Extreme halophilic alcohol dehydrogenase mediated highly efficient syntheses of enantiopure aromatic alcohols

Article abstract of DOI:10.1039/c7ob02299a

Enzymatic synthesis of enantiopure aromatic secondary alcohols (including substituted, hetero-aromatic and bicyclic structures) was carried out using halophilic alcohol dehydrogenase ADH2 from Haloferax volcanii (HvADH2). This enzyme showed an unprecedented substrate scope and absolute enatioselectivity. The cofactor NADPH was used catalytically and regenerated in situ by the biocatalyst, in the presence of 5% ethanol. The efficiency of HvADH2 for the conversion of aromatic ketones was markedly influenced by the steric and electronic factors as well as the solubility of ketones in the reaction medium. Furthermore, carbonyl stretching band frequencies ν (CO) have been measured for different ketones to understand the effect of electron withdrawing or donating properties of the ketone substituents on the reaction rate catalyzed by HvADH2. Good correlation was observed between ν (CO) of methyl aryl-ketones and the reaction rate catalyzed by HvADH2. The enzyme catalyzed the reductions of ketone substrates on the preparative scale, demonstrating that HvADH2 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of pharmaceutical interest.

Full text of DOI:10.1039/c7ob02299a

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Received 00th
January 20xx,
Extreme halophilic alcohol dehydrogenase mediated highly efficient
syntheses of enantiopure aromatic alcohols
a*
a
b*
Diya Alsafadi, Safaa Alsalman and Francesca Paradisi
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Enzymatic synthesis of enantiopure aromatic secondary alcohols (including substituted, hetero‐aromatic and bicyclic
structures) were carried out using the halophilic alcohol dehydrogenase ADH2 from Haloferax volcanii (HvADH2). This
enzyme showed an unprecedented substrate scope and absolute enatioselectivity. The cofactor NADPH was used
catalytically and regenerated in‐situ by the biocatalyst, in the presence of 5% ethanol. The efficiency of HvADH2 for
conversion of aromatic ketones was markedly influenced by the steric and electronic factors as well as the solubility of
ketones in the reaction medium. Furthermore, carbonyl stretching bands frequencies ν (
different ketones to understand the effect of electron withdrawing or donating properties of the ketones substituents on
the reaction rate catalyzed by HvADH2. Good correlation was observed between ν ( ) of methyl aryl‐ketones and the
) have been measured for
reaction rate catalyzed by HvADH2. The enzyme catalyzed the reductions of ketone substrates on the preparative scale,
demonstrating that HvADH2 would be a valuable biocatalyst for the preparation of chiral aromatic alcohols of
pharmaceutical interest.
A variety of chiral metal complexes have been used as catalysts for
enantioselective synthesis of chiral aromatic secondary alcohols4
‐5
Introduction
Chirality is a key factor in the safety and efficacy of many drugs and
thus the production of enantiopure drugs has become increasingly
important in the pharmaceutical industry. Chiral aromatic
secondary alcohols are widely used in synthetic organic and
medicinal chemistry as key intermediates for the synthesis of
however, biocatalytic transformation systems using cell‐free
6
7
enzymes or whole‐cell microorganisms offers advantages with
respect to high catalytic efficiency, mild reaction conditions,
outstanding enantio‐, regio‐ and chemoselectivity and being void of
toxic metals.
1
Alcohol dehydrogenases (ADHs, EC 1.1.1.1) are a class of
+
nicotinamide adenine dinucleotide (phosphate) [NAD(P) ]‐
®
2
various pharmaceutical products such as Zetia (Ezetimibe),
dependent enzymes that catalyse the reversible reduction of
aldehydes and ketones to their corresponding alcohols.8
Asymmetric reduction of the prochiral ketones using ADH is an
®
3
®
1
Prozac (Fluoxetine) and Emend (Aprepitant) (Fig. 1).
9
‐10
important tool for industrial production of enantiopure alcohols.
1
1‐12
Additionally, ADHs can accomplish dynamic kinetic resolution,
1
3‐14
and deracemization of racemic alcohols
of enantiopure alcohols,
as well as racemization
processes by which high yields
1
5‐16
(theoretically up to 100 %) of a single enantiomer can be obtained.
Although several ADHs have been identified from various
organisms, their substrate scope tends to be limited and mutagenesis
has often been necessary to expand the range of substrates.1
Furthermore, cofactor regeneration must be taken into consideration
to minimize the economic impact of the biocatalytic process. As a
consequence, several methods have been developed to regenerate the
cofactor in ADH-catalyzed reactions such as a coupled enzyme
system, or with the use of a second substrate.6
7,18
Steric and electronic properties of substituent groups also play a
major role on the biocatalytic reduction of aromatic ketones. ADHs
1
9
20
from Pichia glucozyma,
Aromatoleum aromaticum
and
Lactobacillus brevis2 have been applied in the asymmetric
reduction of acetophenone derivatives with different size and
electronic properties. The formations of aromatic secondary
alcohols were explained by substrate docking experiment and
1
Fig. 1 Examples of drugs derived from chiral aromatic secondary
alcohols precursors.
interaction energy values,2 σ‐Hammett coefficients
0
19
and IR
aRoyal Scientific Society, Amman 11941, Jordan
Diya.safadi@rss.jo
carbonyl stretching bands. The results have provided valuable
insight into the understanding of how ADHs behave with aromatic
ketones.
21
b
School of Chemistry, University of Nottingham, Nottingham, UK
francesca.paradisi@nottingham.ac.uk
Electronic Supplementary Information (ESI) available: Physico‐chemical data of the
products, HPLC chromatograms, IR spectroscopy study and H NMR spectra
images DOI: 10.1039/x0xx00000x
There are a limited number of ADHs effectively useful in the
synthesis of chiral aromatic secondary alcohols which have been
1
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Journal Name
described in the literature. For instance, alcohol dehydrogenase
from Ralstonia sp. (RasADH) catalyzed the reduction of aromatic
ketones
containing
bulky
substituents
with
high
2
2
enantioselectivity. This type of ADHs has great relevance in
asymmetric synthesis because of its broad substrate scope. ADH
2
3
from Thermus thermophilus HB27 was used in the asymmetric
reduction of aromatic ketones with anti‐Prelog selectivity.
Considering the value of aromatic chiral alcohols, it will be
increasingly important to identify more ADHs that can be employed
in such processes with high enantioselectivity. Extremophiles
represent a promising source of robust biocatalysts with industrial
applications. A number of ADHs from extremophiles have been
identified and studied with respect to their ability to work in harsh
Fig. 2 Reduction of 1‐phenyl‐2‐propanone using HvADH2.
Initially, the effect of pH on the conversion of 1‐phenyl‐2‐
propanone was investigated at pH 6 and pH 8, where HvADH2
27
exhibited the highest activity for the reductive reaction. The
2
4
industrial conditions such as high temperature and non‐aqueous
medium.
reactions were performed either utilizing a substrate‐coupled
approach (5% ethanol) to regenerate the co‐factor or with an
excess of NADPH. In both cases, the best conversion was observed
at pH 8, in fact no reaction was observed at pH 6 in the presence of
2
5‐26
As part of our effort to develop effective ADHs for synthesis of
industrially valuable chiral alcohols, we developed and
characterized four novel alcohol dehydrogenases from halophilic
5
% ethanol and catalytic amount of NADPH likely due to a stability
2
7‐29
bacteria.
In particular; alcohol dehydrogenase from the extreme
issue at the lower pH over an extended period of time.
halophile Haloferax volcanii (HvADH2) showed remarkable activity
For the cofactor regeneration, alcohols that had been used as
substrates in the oxidative reactions were tested in 5%
2
7
and stability in the presence of different organic solvents. For easy
recovery from the reaction mixture and reutilization, HvADH2 was
27
concentrations, including 2‐propanol, 1‐propanol and 1‐butanol,
3
0
also seccesfully immobilized on solid support. The initial substrate
however only ethanol performed well. To determine the optimum
concentration of ethanol as co‐substrate in the reaction, different
concentrations of ethanol (5%–20%) were trialled but already at
screening for this enzyme showed encouraging results towards
2
7
benzyl alcohol which prompted further in depth investigation of
HvADH2 with industrially relevant compounds.
10% the conversion dropped to 60% with respect to the lower
Herein, we report on the application of HvADH2 for the synthesis of
a very broad range of aromatic secondary alcohols. The versatility
of this enzyme and its adaptability to optimized reaction conditions
is uncommon in the examples found to date in the literature. The
effect of the steric, electronic and solubility properties of the
substrates on the reaction catalyzed by HvADH2 is also discussed.
Finally, IR absorption bands of the carbonyl group are measured
and correlated with the reaction rate catalyzed by HvADH2.
amount (results not shown). Likely acetaldehyde accumulating in
the media is toxic to the enzyme and 5% ethanol was chosen for all
subsequent reactions.
To investigate the influence of organic solvents and temperature,
the reduction of 1‐phenyl‐2‐propanone was carried out in the
presence of 5% of organic solvents (dimethyl sulfoxide, methanol
and acetonitrile) at 25°C and 50°C. The reactions were monitored
by HPLC analysis as described in the experimental section. The
maximum conversion of 1‐phenyl‐2‐propanone (95 %) was
observed in the presence of 5% of acetonitrile at 25°C, while the
conversion recorded in DMSO and methanol was lower (62 % and
37 %, respectively) at the same temperature.
Results and discussion
Reduction of aromatic ketone using HvADH2
Aromatic secondary alcohols such as 1‐phenylethanol, 1‐phenyl‐2‐ Substrate scope and stereoselectivity of HvADH2
propanol and 4‐phenyl‐2‐butanol were investigated as possible
substrates for the alcohol dehydrogenase enzyme from the extreme Several aromatic ketones falling into seven structural classes were
halophile Haloferax volcanii (HvADH2) in the oxidation reaction. selected based on their potential for the production of industrially
Preliminary activity assays suggested that HvADH2 accepted valuable chiral aromatic alcohols. Wild‐type HvADH2 was used for
aromatic alcohol substrates with most marked activity observed the enantioselective reduction of each substrate class (Fig. 3).
against 1‐phenyl‐2‐propanol. Therefore, 1‐phenyl‐2‐propanone was The reactions were monitored by HPLC and the purity of the
1
used as the model substrate in order to optimize the reaction product determined by H‐NMR analysis. The percent conversion of
conditions (pH, temperature, organic solvents, and co‐factor ketones and the e.e. values of the produced alcohols are reported
recycling system) in the reductive direction with HvADH2 (Fig 2).
in Table 1.
Set a
Alkyl aryl‐
ketones
Set b
Methyl aryl‐
ketones
Set c
Alpha‐halo aryl‐
ketones
Set d
Beta‐cyano
aryl‐ketones
Set e
Trifluoro
aryl‐ketones
Set f
Acetylpyrazie
Set g
Fused bicyclics
Fig. 3 Various aromatic ketones derivatives and general scheme of their reduction with HvADH2.
2
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Table 1 Results from the reduction of various aromatic ketones
ARTICLE
derivatives using HvADH2
Substrate (set a)
Product
Conv. (%)
ee (%)
Substrate (set c)
Product
Conv. (%)
ee (%)
(
(
(
S)‐1a
S)‐2a
S)‐3a
65
>99 S
(S)‐17c
(S)‐18c
38
>99 S
95
85
>99 S
>99 S
>99.9
>99 S
(
S)‐19c
94
58
>99 S
>99 S
O
Cl
(
S)‐4a
76
0
>99 S
(S)‐20c
F
20
‐
‐‐‐‐‐
(
S)‐21c
56
>99 S
Substrate (set b)
Product
S)‐6b
Conv. (%)
ee (%)
Substrate (set d)
Product
(S)‐22d
Conv. (%)
ee (%)
ꢀ
(
70
>99 S
57
>99 S
(
S)‐7b
76
82
55
>99 S
>99 S
>99 S
(S)‐ 23d
Product
21
>99 S
(
S)‐8b
S)‐9b
Substrate (set e)
Conv. (%)
ee (%)
>99 S
(
S)‐24e
73
(
>
99 S
‐
‐‐‐‐‐‐‐‐‐
0
(
S)‐10b
97
0
>99 S
Substrate (set f)
Product
Conv. (%)
ee (%)
‐
‐‐‐‐‐‐‐‐
S)‐12b
S)‐13b
(
S)‐26f
S)‐27f
16
>99 S
(
(
72
66
62
>99 S
>99 S
>99 S
(
8
>99 S
Substrate (set g)
Product
Conv. (%)
ee (%)
‐
‐‐‐‐‐‐‐‐‐
‐‐‐‐‐‐‐‐‐
0
0
(
S)‐14b
S)‐15b
S)‐16b
‐
(
(
88
25
>99 S
*
Reaction conditions: 4 mM ketone (1‐29) ; 1.5 mM NADP+
and 0.3 mg/mL HvADH2 were dissolved in Tris‐HCl buffer(100
mM, pH 8.0) supplemented with 2 M KCl, 5% ethanol and
% acetonitrile, reaction volume 1.0 mL, temperature 25 °C,
reaction time 36 h; agitation on an orbital shaker (320 rpm).
*% conv was calculated from HPLC analysis.
5
>99 S
ꢀ
*
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HvADH2 follows rigorously Prelog’s rule yielding exclusively (S)‐ HvADH2 was further investigated by monitoring the carbonyl
alcohols (100% e.e.) independently of the substrate tested. Other stretching bands with IR studies (see below).
dehydrogenases/reductases reported in the literature show mixed For the alpha‐halo aryl‐ketones (17‐21), it was noticeable that the
selectivity depending on the substrate. For example, (S)‐1‐ conversion rate for α‐fluorinated ketone 18 with HvADH2 was
phenylethanol dehydrogenase from Aromatoleum aromaticum better than α‐chlorinated ketone 19 and this one was better than α‐
showed strong (S)‐ enantioselectivity however, a racemic mixture of brominated ketone 17. These results is consistent with both steric
1
‐(4‐aminophenyl)ethanol
was
observed
when
4′‐ (atomic size F<Cl<Br) and electronic (high electronegative F atom
2
0
aminoacetophenone 11 was tested. Additionally, acetophenone 1 at α‐position) factors of the groups.
was reduced to (R)‐1‐phenylethanol (opposite enantiomer) with Interestingly, phenacyl chloride 19 was converted faster than
9
5% e.e using benzilketoreductase KRED1‐Pglu isolated from Pichia 2‐chloro‐4′‐fluoroacetophenone 20 and 2‐chloro‐2′,4′‐difluoro‐
19
glucozyma. Alpha‐halo aryl‐ketones such as phenacyl bromide 17 acetophenone 21 with HvADH2. This indicated that the substituents
and phenacyl chloride 19 were reduced to their corresponding (S)‐ on the phenyl ring of α‐halo‐ketone adversely affected the rate of
alcohols using the short‐chain dehydrogenase from Streptomyces enzymatic reaction. Clearly, for substrates 20 and 21 the steric
griseus while, methyl aryl‐ketones such as 4′‐chloroacetophenone 6 effect is predominant in influencing the reaction rate rather than
and 4‐bromoacetophenone 8 were reduced to (R)‐alcohols using the electronic effect. This observation is also confirmed from the
3
1
the same biocatalyst. Interestingly, the stereoselectivity of conversion value of 4‐fluoroacetophenone 7 (76%) which was
HvADH2 was maintained with all aromatic ketones (1‐27) tested in higher than 20 and 21 substrates.
this work.
The choice of solvent played a major role in the enzymatic reaction
Clearly, the efficiency of HvADH2 for conversion of alkyl aryl‐ of HvADH2 with beta‐cyano aryl‐ketones (set d). The reduction of
ketones (set a) were different and markedly influenced by steric benzoylacetonitrile 22 and 4‐bromobenzoylacetonitrile 23, did not
factors such as substrate size and the type of alkyl chain, linear or proceed when the reaction was performed in an aqueous buffer
branched. For liner alkyl chain ketones (1‐4), the smallest substrate containing acetonitrile (optimum co‐solvent). Therefore,
acetophenone 1 showed the lowest conversion (65%). On the other acetonitrile was replaced by dimethyl sulfoxide in an effort to
hand, HvADH2 effectively catalyzed the enantioselective reduction increase the solubility of substrates containing polar cyano group at
of alkyl aryl‐ketones as the alkyl chain between the carbonyl group α‐position. As expected, the conversion yields for the substrates 22
and phenyl group became longer 2 and 3. Notably, good conversion and 23 were greatly improved with the addition of the high polar
(
76%) was observed with the bulkier substrate 4. Thus, bulky groups solvent (DMSO) into the reaction buffer.
might be better stabilized in the enzyme active site in such a way to Enantiopure 1R‐[3,5‐bis(trifluoromethyl)phenyl] ethanol and its (S)‐
increase the catalytic efficiency of the enzyme. In line with our enantiomer are key chiral intermediates in the synthesis of
2
7
33
initial substrate scope investigation, no conversion was observed Aprepitant (a NK‐1 receptor antagonist). The reduction of 24 had
with the substrate containing a branched group 5. It is worth noting been previously reported using different microbial strains with
3
4
that the influence of branched alkyl chain on the ketones reduction varying degree of success. Here, 24e was successfully obtained
by ADH was not observed before in the literatures. with HvADH2 in 73% yield and 100% e.e.
The catalytic activity of HvADH2 on methyl aryl‐ketones substrates 2,2,2‐trifluoroacetophenone 25 was tested as substrate for
set b) was investigated using different meta‐ and para‐ substituted HvADH2. Surprisingly, no conversion was observed with this
of acetophenone derivatives (6‐16) and compared to acetophenone substrate. Acetylpyrazine compounds 26 and 27 were only
(Table 1). minimally active affording low conversions (<20%). HvADH2 like
(
1
3
5
HvADH2 effectively catalyses the reduction of para‐halogenated many ADHs fails in the reduction of fused cyclic ketones such as 28
acetophenone derivatives (6–8), with up to 80% conversion. Para‐ and 29 which are particularly challenging.
methylacetophenone 9 gave satisfactory conversion (55%) and the
best conversion was reported with para‐nitroacetophenone 10 IR carbonyl stretching bands study
which yielded the corresponding (S)‐alcohol almost quantitatively.
In contrast, para‐amionoacetophenone 11 was not reduced by the To understand the electronic effect of aromatic ketones
enzyme, likely due to the strongly electron donating properties of substituents on the reaction rate catalyzed by HvADH2, the
1
9
carbonyl stretching bands for different ketones were measured and
the amino group as also noted by Contente et al. The difference
on the catalytic activity of HvADH2 toward acetophenone plotted against the % conversion of aromatic ketones. The
derivatives could be explained by the electronic effect of the frequency values of the carbonyl bands can be used to predict the
substituents on the reactivity of the carbonyl group. A similar trend carbonyl bond character in the presence of electron withdrawing
2
1
group (EWDG) or electron donating group (EDG). Ketones
containing EWG expected to absorb at low frequency values (low
double‐bond character), whereas EDG suppress the delocalization
was reported on an ADH from L. Brevis and a benzyl reductase
1
9
from P. glucozyma. However, for other ADHs, the electronic
properties of substituted acetophenones were not as significant.32
A more comprehensive study of the influence of the meta‐position of the aryl‐conjugated carbonyl and the carbonyl stretching bands
on the HvADH2 catalytic activity was carried out. In general, observe at high frequency.
acetophenone derivatives with halogenated substituents at meta‐ Good correlation was observed between the IR carbonyl absorption
position (12 and 13) represent suitable substrates, too. However, bands of set b ketone substrates 6‐16 and the reaction rate
the halogenated meta‐substituted acetophenones gave less catalyzed by HvADH2 (Fig. 4). Unexpectedly, the IR absorption
satisfactory results compared with their para‐substituted frequencies of the carbonyl group increased with the reaction rate.
counterpart. This behavior was not observed with methyl, nitro and Therefore, ketones containing EWG absorb at higher frequencies
amino acetophenone substituents (14‐16), which gave better (higher double‐bond character), which is not in line with the
conversion rate at meta‐position than para‐position. The influence evidence of increased electrophilicity of the carbonyl carbon by
of acetophenone substituents on the reaction rate catalyzed by EWG. A similar trend has been previously reported with the
3
6
nucleophilic acyl substitutions of different esters and the
4
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reduction of ketones using ADH from Lactobacillus brevis.21 The Substrates, reagents, and cofactors were purchased either from
authors attributed the high reactivity of esters/ketones to the Sigma Aldrich or Apollo Scientific Ltd, UK. Racemic alcohols were
prepared from the corresponding ketones as follows. Ketones 1‐29
decrease of the esters/ketones ground state resonance stabilization
and this appears to be due to effect of EWG. In contrast, no
correlation was observed between IR carbonyl absorption bands of
ketone substrates from sets (a, c, d, e and f) and the reaction rate
catalyzed by HvADH2 (data not shown). This could be attributed to
the influence of other factors on the reaction rate such as the
substrate solubility and substrate size.
(
15 mmol) were dissolved in methanol (2 mL) at 0˚C. Sodium
borohydride (0.5 equiv) was added and the reaction mixture was
incubated with shaking for 1 h at 0°C. 1M HCl was added dropwise
to maintain the reaction temperature below 5 ˚C. Methanol was
evaporated using a gentle stream of nitrogen. Then, water (5 mL)
was added and the solution was extracted with ethyl acetate (3 x 5
2 4
mL). The organic layers were combined and dried over Na SO . The
solvent was evaporated and the residue was subjected to
chromatography separation.
Protein expression and purification
HvADH2 was homologously overexpressed, using the vector pTA963
and host strain Hfx. volcanii. The vector features an inducible L‐
tryptophan promoter and has a hexahistidine‐tag, which allowed
for easy purification by immobilized metal affinity chromatography
(
IMAC).27 Haloferax volcanii was grown overnight at 45 C on 270
◦
1
6
mL Hv‐YPC medium. Protein overexpression was induced by the
addition of 30 mL (50 mM) L‐tryptophan to final volume 300 mL of
growth medium. After 24 h the culture was harvested by
centrifugation at 4,000 rpm for 10 min. Cell pellets were
resuspended in lysis buffer (50 mM Tris‐HCl, pH 8, containing KCl (3
M) and disodium EDTA (2 mM)). Cells were lysed by sonication until
the suspension was no longer turbid. The cell lysate was clarified by
centrifugation at 18,000 rpm, 4 C for 30 min. After centrifugation,
the supernatant was filtered through a 0.45 µm membrane and
loaded on to a chromatography column filled with Ni Sepharose 6
Fig. 4 Correlation between IR carbonyl stretching bands of
substrates 6‐16 and the % conversion of aromatic ketones in the
reaction catalyzed by HvADH2. The correlation coefficient r = 0.83.
2
Conclusion
◦
There is considerable interest in alcohol dehydrogenases as
biocatalysts in the pharmaceutical industry for their ability to
produce valuable chiral alcohols. In this work, we have shown that
alcohol dehydrogenase from the halophilic archaeon Haloferax Fast Flow resin. The His‐tagged protein eluted when 10 % (5 mM
volcanii (HvADH2) effectively catalyzes the enantioselective disodium EDTA) to 30 % (15 mM disodium EDTA) of elution buffer
reduction of a large selection of aromatic ketones with high
enantiomeric purity to give (S)‐form of aromatic secondary alcohols.
The co‐factor NADPH can be recycled by co‐substrate addition (5%
ethanol).
was applied. The purity of protein samples was assessed by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE).
The conversion of aromatic ketones was different and influenced by
Analytical methods
(i) steric factors, (ii) electronic factors, and (iii) ketone solubility. No
standard correlation parameters could be used to explain the
conversion of aromatic ketones. For example, the conversion of
alkyl aryl‐ketones (set a) were influenced predominantly by sterics
however, the conversion of alpha‐halo aryl‐ketones (set c), was
influenced by both steric and electronic factors. Additionally, the
reduction of cyano aryl‐ketones (set d) was greatly dependant on
solubility of ketones in the reaction medium.
IR absorption band of the carbonyl group was then used to
investigate the effect of aromatic ketones substituents on the
reaction rate catalyzed by HvADH2 and it was concluded that
electron withdrawing groups have positive effect on ketones
reactivity by destabilizing its ground‐state resonance.
The activity of HvADH2 was analyzed spectrophotometrically by
detecting the increase in absorbance at 340 nm, which corresponds
to the formation of the cofactor NADPH, using a Biochrom Libra S50
UV–Visible spectrophotometer. Enantiomeric excess (e.e.) and
conversion rate were determined by normal phase high
performance liquid chromatography (HPLC) using an Agilent 1100
series HPLC with chiral columns as the following: Chiralcel OJ‐H
(
250x4.6 mm and 5µm silica gel), Dr. MAISCH Germany, Reprosil
Chiral‐BM (250x4.6 mm and 5µm silica gel) alternative to OB‐H
column and Phenomenex USA, Lux cellulose‐1 column (250x4.6 mm
and 10µm silica gel) alternative to OD‐H column. H‐NMR spectra
1
were recorded with Bruker 500 MHz−Avance III spectrometer.
The results confirmed the suitability of HvADH2 as a valuable
biocatalyst for the preparation of chiral secondary alcohols of
pharmaceutical interest.
Chemical shifts (δ) are expressed in ppm and coupling constants (J)
20
are expressed in Hz. Optical rotation values ([α]
D
) were measured
on a PolAAr 21 polarimeter at 589 nm in a 1 dm cuvette at 20 °C.
Fourier transform infrared (FT‐IR) spectra for ketones were
obtained in the range 400 cm to 4,000 cm using IR‐Prestige‐21
Shimadzu FT‐IR spectrophotometer.
Experimental
‐1
‐1
Chemicals
All reagents and solvents, unless stated otherwise, were purchased General procedure for reduction of ketones and sample pre‐
as analytical grade.
treatment for HPLC analysis
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Ketone reduction was carried out in 5 mL screw‐capped test tubes 12 I. Karume, M. M. Musa, O. Bsharat, M. Takahashi, S. M.
+
under the following conditions: 4 mM ketone (1‐29), 1.5 mM NADP
and 0.3 mg/mL HvADH2 were dissolved/ suspended in 1 mL of 100
mM Tris‐HCl pH 8.0 supplemented with 2 M KCl, 5% ethanol and
Hamdan and B. E. Ali RSC Adv., 2016, 6, 96616–96622.
C. V. Voss , C. C. Gruber, K. Faber, T. Knaus, P. Macheroux and
Kroutil W, J. Am. Chem. Soc., 2008, 130, 13969–13972.
I. Karume, M. Takahashi, S. M. Hamdan and M. M. Musa,
ChemCatChem, 2016, 8, 1459‐1463.
1
3
4
5%
acetonitrile. For benzoylacetonitrile 22 and 4‐
1
bromobenzoylacetonitrile 23, 5% DMSO was used as co‐solvent
instead of acetonitrile. All reactions were incubated in an orbital
shaker at 25°C, 320 rpm for 96 h.
15 M. M. Musa, J. M. Patel, C. M. Nealon, C. S. Kim, R. S. Phillips,
The reaction mixtures were extracted with ethyl acetate (3×500µL).
The organic layer was dried over anhydrous sodium sulfate and
transferred to a HPLC vial. The ethyl acetate was evaporated using a
gentle stream of nitrogen and the residue was re‐dissolved in 1 mL
hexane. The solubility of 4‐aminoacetophenone 11, 3‐
aminoacetophenone 16 and 2‐acetylpyrazine 26 in hexane was low
and isopropanol was used in this case.
I. Karume, J. Mol. Catal. B: Enzym., 2015, 115, 155‐159.
C. C. Gruber, B. M. Nestl, J. Gross, P. Hildebrant, U. T.
Bornscheuer, K. Faber and W. Kroutil, Chem.–Eur. J., 2007, 13,
1
6
8
271–8276.
17
18
19
C. M. Nealon, T. P. Welsh, C. S. Kim and R. S. Phillips, Arch
Biochem Biophys. 2016, 606, 151–156
Z. Sun, G. Li, A. Ilie and M. T. Reetz, Tetrahedron Lett., 2016,
57, 3648‐3651.
M. L. Contente, I. Serra, L. Palazzolo, C. Parravicini, E. Gianazza,
I. Eberini, A. Pinto, B. Guidi, F. Molinari and D. Romano, Org.
Biomol. Chem., 2016, 14, 3404–3408.
A. Dudzik, W. Snoch, P. Borowiecki, J. Opalinska‐Piskorz, M.
Witko, J. Heider, and M. Szalenie, Appl. Microbiol. Biotechnol.,
2015, 99, 5055–5069
C. Rodriguez, W. Borzecka, J. H. Sattler, W. Kroutil, I. Lavandera
and V. Gotor, Org. Biomol. Chem., 2014, 12, 673–681
I. Lavandera, A. Kern, B. Ferreira‐Silva, A. Glieder, D. S.
Wildeman and W. Kroutil, J. Org. Chem., 2008, 73, 6003–6005.
J. Rocha‐Martín, D. Vega, J. M. Bolivar, A. Hidalgo, J.
Berenguer, J. M. Guisán and F. López‐Gallego, Bioresour.
Technol., 2012, 103, 343–350.
General procedure for reduction of ketones on quantitative scale
The biotransformation on the preparative scale was set up on a 20
mM for each substrate. The reactions were performed as described
before on the analytical scale. The alcohols from set a, b and c were
prepared with HvADH2 in moderate to very good yields (30–90%).
The isolated yields for alcohols from set f and d were low (20 %). To
fully characterize the alcohols the products residue were purified by
preparative TLC using n‐hexane/ethyl acetate (3:1, v/v) as an eluent
Full spectra and analysis are reported in the ESI.
20
21
22
23
Acknowledgments
This research study was financially supported by the Scientific
Research Support Fund (project No. Bas/1/01/2014).
24
D. Zhu, B. A. Hyatt and L. Hua, J. Mol. Catal. B: Enzym., 2009,
5
6, 272–276
25 D. Alsafadi and F. Paradisi, Extremophiles, 2013, 17, 115‐122
L. Olofsson, I. A. Nicholls and S. Wikman, Org. Biomol. Chem.,
005, 3, 750‐75
A. S. Rowan, T. S. Moody, R. M. Howard, T. J. Underwood, I. R. 27 L. M. Timpson, A. K. Liliensiek, D. Alsafadi, J. Cassidy, M. A.
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Orga Pn l iec a s& e Bd oi o nmo ot al ed cj uu sl ta mr Ca rhg ei nms istry
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Alcohol dehydrogenase from the extreme halophile Haloferax volcanii (HvADH2) catalysed asymmetric
reduction of a range of structurally diverse aromatic ketones with co-factor recycling.