Enzyme-catalyzed asymmetric construction of chiral tertiary alcohols via aldol reaction using proteinase

Article abstract of DOI:10.1016/j.molcatb.2016.01.008

A new enzyme-catalyzed asymmetric construction of chiral tertiary alcohols via asymmetric aldol reactions between β,γ-unsaturated α-keto esters and ketones was reported. Proteinase from Aspergillus melleus (AMP) was used as a sustainable biocatalyst. The

Full text of DOI:10.1016/j.molcatb.2016.01.008

Accepted Manuscript
Title: Enzyme-catalyzed asymmetric construction of chiral
tertiary alcohols via aldol reaction using proteinase
Author: Rui Li Zhi-Lin Li Hai-Yan Zhou Yan-Hong He Zhi
Guana
PII:
S1381-1177(16)30008-X
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2016.01.008
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Journal of Molecular Catalysis B: Enzymatic
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3-1-2016
13-1-2016
Please cite this article as: Rui Li, Zhi-Lin Li, Hai-Yan Zhou, Yan-Hong He,
Zhi Guana, Enzyme-catalyzed asymmetric construction of chiral tertiary alcohols
via aldol reaction using proteinase, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2016.01.008
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Enzyme-catalyzed asymmetric construction of chiral tertiary alcohols via aldol
reaction using proteinase
Rui Lia, Zhi-Lin Lia, Hai-Yan Zhoub, Yan-Hong Hea*, Zhi Guana*
a Key Laboratory of Applied Chemistry of Chongqing Municipality, School of
Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR
China
b School of Life Science, Southwest University, Chongqing 400715, PR China
Fax: +86-23-68254091; e-mail: guanzhi@swu.edu.cn for Z. Guan; heyh@swu.edu.cn
for Y-H He
Graphical abstract
Chiral tertiary alcohol was obtained via asymmetric ketone-ketone aldol reaction
using proteinase from Aspergillus melleus (AMP) as a sustainable biocatalyst.
1

Highlights
 Chiral tertiary alcohols were obtained via asymmetric ketone-ketone aldol
reaction.
 Proteinase from Aspergillus melleus (AMP) was used as a sustainable biocatalyst.
 Enzymatic promiscuity was used to construct chiral tertiary alcohols.
 This work expands the application of natural enzyme in organic synthesis.
Abstract
A new enzyme-catalyzed asymmetric construction of chiral tertiary alcohols via
asymmetric aldol reactions between β,γ-unsaturated α-keto esters and ketones was
reported. Proteinase from Aspergillus melleus (AMP) was used as a sustainable
biocatalyst. The best results can be obtained with yields of up to 90%,
diastereoselectives of up to 93:7 dr, and enantioselectivities of up to 70% ee. This
work not only expands the application of enzymatic promiscuity, but also provides
more examples for constructing chiral tertiary alcohols.
Key words: proteinase from Aspergillus melleus, asymmetric aldol reaction, chiral
tertiary alcohol, enzymatic promiscuity, enzyme catalysis
1. Introduction
The structural unit containing chiral tertiary alcohol is important building blocks
of natural products and artificial biologically active molecules [1]. For example,
Fostriecin is found possessing remarkable anti-cancer and anti-fungi properties [2].
Camptothecin (CPT) is a cytotoxic quinoline alkaloid which inhibits the DNA enzyme
topoisomerase I (topo I). It was discovered in 1966 by Wall and Wani et al. in
systematic screening of natural products for anticancer drugs [3]. Taxol 6 was used in
the treatment of ovarian cancer, which was approved by the FDA in 1992 [4].
Efavirenz is Merck’s anti-HIV drug, one of the most structurally attractive
pharmaceuticals, which contains a chiral tertiary alcohol motif [5, 6]. (Figure 1)
2

Although the chiral tertiary alcohols exist widely, it is difficult to achieve these
structures because of the large steric hindrance. Thus, the effective construction of
chiral tertiary alcohols has attracted considerable attention from chemists and become
one of the hot topics in organic synthesis. And so far, there are many encouraging
enantioselective synthesis methods to construct chiral tertiary alcohols. In 1997,
Evans et al. reported bis(oxazolinyl)pyridyl (pybox)3 Cu(II) complexes as chiral
Lewis acid catalysts to catalyze enantioselective aldol additions of enolsilanes to
pyruvate esters to afford tertiary alcohols [7]. In 2005, Bolm’s group used
aminosulfoximines copper complexes to catalyze the same reactions [8]. Many other
asymmetric synthetic methods for tertiary alcohols were also reported via
Cu-catalyzed C-C bond formation such as the reactions of ketones with silyl enolates,
nitromethane, acrylates, allenic esters or zinc acetylides et al. [9]. In 2006, Hoveyda
and co-workers reported enantioselective additions of enolsilanes to α-keto esters
using AgF2 and an amino acid-based ligand that bears a pyridyl Schiff base as a chiral
catalyst [10]. In 2007, Mikami and coworkers reported ketoester-ene reactions of silyl
enol ethers to construct chiral tertiary alcohols by chiral dicationic palladium(II)
complexes [11]. In 2007, Gong’s group used an organic molecule derived from
proline and 6-methyl-2-amino pyridine for the direct aldol reaction of ketones with
α-keto acids, constructing chiral tertiary alcohols [12]. In 2010, Lu and co-workers
reported direct asymmetric aldol reaction of acetone with α-keto esters catalyzed by
primary-tertiary diamine organocatalysts [13]. In 2011, the same group disclosed an
asymmetric organocatalytic MBH reaction between isatins and acrylates using
β-isocupreidine as a catalyst affording chiral tertiary alcohols [14]. In 2012, Yang and
co-workers reported a method of construction of chiral tertiary alcohol stereocenters
via the [2,3]-Meisenheimer rearrangement [15]. In recent years, several groups
reported asymmetric construction of chiral tertiary alcohols via aldol reaction between
β,γ-unsaturated
α-keto
esters
and
cyclohexanone.
So
far,
9-amino(9-deoxy)epi-Cinchona alkaloid/acidic additive [16], chiral primary
amine-imine [17], aniline/acid [18], primary amine/4-nitrophenol [19] and
trans-siloxy-L-proline [20] have been used as the catalysts. In view of the importance
3

of chiral tertiary alcohols, it is still desirable to explore environmental friendly and
sustainable catalysts for construction of these structures.
As a kind of green and environmental benign biocatalysts, enzymes are
increasingly being used in organic synthesis. Enzymes are optimized through
evolution of a specific chemical transformation with specific substrate recognition in
metabolism. Despite this, many enzymes have the ability to catalyze distinctly
different chemical transformation of unnatural substrates, which is called enzyme
promiscuity [21-24]. Enzyme promiscuous behavior can be described as three aspects,
including enzyme reaction condition promiscuity, enzyme substrate promiscuity and
enzyme catalytic promiscuity. Biocatalytic promiscuity has attracted significant
attention from chemists and biochemists [22, 25]. For instance, some elegant works
have testified hydrolases have the ability to catalyze Michael reaction [26, 27],
Mannich reaction [28, 29], Diels-Alder reaction [30], Henry reaction [31, 32], domino
reaction [33, 34], and so on.
Aldol reaction is an important method in C-C bond-forming process in organic
chemistry [35, 36]. Aldol reaction is generally catalyzed by the aldolase in nature;
some brilliant works about aldolase-catalyzed aldol reaction have been reported
[37-41]. However, some hydrolases have been found to have the aldolase activity. In
2003, Berglund and co-workers found lipase B from Candida antarctica (CALB) can
catalyze aldol reaction between linear aldehydes [42]. Until 2008, the first asymmetric
lipase-catalyzed aldol reaction was reported by the Yu group [43]. And our group has
been exploring several enzyme-catalyzed ketone-aldehyde type aldol reactions, using
nuclease p1, lipase and protease [44-47]. Among those investigated enzymes,
proteinase from Aspergillus melleus (AMP) has the best catalytic effect in acetonitrile
in the presence of water [47]. In order to expand the application of enzymes in organic
synthesis to construct chiral tertiary alcohols, and further explore the promiscuity of
enzymes, we attempted to use AMP to catalyze ketone-ketone type aldol reactions.
Generally, asymmetric intermolecular aldol reactions of ketone-ketone type [48, 49]
are less well developed comparing with the aldol reactions of ketone-aldehyde type
and aldehyde-aldehyde type [20], and the results showed that AMP can work for this
4

transformation. Hence, we wish report AMP-catalyzed aldol reaction between
β,γ-unsaturated α-keto esters and various ketones for the construction of chiral tertiary
alcohols.
2. Experimental
2.1 Materials
The proteinase from Aspergillus melleus (AMP) [4 units/mg solid, One unit will
hydrolyze casein to produce color equivalent to 1.0 micromole of tyrosine per minute
o
at pH 7.5 at 37 C (color per Folin Ciocalteu reagent)] was purchased from
Sigma-Aldrich, Shanghai, China (P4032-25G, Lot#SLBF8373V). Unless otherwise
noted, all reagents were purchased from commercial suppliers and used without
further purification. The substrates β,γ-unsaturated α-keto esters (1a-1z) were
prepared according to the literature [50].
2.2 Analytical methods
All reactions were monitored by thin-layer chromatography (TLC, petroleum
ether/ethyl acetate as eluent) with Haiyang GF254 silica gel plates (Qingdao Haiyang
chemical industry Co Ltd, Qingdao, China) using UV light, iodine and vanillic
1
aldehyde as visualizing agents. The H NMR and 13C NMR spectra were recorded
with TMS as the internal standard in CDCl3 on a Bruker 600 MHz instrument at room
temperature. Chemical shifts were reported in ppm from TMS with the solvent
resonance as the internal standard. HRMS were recorded on a Varian 7.0 T
FTICR-MS spectrometer. Melting points were taken on a WPX-4 microscopic
melting point apparatus and were uncorrected (Yice instrument equipment Co Ltd,
Shanghai).
2.3 General procedure for the AMP-catalyzed asymmetric aldol reaction
To a 10 mL round bottom flask was added with β,γ-unsaturated α-keto ester (0.3
mmol), ketone (3.0 mmol), AMP (120 U) and deionized water (0.10 mL). The
5

o
resultant mixture was stirred on a magnetic stirrer at 25 C for the specified time and
monitored by TLC. The reaction was terminated by filtering off the enzyme using
filter paper, and the filter cake was washed with ethyl acetate. Then the solvent in
filtrate was removed under reduced pressure. The residue was purified by silica gel
flash column chromatography using 200-300 mesh silica gel at increased pressure
with petroleum ether/ethyl acetate (12:1-1:1) as eluent to afford the products (Solvent
1
ratios of the eluent mixture are listed in the Supporting Information). H NMR and
13C NMR for all the products and HRMS for unknown compounds are available in the
Supporting Information.
3. Results and discussion
Initially, the aldol reaction between β,γ-unsaturated α-keto ester (1a) and
cyclohexanone (2) was chosen as the model reaction. To confirm the specific catalytic
effect of AMP on the model aldol reaction, some control experiments were performed
(Table 1). When AMP was used as a catalyst in acetonitrile in the presence of water at
25 °C, the product was obtained in a yield of 34% with dr of 64/36 (3/4) and ee of 28%
(for 3) and 69% (for 4) (Table 1, entry 1). In the absence of enzyme, no product was
detected (Table 1, entry 2), which proved that AMP could catalyze the model aldol
transformation. Next, non-enzyme proteins, egg albumin and bovine serum albumin,
were used as catalysts, separately; low yields of 2% and 3% were obtained,
respectively (Table 1, entries 3 and 4), which indicated that the observed results from
AMP-catalyzed model reaction was not simply catalyzed by the amino acid residues
on the surface of protein. Urea as a common denaturation reagent of proteins was
used to pretreat AMP. The reaction with urea-pretreated AMP gave a low yield of 8%
(Table 1, entry 5). As a control, the same amount of urea was also used to catalyze
this reaction; only 1% yield was received (Table 1, entry 6), showing that urea alone
nearly no catalytic effect on the model reaction. These results indicated that the native
structure of AMP is responsible for the reaction. Since AMP is a serine protease,
phenylmethanesulfonyl fluoride (PMSF) as a specific inhibitor of serine protease was
6

used to pretreat AMP [51]. The reaction with PMSF-pretreated AMP only gave a low
yield of 5% (Table 1, entry 7). PMSF alone was proved no catalytic effect on the
reaction (Table 1, entry 8). Because AMP has at least one histidine residue in its
active site [52], as a modifier of the imidazole groups of histidine, diethyl
cyanophosphonate (DEPC) was used to pretreat AMP [53]. DEPC-pretreated AMP
nearly completely lost its catalytic activity for the model reaction; only 1% yield was
detected (Table 1, entry 9). DEPC alone did not show any catalytic activity even after
6 days (Table 1, entry 10). The above control experiments suggested that the native
structure of AMP is responsible for the reaction and the catalysis may take place in the
active site of AMP.
Enzymes have been discovered to maintain their activities in organic solvents
and applied for chemical transformations of organic compounds [54]. Thus, during
the course of our initial investigation, solvents with different log P values were
screened and the results were shown in Table 2. Some polar, non-polar and protic
solvents were examined, and various yields and ees were obtained (Table 2, entries
1-10). In general, the reactions in solvents with log P higher than 2.00 or lower than
-0.24 gave products in low ees (for 3) (Table 2, entries 1, 3, 5, 6 and 8). Relatively
higher ees (for 3) were obtained when log P values of solvents are between 0.49 and
0.96, such as EtOAc, THF, diethyl ether and cyclohexanone (Table 2, entries 2, 4, 7
and 10). However, solvent log P did not show obvious effects on ees for 4. Water as a
solvent was also investigated; the reaction in water gave a low yield and moderate ees
(Table 2, entry 9). Initial reaction rates in different solvents were also tested.
Specifically, the substrate cyclohexanone itself as a solvent gave the best yield of 78%
with 70:30 dr (3/4), 34% ee (for 3) and 64% ee (for 4) with the highest initial reaction
rate of 2.15 (mM/h) (Table 2, entry 10). Considering the yield, cyclohexanone was
selected as the optimal solvent. Thus, the role of cyclohexanone in this
AMP-catalyzed reaction is not only one of the substrates, but a solvent.
7

Temperature has important effect on enzymatic reactions because of its effect on
reaction rate, selectivity and the stability of enzymes. Thus, the effect of different
temperature from 15 oC to 60 oC on the model reaction was explored (Table 3). With
the rise in temperature, the yield of the product increased at first, reaching the highest
o
level of 86% at 35 C (Table 3, entry 5), and then decreased obviously with the
continuous increase of the temperature. It is probably due to the partial denaturation
of enzyme caused by higher temperature. The highest ee of 46% (for 3) was obtained
o
at 15 C (Table 3, entry 1), and then ee values decreased remarkably with increasing
the temperature. When temperature increased from 15 to 35 oC, no obvious change of
ee (for 4) was observed; further rising the temperature led to a serious decrease of ee
value. In general, increasing temperature resulted in a decline of enantioselectivity,
which is in accord with most reported enzyme catalyzed aldol reactions [55-59]. In
terms of the yield and selectivity, 25 oC was selected as the optimal temperature.
In view of the fact that the molar ratios of substrates have great influence on
reactions, the molar ratio of β,γ-unsaturated α-keto ester (1a) to cyclohexanone (2)
was optimized in the AMP-catalyzed aldol reaction (Table 4). Both dr and ee were not
obviously influenced by varying the molar ratio of substrates from 1:2 to 1:34 (Table
4, entries 1-8). However, the yield was significantly improved when the molar ratio of
substrates increased from 1:2 to 1:10 (Table 4, entries 1-3). When the molar ratio is
1:2, only a low yield of 13% was obtained (Table 4, entry 1), but a good yield of 83%
was received at the molar ratio of 1:10 (Table 4, entry 3). Continuously increasing the
molar ratio from 1:10 to 1:34 did not lead to a remarkable change in the yield (Table
4, entries 3-8). Although the best yield of 87% was obtained at 1:20 (Table 4, entry 5),
the slightly better ee was reached at 1:10 with 36% ee (for 3) and 63% ee (for 4)
(Table 4, entry 3). To get better enantioselectivity and for economic consideration,
1:10 was chosen as the optimal molar ratio for the reaction.
It is known that enzymes need essentially bound water in enzymatic reactions
because essential conformation of enzyme for catalytic activity is maintained directly
8

or indirectly by the water molecules through hydrogen bonding and other
non-covalent bonds. The amount of water required to reach the maximal activity of
enzyme differs in different organic solvents [60]. In this investigation, cyclohexanone
was used not only as a substrate but also as a solvent, which is not miscible with water.
Thus, addition of water would form a two-phase medium. To examine the effect of
water addition on the reaction, the amount of water from 0 to 0.60 mL in the reaction
system [1a (57 mg, 0.3 mmol), cyclohexanone 2 (0.31 mL, 3.0 mmol) and AMP (120
U)] was screened (Table 5). The reaction without water only gave a very low yield of
36% with low ee values of 11% (for 3) and 52% (for 4) (Table 5, entry 1). When
increasing the amount of water from 0 to 0.10 mL, the yield increased remarkably
(Table 5, entries 1-3). Further increasing the water led to a decrease of the yield
(Table 5, entries 3-10). However, ee and dr remained almost unchanged for the
reactions with water from 0.05 to 0.60 mL (Table 5, entries 2-10). The best result was
obtained with the yield of 85% and 37% ee (for 3) and 64% ee (for 4) when 0.10 mL
of water was added into the reaction system (Table 5, entry 3), and at this case the
reaction system was still two-phase even in the presence of the substrate
β,γ-unsaturated α-keto ester 1a (57 mg, 0.3 mmol). Therefore, 0.10 mL of water was
selected as the optimized water addition for the reaction.
Next, the enzyme amount was screened. When increasing the amount of AMP
from 20 U to 120 U, both the yield and ee were increased (Table 6, entries 1-6). When
the amount of AMP was increased from 120 U to 160 U, the yield and ee were almost
remained unchanged (Table 6, entries 6-8). Therefore, 120 U of AMP was chosen as
the suitable enzyme amount for the reaction.
Because the yield was low under the unoptimized conditions, to obtain as high
yield as possible, the reaction time was prolonged to 144 h for the reaction (listed in
Table 1, entry 1) based on TLC analysis. In order to compare the effects of different
parameters on the reaction under the same reaction time, all the reactions in the
optimization process were carried out for 144 h (Tables 1-6). To further investigate
9

the influence of time on the reaction, after optimizing a series of conditions, the time
course of the reaction was explored (Table 7). The yield and ee increased with the
reaction time extending (Table 7, entries 1-7). A fast increase of the yield was
investigated during the first 72 h (Table 7, entries 1-3); after that the tendency of the
yield increase slowed down (Table 7, entries 3-7). The best yield of 90% was reached
after 168 h (Table 7, entry 7).
To test the generality and scope of the reaction between β,γ-unsaturated α-keto
esters and cyclohexanone, different β,γ-unsaturated α-keto esters (1) were explored
under the optimal conditions (Table 8). The desired products could be obtained with 1
bearing various substituents, giving yields of up to 90%, drs of up to 82/18 (3/4), and
ees of up to 42% (for 3) and 70% (for 4) (Table 8). Methyl ester and ethyl ester gave
better yields and ees than isopropyl ester and benzyl ester (Table 8, entries 1-4),
probably due to the steric hindrance. β,γ-unsaturated α-keto esters (1) with various
substituents in benzene ring such as F-, Cl-, Br-, CH3O- and CH3- were investigated
(Table 8, entries 5-24). When the substituents of benzene ring were on m- and p-
positions, the ethyl esters gave higher yields than the methyl esters at the same
reaction time (Table 8, entries 5-8, 11-16, 19-24); however, when the substituents
were on o-position, the methyl esters gave higher yields than the ethyl esters (Table 8,
entries 9, 10, 17 and 18). For the methyl esters, the substituents of benzene ring on
o-position gave higher yields than m- and p- positions (Table 8, entries 5, 7, 9, 13, 15
and 17). Moreover, β,γ-unsaturated α-keto ester (1) with Ar as heterocycle pyridine
instead of phenyl ring could also work well in this transformation and good results of
corresponding products were obtained (Table 8, entries 25 and 26). These compounds
we synthesized can be converted to bicyclic lactones containing the unit of
2-hydroxy-γ-butyrolactone via reaction sequences of reduction and lactonization
(Scheme 1) [20, 61], which had been used to assemble natural products and
biologically active compounds [62].
10

To further expand upon the substrate scope of ketones, various ketones including
acyclic and cyclic ketones were also investigated (Table 9). Acetone as a
representative of acyclic ketones was used to react with methyl and ethyl esters (1)
giving low yields and ees (Table 9, entries 1 and 2). The cyclic ketones including
four-membered ring, five-membered ring and pyranoid ring were also investigated;
better results with higher yields and ees were obtained (Table 9, entries 3-8). In
general, when reacting with these cyclic ketones, the ethyl ester gave higher yields but
lower ees than the methyl ester (Table 9 entries 3-8).
According to the literatures [51, 52], AMP is a serine protease and contains at
least one histidine residue in the active site. In 2003, Berglund et al. reported aldol
reaction catalyzed by lipase CALB, containing three amino acid residues Asp, His and
Ser in active site [42]. They mutated the serine for a nonpolar residue alanine. The
wild-type and the mutant enzymes were used to catalyze the aldol reaction; the results
indicated that the mutant had better catalysis activity than the wild. They gave a
possible explanation that in the mutant enzyme the formation of hemiacetal by serine
attacking the substrate was avoided. And they proposed a mechanism, in which
histidine plays a key role for the catalysis of aldol reaction. In the present work, the
control experiments suggested that the native structure of AMP is responsible for the
aldol reaction and the catalysis may take place in the active site of enzyme. Thus,
based on the literatures and our control experiments, we attempted to propose a
possible mechanism of AMP-catalyzed aldol reaction presented in Scheme 2. Firstly,
the imidazole acts as a base to take away a proton from the ketone forming an enolate
ion. Secondly, β,γ-unsaturated α-keto ester accepts the proton from imidazolium
cation and combines the enolate ion forming a new carbon-carbon bond. Finally, the
aldol product is formed, and released from the active site.
4. Conclusion
In conclusion, the report reveals an enzyme-catalyzed method via aldol reaction
11

between β,γ-unsaturated α-keto esters and ketones that provides a simple procedure
constructing chiral tertiary alcohols. Yields of up to 90%, diastereoselectivities of up
to 93:7 dr and enantioselectivities of up to 70% ee were obtained. AMP as an
environment-friendly and sustainable biocatalyst displayed catalytic promiscuity in
this transformation. The chiral tertiary alcohols are important building blocks of
natural products and artificial biologically active molecules, but they are difficult to
be constructed because of the large steric hindrance. We utilized the enzymatic
promiscuity of AMP to achieve a series of chiral tertiary alcohols. Application of
enzyme catalysis in organic synthesis not only develops synthetic methodology but
also expands the new field of enzyme chemistry. Although enantioselectivities were
not ideal in this work, it can be a building block for the future research in this field.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of
China (no. 21276211 and no. 21472152), and the National Training Program of
Innovation and Entrepreneurship for Undergraduates (No. 201510635004).
References
[1] M. Shibasaki, M. Kanai, Chem. Rev. 108 (2008) 2853-2873.
[2] Y. Hayashi, H. Yamaguchi, M. Toyoshima, K. Okado, T. Toyo, M. Shoji, Chem.
Eur. J. 16 (2010) 10150-10159.
[3] M.E. Wall, M.C. Wani, C.E. Cook, K.H. Palmer, J. Am. Chem. Soc. 88 (1966)
3888-3890.
[4] M. C. Wani, S.B. Horwitz, Anti-Cancer. Drug. 25 (2014), 482-487.
[5] J.W. Corbett, S.S. Ko, J.D. Rodgers, L.A. Gearhart, N.A. Magnus, L.T. Bacheler,
S. Diamond, S. Jeffrey, R.M. Klabe, B.C. Cordova, S. Garber, K. Logue, G.L.
Trainor, P.S. Anderson, S.K. Erickson-Viitanen, J. Med. Chem. 43 (2000)
2019-2030.
[6] O. Riant, J. Hannedouche, Org. Biomol. Chem. 5 (2007) 873-888.
12

[7] D.A. Evans, M.C. Kozlowski, C.S. Burgey, D.W.C. MacMillan, J. Am. Chem. Soc.
119 (1997) 7893-7894.
[8] M. Langner, P. Rémy, C. Bolm, Chem. Eur. J. 11 (2005) 6254-6265.
[9] M. Shibasaki, M. Kanai, Chem. Rev. 108 (2008) 2853-2873.
[10] L.C. Akullian, M.L. Snapper, A.H. Hoveyda, J. Am. Chem. Soc. 128 (2006)
6532-6533.
[11] K. Mikami, Y. Kawakami, K. Akiyama, K. Aikawa, J. Am. Chem. Soc. 129 (2007)
12950-12951.
[12] X.Y. Xu, Z. Tang, Y.Z. Wang, S.W. Luo, L.F. Cun, L.Z. Gong, J. Org. Chem. 72
(2007) 9905-9913.
[13] Z.Q. Jiang, Y.X. Lu, Tetrahedron Lett. 51 (2010) 1884-1886.
[14] F.R. Zhong, G.Y. Chen, Y.X. Lu, Org. Lett. 13 (2011) 82-85.
[15] H. Yang, M. Sun, S.G. Zhao, M. Zhu, Y.L. Xie, C.L. Niu, C.L. Li, J. Org. Chem.
78 (2013) 339-346.
[16] P.F. Li, S.H. Chan, A.S.C. Chan, F.Y. Kwong, Adv. Synth. Catal. 353 (2011)
1179-1184.
[17] Z.J. Mao, X. Zhu, A. Lin, W.P. Li, Y. Shi, H.B. Mao, C.J. Zhu, Y.X. Cheng. Adv.
Synth. Catal. 355 (2013) 2029-2036.
[18] Y.M. Deng, L. Liu, R.G. Sarkisian, K. Wheeler, H. Wang, Z.H. Xu, Angew.
Chem. Int. Ed. 52 (2013) 3663-3667.
[19] J.W. Wei, W.G. Guo, X. Zhou, Y. Liu, C. Li, Chin. J. Chem. 32 (2014) 985-990.
[20] C.W. Zheng, Y.Y. Wu, X.S. Wang, G. Zhao, Adv. Synth. Catal. 350 (2008)
2690-2694.
[21] A. Taglieber, H. Hbenreich, J. D. Carballeira, R.J.G. Mondire, M.T. Reetz.
Angew. Chem. Int. Ed. 46 (2007) 8597-8600.
[22] K. Hult, P. Berglund, Trends Biotechnol. 25 (2007) 231-238.
[23] M.S. Humble, P. Berglund, Eur. J. Org. Chem. 2011, 3391-340.
[24] Z. Guan, L.Y. Li, Y.H. He, RSC Adv. 5 (2015) 16801-16814.
[25] O. Khersonsky, C. Roodveldt, D.S. Tawfik, Curr. Opin. Chem. Biol. 10 (2006)
498-508.
13

[26] K.L. Xu, Z. Guan, Y.H. He, J. Mol. Catal., B: Enzym 71 (2011) 108-112.
[27] M. Svedendahl, K. Hult, P. Berglund, J. Am. Chem. Soc. 127 (2005)
17988-17989.
[28] Y. Xue, L.P. Li, Y.H. He, Z. Guan, Sci. Rep. 2 (2012) 761, DOI:
10.1038/srep00761.
[29] K. Li, T. He, C. Li, X.W. Feng, N. Wang, X.Q. Yu, Green Chem. 11 (2009)
777-779.
[30] Y.H. He, W. Hu, Z. Guan, J. Org. Chem. 77 (2012) 200-207.
[31] R.C. Tang, Z. Guan, Y.H. He, W. Zhu, J. Mol. Catal., B: Enzym 63 (2010) 62-67.
[32] N. Gao, Y.L. Chen, Y.H. He, Z. Guan, RSC Adv. 3 (2013) 16850-16856.
[33] C.H. Wang, Z. Guan, Y.H. He. Green Chem. 13 (2011) 2048-2054.
[34] T. He, Q.Q. Zeng, D.C. Yang, Y.H. He, Z. Guan, RSC Adv. 5 (2015)
37843-37852.
[35] T.D. Machajewski, C.H. Wong, Angew. Chem. Int. Ed. 39 (2000) 1352-1374.
[36] R. Mestres, Green Chem. 6 (2004) 583-603.
[37] O. Eyrisch, W.D. Fessner, Angew. Chem., Int. Ed. Engl. 34 (1995) 1639-1641.
[38] P. Clapés, W.D. Fessner, G.A. Sprenger, A.K. Samland, Curr. Opin. Chem. Biol.
14 (2010) 154-167.
[39] W.D. Fessner, V. Helaine, Curr. Opin. Biotechnol. 12 (2001) 574-586.
[40] T.D. Machajewski, C.H. Wong, Angew. Chem. Int. Ed. 39 (2000) 1353-1374.
[41] A. Heine, G. DeSantis, J.G. Luz, M. Mitchell, C.H. Wong, I.A. Wilson, Science,
294 (2001) 369-374.
[42] C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck, P. Berglund, J. Am.
Chem. Soc. 125 (2003) 874-875.
[43] C. Li, X.W. Feng, N. Wang, Y.J. Zhou, X.Q. Yu, Green Chem. 10 (2008)
616-618.
[44] H.H. Li, Y.H. He, Y. Yuan, Z. Guan, Green Chem. 13 (2011) 185-189.
[45] Z, Guan, J.P. Fu, Y.H. He, Tetrahedron Lett. 53 (2012) 4959-4961.
[46] Y.H. He, H.H. Li, Y.L. Chen, Y. Xue, Y. Yuan, Z. Guan, Adv. Synth. Catal. 354
14

(2012) 712-719.
[47] Y. Yi, Z. Guan, Y.H. He, Sci. Chin. Chem. 56 (2013) 939-944.
[48] O. Tokuda, T. Kano, W.G. Gao, T. Ikemoto, K. Maruoka. Org. Lett. 7 (2005)
5103-5105.
[49] S. Konda, Q.S. Guo, M. Abe, H.C. Huang, H. Arman, J.C.G. Zhao, J. Org. Chem.
80 (2015) 806-815.
[50] L. Gremaud, A. Alexakis, Angew. Chem. Int. Ed. 51 (2012) 794-797.
[51] M. Luisetti, P.D. Piccioni, K. Dyne, M. Donnini, A. Bulgheroni, L. Pasturenzi,
A.M. Donnetta, V. Peona, Int. J. Tiss. Reac. XIII 4 (1991) 187-192.
[52] Shinichi Kobayashi, Teijiro Tazawa, Masanori Sasaki, Setsuo Kiryu, Mamoru
Sugiura. Chem. Pharm. Bull. 32 (1984) 3111-3117.
[53] G. Gesecken, Z. Physiol. Chem. 359 (1978) 1086-1087.
[54] K. Griebenow, A.M. Klibanov, J. Am. Chem. Soc. 47(1996) 11695-11700.
[55] L.Y. Li, D.C. Yang, Z. Guan, Y.H. He, Tetrahedron 71 (2015) 1659-1667.
[56] Y.L. Chen, W. Li, Y. Liu, Z. Guan, Y.H. He, J. Mol. Catal., B: Enzym 87 (2013)
83-87.
[57] Z. Guan, J.P. Fu, Y.H. He, Tetrahedron Lett 53 (2012) 4959–4961.
[58] H.H. Li, Y.H. He, Y. Yuan, Z. Guan, Green Chem. 13 (2011) 185-189.
[59] B.H. Xie, W. Li, Y. Liu, H.H. Li, Z. Guan, Y.H. He, Tetrahedron 68 (2012)
3160-3164.
[60] A. Zaks, A.M. Klibanov, J. Biol. Chem. 263 (1988) 8017-8021.
[61] X.Y. Xu, Z. Tang, Y.Z. Wang, S.W. Luo, L.F. Cun, L.Z. Gong, J. Org. Chem. 72
(2007) 9905-9913.
[62] A.G.H. Wee, B. Liu, L. Zhang, J. Org. Chem. 57 (1992) 4404-4414.
15

Figure 1 Some examples of compounds (natural and artificial) carrying chiral tertiary
alcohol.
16

Scheme 1. Transformation of the products to bicyclic lactones.
Scheme 2 Proposed mechanism for the AMP-catalyzed aldol reaction of
β,γ-unsaturated α-keto esters and ketones.
17

Table 1. Control experiments.a
Entr Catalyst
y
Yield
(3+4)
(%)b
34
n.d.k
2
dr
ee (for 3) ee (for 4)
(3/4)b (%)c
(%)d
1
2
3
4
AMP (120 U)
64/36 28
69
--
6
No enzyme
--
--
0
Egg albumin (30 mg)
83/17
82/18
Bovine serium albumin (30 3
0
5
mg)
5
AMP (pretreated with 2.5 M 8
urea)e
67/33 13
61
6
7
Ureaf
1
78/22
63/37
0
0
0
8
AMP
(pretreated
(pretreated
with 5
PMSF)g
PMSFh
AMP
8
9
n.d.k
with 1
--
--
--
63/37 14
59
DEPC)i
DEPCj
10
a
n.d.k
-- --
--
Unless otherwise noted, the reaction was conducted using 1a (0.3 mmol), 2 (0.16
mL, 5 equiv), catalyst, CH3CN (0.90 mL), and deionized water (0.10 mL), and the
mixture was stirred at 25 °C for 144 h.
b Yield and dr were determined by chiral HPLC analysis (Chiralpak AS-H column).
c
Enantiomeric excess of 3a determined by chiral HPLC analysis (Chiralpak AS-H
column).
d Absolute configuration of 4 was not ascertained and ee was determined by chiral
HPLC analysis (Chiralpak AS-H column).
e AMP (120 U) in urea solution (2.5 M) [urea (150 mg) in 1 mL H2O] was stirred at
25 °C for 24 h and then water was removed by lyophilization before use.
f Urea (150 mg) was used instead of AMP.
g AMP (120 U) and PMSF (50 mg) in THF (2 mL) was stirred at 25 °C for 24 h, then
THF was removed under reduced pressure before use.
h PMSF (50 mg) was used instead of AMP.
i AMP (120 U), phosphate buffer solution (NaH2PO4-Na2HPO4, pH 8.02) (1 mL) and
o
DEPC (0.3 mmol) was stirred at 37 C for 2 h and then water was removed by
lyophilization before use.
j DEPC (0.3 mmol) was used instead of AMP.
k n.d.: No product was detected.
18

Table 2. The screening of solvents.a
Entr Solvent
y
Log Yield
dr
ee (for 3) ee (for 4) Initial
P
(3+4)
(%)b
(3/4)b (%)c
(%)d
reaction rate
(mM/h)e
0.03
1
2
3
4
5
Toluene
EtOAc
CHCl3
THF
2.50 14
0.73 16
2.00 17
0.49 25
-0.2 31
4
76/24
75/25 32
67/33
4
27
54
54
65
41
0.09
7
0.08
72/28 46
77/23 21
0.78
EtOH
0.94
6
7
CH3CN
-0.3 34
3
0.85 43
64/36 28
76/24 37
69
52
0.26
0.54
Diethyl
ether
8
n-Hexane
H2O
3.50 45
77/23 29
75/25 36
70/30 34
44
53
64
0.58
0.28
2.15
9
--
25
10
Cyclohexan 0.96 78
one
Reaction conditions: 1a (0.3 mmol), 2 (0.16 mL, 5 equiv), AMP (120 U), solvent
(0.9 mL) and deionized water (0.1 mL) at 25 °C for 144 h.
a
b Yield and dr were determined by chiral HPLC analysis (Chiralpak AS-H column).
c
Enantiomeric excess of 3a determined by chiral HPLC analysis (Chiralpak AS-H
column).
d Absolute configuration of 4 was not ascertained and ee was determined by chiral
HPLC analysis (Chiralpak AS-H column).
e
The initial reaction rate refers to the increase of product concentration (mM/h),
which was detected by HPLC analysis (Chiralpak AS-H column).
19

Table 3. Effect of temperature on the reaction.a
Entry
Temperature
Yield (3+4) dr (3/4)b
ee (for 3) ee (for 4)
(oC)
15
20
25
30
35
40
50
60
(%)b
(%)c
46
38
33
26
16
10
10
5
(%)d
63
58
64
65
59
43
28
7
1
2
3
4
5
6
7
46
60
80
85
86
80
70
66
70/30
71/29
73/27
68/32
68/32
68/32
72/28
77/23
8
a
Reaction conditions: 1a (0.3 mmol), 2 (1.06 mL, 34 equiv), AMP (120 U) and
deionized water (0.10 mL) at specified temperature for 144 h.
b Yield and dr were determined by chiral HPLC analysis (Chiralpak AS-H column).
c
Enantiomeric excess of 3a determined by chiral HPLC analysis (Chiralpak AS-H
column).
d Absolute configuration of 4 was not ascertained and ee was determined by chiral
HPLC analysis (Chiralpak AS-H column).
20

Table 4. The screening for molar ratio of substrates.a
Entry
Molar ratio of Yield (3+4) dr (3/4)b
ee (for 3) ee (for 4)
1a:2
1:2
(%)b
13
61
83
86
87
82
83
80
(%)c
34
37
36
33
32
32
31
30
(%)d
56
58
63
65
64
62
62
60
1
2
3
4
5
6
7
76/24
76/24
74/26
74/26
73/27
74/26
73/27
73/27
1:5
1:10
1:15
1:20
1:25
1:30
1:34
8
a
Reaction conditions: 1a (0.3 mmol), 2 (0.062-1.06 mL, 0.6-10.2 mmol), AMP (120
U) and deionized water (0.10 mL) at 25 oC for 144 h.
b Yield and dr were determined by chiral HPLC analysis (Chiralpak AS-H column ).
c
Enantiomeric excess of 3a determined by chiral HPLC analysis (Chiralpak AS-H
column).
d Absolute configuration of 4 was not ascertained and ee was determined by chiral
HPLC analysis (Chiralpak AS-H column).
21

Table 5. Effect of amount of water on the reaction.a
Entry Amount of water Yield (3+4) dr (3/4)b
ee (for 3) ee (for 4)
(mL)
0
(%)b
36
60
85
81
71
68
63
58
54
47
(%)c
11
(%)d
52
63
64
64
64
65
65
68
66
66
1
2
3
4
5
6
7
8
9
71/29
75/25
75/25
74/26
74/26
74/26
75/25
75/25
75/25
75/25
0.05
0.10
0.15
0.20
0.25
0.30
0.40
0.50
0.60
34
37
36
36
36
36
37
36
36
10
a
Reaction conditions: 1a (0.3 mmol), 2 (0.31 mL, 3.0 mmol), AMP (120 U) and
deionized water (0-0.60 mL) at 25 oC for 144 h.
b Yield and dr were determined by chiral HPLC analysis (Chiralpak AS-H column ).
c
Enantiomeric excess of 3a determined by chiral HPLC analysis (Chiralpak AS-H
column).
d Absolute configuration of 4 was not ascertained and ee was determined by chiral
HPLC analysis (Chiralpak AS-H column).
22

Table 6. The screening of the amount of AMP.a
Entry The amount of Yield (3+4) dr (3/4)b
ee (for 3) ee (for 4)
AMP (U)
20
(%)b
39
48
62
70
81
85
86
87
(%)c
23
23
23
37
38
38
38
38
(%)d
47
44
45
64
63
63
64
63
1
2
3
4
5
6
7
75/25
75/25
75/25
73/27
74/26
73/27
75/25
73/27
40
60
80
100
120
140
160
8
a
Reaction conditions: 1a (0.3 mmol), 2 (0.31 mL, 3.0 mmol), AMP (20-160 U) and
deionized water (0.10 mL) at 25 oC for 144 h.
b Yield and dr were determined by chiral HPLC analysis (Chiralpak AS-H column ).
c
Enantiomeric excess of 3a determined by chiral HPLC analysis (Chiralpak AS-H
column).
d Absolute configuration of 4 was not ascertained and ee was determined by chiral
HPLC analysis (Chiralpak AS-H column).
23

Table 7. The time course of the reaction.a
Entry
Time (h)
Yield
(%)b
40
(3+4) dr (3/4)b
ee (for 3) ee (for 4)
(%)c
24
28
29
35
37
38
38
(%)d
53
1
2
3
4
5
6
24
73/27
73/27
73/27
73/27
74/26
74/26
73/27
48
65
57
72
75
56
96
79
59
120
144
168
82
63
87
63
7
90
64
a
Reaction conditions: 1a (0.3 mmol), 2 (0.31 mL, 3.0 mmol), AMP (120 U) and
deionized water (0.10 mL) at 25 oC.
b Yield and dr were determined by chiral HPLC analysis (Chiralpak AS-H column).
c
Enantiomeric excess of 3a determined by chiral HPLC analysis (Chiralpak AS-H
column).
d Absolute configuration of 4 was not ascertained and ee was determined by chiral
HPLC analysis (Chiralpak AS-H column).
24

Table 8. Substrate scope of β,γ-unsaturated α-keto esters (1).a
Entry Ar
R
Major
Time Yield
dr
ee (for ee
product
(h)
(3+4)
(3/4)c
3)
(for
(%)b
(%)d
4)
(%)e
64
42
38
50
61
50
62
54
70
64
64
52
62
52
65
52
70
54
60
60
67
1
Ph
Me
Et
168
127
112
120
114
114
114
114
96
90
79
31
32
39
50
42
59
64
50
59
61
34
49
40
56
55
37
20
34
53
73/27
81/19
82/18
78/22
69/31
78/22
74/26
74/26
69/31
73/27
69/31
74/26
71/29
73/27
69/31
67/33
69/31
74/26
70/30
72/28
70/30
38
17
6
3a
3b
3c
3d
3e
3f
2
Ph
3
Ph
i-Pr
Bn
Me
Et
4
Ph
12
19
26
31
12
22
23
34
30
28
16
36
18
26
15
29
13
37
5
4-FC6H4
4-FC6H4
3-FC6H4,
3-FC6H4
2-FC6H4
2-FC6H4
3-ClC6H4
3-ClC6H4
4-BrC6H4
4-BrC6H4
3-BrC6H4
3-BrC6H4
2-BrC6H4
2-BrC6H4
6
7
Me
Et
3g
3h
3i
8
9
Me
Et
10
11
12
13
14
15
16
17
18
19
20
21
96
3j
Me
Et
113
113
114
114
113
113
114
114
113
113
113
3k
3l
Me
Et
3m
3n
3o
3p
3q
3r
3s
Me
Et
Me
Et
4-CH3OC6H4 Me
4-CH3OC6H4 Et
3-CH3OC6H4 Me
3t
3u
25

22
23
24
25
26
3-CH3OC6H4 Et
113
119
119
127
127
66
47
52
78
86
70/30
73/27
82/18
69/31
68/32
30
29
21
42
37
57
59
33
65
53
3v
3w
3x
3y
3z
4-CH3C6H4
4-CH3C6H4
3-Pyridyl
Me
Et
Me
Et
3-Pyridyl
a Reaction conditions: 1 (0.3 mmol), 2 (0.31 mL, 3.0 mmol), AMP (120 U), and H2O
(0.10 mL) at 25 oC.
b Yield of the isolated product after chromatography on silica gel.
c The dr was determined by chiral HPLC analysis.
d Enantiomeric excess of 3a-z determined by chiral HPLC analysis (for details, please
see the Supporting Information).
e Absolute configuration of 4 was not ascertained and ee was determined by chiral
HPLC analysis.
26

Table 9. Substrate scope of ketones (5).a
Entr R
y
Major product
Time Yield
dr (6/7)c ee (for
6) (%)d (%)e
ee (for 7)
5
(h)
(6+7)
(%)b
37
1
2
3
4
5
6
7
8
Me
94
--
10
7
--
Et
117
94
29
41
54
48
68
67
73
--
--
Me
Et
58/42
54/46
75/25
77/23
79/21
93/7
21
17
12
4
10
13
28
22
48
14
104
91
Me
Et
117
90
Me
Et
18
2
101
a Reaction conditions: 1 (0.3 mmol), 5 (3.0 mmol), AMP (120 U) and deionized water
(0.10 mL) at 25 oC.
b Yield of the isolated product after chromatography on silica gel.
c The dr was determined by chiral HPLC analysis.
d Enantiomeric excess of 6a-h determined by chiral HPLC analysis (for details, please
see the Supporting Information).
e Absolute configuration of 7 was not ascertained and ee was determined by chiral
HPLC analysis.
27