Enzymatic enantioselective aldol reactions of isatin derivatives with cyclic ketones under solvent-free conditions

Article abstract of DOI:10.1016/j.biochi.2014.01.006

Nuclease p1 from Penicillium citrinum was observed to directly catalyze the asymmetric aldol reactions between isatin derivatives and cyclic ketones with high isolated yields (up to 95%) and moderate to good stereoselectivity (dr up to >99/1, ee up to 82%

Full text of DOI:10.1016/j.biochi.2014.01.006

Biochimie xxx (2014) 1e5
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Research paper
Enzymatic enantioselective aldol reactions of isatin derivatives with
cyclic ketones under solvent-free conditions
*
*
Zhi-Qiang Liu, Zi-Wei Xiang, Zhou Shen, Qi Wu , Xian-Fu Lin
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nuclease p1 from Penicillium citrinum was observed to directly catalyze the asymmetric aldol reactions
between isatin derivatives and cyclic ketones with high isolated yields (up to 95%) and moderate to good
stereoselectivity (dr up to >99/1, ee up to 82%). A series of reaction conditions were investigated in detail,
and the addition of deionized water had a big influence upon the enzyme activity. This case of bio-
catalytic promiscuity not only widens the applicability of nuclease p1 to new chemical transformation in
organic synthesis, but also provides a potentially valuable method to construct pharmaceutically active
compounds in medicinal chemistry.
Received 6 November 2013
Accepted 7 January 2014
Available online xxx
Keywords:
Biocatalysis
Promiscuity
Asymmetric aldol reaction
Solvent free
Ó 2014 Elsevier Masson SAS. All rights reserved.
3-Substituted-3-hydroxyindolin-2-ones
1. Introduction
Wang groups [34e38], however, the intermolecular ketoneeketone
asymmetric aldol addition is rare and remains a significant challenge
Since Klibanov pioneered the anhydrous enzymology in the
early 1980s [1], enzymes as efficient and green biocatalysts in
organic chemistry have gained much attention from chemists and
biochemists due to their high selectivity, mild reaction conditions,
environmental acceptability and potentially regenerable catalysts
[2e6]. Especially, many enzymes have been discovered to catalyze
unnatural reaction completely different from the originally per-
formed, which is called biocatalytic promiscuity [7e11]. In the last
decades, several elegant works on the enzymatic promiscuity have
been reported, such as Michael addition [12e15], Henry reaction
[16,17], Mannich reaction [18e20] and Markovnikov additions
[21,22]. It is still in great demand and interest to develop novel
activities of commercially available enzymes, particularly for the
synthesis of bioactive molecules.
[39e41]. Considering that 3-substituted-3-hydroxyindolin-2-ones
constitute a nucleus of a large variety of natural products and phar-
maceutical candidates [42e47], such as compounds A, B, C and D in
Fig. 1, a few reports about the aldol reactions between isatins and
ketones had been published, but the catalysts were inevitably a base,
an organic molecule and a metal complex [48e52]. In view of green
chemistry, herein we wish to report a concise and straightforward
approach to synthesize 3-alkyl-3-hydroxyindolin-2-ones by eco-
friendly enzymes under solvent-free conditions. To the best of our
knowledge, this is the first enzymatic asymmetric aldol reactions of
isatins and cyclic ketones.
2. Material and methods
The asymmetric aldol reaction emerges as a simple and important
method to form CeC bond in stereocontrolled organic chemistry. In
1974, Hajos and Parrish first described the organocatalyzed aldol re-
action [23], and Shibasaki group reported the direct asymmetric aldol
reaction of aldehydes with unmodified ketones in 1997 [24]. In the
following decades, the aldehydeealdehyde and aldehyde-ketone
aldol reaction had been extensively researched [25e33], more
recently some direct enzymatic asymmetric aldol reactions between
aromatic aldehydes and ketones have been reported by Guan and
2.1. General information for reagents and analytical methods
Amano Lipase M from Mucor javanicus (ꢀ10000 U/g enzyme ac-
tivity, pH 7.0, 40 ꢁC) was purchased from Sigma (Steinheim, Ger-
many). Acidic protease from Aspergillus usamii No 537 (50 U/mg) and
alkaline protease fromBacillus licheniformis No 2709(200 U/mg)were
purchased from Xuemei Enzyme Co. Ltd. (Wuxi, China). Chymopa-
pain isolated from the latexof the unripe fruits of Carica papaya (20 U/
mg, one unit of activity was defined as the amount of enzyme to
produce TCA-soluble hydrolyzed products from casein, which gives
an absorbance value equivalent to 1.0
mg of tyrosine at 275 nm/min at
* Corresponding authors. Tel.: þ86 (0)57187953001; fax: þ86 571 87952618.
E-mail addresses: llc123@zju.edu.cn (Q. Wu), llc123@zju.edu.cn (X.-F. Lin).
30 ꢁC and pH 7.5) and Nuclease P1 from Penicillium citrinum (5 U/mg.
0300-9084/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.biochi.2014.01.006
Please cite this article in press as: Z.-Q. Liu, et al., Enzymatic enantioselective aldol reactions of isatin derivatives with cyclic ketones under
solvent-free conditions, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.01.006

2
Z.-Q. Liu et al. / Biochimie xxx (2014) 1e5
the flask was placed in incubator shaker at 18 ꢁC for 120 h at 200 r/
min. When the reaction was finished, the organic phase was puri-
fied by column chromatography with petroleum ether/ethyl ace-
tate as eluent. Or the products were obtained after solvent in
organic phase volatilized thoroughly.
3. Results and discussion
3.1. The catalytic activity of different enzymes for the asymmetric
aldol reactions of isatins and cyclic ketones
Initial experiments of isatin and cyclohexanone catalyzed by
different enzymes were performed in acetonitrile with 0.1 mL
deionized water (DI). We were delighted to see the enantiose-
lectivity of enzymes for the aldol reaction. The data are summarized
in Table 1, the results indicated that the activity and enantiose-
lectivity of varied biocatalysts were contradictory. For example,
Amano Lipase M from Mucor javanicus (MJL) exhibited excellent
activity with 99% yield, but poor enantioselectivity (Table 1, entry
3), on the contrary, the best enantioselectivity of 56% ee value was
obtained by nuclease p1 from P. citrinum (nuclease p1) under the
same conditions as MJL, but the yield is poor (Table 1, entry 4).
Other proteinase, such as Chymopapain, Acidic protease from
A. usamii (AUAP) and alkaline protease from B. licheniformis (BLAP),
showed lower enantioselectivity than nuclease p1 (Table 1, entries
1, 4 and 5). The size of active center in different enzymes varied a
lot, substrate specificity was understandable here. Based on the
importance of enantioselectivity, nuclease p1 was chosen as the
biocatalyst for the following optimization.
Fig. 1. Bioactive compounds with 3-substituted-3-hydroxyindolin-2-ones skeleton.
The activity was measured in terms of the amount of acid-soluble
nucleotides produced by RNA hydrolysis which is catalyzed by
nuclease p1. One unitofenzyme activity wasdefined asthe amount of
enzyme that produced an increase in the optical density of 1.0 in
1 min at 260 nm) were purchased from Guangxi Nanning Pangbo
Biological Engineering Co. Ltd. (Nanning, China). Unless otherwise
noted, all reagents were obtained from commercial suppliers and
were used without further purification. The 1H and 13C NMR spectra
were recorded with TMS as internal standard using a Bruker AMX-
400 MHz spectrometer. Chemical shifts were expressed in parts per
million and coupling constants (J) in hertz. IR spectra were measured
with a Nicolet Nexus FTIR 670 spectrophotometer. The HPLC analysis
was performed on SHIMADZU LC-20AT system at 254 nm.
2.2. General procedure for the synthesis of racemic products
between isatin derivatives and cyclic ketones
3.2. The effect of reaction media on the asymmetric aldol reactions
of isatins and cyclic ketones
To a stirred solution of 2 mmol isatin derivative and 3 mmol
cyclic ketone in 20 mL methanol was added 5e6 drop diethylamine
and the mixture was left to react for about 4e6 h at room tem-
perature until isatin derivative disappeared, determined by TLC.
Then the solution was concentrated in vacuo and the obtained
mixture was washed in boiling petrol ether/ethyl acetate, recrys-
tallized in them.
Considering the reaction medium has a great effect on the ac-
tivity and the stability of an enzyme, different solvents were
screened for the asymmetric aldol reactions of isatins and cyclic
ketones. Data in Table 2 showed the catalytic activity and stereo-
selectivity were obviously influenced by the reaction media.
Nuclease p1 displayed the best enantioselectivity in acetonitrile
2.3. General procedure for the enzymatic aldol reactions of isatins
and cyclic ketones
Table 2
Solvent screening and controlled experiments.a
To a solution of isatin derivative (0.1 or 0.2 mmol) and DI
(0.15 mL) in cyclic ketone (1 mL) was added nuclease p1 (150 mg),
Table 1
Screening of enzymes on the aldol reaction.a
Entry
Solvent
Yield (%)b
drc
ee (%)d
1
2
3
4
5
6
7
8
9
DMSO
DMF
EG
96
99
60
33
15
e
92/8
96/4
79/21
72/28
87/13
e
5
5
16
34
56
e
Dioxane
Acetonitrile
Toluene
THF
Entry
Enzyme
Yield (%)b
drc
ee (%)d
1
2
3
4
5
Chymopapain
MJL
Nuclease p1
AUAP
57
99
15
54
75
92/8
94/6
87/13
90/10
90/10
37
29
56
32
12
e
e
e
Acetonitrilee
Acetonitrilef
e
e
e
e
e
e
BLAP
a
Conditions: isatin (0.2 mmol), cyclohexanone (1 mmol), acetonitrile (1 mL), DI
(0.1 mL), enzyme (70 mg), 30 ꢁC, 120 h.
a
Conditions: isatin (0.2 mmol), cyclohexanone (1 mmol), acetonitrile (1 mL), DI
(0.1 mL), enzyme (70 mg), 30 ꢁC, 120 h.
b
Determined by chiral HPLC.
The anti/syn ratio, determined by chiral HPLC analysis of the diastereomeric
b
c
Determined by chiral HPLC.
The anti/syn ratio, determined by chiral HPLC analysis of the diastereomeric
c
isomers.
d
isomers.
The anti enantiomeric excess, determined by chiral HPLC analysis.
Denatured with EDTA at 100 ꢁC for 24 h.
d
e
The anti enantiomeric excess, determined by chiral HPLC analysis, absolute
f
configurations assigned by comparison with literature data [48,53,54].
No enzyme.
Please cite this article in press as: Z.-Q. Liu, et al., Enzymatic enantioselective aldol reactions of isatin derivatives with cyclic ketones under
solvent-free conditions, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.01.006

Z.-Q. Liu et al. / Biochimie xxx (2014) 1e5
3
3.4. The effect of substrate molar ratio on the asymmetric aldol
reactions of isatins and cyclic ketones
In order to further enhance the yield and enantioselectivity of
the nuclease p1 catalyzed enantioconvergent aldol reactions of
isatins and cyclic ketones, the effect of substrate molar ratio was
investigated on the model reaction. When the reaction solvent was
acetonitrile, the best result was observed with 1 mmol cyclohexa-
none (Fig. 3), just as we described before. Surprisingly, nuclease p1
showed the highest activity and enantioselectivity in 1 mL cyclo-
hexanone (about 9.6 mmol), which substituted acetonitrile as the
solvent, under this condition the highest ee value of 69% and yield
of 99% were got in the enzymatic reaction. In this two-phase
environment, nuclease p1 was in water so that it could maintain
catalytic activity and tertiary structure for stereoselectivity, there-
fore, good enantioselectivity and high yield were obtained.
Consequently, the enzymatic asymmetric aldol addition between
isatin and cyclic ketone proceeded in solvent-free condition, the
best conditions are: isatin (0.2 mmol), cyclohexanone (1 mL), DI
(0.15 mL), nuclease p1 (150 mg), 18 ꢁC.
Fig. 2. Influence of water content on the nuclease p1 catalyzed asymmetric aldol re-
actions of isatins and cyclic ketones. Conditions: isatin (0.2 mmol), cyclohexanone
(1 mmol), acetonitrile (1 mL), enzyme (70 mg), 18 ꢁC, 120 h.
3.5. The reaction time course of the asymmetric aldol reactions of
isatins and cyclic ketones
(Table 2, entry 5), the best activities were observed in DMSO and
DMF but with very poor enantioselectivity (Table 2, entries 1 and
2). Generally, strong polar solvent like DMSO and DMF had big
influence on the tertiary structure of enzymes, this may result in
poor enantioselectivity. Nevertheless, no product was observed in
toluene (Table 2, entry6), this was probably because of low solu-
bility of isatin in toluene. And nuclease p1 showed no activity to
this aldol reaction in THF (Table 2, entry7). Next, some controlled
experiments were evaluated to verify the catalytic specificity of
nuclease p1 on the aldol reaction of isatin and cyclohexanone.
Because nuclease p1 is a zinc-dependent enzyme, EDTA (ethylene
diamine tetraacetic acid) was used to destroy the active site and
denature the enzyme. As we expected, no products were observed
with denatured nuclease p1 or in the absence of enzyme (Table 2,
entries 8 and 9). These results suggested that the tertiary structure
of this nuclease p1 was essential for the reaction.
Finally, we studied the reaction time course of the aldol addition
between isatin and cyclohexanone under solvent-free condition. It
could be seen from Fig. 4, the ee value almost kept constant from
63% to 69% during the whole reaction phase. Nevertheless, the
yields had an obvious increase as the reaction time went by, and
reached the platform after 96 h. This meant that substrates quickly
entered the active site of enzyme and fitted in it, but product
releasing was slow, the reaction completed thoroughly after 4 days.
3.6. Scope and limitation of the enzymatic asymmetric aldol
reactions of isatins and cyclic ketones
With the optimal reaction conditions in hand, we studied the
substrate scope and the generality of the enzymatic asymmetric
aldol reactions of isatins and cyclic ketones, various isatin de-
rivatives and cyclic ketones were investigated. As shown in Table 3,
a wide range of substrates could participate in the reaction to form
3-hydroxyindolin-2-ones in moderate to excellent yields. The
3.3. The influence of water content on the asymmetric aldol
reactions of isatins and cyclic ketones
The role of water is crucial to the enzyme, not only it affects the
enantioselectivity and activity of enzymes, but it acts as “molecular
lubricant” to change the conformational flexibility [55e61].
Therefore, it is very necessary to confirm the optimum water con-
tent in this system. A series of water contents was inspected in
nuclease p1-catalyzed asymmetric aldol reaction in 1 mL MeCN. As
shown in Fig. 2, the yield and ee value were considerably affected by
the water content. Almost no reaction proceeded when the water
content was less than 0.05 (Vwater/VMeCN), which fully illustrated
the vital function of water in this enzymatic reaction. Nuclease p1
simultaneously exhibited the best enantioselectivity (63%) and
yield (18%) at water content 0.15 (Vwater/VMeCN) respectively. As the
water content increased, both the yield and ee value obviously
dropped. Therefore, water content 0.15 (Vwater/VMeCN) was chosen
for the following optimization. We then examined the impact of
enzyme loading on the aldol reaction of isatin (0.2 mmol) with
cyclohexanone (1 mmol) under the water content 0.15 (Vwater
/
VMeCN). When the amount of enzyme increased to 150 mg, a major
enhancement in yield was observed from 18% to 34%. Further in-
crease had no effect on the asymmetric aldol reaction in ee and
yield. So we chose the enzyme loading of 150 mg for the further
studies.
Fig. 3. Influence of cyclohexanone amount on the nuclease p1 catalyzed asymmetric
aldol reaction. Conditions: isatin (0.2 mmol), DI (0.15 mL), enzyme (150 mg), 18 ꢁC,
120 h.
Please cite this article in press as: Z.-Q. Liu, et al., Enzymatic enantioselective aldol reactions of isatin derivatives with cyclic ketones under
solvent-free conditions, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.01.006

4
Z.-Q. Liu et al. / Biochimie xxx (2014) 1e5
ee and the best diastereoselectivity of >99:1 were achieved when
4-bromoisatin acted as aldol acceptor (Table 3, entry 9), probably
because the substituent on position 4 is so close to hydroxyl group
that facilitate the configuration forming. Gratifyingly, heterocyclic
ketone could also act as aldol donor in the asymmetric aldol re-
actions of isatins and cyclic ketones providing with good results
(Table 3, entries 11e12).
4. Conclusions
In summary, we have developed the first enzymatic direct
asymmetric aldol reactions of isatin derivatives with cyclic ketones
under mild conditions. The influence of some reaction parameters
including enzymes, solvents, water content, the amount of cyclic
ketone and reaction time were investigated in detail. Furthermore,
a wide range of substituted isatin could successfully react with five-
, six-membered cyclic ketones in the promiscuous nuclease p1-
catalyzed reaction, and a series of 3-hydroxyindolin-2-ones were
obtained in moderate to excellent diastereoselectivity (up to 99:1)
with moderate to high enantioselectivity (up to 82% ee). This
demonstration of enzymatic asymmetric aldol reactions of isatins
and cyclic ketones not only expands the application of nuclease p1
in organic synthesis but also provides an alternative method to
prepare optical 3-alkyl-3-hydroxyindolin-2-ones compounds.
Fig. 4. The time course of the enzymatic asymmetric aldol reaction. Conditions: isatin
(0.2 mmol), cyclohexanone (1.0 mmol), DI (0.15 mL), enzyme (150 mg), 18 ꢁC.
diastereoselectivity and enantioselectivity greatly depended on the
structure of ketones. In general, nuclease P1 exhibited better ac-
tivity and selectivity toward six-membered cyclic ketone than
cyclopentanone (Table 3, entries 4e12 and entries 1e3), which
indicated that the former is more favorable for the active site of this
enzyme. The electronic nature and the size of the substituents on
the isatin phenyl ring showed little influence on the reaction.
Almost all were accepted with high dr value and good ee value
(Table 3, entries 5e10), for example, the 5-position substituted
isatin derivatives were all able to be accepted by the enzyme,
similar activity (yield >89%) and selectivity (dr > 90:10, ee > 62)
were observed (Table 3, entries 4e8). The substituent on position 7
gave almost the same results in yield and selectivity as position 5
(Table 3, entry 10 vs 6). Notably, the best enantioselectivity of 82%
Acknowledgments
The financial support from the National Natural Science Foun-
dation of China (no. 21072172, 21272208) and Ph.D. Programs
Foundation of Ministry of Education of China (20110101110008) are
gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biochi.2014.01.006.
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Table 3
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57/43
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90/10
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Please cite this article in press as: Z.-Q. Liu, et al., Enzymatic enantioselective aldol reactions of isatin derivatives with cyclic ketones under
solvent-free conditions, Biochimie (2014), http://dx.doi.org/10.1016/j.biochi.2014.01.006