Dramatic enhancement of enantioselectivity of biotransformations of β-hydroxy nitriles using a simple O-benzyl protection/docking group

Article abstract of DOI:10.1021/ol0610688

Catalyzed by the Rhodococcus erythropolis AJ270 whole cell catalyst, the O-benzylated β-hydroxy alkanenitriles underwent remarkably high enantioselective biotransformations, whereas the biotransformations of free β-hydroxy alkanenitriles gave very low ena

Full text of DOI:10.1021/ol0610688

ORGANIC
LETTERS
2006
Vol. 8, No. 15
3231-3234
Dramatic Enhancement of
Enantioselectivity of Biotransformations
of
â-Hydroxy Nitriles Using a Simple
O-Benzyl Protection/Docking Group
Da-You Ma, Qi-Yu Zheng, De-Xian Wang, and Mei-Xiang Wang*
Beijing National Laboratory for Molecular Sciences, Institute of Chemistry,
Chinese Academy of Sciences, Beijing 100080, China
mxwang@iccas.ac.cn
Received May 2, 2006
ABSTRACT
Catalyzed by the Rhodococcus erythropolis AJ270 whole cell catalyst, the O-benzylated
â
-hydroxy alkanenitriles underwent remarkably high
enantioselective biotransformations, whereas the biotransformations of free â-hydroxy alkanenitriles gave very low enantioselectivity. The
easy manipulations of O-protection and O-deprotection, excellent chemical and enantiomeric yields of biotransformations, along with the
scalability render this enzymatic transformation attractive and practical for the synthesis of highly enantiopure
their amide derivatives.
â-hydroxy alkanoic acids and
Molecular recognition of an enzyme toward a substrate is a
crucial step in enantiospecific or highly enantioselective
biocatalytic transformations.1 When a non-natural substrate
is used, a general practice in synthetic biotransformations,
low enantioselectivity, and slow reaction is always encoun-
tered because of nonspecific interaction between the enzyme
and the substrate. To circumvent these problems, structural
modifications of enzymes by means of site-directed mu-
tagenesis2 and directed evolution,3 etc. have enjoyed the
success in upgrading the performance of enzymes. As an
alternative to biotechnological approaches, substrate engi-
neering has been shown to be useful in improving the
efficiency and selectivity of biocatalysis. Substrate engineer-
ing, in contrast to protein engineering, modifies the structure
of small organic molecular substrates to best fit into the active
site of the enzyme. The nonbiological approaches are
therefore relatively simple, easy to handle, and cost-effective.
One excellent example of substrate engineering has been
demonstrated by Griengl, who introduced the docking or
protecting group concept in biohydroxylation reaction of non-
natural substrates.4-6 Despite the new concept, successful
applications are rare in terms of catalytic efficiency and,
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27.
10.1021/ol0610688 CCC: $33.50
© 2006 American Chemical Society
Published on Web 06/22/2006

particularly, enantioselectivity. In most cases of biohydroxy-
lation, even with the use of a chiral protecting group, the
enantiomeric excess values of products hardly exceed
90%.4-7
thesis, and synthetic methods involving catalytic asymmetric
hydrogenation,14a,15 reduction,16 Mukaiyama-aldol reaction,17
and Reformatsky reaction18 have been developed. Biocata-
lytic reduction of â-ketoesters,19 kinetic resolution of second-
ary alcohols,20 and deracemization21 have proved useful, as
well. Considering the easy availability of â-hydroxy al-
kanenitriles, which can be readily prepared from condensa-
tion of acetonitrile and various aldehydes,22 we envisioned
that highly efficient and enantioselective biotransformations
of nitriles would provide a convenient and unique approach
to optically active â-hydroxy carboxylic acids and their amide
derivatives. This has led us to undertake the current study,
and herein we report a successful docking/protecting group
strategy to dramatically enhance the enantioselectivity of
biotransformations of nitriles. It has been reported23 that
enzyme-catalyzed kinetic resolution of â-substituted nitriles
gave generally unsatisfactory enantioselectivity. Desymme-
trization of prochiral 3-hydroxyglutaronitrile yielded poor
enantiocontrol, and the enantioselectivity was improved by
using either a 3-benzylated substrate24 or an engineered
nitrilase.25
Biotransformations of nitriles, either through a direct con-
version from a nitrile to a carboxylic acid catalyzed by a nitri-
lase or through the nitrile hydratase-catalyzed hydration of
a nitrile followed by the amide hydrolysis catalyzed by the
amidase,8 are an effective and environmentally benign method
for the production of carboxylic acids and their amide deriva-
tives.9 Recent studies have demonstrated that biotransforma-
tions of nitriles complement the existing asymmetric chemi-
cal and enzymatic methods for the synthesis of chiral carbox-
ylic acids and their derivatives bearing an R-stereocenter.10-13
Optically active R-unsubstituted â-hydroxy carboxylic
acids and their derivatives are key intermediates in the
synthesis of natural products and biologically important
compounds.14 Much effort has been devoted to their syn-
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3232
Org. Lett., Vol. 8, No. 15, 2006

Incubated with Rhodococcus erythropolis AJ27012 micro-
bial whole cells under mild conditions, racemic â-hydroxy
alkanenitriles 1a and 1c were transformed into the corre-
sponding amides and acids within a very short time.
Unfortunately, the enantiocontrol of the reaction was very
disappointing (entries 1 and 2, Table 1). This result was not
intrinsically UV-active protection group is beneficial because
it will facilitate the detection and visualization of both
substrates and products and, therefore, simplify the monitor-
ing of the biotransformation. Taking the aforementioned
factors into consideration, the benzyl group was finally
selected as the protection group. Gratifyingly, the introduc-
tion of a benzyl protection group led to a dramatic enhance-
ment of enantioselectivity of the biotransformations, although
the protection group was remote from the functional group.
As illustrated in Table 2, all of the â-benzyloxy alkaneni-
Table 1. Biotransformations of â-Hydroxy Alkanenitriles 1a
Table 2. Biotransformations of â-Benzyloxy Alkanenitriles 4a
concn time 5 (yield %)b 6 (yield %)b
entry
4
R
(mmol) (h)
(ee %)c
(ee %)c
Ed
concn
(mmol) (min)
time 2 (yield %)b 3 (yield %)b
1
a
Me
Et
be Et
2
1.75 R-5a (47)
(92.0)
S-6a (49)
(86.2)
S-6b (47)
(83.4)
S-6b (49)
(92.4)
R-6c (47)
(91.0)
S-6d (48)
(86.4)
R-6e (48)
(93.8)
R-6f (47)
(94.4)
R-6g (51)
(85.4)
43
entry
1
1
a
R
(ee %)c
(ee %)
Me
2
2
2
20
20
50
R-2a (36)
(12.0)
S-3a (12)
(17.8)d
2
3
5
4
6
7
8
b
2
5.5 R-5b (46)
(95.4)
39
85
2
c
g
iPr
S-2c (41)
(2.0)
R-3c (31)
(3.0)e
12
2
10
R-5b (41)
(94.4)
3f
c-Pen
S-2g (13)
(11.5)
R-3g (32)
(6.4)d
c
d
e
f
iPr
25.5 S-5c (47)
(95.0)
78
a Biotransformation was carried out in a suspension of Rh. erythropolis
AJ270 cells (2 g wet weight) in phosphate buffer (50 mL, pH 7.0) at 30
°C. b Isolated yield. c Determined by chiral HPLC analysis of the corre-
sponding â-benzyloxy alkaneamides. d Determined by chiral HPLC analysis
of the corresponding methyl ester of â-benzyloxy alkanoic acid. e Deter-
mined by chiral HPLC analysis of the benzyl ester. f Nitrile (49% yield,
2.8% ee) was recovered.
allyl
c-Pr
c-Bu
c-Pen
2
28
21
24
70
R-5d (47)
(91.0)
S-5e (47)
(92.4)
S-5f (49)
(95.6)
S-5g (48)
(95.4)
41
2
106
127
45
2
g
1
a Biotransformation was carried out in a suspension of Rh. erythropolis
AJ270 cells (2 g wet weight) in phosphate buffer (50 mL, pH 7.0) at 30
°C. b Isolated yield. c Determined by chiral HPLC analysis. d E value was
calculated according to ref 27. e A suspension of 5 g wet weight cells in
125 mL of phosphate buffer was used, and nitrile (10% yield, 93.0% ee)
was recovered.
unexpected, however, as the movement of the stereocenter
from the R to â position to the functional group of the
substrate generally has a detrimental effect on enantioselec-
tivity of biocatalytic resolution of nitriles.23 Further metabo-
lization and hydrophilic nature of the polar products posed
further problems in the isolation of â-hydroxy carboxylic
acids and amides from aqueous reaction media. Increasing
the bulkiness of the substrate led to a sluggish conversion
of nitrile 1g (entry 3, Table 1). As indicated by the
enantiomeric excess values of amide and acid products and
of the nitrile recovered (Table 1), neither the nitrile hydratase
nor the amidase exhibited even moderate enantioselectivity
against â-hydroxy alkanenitriles and alkaneamides, respec-
tively.
triles 4,26 prepared readily from the benzylation of â-hydroxy
alkanenitriles 1, underwent effective biotransformations to
afford optically active â-benzyloxy alkanoic acids 6 and
amides 5 with enantiomeric excess values up to 95%.
Comparison of the biotransformations of 4a, 4c, and 4g with
those of 1a, 1c, and 1g and protection of the hydroxyl by a
benzyl group led to a dramatic increase of enantioselectivity
with enantiomeric ratio E27 ranging from 43 to 78. For the
substrates, such as 4e and 4f, that contains a cyclopropyl
and cyclobutyl moiety, respectively, biotransformations gave
excellent enantioselectivity with an E over 100 (entries 6
and 7, Table 2). In addition to high reaction efficiency and
enantioselectivity, an extra advantage of utilizing the benzyl
protection group is the prohibition of product metabolization
and the increase of molecular hydrophobicity, both leading
to the isolation of products in high yields. Furthermore,
biotransformations of O-benzylated â-hydroxy alkanenitriles
To increase the enantioselectivity of the biotransforma-
tions, and also to circumvent the problems, such as further
metabolization and hydrophilicity of â-hydroxy alkanoic
acids, we decided to protect the â-hydroxy group of the
substrates. The following criteria should be considered while
choosing a protecting group. First, both the introduction and
the removal of a protection group should be readily carried
out under mild conditions and cause no contamination to
the products. Second, under the deprotection conditions, no
racemization is allowed. In addition, because a whole cell
biocatalyst is used, the protection group should be stable and
resistant to other enzymes, such as esterases. Furthermore,
large protection groups are not favorable because they result
in the deficiency of biocatalysis. Last but not least, an
(26) Walls, B. O. F.; Barrios, F. Y. H.; Sanchez-Obregon, R.; Pinelo, L.
Synth. Commun. 1993, 23, 749.
(27) (a) Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem.
Soc. 1982, 104, 7294. (b) Program “Selectivity” by Faber, K.; Hoenig, H.
http://www.cis.TUGraz.at/orgc/.
Org. Lett., Vol. 8, No. 15, 2006
3233

allowed practical synthesis of highly enantiopure â-hydroxy
alkanoic acids and their amide derivatives, which was
exemplified by the multigram biotransformation of 4b (entry
3, Table 2). Finally, the protection group was conveniently
removed through catalytic hydrogenolysis giving rise to the
highly enantiopure â-hydroxy carboxylic acid and amide in
quantitative yield (Scheme 1). To demonstrate the further
structural information of the enzymes. Nevertheless, much
more efficient chiral recognition of the enzymes toward
protected â-hydroxy alkanenitriles or amides than free
â-hydroxy alkanenitriles or amides might account for the
increase of enantioselectivity. In other words, it is most likely
that the â-benzyl protection group acts as an excellent
docking group, leading to the enhancement of enantioselec-
tive interaction of the substrate with the chiral pocket of the
enzyme (Figure 1).
Scheme 1. Enhancement of Enantioselectivity of
Biotransformations of 1 Using a Benzyl Protection Group
Figure 1. Schematic illustration of enhancement of enantioselec-
tivity through a benzyl protection/docking group.
In summary, we have shown that the introduction of a
simple and remote protection/docking benzyl group dramati-
cally increases the enantioselectivity of the biotransforma-
tions of â-hydroxy alkanenitriles. The easy protection and
deprotection manipulations, high chemical yields, and excel-
lent enantioselectivity along with the scalability render this
microbial transformations attractive and practical for the
synthesis of highly enantiopure â-hydroxy alkanoic acids and
their amide derivatives. The protection/docking strategy may,
in general, provide a simple and cost-effective method to
improve the enantioselectivity and efficiency of biocatalysis,
even if such a docking/protection group is far away from
the reactive functional group. Studies toward the understand-
ings of the remarkable protection/docking effect and toward
the further improvement of enantioselectivity using a modi-
fied benzyl group in the biotransformations of nitriles are
actively being investigated in this laboratory.
advantage of protection strategy, the biotransformation
product 5d was directly employed as the reactant to prepare,
via iodolactonization,13e,24c lactone 8 which is the essential
intermediate for the construction of effective cholesterol-
lowering agents Lipitor (atorvastatin) and Crestor (rosuvas-
tatin) (see Supporting Information).28
The dramatic enhancement of enantioselectivity of the
biotransformation of â-benzyloxy alkanenitriles 4 is extraor-
dinarily intriguing, especially when we consider the intro-
duction of a protecting benzyl group at the hydroxyl oxygen,
a position being remote from the reaction site, such as a
cyano or an amido group. It is difficult at this current stage
to rationalize the effect of a â-positioned benzyl protection
group on the enantioselectivity because of the lack of
Acknowledgment. The project was supported by the 973
Program (2003CB716005), the National Natural Science
Foundation of China, and the Chinese Academy of Sciences.
Supporting Information Available: Experimental details
of biotransformation of nitriles, copies of 1H and 13C NMR
spectra of compounds 2, 3, and 5-8. This material is
available free of charge via the Internet at http://pubs.acs.org.
(28) Greenberg, W. A.; Varvak, A.; Hanson, S. R.; Wong, K.; Huang,
H.; Chen, P.; Burk, M. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 5788
and references therein.
OL0610688
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