Rapid preparation of (methyl)malonyl coenzyme A and enzymatic formation of unusual polyketides by type III polyketide synthase from Aquilaria sinensis

Article abstract of DOI:10.1016/j.bmcl.2015.01.045

(Methyl)malonyl coenzyme A was rapidly and effectively synthesized by a two-step procedure involving preparation of N-hydroxysuccinimidyl (methyl)malonate from (methyl)Meldrum's acid, and followed by transesterification with coenzyme A. The synthesized (methyl)malonyl coenzyme A could be well accepted and assembled to 4-hydroxy phenylpropionyl coenzyme A by type III polyketide synthase from Aquilaria sinensis to produce dihydrochalcone and 4-hydroxy-3,5-dimethyl-6-(4-hydroxyphenethyl)-2H-pyrone as well as 4-hydroxy-3,5-dimethyl-6-(5-(4-hydroxyphenyl)-3-oxopentan-2-yl)-2H-pyrone.

Full text of DOI:10.1016/j.bmcl.2015.01.045

Bioorganic & Medicinal Chemistry Letters 25 (2015) 1279–1283
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rapid preparation of (methyl)malonyl coenzyme A and enzymatic
formation of unusual polyketides by type III polyketide synthase
from Aquilaria sinensis
a,b,c
, Xiao-Hui Wang , Xiao Liu , She-Po Shi , Peng-Fei Tu a,
⇑
a
a
a,
⇑
Bo-Wen Gao
a
Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China
Baotou Medical College, Baotou 014060, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
(Methyl)malonyl coenzyme A was rapidly and effectively synthesized by a two-step procedure involving
preparation of N-hydroxysuccinimidyl (methyl)malonate from (methyl)Meldrum’s acid, and followed by
transesterification with coenzyme A. The synthesized (methyl)malonyl coenzyme A could be well
accepted and assembled to 4-hydroxy phenylpropionyl coenzyme A by type III polyketide synthase from
Aquilaria sinensis to produce dihydrochalcone and 4-hydroxy-3,5-dimethyl-6-(4-hydroxyphenethyl)-2H-
pyrone as well as 4-hydroxy-3,5-dimethyl-6-(5-(4-hydroxyphenyl)-3-oxopentan-2-yl)-2H-pyrone.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 25 August 2014
Revised 7 January 2015
Accepted 21 January 2015
Available online 29 January 2015
Keywords:
Meldrum’s acid
Malonyl-CoA
Methylmalonyl-CoA
Type III polyketide synthase
Aquilaria sinensis
Malonyl coenzyme A (malonyl-CoA) and methylmalonyl coen-
zyme A (methylmalonyl-CoA) are universally used as chain
and protein preparation, and always accompanying with low yield
of malonyl-CoA. Besides of enzymatic synthesis of malonyl-CoA,
extenders by polyketide synthases (PKSs) to synthesize a large
chemical synthesis of (methyl)malonyl-CoA by transesterification
number of structurally diverse polyketides1
–10
exhibiting a vast
of (methyl)malonic monothiophenyl ester
28–30
or S-malonyl-N-
3
1
array of biological and pharmacological activities such as antibac-
decanoyl cysteamine with Coenzyme A have also been reported.
However, these methods are hard to differentiate the two ends of
(methyl)malonic acid, which results in the preparation of monothi-
ophenyl ester or S-malonyl-N-decanoyl cysteamine nonselective,
and multiple steps for purification of the committed intermediate
are needed. It has been reported that malonyl-CoA could be
directly synthesized by stirring coenzyme A in cold malonyl
1
1,12
13,14
15
16
terial,
nosuppressive properties. As the most common chain extenders,
methyl)malonyl-CoA could be repeatedly assembled to a start
antifungal,
anticancer, antioxidation, and immu-
17
(
coenzyme A thioester by PKSs via decarboxylative Claisen conden-
sation to construct a linear poly-b-ketone intermediate which
could finally format structurally varied polyketides by sequential
1
8–
32
modifications such as reduction, cyclization and aromatization.
dichloride, while the low yield of malonyl-CoA made this method
2
0
Owing to the wide range of substrate tolerance and unordinary
not practically useful to prepare large amount of malonyl-CoA.
Therefore, it is still a challenge to rapidly and cost effectively syn-
thesize large amount of (methyl)malonyl-CoA.
flexibility of structural modification, PKSs are ideal biocatalysts for
construction of unusual molecule libraries for screening of new
2
1,22
drug candidates,
methyl)malonyl-CoA prohibits large scale in vitro synthesis of
these unusual molecules. In previous reports, enzymes related to
while the high cost of commercially available
Here we report a more rapid and cost effective method to syn-
thesize (methyl)malonyl-CoA with high yield (more than 90%
according to the amount of coenzyme A used). The two-step
procedure involves preparation of N-hydroxysuccinimidyl
(methyl)malonate via N-hydroxysuccinimide nucleophilic attack-
ing commercially available (methyl)Meldrum’s acid, and followed
by transesterification with coenzyme A. Notably, the purity of
the synthesized (methyl)malonyl-CoA could reach to more than
(
2
3
the formation of malonyl-CoA such as acetyl-CoA carboxylase
and malonyl-CoA synthase2
4–27
have been cloned and applied to
enzymatically synthesize malonyl-CoA. However, enzyme cata-
lyzed synthesis of malonyl-CoA involves laborious gene cloning
9
2
0% after purification by a simple procedure using Sephadex LH-
0 column chromatography. Enzymatic reaction demonstrated
⇑
Corresponding authors. Tel./fax: +86 10 64286350.
E-mail addresses: shishepo@163.com (S.-P. Shi), pengfeitu@163.com (P.-F. Tu).
http://dx.doi.org/10.1016/j.bmcl.2015.01.045
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

1
280
B.-W. Gao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1279–1283
R
O
O
N
O
O
O
O
O
O
O
toluene
Coenzyme A
PH 8.0
+
HO
HO
O
N
O
HO
O
O
SCoA
1
40 °C 4 h
R
R
1
1
a: R=H
b: R=-CH3
2
3a: R=H
3b: R=-CH3
5a: R=H
5b: R=-CH3
NH2
N
N
HO
O
O
O
O
P
N
N
Coenzyme A = HS
O
O
O
N
N
H
O
P
H
OH
OH
O OH
HO
HO
P
O
4
Scheme 1. Strategy for (methyl)malonyl-CoA synthesis.
O
O
SEnz
O
HO
OH
O
OH
OH
OH
CoAS
OH
(
(
A)
B)
SCoA
AsCHS
+
+
3
2
x
O
HO
HO
O
O
O
OH
7
O
6
(920, 100%) OH
O
CH
3
O
O
SCoA
CoAS
OH AsCHS
EnzS HO
O
H
x
x
O
H
3
C
CH
3
3
C
CH
3
OH
(127, 13.8%)
O
6
8
OH
OH
CH
3
O
CH3
CH3
O
O
(
(
C)
D)
HO
O
EnzS
O
H
3
SCoA
CoAS
CoAS
OH
AsCHS
AsCHS
+
3
O
O
H
3
C
CH
3
HO
HO
O
O
C
CH
3
OH
6
0
9
(134, 14.6%)
O
O
SEnz
O
HO
OH
OH
OH
OH
SCoA
+
+
3
3
x
x
O
O
O
OH
O
O
1
1
1 (710, 77.2%)
O
O
SEnz
HO
OH
CoAS
OH
(
E)
SCoA
AsCHS
AsCHS
O
O
O
O
O
OH
O
13 (641, 69.7%)
12
OH
OH
O
CH
3
EnzS HO
O
H
O
O
SCoA
CoAS
OH
+
2
x
(
(
F)
HO
HO
O
O
O
H
3
C
CH
3
3
C
CH
3
3
OH
O
1
0
14 (66, 7.2%)
OH
OH
CH
3
O
CH3
CH
O
O
CoAS
OH
AsCHS
EnzS HO
O
SCoA
G)
+
3
x
O
O
O
3
H C
CH
3
H
3
C
CH
3
OH
O
1
0
15 (89, 9.7%)
Scheme 2. Proposed mechanism for enzymatic formation of varied polyketides catalyzed by AsCHS. The numbers in each parenthese are the values of absolute peak area of
each enzymatic product in HPLC and percentage relative to the maximum peak area of naringenin chalcone (7).

B.-W. Gao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1279–1283
1281
that (methyl)malonyl-CoA prepared by the present method could
be well accepted by type III PKS from Aquilaria sinensis to produce
structurally unusual polyketides such as dihydrochalcone from the
condensation of 4-hydroxy phenylpropionyl-CoA with three mole-
cules of prepared malonyl-CoA, and 4-hydroxy-3,5-dimethyl-6-(4-
hydroxyphenethyl)-2H-pyrone from the condensation of 4-
hydroxy phenylpropionyl-CoA with two molecules of prepared
methylmalonyl-CoA as well as 4-hydroxy-3,5-dimethyl-6-(5-(4-
hydroxyphenyl)-3-oxopentan-2-yl)-2H-pyrone from the conden-
sation of 4-hydroxy phenylpropionyl-CoA with three molecules
of prepared methylmalonyl-CoA.
95%. The purified malonyl-CoA was identified by comparing its
retention time in the HPLC chromatogram and UV spectrum to
those of the authentic malonyl-CoA. The retention time of syn-
thetic malonyl-CoA in HPLC chromatogram was completely identi-
cal to that of the authentic malonyl-CoA. Additionally, the UV
spectrum of the synthesized malonyl-CoA indicated superposable
feature to that of the authentic malonyl-CoA. ESI-MS spectrum
showed the presence of three quasimolecular ion peaks at m/z
+
+
+
854 [M+H] , 876 [M+Na] , and 892 [M+K] which provided further
powerful evidence for the identification of the synthetic malonyl-
CoA (Supplementary data).
Meldrum’s acid (1a) and N-hydroxysuccinimide (2) were
refluxed in toluene for 4 h, the reaction mixture was completely
removed the solvent under reduce pressure, and the residue was
recrystallized in absolute ethanol to afford the key intermediate
N-hydroxysuccinimidyl malonate (3a). The purified intermediate,
N-hydroxysuccinimidyl malonate, was subsequently stirred with
coenzyme A (4) in weak alkaline aqueous solution (pH = 8.0) to
yield malonyl-CoA (5a) (Scheme 1). The reaction progress was
monitored by HPLC showing the appearance of malonyl-CoA
accompanied by the disappearance of coenzyme A. The reaction
was terminated when the free coenzyme A completely disappeared
after 12 h later, and the reaction mixture was purified by Sephadex
LH-20 column chromatography to afford malonyl-CoA. HPLC anal-
ysis suggested that excess N-hydroxysuccinimidyl malonate in the
reaction mixture could be completely removed by Sephadex LH-20
column chromatography, and the purity of malonyl-CoA reached to
As a commercially available and commonly used substrate for
3
3–35
acylation chemistry,
Meldrum’s acid could specifically
differentiate the two ends of malonic acid, and thus leading to sig-
nificantly increase the yield of the key intermediate N-hydroxy-
succinimidyl malonate. Furthermore, the moderate reaction
conditions and the convenient purification process avoided the
unexpected loss and hydrolysis of malonyl-CoA.
Using the same strategy, methylmalonyl-CoA could also be syn-
thesized from methylMeldrum’s acid (1b) (Scheme 1). In the ESI-
MS spectrum of the synthesized methylmalonyl-CoA (5b), quasi-
+
+
molecular ion peaks at m/z 868 [M+H] and 978 [M+5NaÀ4H]
were observed which was identical to the data reported by Pad-
3
0
makumar. Comparing the NMR data of the synthesized methyl-
malonyl-CoA with those of the free coenzyme A, a methyl group
presented at d 1.37 (d, J = 7.6 Hz) and the sulfureted methylene
group presented at relative downfield d 3.11 (t, J = 6.2 Hz) in the
mAU
100
A
11
80
60
40
20
0
220 240 260 280 300 320 340 360 380nm
0
2
4
6
8
10
12
14
16
18
min
mAU
B
300
250
200
150
100
OH
HO
OH
50
0
OH
O
4
2
20 240 260 280 300 320 340 360 380nm
0
2
6
8
10
12
14
16
18
min
mAU
60
1
C
13
140
1
20
00
1
8
0
0
0
0
6
4
2
0
220 240 260 280 300 320 340 360 380nm
0
2
4
6
8
10
12
14
16
18
min
mAU
D
3
50
3
00
HO
OH
O
250
2
1
1
00
50
00
5
0
0
OH
220 240 260 280 300 320 340 360 380 nm
0
2
4
6
8
10
12
14
16
18
min
Figure 1. The HPLC elution profiles and UV spectra of AsCHS enzyme reaction products and the authentic compounds. The enzyme product from malonyl-CoA and 4-hydroxy
phenylpropionyl-CoA (A), the authentic compound of phloretin (B), the enzyme product from malonyl-CoA and benzoyl-CoA (C), and the authentic compound of 2,4,6-
trihydroxybenzophenone (D).

1
282
B.-W. Gao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1279–1283
OH
OH
CH3
O
O
O
O
mAU
40
O
mAU
H C
CH3
H C
CH
3
3
3
3
3
2
2
1
1
5
0
5
0
5
0
OH
OH
ꢀꢂ
3
2
0
0
ꢀ
ꢁ
10
0
5
0
2
20 240 260 280 300 320 340 360 380nm
2
20 240 260 280 300 320 340 360 380nm
ꢀ
ꢁ
ꢀ
ꢂ
ꢃ
ꢄ
ꢁ
ꢅ
ꢆ
ꢀꢃ
ꢀꢄ
ꢀꢁ
ꢀꢅ
ꢀꢆ
PLQ
Figure 2. The HPLC elution profile and UV spectra of the AsCHS enzyme reaction products 14 and 15 from 4-hydroxy phenylpropionyl-CoA and methylmalonyl-CoA.
1
H NMR spectrum as well as the signals presented at d 14.3 and
01.8 in the C NMR spectrum of the synthesized methylmalo-
Remarkably, When AsCHS was incubated with the synthesized
methylmalonyl-CoA and 4-hydroxy phenylpropionyl-CoA, the
enzymatic reaction generated a 1.3:1 mixture of two products hav-
1
3
2
nyl-CoA suggested that a methylmalonic acid moiety was success-
3
6
+
fully introduced into the molecular by transesterification
Supplementary data).
In order to test the activity of the synthesized (methyl)malonyl-
ing different parent ion peaks at m/z 261.1114 [M+H] and
317.1379 [M+H] in the LCMS-IT-TOF spectra (Fig. 2). The 1
+
H
(
NMR spectrum of 14 showed the presence of two methyl singlets
at d 1.90 (3H, s) and 1.72 (3H, s), two methylene triplets at d
2.85 (2H, t, J = 6.8 Hz) and 2.78 (2H, t, J = 6.8 Hz), and four aromatic
protons at d 6.96 (2H, d, J = 8,4 Hz,) and 6.67 (2H, d, J = 8.4 Hz),
CoA, a type III polyketide synthase (AsCHS) was cloned from Aqui-
laria sinensis. AsCHS shares 77.4% amino acid sequences identity to
those of chalcone synthase from Medicago sativa37 and the highly
conserved Cys-His-Asn cytalytic triad of type III polyketide syn-
thase could also be observed in AsCHS. Phylogenetic analysis
revealed that AsCHS grouped with other chalcone-producing type
III polyketide synthases, including Medicago sativa CHS2, Rheum
palmatum CHS2, Scutellaria baicalensis CHS, and Ruta graveolensis
CHS (Supplementary data). The Chalcone-producing function of
AsCHS was confirmed by enzymatic formation of naringenin chal-
cone (7) from the condensation of 4-coumaroyl-CoA (6) with three
molecules of malonyl-CoA. Additionally, when methylmalonyl-CoA
was used as the chain extender, AsCHS could catalyze the conden-
sation of 4-coumaroyl-CoA with two or three molecules of methyl-
malonyl-CoA to produce two known polyketides, (E)-4-hydroxy-6-
1
3
respectively. The C NMR spectrum of 14 showed the presence
of two methyl carbons at d 8.5 and 7.5, carbons attributed to 4-
hydroxy phenylethyl group at d 155.4, 131.0, 129.0, 114.7, 32.6,
and 32.2, and the presence of carbons due to lactone ring at d
168.1, 167.4, 157.4, 109.3 and 97.1, respectively. In the NMR spec-
tra of 15, the signals due to 4-hydroxy phenylethyl moiety and 3,5-
dimethyl-4-hydroxy lactone moiety were typically observed. In
addition, a methyl doublet at d 1.31 (3H, d, J = 6.7 Hz), and a
1
methine multiplet at d 3.88 (1H, m) in the H NMR spectrum of
15, and a methyl carbon at d 12.0, a methine carbon at d 48.3,
1
3
and a carbonyl carbon at d 207.0 in the C NMR spectrum of 15
were presented. By analysis of the spectroscopic data including
1
13
(
4-hydroxystyryl)-3,5-dimethyl-2H-pyrone (8) and (E)-4-hydroxy-
H, C, HSQC, and HMBC, the structures of the new polyketides
6
-(5-(4-hydroxyphenyl)-3-oxopent-4-en-2-yl)-3,5-dimethyl-2H-
14 and 15 were unambiguously elucidated as 4-hydroxy-3,5-
dimethyl-6-(4-hydroxyphenethyl)-2H-pyrone 14 and 4-hydroxy-
3,5-dimethyl-6-(5-(4-hydroxyphenyl)-3-oxopentan-2-yl)-2H-pyr-
38
pyrone (9), respectively (Scheme 2).
When AsCHS was incubated with the synthesized malonyl-CoA
and 4-hydroxy phenylpropionyl-CoA (10), a dihydrochalcone with
molecular weight m/z 274 was produced. The structure of the
dihydrochalcone was unambiguously identified as phloretin (11)
by analyzing its ESI-MS data and comparing with authentic com-
pound using HPLC (Fig. 1A and B) (Scheme 2). Phloretin, as a key
intermediate of the biosynthetic pathway of flavonoids in plants,
has not been isolated from A. Sinensis, but commonly occurred in
the peel of juicy fruit such as apple and strawberries with remark-
ably antioxidant activity.16 On the other hand, AsCHS could cata-
lyze the formation of 2,4,6-trihydroxybenzophenone (13) from
the condensation of benzoyl-CoA (12) and three molecules of mal-
onyl-CoA (Scheme 2) (Fig. 1C and D). Notably, benzophenone and
4
0,41
one 15, respectively
(Scheme 2) (Supplementary data).
In summary, the present report provides a rapid and cost
effective method to synthesize large amount of (methyl)malonyl-
CoA, and exemplary synthesized varied polyketides by a type III
PKS from Aquilaria sinensis, which could be meaningful and
luminous for in vitro construction of unusual molecule libraries
using PKSs.
Acknowledgments
We are thankful to the financial supported by Beijing Natural
Science Foundation (No. 5132022) and the Program for New Cen-
tury Excellent Talents in University (No. NCET-11-0604) and grad-
uate students independent subject of Beijing University of Chinese
Medicine (No. 2013-JYBZZ-XS-081).
its glycosides such as iriflophenone 3-C-b-
ophenone 2-O- -rhamnoside have been already isolated from
the leaves of A. Sinensis.
D-glucoside and irifl-
a-L
3
9

B.-W. Gao et al. / Bioorg. Med. Chem. Lett. 25 (2015) 1279–1283
1283
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Supplementary data
Supplementary data associated with this article can be found, in
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1
8
(
3
68 [M+H] , 978 [M+5Na-4H] . H NMR (500 MHz, D
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R
2
t
= 11.2 min,
61.1114; calcd 261.1121.
k
max 297 nm. 1 HRESIMS (positive): found for [C15
16 4
H O ]
1
1
1
1
H
NMR (500 MHz, CD OD):
3
d
6.96 (2H, d,
2
J = 8,4 Hz), 6.67 (2H, d, J = 8.4 Hz), 2.85 (2H, t, J = 6.8 Hz), 2.78 (2H, t,
13
J = 6.8 Hz), 1.90 (3H, s), 1.72 (3H, s). C NMR (125 MHz, CD
67.4, 157.4, 155.4, 131.0, 129.0, 114.7, 109.3, 97.1, 32.6, 32.2, 8.5, 7.5.
1. 4-Hydroxy-3,5-dimethyl-6-(5-(4-hydroxyphenyl)-3-oxopentan-2-yl)-2H-pyrone
3
OD): d 168.1,
3
1
4
(
15): HPLC-UV: R = 11.9 min, kmax 297 nm. HRESIMS (positive): found for
t
+
1
[
18 20 5 3
C H O ] 317.1379; calcd 317.1384. H NMR (500 MHz, CD OD): d 6.92 (2H,
252, 675.
d, J = 8,4 Hz), 6.43 (2H, d, J = 8.4 Hz), 3.88(1H, m), 2.71 (4H, m), 1.94 (3H, s),
2
2
0. Austin, M. B.; Noel, J. P. Nat. Prod. Rep. 2003, 20, 79.
1. Kwon, S. J.; Lee, M. Y.; Ku, B.; Sherman, D. H.; Dordick, J. S. ACS Chem. Biol. 2007,
1
1
7
.90 (3H, s), 1.31 (3H, d, J = 6.7 Hz). 13C NMR (125 MHz, CD
67.0, 155.2, 154.9, 131.5, 128.8, 114.7, 111.4, 97.6, 48.3, 42.0, 28.7, 12.0, 8.9,
.6.
3
OD): d 207.0, 169.7,
2
, 419.
2
2. Katsuyama, Y.; Nobutaka, F.; Miyahisa, I.; Sueharu, H. Chem. Biol. 2007, 14, 613.