Regio- and stereospecific prenylation of flavonoids by Sophora flavescens prenyltransferase

Article abstract of DOI:10.1002/adsc.201300196

Prenylflavonoids are valuable natural products that are widely distributed in plants. They often possess divergent biological properties, including phytoestrogenic, anti-bacterial, anti-tumor, and anti-diabetic activities. The reaction catalyzed by prenyltransferases represents a Friedel-Crafts alkylation of the flavonoid skeleton in the biosynthesis of natural prenylflavonoids and often contributes to the structural diversity and biological activity of these compounds. However, only a few plant flavonoid prenyltransferases have been identified thus far, and these prenyltransferases exhibit strict substrate specificity and low catalytic efficiency. In this article, a flavonoid prenyltransferase from Sophora flavescens, SfFPT, has been identified that displays high catalytic efficiency with high regiospecificity acting on C-8 of structurally different types of flavonoid (i.e., flavanone, flavone, flavanonol, and dihydrochalcone, etc.). Furthermore, SfPFT exhibits strict stereospecificity for levorotatory flavanones to produce (2S)-prenylflavanones. This study is the first to demonstrate the substrate promiscuity and stereospecificity of a plant flavonoid prenyltransferase in vitro. Given its substrate promiscuity and high catalytic efficiency, SfFPT can be used as an environmentally friendly and efficient biological catalyst for the regio- and stereospecific prenylation of flavonoids to produce bioactive compounds for potential therapeutic applications. Copyright

Full text of DOI:10.1002/adsc.201300196

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DOI: 10.1002/adsc.201300196
Regio- and Stereospecific Prenylation of Flavonoids by Sophora
flavescens Prenyltransferase
Ridao Chen,a Xiao Liu,a Jianhua Zou,a Yunze Yin,a Bin Ou,a Jianhua Li,a
Ruishan Wang,a Dan Xie,a Peicheng Zhang,a and Jungui Daia,*
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking
Union Medical College & Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, Peopleꢀs
Republic of China
Fax : (+86)-10-6301-7757; phone: (+86)-10-6316-5195; e-mail: jgdai@imm.ac.cn
Received: March 13, 2013; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201300196.
Abstract: Prenylflavonoids are valuable natural (i.e., flavanone, flavone, flavanonol, and dihydrochal-
products that are widely distributed in plants. They cone, etc.). Furthermore, SfPFT exhibits strict stereo-
often possess divergent biological properties, includ- specificity for levorotatory flavanones to produce
ing phytoestrogenic, anti-bacterial, anti-tumor, and (2S)-prenylflavanones. This study is the first to dem-
anti-diabetic activities. The reaction catalyzed by onstrate the substrate promiscuity and stereospecific-
prenyltransferases represents a Friedel–Crafts alkyla- ity of a plant flavonoid prenyltransferase in vitro.
tion of the flavonoid skeleton in the biosynthesis of Given its substrate promiscuity and high catalytic ef-
natural prenylflavonoids and often contributes to the ficiency, SfFPT can be used as an environmentally
structural diversity and biological activity of these friendly and efficient biological catalyst for the
compounds. However, only a few plant flavonoid regio- and stereospecific prenylation of flavonoids to
prenyltransferases have been identified thus far, and produce bioactive compounds for potential therapeu-
these prenyltransferases exhibit strict substrate spe- tic applications.
cificity and low catalytic efficiency. In this article,
a flavonoid prenyltransferase from Sophora flaves-
cens, SfFPT, has been identified that displays high Keywords: enzyme catalysis; flavonoid prenylation;
catalytic efficiency with high regiospecificity acting prenyltransferase; regioselectivity; Sophora flaves-
on C-8 of structurally different types of flavonoid cens; stereoselectivity
Introduction
flavonoids. However, the study of plant membrane
proteins is challenging; thus, only five flavonoid pre-
Prenylflavonoids are valuable natural products that nyltransferases have been characterized at the molec-
are mainly found within a few families, including the ular level to date.[3–6] Three of these prenyltransfer-
Leguminosae, Moraceae, Cannabaceae, Guttiferae,
ACHTUNGTRENaNUNG ses were identified in Sophora flavescens (i.e.,
Umbelliferae, and Euphorbiaceae. They exhibit vari- SfN8DT-1, SfG6DT, and SfiLDT), while the other
ous biological properties, including phytoestrogenic, two were isolated from Glycine max (i.e., G4DT) and
anti-bacterial, anti-tumor, and anti-diabetic activi- Lupinus albus (i.e., LaPT1). SfN8DT-1, the first flavo-
ties.[1] The prenylation of flavonoids contributes sig- noid-specific prenyltransferase to be identified, was
nificantly to the structural variation of plant secon- shown to be responsible for the prenylation of a very
dary metabolites and enhances the bioactivity and few select flavanones (i.e., naringenin, liquiritigenin,
bioavailability of these plant secondary metabolites; and hesperetin) at the C-8 position. SfG6DT was
this enhancement is likely a result of the presence of found to specifically prenylate the isoflavone genis-
the lipophilic prenyl side chain, which increases the tein at the C-6 position. The chalcone-specific prenyl-
membrane permeability and/or enhances the binding transferase SfiLDT was shown to prenylate isoliquiri-
affinity of target proteins of these compounds.[2]
tigenin. G4DT was discovered to specifically act on
The enzymatic reaction catalyzed by prenyltransfer- a pterocarpan substrate, glycinol. Finally, LaPT1 was
ase is crucial for the biosynthesis of bioactive prenyl- found to prenylate the B-rings of isoflavones, such as
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genistein and 2’-hydroxygenistein. These membrane- provide insight into the enzymatic promiscuity and re-
bound prenyltransferases exhibit strict substrate spe- action selectivity of plant flavonoid prenyltransferas-
cificities, as they only accept dimethylallyl diphos- es, which would then explain the structural diversity
phate (DMAPP) as a prenyl donor and a few similar- of the prenylflavonoids found in nature. The prenyla-
ly structured substrates as prenyl acceptors. Given tion of structurally different flavonoids by the same
this substrate specificity, these plant prenyltransferas- enzyme illustrates the economical use of a “combina-
es are quite different from the soluble prenyltransfer- torial biosynthetic” strategy in nature to create struc-
ases found in fungi, bacteria, and actinomycetes, tural diversity. The results indicate that SfFPT may
which display remarkable substrate tolerance and no- contribute to the biosynthesis of diverse prenylflavo-
table catalytic promiscuity.[7–10]
noids in S. flavescens. Moreover, SfFPT can be used
Because plants usually do not have biosynthetic as an environmentally friendly and efficient biological
gene clusters like microorganisms do and given the catalyst for the regio- and stereospecific prenylation
transmembrane properties of the prenyltransferases, of flavonoids, which have potential therapeutic appli-
the detailed function of a plant prenyltransferase is cations in humans.
much more difficult to elucidate. In addition, the pre-
nyltransferases identified thus far exhibit strict sub-
strate specificity and low catalytic efficiency, which Results and Discussion
significantly restricts their use in the structural deriva-
tion of flavonoids to produce biologically active com- The Molecular Cloning of a Flavonoid
pounds for therapeutic applications.
Prenyltransferase cDNA
The medicinal plant S. flavescens can produce di-
verse prenylated flavonoids, including different types S. flavescens plants, as well as cultured cells from
of flavonoids with prenyl or lavandulyl group substitu- these plants, can produce various types of prenylated
tions (i.e., flavanones, flavanonols, flavonols, and chal- flavonoids, such as prenylated flavonol, flavanone, fla-
cones, etc.).[11–13] It has previously been demonstrated vanonol, and chalcone. The existence of flavonoid
that enzyme promiscuity plays a role in protein evolu- prenyltransferases and the elucidation of their catalyt-
tion, as well as in the biosynthesis of natural products ic abilities were first demonstrated by an analysis of
for plant defense, where multiple functions have been the enzymatic prenylation activity of the microsomal
associated with a single enzyme.[14,15] Thus, it can be fraction prepared from cultured cells of S. flavescens.
hypothesized that either diverse flavonoid prenyl- Diverse prenyltransferase activities were observed in
transferases with strict substrate specificities solely the microsomal fraction. Specifically, the results indi-
exist in S. flavescens or some flavonoid prenyltransfer- cated that the microsomal fraction can catalyze the
ases in S. flavescens with broad substrate spectra have prenylations of pinocembrin (1), naringenin (2),
yet to be discovered; both hypotheses would allow S. chrysin (3), and taxifolin (4) in vitro, which further
flavescens to biosynthesize various prenylated flavo- supported the hypothesis that either diverse flavonoid
noids.
prenyltransferases or some flavonoid prenyltransfer-
In this study, a flavonoid prenyltransferase gene ases with broad substrate specificity were present in
from S. flavescens, SfFPT, was cloned and successfully S. flavescens (Supporting Information, Figure S1).
expressed heterologously in yeast (Saccharomyces cer- Based upon these results, we focused on the identifi-
evisiae). A sequence alignment analysis indicated that cation of candidate prenyltransferases that could be
SfFPT may be a paralogue of the previously reported responsible for the prenylation of different flavonoids
flavonoid prenyltransferase SfN8DT-1 (93% identity). and investigated the use of these candidates in the en-
Interestingly, SfFPT recognized a diverse array of fla- zymatic synthesis of prenylflavonoids in vitro. As
vonoid structure types as prenyl acceptors, including a result, a candidate cDNA, SfFPT (GenBank acces-
flavanone, flavone, flavanonol, and dihydrochalcone; sion KC513505), was cloned from the cultured cells of
furthermore, the regio- and stereospecificity of the S. flavescens by homology-based RT-PCR. A 1665 bp
transferred prenyl group into the flavonoid skeleton full-length cDNA containing a 1224 bp open reading
was performed with high catalytic efficiency when frame (ORF) and encoding a 407 amino acid polypep-
DMAPP was used as a prenyl donor. Meanwhile, tide was obtained using the rapid amplification of
SfFPT could also accept geranyl diphosphate (GPP) cDNA ends (RACE) method. The deduced protein
as a prenyl donor in some cases.
sequence of SfFPT showed very high sequence simi-
To the best of our knowledge, this study is the first larity to those of S. flavescens flavonoid prenyltrans-
demonstration of a plant flavonoid prenyltransferase ferases that were previously reported (Supporting In-
that exhibits broad substrate specificity. The heterolo- formation, Figure S2); in particular, it shared a se-
gous over-expression of SfFPT could provide a con- quence identity of 93% with SfN8DT-1. In addition,
venient approach for the production of diverse, bioac- a molecular evolutionary genetics analysis using Clus-
tive prenylated flavonoids by enzymatic synthesis and tal X[16] and MEGA5[17] indicated that SfFPT be-
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Regio- and Stereospecific Prenylation of Flavonoids by Sophora flavescens Prenyltransferase
theoretical pI of 9.2, as predicted by ExPASy
(http://web.expasy.org/cgi-bin/protparam/protparam).
It was also indicated that the protein would exhibit in-
creased stability and activity under basic conditions.
The Functional Characterization of Recombinant
SfFPT
The coding region of SfFPT was cloned into a yeast
expression vector, pESC, under the control of the
GAL10 yeast promoter to yield pESC-SfFPT. After
verification of the sequence, the plasmid was intro-
duced into YPH499 yeast for expression analysis. The
microsomal fraction of the recombinant yeast was
prepared for an enzymatic activity analysis using
400 mM DMAPP as a prenyl donor and 400 mM pino-
cembrin, naringenin, chrysin, or taxifolin as prenyl ac-
ceptors, which were chosen because the prenylated
derivatives were found in both the primary plant and
cultured cells of S. flavescens. Recombinant SfFPT ex-
Figure 1. The phylogenetic relationships between SfFPT and
related prenyltransferases from plants. The results were cal-
culated using the MEGA version 5 software. Method: hibited clear prenyltransferase activity in the enzy-
Neighbor-joining, Bootstrap: 1000. The branch lengths rep-
resent relative genetic distances. The protein sequences and
corresponding accession numbers that were used for this
comparison are as follows: SfFPT, KC513505; SfN8DT-1,
AB325579; SfN8DT-2, AB370330; SfL17a, AB371287;
SfL17b, AB370329; SfG6DT, BAK52291; SfiLDT,
AB604223; GmG4DT, AB434690; LaPT1, JN228254;
HIPT1, AB543053; AtVTE2-2, DQ231060; GmVTE2-2,
DQ231061; OsHGGT, AY222862; HvHGGT, AY222860;
TaHGGT, AY222861; CpVTE2-1, DQ231058; AtVTE2-1,
matic reaction analyses (Figure 2). It can regioselec-
tively and efficiently catalyze the prenylation of pino-
cembrin (in 58.8% yield), naringenin (in 62.6% yield),
chrysin (in 16.4% yield), and taxifolin (in 33.5%
yield) at the C-8 position. In contrast, no prenyltrans-
ferase activity was observed when microsomes isolat-
ed from yeast cells transformed with an empty vector
or boiled recombinant protein was used; furthermore,
no reactivity was observed when DMAPP, a prenyl
AY089963; ApVTE2-1, DQ231057; GmVTE2-1, DQ231059; acceptor or Mg2+ was absent from the enzymatic reac-
TaVTE2-1, DQ231056; ZmVTE2-1, DQ231055.
tion (Supporting Information, Figure S3).
Previous studies reported that the catalytic activity
of plant flavonoid prenyltransferases is dependent on
longed to the S. flavescens flavonoid prenyltransferase divalent cations.[3,4] Further investigations in this study
clade and exhibited close evolutionary relationships revealed that a variety of divalent cations can contrib-
with the other prenyltransferases (Figure 1).
ute to the activity of SfFPT; specifically, while Mg2+
As with previously characterized flavonoid prenyl- (100%) is the most effective, Ba2+ (84.3%), Ca2+
transferases (i.e., SfN8DT-1, G4DT, SfG6DT, and (83.6%), Fe2+ (55.1%), Co2+ (45.9%), Cu2+ (40.6%),
LaPT1), the SfFPT polypeptide was predicted to con- Zn2+ (36.8%), and Mn2+ (36.6%) also contribute to
tain eight transmembrane a-helices using TMHMM SfFPT activity. The optimal pH for enzyme activity
2.0 (http://www.cbs.dtu.dk/services/TMHMM/). In ad- was approximately 9.0 and is likely related to the
dition, SfFPT was predicted to localize to plastids basic protein characteristics of SfFPT, which has a the-
using WoLF PSORT (http://wolfpsort.org/), which is oretical pI of 9.2. The optimal reaction temperature
similar to the other known flavonoid prenyltransferas- was approximately 408C, and the catalytic activity of
es. However, the predicted polypeptide sequence did SfFPT decreased rapidly when the reaction tempera-
not contain a transit peptide at the N-terminal end, as ture increased above 608C, which is likely due to the
assessed by ChloroP 1.1 (http://www.cbs.dtu.dk/serv- denaturation of the enzyme (Supporting Information,
ices/ChloroP/), TargetP 1.1 (http://www.cbs.dtu.dk/ Figure S4).
services/TargetP/), iPSORT (http://ipsort.hgc.jp/), and
the SignalP 4.0 Server (http://www.cbs.dtu.dk/services/
SignalP/). Two characteristically conserved motifs, The Prenylations of Flavonoids by Recombinant
NQꢂ ꢂDꢂ ꢂ ꢂD and KDACHTNUGTRNEUNG(I/L)ꢂDꢂ(E/D)GD, of fla- SfFPT
vonoids and homogentisate prenyltransferase were
also present in the polypeptide sequence of SfFPT. In To analyze the prenyl acceptor specificity of recombi-
addition, SfFPT might be a basic protein with a nant SfFPT, various putative, aromatic substrates
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Figure 2. The functional characterization of recombinant SfFPT with DMAPP as a prenyl donor and pinocembrin (1), narin-
genin (2), chrysin (3), or taxifolin (4) as prenyl acceptors. The existence of flavonoid prenyltransferases and the elucidation
of their catalytic abilities in S. flavescens were demonstrated by analyzing the microsomal fraction enzyme reactivity of cul-
tured cells (Supporting Information, Figure S1).
(400 mM concentration) were utilized, including vari- cules were not discovered in the plant S. flavescens.
ous types of flavonoids and structurally simple phe- The apparent Michaelis–Menten constants (Km) were
nols that have flavonoid-like structures; DMAPP determined for eleven flavonoids using Lineweaver–
(400 mM) was used as a prenyl donor. Analyses of the Burk plots (Table 1); the Michaelis–Menten constant
enzymatic products revealed the significant substrate was not determined for sakuranetin because of its low
promiscuity of SfFPT. In addition to the four previ- conversion rate. The results indicate that SfFPT exhib-
ously tested flavonoids (i.e., pinocembrin, naringenin, ited substantially stronger affinities and higher cata-
chrysin, and taxifolin), SfFPT could also catalyze the lytic efficiencies with various flavonoids than other
prenylation of eight other flavonoids, including seven previously reported plant flavonoid prenyltransferas-
flavanones, specifically liquiritigenin (5, in 45.8% es. For example, the apparent Km values of naringenin
yield), steppogenin (6, in 29.9% yield), eriodictyol (7, and DMAPP (naringenin was used as a prenyl accept-
in 63.4% yield), hesperetin (8, in 45.1% yield), sakur- or) for SfFPT were calculated to be 0.236 mM and
anetin (9, in 3.0% yield), isosakuranetin (10, in 37.9% 0.105 mM compared with 55 mM and 106 mM for
yield), and tsugafolin (11, in 18.3% yield), and one di- SfN8DT-1, respectively.
hydrochalcone, phloretin (12, in 22.1% yield)
Given the high efficiency and conversion rate of
(Figure 3; Supporting Information, Figures S5a and flavonoid prenyltransferase SfFPT, most of the enzy-
S5b). The prenylated products for most of these mole- matic products could be efficiently prepared for struc-
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Regio- and Stereospecific Prenylation of Flavonoids by Sophora flavescens Prenyltransferase
Figure 3. The prenylation of flavonoids by recombinant SfFPT.
tural elucidation. Eleven flavonoids were used in DAD/ESI-MSn analysis (Supporting Information, Fig-
larger scale reactions, and the prenylated products ure S6).
were purified by semi-preparative RP-HPLC. Identifi-
The substrate promiscuity of SfFPT was further
cation of the enzymatic products was carried out by demonstrated by prenyl donor specificity analyses.
comparison of their NMR and MS data with those of The prenyl donor specificity of SfFPT was tested
know compounds in the published literature (see Ex- using DMAPP, GPP, and FPP as prenyl donors and
perimental Section). In addition, the prenylated prod- pinocembrin, naringenin, liquiritigenin, steppogenin,
uct of sakuranetin was identified using an HPLC- eriodictyol, hesperetin, sakuranetin, isosakuranetin,
tsugafolin, taxifolin, phloretin, and chrysin were used
as prenyl acceptors. The results indicated that SfFPT
could also recognize GPP as a prenyl donor and cata-
lyze the geranylation of pinocembrin, isosakuranetin,
and naringenin (Figure 3). The prenylation products
were identified using HPLC-DAD/ESI-MSn analyses.
(Supporting Information, Figures S7–S9). This study is
the first to report that a plant flavonoid prenyltrans-
ferase can accept both DMAPP and GPP as prenyl
donors.
Table 1. The apparent Km values of recombinant SfFPT for
various flavonoids.
Substrate
Km
Substrate[a]
Km
Conversion
[mM]
[mM] yields [%]
pinocembrin
naringenin
0.586 pinocembrin
0.236 naringenin
0.120 58.8
0.105 62.6
liquiritigenin 0.092 liquiritigenin 0.128 45.8
steppogenin
eriodictyol
hesperetin
0.013 steppogenin
0.092 eriodictyol
0.228 hesperetin
0.031 29.9
0.125 63.4
0.140 45.1
isosakuranetin 0.207 isosakuranetin 0.047 37.9
The Substrate Structure–Enzyme Selectivity
Relationship of SfFPT
tsugafolin
taxifolin
phloretin
chrysin
0.032 tsugafolin
0.114 taxifolin
0.045 phloretin
0.020 chrysin
0.446 18.3
0.869 33.5
0.102 22.1
2.030 16.4
The prenylation of natural prenylflavonoids always
occurs with the substitution of hydroxy group(s) or
methoxy group(s) at the flavanone skeleton, which
[a]
Apparent Km value for DMAPP.
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Figure 4. The relative enzyme activities of recombinant SfFPT on various flavanones. N.D.: not detected.
may affect the biological activities of these com- C-8 position by SfFPT, and improved the reactivity
pounds.[18,19] On the other hand, different substitution and regioselectivity of the prenyl group substitution
patterns and the substitution of different groups at at the C-8 position of the flavanone skeleton, which
the flavanone skeleton may affect the substrate selec- was in accordance with previous reports.[20,21] In con-
tivity of a prenyltransferase. Through substrate selec- trast to hydroxy groups, methoxy groups at the C-7/5
tivity analyses, it was revealed that SfFPT mainly pre- positions reduced the prenylation efficiency of SfFPT.
nylates flavanones at the C-8 position. To provide in- Because the prenylation rate of naringenin (2) and
sight into the “substrate structure–enzyme selectivity the yield of its prenylated product by SfFPT were
relationship” and to provide valuable strategies for higher than those of tsugafolin (11) and sakuranetin
the further chemo-enzymatic synthesis of active pre- (9), respectively, and because the prenylation rate of
nylflavonoids, the substrate selectivity of SfFPT for tsugafolin (11) was higher than the rate of sakurane-
fourteen flavanones, including flavanone (13), pino- tin (9), it can be concluded that a hydroxy group sub-
cembrin, pinostrobin (14), naringenin, sakuranetin, stituted at the C-7 position is crucial for a prenylation
tsugafolin, isosakuranetin, eriodictyol, steppogenin, at the C-8 position by SfFPT. This conclusion is also
hesperetin, robtin (15), liquiritigenin, naringin (16), confirmed by the fact that SfFPT could not prenylate
and hesperiden (17), was compared (Figure 4). The pinostrobin (14) (a methoxy group is substituted at
following conclusions were preliminarily made about the C-7 position of pinocembrin), naringin (16, an O-
the effects of different substitution groups on the pre- glycosylation group is at the C-7 position of naringe-
nylation and substrate selectivity of SfFPT. nin), and hesperiden (17, an O-glycosylation group is
Firstly, hydroxy groups at the C-7/5 positions on the at the C-7 position of hesperetin).
A-ring enhanced the electron density around the C-8
Secondly, hydroxy groups at the C-3’/4’ positions on
position, which was crucial for the prenylation of the the B-ring increased the prenylation efficiency of
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SfFPT. For example, the conversion rate of eriodictyol prenyl acceptors, and the products were analyzed by
(7) by SfFPT was higher than that of naringenin (2), chiral chromatography. The results indicated that the
and the conversion rate of naringenin was higher than pair of optical isomers can transform into each other
that of pinocembrin (1). In addition, flavanones with in the reaction buffer without recombinant SfFPT. On
hydroxy groups at the C-3’,4’,5’ positions on the B- the other hand, a dextrorotatory product [i.e., (+)-8-
ring cannot be accepted as substrates given that liquir- dimethylallylnaringenin] was not detected when
itigenin (5), and not robtin (15), can be prenylated by (+)-naringenin was incubated with SfFPT and
SfFPT. Similar results were observed in the enzymatic DMAPP (Figure 5). These results demonstrate that
reaction of taxifolin (dihydroxy groups at the C-3’,4’ SfFPT is stereospecific for levorotatory flavanone.
positions) and dihydromyricetin (trihydroxy groups at Similar results were also observed when optical iso-
the C-3’,4’,5’ positions).
Thirdly, a flavanone with a methoxy group on the
B-ring was less effectively converted by SfFPT than
flavanones with a hydroxy group. For example, the
conversion rate of eriodictyol (7) was higher than that
of hesperetin (8), and the conversion rate of naringe-
nin (2) was higher than that of isosakuranetin (10).
Fourthly, overall, the conversion rate of flavanones
by SfFPT was influenced by the hydroxy or methoxy
group substitution as follows: hydroxy groups in the
C-7/5 positions on the A-ring and the C-3’/4’ positions
on the B-ring had a beneficial effect on the prenyla-
tion of flavanones by SfFPT, while methoxy groups in
these positions obviously decreased the conversion
rate.
The Regiospecific and Stereospecific Prenylation of
Flavonoids by SfFPT
While various flavonoids, including flavanone, fla-
vone, dihydrochalcone, and flavonol, were accepted
as substrates by SfFPT, a prenyl side chain was regio-
selectively introduced into the skeleton of the flavo-
noids only at the C-8 position. In contrast, when step-
pogenin was incubated with SfFPT, DMAPP, and
MgCl2 in Tris-HCl buffer (pH 9.0), both C-6 and C-8
prenylated products were simultaneously detected.
These findings are consistent with previously reported
results that steppogenin was prenylated by the micro-
somal fraction of S. flavescens cultured cells.[13] How-
ever, further investigation of this reaction revealed
that 6-dimethylallylsteppogenin and 8-dimethylallyl-
steppogenin can easily and automatically transform
into each other in the reaction buffer without enzyme.
Thus, 6-prenylsteppogenin is unlikely to be a direct
enzymatic product of steppogenin when catalyzed by
SfFPT. These results strongly suggest that SfFPT is
strictly regiospecific for the prenylation of flavonoids
at the C-8 position.
Interestingly, regardless of whether optically active
or optically inactive flavanones were incubated with
recombinant SfFPT, the enzyme products were always
determined to be in the levorotatory form (2S). These
findings prompted us to investigate the stereoselectiv-
Figure 5. Chiral HPLC analyses of enzymatic reaction prod-
ucts of recombinant SfFPT. (A) (À)-Naringenin (2) was
used as a prenyl acceptor. (B) (+)-Naringenin (2’) was used
as a prenyl acceptor. (C) Control reaction without recombi-
nant enzyme. (D) Authentic (+)-8-dimethylallylnaringenin
ity of recombinant SfFPT. (+)-Naringenin and (À)-
naringenin, a pair of optical isomers, were used as (2a’). (E) Authentic (À)-8-dimethylallylnaringenin (2a).
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mers of other flavanones (e.g., pinocembrin and hes- Chemicals
peretin, data not shown) were employed as prenyl ac-
The prenyl donors dimethylallyl diphosphate (DMAPP),
geranyl diphosphate (GPP), and farnesyl diphosphate (FPP)
were chemically synthesized as previously reported.[24] Most
of the tested aromatic substrates were purchased from
Sigma–Aldrich (St. Louis, MO, USA) and BioBioPha
(Kunming, Yunnan, China). We sincerely thank Prof. Min
Ye from Peking University for the gift of liquiritigenin for
enzymatic activity analyses. Optically active flavanones [i.e.,
(À)-naringenin, (+)-naringenin, (À)-8-dimethylallylnaringe-
nin, and (+)-8-dimethylallylnaringenin] were isolated from
their optically inactive enantiomers by chiral HPLC, and
their enantiomeric excess values were over 99%.
HPLC grade methanol was purchased from Merck
(Darmstadt, Germany); water was purified and deionized
by a water purification system from Tautobiotech (Shanghai,
China). The formic acid used in this study was of HPLC
grade (Mreda, Columbia, USA). All the other chemicals
used in this study were of analytical grade for laboratory
use.
ceptors. It is well-known that the efficient synthesis of
optically pure flavanones by chemical asymmetric cat-
alysis methods is highly attractive to chemists. How-
ever, this type of synthesis has faced many difficulties
and challenges in this field.[22] The chemical synthesis
of optically pure prenylflavanones has not been re-
ported thus far. Interestingly, SfFPT exhibits a desired
reaction specificity and high catalytic efficiency, which
makes it a useful tool for the production of bioactive
chiral prenylflavanones. These results suggest a power-
ful method for the preparation of optical prenylflava-
nones by dynamic resolution and SfFPT prenylation.
Conclusions
The prenyl group substitution at the flavonoid skele-
ton contributes to the structural diversity and biologi-
cal activity of natural flavonoids in the plant kingdom.
The enzymatic reaction catalyzed by a prenyltransfer-
ase is crucial for the biosynthesis of these diverse pre-
nylated flavonoids. However, only a few plant flavo-
noid prenyltransferases have been identified, and they
exhibit strict substrate specificity and low catalytic ef-
Analytical Methods
The analyses of substrate specificity and the determinations
of the conversion rates were performed on an Agilent 1200
series HPLC system (Agilent Technologies, Bçblingen, Ger-
many) coupled with an LCQ Fleet ion trap mass spectrome-
ficiency. The present work describes the identification ter (Thermo Electron Corp., San Jose, CA, USA) equipped
with an electrospray ionization (ESI) source.
of a flavonoid prenyltransferase SfFPT from S. flaves-
cens. We discovered that SfFPT exhibits significant
substrate promiscuity in vitro and has an unprecedent-
ed ability to recognize diverse flavonoids as prenyl ac-
ceptors when DMAPP is used as a prenyl donor;
SfFPT could also accept GPP as a prenyl donor in
some cases, which has not yet been found in the re-
ported plant flavonoid prenyltransferases. Intriguingly,
SfPFT exhibits strict stereospecificity for levorotatory
The LC chromatograph consisted of a quaternary pump
equipped with an on-line solvent degasser unit, an auto sam-
pler, a column temperature controller and a diode-array de-
tector coupled with an analytical workstation. HPLC analy-
ses were performed on a Shiseido capcell pak C18 MG II
column (250 mmꢂ4.6 mm i.d., 5 mm, Shiseido Co., Ltd.,
Tokyo, Japan) at a flow rate of 1 mLminÀ1, and the column
temperature was maintained at 308C. The injection volume
was 10 mL. The mobile phase was a gradient elution of sol-
flavanones. The results indicate that SfFPT may not vents A (i.e., 0.1% formic acid aqueous solution) and B
(i.e., methanol). The gradient program was as follows: 0–
25 min, a linear change from A:B (50:50, v/v) to A:B
(0:100, v/v), and then an isocratic elution for 10 min. The re-
equilibration duration was 10 min between individual runs.
For HPLC-MS analyses, ultra-high purity helium (He)
was used as the collision gas, and high purity nitrogen (N2)
was used as the nebulizing gas. The optimized ESI source
parameters were as follows: sheath gas flow rate, 20 (arbi-
trary units); auxiliary gas flow rate, 5 (arbitrary units); spray
voltage, 5.0 kV; capillary temperature, 3508C; source colli-
sion-induced decomposition (CID), 35 V; tube lens offset
voltage, À75 V. The spectra were recorded in the 100–
1000 m/z range for a full scan MS analysis. The split ratio of
effluent from the LC to ion source was 2:1. The data were
analyzed using XCalibur software.
The resolution of flavanone enantiomers and the determi-
nation of enantiomeric excess values were carried out utiliz-
ing a Chiralpak AD-H column (250 mmꢂ4.6 mm i.d., 5 mm,
Daicel Chemical Industries Ltd., Tokyo, Japan). The mobile
phase consisted of n-hexane and 2-propanol (87:13, v/v).
Resolution analyses were carried out at 308C at a flow rate
of 1.1 mLminÀ1 with detection at 290 nm (UV).
only contribute to the biosynthesis of diverse prenyl-
flavonoids in S. flavescens, but also can be used as an
efficient biological catalyst for the regio- and stereo-
specific prenylation of flavonoids, which have poten-
tial therapeutic applications in humans.
Experimental Section
Plant Material and Culture Method
S. flavescens cultured cells were maintained in Murashige
and Skoogꢀs basal medium[23] supplemented with 0.5 mgLÀ1
a-naphthaleneacetic acid (NAA), 0.5 mgLÀ1 6-benzylamino-
purine (6-BA), 0.2 mgLÀ1 2,4-dichlorophenoxyacetic acid
(2,4-D), 30 gLÀ1 sucrose, and 5 gLÀ1 agar. Cell suspension
cultures were performed in 500-mL flasks containing
125 mL of the same medium without agar. Methyl jasmo-
nate (MJ) solution in EtOH (100 mM) was added to the
medium (at a final concentration of 100 mM) on the 12th day
of subculture to induce prenyltransferase gene expression.
8
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Regio- and Stereospecific Prenylation of Flavonoids by Sophora flavescens Prenyltransferase
10 mM MgCl2, 400 mM prenyl donors, 400 mM prenyl accept-
cDNA Cloning
ors, and 0.1–0.2 mg of recombinant yeast microsomal protein
in a total volume of 200 mL. After incubation for 30 min at
408C, the reaction was terminated by the addition of 400 mL
of EtOAc and a 2 min vortex step. After the removal of the
protein by centrifugation at 12,000ꢂg for 15 min, the organ-
ic layer was evaporated until dry, and the product was dis-
solved in 200 mL MeOH for HPLC-MS analysis. For quan-
tification, three parallel assays were routinely carried out.
To test the necessity of divalent cations for SfFPT activity,
MgCl2, BaCl2, CaCl2, FeCl2, CoCl2, CuCl2, ZnCl2, and MnCl2
were used individually. To study the optimal pH, the enzy-
matic reaction was performed in various reaction buffers
(pH 6.0–12.0). To assay for the optimal reaction tempera-
ture, the reaction mixtures were incubated at different tem-
peratures (40–808C).
Twelve-day-old cell cultures of S. flavescens treated with MJ
for 24 h were used for total RNA preparations; the RNA
was isolated using the RNA prep pure Plant Kit (TianGen
Biotech, China). Total RNA was reverse-transcribed using
SMARTScribe reverse transcriptase (Clontech, USA). A
core cDNA fragment encoding a flavonoid prenyltransferase
was cloned by homology-based RT-PCR using TransStart
FastPfu DNA Polymerase (TransGen Biotech, China) and
a degenerate primer pair, MC-GP-F/MC-GP-R, that was de-
signed using the conserved sequences of plant flavonoid pre-
nyltransferases (MC-GP-F: 5’-THGARATAGACAAGRTWAA
CAAGCCIKATCTTCC-3’; MC-GP-R: 5’-CYCCTTCMATRT
CAGGTATATCCTTRAAYAATGC-3’). The 3’-end and 5’-end
amplifications were carried out using the Smart RACE
cDNA Amplification Kit (Clontech, USA) according to the
manufacturerꢀs protocol. The full-length cDNA of SfFPT
was isolated by RT-PCR using gene-specific primer pairs.
Apparent Km Value Assays
To determine the apparent Km values, 160 mg of recombinant
yeast microsomes in a total volume of 200 mL at 408C for
30 min, were incubated with either various concentrations of
prenyl acceptors (20–400 mM) and a fixed concentration of
DMAPP (800 mM) or various concentrations of DMAPP
(20–400 mM) and a fixed concentration of prenyl acceptors
(800 mM). All experiments were performed in triplicate, and
enzyme activity was evaluated by the production of prenyl-
flavonoids (nmol) per mg of microsomal protein per minute.
Apparent Km values were calculated from Lineweaver–Burk
plots using the Hyper32 software (http://homepage.ntl-
world.com/john.easterby/hyper32.html).
The Expression of the cDNA
The coding region of SfFPT was amplified using N-terminal
and
C-terminal
primers:
5’-GTCGAATTCATGGGTTC
TATGCTTCTTGCATCTTTTCC-3’ (the EcoR I site is under-
lined) and 5’-ATTTGCGGCCGCTCATCTAAACAAAGGTATGAG
GAAG-3’ (the Not I site is underlined). The amplified cDNA
was cloned into the EcoR I/Not I site of the expression
vector pESC (Stratagene, La Jolla, CA, USA) via the clon-
ing vector pEASY-B (TransGen Biotech, China). After the
verification of the sequence, the plasmid was transformed
into YPH499 yeast for heterologous expression.
Enzyme Preparation
Substrate Specificity Assays
Recombinant yeast transformed with foreign plasmid was
stored in SD-His dropout medium prior to incubation at
308C in a shaking incubator (200 rpm) overnight. Cells were
collected by centrifugation at 1,500ꢂg for 5 min at room
temperature, and the cell pellet was resuspended in SG-His
dropout medium to achieve an OD600 of 1.0 to induce gene
expression. After another incubation at 288C for 24 h, the
cells were harvested by centrifugation at 1,500ꢂg for 5 min
at 48C, and the cell pellet was resuspended in 100 mM Tris-
HCl buffer (pH 9.0) containing 1 mM PMSF. The cell pellet
was homogenized with acid-washed glass beads (425–600 mm
size, Sigma–Aldrich, St. Louis, MO, USA). The mixture was
vortexed for 30 s followed by 30 s on ice, and this cycle was
repeated eight times for a total of 8 min to lyse the cells.
The mixture was centrifuged at 10,000ꢂg for 15 min. Then,
the supernatant was further ultracentrifuged at 160,000ꢂg
for 90 min at 48C to pellet the microsome fraction. After
two washes with 100 mm Tris-HCl buffer (pH 9.0), the mem-
brane fraction was resuspended in the same buffer. The pro-
tein content was determined using the Bradford method.[25]
The microsomal enzyme preparation and reaction procedure
of cultured cells is provided in the Supporting Information
(Supplemental Experimental Procedures).
To study the substrate specificity of SfFPT, seventy-two aro-
matic substrates were used as prenyl acceptors, including fla-
vone, chrysin, apigenin, luteolin, diosmetin, baicalein, hispi-
dulin, acacetin-7-O-b-d-rutinoside, fisetin, kaempferol,
morin hydrate, quercetin, myricetin, kumatakenin A, (À)-
flavanone, (Æ)-pinocembrin, (À)-pinocembrin, (+)-pinocem-
brin, (Æ)-naringenin, (À)-naringenin, (+)-naringenin, (À)-
pinostrobin, (À)-sakuranetin, (À)-tsugafolin, (À)-isosakura-
netin, (À)-eriodictyol, (À)-steppogenin, (À)-hesperetin, (À)-
robtin, (À)-liquiritigenin, (À)-5,6,7-trimethoxyflavanone,
(À)-hesperiden, (À)-naringin, (+)-taxifolin, dihydromyrice-
tin, dihydrokaempferol-7-O-b-d-glucoside, daidzein, formo-
nonetin, genistein, orobol, 2’-hydroxygenistein, sophorico-
side, genistin, 2-hydroxychalcone, 4-hydroxychalcone, 2’-hy-
droxychalcone, 4’-hydroxychalcone, 2’,4’,6’-trihydroxychal-
cone, isoliquiritigenin, phloretin, 4,4’-dihydroxybenzophe-
none,
2,4-dihydroxybenzophenone,
(À)-maackiain,
norathyriol, (+)-catechin, l-epicatechin, resveratrol, 4-hy-
droxybenzoic acid, 4-hydroxybenzaldehyde, 2,4-dihydroxy-
benzoic acid, 2,5-dihydroxybenzoic acid, 3,5-dihydroxyben-
zoic acid, 2,6-dihydroxybenzoic acid, 2,3-dihydroxybenzoic
acid, 3,4-dihydroxybenzoic acid, 2,4,6-trihydroxybenzoic
acid, 3,4-dihydroxyphenylacetic acid, 2,5-dihydroxyphenyl-
acetic acid/homogentisic acid, 4-hydroxyphenylpyruvic acid,
caffeic acid, 4-chromanol, and 4-chromanone.
Enzyme Reactions
The prenyl donor specificity of SfFPT was tested with
DMAPP, GPP, and FPP when pinocembrin, naringenin, liq-
uiritigenin, steppogenin, eriodictyol, hesperetin, sakuranetin,
Standard enzyme assays were performed in 100 mM Tris-
HCl buffer (pH 9.0). The reaction mixtures contained
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isosakuranetin, tsugafolin, taxifolin, phloretin, and chrysin
were used as prenyl acceptors in individual reactions.
J=7.2 Hz, H-2’’), 1.62 (3H, s, H-4’’), 1.61 (3H, s, H-5’’);
13C NMR (acetone-d6, 150 MHz): d=80.29 (C-2), 44.55 (C-
3), 190.88 (C-4), 116.39 (C-4a), 126.25 (C-5), 110.43 (C-6),
162.47 (C-7), 115.23 (C-8), 161.94 (C-8a), 131.561 (C-1’),
128.75 (C-2’), 115.99 (C-3’), 158.36 (C-4’), 115.99 (C-5’),
128.75 (C-6’), 22.76 (C-1’’), 123.18 (C-2’’), 131.56 (C-3’’),
17.90 (C-4’’), 25.86 (C-5’’).[28]
The Preparation and Identification of the Reaction
Products
To prepare the enzymatic products for structural elucida-
tion, each reaction mixture containing 10 mM MgCl2, 2 mM
prenyl donor, 4 mM DMAPP, and 5 mg of recombinant
yeast microsomal protein in a total volume of 5 mL was in-
cubated for 2 h at 408C. The enzymatic product was extract-
ed by EtOAc (3ꢂ10 mL). The organic layers were combined
and concentrated under vacuum. The residue was further
dissolved in MeOH, and the enzymatic products were puri-
fied by reversed-phase HPLC. The structures of the enzy-
matic products were characterized by MS and NMR spectral
and polarimetric analyses.
Positive ESI mass spectra were obtained from an LCQ
Fleet ion trap mass spectrometer. Optical rotations were de-
termined using a Perkin–Elmer Model 341 LC polarimeter
(Perkin–Elmer, Waltham, MA, USA). 1H NMR and
13C NMR spectra were recorded on Varian NMR System
600, 500, and 400 spectrometers (Varian Inc., Palo Alto,
CA, USA).
8-Dimethylallylsteppogenin (6a): ESI-MS: m/z=357 [M+
H]+; [a]2D0: À16.7 (c 0.03, MeOH); 1H NMR (acetone,-d6
600 MHz): d=5.67 (1H, dd, J=12.6, 3.0 Hz, H-2), 3.12 (1H,
dd, J=17.0, 13.2 Hz, H-3a), 2.83 (1H, dd, J=17.0, 3.0 Hz,
H-3b), 6.03 (1H, s, H-6), 6.47 (1H, d, J=2.4 Hz, H-3’), 6.42
(1H, dd, J=8.4, 2.4 Hz, H-5’), 7.33 (1H, d, J=8.4 Hz, H-6’),
3.24 (2H, d, J=6.6 Hz, H-1’’), 5.20 (1H, m, H-2’’), 1.59 (3H,
s, H-4’’), 1.61 (3H, s, H-5’’), 12.16 (1H, br s, 5-OH).[29]
6-Dimethylallylsteppogenin (6a’): ESI-MS; m/z=357
1
[M+H]+; [a]2D0: +16.7 (c 0.03, MeOH); H NMR (acetone-
d6, 600 MHz): d=5.67 (1H, dd, J=13.2, 3.0 Hz, H-2), 3.14
(1H, dd, J=17.4, 13.2 Hz, H-3a), 2.70 (1H, dd, J=17.4,
3.0 Hz, H-3b), 6.04 (1H, s, H-8), 6.47 (1H, d, J=2.4 Hz, H-
3’), 6.42 (1H, dd, J=8.4, 2.4 Hz, H-5’), 7.30 (1H, d, J=
8.4 Hz, H-6’), 3.25 (2H, d, J=6.6 Hz, H-1’’), 5.25 (1H, t, J=
6.6 Hz, H-2’’), 1.75 (3H, s, H-4’’), 1.64 (3H, s, H-5’’), 12.51
(1H, br s, 5-OH); 13C NMR (acetone-d6, 150 MHz): d=
75.22 (C-2), 42.75 (C-3), 197.58 (C-4), 103.42 (C-4a), 162.27
(C-5), 108.91 (C-6), 162.44 (C-7), 95.39 (C-8), 159.46 (C-8a),
117.61 (C-1’), 156.28 (C-2’), 103.42 (C-3’), 159.46 (C-4’),
107.80 (C-5’), 128.94 (C-6’), 21.65 (C-1’’), 123.77 (C-2’’),
131.01 (C-3’’), 17.81 (C-4’’), 25.84 (C-5’’).[30]
8-Dimethylallyleriodictyol (7a): ESI-MS: m/z=357 [M+
H]+; [a]2D0: À32.9 (c 0.09, MeOH); 1H NMR (acetone-d6,
600 MHz): d=5.38 (1H, dd, J=12.6, 3.0 Hz, H-2), 3.09 (1H,
dd, J=17.4, 12.6 Hz, H-3a), 2.73 (1H, dd, J=17.4, 3.0 Hz,
H-3b), 6.02 (1H, s, H-6), 7.05 (1H, d, J=1.2 Hz, H-2’), 6.89
(1H, d, J=8.4 Hz, H-5’), 6.87 (1H, dd, J=8.4, 1.2 Hz, H-6’),
3.22 (2H, d, J=7.2 Hz, H-1’’), 5.20 (1H, t, J=7.2 Hz, H-2’’),
1.61 (3H, s, H-4’’), 1.62 (3H, s, H-5’’), 12.15 (1H, br s, 5-
OH); 13C NMR (acetone-d6, 150 MHz): d=79.76 (C-2),
43.58 (C-3), 197.46 (C-4), 103.19 (C-4a), 165.31 (C-5), 96.37
(C-6), 163.01 (C-7), 108.36 (C-8), 161.05 (C-8a), 131.89 (C-
1’), 116.03 (C-2’), 146.06 (C-3’), 146.29 (C-4’), 114.67 (C-5’),
118.99 (C-6’), 22.30 (C-1’’), 123.75 (C-2’’), 131.18 (C-3’’),
17.89 (C-4’’), 25.92 (C-5’’).[31]
8-Dimethylallylhesperetin (8a): ESI-MS: m/z=371 [M+
H]+; [a]2D0: À46.9 (c 0.13, MeOH); 1H NMR (MeOH-d4,
500 MHz): d=5.24 (1H, dd, J=12.5, 3.0 Hz, H-2), 2.98 (1H,
dd, J=17.0, 12.5 Hz, H-3a), 2.66 (1H, dd, J=17.0, 3.0 Hz,
H-3b), 5.86 (1H, s, H-6), 6.91 (1H, d, J=1.5 Hz, H-2’), 6.88
(1H, d, J=8.5 Hz, H-5’), 6.86 (1H, dd, J=8.5, 1.5 Hz, H-6’),
3.15 (1H, dd, J=14.0, 7.5 Hz, H-1’’a), 3.10 (1H, dd, J=14.0,
7.5 Hz, H-1’’b), 5.09 (1H, t, J=7.5 Hz, H-2’’), 1.57 (3H, s,
H-4’’), 1.53 (3H, s, H-5’’), 3.81 (3H, s, 4’-OCH3); 13C NMR
(MeOH-d4, 125 MHz): d=80.08 (C-2), 44.05 (C-3), 197.86
(C-4), 109.18 (C-4a), 166.56 (C-5), 96.54 (C-6), 163.18 (C-7),
112.54 (C-8), 161.44 (C-8a), 133.51 (C-1’), 114.57 (C-2’),
147.79 (C-3’), 149.27 (C-4’), 114.57 (C-5’), 118.87 (C-6’),
22.50 (C-1’’), 123.98 (C-2’’), 131.61 (C-3’’), 17.89 (C-4’’),
25.95 (C-5’’), 56.46 (4’-OCH3).[32]
8-Dimethylallylpinocembrin (1a): ESI-MS: m/z=325
1
[M+H]+; [a]2D0: À68.4 (c 0.19, MeOH); H NMR (acetone-
d6, 500 MHz): d=5.56 (1H, dd, J=12.5, 3.0 Hz, H-2), 3.12
(1H, dd, J=17.0, 12.5 Hz, H-3a), 2.84 (1H, dd, J=17.0,
3.0 Hz, H-3b), 6.04 (1H, s, H-6), 7.58 (1H, d, J=7.0 Hz, H-
2’), 7.46 (1H, dd, J=7.0, 1.5 Hz, H-3’), 7.38 (1H, m, H-4’),
7.46 (1H, dd, J=7.0, 1.5 Hz, H-5’), 7.58 (1H, d, J=7.0 Hz,
H-6’), 3.27 (2H, d, J=7.0 Hz, H-1’’), 5.20 (1H, m, H-2’’),
1.70 (3H, s, H-4’’), 1.68 (3H, s, H-5’’), 12.11 (1H, s, 5-OH);
13C NMR (acetone-d6, 125 MHz): d=79.75 (C-2), 43.60 (C-
3), 197.13 (C-4), 103.36 (C-4a), 160.89 (C-5), 96.48 (C-6),
164.95 (C-7), 108.38 (C-8), 163.01 (C-8a), 140.38 (C-1’),
129.44 (C-2’), 127.13 (C-3’), 129.44 (C-4’), 127.13 (C-5’),
129.26 (C-6’), 22.27 (C-1’’), 123.67 (C-2’’), 131.26 (C-3’’),
17.84 (C-4’’), 25.85 (C-5’’).[26]
8-Dimethylallylnaringenin (2a): ESI-MS: m/z=341 [M+
H]+; [a]2D0: À33.8 (c 0.08, MeOH); 1H NMR (acetone-d6,
600 MHz): d=5.44 (1H, dd, J=12.5, 3.0 Hz, H-2), 3.13 (1H,
dd, J=17.0, 12.5 Hz, H-3a), 2.75 (1H, dd, J=17.0, 3.0 Hz,
H-3b), 6.01 (1H, s, H-6), 7.40 (1H, d, J=8.5 Hz, H-2’), 6.90
(1H, d, J=8.5 Hz, H-3’), 6.90 (1H, d, J=8.5 Hz, H-5’), 7.40
(1H, d, J=8.5 Hz, H-6’), 3.21 (2H, d, J=7.0 Hz, H-1’’), 5.19
(1H, m, H-2’’), 1.70 (3H, s, H-4’’), 1.68 (3H, s, H-5’’), 8.47
(1H, s, -OH), 9.50 (1H, s, -OH), 12.13 (1H, s, 5-OH);
13C NMR (acetone-d6, 150 MHz): d=79.73 (C-2), 43.44 (C-
3), 197.56 (C-4), 103.34 (C-4a), 161.10 (C-5), 96.39 (C-6),
164.88 (C-7), 108.30 (C-8), 162.98 (C-8a), 131.11 (C-1’),
128.82 (C-2’), 116.15 (C-3’), 158.57 (C-4’), 116.15 (C-5’),
128.82 (C-6’), 22.25 (C-1’’), 123.18 (C-2’’), 131.19 (C-3’’),
17.84 (C-4’’), 25.86 (C-5’’).[27]
8-Dimethylallylliquiritigenin (5a): ESI-MS: m/z=325
1
[M+H]+; [a]2D0: À90.0 (c 0.05, MeOH); H NMR (acetone-
d6, 600 MHz): d=5.43 (1H, dd, J=13.2, 3.0 Hz, H-2), 2.99
(1H, dd, J=16.8, 13.2 Hz, H-3a), 2.68 (1H, dd, J=16.8,
3.0 Hz, H-3b), 7.57 (1H, d, J=8.4 Hz, H-5), 6.61 (1H, d, J=
8.4 Hz, H-6), 7.41 (1H, d, J=8.4 Hz, H-2’), 6.89 (1H, d, J=
8.4 Hz, H-3’), 6.89 (1H, d, J=8.4 Hz, H-5’), 7.41 (1H, d, J=
8.4 Hz, H-6’), 3.32 (2H, br d, J=7.2 Hz, H-1’’), 5.22 (1H, t,
8-Dimethylallylisosakuranetin (10a): ESI-MS:ACTHNUGRTNEUNG(m/z=355
1
AHCTUNGTRENNUNG
600 MHz): d=5.50 (1H, dd, J=12.6, 3.0 Hz, H-2), 3.15 (1H,
dd, J=17.4, 12.6 Hz, H-3a), 2.78 (1H, dd, J=17.4, 3.0 Hz,
10
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Regio- and Stereospecific Prenylation of Flavonoids by Sophora flavescens Prenyltransferase
H-3b), 6.04 (1H, s, H-6), 7.51 (1H, d, J=8.4 Hz, H-2’), 7.00
(1H, d, J=8.4 Hz, H-3’), 7.00 (1H, d, J=8.4 Hz, H-5’), 7.51
(1H, d, J=8.4 Hz, H-6’), 3.23 (2H, d, J=7.2 Hz, H-1’’), 5.20
(1H, d, J=7.2 Hz, H-2’’), 1.62 (3H, s, H-4’’), 1.61 (3H, s, H-
5’’), 3.83 (3H, q, -OCH3), 9.58 (1H, s, -OH), 12.14 (1H, s, 5-
OH); 13C NMR (acetone-d6, 150 MHz): d=79.55 (C-2),
43.44 (C-3), 197.45 (C-4), 103.30 (C-4a), 160.99 (C-5), 96.35
(C-6), 164.92 (C-7), 108.29 (C-8), 162.96 (C-8a), 131.20 (C-
1’), 128.69 (C-2’), 114.72 (C-3’), 160.83 (C-4’), 114.72 (C-5’),
128.69 (C-6’), 22.23 (C-1’’), 123.65 (C-2’’), 132.20 (C-3’’),
17.84 (C-4’’), 25.86 (C-5’’), 55.57 (4’-OCH3).[33]
8-Dimethylallyltsugafolin (11a): ESI-MS: m/z=355 [M+
H]+; [a]2D0: À28.9 (c 0.05, MeOH); 1H NMR (acetone-d6,
600 MHz): d=5.34 (1H, dd, J=12.6, 3.0 Hz, H-2), 2.90 (1H,
dd, J=16.8, 12.6 Hz, H-3a), 2.60 (1H, dd, J=16.8, 3.0 Hz,
H-3b), 6.23 (1H, s, H-6), 7.39 (1H, d, J=8.4 Hz, H-2’), 6.89
(1H, d, J=8.4 Hz, H-3’), 6.89 (1H, d, J=8.4 Hz, H-5’), 7.39
(1H, d, J=8.4 Hz, H-6’), 3.26 (2H, d, J=6.6 Hz, H-1’’), 5.21
(1H, d, J=6.6 Hz, H-2’’), 1.61 (3H, s, H-4’’), 1.60 (3H, s, H-
5’’), 3.73 (3H, q, -OCH3); 13C NMR (acetone-d6, 150 MHz):
d=76.47 (C-2), 46.25 (C-3), 188.54 (C-4), 106.07 (C-4a),
161.16 (C-5), 93.79 (C-6), 162.80 (C-7), 108.97 (C-8), 162.50
(C-8a), 130.92 (C-1’), 128.64 (C-2’), 115.98 (C-3’), 158.29 (C-
4’), 115.98 (C-5’), 128.64 (C-6’), 22.54 (C-1’’), 123.99 (C-2’’),
131.62 (C-3’’), 18.87 (C-4’’), 25.87 (C-5’’), 55.81 (5-OCH3).[34]
8-Dimethylallyltaxifolin (4a): ESI-MS: m/z=373 [M+
H]+; 1H NMR (DMSO-d6, 600 MHz): d=4.90 (1H, d, J=
10.8 Hz, H-2), 4.40 (1H, d, J=10.8 Hz, H-3), 5.94 (1H, s, H-
6), 6.87 (1H, d, J=1.2 Hz, H-2’), 6.74 (1H, d, J=8.4 Hz, H-
5’), 6.70 (1H, dd, J=8.4, 1.2 Hz, H-6’), 3.01 (2H, d, J=
7.2 Hz, H-1’’), 5.07 (1H, m, H-2’’), 1.56 (3H, s, H-4’’), 1.54
(3H, s, H-5’’); 13C NMR (DMSO, 150 MHz): d=82.89 (C-2),
71.20 (C-3), 196.70 (C-4), 100.04 (C-4a), 160.99 (C-5), 95.63
(C-6), 159.17 (C-7), 107.04 (C-8), 158.00 (C-8a), 130.10 (C-
1’), 115.22 (C-2’), 144.83 (C-3’), 145.53 (C-4’), 115.01 (C-5’),
119.06 (C-6’), 21.24 (C-1’’), 122.59 (C-2’’), 128.39 (C-3’’),
17.50 (C-4’’), 25.48 (C-5’’).[35]
Accession Numbers
The sequence of SfFPT was submitted to GenBank under
the accession number KC513505.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 81273405), the Fun-
damental Research Funds for the Central Universities (Grant
No. 2012N06), the Program for Doctorate Education (Grant
No. 20111106110029), and the National Science & Technolo-
gy Major Project ’Key New Drug Creation and Manufactur-
ing’, China (No. 2012ZX09301002-001-005).
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7.8 Hz, H-b), 3.33 (2H, t, J=7.8 Hz, H-a), 6.07 (1H, s, H-
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3’), 7.61 (1H, m, H-4’), 7.61 (1H, m, H-5’), 8.03 (1H, m, H-
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-OH), 12.75 (1H, s, -OH); 13C NMR (DMSO-d6, 100 MHz):
d=163.1 (C-2), 104.9 (C-3), 182.1 (C-4), 103.9 (C-4a), 159.1
(C-5), 98.5 (C-6), 161.9 (C-7), 106.2 (C-8), 154.6 (C-8a),
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Regio- and Stereospecific Prenylation of Flavonoids by
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Sophora flavescens Prenyltransferase
Adv. Synth. Catal. 2013, 355, 1 – 13
Ridao Chen, Xiao Liu, Jianhua Zou, Yunze Yin, Bin Ou,
Jianhua Li, Ruishan Wang, Dan Xie, Peicheng Zhang,
Jungui Dai*
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