Production of (S)-β-Nitro Alcohols by Enantioselective C?C Bond Cleavage with an R-Selective Hydroxynitrile Lyase

Article abstract of DOI:10.1002/cbic.201800416

Hydroxynitrile lyase (HNL)-catalysed stereoselective synthesis of β-nitro alcohols from aldehydes and nitroalkanes is considered an efficient biocatalytic approach. However, only one S-selective HNL—Hevea brasiliensis (HbHNL)—exists that is appropriate for the synthesis of (S)-β-nitro alcohols from the corresponding aldehydes. Further, synthesis catalysed by HbHNL is limited by low specific activity and moderate yields. We have prepared a number of (S)-β-nitro alcohols, by kinetic resolution with the aid of an R-selective HNL from Arabidopsis thaliana (AtHNL). Optimization of the reaction conditions for AtHNL-catalysed stereoselective C?C bond cleavage of racemic 2-nitro-1-phenylethanol (NPE) produced (S)-NPE (together with benzaldehyde and nitromethane, largely from the R enantiomer) in up to 99 % ee and with 47 % conversion. This is the fastest HNL-catalysed route known so far for the synthesis of a series of (S)-β-nitro alcohols. This approach widens the application of AtHNL for the synthesis not only of (R)- but also of (S)-β-nitro alcohols from the appropriate substrates. Without the need for the discovery of a new enzyme, but rather by use of a retro-Henry approach, it was used to generate a number of (S)-β-nitro alcohols by taking advantage of the substrate selectivity of AtHNL.

Full text of DOI:10.1002/cbic.201800416

Accepted Article
Title: Preparation of (S)-β-nitro alcohols by a (R)-selective HNL via
enantioselective C-C bond cleavage
Authors: Santosh Kumar Padhi and D H Sreenivasa Rao
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Preparation of (S)-β-nitro alcohols by a (R)-selective HNL via
enantioselective C-C bond cleavage
D.H. Sreenivasa Rao, [a] and Santosh Kumar Padhi*[a]
Abstract: Hydroxynitrile lyase (HNL) catalysed stereoselective
synthesis of β-nitro alcohols is considered as an efficient biocatalytic
approach. However, there exist only one (S)-selective HNL i.e. Hevea
brasiliensis (HbHNL) to synthesize (S)-β-nitro alcohols from
corresponding aldehydes. Further, HbHNL synthesis is limited by low
specific activity and moderate yield. We prepared a number of (S)-β-
nitro alcohols using a (R)-selective HNL from Arabidopsis thaliana
(AtHNL). Optimization of the reaction conditions of AtHNL catalyzed
stereoselective C-C bond cleavage of racemic 2-ntro-1-phenylethanol
(NPE) produced (S)-NPE up to 99% ee and 47% conversion. This is
the fastest HNL catalyzed route known so far to synthesize a series
of (S)-β-nitro alcohols. This approach widens the application of AtHNL
not only to synthesize (R)- but also (S)-β-nitro alcohols starting with
appropriate substrate. Without discovering a new enzyme, rather
using a retro-Henry approach, it synthesized a number of (S)-β-nitro
alcohols by taking the advantage of the substrate selectivity of AtHNL.
stereoselective C-C bond formation between a carbonyl centre
and nitroalkane (Scheme 1).
O
OH
O
OH
*
O
*
R'
Lipase
+
NO2
+
NO2
NO2
R1
R1
R1
R1
OH
HNL
R2CH2NO2
R2
*
*
H
R1
NO2
Scheme 1. (top) Lipase catalyzed kinetic resolution of a racemic β-nitroalcohol.
R1 is an alkyl group and R’ is part of the acylating agent. (bottom) HNL catalyzed
synthesis of chiral β-nitroalcohols. R1 is an alkyl group and R2 is usually H or
another alkyl group.
Introduction
Kinetic resolution of racemic β-nitro alcohols has been reported
with several lipases. Kitayama et al reported stereoselective
transesterification of four aliphatic β-nitroalcohols using lipase
from Pseudomonas sp. (Amano AK) in organic solvents but found
highest 78% ee with two (S) β-nitroalcohols.[15] Sorgedragor et al
screened different lipases in the kinetic resolution of 1-nitro-2-
pentanol and found highest E value with Novozym 435 when
succinic anhydride was used for acylation in presence of
TBME.[16] They found the (S)-enantiomers as succinic esters in 43
to 97% ee in 24 h. However this process requires one more step
to deprotect the succinyl group to prepare (S)-β-nitroalcohols.
CALB catalyzed kinetic resolution of 4,4-diethoxy-1-nitrobutan-2-
ol using vinyl butyrate as acyl donor in DIPE produced its (R)-
enantiomer in 92% ee and 50% conversion in 7 days.[17] Milner et
al carried out the kinetic resolution of 2-nitrocyclohexanol to
prepare its all four diastereomers.[18] This method used hydrolase-
catalyzed transesterification of 2-nitrocyclohexanol and
hydrolase-catalyzed hydrolysis of 2-nitrocyclohexyl acetate but in
24 h.[18] Xu et al performed a two step biocatalytic reaction that
involved D-aminoacylase-catalyzed synthesis of racemic β-
nitroalcohols and its kinetic resolution by immobilized lipase
PS.[19] They prepared seven (S)-β-nitroalcohols in 84 to 97% ee
and 46 to 53% conversion in 12 h. Li et al demonstrated
enantioselective transesterification of racemic β-nitroalcohols
using Burkholderia cepacia lipase.[20] They obtained the acyl
esters of seven (S)-β-nitroalcohols in 81 to 99% ee in 12 h.
Enantiopure β-nitro alcohols are versatile synthetic intermediates.
They can be transformed into various fine chemicals and chiral
intermediates e.g. dehydration to conjugated nitro alkenes,
reduction to vicinal amino alcohols, denitration to alcohols,
oxidation to nitro carbonyl compounds and Nef reaction to α-
hydroxy carbonyl compounds etc.[1–3] Nitroaldol reduced products
i.e. β-amino alcohols are structural intermediates of many
pharmaceuticals, such as (−)-arbutamine,[4] ritonavir,[5] (R)-
salmeterol,[6] pindolol,[7] propranolol[8] and epinephrine[9] and
fungicides.[10] Further they are important chiral building blocks in
the synthesis of several bioactive molecules e.g.
codonopsinine,[11] spisulosine,[12] taxotere[13] and nummularine
F.[14]
Synthesis of optically pure β-nitro alcohols are reported using
chemical and biocatalytic methods. Biocatalytic synthesis has
advantages over chemical catalyst i.e. high regio- and
stereoselectivity, mild reaction conditions, biodegradable catalyst
etc. Two major biocatalytic approaches used to synthesize them
are (i) kinetic resolution of racemic β-nitro alcohols, and (ii)
[a]
D.H. Sreenivasa Rao, Dr. Santosh Kumar Padhi.
Biocatalysis and Enzyme Engineering Laboratory, Department of
Biochemistry, School of Life Sciences, University of Hyderabad,
Hyderabad – 500 046, India
E-mail: skpsl@uohyd.ernet.in
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Kuhbeck et al prepared racemic β-nitroalcohols using Ca2+-
alginate hydrogel beads in DMSO and subjected to immobilized
lipase PS catalyzed kinetic resolution to prepare five (S)-β-
nitroalcohols in 31 to 41% yield and >95% ee.[21] This analysis
clearly indicates that lipase catalyzed kinetic resolution of racemic
-nitroalcohols suffers with either long reaction time i.e. 12 to 24
h (in some cases up to 7 days)[17–21] or an additional step to
deprotect the acyl derivative product. Ramstrӧm and coworkers
have demonstrated dynamic kinetic resolution approach in one-
pot synthesis of enantioenriched β-nitro alcohol derivatives using
triethyl amine and Pseudomonas cepacia (PS-C I).[22] However,
this method synthesized (R)-products in 2 to 4 days reaction time
and in the form of acyl derivatives.
synthesize enantioenriched β-nitro alcohols of opposite
stereopreference of the HNL. We used a simlar approach but a
different HNL to synthesize (S)-β-nitro alcohols.
Here we report for the first time AtHNL catalysed stereoselective
C-C bond cleavage of racemic β-nitro alcohols in the preparation
of a number of (S)-β-nitro alcohols i.e. opposite stereo preference
of AtHNL (Scheme 2). We exploited this cleavage reaction and
optimized its reaction conditions that produced (S)-β-nitro
alcohols with ee up to 99% and 47% conversion in 3 h.
OH
O
NO2
NO2
HNL catalyzed stereoselective synthesis of β-nitro alcohols is
considered to be an efficient biocatalytic approach because it is a
single step transformation that uses easily available aldehydes as
substrates and has the potential to achieve 100% yield. So far
there exist three (R)-selective HNLs i.e. Arabidopsis thaliana
R
R
R1
H
1 (R)
AtHNL
+
+
OH
OH
(AtHNL),[23]
Granulicella
tundricola
(GtHNL),[24]
and
Acidobacterium capsulatum (AcHNL)[24], while only one (S)-
selective HNL i.e. Hevea brasiliensis (HbHNL)[25,26] to synthesize
corresponding stereoselective β-nitro alcohols. HbHNL is the first
HNL to be reported for nitroaldolase activity.[25] However HbHNL
catalyzed synthesis of chiral Henry products is limited by low
specific activity (Table S2). Further the yield and ee for (S)-2-ntro-
1-phenylethanol was reported to be moderate i.e. 63% and 92%
ee respectively.[25] When ee of the products was increased by
lowering the pH of the reaction, corresponding yield was
decreased.[26] Therefore both lipase and HNL catalyzed methods
for the synthesis of (S)-β-nitro alcohols are limited with not only
long reaction time but also moderate yield/conversion to products.
Devamani et al reported nitroaldol activity in the ancestral enzyme
HNL1 which showed (S)-selectivity in the synthesis of 2-ntro-1-
phenylethanol.[27] Yu et al described acyl-peptide releasing
enzyme from Sulfolobus tokodaii (ST0779) catalyzed Henry
reaction. However the absolute configuration of the Henry
products have not been mentioned.[28]
NO2
R
1 (S)
1 (S)
Scheme 2. AtHNL catalyzed stereoselective cleavage of racemic β-nitro
alcohols.
This is the first biocatalytic approach to synthesize (S)-Henry
products using a (R)-selective HNL. This route has advantages
over the existing biocatalytic routes for synthesis of chiral β-nitro
alcohols. We believe this retro-Henry reaction approach is the
fastest HNL catalyzed approach to synthesize a series of (S)-β-
nitro alcohols known so far. It uses HNL for a C-C bond cleavage
reaction which is closer toward the natural reaction of a HNL than
a C-C bond formation. This approach widens the application of
AtHNL not only to synthesize (R)- but also to prepare (S)-β-nitro
alcohols starting with appropriate substrate. It gives opportunity to
synthesize a number of (S)-β-nitro alcohols by exploiting the
substrate selectivity of AtHNL without discovering a new enzyme
but using a different approach.
Compared to the synthesis, HNLs show a higher rate in the
cleavage of cyanohydrins.[29] A similar effect has been observed
with β-nitro alcohols.[30] Kinetic studies of HbHNL catalyzed Henry
reaction reported kcat of 0.013 s-1 for the synthesis (S)-NPE while
0.16 s-1 for the cleavage of racemic NPE into benzaldehyde and
nitromethane.[30] Therefore exploiting the cleavage reaction rather
than the synthesis, to prepare enantiopure NPE appears to be
beneficial. Yuryev et al showed HbHNL catalyzed retro Henry
reaction in the preparation of (R)-NPE from its racemic
counterpart.[31] Their study was limited to finding a process where
they could minimize benzaldehyde inhibition, thus they reported
the use of HCN that converted benzaldehyde to mandelonitrile.
Further, they have not investigated nor showed any scope of their
method to synthesize various (R)-β-nitro alcohols. However, it
suggests that the cleavage reaction could be advantageous to
Results and Discussion
Prior to exploring stereoselective C-C bond cleavage by AtHNL
as a method to prepare enantioenriched β-nitro alcohols, the
potential of this method was evaluated by determining the kinetic
parameters using racemic NPE as substrate. Due to unavailability
of (R)-NPE commercially, racemic substrate was used in the
kinetic experiments. NPE concentrations ranging from 0.2 to 4
mM was used in this cleavage reaction along with purified AtHNL.
Formation of benzaldehyde due to stereoselective cleavage of
NPE was monitored at 280 nm. Michaelis-Menten plot (Fig 1)
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showed Km: 0.012 mM, kcat: 30.8 min-1, kcat/Km: 2571 min-1 mM-1
and Vmax: 1.1 U/mg. AtHNL catalysed synthesis of NPE appeared
to be even slower for which kinetic parameters were not
measured. Purified AtHNL was also used to determine kinetic
parameters using mandelonitrile (MN) as substrate. This was
performed to measure AtHNL’s biocatalytic potential and also to
compare its catalytic efficiency between NPE cleavage vs MN.
The kinetic parameters of MN cleavage are found to be, Km: 2 mM,
kcat: 4424 min-1, kcat/Km: 2212 min-1 mM-1 and Vmax: 158 U/mg (Fig
S 1).
produces (S)-2-nitro-1-phenylethanol and hence AtHNL catalyzed
stereoselective cleavage appears to be catalytically efficient
method for the production of (S)-β-nitro alcohols than HbHNL
catalyzed synthesis. This higher efficiency has motivated us to
explore AtHNL catalyzed stereoselective cleavage as a method
to prepare (S)-β-nitro alcohols.
Different biocatalytic parameters for the stereoselective cleavage
were optimized using racemic NPE as substrate to obtain
highest % conversion and enantiomeric excess (ee) of its (S)-
enantiomer. AtHNL catalyzed enantioselective cleavage of
racemic NPE at different pH was determined to know the effect of
different pH of the buffer in this biotransformation. As below pH
5.0, wild type AtHNL is reported to be less stable[33], we have
selected the pH range from 5.0 to 6.0. This experiment showed
not much difference in the % ee (96.5 to 97.1) and production
(48.1 to 46.1) of (S)-NPE at five different pHs between 5.0 and
6.0 (Fig 2). The optimum pH was taken as 5.0 where 96.54% ee
and 48.1% conversion of (S)-NPE was observed. AtHNL
catalyzed synthesis of (R)-cyanohydrin has been reported at pH
5.0[34] and (R)-β-nitroalcohol at pH 7.[23]
Figure 1. Michaelis Menten plot for the cleavage of rac NPE by AtHNL
Comparison of Vmax and kcat for the above two reactions shows
that, MN cleavage is faster than NPE cleavage. Similar
observation has been made in case of HbHNL.[32] Nitroaldol
cleavage is usually a slower reaction compared to cyanohydrin
cleavage by HNLs. Among the four HNLs i.e. AtHNL, HbHNL,
AcHNL and GtHNL known to catalyse enantioselective synthesis
of nitroaldols[23–26], kinetic studies for synthesis or cleavage of
nitroaldol have not been reported for AcHNL and GtHNL. Between
AtHNL and HbHNL, the rate and catalytic efficiency of retro-Henry
reaction are high for AtHNL. kcat of the cleavage reaction by
AtHNL was found to be three fold higher than HbHNL i.e. 30 min1
vs 0.16 s1, although the stereo preference of both the enzymes
differ.[30] Catalytic efficiency of NPE cleavage by HbHNL was
reported 3.8 min-1mM-1 only vs 2571 min-1mM-1 by AtHNL (this
study). Not only wild type, even if the nitroaldol activity by
engineered HbHNL is compared, maximum specific activity of the
best mutant i.e. L121Y-F125T-L146M, for cleavage of racemic 2-
nitro-1-phenylethanol is 0.71 U/mg.[32] As the synthesis reaction is
slower than the cleavage one, therefore preparation of (S)-β-nitro
alcohols by engineered HbHNL would have lower specific activity
than 0.71 U/mg. In contrary AtHNL has showed 1.1 U/mg for the
cleavage of racemic 2-nitro-1-phenylethanol. This process
Figure 2. Effect of pH on enantioselective cleavage of rac NPE
AtHNL catalyzed stereoselective cleavage of racemic NPE was
performed at different substrate concentrations. The
concentration of NPE was varied from 0.81 to 6.50 mM in the
biotransformation (Fig 3). At higher substrate concentration,
decrease in enantiopurity of product was observed. This decrease
in ee could be due to product inhibition. Increase in the formation
of benzaldehyde at higher NPE concentration may be a possible
reason for this. A similar argument is done by Yuryev et al in case
of retro-Henry reaction catalyzed by HbHNL.[31] We have selected
3.25 mM as the optimum substrate concentration where 93.79%
ee and 38.94% conversion of (S)-NPE was found.
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water content of the biotransformation that influences
stereoselectivity of the enzyme varies in each set of experiment
due to the vary in volume of enzyme used based on their
concentration.
Figure 3. Effect of substrate concentration on enantioselective cleavage of rac
NPE.
Asano and coworkers reported the effect of different organic
solvents in the AtHNL catalyzed synthesis of (R)-β-nitro
alcohols.[23] They observed highest % ee and yield in case of n-
butylacetate among diethyl ether, diisopropyl ether (DIPE), tert-
butyl methyl ether (TBME), ethyl acetate, hexane, cyclohexane,
toluene and xylene. We have selected most of these organic
solvents except ethyl acetate which is similar to n-butylacetate, to
examine their effect on the AtHNL catalyzed enantioselective
cleavage of rac NPE. The best result was obtained in case of
toluene that showed the highest enantiomeric excess i.e. 96.3%
of (S)-NPE (Fig 4). Use of tetrahydrofuran (THF) did not contribute
toward the enantioselectivity of product while hexane has resulted
in poor conversion. DIPE (81%), toluene (96.3%) and n-butyl
acetate (76.2%) has showed moderate to high % ee of product.
Earlier reports shows the use of toluene in biocatalysis to prepare
enantiopure β-nitroalcohols.[19,23] Lipases have been reported to
show high activity when toluene is used as a solvent in the kinetic
resolution of β-nitro alcohols and (S)-1-chloro-3-(4-(2-
methoxyethyl)phenoxy) propan-2-ol.[19,35] Biocatalysis without any
organic solvent resulted in 93.6% ee and 35% conversion of (S)-
NPE. Thus, comparison of stereoselective cleavage of racemic
NPE to its (S)-enantiomer by AtHNL in different organic solvents,
with the reaction without solvent (only buffer) showed marginal
improvement in both % ee and conversion by organic solvent.
This result is similar to our recent findings that BmHNL catalysed
biotransformation in organic solvent has marginally increased the
ee of cyanohydrins [manuscript under revision], and thus we have
selected toluene as the best organic solvent and further
optimisations were planned. Previous two experiments were
conducted using DPIE as organic solvent, however both Fig 1 and
2 showed higher % ee of (S)-NPE compared to Fig 3. This vary in
enantiopurity of product could be because (a) enzymes of
different batches were used and hence the purity may vary, (b)
Figure 4. Effect of different organic solvents on the enantioselective cleavage
of rac NPE. DIPE: diisopropyl ether, HEX: hexane, TOL: toluene, TBME: tert-
butyl methyl ether, THF: tetrahydrofuran, nBA: n-butyl acetate.
The effect of amount of enzyme in the biocatalysis was studied by
using 0.01 to 0.3 µmol (12.5 to 200 units) of pure AtHNL. The ee
of (S)-NPE was attained maximum at 0.15 mM of AtHNL. Further
increase in enzyme amount had showed negligible improve in %
ee of product (Fig. 5). The optimum amount of enzyme was
selected as 0.15 µmol where 99.73% ee and 53.94% conversion
was observed.
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Figure 5. Effect of amount of enzyme on the enantioselective cleavage of rac
NPE.
reaction time as well as low specific activity of HbHNL catalyzed
synthesis of (S)-β-nitro alcohols[30,31], and (ii) low nitroaldol
cleavage specific activity of HbHNL variants (synthesis reaction
should have even lower activity), clearly indicates that the current
approach is the fastest HNL catalysed route known so far to
synthesize (S)-β-nitro alcohols.
Although use of biphasic systems are known to enhance
selectivity of enzymes but stability of enzyme in presence of
organic solvents usually decreases. Excess organic solvent may
denature the enzyme. Therefore it is necessary to find out the
content of organic solvent that would be good enough to provide
highest selectivity in a biocatalysis. We tried to find out the %
volume of toluene in the enantioselective cleavage of racemic
NPE. The % volume of toluene was varied from 0 to 65, of the
overall reaction. There was not much difference in % ee of (S)-
NPE i.e. 97.4 to 98% when 35 to 65% of toluene was used,
whereas high conversion 45.77% was observed with 65%. Hence
65% of toluene was chosen as optimum solvent content to pursue
further optimization experiments.
Figure 7. Time course of (S)-NPE preparation under optimized reaction
conditions.
Using the optimized biocatalytic conditions substrates other than
racemic NPE were used in the enantioselective cleavage by
AtHNL to prepare their corresponding (S)-β-nitro alcohols.
Several racemic β-nitro alcohols having substituents at different
positions of the aromatic ring were used to explore the catalytic
potential of the enzyme as well as to measure the efficacy of the
method. Along with NPE, 2-nitro-1-(3-methoxyphenyl)ethanol and
2-nitro-1-(3-methylphenyl)ethanol resulted in high ee (99%) with
35 to 47% conversion to their corresponding (S)-enantiomers.
The high % ee and E value with these two substrates indicates
AtHNL’s preference for meta substituted aromatic β-nitro alcohols.
AtHNL has been reported to show high % ee of product in the
synthesis of similar meta substituted aromatic β-nitro alcohols
from corresponding aldehydes.[23] However with 3,4,5-trimethoxy
benzaldehyde and 3-hydroxybenzaldehyde, neither chiral
cyanohydrin nor β-nitro alcohol synthesis using AtHNL has been
reported. For these two aldehydes, we report here for the first time
Figure 6. Effect of different ratio of toluene on the enantioselective cleavage of
rac NPE.
AtHNL catalyzed enantioselective cleavage of racemic NPE was
monitored at different time intervals. An increase in % ee of (S)-
NPE was observed with time (Fig 7). Although at 2 h, 97% ee of
(S)-NPE was observed, at 3 h it reached 99.1% ee and 46.85%
conversion. The increased % ee at longer reaction time is due to
the cleavage of (R)-NPE to benzaldehyde and hence it increases
the % ee of unreacted (S)-NPE. During this optimization study,
we have quantified all the components of biocatalysis e.g.
benzaldehyde, (R)-NPE, and (S)-NPE and found both % ee and
conversion at various time points. A clear trend of increase in
benzaldehyde up to ~ 50%, and decrease in concentration of (R)-
NPE from 50 to 0% was observed in 3 h. (S)-NPE cleavage
(represented as % conversion) was very slow and only 3% of its
loss was noticed in 3 h. The conversion of total NPE was found to
be 46.85% at 3 h. Comparison of this result with the (i) long
AtHNL
catalyzed
synthesis
of
(S)-2-nitro-1-(3,4,5-
trimethoxyphenyl)ethanol, entry 4 of table 1 in 88% ee, 43%
conversion and (S)-2-nitro-1-(3-hydroxyphenyl)ethanol, entry 5 of
table 1 in 81% ee, 19% conversion. Among the para substituted
aromatic β-nitro alcohols tested with AtHNL, 2-nitro-1-(4-
methylphenyl)ethanol was converted to its (S)-enantiomer in 85%
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ee and 45% conversion. The 2-nitro-1-(4-methoxyphenyl)ethanol,
however resulted in moderate (44%) ee of its (S)-enantiomer. In
case of 2-nitro-1-(4-nitrophenyl)ethanol, very poor ee was
observed i.e. 1%. The probable reason for this varied
enantioselectivity for the para substituted aromatic substrates is
not understood. AtHNL has been reported to synthesize (R)--
cyanohydrin of p-methoxy benzaldehyde in 68% ee but not with
p-methylbenzaldehyde.[34] Further, AtHNL has been reported to
synthesize (R)-β-nitro alcohols of 4-methoxybenzaldehyde in
79% ee and 2% yield and 4-methylbenzaldehyde in 94% ee and
11% yield.[23] However AtHNL has not tested in the synthesis of
either (R)--cyanohydrin or (R)-β-nitro alcohol using 4-
nitrobenzaldehyde as substrate. Effect of di- and tri-substituted
aromatic substrates has not been studied earlier with AtHNL
either for enantioselective synthesis of -cyanohydrins or for β-
nitro alcohols. Our studies showed mixed results for three such
compounds. While substrate 4 having substituents in three
positions of the aromatic ring has resulted in 88% ee of its (S)-
enantiomer, substrates 9 and 10 (table 1) having three and two
methoxy substituents respectively have showed poor
enantioselectivity. One of the probable difference between
substrates 9 and 10 with 4 is, they are ortho substituted. However
it is not clear whether ortho substitution is the reason for this poor
enantioselectivity because earlier AtHNL has been reported to
synthesize (R)--cyanohydrin or (R)- β-nitro alcohol in high % ee
with ortho substituted aromatic aldehydes.[23,34]
6
4-Me
7
6
6
6
6
85
44
1
45
41
47
49
47
25
6
7
4-OMe
8
4-NO2
1
9
2,3,4-triOMe
2,5-diOMe
3
5
10
22
7
푆
[a] % conversion or % c = [
]*100. R: % area
푅+푆+퐴푙푑∗푐표푛푣푒푟푠푖표푛 푓푎푐푡표푟
of (R)-β-nitroalcohol, S: % area of (S)- β-nitroalcohol and Ald: %
area of aldehyde in the biocatalytic product mixture; conversion
factor: area of 1 mM racemic β-nitroalcohol /area of 1mM aldehyde.
[b] % ee =[ (푆−푅)]*100, [c] E was calculated by Sih’s equation (see SI).
(푆+푅)
Preparative scale synthesis of (S)-NPE was carried out
using purified AtHNL as described in the experimental
section. At the end of 3 h, the biocatalytic product
mixture
obtained
was
purified
by
column
chromatography that produced (S)-NPE in 54% yield
(with hexane as impurity) and 93% ee.
Conclusions
OH
OH
O
Biocatalytic application of (R)-selective AtHNL was exploited in
the synthesis of (S)-β-nitroalcohols. Retro Henry reaction by
AtHNL has successfully demonstrated as a new route to prepare
(S)-β-nitroalcohols from their racemic counterparts. Measurement
of kinetic parameters of the cleavage of rac NPE by AtHNL has
revealed Km: 0.012 mM, kcat: 30 min-1, kcat/Km: 2571 min-1 mM-1
and Vmax: 1.1 U/mg. This kcat is found to be three fold higher and
kcat/Km is more than 75 fold higher than the corresponding reaction
by HbHNL. Optimization of various biocatalytic reaction
parameters of the stereoselective C-C bond cleavage by wild type
AtHNL using racemic NPE as the substrate was performed to find
out optimal reaction conditions. Under optimized biocatalytic
reaction conditions, this transformation resulted in 99% ee (S) and
47% conversion of the NPE, with E value of 84. Ten racemic β-
nitro alcohols having substituents at different positions of the
aromatic ring were used to prepare their corresponding (S)-β-nitro
alcohols with varied enantioselectivity. This proves not only the
broad substrate selectivity of AtHNL but also the efficacy of the
method. Preparative scale synthesis has produced (S)-NPE in
54% yield and 93% ee. We have demonstrated that this is the
fastest HNL catalyzed route known so far to synthesize a series
of (S)-β-nitro alcohols (Table S2). Along with this method, AtHNL
now can be used not only to synthesize (R)- but also (S)-β-nitro
alcohols starting with appropriate substrate. This method of retro-
AtHNL
NO2
NO2
+
H
(S)
racemic
R
R
R
Scheme 3. AtHNL catalyzed preparation of enantioenriched (S)-β-nitro alcohols
from corresponding racemic β-nitro alcohols.
Table 1. Preparation of (S)-β-nitro alcohols by AtHNL catalyzed
stereoselective cleavage of corresponding racemic substrates.
S. No
R
Time (h)
% ee[b]
% conversion[a]
E[c]
1
2
3
4
5
H
3
6
4
6
5
99
99
99
88
81
47
41
35
43
19
84
30
20
19
3
3-OMe
3-Me
3,4,5-triOMe
3-OH
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of the data to Michaelis-Menten equation was done using Solver function
of Microsoft excel. In case of steady state kinetics of NPE, 0.025, 0.05, 0.1,
0.3, 0.5, 1, 2, and 4 mM racemic NPE and 0.5 mg/mL enzyme
concentration was used. The other reaction conditions and best fit of the
data to Michaelis-Menten equation were same as mandelonitrile kinetic
experiments.
Henry reaction to prepare opposite enantioselective products can
be extrapolated to other HNLs.
Experimental Section
Materials: The recombinant AtHNL gene in pET28a plasmid was
synthesized and purchased from Abgenex Pvt. Ltd, India. Culture media
and ampicillin were purchased from HiMedia laboratory Pvt. Ltd, India.
Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was purchased from BR-
BIOCHEM Pvt. Ltd, India. Aldehydes, nitromethane and mandelonitrile
were purchased from Sigma Aldrich, AVRA, SRL and Alfa-Aesar. HPLC
grade solvents were obtained from RANKEM, Molychem, FINAR, and SRL,
India. Chemicals purchased were used without purification.
Optimization of biocatalysis parameters for enantioselective
cleavage of racemic NPE:
Effect of pH: The reaction mixture contained 94 units of purified AtHNL in
20 mM KPB pH 7, 2 µmol of NPE, 0.37 ml (equal volume with respect to
enzyme) of 50 mM citrate phosphate buffer of varied pH ranging from 5.0
to 6.0 and 0.4 ml (35% v/v) DIPE. In the control, enzyme was replaced by
equal volume of 20 mM KPB. Reaction mixture was shaken at 1,000 rpm
for 3 h at 30 oC in an incubator shaker. A 100 µl of aliquot from the organic
layer was added to 700 µl of hexane:2-propanol = 9:1, centrifuged at
15,000×g, 4 oC for 5 min. A 20 uL of the organic layer was analyzed in a
HPLC using Chiralpak IB chiral column. HPLC conditions: n-hexane: 2-
propanol = 90:10 (v/v); flow rate: 1 mL/min; absorbance: 210 nm. The
retention times of benzaldehyde, (R)-NPE, and (S)-NPE are 4.6, 9.7, and
10.8 min respectively.[23]
Expression and purification of AtHNL: Expression and purification of
AtHNL was carried out using the method reported by Asano and co-
workers.[23] Briefly, the recombinant AtHNL gene in pET28a plasmid was
transformed into E. coli BL21DE3 competent cells. Primary culture was
prepared by inoculating a loop of transformed E. coli cells in 20 mL of LB
broth containing 50 µg/mL of kanamycin grown for 12 hours in an incubator
shaker at 37°C. Secondary culture was prepared by adding 1% (20 mL) of
grown E. coli cells in 2 L of LB broth containing 50 µg/mL of kanamycin
and incubated at 37°C until the OD reached ~0.5. The cells were then
induced with 0.5 mM IPTG and incubated at 30°C for 6 h. Cells were
harvested at 10000 rpm for 15 min at 4°C and the cell pellet was
suspended in 20 mM potassium phosphate buffer (KPB), pH 7. All the
purification steps were done at 4°C. The cell suspension was disrupted by
sonication. Disrupted cells were centrifuged at 10000 rpm for 45 min. The
supernatant and pellet were analysed by mandelonitrile cleavage assay to
confirm HNL activity in soluble fraction. The supernatant was loaded into
a Ni-NTA agarose column pre-equilibrated with twice its volume of binding
buffer (20 mM imidazole, 300 mM sodium chloride, 20 mM KPB, pH 7).
The column was subsequently washed with three supernatant volumes of
wash buffer [50 mM imidazole, 300 mM sodium chloride, 20 mM KPB, pH
7), and finally eluted with one supernatant volumes of elution buffer (500
mM imidazole, 300 mM sodium chloride, 20 mM KPB, pH 7). The eluted
protein solution was dialyzed in KPB buffer for 3 h, 3 times and later used
for biocatalysis. (SDS-PAGE in Fig S2).
Effect of substrate concentration: Purified AtHNL, 94 units in 20 mM
KPB pH 7, 0.4 ml (32.5% v/v) of 50 mM citrate phosphate buffer pH 5.0,
0.43 ml (35% v/v) of DIPE and racemic NPE ranging from 0.7 to 6.1 mM
were taken in a 2 mL micro tube and shaken at 1000 rpm for 5 h at 30 oC
in an incubator shaker. Aliquot extraction and analysis was done according
to the method described in the above paragraph.
Effect of organic solvents: To a reaction mixture containing 94 units of
purified AtHNL in 20 mM KPB pH 7, and 4 µmol of NPE, 0.38 ml of 50 mM
citrate phosphate buffer pH 5, 0.4 ml of organic solvent was added.
Separate experiments carried out using different organic solvents e.g.
DIPE, hexane, toluene, TBME, THF, and n-butyl acetate. Each reaction
mixture was shaken at 1,000 rpm for 3 h at 30 oC in an incubator shaker.
Aliquot extraction and analysis was done according to the method
described above.
Effect of amount of enzyme: The reaction was performed in a 5 ml glass
vial. Reaction mixture contained corresponding amount of purified AtHNL
in 20 mM KPB pH 7 ranging from 0.01 to 0.3 µmol (12.5 to 200 units), 3
µmol of NPE, 32.5% (v/v) of 50 mM citrate phosphate buffer pH 5, and
35% (v/v) of toluene. Reaction mixture was shaken at 1000 rpm for 4 h at
30 oC in an incubator shaker. Aliquot extraction and analysis was done
according to the method described above.
HNL assay and steady state kinetics: AtHNL activity was measured by
monitoring the continuous formation of benzaldehyde from racemic
mandelonitrile at 280 nm in a spectrophotometer.[29] The reaction was
performed in a 96 well microtiter plate. Each well of the plate consisted of
160 µl of 50 mM citrate phosphate buffer, pH 5, 20 µl of purified AtHNL (1
mg/mL), and 20 µl of 67 mM mandelonitrile solution prepared in 5 mM
citrate phosphate buffer, pH 3.15. The activity was calculated using molar
extinction coefficient of benzaldehyde (1376 M-1 cm-1). One unit of HNL
activity is defined as the amount of enzyme which produced 1 µmol of
benzaldehyde from mandelonitrile per minute. All measurements were
performed in triplicates. Control experiment had all the components of
reaction except the enzyme was replaced by its corresponding buffer.
Steady state kinetic of AtHNL was performed with 1, 2, 4, 6, 8, 10, 12, 14,
16, 18, 20 and 24 mM racemic mandelonitrile using the above cleavage
assay, however the volume of assay buffer, enzyme (0.5 mg/mL) and
substrate were taken as 140, 20 and 40 µl respectively. The reaction was
Effect of organic solvent content: The reaction was performed in a 5 ml
glass vial. Reaction mixture contained 133 units of purified AtHNL in 20
mM KPB pH 7, 4 µmol of NPE, 0.62 ml of 50 mM citrate phosphate buffer
pH 5, and corresponding volume of toluene ranging from 0-65% (v/v).
Reaction mixture was shaken at 1000 rpm for 4 h at 30 oC in an incubator
shaker. Aliquot extraction and analysis was done according to the method
described above.
Study of time course of the reaction under optimized reaction
conditions: A reaction mixture containing 133 units of purified AtHNL in
20 mM KPB pH 7, 4 µmol of NPE, 0.59 ml (17.5% v/v) of 50 mM citrate
monitored for
1 min. Absorbance of control resulted due to the
spontaneous reaction was subtracted from the enzymatic reaction. Best fit
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for 5 min. A 20 uL of the organic layer was analyzed in a HPLC (Agilent)
using Chiralpak IB chiral column using HPLC conditions described
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46.3 ml (17.5% v/v) of 50 mM citrate phosphate buffer pH 5, and 172 ml
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Keywords: AtHNL • β-nitro alcohol • asymmetric synthesis •
stereoselective C-C bond cleavage • biocatalysis.
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Entry for the Table of Contents (Please choose one layout)
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Cleavage is faster than synthesis:
AtHNL catalysed stereoselective
cleavage of racemic β-nitro alcohols
produced corresponding (S)-
D.H. Sreenivasa Rao [a] and Santosh
Kumar Padhi*
Page No. – Page No.
enantiomers with up to 99% ee and
47% conversion in 3-7 h.
Preparation of (S)-β-nitro alcohols by
a (R)-selective HNL via
stereoselective C-C bond cleavage
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Author(s), Corresponding Author(s)*
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Title
Text for Table of Contents
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