Nuclease p1: A new biocatalyst for direct asymmetric aldol reaction under solvent-free conditions

Article abstract of DOI:10.1039/c0gc00486c

The unnatural ability of nuclease p1 from Penicillium citrinum was first discovered to catalyze asymmetric aldol reactions between aromatic aldehydes and cyclic ketones under solvent-free conditions. The excellent enantioselectivities of up to 99% ee and

Full text of DOI:10.1039/c0gc00486c

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PAPER
Nuclease p1: a new biocatalyst for direct asymmetric aldol reaction under
solvent-free conditions†
Hai-Hong Li,‡ Yan-Hong He,‡ Yi Yuan and Zhi Guan*
Received 26th August 2010, Accepted 3rd November 2010
DOI: 10.1039/c0gc00486c
The unnatural ability of nuclease p1 from Penicillium citrinum was first discovered to catalyze
asymmetric aldol reactions between aromatic aldehydes and cyclic ketones under solvent-free
conditions. The excellent enantioselectivities of up to 99% ee and high diastereoselectivities of up
to >99 : 1 (anti/syn) were achieved. This nuclease p1 catalyzed reaction provided a novel case for
new activities of existing enzymes, which widens the applicability of this biocatalyst in organic
synthesis.
examples of aldol reactions catalyzed by enzymes, besides
1. Introduction
aldolases11 that are the natural enzymes for aldol reactions.
Biocatalytic processes have often proven to be economically fea-
Berglund and co-workers once used mutant CAL-B (lipase from
sible, ecologically advantageous and more sustainable than cur-
Candida antarctica) to catalyze aldol additions in 2003.12 Wang
rent chemical technologies because of the intrinsic advantages of
and Yu also reported lipase catalyzed aldol reactions in 2008.13
biocatalysts in higher reaction selectivity, milder reaction con-
However, of those reactions reported to date for which ee values
ditions and potential use of inexpensive regenerable resources.1
are reported, the best ee value so far was only 43.6% with low
However, there are some drawbacks of biocatalysis. For example,
yield of 11.7%.13 To the best of our knowledge, other hydrolase
there is only limited availability of enzymes, and enzymes only
catalyzed aldol addition has not been reported. Therefore, the
feature limited substrate specificity.2 Therefore, the discovery
development of highly enantioselective aldol reactions catalyzed
of novel unnatural activities of existing enzymes to widen the
by enzymes is still in great demand.
applicability of existing enzymes is significant. Recently, research
in the area of biocatalytic promiscuity has attracted significant
attention from chemists and biochemists.3 Hilvert et al. reported
Nuclease P1 (EC 3.1.30.1) from Penicillium citrinum belongs
to a family of zinc-dependent endonucleases consisting of 270
amino acid residues with two disulfide bonds which cleaves
the use of minimal modifications at enzyme active sites to expand
single-stranded RNA and DNA into 5-mononucleotide.14 We
their catalytic repertoires, including targeted mutagenesis and
found that nuclease p1 also had the ability to catalyze asymmet-
the addition of new reactive functionalities.4 Danson and Bull
et al. reported structurally informed site-directed mutagenesis
of a stereochemically promiscuous aldolase to afford stereo-
chemically complementary biocatalysts.5 Griengl et al. reported
ric aldol reactions between aromatic aldehdes and cyclic ketones
with excellent stereoselectivity and high diastereoselectivities
under solvent-free conditions. This finding provided a novel
example of unnatural activities of existing enzymes.
hydroxynitrile lyase from Hevea brasiliensis catalyzed nitroaldol
reactions.6 More recently, Zhang and Chen et al. reported lipase-
catalysed tandem Knoevenagel condensation and esterification
2. Results and discussion
with alcohol cosolvents.7 In addition, some other elegant works
Asymmetric direct aldol reaction of 4-cyanobenzaldehyde and
on enzyme catalyzed promiscuity have also been reported.8
cyclohexanone catalyzed by nuclease p1 was first carried out as a
Catalytic aldol reactions are among the most useful synthetic
model experiment to find out the optimum reaction conditions,
methods for highly stereocontrolled asymmetric synthesis.9
such as the solvent, the ratio of substrates, the enzyme loading,
There are many reports about enantioselective aldol reactions
the water content and the temperature. Based on the concept that
catalyzed by small organic molecules,10 but there are few
not only do enzymes work in anhydrous organic media, but they
acquire remarkable properties such as enhanced stability, altered
School of Chemistry and Chemical Engineering, Southwest University,
Chongqing, 400715, P. R. China. E-mail: guanzhi@swu.edu.cn;
Fax: +86-23-68254091; Tel: +86-23-68254091
† Electronic supplementary information (ESI) available: General meth-
ods and the data of HPLC and 1H NMR. See DOI: 10.1039/c0gc00486c
‡ Hai-Hong Li and Yan-Hong He contributed equally to this work
substrate and enantiomeric specificities, molecular memory, and
the ability to catalyze unusual reactions which are impossible
in aqueous media,15 the catalytic activity of nuclease p1 was
evaluated in different solvents including solvent-free conditions
(Table 1). The results clearly indicated that the catalytic activity
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Table 1 Solvent screening and control experimentsa
Entry Solvent
Time/h Yield [%]b d.r.c
ee[%]d
1
2
3
4
5
6
7
8
9
DMSO
DMF
96
96
96
96
96
96
96
96
72
53
30
28
27
21
20
18
3
64 : 36 54
72 : 28 55
85 : 15 81
74 : 26 64
79 : 21 67
42 : 58 65
66 : 34 61
57 : 43 42
Solvent-freee
THF
CH3CN
Cyclohexane
CH2Cl2
H2O
Solvent-free (nuclease p1 96
—
—
denatured with EDTAf)
10
Solvent-free (no enzyme) 168
nr
—
—
Fig. 1 Effect of the molar ratio of substrates on the nuclease p1
catalyzed aldol reaction. Conditions: 4-cyanobenzaldehyde (1.0 mmol),
cyclohexanone (4–25 mmol) and nuclease p1 (200 mg) at 20 ◦C for 90 h.
a All reactions except the reactions under solvent-free conditions were
carried out using 4-cyanobenzaldehyde (200 mg, 1.5 mmol), cyclohex-
anone (1.0 ml, 10 mmol), nuclease p1 (200 mg), H2O (0.75 ml) and
organic solvent (5 ml) at 20 ◦C. b Yield of the isolated product after
chromatography on silica gel. c The d.r. was the anti/syn ratio, which
was determined by 1H NMR analysis of the diastereomeric isomers.
d Enantiomeric excess (anti) was determined by HPLC analysis using
a chiral column; relative and absolute configurations of the products
We then examined the influence of enzyme loading on
the aldol reaction of cyclohexanone (5.0 mmol) and 4-
cyanobenzaldehyde (1.0 mmol) under solvent-free conditions.
From Fig. 2, it can be seen that the catalytic activity and the
stereoselectivity of nuclease p1 were significantly influenced by
the enzyme loading. When the enzyme loading was increased
to 200 mg, a major enhancement in ee and yield was observed.
However, no evident improvement of ee was observed though
there was a slight enhancement in yield on increasing further
the amount of enzyme. So the enzyme loading of 200 mg was
chosen for the further studies under solvent-free conditions.
1
were determined by comparison with the known H NMR and chiral
HPLC analysis. e Reaction conditions: 4-cyanobenzaldehyde (200 mg,
1.5 mmol), cyclohexanone (1.0 ml, 10 mmol) and nuclease p1 (200 mg)
at 20 ◦C. f Pre-treated with EDTA at 30 ◦C for 24 h.
and the stereoselectivity of nuclease p1 were significantly
influenced by the reaction media. Among the catalytic systems
examined, the nuclease p1 exhibited the best catalytic activity
and moderate stereoselectivity in DMSO (Table 1, entry 1).
However, the enzyme showed the best diastereo- and enantio-
selectivities under solvent-free conditions (Table 1, entry 3).
Thus, we chose solvent-free as the optimum conditions for the
asymmetric direct aldol reaction.
In order to verify the specific catalytic effect of nuclease p1
on the aldol reaction, we performed some control experiments
under solvent-free conditions (Table 1). Just as we expected,
the aldol reaction between 4-cyanobenzaldehyde and cyclohex-
anone in the absence of enzyme under solvent-free conditions
showed no adduct after 168 h (Table 1, entry 10). In addition,
since nuclease p1 is a zinc-dependent enzyme, EDTA (ethylene
diamine tetraacetic acid) was used to denature the enzyme. We
found that the aldol reaction with EDTA-denatured nuclease p1
only gave 3% yield of aldol product after 96 h, which excluded
the possibility that the reaction was caused by impurities of the
enzyme or the catalysis simply arose from amino acids of the
protein. On the other hand, it can be assumed that the tertiary
structure of the enzyme was essential in the process. Thus, we
validated that nuclease p1 catalyzed the direct aldol reaction.
Next, the effect of molar ratio of substrates on the nuclease
p1 catalyzed aldol reaction was investigated under solvent-free
conditions (Fig. 1). The best ee value of 82% with yield of
32% was obtained when the molar ratio of cyclohexanone to
4-cyanobenzaldehyde was 5 : 1. No positive effect was observed
on ee as well as on yield while increasing the amount of
cyclohexanone. Thus, the 5 : 1 molar ratio of ketone to aldehyde
was chosen as the optimal ratio for further studies.
Fig. 2 Influence of the enzyme loading on the nuclease p1 catalyzed
aldol reaction. Conditions: 4-cyanobenzaldehyde (1.0 mmol), cyclohex-
anone (5.0 mmol) and nuclease p1 (50–400 mg) at 25 ◦C for 96 h.
Water concentration affects both the enantioselectivity and
activity of enzymes.16 In order to ascertain the role played by
water, we carried out the reaction in different water content
under solvent-free conditions. As shown in Fig. 3, the yield and
ee value of the aldol reaction could be evidently effected by water
content. The nuclease p1 exhibited the highest enantioselectivity
at water content of 0.15 (wwater/wenzyme), and under this conditions
the enzymatic reaction reached the highest ee value of 84%
(yield of 35%). Once the concentration of water surpassed 0.15
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Fig. 4 Influence of temperature on the nuclease p1 catalyzed aldol
Fig. 3 Influence of water content on the nuclease p1 catalyzed aldol
reaction. Conditions: 4-cyanobenzaldehyde (1.0 mmol), cyclohexanone
reaction. Conditions: 4-cyanobenzaldehyde (1.0 mmol), cyclohexanone
(5.0 mmol), nuclease p1 (200 mg), temperature (5–70 ◦C) and deionized
water (30 mg) for 96 h.
(5.0 mmol), nuclease p1 (200 mg), deionized water (0–2.0, wwater/wenzyme
at 25 ◦C for 107 h.
)
showed the best enantioselectivity of 91% ee at 15 ◦C, however, it
required a higher temperature (45 ◦C) to exhibit its best activity.
In order to get the best enantioselectivity, we chose 15 ◦C as the
optimal temperature.
With the optimal reaction conditions in hand, we further
studied the substrate scope and the generality of the nuclease
p1 catalyzed asymmetric direct aldol reaction. Various cyclic
ketones and substituted benzaldehydes were investigated under
solvent-free conditions (Table 2). It can be seen that a wide
range of substrates could participate in the reaction. Five-,
six- and seven-membered cyclic ketones as aldol donors could
(wwater/wenzyme), the ee value decreased. However, the highest en-
zyme activity was reached at water content of 0.70 (wwater/wenzyme),
which gave the best yield of 46% (76% ee), and higher water
content led to a decrease of yield. Therefore, to obtain the best
ee, we chose the water content of 0.15 (wwater/wenzyme) for the aldol
reaction.
Temperature plays an important role in enzyme catalyzed
reactions, due to its effects on the selectivity and rate of the
reaction, and also on the stability of the enzyme. We then studied
the effect of temperature on the reaction (Fig. 4). The enzyme
Table 2 Substrate scope of the nuclease p1 catalyzed aldol reactiona
Entry
R
Prod.
n
Time/h
Yield [%]b
d.r.c
ee[%]d (anti)
1
2
3
4
5
6
7
8
4-MeC6H4
C6H5
4-ClC6H4
2-ClC6H4
3-ClC6H4
2,4-Cl2C6H3
2,6-Cl2C6H3
4-BrC6H4
4-CNC6H4
4-CF3C6H4
2-NO2C6H4
3-NO2C6H4
4-NO2C6H4
4-CNC6H4
4-NO2C6H4
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
2
2
2
2
2
2
2
2
2
2
2
2
1
1
3
144
144
144
240
158
240
240
144
165
158
165
165
165
144
165
25
20
21
38
24
31
37
17
43
26
21
28
55
39
39
80 : 20
70 : 30
87 : 13
94 : 6
87 : 13
93 : 7
>99 : 1
90 : 10
86 : 14
87 : 13
98 : 2
>99
85
91
93
92
90
81
93
91
9
10
11
12
13
14
15
90
89
92
91 : 9
70 : 30
64 : 36
50 : 50
79/33e
82/47f
49
a For the general procedure see ref. [17]. b Yield of the isolated product (anti + syn) after chromatography on silica gel. c The d.r. was the anti/syn ratio,
which was determined by 1H NMR analysis. d Enantiomeric excess was determined by HPLC analysis using a chiral column; absolute configurations
of the products were determined by comparison with the known chiral HPLC analysis 18 (see the ESI†). e anti (79% ee), syn (33% ee). f anti (82% ee),
syn (47% ee).
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be accepted by the enzyme. Generally, the enzyme exhibited
better diastereoselectivity and enantioselectivity with cyclo-
hexanone (Table 2, entries 1–12) than with cyclopentanone
and cycloheptanone (Table 2, entries 13–15). Furthermore,
both electron-donating and electron-withdrawing substituents
of aromatic aldehydes were tolerated. Notably, this nuclease
p1 catalyzed asymmetric direct aldol reaction exhibited high
selectivity. The best enantioselectivity of >99% ee (Table 2,
entry 1) and the best diastereoselectivity of >99 : 1 (anti/syn)
(Table 2, entry 7) were achieved. Moreover, the effect of sterically
hindered substituents on benzaldehydes had a great impact on
the diastereoselectivity of the reaction. When reacting with
cyclohexanone, substituted benzaldehydes (Table 2, entries 1
and 3–12) gave better diastereoselectivity than benzaldehyde
(Table 2, entry 2), substituents in the 2-position gave higher
dr values (Table 2, entries 4, 6, 7 and 11), and the best
diastereoselectivity of >99 : 1 was achieved by using the most
hindered substrate 2,6-dichlorobenzaldehyde (Table 2, entry 7).
Interestingly, anti isomers were received as the major products
by using cyclohexanone and cyclopentanone, but no diastereos-
electivity was observed by using cycloheptanone. The nuclease
p1 catalyzed-aldol reaction seems to prefer cyclohexanone than
cyclopentanone and cycloheptanone. It is also worthy to note
that this enzyme had a moderate to excellent enantioselectivity
for anti isomers, but low or no enantioselectivity for syn isomers.
Maybe the catalytic site of the nuclease p1 had a specific
selectivity for the aldol reaction. The yield of the aldol reaction
catalyzed by nuclease p1 is still low and the reaction mechanism
is unclear at the moment. Further efforts to deal with this
problem are currently underway in our laboratory.
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3. Conclusion
In summary, we have succeeded in obtaining enantiomeric aldol
products by using enzyme nuclease p1 as a new biocatalyst.
A wide range of cyclic ketones and substituted benzaldehydes
could be accepted by the enzyme. In most cases, excellent ee val-
ues and good d.r. were obtained without any additive. Notably,
this is the first highly stereoselective aldol reaction catalyzed
by enzymes besides aldolases. This biocatalytic reaction was
performed under mild and solvent-free conditions. Compared
with current chemical technologies, the nuclease p1 catalyzed
direct asymmetric aldol reaction is more economically feasible,
ecologically advantageous and sustainable by using inexpensive
regenerable resources. It is also a novel case of unnatural
activities of existing enzymes, and might be a potential synthetic
method for organic chemistry.
Acknowledgements
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Financial support from Natural Science Foundation Project of
CQ CSTC (2009BA5051) is gratefully acknowledged.
References
17 General procedure: A test tube was charged with nuclease p1 (5
U/mg) (200 mg), aldehyde (1.0 mmol), to which the deionized
water (30 mg) and ketone (5.0 mmol) were introduced. The resulting
mixture was stirred for the specified amount of time at 15 ◦C. The,
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reaction was terminated by filtering the enzyme. CH2Cl2 was used
to wash the filter paper to assure that products obtained were all
dissolved in the filtrate. 20 ml of water was then added to the filtrate,
and the filtrate was extracted three times with 20 ml of CH2Cl2.
The combined extracts were dried over anhydrous Na2SO4, and
the solvents were then removed under reduced pressure. The crude
products were purified by column chromatography with petroleum
ether/ethyl acetate as eluent.
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