Promiscuous enzyme-catalyzed cascade reaction: Synthesis of xanthone derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.06.034

Based on the screening of biocatalysts and reaction conditions including organic solvent, water content, lipase loading, reaction temperature and time, lipase TLIM exhibited the prominent promiscuity for the Knoevenagel-Michael cascade reactions of 1, 3-diketones with aromatic aldehydes to synthesize xanthone derivatives. This procedure provides satisfactory advantages such as environmental begin, simple work-up, generality, obtaining in excellent yields (80–97%), and potential for recycling of biocatalyst.

Full text of DOI:10.1016/j.bioorg.2018.06.034

Accepted Manuscript
Promiscuous Enzyme-catalyzed Cascade Reaction: Synthesis of Xanthone De-
rivatives
Yajie Fu, Bingbing Fan, Hongyue Chen, He Huang, Yi Hu
PII:
S0045-2068(18)30405-X
https://doi.org/10.1016/j.bioorg.2018.06.034
YBIOO 2414
DOI:
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
26 April 2018
12 June 2018
29 June 2018
Please cite this article as: Y. Fu, B. Fan, H. Chen, H. Huang, Y. Hu, Promiscuous Enzyme-catalyzed Cascade
Reaction: Synthesis of Xanthone Derivatives, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.06.034
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Promiscuous Enzyme-catalyzed Cascade Reaction: Synthesis
of Xanthone Derivatives
Yajie Fu, Bingbing Fan, Hongyue Chen, He Huang, Yi Hu*
(State Key Laboratory of Materials-Oriented Chemical Engineering, School of
Pharmaceutical Sciences, Nanjing Tech University, Nanjing 210009, China)
Abstract
Based on the screening of biocatalysts and reaction conditions including organic
solvent, water content, lipase loading, reaction temperature and time, lipase TLIM
exhibited the prominent promiscuity for the Knoevenagel-Michael cascade reactions
of 1, 3-diketones with aromatic aldehydes to synthesize xanthone derivatives. This
procedure provides satisfactory advantages such as environmental begin, simple
work-up, generality, obtaining in excellent yields (80-97%), and potential for
recycling of biocatalyst.
Keywords lipase, Knoevenagel condensation, aromatic aldehyde, xanthone, catalysis
1 Introduction
Xanthone derivatives comprise key structural unit in a variety of natural products and
are found to possess significant pharmacological and biological activities[1-3]. Besides,
these compounds can be applied in the field of pH sensitive fluorescent material[4],
laser technology[5] and dyestuff[6]. Owing to its important contributions in different
areas, these compounds are getting an upsurge of interest in recent years. Xanthones
are generally obtained from Knoevenagel condensation and Michael addition of 1,
3-diketones with aromatic aldehydes. Various catalysts for synthesizing xanthones
have been reported such as phase transfer catalysts TEBA[7], CsF[8], urea under
ultrasound[9], nano zeolites Fe/NaY[10], supercritical diethyl ether[11], natural
phosphates NP[12], taurine[13], acidic ionic liquids under microwave irradiation[14]
,
magnetic
nanoparticles
CuFe2O4@SiO2‐ OP2O5H[15]
,
MCM-41@Schiff
Base-Mn(OAc)2[16], and Oleylamine[17]. However, these methods are usually restricted
by volatile toxic organic solvents needed, expensive and non reusable catalysts reused,
impractical for large scale production, and sometimes only moderate yields obtained.
In recent years, enzymes have gained a wide interest of researchers for their
increasingly important role in organic synthesis due to their high efficiency and
environmental friendliness[18, 19]. It is well reported that enzymes play an important
catalytic role in various C-C bond forming reactions such as Aldol condensation,
Mannich reaction, Henry reaction, Knoevenagel condensation, Michael addition,
Baylis-Hillman reaction and so on[20, 21]. Ye et al[22] reported the use of “Amano”

lipase DF to catalyze the Knoevenagel-Michael addition cascade reaction in the
synthesis of xanthone derivatives. However, the protocol suffers some limitations
such as the use of strong polar solvent DMF which posed difficulty in the work-up
procedure, requirement of excessive aromatic aldehyde and the consumption of
relatively large amount of enzyme which couldn't be reused in this protocol.
Continuing our interest in enzyme-catalyzed C-C bond forming reactions[23, 24], we,
herein, reported the use of
immobilized lipase TLIM as catalyst in the
Knoevenagel-Michael cascade reactions of 1, 3-diketones with aromatic aldehydes to
synthesize xanthone derivatives (Scheme 1).
Scheme 1. Enzymatic reaction of aromatic aldehydes with 1, 3-diketones
2. Materials and Methods
Porcine pancreas lipase (PPL), Amano Lipase PS from Burkholderia cepacia (BCL)
and Candida rugosa lipase (CRL) were purchased from Sigma. Pancreatin from
porcine pancreas (PPP) was purchased from Aladdin Industrial Corporation. TLIM
（Lipase from Thermomyces lanuginosus, immobilized on particle silica gel）, Papain
and, Novozym 435 (lipase B from Candida antarctica, immobilized on a
macroporous acrylic resin) were purchased from Novo Nordisk. Lipase XHlip-F was
given by XHSynbio. Lipase lipoprotein from Aspergillus niger (LPL) was purchased
from Ningxia Sunson group corporation. Lipase DF was donated by Amano Enzyme
China Ltd. Other reagents were commercially available and were used without further
purification.
The melting points were determined on a WRS-1B digital melting point instrument
1
and were not corrected. The H NMR and 13C NMR spectra were measured on a
Bruker Advance 2B 400 MHz instrument with CDCl3 as solvent and TMS as internal
standard. The HRMS were measured on Agilent LC/MS mass spectrometer. The
progress of the reaction was monitored by TLC using pre-coated Haiyang GF254
silica gel plates. HPLC data was obtained using Dionex Liquid Chromatography
(Amethyst C18-H (4.6×250mm, 5μm), formic acid with 0.1% water/methanol
(v:v=2/3-3/7,15min, radiant elution), 45℃, 254nm).

2.1 General procedure for the synthesis of xanthone derivatives
A mixture of 1, 3-cyclohexanedione (2mmol), aromatic aldehyde (1mmol) and TLIM
(50mg) in n-hexane (5 mL) was stirred at 35℃. The progress of the reaction was
monitored by TLC (dichloromethane: ethyl acetate = 2:1). Upon completion of the
reaction, the reaction mixture was filtered, and the residue obtained was subsequently
dissolved in an appropriate amount of dichloromethane to separate the product and
TLIM. By simple filtration, the immobilized biocatalyst TLIM was recovered and
subjected to the next run directly, the filtrate was evaporated to obtain the crude
product which was further purified by recrystallization using 95% ethanol.
3. Results and discussion
3.1 Optimization of enzyme sources for the Knoevenagel -Michael cascade reactions
Scheme 2. Enzymatic reaction of p-chlorobenzaldehyde with 1, 3-cyclohexanedione
Table1. Effect of enzyme sources on the yield of 3a
Entrya
Enzyme
Yield(%)b
Entrya
Enzyme
Yield(%)b
1
2
3
4
5
6
No enzyme
TLIM
trace
53
7
8
CRL
Lipase DF
LPL
33
46
40
30
28
25
BCL
32
9
XHlip-F
Novozym 435
PPL
41
10
11
12
PPP
43
Papain
TLIMc
45
a Reaction conditions: lipase (50 mg), 4-chlorobenzaldehyde (1mmol), cyclohexane-1,3-dione (2mmol),
CH2Cl2 (5mL),35℃,18 h.
b HPLC yield.
c Denatured TLIM was obtained by treating with acetone for 24 h.
In an initial study, we chose p-chlorobenzaldehyde and 1, 3-cyclohexanedione as the
model
substrates
to
generate

2,2'-((4-chlorophenyl)methylene)bis(3-hydroxycyclohex-2-en-1-one) (3a), as shown
in Scheme 2. Our previous study showed that LPL and PPL efficiently catalyzed the
Knoevenagel condensation[23, 24]. Ye et al reported that Lipase DF posses the efficient
biocatalytic promiscuity for the Knoevenagel -Michael addition cascade reaction in
the synthesis of xanthone derivatives[25]. Different from the reported results, TLIM
(Table 1, entry 2) showed a relatively better catalytic efficiency than PPL, Lipase DF
and LPL (Table 1, entries 6-9). To explore the effect of specific structure of the lipase
on its catalytic efficiency, we conducted the reaction using PPP (Table 1, entry 10),
papain (Table 1, entry 11) and denatured TLIM (Table 1, entry 12) as catalyst. It
turned out that all these three enzymes showed lower catalytic efficiency compared
with lipases investigated before (Table1, entries 2-9). From these results, it could be
speculated that the specific structure of lipase imparts significant effect on the
catalytic performance during this reaction.
Table2. Effect of solvent source and water content on the yield of 3a
Entrya
Solvent
Yield(%)b
Dielectric constant
LogP
1
2
H2O
Isopropanol
Methylbenzene
CH2Cl2
66
49
48
53
35
56
56
64
95
82
74
78.5
19.9
2.4
-
0.4
2.5
1.0
0.4
0.2
-1.5
-0.6
3
3
4
8.9
5
THF
7.5
6
Acetonitrile
DMSO
37.5
47.2
38.3
1.9
7
8
DMF
9
n-Hexane
n-Hexanec
n-Hexaned
10
11
1.9
3
1.9
3
a Reaction conditions: TLIM(50 mg), 4-chlorobenzaldehyde (1mmol), cyclohexane-1,3-dione (2mmol),
solvent (5mL),35℃,18 h
b HPLC yield.
c n-hexane(3.5mL),water(1.5mL).
d n-hexane(2.5mL),water(2.5mL).

3.2 Optimization of solvent and water content for the Knoevenagel -Michael cascade
reactions
In the non-aqueous enzymatic reactions, it is reported that LogP value of the reaction
medium influences the catalytic activity[26]. It is generally believed that the catalytic
activity of an enzyme increases as the solvent LogP increases[27]. However, our results
were not found consistent with this belief as shown in Table 2. We assume that the
solubility of the substrates and products in the desired solvent also affects the reaction
rate. Table 2 showed up the joint action of solubility and LogP. Subsequently, we
chose the combination of TLIM and n-hexane to screen for the most suitable water
content. It is known that water is important for conformational flexibility of enzyme
and the biocatalytic efficiency of the enzyme increases with increase in water content
consumed by the reaction system[28, 29]. We, however, discovered that the product
yield decreased from 95% to 74% (Table 2, entries 9- 11) with the addition of water,
which is consistent with the result of Knoevenagel condensation of aromatic
aldehydes with acyclic active methylene compounds catalyzed by LPL[24]
.
3.3 Optimization of enzyme quantity, reaction time and temperature for the
Knoevenagel -Michael cascade reactions
Table3. Effects of enzyme amount and reaction time on the yield of 3a
Entrya
1
Enzyme amount(mg)
30
Time (h)
18
Temperature(℃)
Yield (%)b
78
35
2
3
4
5
6
70
50
50
50
50
18
18
14
16
24
35
35
35
35
35
90
95
79
93
93
a Reaction conditions: TLIM, 4-chlorobenzaldehyde (1mmol), cyclohexane-1,3-dione (2mmol),
n-hexane (5mL).
b HPLC yield.

25
30
35
40
45
50
55
96
94
92
90
88
86
84
82
80
78
25
30
35
40
45
50
55
Temperature (℃)
Fig.1. Effects of temperature on the yield of 3a. Reaction conditions: TLIM (50mg),
4-chlorobenzaldehyde (1mmol), cyclohexane-1, 3-dione (2mmol), n-hexane (5mL), 18h. HPLC
yield.
The concentration of enzyme exerts significant effect on the proximity of substrate
and enzyme. As shown in Table 3 (Entries 1-3), the optimum concentration of enzyme
used was 50mg, as compared to use of 200mg enzyme for 1mmol aromatic aldehydes
in the Lipase DF-catalyzed reaction system[25]. It was observed that the yield kept
increasing until the reaction time reached 18 hours (Table 3, entries 3-5). After 18
hours, the reaction yield was found nearly constant (Table 3, entry 6). The reaction
temperature also exerted very important influence on the activity of enzyme (Fig.1.).
Under these experimental conditions, the optimal reaction temperature of the enzyme
was selected as 35°C.
3.4 TLIM-catalyzed synthesis of oxindanedione derivatives
After the optimal reaction conditions were determined, the substrate applicability of
Knoevenagel-Michael cascade reactions catalyzed by lipase TLIM was further
investigated (Table 4). TLC was used to monitor the progress of reactions. It was
observed that aromatic aldehydes with electron-withdrawing substituents reacted
more readily with 1, 3-diketones as compared to aromatic aldehydes with
electron-donating substituents to generate product 3 in excellent yields (80%-97%). 1,
3 - cyclohexanedione showed higher reactivity than dimedone. Also, it was
noteworthy that satisfactory results were obtained for heterocyclic aldehydes like
2-thiophenealdehyde (Table 4, entry 18) and pyridine-2-carbaldehyde (Table 4, entry
26), as well as with aromatic aldehydes having large steric hindrance such as
4-tert-butylbenzaldehyde (Table 4, entry 13) and 2, 6-dichlorobenzaldehyde (Table 4,

entry 25). Unexpectedly, the reaction of 2, 6-dichlorobenzaldehyde and
pyridine-2-carbaldehyde with 1,3-cyclohexanedione produced 4a and 4b respectively,
which were usually obtained in presence of acid and at relatively higher temperature[7,
12,
30]. Benzaldehydes with hydroxyl group in orbit position reacted with 1,
3-cyclohexanedione to generate 5 (Table 4, entries 27-28), which could be attributed
to the dehydration reaction of phenolic hydroxyl and enol hydroxyl groups. This
result was different from the reaction of dimedone with aromatic aldehydes in water
without catalyst[31] while found similar to the reaction of dimedone with aromatic
aldehydes catalyzed by TEBA and p-toluenesulfonic acid[7]. Moreover, the developed
method was applied to a gram-scale synthesis and high yield was obtained (Table 4,
entry 29). Finally, recyclability and reusability of TLIM were investigated for the
synthesis of desired product 3a in a good yield (Table 4, entries 30-31).
Table 4. Comparing the yield of xanthenediones catalyzed by lipase TLIM
Entrya
1
X
Product
Time/h
Yield/%b
4-Cl
4-Br
3a
3b
3c
3d
3e
3f
12
18
22
14
18
18
32
14
18
16
20
16
24
14
20
95
95
91
97
87
95
82
94
93
88
84
90
82
93
92
2
3
3-NO2
4-NO2
3-Cl
4
5
6
4-F
7
2,4-di-Cl
4-CN
3g
3h
3i
8
9
H
10
11
12
13
14
15
4-OCH3
4-CH3
4-OH
3j
3k
3l
4-C4H9
4-CF3
3m
3n
3o
4-OH-3-OCH3

Entrya
X
Product
Time/h
Yield/%b
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
2-F
2-NO2
2-Thienyl
4-NO2
4-Cl
3p
3q
3r
14
18
13
18
18
14
20
24
30
32
30
14
20
18
18
18
94
85
85
96
93
92
86
90
82
80
87
85
92
94c
85d
82e
3s
3t
2-F
3u
3v
3w
3x
4a
4b
5a
5b
3a
3a
3a
2-NO2
H
4-CH3
2,6-di-Cl
2-pyridyl
2-OH
2-OH-3-OCH3
4-Cl
4-Cl
4-Cl
a Reaction conditions: TLIM (50mg), aldehyde (1mmol), 1,3-diketones(2mmol), n-hexane(5mL), 35℃.
b Isolated yield.
c
Reaction conditions: TLIM (500mg), p-chlorobenzaldehyde (10mmol), 1,3-cyclohexanedione
(20mmol), n-Hexane(50mL), 35℃, isolated yield.
d HPLC yield of 3a (run 2).
e HPLC yield of 3a (run 3).
3.5 Possible mechanism for the synthesis of oxindanedione derivatives catalyzed by TLIM
Enzymatic synthesis of xanthone derivatives was carried out using Knoevenagel
condensation-Michael addition as two-step tandem reaction. A possible mechanism
was proposed in Scheme 3: Firstly, 1, 3-cyclohexanedione or dimedone interacted
with Asp-His residue of lipase TLIM to form an enol anion, which was stable in the

oxygen ion pore of the lipase TLIM. Further, the enol anion attacked the aromatic
aldehyde to undergo Knoevenagel condensation. As a result, an intermediate was
obtained, which underwent Michael addition with the next enolate anion to produce
the desired xanthone derivatives.
Scheme 3. Possible mechanism for the synthesis of oxindanedione derivatives catalyzed by TLIM
Spectroscopic data for representative products
2,2'-((4-chlorophenyl)methylene)bis(3-hydroxycyclohex-2-en-1-one) (3a): White
solid; mp:203-205℃; 1H NMR (400 MHz, CDCl3) δ 12.33 (s, 1H), 12.03 (s, 1H), 7.21
(d, J = 8.5 Hz, 2H), 7.02 (d, J = 8.0 Hz, 2H), 5.40 (s, 1H), 2.68 – 2.52 (m, 4H), 2.50 –
2.33 (m, 4H), 2.02 (dt, J = 13.6, 7.6 Hz, 4H); 13C NMR (100 MHz, CDCl3) δ 192.27,
190.90, 136.49, 131.57, 128.27, 127.95, 116.19, 33.49, 32.99, 32.57, 20.07;
HRMS(ESI-TOF) m/z [M+Na]+：369.0864，found: 369.0880.
9-(2,6-dichlorophenyl)-3,4,5,6,7,9-hexahydro-1H-xanthene-1,8(2H)-dione
(4a):
1
White solid; mp:260-262℃; H NMR (400 MHz, CDCl3) δ 7.35 (d, J = 7.8 Hz, 1H),
7.09 (d, J = 7.8 Hz, 1H), 6.99 (t, J = 8.0 Hz, 1H), 5.52 (s, 1H), 2.65 – 2.46 (m, 4H),
13
2.36 – 2.28 (m, 4H), 2.06 – 1.90 (m, 4H); C NMR (100 MHz, CDCl3) δ 196.74,
165.36, 136.26, 129.53, 128.60, 127.77, 113.37, 37.02, 30.29, 27.15, 20.29;
HRMS(ESI-TOF) m/z [M+Na]+：385.0369, found: 385.0383.
9-(2-hydroxy-6-oxocyclohex-1-en-1-yl)-2,3,4,9-tetrahydro-1H-xanthen-1-one
(5a):
White solid; mp:230-232℃; 1H NMR (400 MHz, CDCl3) δ 10.86 (s, 1H), 7.15 (dq, J
= 7.9, 3.9 Hz, 1H), 7.04 – 6.99 (m, 3H), 4.64 (s, 1H), 2.76 (dt, J = 17.8, 4.7 Hz, 1H),
2.65 – 2.48 (m, 3H), 2.42 (ddd, J = 17.0, 11.4, 5.5 Hz, 2H), 2.35 – 2.15 (m, 2H), δ
2.11 – 1.87 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ 196.62, 167.25, 150.03,
128.88, 127.31, 126.19, 124.71, 121.86, 121.84, 120.03, 115.69, 112.42, 37.13, 34.25,
27.72, 20.86, 20.75. HRMS(ESI-TOF) m/z [M+Na]+：333.1097, found: 333.1110.

4. Conclusion
In summary, an efficient enzymatic method for the synthesis of xanthone derivatives
was developed using Knoevenagel-Michael cascade reactions of 1, 3-diketones with
aromatic aldehydes. The use of separable solvent, recyclable biocatalyst, high yields
and the ability to be scaled up, are the important attributes of the present protocol. The
present methodology has extended the potential use of TLIM in organic and
pharmaceutical synthesis.
Acknowledgments
This research was financially supported by the National Natural Science Foundation
of China (No.21676143), the self-owned research project from key laboratory of
Materials-Oriented Chemical Engineering (No. ZK201603), the Jiangsu Synergetic
Innovation Center for Advanced Bio-Manufacture, and the Qing Lan Project of
Jiangsu Province.

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Abstract
Based on the screening of biocatalysts and reaction conditions including organic
solvent, water content, lipase loading, reaction temperature and time, lipase TLIM
exhibited the prominent promiscuity for the Knoevenagel-Michael cascade reactions
of 1, 3-diketones with aromatic aldehydes to synthesize xanthone derivatives. This
procedure provides satisfactory advantages such as environmental begin, simple
work-up, generality, obtaining in excellent yields (80-97%), and potential for
recycling of biocatalyst.
Scheme 1. Enzymatic reaction of aromatic aldehydes with 1, 3-diketones
Scheme 2. Enzymatic reaction of p-chlorobenzaldehyde with 1, 3-cyclohexanedione
Table1. Effect of enzyme sources on the yield of 3a

Entrya
Enzyme
Yield(%)b
Entrya
Enzyme
Yield(%)b
1
2
3
4
5
6
No enzyme
TLIM
trace
53
7
8
CRL
Lipase DF
LPL
33
46
40
30
28
25
BCL
32
9
XHlip-F
Novozym 435
PPL
41
10
11
12
PPP
43
Papain
TLIMc
45
a Reaction conditions: lipase (50 mg), 4-chlorobenzaldehyde (1mmol), cyclohexane-1,3-dione (2mmol),
CH2Cl2 (5mL),35℃,18 h.
b HPLC yield.
c Denatured TLIM was obtained by treating with acetone for 24 h.
Table2. Effect of solvent source and water content on the yield of 3a
Entrya
Solvent
Yield(%)b
Dielectric constant
LogP
1
2
H2O
Isopropanol
Methylbenzene
CH2Cl2
66
49
48
53
35
56
56
64
95
82
74
78.5
19.9
2.4
-
0.4
2.5
1.0
0.4
0.2
-1.5
-0.6
3
3
4
8.9
5
THF
7.5
6
Acetonitrile
DMSO
37.5
47.2
38.3
1.9
7
8
DMF
9
n-Hexane
n-Hexanec
n-Hexaned
10
11
1.9
3
1.9
3
a Reaction conditions: TLIM(50 mg), 4-chlorobenzaldehyde (1mmol), cyclohexane-1,3-dione (2mmol),
solvent (5mL),35℃,18 h

b HPLC yield.
c n-hexane(3.5mL),water(1.5mL).
d n-hexane(2.5mL),water(2.5mL).
Table3. Effects of enzyme amount and reaction time on the yield of 3a
Entrya
1
Enzyme amount(mg)
30
Time (h)
18
Temperature(℃)
Yield (%)b
78
35
2
3
4
5
6
70
50
50
50
50
18
18
14
16
24
35
35
35
35
35
90
95
79
93
93
a Reaction conditions: TLIM, 4-chlorobenzaldehyde (1mmol), cyclohexane-1,3-dione (2mmol),
n-hexane (5mL).
b HPLC yield.
25
30
35
40
45
50
55
96
94
92
90
88
86
84
82
80
78
25
30
35
40
45
50
55
Temperature (℃)
Fig.1. Effects of temperature on the yield of 3a. Reaction conditions: TLIM (50mg),
4-chlorobenzaldehyde (1mmol), cyclohexane-1, 3-dione (2mmol), n-hexane (5mL), 18h. HPLC
yield.

Table 4. Comparing the yield of xanthenediones catalyzed by lipase TLIM
Entrya
1
X
Product
Time/h
Yield/%b
4-Cl
4-Br
3a
3b
3c
3d
3e
3f
12
18
22
14
18
18
32
14
18
16
20
16
24
14
20
14
18
13
18
18
14
20
95
95
91
97
87
95
82
94
93
88
84
90
82
93
92
94
85
85
96
93
92
86
2
3
3-NO2
4-NO2
3-Cl
4
5
6
4-F
7
2,4-di-Cl
4-CN
3g
3h
3i
8
9
H
10
11
12
13
14
15
16
17
18
19
20
21
22
4-OCH3
4-CH3
4-OH
3j
3k
3l
4-C4H9
4-CF3
4-OH-3-OCH3
2-F
3m
3n
3o
3p
3q
3r
2-NO2
2-Thienyl
4-NO2
4-Cl
3s
3t
2-F
3u
3v
2-NO2

Entrya
X
Product
Time/h
Yield/%b
23
24
25
26
27
28
29
30
31
H
4-CH3
3w
3x
4a
4b
5a
5b
3a
3a
3a
24
30
32
30
14
20
18
18
18
90
82
2,6-di-Cl
2-pyridyl
2-OH
80
87
85
2-OH-3-OCH3
4-Cl
92
94c
85d
82e
4-Cl
4-Cl
a Reaction conditions: TLIM (50mg), aldehyde (1mmol), 1,3-diketones(2mmol), n-hexane(5mL), 35℃.
b Isolated yield.
c
Reaction conditions: TLIM (500mg), p-chlorobenzaldehyde (10mmol), 1,3-cyclohexanedione
(20mmol), n-Hexane(50mL), 35℃, isolated yield.
d HPLC yield of 3a (run 2).
e HPLC yield of 3a (run 3).
Scheme 3. Possible mechanism for the synthesis of oxindanedione derivatives catalyzed by TLIM

An efficient enzymatic method for the synthesis of xanthone derivatives was
developed using Knoevenagel-Michael cascade reactions of 1, 3-diketones with
aromatic aldehydes. The use of separable solvent, recyclable biocatalyst, high yields
and the ability to be scaled up, are the important attributes of the present protocol. The
present methodology has extended the potential use of TLIM in organic and
pharmaceutical synthesis.