Chymopapain-catalyzed direct asymmetric aldol reaction

Article abstract of DOI:10.1002/adsc.201100555

Chymopapain, a cysteine proteinase isolated from the latex of the unripe fruits of Carica papaya, displays a promiscuous activity to catalyze the direct asymmetric aldol reactions of aromatic and heteroaromatic aldehydes with cyclic and acyclic ketones in

Full text of DOI:10.1002/adsc.201100555

FULL PAPERS
DOI: 10.1002/adsc.201100555
Chymopapain-Catalyzed Direct Asymmetric Aldol Reaction
Yan-Hong He,a,b Hai-Hong Li,a,b Yan-Li Chen,a Yang Xue,a Yi Yuan,a
and Zhi Guana,*
a
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, Peopleꢀs Republic of China
Fax : (+86)-23-6825-4091; e-mail: guanzhi@swu.edu.cn
Yan-Hong He and Hai-Hong Li contributed equally to this work
b
Received: July 14, 2011; Revised: October 27, 2011; Published online: February 23, 2012
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201100555.
Abstract: Chymopapain, a cysteine proteinase isolat- 96% ee and high diastereoselectivities of up to
ed from the latex of the unripe fruits of Carica >99:1 (anti/syn) were achieved. The novel catalytic
papaya, displays a promiscuous activity to catalyze promiscuity of chymopapain widens the applicability
the direct asymmetric aldol reactions of aromatic of this biocatalyst in organic synthesis.
and heteroaromatic aldehydes with cyclic and acyclic
ketones in acetonitrile in the presence of a phosphate Keywords: aldol reaction; asymmetric synthesis; chy-
buffer. The excellent enantioselectivities of up to mopapain; enzyme catalysis; promiscuity
Introduction
group using heterobimetallic multifunctional catalysts
of the type LnLi3tris[(R)-binaphthoxide].[11] In recent
Since enzymes were discovered to be active in nearly years, numerous successful organocatalysts and metal
anhydrous organic media by Klibanov in the early catalysts for direct asymmetric aldol reactions have
1980s,[1] particularly as it has been found that the been described with high efficiency and enantioselec-
nature of the solvent can influence enzyme activity tivity,[12] yet the development of sustainable and cost-
and selectivity,[2] enzymes as biocatalysts in organic efficient catalysts for the asymmetric aldol reaction
media have attracted significant attention due to their still remains a significant challenge.[13] It is well
high selectivity, mild reaction conditions and environ- known that a number of aldol reactions occur in the
mental benign quality.[3] However, there is only a limit- living cells catalyzed by aldolases. There are some ele-
ed number of enzymes existing in nature, and the gant reports about aldolase-catalyzed aldol reac-
commercial availability of enzymes is also limited.[4] tions.[14] However, there are only a few reports of
Thus, it is a significant challenge to discover the enzy- aldol reactions catalyzed by enzymes besides
matic promiscuity of each available enzyme. In recent aldolases. Berglund and co-workers reported the first
years, a growing number of enzymes have demon- example of hydrolase-catalyzed aldol reaction in
strated their new activities with unnatural substrates 2003.[15] They used CAL-B (lipase from Candida ant-
in organic media.[5] Among those promiscuous en- arctica) and the Ser105Ala mutant CAL-B to catalyze
zymes, hydrolases are considered to be some of the aldol additions of aliphatic aldehydes (propanal or
most useful due to their good stability, broad range of hexanal). They found that the mutant had better ac-
substrate compatibility and high efficiency of forming tivity than the wild-type lipase, but both of them
various chemical bonds.[6] Several novel examples of showed quite low activities (the reaction took almost
catalytic promiscuity of hydrolases have been report- 2 months) and the enzymatic process was not enantio-
ed, such as Michael additions,[7] Markovnikov addi- selective. Wang and Yu et al. reported the first lipase-
tions,[8] direct Mannich reactions,[9] and Henry reac- catalyzed asymmetric aldol reaction in 2008.[16] They
tions.[10]
used PPL (lipase from porcine pancreas) to catalyze
The asymmetric aldol reaction is one of the most the asymmetric aldol reaction between acetone and 4-
important carbon-carbon bond-forming reactions in nitrobenzaldehyde in the presence of water, and the
organic synthesis. In 1997, the first chemocatalytic best enantioselectivity obtained was 43.6% ee. Again
direct asymmetric aldol reaction of aldehydes with in 2010, Wang and Yu et al. reported pepsin-catalyzed
unmodified ketones was realized by the Shibasaki asymmetric aldol reactions of acetone and 4-nitroben-
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Adv. Synth. Catal. 2012, 354, 712 – 719

Chymopapain-Catalyzed Direct Asymmetric Aldol Reaction
zaldehyde in the presence of water, which gave an Results and Discussion
enantioselectivity of 44% ee.[17] Recently, we reported
that the nuclease p1 from Penicillium citrinum cata- Initially, the aldol reaction between 4-cyanobenzalde-
lyzed the direct asymmetric aldol reaction between ar- hyde and cyclohexanone was used as a model reac-
omatic aldehydes and cyclic ketones under solvent- tion. Since the reaction media have a great effect on
free conditions. The products were obtained in yields the catalytic activity and the stability of an enzyme, in
of 17–55% with 49–99% ee.[18] We also found that particular, on enzyme enantioselectivity and regiose-
BLAP (alkaline protease from Bacillus licheniformis) lectivity,[21] the catalytic activity of chymopapain in
could catalyze the direct asymmetric aldol reactions the aldol reaction was evaluated in different solvents
between aromatic aldehydes and cyclic ketones in (Table 1). It could be seen that the catalytic activity
DMSO in the presence of water. The products were and the steroselectivity of chymopapain were obvi-
obtained in yields of 28–92% with 22–99% ee.[19]
ously influenced by different media. Chymopapain ex-
In continuation of our work with enzymatic syn- hibited the best catalytic activity and moderate ste-
thetic promiscuity, we report herein another new bio- reoselectivity in DMSO (Table 1, entry 1), and the
catalyst for the direct asymmetric aldol reaction. Chy- enzyme showed the best enantioselectivity of 79% ee
mopapain (EC 3.4.22.6) is the most abundant cysteine in CH2Cl2 with low diastereoselectivity (Table 1,
proteinase, and indeed protein, in the latex of the entry 7). In consideration of both diastereo- and enan-
unripe fruit of Carica papaya.[20] It is a medication tioselectivities, we chose MeCN as a suitable solvent
used to treat slipped (herniated) lower lumbar discs for the asymmetric direct aldol reaction, which gave
in the spine. We have now found that chymopapain the best dr of 77:23 and a moderate ee of 76%
also has the ability to catalyze the direct asymmetric (Table 1, entry 5) among the tested solvents.
aldol reactions of aromatic or heteroaromatic alde-
Next, we performed some control experiments to
hydes with cyclic ketones or acetone giving moderate verify the specific catalytic effect of chymopapain on
to good enantioselectivities and diastereoselectivities. the aldol reaction (Table 1, entries 8–13). The reaction
Wide substrate scopes were also investigated. This of 4-cyanobenzaldehyde with cyclohexanone in the
finding provides a novel example of enzymatic pro- absence of enzyme in MeCN at 258C only gave trace
miscuity.
amounts of adduct even after 4 days (Table 1,
entry 8). Then, the reactions catalyzed by the non-
Table 1. The effect of solvents on the chymopapain-catalyzed direct asymmetric aldol reaction and control experiments.[a]
Entry
Solvent
Time [h]
Yield [%][b]
dr[c]
ee [%][d]
1
2
3
4
5
6
7
8
DMSO
TBME
cyclohexane
THF
MeCN
H2O
CH2Cl2
MeCN (no enzyme)
MeCN (bovine serum albumin)
MeCN (egg white albumin)
MeCN (chymopapain denatured with urea[e])
MeCN (chymopapain inhibited with MMTS[f])
MeCN (papain)
89
89
89
89
89
89
89
96
130
130
96
96
89
50
17
19
30
21
18
18
trace
38
55:45
65:35
57:43
65:35
77:23
64:36
61:39
–
40
70
70
69
76
57
79
–
0
0
–
–
9
60:40
60:40
–
10
11
12
13
34
trace
trace
7
–
66:34
52
[a]
All reactions were carried out using 4-cyanobenzaldehyde (131 mg, 1.0 mmol), cyclohexanone (294 mg, 3 mmol,), enzyme
(200 mg), H2O (0.09 mL) and organic solvent (1.0 mL) at 258C.
Yield of the isolated product after chromatography on silica gel.
[b]
[c]
[d]
The dr is the anti/syn ratio, which was determined by HPLC analysis of the diastereomeric isomers.
The ee (anti) was determined by HPLC analysis using a chiral column; relative and absolute configurations of the prod-
1
ucts were determined by comparison with the known H NMR and chiral HPLC analysis results.
[e]
[f]
Pre-treated with urea at 1008C for 24 h.
Pre-treated with MMTS at 258C for 24 h.
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Yan-Hong He et al.
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Table 2. The influence of water content on the chymopa-
enzyme proteins bovine serum albumin (BSA) and
egg white albumin were also conducted, which gave
the products in yields of 38% and 34%, respectively,
however, no enantioselectivity was observed in these
two cases (Table 1, entries 9 and 10). This indicated
that non-enzyme proteins also had the ability to cata-
lyze the aldol reaction, but did not exhibit any enan-
tioselectivity for aldol products. Next, the experiment
with urea-denatured chymopapain only gave a trace
of product after 4 days (Table 1, entry 11). This sug-
gested that the tertiary structure of chymopapain was
essential in the process. Besides, a complete inhibition
of the catalytic activity of chymopapain in the aldol
reaction was observed by using the cysteine protease
inhibitor MMTS (methyl methanethiosulfonate),[22]
and only a trace of product was observed on TLC
(Table 1, entry 12). These experiments suggested that
the direct asymmetric aldol reaction must take place
on the catalytic site of chymopapain, and the catalysis
did not simply arise from the amino acid residues on
the surface of chymopapain. In addition, papain is
also a cysteine protease present in the Carica papaya
extracts, and similarly to chymopapain it is also inhib-
ited by MMTS. In order to exclude the possibility
that the chymopapain preparation had also papain ac-
tivity that catalyzed the reaction, the experiment with
papain in MeCN was conducted which gave a low
pain-catalyzed direct asymmetric aldol reaction.[a]
Entry Water content
[water/MeCN, v/v]
Yield [%][b] dr[c]
ee [%][d]
1
2
3
4
5
6
7
8
0
21
25
26
27
28
32
31
28
24
24
22
62:38 45
69:31 59
0.03
0.06
0.09
0.12
0.15
0.20
0.25
0.30
0.40
0.50
69:31 65
72:28 73
73:27 77
68:32 71
61:39 59
69:31 55
68:32 53
67:33 51
62:38 46
9
10
11
[a]
All reactions were carried out using 4-cyanobenzalde-
hyde (131 mg, 1.0 mmol), cyclohexanone (294 mg,
3.0 mmol), chymopapain (200 mg), deionized water (0–
0.50, water/MeCN, v/v) and MeCN (1.0 mL) at 258C for
98 h.
Yield of the isolated product after chromatography on
silica gel.
The dr is the anti/syn ratio, which was determined by
HPLC analysis of the diastereomeric isomers.
The ee (anti) was determined by HPLC analysis using
a chiral column.
[b]
[c]
[d]
yield of 7% with 66:34 dr and 52% ee (Table 1, enantioselectivity, the water content of 0.12 (water/
entry 13). The result indicated that papain indeed had MeCN, v/v) was chosen for the chymopapain-cata-
activity in aldol reaction, but much lower than chymo- lyzed aldol reaction.
papain. Thus, we confirmed that chymopapain cata-
lyzed the direct asymmetric aldol reaction.
Temperature is another important factor affecting
the enzyme stability, selectivity and reaction rate.
To further optimize the chymopapain-catalyzed Then, to further characterize the activity and selectivi-
direct asymmetric aldol reaction, the other main fac- ty of chymopapain in the aldol reaction, the influence
tors which affect the enzymatic reactions such as of temperature was investigated. As shown in Table 3,
water content of the reaction medium, temperature the chymopapain-catalyzed aldol reaction exhibited
and addition of the buffer were investigated, respec- the best diastereoselectivity and enantioselectivity at
tively. The reaction of 4-cyanobenzaldehyde with cy- 308C, which gave a yield of 27% with 76:24 dr (anti/
clohexanone was still used as a model reaction.
syn) and 78% ee (for anti isomer) (Table 3, entry 4).
The role of water content in the reaction medium is However, a higher temperature (408C) was required
crucial, as it dramatically influences the activity, sta- for chymopapain to display its best activity for the
bility of the enzymes and, probably, their conforma- aldol reaction. In order to get the best enantioselec-
tional flexibility.[23] Thus, the control of this parameter tivity, we chose 308C as the optimal temperature.
was proven to be vital. The water contents from 0–
Next, we investigated the time course of the chy-
0.50 (water/solvent, v/v) in MeCN were screened for mopapain-catalyzed aldol reaction between 4-cyano-
the chymopapain-catalyzed direct asymmetric aldol benzaldehyde and cyclohexanone. As shown in
reaction (Table 2). It could be seen that the water Table 4, the enantioselectivity almost kept a constant
content greatly affected the activity and selectivity of value of approximately 74% ee during the whole
chymopapain for the aldol reaction. Chymopapain ex- phase of the reaction. Similarly the diastereoselectiv-
hibited the best enantioselectivity and diastereoselec- ity also almost kept a constant value of 75:25 dr aside
tivity at the water content of 0.12 (water/MeCN, v/v), from two exceptions. The reaction progressed at
which gave the product in a yield of 28% with 73:27 a nearly constant rate from 6 h to 28 h (Table 4, en-
dr (anti/syn) and 77% ee (for anti isomer) (Table 2, tries 1–3). After that the reaction rate decreased evi-
entry 5). However, the best enzyme activity was dently. Only 26% yield was obtained after 101 h
reached at the water content of 0.15, which gave (Table 4, entry 7), and prolonging the reaction time to
a yield of 32% with lower selectivity (Table 2, 125 h did not increase the yield (Table 4, entry 8). In
entry 6). To obtain the best diastereoselectivity and order to verify whether a thermodynamic equilibrium
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Adv. Synth. Catal. 2012, 354, 712 – 719

Chymopapain-Catalyzed Direct Asymmetric Aldol Reaction
Table 3. The influence of temperature on the chymopapain-
was the reason for the limitation to 26% yield, the
analogous retro-aldol reaction was conducted under
the same reaction conditions starting from the aldol
product (A) which was prepared by the aldol reaction
of 4-cyanobenzaldehyde and cyclohexanone catalyzed
by sodium bicarbonate. We found that chymopapain
could catalyze the retro-aldol reaction (Scheme 1).
However, only 12% of aldol product (A) was convert-
ed after 100 h, and only 6% yield of 4-cyanobenzalde-
hyde was obtained while a small amount of eliminat-
ed product (B) was observed. The dr for the aldol
product (A) was 58:42 (anti/syn) before the retro-
aldol reaction, but we did not check the dr of the re-
maining A after the reaction because some aldol
product (A) was converted to eliminated product (B).
The experiment clearly indicated that a thermodynam-
ic equilibrium was not the reason for the limitation to
26% yield because the analogous retro-aldol reaction
could not reach the same product/substrate mixture.
Then the other potential explanations for the limita-
tion in conversion might be deactivation and destabi-
lization of the enzyme. To confirm these explanations
we further conducted the experiment of chymopa-
pain-catalyzed aldol reaction between 4-cyanobenzal-
dehyde and cyclohexanone. The reaction was first per-
formed under the same reaction conditions as the ex-
periment above (listed in Table 4, entry 7) for 100 h;
after that another 100 mg of chymopapain as extra
amount of catalyst were added to the reaction mix-
ture, which then was stirred for another 100 h before
terminating the reaction. In this case, the aldol prod-
uct was obtained in a much better yield of 52% with
71:29 dr (anti/syn) and 75% ee (for anti isomer). The
fact that addition of the catalyst could enhance the
yield of the aldol reaction further demonstrated that
a thermodynamic equilibrium was not the reason for
the limitation in conversion. At the same time, it sug-
gested that deactivation and destabilization of chymo-
papain may be the explanations.
catalyzed direct asymmetric aldol reaction.[a]
Entry Temperature [oC] Yield [%][b] dr[c]
ee [%][d]
1
2
3
4
5
6
7
15
20
25
30
35
40
50
6
66:34 67
11
25
27
35
38
25
72:28 71
67:33 70
76:24 78
74:26 72
70:30 66
60:40 45
[a]
All reactions were carried out using 4-cyanobenzalde-
hyde (131 mg, 1.0 mmol), cyclohexanone (294 mg,
3.0 mmol), chymopapain (200 mg), deionized water
(0.12 mL) and MeCN (1.0 mL) at temperature (15–508C)
for 94 h.
Yield of the isolated product after chromatography on
silica gel.
The dr is the anti/syn ratio, which was determined by
HPLC analysis of the diastereomeric isomers.
The ee (anti) was determined by HPLC analysis using
a chiral column.
[b]
[c]
[d]
Table 4. The time course of the chymopapain-catalyzed
aldol reaction.[a]
Entry
Time [h]
Yield [%][b]
dr[c]
ee [%][d]
1
2
3
4
5
6
7
8
6
5
60:40
75:25
74:26
76:24
68:32
76:24
75:25
75:25
73
72
74
74
75
74
75
74
17
28
41
53
77
101
125
11
17
20
22
24
26
26
[a]
All reactions were carried out using 4-cyanobenzalde-
hyde (131 mg, 1.0 mmol), cyclohexanone (294 mg,
3.0 mmol), chymopapain (200 mg), deionized water
(0.12 mL) and MeCN (1.0 mL) at 308C.
Yield of the isolated product after chromatography on
silica gel.
The dr is the anti/syn ratio, which was determined by
HPLC analysis of the diastereomeric isomers.
The ee (anti) was determined by HPLC analysis using
a chiral column.
[b]
[c]
[d]
Next, the pH value of the reaction medium plays
a significant role in maintaining the stability and cata-
lytic activity of enzymes. We then used phosphate
buffer (pH from 4.60 to 7.84) to replace the optimized
water content in the reaction system (buffer/MeCN=
0.12, v/v) to obtain the optimum reaction conditions
(Table 5). It could be seen that pH had obvious ef-
fects on the chymopapain-catalyzed direct asymmetric
Scheme 1. The chymopapain-catalyzed analogous retro-aldol reaction. Reaction conditions: A (1.0 mmol, anti/syn 58:42,),
chymopapain (200 mg), MeCN (1.0 mL) and deionized water (0.12 mL) at 308C for 100 h. Yield refers to the isolated prod-
uct after chromatography on silica gel.
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Yan-Hong He et al.
FULL PAPERS
Table 5. The influence of pH conditions (phosphate buffer)
on the chymopapain-catalyzed direct asymmetric aldol reac-
tion.[a]
substituents on benzaldehydes had a great impact on
the diastereoselectivity and the yield of the reaction.
When reacting with cyclohexanone, the benzalde-
hydes with a substituent in the o-position gave higher
dr values but lower yields (Table 6, entries 4 and 10)
than those with a substituent in the m- or p-position
(Table 6, entries 3, 5, 9 and 11). The most hindered
substrate 2,6-dichlorobenzaldehyde gave the best dia-
stereoselectivity of >99:1 dr (Table 6, entry 7). The
anti-isomers were obtained as the major products by
using cyclohexanone as an aldol donor (Table 6, en-
tries 1–11 and 15), but there was rarely any diastereo-
selectivity observed on using cyclopentanone and cy-
cloheptanone as the donors (Table 6, entries 12 and
13). Besides, furfural was also accepted by chymopa-
pain as a substrate, which gave a low yield with mod-
erate diastereo- and enantioselectivity (Table 6,
entry 15). However, chymopapain showed a poor ac-
tivity and selectivity towards acetone (Table 6,
entry 14). It seems likely that the chymopapain-cata-
lyzed aldol reaction prefers cyclic ketones over acyclic
ketones. It is also worthy to note that chymopapain
had a different degree of enantioselectivity for anti
isomers, but low or no enantioselectivity for syn iso-
mers. These results implied that the catalytic site of
chymopapain had a specific substrate selectivity as
well as stereoselectivity in direct aldol reactions.
Entry
pH
Yield [%][b]
dr[c]
ee [%][d]
1
2
3
4
5
6
7
8
4.60
4.91
5.11
5.35
5.85
6.17
6.45
6.74
7.06
7.36
7.63
7.84
37
45
42
41
41
40
39
38
38
38
36
35
72:28
72:28
73:27
67:33
72:28
73:27
72:28
68:32
73:27
76:24
73:27
74:26
70
78
77
75
74
74
74
74
74
68
63
60
9
10
11
12
[a]
All reactions were carried out using 4-cyanobenzalde-
hyde (131 mg, 1.0 mmol), cyclohexanone (294 mg,
3.0 mmol), chymopapain (200 mg), phosphate buffer
(pH 4.60–7.84, 0.12 mL) and MeCN (1.0 mL) at 308C, for
131 h.
Yield of the isolated product after chromatography on
silica gel.
The dr is the anti/syn ratio, which was determined by
HPLC analysis of the diastereomeric isomers.
the ee (anti) was determined by HPLC analysis using
a chiral column.
[b]
[c]
[d]
Based on the above results and the viewpoint, we
aldol reaction. Chymopapain showed the best activity attempted to propose a mechanism for the chymopa-
and enantioselectivity in the presence of phosphate pain-catalyzed aldol reaction. The experiments cata-
buffer (pH 4.91, buffer/MeCN=0.12, v/v), which gave lyzed by denatured enzyme indicated that the specific
the product in yield of 45% with 72:28 dr (anti/syn) natural fold of chymopapain is responsible for its abil-
and 78% ee (for anti isomer) (Table 5, entry 2). Al- ity to catalyze the direct asymmetric aldol reaction.
though the addition of phosphate buffer could not en- Also, the control experiment with MMTS-inhabited
hance the enantioselectivity, it increased the yield ob- chymopapain implied that the aldol reaction must
viously. Therefore, we chose the phosphate buffer take place on the catalytic site of chymopapain. Fur-
(pH 4.91, buffer/MeCN=0.12, v/v) as the optimum thermore, according to the X-ray structure of chymo-
conditions for aldol reaction.
papain determined by Maes and co-workers,[25] the
Subsequently, we studied the substrate scope and polypeptide chain is folded into two domains of
the generality of the chymopapain-catalyzed direct roughly the same size but with different conforma-
asymmetric aldol reaction. Various aromatic or heter- tions. One domain (the L-domain) is mainly a-helical;
oaromatic aldehydes and ketones were investigated in the other domain (the R-domain) essentially consists
MeCN in the presence of the phosphate buffer of extensive antiparallel b-sheet interactions. The
(pH 4.91, buffer/MeCN=0.12, v/v) at 308C (Table 6). active site is located at the interface between these
A wide range of substrates could participate in the re- domains. The active site Cys-25 residue is part of the
action. Five-, six- and seven-membered cyclic ketones L1 a-helix at the surface of the L-domain, while the
and acetone as aldol donors could be accepted by the His-159 is in a b-sheet at the surface of the R-domain
enzyme. Generally, chymopapain exhibited better dia- of the enzyme. The active site residues (Gln-19, Cys-
stereoselectivity and enantioselectivity with cyclohex- 25, His-159 and Asn-179) are involved in the catalytic
anone (Table 6, entries 1–11 and 15) than with cyclo- process. Their study also revealed that the structure
pentanone, cycloheptanone and acetone (Table 6, en- of chymopapain is extremely similar to that of papain,
tries 12–14). The best diastereoselectivity of >99:1 dr and differences in backbone conformation were found
(anti/syn) (Table 6, entry 7) and the best enantioselec- only for two loops at the surface of the protein, far
tivity of 96% ee (Table 6, entry 1) were achieved. away from the active site cleft. Moreover, according
Moreover, the aldehydes with election-withdrawing to Hillier and co-workersꢀ predictions about the cata-
groups gave higher yields than those with electron-do- lytic mechanism of papain,[26] and based on Berglund
nating groups. Also, the effect of steric hindrance of and co-workersꢀ mechanism of serine hydrolase
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Adv. Synth. Catal. 2012, 354, 712 – 719

Chymopapain-Catalyzed Direct Asymmetric Aldol Reaction
Table 6. Substrate scope of the chymopapain-catalyzed direct asymmetric aldol reactions.[a]
Entry
R
R1, R2
No.
Time [h]
Yield [%][b]
dr (anti/syn)[c]
ee [%][d] (anti)
1
2
3
4
5
6
7
8
4-MeC6H4
4-BrC6H4
4-ClC6H4
2-ClC6H4
3-ClC6H4
2,4-Cl2C6H3
2,6-Cl2C6H3
4-CNC6H4
4-NO2C6H4
2-NO2C6H4
3-NO2C6H4
4-NO2C6H4
4-NO2C6H4
4-NO2C6H4
2-furanyl
N
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
240
240
240
168
168
240
168
240
240
168
168
240
240
120
168
23
29
47
30
32
43
39
60
69
24
41
73
19
12
16
63:37
81:19
69:31
86:14
65:35
80:20
>99:1
72:28
63:37
87:13
87:13
56:44
47:53
–
96
84
75
79
93 (51)[e]
81
58
76
9
78 (70)[f]
82
10
11
12
13
14
15
86
52 (34)[g]
26
14
63:37
61 (44)[h]
[a]
For the general procedure see Experimental Section.
Yield of the isolated product (anti+syn) after chromatography on silica gel.
The dr was determined by HPLC analysis of the diastereomeric isomers.
[b]
[c]
[d]
The ee was determined by HPLC analysis using a chiral column; absolute configurations of the products were determined
1
by comparison with the known H NMR and chiral HPLC analysis results[24] (see the Supporting Information).
[e]
[f]
anti (93% ee), syn (51% ee).
anti (78% ee), syn (70% ee).
anti (52% ee), syn (34% ee).
anti (61% ee), syn (44% ee).
[g]
[h]
CALB-catalyzed aldol reaction,[15] we hypothesized equilibrium between the neutral (thiol-imidazole) and
the following mechanism of the chymopapain-cata- the ion-pair (thiolate-imidazolium) of the Cys-His
lyzed direct aldol reaction (Scheme 2).
couple. Firstly, the carbonyl of the substrate ketone is
The active site of chymopapain consists of a catalyt- coordinated in the Asn-His dyad and the oxyanion
ic triad formed by Cys, His and Asn, and there is an hole in the active site. Secondly, a proton is trans-
Scheme 2. Proposed mechanism for the chymopapain-catalyzed aldol reaction.
Adv. Synth. Catal. 2012, 354, 712 – 719
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
717

Yan-Hong He et al.
FULL PAPERS
Carica papaya (650 U/mg, one unit of activity was defined
as the amount of the enzyme to produce TCA-soluble hy-
drolysis products from casein, which gives an absorbance
value equivalent to 1.0 mg of tyrosine at 275 nmminÀ1 at
378C and pH 7.0) 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 purifi-
cation.
ferred from the ketone to the His residue and an eno-
late ion is formed. Thirdly, another substrate aldehyde
accepts the proton from imidazolium cation and com-
bines the ketone forming a new carbon-carbon bond.
Eventually, the product is released from the oxyanion
hole, and separates from the active site.
Conclusions
Supporting Information
In summary, here we have reported a chymopapain-
catalyzed direct asymmetric aldol reaction. Several
important factors including solvent, water content,
temperature and the addition of phosphate buffer
were examined to optimize the biocatalytic process. A
wide range of ketones including five-, six- and seven-
membered cyclic ketones and acetone as aldol donors
could be accepted by the enzyme to react with differ-
ent aromatic or heteroaromatic aldehydes. In most
cases, chymopapain showed a moderate to good enan-
tioselectivity and diastereoselectivity. Compared with
current chemical technologies, the chymopapain-cata-
lyzed direct asymmetric aldol reaction is more eco-
nomically feasible and sustainable by using inexpen-
sive regenerable resources. This case of biocatalytic
promiscuity not only expands the application of chy-
mopapain to new chemical transformations, but also
could be developed into a potentially valuable
method for organic synthesis.
General methods, the influence of some reaction conditions
on the chymopapain-catalyzed direct asymmetric aldol reac-
1
tion shown in figures, HPLC data, H NMR and 13C NMR
are available as Supporting Information.
Acknowledgements
Financial support from Natural Science Foundation Project
of CQ CSTC (2009A5051) is gratefully acknowledged.
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