Lipase catalysed resolution of nitro aldol adducts

Article abstract of DOI:10.1016/j.tetasy.2004.02.025

The kinetic resolution of a range of 1-nitro-2-alkanols by lipase-catalysed esterification using various lipases and succinic anhydride as an acyl donor has been studied. E values of up to 100 were obtained with Novozym 435 in the resolution of 1-nitro-2-pentanol with succinic anhydride in TBME. Acylation with succinic anhydride proved much more enantioselective than with vinyl acetate.

Full text of DOI:10.1016/j.tetasy.2004.02.025

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 1295–1299
Lipase catalysed resolution of nitro aldol adducts
Menno J. Sorgedrager, Rita Malpique, Fred van Rantwijk and Roger A. Sheldon*
Laboratory of Biocatalysis and Organic Chemistry, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands
Received 21 January 2004; accepted 26 February 2004
Abstract—The kinetic resolution of a range of 1-nitro-2-alkanols by lipase-catalysed esterification using various lipases and succinic
anhydride as an acyl donor has been studied. E values of up to 100 were obtained with Novozym 435 in the resolution of 1-nitro-2-
pentanol with succinic anhydride in TBME. Acylation with succinic anhydride proved much more enantioselective than with vinyl
acetate.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The products of Henry reactions are secondary alcohols;
hence, they are suitable substrates for resolution by
lipase catalysed enantioselective acylation (see Fig. 1).
The lipase mediated kinetic resolution of secondary
alcohols has been widely studied over the past 20 years4
and has become a common synthetic and industrial
methodology for producing chiral compounds as pure
enantiomers.5;6
The aldol reaction is one of the most important methods
for C–C bond formation.1 In the related nitro aldol
condensation, the Henry reaction, the coupling of a nitro
alkane to an aldehyde or ketone results in the corre-
sponding b-nitro alcohol. Such chiral nitro alcohols are
of interest as building blocks in organic synthesis as they
can be converted into chiral b-hydroxy amines via
reduction of the nitro group. Alternatively, further
carbon–carbon bond formation on the a-carbon of the
nitro group can lead to a wide variety of other useful
intermediates.2;3 The control of the stereochemistry is of
importance for many synthetic purposes, especially for
pharmaceutical and agricultural applications.
The choice of the acyl donor in such kinetic resolutions
requires careful consideration because the transesterifi-
cation equilibrium should be entirely on the side of the
product.4 The often-used vinyl esters, which react irre-
versibly because the liberated vinyl alcohol isomerises
into acetaldehyde, satisfy this requirement.4
OH
NO2
H3C
Base
+
NO2
O
1c
2c
O
OH
OH
O
R
NO2
Lipase
+
NO2
NO2
Acyl
donor
2c
(R)-2c
(S)-3c
R = CH3
C2H4COOH
Figure 1. Nitro aldol synthesis of 1-nitro-2-pentanol followed by lipase catalysed resolution.
* Corresponding author. Tel.: +31-15-278-2683; fax: +31-15-278-1415; e-mail: r.a.sheldon@tnw.tudelft.nl
0957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.02.025

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M. J. Sorgedrager et al. / Tetrahedron: Asymmetry 15 (2004) 1295–1299
Table 1. The performance of different lipases in the resolution of
1-nitro-2-pentanola
trolled but limited by the rate of diffusion in and out of
the particles. This could lead to lower conversion and
enantioselectivity.
Enzyme
Conversion
(%, 24 h)
Ees (%)
Eep (%)
E
In the presence of CaLB, the (S)-enantiomer was pref-
erentially converted, as predicted by the Kazlauskas
rule.15 Two preparations of Burkholderia cepacia lipase
(Amano 1 and ChiroCLECTM PC), in contrast, showed
a slight preference for the (R)-enantiomer of the reac-
tant. The activities of the other lipases tested were much
lower and with the exception of C. antarctica lipase A
(CaLA), the enantioselectivities were also poor.
Novozym 435
CaLB CLEC
CaLB CLEA
CaLA SP 526
BcL (Amano 1)
BcL CLEC
CrL
54
11
7
92
8
93
67
5
82 (S)
5 (S)
1 (S)
12 (R)
4 (R)
4 (R)
1 (R)
1 (S)
––
0
10
45
54
3
11
34
59
2
82
46
37
14
3
CrL CLEC
ClL
RmL SP 524
8
1
Nrb
Nr
––
––
––
––
––
In view of the above results, we selected Novozym 435
for our further investigations. We next compared the
performance of the acyl donors vinyl acetate and suc-
cinic anhydride in the resolution of 2c. These reactions
were performed in a range of solvents because it is
known that the outcome often critically depends on the
nature of the solvent. We found (see Table 2) that the
acylation with succinic anhydride took place with high
enantioselectivity in tert-butyl methyl ether (TBME) or
diisopropyl ether (DIPE) as the reaction medium.
However when the reaction was performed in 1,2-di-
methoxyethane (DME), it proceeded sluggishly and with
low enantioselectivity.
CaLB: C. antarctica lipase B; CaLA: C. antarctica lipase A; RmL:
R. miehei lipase; CrL: C. rugosa lipase; ClL: C. lipolytica lipase, BcL:
B. cepacia lipase.
a Reaction conditions: 1 mL diisopropyl ether, 100 mM 1-nitro-2-
pentanol, 100 mM succinic anhydride, 25 mg enzyme or 5 mg CLEC
or CLEA.
b No reaction.
The resolution of some b-nitro alcohols via transesteri-
fication with vinyl acetate as the acyl donor has been
reported previously,3;7;8 but the separation of the enan-
tiomerically enriched ester and alcohol is often laborious.
Acylation with a cyclic anhydride, which also reacts
irreversibly, results in a half ester that can be readily
extracted from the reaction mixture. This potential ben-
efit for reaction work-up procedures has been reported
for the resolution of several secondary alcohols.9–14
Nitromethane and acetonitrile likewise proved to be
unsuitable for this reaction. No adequate resolution of
2c upon acylation with vinyl acetate could be obtained
in any solvent.
Herein we report the applicability of succinic anhydride
as an acyl donor in the lipase-mediated resolution of a
number of alkyl- and phenylalkyl substituted nitro
alcohols. Furthermore the effects of the lipase and
reaction medium will also be discussed.
2.2. Substrate specificity and enantiodiscrimination
A range of nitro alcohols, resulting from aldol conden-
sation of aliphatic 1a–c and aromatic aldehydes 1a–f
with nitromethane, was studied in the lipase-mediated
resolution with succinic anhydride. The selectivity of
Novozym 435 for these substrates followed the
Kazlauskas rule15 (see Fig. 2). This model intrinsically
implies that the enantiodiscrimination of second-
ary alcohols by a lipase is dominated by steric interac-
tions. The presence of a nitro group, which also
interacts electrostatically, could affect the enantiodis-
crimination.
2. Results and discussion
2.1. Resolution of nitro alcohols: lipases, acyl donors,
solvents
The acylation of 1-nitro-2-pentanol 2c (Fig. 1), which we
selected as a suitable test reactant, was performed in the
presence of a range of microbial lipases (see Table 1).
The reaction proved fast and enantioselective when
performed in the presence of Novozym 435, an immo-
bilised preparation of Candida antarctica lipase B
(CaLB). Two cross-linked preparations of CaLB, the
cross-linked enzyme aggregate (CLEA) and the cross-
linked enzyme crystal (ChiroCLECTM CaB) were much
less active, although comparable amounts of units were
used, with the enantioselectivity being low. In these
hydrophilic particles, with a high density of active pro-
tein, the reaction is probably no longer kinetically con-
Such effects would become visible when both enantio-
topic groups are similar in size. We found, however, that
the enantiomeric discrimination of 2b (R ¼ Et, see Fig.
3) was much lower that that of 2a or c (R ¼ Me or Pr,
respectively, see Table 3).
From these results we can see that the enantiodiscrimi-
nation of the compound by CaLB is dominated by steric
interactions and that electrostatic effects are at best
minor. The longer alkyl 2c and the phenyl alkyl substi-
tuted 2d and f substrates all showed (S)-selectivity in
accordance with the predictive model shown in Figure 2.
Surprisingly, there was no reaction observed with 3-
phenyl-1-nitro-2-propanol 2e when succinic anhydride
was used as the acyl donor. We have no plausible
explanation for this observation.
1 unit will liberate 1 lmol of acetic acid per minute from triacetin.

M. J. Sorgedrager et al. / Tetrahedron: Asymmetry 15 (2004) 1295–1299
1297
Table 2. Resolution of 1-nitro-2-pentanol in the presence of CaLB; effect of the acyl donor and the solventa
Solvent
Succinic anhydride
Vinyl acetate
Ees (%)
Conversion (%)
Ees (%)
Eep (%)
E
Conversion (%)
Eep (%)
E
MeNO2
ACN
<1
8
0
2
1
100
76
––
3
Ndb
37
1
Nd
38
45
Nd
52
3
2
7
Nd
5
DME
TBME
DIPE
4
3
42
54
70
92
95
93
100
82
46
37
41
15
35
25
3
2
a Reaction conditions: 1 mL diisopropyl ether, 100 mM 1-nitro-2-pentanol, 100 mM acyl donor, 25 mg Novozym 435.
b Not determined.
H
OH
the corresponding nitroalkene was observed. Elimina-
tion of acetic acid occurred more readily than that of
succinic acid. No detectable alkene formation from the
aliphatic nitroalcohols and their esters was observed
under the reaction conditions.
M
L
Figure 2. Steric model for the preferentially converted enantiomer of
secondary alcohols by a lipase.
Overall, much higher enantioselectivities were found in
the resolution of 1-nitro-2-alkanols when succinic
anhydride was used as the donor when compared with
vinyl acetate.
During the resolution of the aromatic nitro alcohols,
some spontaneous elimination of carboxylic acid into
O
OH
OH
Lipase
OH
O
NO2
+
O
+H3C
NO2
NO2
R
Succinic
anhydride
R
R
NO2
O
R
O
O
O
OH
OH
O
NO2
O
NO2
O
(R)-3a
(S)-3d
O
O
O
OH
OH
OH
OH
O
NO2
O
O
NO2
O
O
(R)-3b
(S)-3e
O
O
O
O
NO2
NO2
(S)-3c
(S)-3f
Figure 3. Preferentially formed enantiomers by Novozym 435 of 1-nitro-2-alkanols.
Table 3. Resolution of 1-nitro-2-alkanols with succinic anhydride and vinyl acetate in diisopropyl ethera
R
Succinic anhydride
Vinyl acetate
Ees (%)
Conversion (%, 24 h)
Ees (%)
Eep (%)
E
Conversion (%, 24 h)
Eep (%)
E
CH3
C2H5
39b
47b
54
57
44
92
3
67
43
93
75
––
97
28 (R)
4 (R)
82 (S)
7 (S)
––
30
72
2
1
5
Ndc
25
1
(R)
1d (R)
C3H7
C6H5
37
15
13
34
21
2 (S)
4e
13e
53e
56e
100
Ndc
100
20d (S)
2d (S)
2d (S)
(C6H5)CH2
(C6H5)C2H4
Nr
42
––
71
96 (S)
a Reaction conditions: 1 mL diisopropyl ether, 100 mM substrate, 100 mM succinic anhydride, 25 mg Novozym 435.
b Reaction finish after 5 h.
c Not determined.
d Calculated only with ees.
e Substrate and product degradation to the corresponding alkene.

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M. J. Sorgedrager et al. / Tetrahedron: Asymmetry 15 (2004) 1295–1299
3. Conclusions
4.3.3. 1-Nitro-2-pentanol 2c. The reaction was carried
out on 1 M scale. Isolated yield 69%. 1H NMR
(300 MHz, CDCl3) d (ppm): 1.0 (3H, t), 1.6 (2H, m), 3.0
(1H, s), 4.3 (1H, m), 4.5 (2H, m).
Succinic anhydride, besides having potential benefits for
reaction work-up, was shown to be an efficient acyl
donor for the resolutions of b-nitro alcohols. Much
higher E values can be achieved compared to other
common acyl donors such as vinyl acetate. The best
results, with regard to both rate and enantioselectivity,
were observed in tert-butyl methyl ether and diisopropyl
ether.
4.4. General synthesis of aromatic nitro alcohols 2d–f
Aldehydes 1d–f (100 mmol) and KOH (1 mL, 1 M solu-
tion) were added to nitromethane (20 mL) at 0–5 °C.
After 1 h, the mixture was brought to room temperature
and then left to stir for another 2–4 h. The reactions
were followed by TLC using ether–petroleum ether 1:1
as the eluent. The pH was adjusted to 7 and the excess of
nitromethane evaporated. Ether was added and washed
successively with acidic water, saturated NaHCO3
solution and water. The organic phase was dried over
MgSO4 and the solvent evaporated to yield the crude
product.
4. Experimental
4.1. Instruments
HPLC analyses were performed on a Chiralcel OD
column with a flow rate of 0.6 mL/min and an eluent
consisting of hexane–isopropyl alcohol–trifluoroacetic
acid (95:5:0.1) for the aliphatic nitro alkanols and hex-
ane–isopropyl alcohol–trifluoroacetic acid (80:20:0.1)
for the aromatic nitro alkanols. Detection was per-
formed with a Waters 486 UV detector at 215 nm.
4.4.1. 2-Phenyl-1-nitro-2-ethanol 2d. The crude product
was purified by distillation giving a yellow liquid. Iso-
1
lated yield 57%. H NMR (300 MHz, CDCl3) d (ppm):
3.2 (1H, s), 4.5 (2H, m), 5.4 (1H, q), 7.3 (5H, m). 13C
NMR (75 MHz, CDCl3) d (ppm): 70.9, 81.1, 125.9,
128.9, 129, 138.2.
4.2. Materials
4.4.2. 3-Phenyl-1-nitro-2-propanol 2e. The crude product
was purified by distillation giving a yellow liquid. Iso-
All chemicals were of analytical purity and obtained
from Sigma–Aldrich. The lipase from Candida rugosa
was obtained from Sigma–Aldrich. Candida lypolitica
lipase was bought from Fluka. Novozym 435 (C. ant-
arctica lipase B on Lewatit E), SP524 (Rhizomucor
miehei lipase), SP526 (C. antarctica lipase A) were kindly
donated by Novozymes. Cross-linked enzyme crystals
(CLECs) were donated by Altus Biologics; CaLB CLEA
was donated by CLEA Technologies. The 1-nitro-2-
alkanols were synthesised as described below.
1
lated yield 23%. H NMR (300 MHz, CDCl3) d (ppm):
2.8 (2H, m), 4.4 (2H, m), 4.5 (1H, q), 7.3 (5H, m). 13C
NMR (75 MHz, CDCl3) d (ppm): 40.4, 69.5, 79.7, 127.3,
128.9, 129.4, 135.9.
4.4.3. 4-Phenyl-1-nitro-2-butanol 2f. The product was
recrystallised from diisopropyl ether resulting in a white
solid. Isolated yield 56%. 1H NMR (300 MHz, CDCl3) d
(ppm): 1.8 (2H, m), 2.6 (1H, d), 2.85 (2H, m), 4.35 (1H,
m), 4.45 (2H, m), 7.3 (5H, m). 13C NMR (75 MHz,
CDCl3) d (ppm): 31.3, 35.1, 67.7, 80.5, 126.3, 128.4,
128.6, 140.6.
4.3. General synthesis of aliphatic nitro alcohols 2a–c
Aldehydes 1a–c (100 mmol) and KOH (1 mL, 1 M solu-
tion) were added to nitromethane (10 mL) at 0–5 °C.
After 1 h, the mixture was brought to room temperature
and left for another 2–4 h. The reactions were followed by
TLC using ether–petroleum ether 3:2 as the eluent.
Dichloromethane was then added and this mixture washed
successively with 5% aqueous HCl and saturated aqueous
NaHCO3. The organic phase was dried over MgSO4 and
the solvent then evaporated. The crude product was
purified by distillation, giving a colourless liquid.
4.5. Lipase catalysed resolutions
The lipase catalysed acylation reactions were performed
in solvent (1 mL) at room temperature, with 100 mM
nitro alcohol, an equivalent amount of acyl donor and
25 mg of the various enzyme preparations. When
CLECs or CLEAs were used, 5 mg of enzyme was added
instead. Except for the enzyme screening, all reactions
were carried out with Novozym 435. Trimethoxyben-
zene (2.5 g/L) was used as the internal standard. The
reactions were monitored by chiral HPLC.
4.3.1. 1-Nitro-2-propanol 2a. Isolated yield 46%. 1H
NMR (300 MHz, CDCl3) d (ppm): 1.3 (3H, d), 3.2 (1H,
s), 4.4 (2H, m), 4.5 (1H, m). 13C NMR (75 MHz, CDCl3)
d (ppm): 19.8, 65.0, 81.6.
Acknowledgements
4.3.2. 1-Nitro-2-butanol 2b. Isolated yield 48%. 13C
NMR (75 MHz, CDCl3) d (ppm): 5.6, 26.9, 70.1, 80.5.
The authors wish to thank Novozymes (Bagsvaerd,
Denmark), Altus Biologics (Cambridge, MA) and

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