Enzymatic synthesis of optical pure β-nitroalcohols by combining d-aminoacylase-catalyzed nitroaldol reaction and immobilized lipase PS-catalyzed kinetic resolution

Article abstract of DOI:10.1039/c1gc15417f

An enzymatic method which combines d-aminoacylase catalyzed nitroaldol reaction and lipase PS-IM catalyzed acyl resolution reaction was constructed to obtain optically pure β-nitroalcohols in organic media under mild conditions. A series of β-nitroalcohols were synthesized and then resolved, with excellent ee values (>95%) and yields mostly, giving enantiomerically pure acetate and alcohol of the opposite configuration simultaneously.

Full text of DOI:10.1039/c1gc15417f

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Enzymatic synthesis of optical pure b-nitroalcohols by combining
D-aminoacylase-catalyzed nitroaldol reaction and immobilized lipase
PS-catalyzed kinetic resolution†
Fan Xu, Junliang Wang, Bokai Liu, Qi Wu* and Xianfu Lin*
Received 15th April 2011, Accepted 23rd June 2011
DOI: 10.1039/c1gc15417f
An enzymatic method which combines D-aminoacylase cat-
alyzed nitroaldol reaction and lipase PS-IM catalyzed acyl
resolution reaction was constructed to obtain optically pure
b-nitroalcohols in organic media under mild conditions. A
series of b-nitroalcohols were synthesized and then resolved,
with excellent ee values (>95%) and yields mostly, giving
enantiomerically pure acetate and alcohol of the opposite
configuration simultaneously.
(ee) values.5 Then they used the same enzyme to catalyze the
asymmetric retro-Henry reaction and introduced this method
to a kinetic resolution of racemic b-nitroalcohols in order to get
the unreacted substrate.8 Hydroxynitrile lyase from Arabidopsis
thaliana catalyzed R-selective asymmetric Henry reaction was
also reported by Asano and Fuhshuku recently.9 Ramstro¨m and
his co-workers developed a one-pot dynamic kinetic resolution
(DKR) of b-nitroalcohols without b-hydrogen, using argon
as protective atmosphere, which produced the corresponding
acetate of b-nitroalcohols of R configuration with excellent ee
values and high yields.4 There are also several articles focusing on
the biocatalytic way to obtain racemic b-nitroalcohols as well. D-
Aminoacylase,10 protein-glutamine g-glutamyltransferase,11 and
protein bovine serum albumin (BSA)12 are all reported to be
capable of catalyzing Henry resolution efficiently, however, with
no enantiomeric selectivity being observed.
Enzymatic kinetic resolution (KR)13 is a common and power-
ful tool to obtain enantiomerically pure products. As performing
DKR could only get product with only one configuration,
either R or S, depending on the nature of the catalyst, a well-
designed KR is able to give optically pure product and unreacted
substrate of opposite configuration, with nearly 50% yields (fully
completed) and excellent ee values, while both of them could be
taken into the next research step and contribute to the medicine
or biochemistry studies. Considering the difficulty of performing
a direct enzymatic asymmetric Henry reaction, and the advan-
tage of applying the KR process, developing a protocol which
combines an enzymatic Henry reaction and a KR process could
fulfil both the industrial or academic needs and the environment
requirements. Since our team developed an efficient enzymatic
Henry reaction which could produce racemic b-nitroalcohols
bearing b-hydrogen,10 in further research, we have studied the
enzymatic kinetic resolution of a series synthesized racemic b-
nitroalcohols, and construct this two-step enzymatic method
which combines D-aminoacylase catalyzed nitroaldol reaction
and immobilized lipase PS catalyzed kinetic resolution under
very mild reaction condition without protective atmosphere
(Scheme 1), giving both the enantiomerically pure substrate and
product simultaneously.
Nitroaldol (Henry) reaction is an well-developed and atom-
economical carbon-carbon bond forming reaction and serves
well in organic synthesis to construct complex compounds, as
its product, b-nitroalcohol, is a versatile synthetic intermediate
which could be further transformed into a-hydroxyl carboxylic
acids, b-amino-alcohols, b-nitro-ketones, and other important
building blocks for synthesizing insecticides, fungicides, antibi-
otics and some useful natural compounds.1 Increasing effort has
been made to obtain optically pure b-nitroalcohols in a mild and
efficient way. Diverse catalysts such as chiral metal complexes,2
small organic molecules,3 enzymes,4,5 etc. have been reported
to be capable of producing b-nitroalcohols or their derivatives
with good to excellent ee values, through an asymmetric Henry
reaction6 or a dynamic kinetic resolution (DKR) pathway.
Biocatalysts play an important role in many industrial syn-
theses aiming for chiral molecules.7 The advantage of using
biocatalysts or enzymes includes good environment affinity, high
activity, high regioselectivity and excellent enantioselectivity.
The fact that many kinds of enzymes have been commercial-
ized successfully with reasonable costs expands their range of
application. However, the enzymatic ways to obtain chiral b-
nitroalcohols are barely reported so far. Here are some impor-
tant examples. Herfried Griengl and co-workers found that the
hydroxynitrile lyase from hevea brasiliensis could catalyze the
asymmetric Henry reaction, giving b-nitroalcohols of S con-
figuration with moderate yields and good enantiomeric excess
Department of Chemistry, Zhejiang University, Hangzhou 310027,
Zhejiang, PR China. E-mail: llc123@zju.edu.cn; Fax: +86 571
87952618; Tel: +86 571 87951588
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1gc15417f
Initially, a series of racemic b-nitroalcohols were synthesized
using D-aminoacylase as the catalyst and DMSO as the solvent.
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Table 1 Enzyme activity study of kinetic resolution of 2aa
d
Entry Enzyme source eesc (%)b eep (%)b Conv. (%)b Yield (%)b
1
2
3
4
5
6
7
8
No Cat.
PS-SD
PS-CI
PS-IM
CAL-B
—
—
98
>99
10
—
—
—
—
98
97
—
—
—
—
99
—
—
—
—
—
96
99
—
n.d.
2
51
50
9
n.d.
<1
2
28
1
<1
n.d.
n.d.
n.d.
40
49
n.d.
R
Lypozymeꢀ
6
MJL
n.d.
n.d.
n.d.
50
49
n.d.
HPL
9
DA
10
11
12
PS-C Ie
PS-IMe
BSA
a Reaction conditions: racemic compound 2a 0.1 mmol, enzyme 50 mg,
VA 0.5 mmol, t-amyl alcohol 0.5 ml, 25 ◦C, 24 h. b Determined by HPLC
analysis using AD-H column. c The ee value of the unreacted substrate
(b-nitroalcohol). d The ee value of the products. e Reaction conditions:
racemic compound 2a 0.1 mmol, enzyme 50 mg, VA 0.5 mmol, toluene
0.5 ml, 25 ◦C, 12 h.
way) being used as standards. Of all the enzymes tested,
immobilized lipases from Burkholderia cepacia (PS-C I and PS-
IM) showed high activity and enantioselectivity (entry 3 and
4, Table 1), with almost 100% ee value and 50% conversion
of the substrates. In contrast, other enzymes commonly used
in the resolution of other secondary alcohol substrates didn’t
show any remarkable catalytic activity. No obvious reactions
occurred when BSA was used as the catalyst, so the enzymatic
tertiary structure might play a critical role to the reaction. Free
lipase PS (PS-SD) (entry 2, Table 1) did not show a good reaction
activity either, which might be caused by the alteration of the
conformation of the activity centre under the reaction condition,
while the immobilized enzyme’s conformation was stabled by the
vector and therefore maintained a good activity. However, no
obvious peek of product is observed form the HPLC spectrum
of the PS-C I catalyzed reaction, but the one of 4¢-nitro-b-
nitrostyrene (entry 3, Table 1). The HPLC spectrum of the PS-
IM catalyzed reaction shows peeks of both acylated product and
4¢-nitro-b-nitrostyrene. (entry 4, Table 1) It indicates that the
acylated product, which is fragile and sensible just as mentioned
above, underwent an elimination reaction under that reaction
condition and this elimination reaction could be controlled by
tuning the reaction condition. Luckily, we found that when
toluene was used as the solvent, the elimination reaction was
inhibited dramatically (Entry 10, 11, Table 1). Within 12 h, the
reaction using PS-IM as the catalyst was nearly completed with
49% conversion, 49% yield, 97% ees and 99% eep, while the peak
of 4¢-nitro-b-nitrostyrene was hardly observed from the HPLC
spectrum. According to Ramstro¨m’s work, the configuration of
the product is assigned to R while that of the unreacted substrate
is assigned to S.
Scheme 1 D-Aminoacylase-catalyzed nitroaldol reaction combined
immobilized lipase PS-catalyzed kinetic resolution.
The reactions were carried under 50 ◦C and monitored by
TLC. As we expected, b-nitroalcohols with the aromatic ring
substituted by electron-withdrawing groups could be produced
with good yields. Compounds 2a to 2c were isolated with more
than 80% yields in half an hour, while compounds 2d to 2f could
still be obtained with more than 50% yields in 5 h. However, b-
nitroalcohols bearing electron-donating groups showed lowered
reaction activity, giving yields between 12% and 33%. The para-
and meta-substituted aldehydes reacted a little better than the
ortho-substituted aldehydes due to the steric hindrance between
the substituted group and the nitromethane. The products were
detected to undergo an elimination reaction and be transformed
into more thermo-stable b-nitrostyrenes at prolonged reac-
tion time, while b-nitroalcohols with electron-donating groups
eliminated much more seriously than the ones with electron-
withdrawing groups. Later it was also observed that acylated
b-nitroalcohols, the products of the KR reaction, would be
eliminated easier than the corresponding b-nitroalcohols under
the same reaction condition. Due to this unwanted side reaction,
the reaction condition of KR has to be as mild as possible. High
reaction temperature, basic additives, and high polar solvents
have to be avoided. So Ramstro¨m’s DKR procedure may not be
applied on this kind of b-nitroalcohols, the acylated products
will be eliminated completely in the presence of Et3N.
The KR work was started by the study of the catalytic activity
of a series commercial enzyme source to the asymmetric acyl
reaction. We chose 2-nitro-1-(4-nitrophenyl) ethanol (2a) to
be the substrate of the model reaction, with commonly used
vinyl acetate (VA) as the acyl donor, and t-amyl alcohol as the
reaction media. The reaction was carried out at 25 ◦C for 24 h.
The result of the reaction was analyzed by chiral HPLC, with
the racemic b-nitroalcohols and their corresponding racemic
acylated products (which were synthesized by non-enzymatic
Encouraged by the excellent experiment results, we applied
this KR reaction condition to the synthesized b-nitroalcohols.
To our delight, the result is excellent for most of the b-
nitroalcohols. All the reactions of the b-nitroalcohols with para-
or meta-substituents were almost completed, with nearly 50%
conversions and yields, as well as mostly more than 200 enantios-
electivity (E). b-Nitroalcohols with electron withdrawing groups
shows excellent ees, which could reach up to 97% (entry 1 and
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Table 2 Kinetic resolution of b-nitroalcoholsa
Acknowledgements
Entry
R
ees (%)b eep (%)b Conv. (%)b Yield (%)b
E
This work was supported by the National Natural Science
Foundation of China (No.20872130).
1
2
3
4
5
6
7
8
9
p-NO2
m-NO2
o-NO2
p-Cl
m-Cl
o-Cl
H
97
95
—
97
91
—
95
84
>99
99
—
98
98
—
96
99
98
50
49
2
46
47
<1
48
53
52
48
49
<1
46
47
<1
47
48
49
>200
>200
—
>200
>200
—
155
>200
>200
Notes and references
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p-CH3
p-OCH3 84
a Reaction conditions: racemic b-nitroalcohol 0.1 mmol, PS-IM 50 mg,
0.5 mmol VA, toluene 0.5 ml, 25 ◦C, 12 h. b Determined by HPLC
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This journal is
The Royal Society of Chemistry 2011
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