Biocatalytic promiscuity: Lipase-catalyzed asymmetric aldol reaction of heterocyclic ketones with aldehydes

Article abstract of DOI:10.1016/j.tetlet.2012.07.007

The new promiscuous activity of lipase from porcine pancreas, type II (PPL II), has been observed to catalyze the direct asymmetric aldol reaction of heterocyclic ketones with aromatic aldehydes. PPL II showed favorable catalytic activity and had a good adaptability to different substrates in the reaction. The enantioselectivities of up to 87% ee and diastereoselectivities of up to 83:17 (anti/syn) were achieved. It is interesting that PPL II possesses the function of aldolase in organic solvents.

Full text of DOI:10.1016/j.tetlet.2012.07.007

Tetrahedron Letters 53 (2012) 4959–4961
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Tetrahedron Letters
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Biocatalytic promiscuity: lipase-catalyzed asymmetric aldol reaction
of heterocyclic ketones with aldehydes
⇑
⇑
Zhi Guan , Jian-Ping Fu, Yan-Hong He
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The new promiscuous activity of lipase from porcine pancreas, type II (PPL II), has been observed to cat-
alyze the direct asymmetric aldol reaction of heterocyclic ketones with aromatic aldehydes. PPL II
showed favorable catalytic activity and had a good adaptability to different substrates in the reaction.
The enantioselectivities of up to 87% ee and diastereoselectivities of up to 83:17 (anti/syn) were achieved.
It is interesting that PPL II possesses the function of aldolase in organic solvents.
Received 1 April 2012
Revised 19 June 2012
Accepted 3 July 2012
Available online 7 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Biocatalytic promiscuity
Lipase
Aldol reaction
Heterocyclic ketones
Asymmetry
Biocatalysis is an efficient and green tool for modern organic
synthesis due to its high selectivity, mild reaction conditions, and
potential use of inexpensive regenerable resources.1 Especially,
biocatalytic promiscuity, the flexibility of the reactions catalyzed
by enzymes, using the same or different, or induced active site
but performing overall different chemical transformations which
usually differ in the type of bond formation or cleavage and in
the catalytic mechanism of bond making or breaking,2 has at-
tracted much attention and expanded rapidly in recent years.3 It
is very significative to profile the novel unnatural activities of
existing enzymes systematically since it might lead to improve-
ments in existing catalytic methods and provide novel synthesis
pathways which are currently not available.3b Some elegant works
of biocatalytic promiscuity including aldol condensations,4 Man-
nich reactions,5 Michael additions,6 Markovnikov additions,7 and
Henry reactions8 have been reported in the last decades.
to construct heterocyclic natural products. However, only limited
reports about enantioselective aldol reactions of heterocyclic ke-
tones with aldehydes catalyzed by organocatalysts are available.11
Moreover, more environmentally friendly and sustainable biocata-
lytic methods are still to be explored. Wang and Yu and co-workers
reported the first lipase-catalyzed asymmetric aldol reaction be-
tween acetones and aldehydes in ‘wet’ acetone.12 Recently, our
group has reported direct asymmetric aldol reactions between
cyclohexanone and aromatic aldehydes catalyzed by Nuclease p1
from Penicillium citrinum, alkaline protease from Bacillus lichenifor-
mis, chymopapain from Carica papaya, and acidic protease from
Aspergillus usamii respectively.13 However, to the best of our
knowledge, the enzymatic asymmetric aldol reactions of heterocy-
clic ketones have not been reported yet. Herein we hope to report
the first direct asymmetric aldol reaction of heterocyclic ketones
with aldehydes catalyzed by PPL II.
Carbon–carbon bond-forming reactions are fundamental in or-
ganic synthesis. The aldol reaction is recognized as one of the most
powerful methods for the construction of new carbon–carbon
bonds. Therefore, the controls of both the absolute and the relative
configuration of the aldol products are of paramount importance
for the synthesis.9 There are many reports about enantioselective
aldol reactions catalyzed by small organic molecules and metal
complexes.10 Despite the plentiful variety of aldol acceptors, the
range of donors has remained narrow. Meanwhile the aldol prod-
ucts of heterocyclic ketone donors are important intermediates
In initial research, the aldol reaction of ketone 1a and 4-nitro-
benzaldehyde 2a was used as the model reaction (Table 1). Eight
hydrolases which are stable and commercially available had been
investigated to find the most suitable enzyme to catalyze the direct
aldol reaction of heterocyclic ketones. The best result, 59% ee and
43% yield, was achieved by using PPL II as catalyst after 120 h in
mixed solvents [H2O/(H2O+CH3CN) = 1:10, v/v] at 25 °C (Table 1,
entry 1). Meanwhile, some tested hydrolases also showed catalytic
activities. Among them, protease from streptomces griseus gave a
yield of 46% but no enantioselectivity was observed (Table 1, entry
2). Lipase from wheat germ gave 28% yield and 12% ee (Table 1, en-
try 3). Amano lipase A from Aspergillus niger provided 26% yield
and 13% ee (Table 1, entry 4). Trypsin from porcine pancreas gave
⇑
Corresponding authors. Fax: +86 23 68254091.
E-mail addresses: guanzhi@swu.edu.cn (Z. Guan), heyh@swu.edu.cn (Y.-H. He).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.07.007

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Z. Guan et al. / Tetrahedron Letters 53 (2012) 4959–4961
Table 1
The catalytic activities and stereoselectivities of different hydrolases to the aldol reactiona
O
O
OH
CHO
hydrolase
+
+
syn-isomer
N
NO2
25 °C, solvent/H2O
N
NO2
Boc
Boc
1a
anti-3a
2a
Entry
Enzyme
Time (h)
Yieldb (%)
drc (anti:syn)
% eed (anti)
1
2
3
4
5
6
7
8
Lipase from porcine pancreas, type II (PPL II)
Protease from Streptomces griseus
Lipase from wheat germ
Amano Lipase A from Aspergillus niger
Trypsin from porcine pancreas
Lipase from Candida rugosa
Lipase from Rhizopus niveus
Protease from Bacillus sp.
120
120
120
120
120
144
144
144
168
120
120
120
48
43
46
28
26
60:40
32:68
54:46
40:60
57:43
—
—
—
—
59
0
12
13
19
—
—
—
—
5
45
Trace
Trace
Trace
nr
43
9
9
no enzyme
10
11
12
13
Bovine serum albumin (B.S.A.)
PPL II denatured with ureae
PPL II inhibited with PMSFf
37:63
35:65
—
20
—
0
Trace
61
g
NaHCO3
31:69
a
The reaction was performed by employing ketone 1a (0.25 mmol), 4-nitrobenzaldehyde 2a (0.50 mmol), enzyme (50 mg) in 1 mL mixed solvents [H2O/
(H2O+CH3CN) = 1:10, v/v] at 25 °C.
b
Isolated yield after silica gel chromatography.
c
Determined by chiral HPLC.
d
Determined by chiral HPLC, and the absolute configuration was determined by comparison with literature.11a
e
Pre-treated with 50 mg urea in 1 mL water (0.83 M) at 100 °C for 24 h.
Pre-treated with 50 mg PMSF in 1 mL THF (0.29 M) at 25 °C for 24 h.
The reaction was performed by employing ketone 1a (0.25 mmol), 4-nitrobenzaldehyde 2a (0.25 mmol), NaHCO3 (20 mg) in 1 mL EtOH.
f
g
Table 2
45% yield and 19% ee (Table 1, entry 5). However, lipase from
Candida rugosa, lipase from Rhizopus niveus, and protease from
Bacillus sp displayed almost no activity for the aldol reaction and
just trace aldol products were observed (Table 1, entries 6–8). As
seen from Table 1, in order to verify that enzyme was necessary
and the reaction took place at the active site of enzyme, some con-
trol experiments were carried out, and the significative results
were observed. In the absence of enzyme no detectable products
were obtained for the model aldol reaction even after 168 h (Ta-
ble 1, entry 9), which indicated that catalyst was essential for the
reaction. When the reactants were incubated with urea-denatured
PPL II, just 9% yield was obtained (Table 1, entry 11), which sug-
gested the tertiary structure of the enzyme might also be neces-
sary. Then PPL II was pre-treated with PMSF (phenylmethyl
sulfonylfluoride, an irreversible serine protein inhibitor), which
gave almost no product after 120 h (Table 1, entry 12). It might
be concluded that the catalytic site of PPL II contributed to the bio-
catalytic promiscuity.14 Moreover, the reaction catalyzed by non-
enzyme protein bovine serum albumin (B.S.A.) gave product in
43% yield with only 5% ee (Table 1, entry 10), which showed that
non-enzyme protein also had the ability to catalyze aldol reaction,
but it almost did not have the enantioselectivity for aldol products.
These results implied that the reaction must take place in a specific
fashion on the catalytic site of PPL II. Therefore, PPL II was chosen
as a catalyst for the aldol reaction in our study.
The reaction medium has been recognized to be one of the most
important factors influencing the enzymatic reactions.15 Thus, we
surveyed the reaction in eight organic solvents in the presence of
water and the results are shown in Table 2. The results clearly indi-
cated that the catalytic activity and the stereoselectivity of PPL II
were significantly influenced by the reaction media. Generally,
the reaction in high-polarity solvents such as DMF and DMSO gave
higher yields (Table 2, entries 7 and 8) than in low-polarity sol-
vents such as MTBE and xylene (Table 2, entries 2 and 3). The ste-
reoselectivities including diastereo- and enantioselectivities had
no clear correlation with the polarities of solvents. It could be seen
Solvent screening of PPL II-catalyzed direct asymmetric aldol reactiona
Entry
Solvent
Yieldb (%)
drc (anti:syn)
% eec (anti)
1
2
3
4
5
6
7
8
CH3CN
MTBE
Xylene
1,4-Dioxane
THF
i-PrOH
DMSO
DMF
43
31
21
30
43
39
46
52
60:40
52:48
56:44
59:41
55:45
51:49
44:56
47:53
59
50
43
40
36
18
16
15
a
The reaction was performed by employing ketone 1a (0.25 mmol), 4-nitro-
benzaldehyde 2a (0.50 mmol), PPL II (50 mg) in 1 mL mixed solvents [H2O/
(H2O+solvent) = 1:10, v/v] at 25 °C for 120 h.
b
Isolated yield after silica gel chromatography.
Determined by chiral HPLC.
c
that PPL II gave the best yield of 52% but the lowest enantioselec-
tivity of 15% ee in DMF (Table 2, entry 8). However, the best diaste-
reoselectivity (60:40/anti:syn) and enantioselectivity (59% ee) were
obtained in CH3CN (Table 2, entry 1). In order to pursue asymmet-
ric aldol condensation, CH3CN was chosen as the reaction medium
for further investigation.
In order to optimize the reaction conditions, the influences of
water content in the reaction medium, the molar ratio of sub-
strates, enzyme concentration, and temperature were investigated.
The time course of PPL II-catalyzed aldol reaction was also plotted.
The optimal temperature of 30 °C, the water content of 0.10 [H2O/
(H2O+CH3CN) = 1:10, v/v], the 1:1 molar ratio of ketone to alde-
hyde, and the enzyme concentration of 50 mg/ml were chosen as
the optimized reaction conditions. For details, please consult the
Supplementary data.
Having established optimized reaction conditions, we probed
the generality of the PPL II-catalyzed aldol reaction. Various aro-
matic aldehydes and heterocyclic ketones were investigated. The
results were summarized in Table 3. It could be seen that the
PPL-catalyzed aldol addition of heterocyclic ketones with aromatic

Z. Guan et al. / Tetrahedron Letters 53 (2012) 4959–4961
4961
Table 3
Substrate scope of PPL II-catalyzed direct asymmetric aldol reaction of aromatic aldehydes and heterocyclic ketonesa
O
OH
O
O
PPL II
H
+
syn-isomer
+
R
R
CH3CN/H2O
30 °C
X
X
anti-3
1
2
Entry
R
X
Product
Time (h)
Yieldb (%)
drc (anti:syn)
% eed (anti)
1
2
3
4
5
6
7
8
4-NO2
3-NO2
2-NO2
3-Br
4-NO2
3-NO2
2-NO2
4-CN
4-NO2
3-NO2
2-NO2
4-CN
NBoc
NBoc
NBoc
NBoc
O
O
O
O
S
3a
3b
3c
3d
3e
3f
3g
3h
3i
120
120
120
120
120
120
144
144
120
120
144
144
49
45
36
38
56
50
43
35
41
34
31
32
62:38
32:68
53:47
47:43
38:62
37:63
48:52
34:66
51:49
80:20
83:17
54:46
62
45
87
65
46
50
62
62
43
72
74
61
9
10
11
12
S
S
S
3j
3k
3l
a
b
c
The reactions were performed by employing aldehyde (0.25 mmol), ketone (0.25 mmol), PPL II (50 mg) in 1 mL mixed solvents [H2O/(H2O+CH3CN) = 1:10, v/v] at 30 °C.
Isolated yield after silica gel chromatography.
Determined by chiral HPLC.
d
Determined by chiral HPLC, and the absolute configurations were determined by comparison with literature (for details, please consult the Supplementary data).
aldehydes gave the desired products in reasonable yields (31–56%)
and enantioselectivities (up to 87% ee) (Table 3, entries 1–12). We
investigated the electronic effect and steric effect of the substitu-
ents in aromatic aldehydes on the reaction. This procedure worked
well when benzaldehydes with electron-withdrawing groups were
employed (Table 3, entries 1–12). On the contrary, only trace prod-
ucts were obtained when benzaldehydes with electron-donating
groups, such as methyl and methoxyl, were employed (data were
not shown). This could be explained that electron-withdrawing
groups enhance the electrophilicity of carbonyl carbons in alde-
hydes which facilitates the reaction, while electron-donating
groups lessen the electrophilicity. Otherwise, the sterically
hindered substituents in benzaldehydes had a great impact on
the stereoselectivity and yield of the reaction. For instance, among
the 2-, 3-, and 4-nitrobenzaldehydes, the most sterically hindered
2-nitrobenzaldehyde provided the best enantioselectivity but the
lowest yield (Table 3, entries 3, 7, and 11), while the least sterically
hindered 4-nitrobenzaldehyde provided the best yield but rela-
tively low enantioselectivity (Table 3, entries 1, 5, and 9). We spec-
ulated that the large steric hindrance limited the attack direction of
ketone.
References and notes
1. (a) Schmid, A.; Dordick, J. S.; Hauer, B.; Kiener, A.; Wubbolts, M.; Witholt, B.
Nature 2001, 409, 258–268; (b) Pollard, D. J.; Woodley, J. M. Trend Biotechnol.
2007, 25, 66–73; (c) Aleu, J.; Bustillo, A. J.; Galan, R. H.; Collado, I. G. Curr. Org.
Chem. 2006, 10, 2037–2054; (d) Panke, S.; Held, M.; Wubbolts, M. Curr. Opin.
Chem. Biol. 2004, 15, 272–279; (e) Knowles, J. R. Nature 1991, 350, 121–124.
2. Kazlauskas, R. J. Curr. Opin. Chem. Biol. 2005, 9, 195–201.
3. (a) Bornscheuer, U. T.; Kazlauskas, R. J. Angew. Chem., Int. Ed. 2004, 43, 6032–
6040; (b) Hult, K.; Berglund, P. Trend Biotechnol. 2007, 25, 231–238; (c) Busto,
E.; Fernández, V. G.; Gotor, V. Chem. Soc. Rev. 2010, 39, 4504–4523.
4. Branneby, C.; Carlqvist, P.; Magnusson, A.; Hult, K.; Brinck, T.; Berglund, P. J. Am.
Chem. Soc. 2003, 125, 874–875.
5. (a) Li, K.; He, T.; Li, C.; Feng, X. W.; Wang, N.; Yu, X. Q. Green Chem. 2009, 6, 777–
779; (b) Chai, S. J.; Lai, Y. F.; Zheng, H.; Zhang, P. F. Helv. Chim. Acta 2010, 93,
2231–2236.
6. (a) Svedendahl, M.; Hult, K.; Berglund, P. J. Am. Chem. Soc. 2005, 127, 17988–
17989; (b) Xu, J. M.; Zhang, F.; Liu, B. K.; Wu, Q.; Lin, X. F. Chem. Commun. 2007,
27, 2078–2080; (c) Cai, J. F.; Guan, Z.; He, Y. H. J. Mol. Catal. B Enzym. 2011, 68,
240–244.
7. (a) Wu, W. B.; Wang, N.; Xu, J. M.; Wu, Q.; Lin, X. F. Chem. Commun. 2005, 18,
2348–2350; (b) Lou, F. W.; Liu, B. K.; Wu, Q.; Liu, D. S.; Lin, X. F. Adv. Synth.
Catal. 2008, 350, 1959–1962; (c) Wu, W. B.; Xu, J. M.; Wu, Q.; Liu, D. S.; Lin, X. F.
Adv. Synth. Catal. 2006, 348, 487–492.
8. (a) Tang, R. C.; Guan, Z.; He, Y. H.; Zhu, W. J. Mol. Catal. B Enzym. 2010, 63, 62–
67; (b) Wang, J. L.; Li, X.; Xie, H. Y.; Liu, B. K.; Lin, X. F. J. Biotechnol. 2010, 14,
240–243.
9. Sasai, H.; Suzuki, T.; Itoh, N. Tetrahedron Lett. 1993, 34, 851–854; (b) Abiko, A.;
Liu, J.; Masamune, S. J. Am. Chem. Soc. 1997, 119, 2586–2587.
10. (a) List, B.; Richard, A. L.; Barbas, C. F., III J. Am. Chem. Soc. 2000, 122, 2395–
2396; (b) Suri, J. T.; Ramachary, D. B.; Barbas, C. F., III Org. Lett. 2005, 7, 1383–
1385; (c) Yamada, Y. M. A.; Yoshikawa, N.; Sasai, H.; Shibasaki, M. Angew. Chem.
1997, 109, 1942–1944; (d) Ariza, X.; Garcia, J.; Romea, P.; Urpi, F. Synthesis
2011, 14, 2175–2191.
11. (a) Chen, J. R.; Li, X. Y.; Xing, X. N.; Xiao, W. J. J. Org. Chem. 2006, 71, 8198–8202;
(b) Pihko, P. M.; Laurikainen, K. M.; Usano, A.; Nyberg, A. I.; Kaavi, J. A.
Tetrahedron 2006, 62, 317.
In conclusion, we herein report a lipase (PPL II)-catalyzed direct
asymmetric aldol reaction of heterocyclic ketones with aromatic
aldehydes in CH3CN/H2O. A series of substrates participated in
the reaction. Although the yields and stereoselectivities are not
satisfied, it is interesting that PPL II possesses the function of aldol-
ase in organic solvents. This novel process also provides an exam-
ple for exploring environmentally friendly enzyme-catalyzed
synthetic route for organic chemistry in nonaqueous media.
12. Li, C.; Feng, X. W.; Wang, N.; Zhou, Y. J.; Yu, X. Q. Green Chem. 2008, 10, 616–
618.
Acknowledgments
13. (a) Li, H. H.; He, Y. H.; Yuan, Y.; Guan, Z. Green Chem. 2011, 13, 185–189; (b) Li,
H. H.; He, Y. H.; Guan, Z. Catal. Commun. 2011, 12, 580–582; He, Y. H.; Li, H. H.;
Chen, Y.; Li; Xue, Y.; Yuan, Y.; Guan, Z. Adv. Synth. Catal. 2012, 31, 712–719; (d)
Xie, B. H.; Li, W.; Liu, Y.; Li, H. H.; Guan, Z.; He, Y. H. Tetrahedron 2012, 68,
3160–3164.
Financial support from Natural Science Foundation Project of
CQ CSTC (2009BA5051) is gratefully acknowledged.
14. James, G. T. Anal. Biochem. 1978, 86, 574–579.
15. (a) Klibanov, A. M. Trends Biochem. Sci. 1989, 14, 141–144; (b) Tawaki, S.;
Klibanov, A. M. J. Am. Chem. Soc. 1992, 114, 1882–1884.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
07.007.