, 2a and 3a, respectively, in Table 1).
Conversions between 88 and 97 % were observed within only
30 minutes (Figure 2). The yields after 1 h of reaction time
ranged between 70 and 87%. Next to the formation of the main
(S)-product very small amounts of the (R)-product were
detected, leading to decent enantiomeric excesses (ee-values)
(Table 2). Taking both chiral products into account the mass
balance of the reactions is imbalanced by 7-19 % on the
product side. This divergence between the measured decrease
of substrate and increase of product may be explained by the
substrate evaporation and adsorption to the reaction vessel, and
a light-induced side-reaction of diketones as described by
Maekinen and co-workers.[28]
After completion of the reaction after one hour, the reactants
were retrieved by a four-fold extraction with equal volumes of
ethyl acetate. Purification of the reaction products by column
chromatography yielded highly pure fractions of the 2-hydroxy-
alkan-3-ones. After solvent removal 1H-NMR analysis was
conducted showing no major impurities in the reaction products
(supporting material, Figures S4-6). Hence, for the investigated
group of substrates, the reaction is chemoselective regarding
the reduction of diketones to hydroxy ketones, and
regioselective regarding the carbonyl function on C2-position.
In addition, the reaction is stereoselective, yielding ee-values
between 89 and 95% (Table 2). A significant effect of the
reaction parameters temperature, pH-value und cosubstrate
concentration was not observed (Figure S7).
Figure 2. Progress of 2,3-alkanedione reduction and corresponding
product formation in preparative scale biotransformations. A) 2,3-
pentanedione, B) 2,3-hexanedione, C) 2,3-heptanedione.
Performance of cofactor regeneration by using isopropyl
alcohol as co-substrate effectively supplied the reaction with
reduced cofactor. Turnover numbers (TONs) of NADH were
300-450 (Table 3), which is a reasonable range. Improvement
would certainly be possible by decreasing NADH
concentration, but most probably at the cost of reaction rates.
The TON of the enzyme was in the order of 105, which is
excellent and a good measure for the efficiency of the
catalyst.[29] The procedure yielded 360–600 mg of the pure (S)-
α-hydroxy ketones, corresponding to isolated yields between
36–46%. On the one hand, these low values may be explained
by the factors already discussed for the reaction yield. On the
other hand the purification of the products by silica gel
chromatography was not optimized and specifically difficult for
the short hydrophilic substrates. Nevertheless, high space time
yields (STY) between 43 and 72 g·L-1·d-1 were achieved (Table
3), mainly due to the fast conversion of substrates.
2 | J. Name., 2012, 00, 1-3
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Table 2. Conversion, reaction yield, enantiomeric excess and isolated yield of 2,3-alkanedione reduction with CPCR2 on a preparative scale. a)
Substrate consumption after 1 h, measured via GC; b) Product concentration after 1 h, measured via GC.
Conversiona)
Reaction
Yieldb) [%]
Yield
[mg]
Isolated
Entry
(1b)
Product
ee [%]
[%]
Yield [%]
97.1 ± 0.2
97.7 ± 0.1
89.5 ± 0.1
75.5 ± 1.2
87.0 ± 0.5
70.4 ± 0.3
94.8 ± 1.2
362
529
601
36.2
46.3
46.9
(2b)
(3b)
93.5 ± 1.3
88.7 ± 0.8
For determination of the absolute configuration of the main
STY [gL-
1d-1]
Product
TON NADH
TON CPCR2
products,
Mosher´s esters of the reaction products were
synthesised and analysed via 1H-NMR[30]. This method is based
on an anisotropic effect of a phenyl group in the derivatization
agent MTPA on the chemical shift of the protons in the
substituents of a target hydroxyl compound. Both, the (R)- and
(S)-MTPA esters of each reaction product were synthesized and
1H-NMR spectra were recorded. The differences between the
1b
2b
3b
2.8 x 105
4.2 x 105
3.3 x 105
43
64
72
297
447
351
Table 3. Process relevant data from preparative scale
biotransformations of diketones to the corresponding chiral α-hydroxy
chemical shifts for protons, in the same position in both esters, ketones. Turnover numbers (TON) for biocatalyst and cofactor, and
space time yield (STY) of the reaction.
are descried as Δδ values. For all reaction products, these Δδ
values could only be determined for the protons on C1-position,
ranging between -0.05 and -0.06 ppm (Table 4). Considering
the spatial position of the MTPA-phenyl group, the position of
the small substituent relative to the large substituent could be
determined, yielding (S)-configuration at the hydroxy group.[30]
Results of the analysis are illustrated in the supporting
information (Figures S8, S9 and S10).
Summarising, the asymmetric reduction of three different 2,3-
alkanediones with CPCR2 yielded the corresponding (S)-2-
hydroxy-alkane-3-ones with good enantiomeric excesses. Due
to a considerably small substrate binding pocket[25], the
substrate specificity of CPCR2 restricts its application to
compounds with a carbonyl group on C2-position, which on the
other hand allows
a highly regioselective reduction of
The optical rotation of the reaction products was measured
applying the conditions described by Martre and coworkers
([α]25D (c=0.9, chloroform)[11]). With +43.8° the optical rotation
of (S)-2-hydroxy-pentane-(3)-one is in good agreement with
+48°, determined in the reference study[11](Table 4). With
+52.0° for (S)-2-hydroxy-hexane-(3)-one and +59.2° (S)-2-
hydroxy-heptane-(3)-one, the reaction products exhibited
increasing optical rotations with increasing size of the large
substituent. For (S)-2-hydroxy-hexane-(3)-one the measurement
was repeated under the conditions applied by Bortolini and
asymmetric diketones. In addition, no consecutive reduction of
the hydroxy ketone to the corresponding diol was observed as
described in previous studies with ADHs.[20,21] Here again the
limitation of the substrate scope of the enzyme is turned into an
advantage since the reaction selectively stops at the hydroxy
ketone level. Thus, low molecular weight α-hydroxy ketones,
containing the hydroxy function on C2-position, may be
synthesised chemo-, regio- and stereoselectively utilising
CPCR2.
In contrast, the application of lipases for the synthesis of
aliphatic hydroxy ketones shows improving enantiomeric
excesses with an increasing size of both substituents.[19] This is
also true for lyases, where symmetric α-hydroxy ketones with
C5-substitutents were synthesized with the highest
enantiomeric excesses.[17] Thus, the comparably small substrate
scope of the asymmetric diketone reduction with CPCR2 is
compensated by the fact that the products obtained in this study
are difficult to synthesize applying other enzymatic approaches.
coworkers ([α]20 (c=2.5, chloroform)[23]) The measured value
D
of +55.2° is in good agreement with the reference (57°[23]). In
case of (S)-2-hydroxy-heptane-(3)-one, no reference value was
detected. Thus, where available, the measured optical rotations
were comparable to literature, with a tendency to lower values.
This may be explained by enantiomeric impurities resulting
from the biotransformation.
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Reactions were performed at room temperature in a shaker
(Vibramax 110, Heidolph, Germany) at 1000 rpm for 30 min.
125 µL samples were withdrawn every 10 min and extracted
with 125 µL ethyl acetate. Product concentrations were
determined via chiral GC.
Product Hydroxy
ketone
(
R
)-
(
S
)-
ΔδSR
Optical
rotation
Ester
1.43
1.43
Ester
1.49
1.48
1b
2b
1.34
1.34
-0.06
-0.05
+43.8° ± 1.3a)
+52.0° ± 1.3 a)
/
+55.2° ± 2.5 b)
+59.2° ± 1.9 a)
3b
1.34
1.43
1.49
-0.05
Preparative Biotransformation
Table 4. Chemical shifts and ΔδSR values (in ppm) for hydrogen atoms
at C1-position of hydroxy ketones and corresponding Mosher´s esters.
a) measured at [α]25 (c=0.9, chloroform) b) measured at [α]20 (c=2.5,
Preparative biotransformations were conducted in 200 mL-scale
in 500 mL shake-flasks mixed with a magnetic stir bar (80 rpm)
at room temperature. The reaction mixture contained: CPCR2
8.5 µg·mL-1, substrate 50 mmol·L-1 (1(a): 961 mg, 2(a): 1066
mg, 3(a): 1179 mg), NADH 100 µmol·L-1, isopropyl alcohol 5
% (v/v), TEA (100 mmol·L-1, pH 7.3), dithiothreitol (DDT) 4
mmol·L-1. Like for activity assays isopropyl alcohol acts as
cosubstrate for cofactor regeneration. 5 % (v/v) isopropyl
alcohol corresponds to a concentration of 650 mmol·L-1
yielding a 13-fold excess of cosubstrate over the substrate. All
components except for CPCR2 were premixed under the given
conditions until the substrate was dissolved completely. Then
the reaction was initiated by the addition of CPCR2. The
biotransformations were stirred for one hour and samples were
withdrawn at definite time intervals (15 min) and extracted with
one sample volume of ethyl acetate. Conversion of substrates
and synthesis of products was assessed by GC.
D
D
chloroform)
Experimental section
If not stated otherwise all chemicals were purchased from
Sigma-Aldrich (St. Louis, USA). All reactions were performed
in aqueous solution.
Enzyme preparation and characterization
CPCR2 was prepared according to a modified protocol of
Jakoblinnert et al..[27] Fermentation was performed in 1 L scale
in LB medium with ampicillin (100 µg·mL-1) in a stirred tank
reactor (Biostat B Plus, Sartorius, Germany). The medium was
inoculated from a preculture of E.coli BL21 (DE3) containing
the plasmid pET22b(+):CPCR2 to a final OD600 of 0.1.
Temperature (37°C), pH (7.0) and dissolved oxygen (50%)
were controlled automatically. After reaching an OD600 of 0.8,
the temperature was reduced to 20°C and protein expression
Product purification and analytics
Reaction products were isolated by four consecutive extractions
with 200 mL of ethyl acetate. Purification was performed via
column chromatography using silica gel 60 (Sigma Aldrich,
Germany) as solid phase and a solvent mixture of n-hexane and
ethyl acetate at a ratio of 4:1 (compounds 2,3) and 1:1
(compound 1), respectively. Purification was monitored via GC
and thin layer chromatography (TLC) in combination with
cerium sulfate staining.
GC analysis of the reaction products was performed on a
Shimadzu GC2010 gas chromatograph with flame ionization
detector (FID) and an AOC20i Autosampler. For separation of
all chiral compounds Hydrodex γ DIMOM (Macherey-Nagel,
Germany) was applied as solid phase using different
temperature gradients for each compound. The carrier gas N2
was applied with a pressure of 70 kPa. 2 µL of the extracted
samples were injected to the column without split flow.
Exemplary chromatograms are provided in the supporting
information (Figures S1-S3).
was
induced
with
IPTG
(Isopropyl
β-D-1-
thiogalactopyranoside) at a final concentration of 0.1 mmol·L-1.
After 4 hours incubation, cells were harvested by centrifugation
(10 min, 5000 g, 4°C).
For cell lysis, the cell pellet was resuspended in 30 mL
triethanolamin (TEA) buffer (100 mmol·L-1, pH = 8.0) adding
50 µg·mL-1 DNaseI (Applichem GmbH, Germany), 500
µg·mL-1 lysozyme and 1 mmol·L-1 phenylmethylsulfonyl
fluoride (PMSF). The lysate was cleared by centrifugation (20
min, 16000 g, 4°C). CPCR2 was purified by StrepTag affinity
chromatography applying a fast protein liquid chromatography
system (Äkta 920, General Electrics, USA) equipped with a 5
mL StrepTrap® column (General Electrics, USA) according to
modified manufacturer’s instructions. As washing buffer, a
TEA buffer (100 mmol·L-1, pH = 8.5) with 500 mmol·L-1
sodium chloride, 10% (v/v) glycerol, and 1 mmol·L-1 ethylene
diamine tetra acetic acid (EDTA) was applied. For elution a
TEA buffer (100 mmol·L-1, pH = 8.5) with 500 mmolL-1 NaCl,
10% (v/v) glycerol, and 2.5 mmol·L-1 desthiobiotin was used.
CPCR2 concentration for determination of specific activities
was quantified via BCA-assay (Pierce, USA) according to the
manufacturer’s instructions. Enzyme activity assays were
performed at 25°C with 6.2 µg·mL-1 CPCR2 in 1 mL TEA
buffer (100 mmol·L-1, pH 7.3) applying 3 mmol·L-1 substrate.
Reactions were started by addition of nicotinamide adenine
dinucleotide (NADH) to a final concentration of 250 µmol·L-1.
For all substrates the initial reaction rate was measured
photometrically at 340 nm for 2 minutes. Measurements were
performed in triplicates.
2,3-Pentanedione: A temperature gradient from 95-115°C was
applied with a ramp of 2°C min-1. Retention times were
determined at 3.3 min for 2,3-pentanedione 1(a), 5.6 min for
(R)-2-hydroxy-pentan-(3)-one and 7.8 min for (S)-2-hydroxy-
pentan-(3)-one 1(b) (supporting information, Figure S1).
2,3-Hexanedione: A temperature gradient from 98-122°C was
applied with a ramp of 2°C min-1. Retention times were
determined at 3.8 min for 2,3-hexanedione 2(a), 8.6 min for
(R)-2-hydroxy-hexan-(3)-one and 9.2 min for (S)-2-hydroxy-
hexan-(3)-one 2(b) (supporting information, Figure S2).
2,3-Heptanedione: A temperature gradient from 105-125°C was
applied with a ramp of 2°C min-1. Retention times were
determined at 4.4 min for 2,3-heptanedione 2(a), 9.1 min for
(R)-2-hydroxy-heptan-(3)-one and 9.4 min for (S)-2-hydroxy-
heptan-(3)-one 2(b) (supporting information, Figure S3).
For determination of stereoselectivity, activity assays were
performed on 500 µL-scale. The reaction mixture, containing
25 mmol·L-1 of substrate (1(a): 480 ng, 2(a): 533 ng, 3(a): 590
ng), 250 µmol L-1 NADH, 5% (v/v) isopropyl alcohol and 4
mmol·L-1 dithiothreitol (DDT) in TEA buffer (100 mmol·L-1,
pH 7.3), was prepared and preincubated at 25°C for 5 min.
Conversion was started by addition of 1 µg·mL-1 CPCR2.
1
Reaction products were identified via H-NMR spectroscopy
(500 MHz, Bruker DRX-500) using chloroform-d (1H: δ 7.26).
1H-NMR spectra are provided in the supporting information
(Figures S4-S6).
4 | J. Name., 2012, 00, 1-3
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(S)-2-Hydroxy-pentan-3-one (1b): [α]25 = +43.8° ± 1.3 (c=0.9,
D
Notes and references
chloroform) Yellowish oil, (1H-NMR, 500 MHz) δ (ppm): 4.19
(1H, q, j = 7.08, CH-OH), 3.40 (1H, bs, OH), 2.45 (2H, m, Chair of Molecular Biotechnology, Institute of Microbiology, Technische
Universität Dresden, Germany.
CH2), 1.32 (3H, d, j = 7.07, CH3), 1.05 (3H, t, j = 7.32, CH3),
(supporting information, Figure S4)
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
Reference: [α]25D = +48° (c=0.9, chloroform)[11]
(S)-2-Hydroxy-hexan-3-one (2b): [α]25 = +52.0° ± 1.3 (c=0.9,
D
chloroform), [α]20 = +55.2° ± 2.5 (c=2.5, chloroform)
D
1
2
3
4
5
6
7
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Yellowish oil, (1H-NMR, 500 MHz) δ (ppm): 4.23 (1H, q, j =
7.08, CH-OH), 3.52 (1H, bs, OH), 2.45 (2H, m, CH2), 1.66
(2H, m, CH2), 1.38 (3H, d, j = 7.08, CH3), 0.93 (3H, t, j = 7.31,
CH3), (supporting information, Figure S5)
Reference: [α]20D = +57° (c=2.5, chloroform)[23]
(S)-2-Hydroxy-heptan-3-one (3b): [α]25 = +59.2° ± 1.9 (c=0.9,
D
chloroform) Yellow oil, (1H-NMR, 500 MHz) δ (ppm): 4.22
(1H, q, j = 7.09, CH-OH), 3.56 (1H, bs, OH), 2.45 (2H, m,
CH2), 1.60 (2H, m, CH2), 1.35 (3H, d, j = 7.14, CH3), 1.31
(2H, m, CH2), 0,89 (3H, t, j = 7.35, CH3), (supporting
information, Figure S6)
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The absolute configuration of reaction products was determined
via 1H-NMR analysis of Mosher´s esters of the products
according to the protocol of Hoye and coworkers (Procedure:
1.b).[30] In this method, chiral alcohols are esterified with both
enantiomers of a phenyl substituted chiral acid. Electrostatic
shielding effects lead to a shift in the 1H-NMR-spectrum, which
can be correlated with the absolute configuration of the
analysed alcohol. The reaction was performed in
dichloromethane-d (1H: δ 5.32) on 50 µg-scale. The synthesis
of (S)-Mosher´s esters of hydroxy ketones was performed by
esterification with the (R)-MTPA-Cl ((R)-(−)-α-methoxy-α-
4
15 R. Mikolajek, A. C. Spiess, M. Pohl, L. Sylvain, J. Büchs,
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16 R. Mikolajek, A. C. Spiess, M. Pohl, J. Büchs, Biotechnol.
Prog., 2009, 25(1), 132-138;
(trifluoro-methyl)phenylacetyl
chloride,
Sigma-Aldrich,
Germany), while esterification with the (S)-MTPA-Cl gives rise
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Acknowledgements
Deutsche Forschungsgemeinschaft (DFG) is gratefully
acknowledged for financial support within research project AN
387/5-1. We also thank the collaborative research network
BioNoCo (GRK1166) for scientific support and fruitful
discussion.
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