Modifying the acylation of flavonols in Petunia hybrida

Article abstract of DOI:10.1016/j.phytochem.2008.04.014

The potential for chemically-regulating the acylation of natural products in whole plants has been determined by treating petunia leaves with phenylpropanoid acyl donors supplied as the respective methyl esters. Treatment with derivatives of the naturally-occurring acylating species caffeic acid resulted in a general increase in flavonol derivatives, notably caffeoylated quercetin-3-O-diglucoside (QDG) and kaempferol-3-O-diglucoside (KDG). Similarly, methyl ferulate increased the content of feruloylated KDG 40-fold. Treatment with methyl coumarate resulted in the appearance of a coumaroylated derivative of quercetin-3-O-glucuronyl-glucoside (QGGA). When the feeding studies were repeated with the equivalent phenylpropanoid isosubstituted with fluorine groups a semi-synthetic 4-fluorocinnamoyl ester of QGGA was observed. Our results demonstrate the potential to regulate the acylation of flavonols and potentially other natural products by treating whole plants with methyl esters of natural and unnatural acyl donors.

Full text of DOI:10.1016/j.phytochem.2008.04.014

Phytochemistry 69 (2008) 2016–2021
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Modifying the acylation of flavonols in Petunia hybrida
*
Oliver D. Cunningham, Robert Edwards
Centre for Bioactive Chemistry, Durham University, Durham DH1 3LE, UK
Centre for Bioactive Chemistry, School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The potential for chemically-regulating the acylation of natural products in whole plants has been deter-
mined by treating petunia leaves with phenylpropanoid acyl donors supplied as the respective methyl
esters. Treatment with derivatives of the naturally-occurring acylating species caffeic acid resulted in a
general increase in flavonol derivatives, notably caffeoylated quercetin-3-O-diglucoside (QDG) and
kaempferol-3-O-diglucoside (KDG). Similarly, methyl ferulate increased the content of feruloylated
KDG 40-fold. Treatment with methyl coumarate resulted in the appearance of a coumaroylated derivative
of quercetin-3-O-glucuronyl-glucoside (QGGA). When the feeding studies were repeated with the equiv-
alent phenylpropanoid isosubstituted with fluorine groups a semi-synthetic 4-fluorocinnamoyl ester of
QGGA was observed. Our results demonstrate the potential to regulate the acylation of flavonols and
potentially other natural products by treating whole plants with methyl esters of natural and unnatural
acyl donors.
Received 6 February 2008
Received in revised form 4 April 2008
Available online 3 June 2008
Keywords:
Acyltransferases
Fluoroacylation
Phenylpropanoids
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
responsible for these modifications have recently been the subject
of considerable research effort (reviewed by D’Auria, 2006). Typi-
Acylation, the esterification of a hydroxyl acceptor by an acid
donor, is a commonly observed modification of plant secondary
metabolites leading to products with altered physical and biologi-
cal properties. Acylating moieties include acetyl, malonyl, benzoyl,
and phenylpropanoyl groups, with the acceptors varying from sim-
ple sugars to complex alkaloids (Ghangas, 1999; Walker and Cro-
teau, 2000). As a well studied example, many flavonoids undergo
acylation, most commonly with malonic acid or the phenylpropa-
noids coumaric, caffeic or ferulic acid (Bloor and Abrahams,
2002; Fujiwara et al., 1998). Flavonoid acceptors include anthocy-
anins, isoflavones, flavones and flavonols (Nielsen et al., 1993;
Stochmal et al., 2001; Suzuki et al., 2001). While the exact roles
of acylation are not always known, malonylation is known to direct
flavonoid glycoside metabolites for storage in the vacuole (Boller
and Wiemken, 1986), while esterification of anthocyanins with
phenylpropanoids promotes intramolecular aggregation or stack-
ing, which protects the oxonium ion from decomposition (Ceval-
los-Casals and Cisneros-Zevallos, 2004). With respect to novel
biological activities, acylation of flavonoids can result in changes
in pigmentation (Bloor, 2001), insect antifeedant activity
(Harborne and Williams, 1998) and antioxidant properties (Alluis
and Dangles, 1999). In addition, the pharmacological activities of
several plant secondary metabolites are due to specific acylations
by aromatic donors (Ma et al., 2005). As such the acyltransferases
cally, the acylation of complex natural products proceeds via a
two step pathway in which the donor acid is activated through
the formation of the respective CoA thioester through the action
of an ATP-dependent CoA ligase, followed by acyl-transfer to the
acceptor through the action of a BAHD acyltransferase (D’Auria,
2006). Other acyltransferases operating in plant secondary metab-
olism include acyl-glucose dependent enzymes, which belong to
the SCPL enzyme family (Fraser et al., 2007).
With our background in foreign compound metabolism in
plants, we have become interested in the possibility that xenobi-
otic acids can be mistaken for natural products and enter plant sec-
ondary metabolism as acyl donors. Such semi-synthetic acylated
products have the potential to have novel biological activities, as
demonstrated with fluoroacylated derivatives of the anticancer
taxane drugs (Ojima, 2004). Fluorine-substitution is particularly
effective in regulating the properties of bioactive molecules, due
to the combination of the element’s unique electronic properties
and non-intrusive size (Smart, 2001). While such fluoroacylated
derivatives can be prepared synthetically, there are potentially
many advantages in using biotransformation reactions to achieve
regio- and stereo-selective incorporation into natural product
acceptors. Significantly, halogenated xenobiotic acids are known
to enter acylation pathways in plant primary metabolism, with
fatty acid derivatives of 2,4-dichlorophenoxyacetic acid accumu-
lating as triglycerides in coconut (Lopez-Villalobos et al., 2004).
With this in mind, we wanted to see if the acyl-incorporation of
halogenated acids could be demonstrated in secondary metabo-
* Corresponding author. Tel.: + 44 191 334 1318; fax: +44 191 334 1201.
E-mail address: robert.edwards@durham.ac.uk (R. Edwards).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.04.014

O.D. Cunningham, R. Edwards / Phytochemistry 69 (2008) 2016–2021
2017
lism using commonly encountered acceptor species such as flavo-
noids. The acylation of flavonols in the leaves of petunia (Petunia
hybrida L.) plants was selected as a suitable test system, as the
respective natural products are well characterized and include a
range of aromatic and aliphatic esters of glycosides of quercetin
and kaempferol (Bloor et al., 1998). The experimental strategy
has been to treat plants with caffeic and ferulic acid which are
known to serve as acyl donors and then test the system with other
naturally-occurring phenylpropanoids which are not normally
incorporated. Having established the tractability of the acylating
pathway, the plants were then fed with synthetic phenylpropa-
noids, where ring substituents have been replaced with fluorine
atoms and the incorporation into the acylated flavonol pool
determined.
accumulated a range of UV-absorbing metabolites with the profiles
obtained being essentially identical in individual plants. The indi-
vidual UV-absorbing metabolites were identified with reference
to published data (Cuyckens et al., 2003), using a combination of
their UV–Vis absorbance spectra determined using photodiode ar-
ray (PDA) detection and MS analysis of parent and fragment ions.
The structures of the compounds identified are shown (Fig. 1)
and are numbered in bold with reference to the respective peaks
resolved by HPLC (Fig. 2A). The individual compounds were then
quantified based on their absorbance at 330 nm, using a standard
curve prepared from quercetin-3-O-diglucoside. By quantifying at
330 nm the concentration of the parent flavonoids was deter-
mined, with minimal interference from the acylating groups. The
concentrations of the metabolites present were then compared in
the plants treated with high level illumination ( detergent treat-
2. Results
ment) with plants grown under a conventional (80 lEin-
stein mꢀ2 sꢀ1) lighting regime (Table 1). The major UV-absorbing
metabolites identified in the light-stressed leaves were derivatives
of the flavonols quercetin-3-O-diglucoside (QDG) and kaempferol-
3-O-diglucoside (KDG), respectively. The relative abundances of
the individual compounds were similar in all extracts, though their
overall contents were approximately tripled by the high light treat-
ment (Table 1). Spraying with detergent had no significant effect
on the accumulation of the metabolites, as compared with the un-
sprayed controls (data not shown), so the results shown in Table 1
are derived from the plants sprayed with formulation. QDG accu-
mulated as a major metabolite in its unmodified form (1), as well
as being acylated with caffeic acid (QDG-Caf; 3). In contrast, non-
2.1. Acylated flavonols in petunia leaves
To facilitate the active accumulation of flavonoids, petunia
plants were exposed to high level illumination (160 lEin-
stein mꢀ2 sꢀ1) for 24 h prior to harvest. High level irradiance with
the attendant exposure to UV light is known to provoke a major in-
crease in the content of phenolic metabolites in a range of plant
species (Himer et al., 2001; Li et al., 1993). The plants were either
otherwise untreated, or sprayed with 0.1% (v/v) Triton X-100 to
simulate the feeding conditions to be used later in the project with
the potential acyl donors. As determined by HPLC, leaf extracts
R4
Flavonol Metabolite
HO
Acyl Donor
A
C
OH
O
O
O
OH
R3
O
OH
HO
HO
HO
O
O
R1
R2
R6
O
HO
HO
R5
B
D
Acyl Group
R1
R2
R3
Metabolite
R4
R5
R6
Coumaroyl
(4Ca)
QDG (1)
OH
OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
O4Ca
H
OH
H
KDG (2)
H
OH
H
OH
Caffeoyl
(Caf)
OH
OH
OH
OH
H
H
QDG-Caff (3)
KDG-Caff (4)
KDG-Fer (5)
OCaf
OCaf
OFer
Feruloyl
(Fer)
OMe
OMe
H
Sinapoyl
(Sin)
OMe
QGGA-Coum (6)
QGGA-Fca (7)
OH
OH
COOH
COOH
Fluorocinnamoyl
(Fca)
OFca
H
F
F
F
F
F
H
H
F
O
OH
OH
OH
Difluorocinnamoyl
(Dif)
O
HO
HO
R =
Trifluorocinnamoyl
(Trif)
Fig. 1. The UV absorbing metabolites identified and acyl donors used in the studies with petunia. The templates of the flavonol acceptors are shown (A), along with the
respective acylated metabolites (B), which are numbered in bold and refer to the peaks shown in Fig. 2. The phenylpropanoid metabolite rosmarinic acid (R) is also shown.
The template of the acyl donors used is shown (C) and their specific substituents (D). K = kaempferol, Q = quercetin, DG = diglucoside, GGA = glucosyl-glucuronide.

2018
O.D. Cunningham, R. Edwards / Phytochemistry 69 (2008) 2016–2021
described in earlier studies (Bloor et al., 1998). However, in con-
A
trast to these studies, we were unable to detect any 600-O-malony-
lated derivatives of QDG. Based on previously published data
(Bloor et al., 1998) the substitutions with the acyl groups most
likely occurred at the 2-O-position of the terminal glucose substi-
tuent (Fig. 1A and B).
3.0
2.0
1.0
0.0
Sprayed with
Detergent
2.2. Feeding studies with phenylpropanoids
3
4
1
R
2
The petunia leaves were first sprayed with caffeic acid, as this
was the most abundantly incorporated acyl group in the flavonol
pool (Table 1). The caffeoyl compound was applied either as its free
acid or as the corresponding methyl caffeate ester. The application
of the methylated derivative led to over a 10-fold increase in
the pool size of QDG-Caf and a 3-fold enhancement in KDG-Caf
(Table 1). In addition, feeding with methyl caffeate also increased
the pool sizes of all the other UV-absorbing metabolites except
for QDG. In addition to the originally identified compounds, feed-
ing with methyl caffeate led to the appearance of a series of
unidentified polar UV-absorbing metabolites (Fig. 2B). Applications
of the methyl ester led to an overall 61% enhancement in incorpo-
ration into QDG-Caf as compared with feeding with free caffeic
acid. All future feeding studies with the phenylpropanoids there-
fore used the respective methyl esters.
The plants were then sprayed with the methyl esters of couma-
ric, ferulic and sinapic acids. Concentrating on the major effects
determined on the HPLC profiles of the flavonols, treatment with
the other natural acyl donor, ferulic acid, resulted in a selective
40-fold enhancement in KDG-Fer (Table 1). Feeding with methyl
sinapate had no effect on the flavonol content (data not shown),
while feeding with methyl coumarate (Fig 2B; Table 2) resulted
in a large enhancement in the levels of KDG and its feruloyl ester.
In addition a new metabolite, compound 6, was determined
(Fig. 2B). Based on MS fragmentation analysis (Fig. 3), this
compound was tentatively identified as quercetin-3-O-(5000-O-
coumaroyl-glucuronyl(1 ? 2)glucoside) (QGGA-Coum; 6). This
5
B
C
3.0
2.0
1.0
0.0
O
O
4
R
5
HO
1
6
2
3.0
2.0
1.0
0.0
7
2
O
4
O
3
F
1
R
5
12.00
14.00
16.00
Time (minutes)
18.00
Fig. 2. UV-absorbing metabolites extracted from Petunia leaves exposed to high
level illumination for 24 h in the presence of (A) formulation alone; (B) 1 mM
methyl coumarate acid; and (C) 1 mM methyl 4-fluorocinnamate. The numbered
peaks refer to the compounds identified in Fig. 2.
Table 2
Determination of parent mass ions and yield of synthesized methyl esters of
phenylpropanoates
Methyl ester
Expected [M+H]+ (Da)
Obtained (ES+) (Da)
Yield (%)
Coumarate
Ferulate
Caffeate
Sinapate
Fluorocinnamate
Difluorocinnamate
Trifluorocinnamate
179.1
179.2
209.1
195.1
239.2
181.2
199.1
217.1
93
89
92
91
86
95
86
acylated KDG (2) was a relatively minor component of the total
kaempferol pool, with the respective caffeoyl-ester (KDG-Caf; 4)
dominating. In addition, KDG also accumulated to a lesser extent
as the feruloyl ester (KDG-Fer; 5). In addition to these flavonol
derivatives, the caffeic acid derivative rosmaric acid (Fig 1B; R)
was also identified as a phenolic metabolite in petunia foliage, as
209.07
195.06
239.08
181.06
199.05
217.04
Table 1
The effect of exogenously-fed methyl esters of phenylpropanoids upon the accumulation of acylated metabolites in the foliage of Petunia hybrida
Compound
Control LL
Control HL
Phenylpropanoid feeding treatment
4Ca
Fca
Caf
Fer
Dif
Concentration (nmol gꢀ1 FW)
*
QDG (1)
72 16.8
200 21.0
62 8.4
235 19.9
338 64.0
37 1.4
191 20.7
257 34.3
59 10.3
326 39.4
511 29.0
81 18.7
127 12.5
148 9.4
*
263 14..8
64 4.8
140 7.2
*
*
KDG (2)
25 5.7
49 10.9
96 5.0
22 6.8
124 12.1
118 10.3
110 6.9
*
*
*
*
QDG-Caff (3)
KDG-Caff (4)
KDG-Fer (5)
QGGA-Coum (6)
QGGA-Fca (7)
R
46 11.9
2891 143.2
16 1.2
*
366 11.6
1018 24.2
469 32.2
1509 48.7
510 13.9
*
*
*
*
239 9.7
121 15.6
0
0
0.0
0.0
0.0
*
*
*
0
0
0.0
0.0
0
0
0.0
0.0
220 18.8
59 7.7
144 10.2
0
0.0
0.0
*
0
0.0
15 0.2
0
0.0
0
0
*
*
*
*
79 10.8
140 28.6
368 9.6
217 2.8
328 23.1
53 6.2
15 4.5
Untreated plants were grown under ambient lighting (= Con LL) or were sprayed with formulated treatments containing 1 mM of the methyl esters then exposed to high level
lighting for 24 h. Con HL refers to plants treated under high light with formulant solution only. Values are the mean of two biological replicates standard deviation
*
(
= values significantly greater than control (P < 0.05)).

O.D. Cunningham, R. Edwards / Phytochemistry 69 (2008) 2016–2021
2019
OH
OH
A
B
OH
OH
HO
O
O
HO
O
O
O
O
OH
HO
HO
HO
OH
HO
HO
HO
O
O
O
O
O
O
O
HO
HO
O
HO
HO
F
HO
O
O
O
O
HO
HO
Metabolite 6
Metabolite 7
C
Metabolite 6
Metabolite 7
OH
D
OH
OH
OH
O
O
OH
HO
O
O
O
O
O
O
HO
HO
OH / F
OH
OH
(M + 2H)+ = 287
(M + H)+ = 339
(M)+ = 147 (OH) / 149 (F)
OH
OH
OH
OH
HO
O
O
O
O
O
OH
OH
OH
O
OH
(M + 2H)+ = 449
(M)+ = 177
= Proposed fragmentations
Fig. 3. The identification of two novel and analogous acylated derivatives of quercetin-3-O-glucuronyl-glucoside showing the incorporation of coumaric acid (A) and 4-
fluorocinnamic acid (B) with characteristic fragmented mass ions determined by MS shown (C) along with the identities of the assigned mass ions (D).
identification was based on the parent mass ion and generation of
fragments corresponding to quercetin (287 and 177 Da), quercetin
glucosyl (449 Da) and coumaroyl (147 Da), as found in previous
studies on flavonol glycoside fragmentation (Cuyckens et al.,
2003). In addition, a fragment corresponding to glucuronyl-gluco-
syl (339 Da) was also identified. While glucosyl-glucuronic acid
conjugates of flavonols, have not been previously described in
petunia, similar diglycosides are known in other plants and the
proposed structure of the metabolite, as derived from MS fragmen-
tation experiments, is consistent with those described in the liter-
ature (Sawada et al., 2005). A careful examination of the plants
treated in high light only, showed that this compound was in fact

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O.D. Cunningham, R. Edwards / Phytochemistry 69 (2008) 2016–2021
a natural constituent of the flavonol pool in Petunia, albeit as a
trace metabolite. A re-examination of the other feeding experi-
ments showed that this coumaroylated metabolite also accumu-
lated in plants fed with methyl caffeate (Table 2).
acylated metabolites can be usefully enhanced by spraying whole
plants with methylated donor acids and (b) that these feeding
studies can usefully incorporate unnatural fluorinated acids to derive
novel semi-synthetic esters. Previous studies in suspension-
cultured cells of wild carrot (Daucus carota spp. carota) have shown
that it is possible to manipulate the acylation of anthocyanin gly-
cosides by feeding with naturally-occurring benzoic and phenyl-
propanoid acids to form novel ester species (Baker et al., 1994;
Dougall et al., 1998). Additionally, in the later studies incorporation
of halogenated cinnamic acids into the anthocyanin pool was also
observed (Dougall et al., 1998). From an applied perspective, stud-
ies in Taxus cultures have shown that feeding with benzoic acid
acyl donors can enhance the content of the bioactive acylated tax-
anes (Fett-Neto et al., 1994). Potential application also exists in
other medicinal plant species which are known to accumulate
acylated bioactive compounds, such as Rauvolfia (Bayer et al.,
2004) or Catharanthus (St-Pierre et al., 1998). Our results show that
it is also possible to achieve useful enhancement in yields (mM) of
acylated flavonoids in whole plants without the need to generate
and maintain suspension cultures. In addition to boosting the
content of useful natural acylated secondary metabolites, the sim-
plicity of our treatment suggests that it would be relatively
straight-forward to screen large numbers of plant species for their
ability to incorporate fluoroacyl donors into their natural products
pools, thereby extending chemical diversity and potential biologi-
cal activity. The potential for the incorporation of fluoroacyl groups
is particularly attractive, based on the associated novel biological
properties associated with such semi-synthetic plant secondary
products (Ojima, 2004).
The enhancement of the acylated flavonol pool following
feeding with phenylpropanoid donors is also of interest in under-
standing how these pathways are regulated. The scale of the
enhancement in the acylated flavonol pool on feeding caffeoyl
phenylpropanoids is strongly suggestive of metabolic regulation
by positive feed-back from the end products of the pathway. This
conclusion can be made as the scale of the accumulation of the
acylated flavonol glycosides greatly exceeded the depletion in the
levels of the KDG and QDG intermediates, implying that the pool
was being replenished by de novo flavonol synthesis. Drawing
analogies with what is known of the detoxification of other exoge-
nously supplied compounds in plants, a short-term response to the
influx of xenobiotic acids is to biotransform them to the respective
esters, classically using sugar acceptors (Cole and Edwards, 2000).
Our results with caffeic acid and ferulic acid in Petunia suggest that
these compounds are recognized by the endogenous CoA ligases
and acyltransferases and removed from the cell by incorporation
into the flavonol glycosides. In this respect the QDG and KDG are
serving the functions as glycoside acceptors which can then be
recycled and re-used as required following deacylation. This model
for detoxifying exogenously-supplied acids may suggest that
xenobiotics could become incorporated into a diverse range of
plant esters. The range of plants which can incorporate xenobiotic
acids into their acylated natural products, the regulation of the
respective pathways and the in vitro biosynthesis of xenobiotic
natural compounds is currently under further investigation.
Having established the perturbation in flavonol acylation fol-
lowing feeding with naturally-occurring phenylpropanoids, the
study was extended by spraying the plants with the methyl esters
of 4-fluoro- (Fca), 3,4-difluoro- (Dif), and 3,4,5-trifluoro- (Trif)
substituted cinnamoyl acid derivatives. The mono- and tri-fluori-
nated compounds did not significantly perturb the accumulation
of the endogenous metabolites, while feeding with 3,4-difluorocin-
namic acid led to a selective decline in QDG-Caf, KDG-Fer and ros-
marinic acid. To look for incorporation of the fluoroacyl donors into
novel flavonols, for each feeding study, the eluting metabolites
were fragmented by MS and monitored for evidence of the fluor-
cinnamoyl species. This is effectively a sensitive means of looking
for fluoroacylation due to the predictable cleavage of the phenyl-
propanoid ester during ionization of the parent acylated flavonol
(Fig. 3). Using this approach we were unable to find any evidence
for incorporation of 3,4-difluorocinnamic acid (167 Da) or 3,4,5,-
trifluorocinnamic acid (185 Da). However, a compound fragmenting
to release a 149 Da fragment characteristic of 4-fluorocinnamoy-
lated compounds was identified in a minor peak eluting just after
KDG (Fig. 2C). MS analysis of this compound (7) (Fig 3), subse-
quently identified it as quercetin-3-O-(5000-O-4-fluorocinnamoyl-
glucuronyl(1 ? 2)glucoside) (QGGA-Fca; 7), with the compound
accumulating to a final concentration of 15 nmol gꢀ1 FW (Table 2).
With respect to the levels of incorporation and recovery of the
applied methylated-acyl donors; in the absence of isotopic labeling
these could not be calculated with any confidence for the natu-
rally-occurring phenylpropanoids. For example, whereas only
approximately 200 nmol of methyl caffeate was sprayed onto the
plants, the large increase in the levels of caffeoylated flavonols
could account for over four times the applied dose. The one excep-
tion was with 4-fluorocinnamate, where a final level of incorpora-
tion of 7.5% into the acylated flavonol pool could be calculated
(Table 1). Intriguingly, in all cases methanolic washes of the leaf
surface 24 h after application failed to recover any of the methyl
esters. This, together with the absence of further novel absorbing
peaks in the HPLC profiles of the leaf extracts (Fig. 2), suggested
that the applied phenylpropanoids had either been metabolised
to non-UV-absorbing soluble compounds or incorporated into
insoluble matrices.
3. Discussion
Our results demonstrate that the accumulation of acylated
flavonols in the foliage of petunia can be selectively regulated by
simply spraying the plants with the acyl donor species supplied
as the respective methyl ester. This was most dramatically demon-
strated in the major increases in the content of endogenous feru-
loylated and caffeoylated QDG and KDG achieved by feeding with
methyl ferulate and methyl caffeate respectively. In fact feeding
with methyl caffeate caused an accumulation of all of the acylated
flavonols. While methyl sinapate was not incorporated, treatment
with the coumarate ester led to the accumulation of the novel acyl-
ated flavonol derivative QGGA-Coum. The acylating pathway in
petunia also had a limited capacity to accept non-natural phenyl-
propanoids. Thus, when the plants were fed with methyl 4-fluoro-
cinnamate, the respective phenylpropanoid entered the flavonol
acylation pathway to give rise to relatively small amounts of the
semi-synthetic ester QGGA-Fca.
4. Experimental
4.1. Synthesis of methyl phenylpropanoate esters
All chemicals were analytical grade and obtained from Sigma–
Aldrich (Poole, Dorset, UK). The cinnamic acid derivatives
(0.91 mmol) was dissolved in dry dichloromethane (5 ml) and
These simple observations may be of great utility in selectively
enhancing the content of valuable acylated natural products in
medicinal plants. Our results suggest that (a) levels of endogenous
dimethylformamide (4
ll) added. Oxalyl chloride (1.82 mmol)
was subsequently added drop-wise, followed by methanol

O.D. Cunningham, R. Edwards / Phytochemistry 69 (2008) 2016–2021
2021
Bayer, A., Ma, X.Y., Stockigt, J., 2004. Acetyltransfer in natural product biosynthesis –
functional cloning and molecular analysis of vinorine synthase. Bioorg. Med.
Chem. 12, 2787–2795.
Bloor, S.J., 2001. Deep blue anthocyanins from blue Dianella berries. Phytochemistry
58, 923–927.
Bloor, S.J., Abrahams, S., 2002. The structure of the major anthocyanin in Arabidopsis
thaliana. Phytochemistry 59, 343–346.
Bloor, S.J., Bradley, J.M., Lewis, D.H., Davies, K.M., 1998. Identification of flavonol and
anthocyanin metabolites in leaves of petunia ‘Mitchell’ and its LC transgenic.
Phytochemistry 49, 1427–1430.
(2.73 mmol) and the solution stirred for 30 min. The reaction mix-
ture was washed with water (3 ꢁ 10 ml) and dried over magne-
sium sulphate. Concentration in vacuo afforded the corresponding
cinnamate derivative as a pale yellow–brown oil. The yield and
identity of the corresponding methyl esters was determined by
HPLC-MS (Table 2).
4.2. Plant experiments
Boller, T., Wiemken, A., 1986. Dynamics of vacuolar compartmentalisation. Ann.
Rev. Plant Physiol. 37, 137–164.
P. hybrida (‘Grandiflora Selection Mixed’ F1) seeds were pur-
chased from Mr Fothergill’s Seeds Ltd. (Kentford, Suffolk, UK) and
sown onto multipurpose potting compost. Plants were maintained
Cole, D.J., Edwards, R., 2000. Secondary metabolism of agrochemicals in plants. In:
Roberts, T.R. (Ed.), Metabolism of Agrochemicals in Plants, John Wiley and Sons,
Chichester, pp. 107–154.
Cevallos-Casals, B.A., Cisneros-Zevallos, L., 2004. Stability of anthocyanin-based
aqueous extracts of Andean purple corn and red-fleshed sweet potato compared
to synthetic and natural colorants. Food Chem. 86, 69–77.
Cuyckens, F., Shahat, A.A., Van den Heuvel, H., Abdel-Shhafeek, K.A., El-Messiry,
M.M., Seif-El Nasr, M.M., Pieters, L., Vlietinick, A.J., Claeys, M., 2003. The
application of liquid chromatography-electrospray ionization mass
spectrometry and collision-induced dissociation in the structural
characterization of acylated flavonol O-glycosides from the seeds of
Carrichtera annua. Eur. J. Mass Spectrosc. 9, 409–420.
D’Auria, J.C., 2006. Acyltransferases in plants: a good time to be BAHD. Curr.
Opinion. Plant Biol. 9, 31–340.
Dougall, D.K., Baker, D.C., Gakh, E.G., Redus, M.A., Whittemore, N.A., 1998.
Anthocyanins from wild carrot suspension cultures acylated with supplied
carboxylic acids. Carb. Res. 310, 177–189.
in a growth room for 28 days under a regime of 16 h light (80 lEin-
stein mꢀ2 sꢀ2, 24 °C), 8 h dark (22 °C). Plants were sprayed for-
mulation consisting of an aqueous solution of Triton-X 100
detergent (0.1% v/v) containing the phenylpropanoid derivatives
at a final concentration of 1 mM. The solution was applied as a fine
spray at a rate of 25 ml per seed tray (800 cm2), with each tray con-
taining 24 plants. After spraying, the plants were immediately
transferred into a Sanyo versatile environmental test chamber set
to 24 h lighting (160 l
Einstein mꢀ2 sꢀ2, at 17 °C). After a 24 h treat-
ment, their leaf tissue was weighed and frozen in liquid nitrogen
Fett-Neto, A.G., Melanson, S.J., Nicholson, S.A., Pennington, J.J., DiCosmo, F., 1994.
Improved taxol yield by aromatic carboxylic acid and amino acid feeding to cell
cultures of Taxus cuspidata. Biotech. Bioeng. 44, 967–971.
Fraser, C.M., Thompson, M.G., Shirley, A.M., Ralph, J., Schoenherr, J.A., Sinlapadech,
T., Hall, M.C., Chapple, C., 2007. Related serine carboxypeptidase-like
Sinapoylglucose acyltransferases display distinct but overlapping substrate
specificities. Plant Phys. 144, 1986–1999.
Fujiwara, H., Tanaka, Y., Fukui, Y., Ashikari, T., Yamaguchi, M., Kusumi, T., 1998.
Purification and characterization of anthocyanin 3-aromatic acyltransferase
from Perilla frutescens. Plant Sci. 137, 87–94.
Ghangas, G.S., 1999. A survey of the nature of glucose acylation reactions in plant
extracts. Phytochemistry 52, 785–792.
Harborne, J.B., Williams, C.A., 1998. Anthocyanins and other flavonoids. Nat. Prod.
Rep. 15, 631–652.
Himer, A.A., Veit, S., Seitz, H.U., 2001. Regulation of anthocyanin biosynthesis in UV-
A irradiated cell cultures of carrot and in organs of intact carrot plants. Plant Sci.
161, 315–322.
Li, J.Y., Oulee, T.M., Raba, R., Amundson, R.G., Last, R.L., 1993. Arabidopsis flavonoid
mutants are sensitive to UV-B radiation. Plant Cell 5, 171–179.
Lopez-Villalobos, A., Hornung, R., Dodds, P.F., 2004. Hydrophobic metabolites of 2,4-
dichlorophenoxyacetic acid (2,4-D) in cultured coconut tissue. Phytochemistry
65, 2763–2774.
Ma, X.Y., Koepke, J., Panjikar, S., Fritzsch, G., Stockigt, J., 2005. Crystal structure of
vinorine synthase, the first representative of the BAHD superfamily. J. Biol.
Chem. 280, 13576–13583.
prior to storage at ꢀ80 °C.
4.3. Extraction and analysis of metabolites
Plant samples (1 g) were extracted in ice-cold methanol (3 ml)
using a pestle and mortar and acid-washed sand as an abrasive.
After centrifugation (16,000g, 5 min), the extract was concentrated
to 1 ml under reduced pressure. Chloroform (1 ml) and distilled
water (200
fuged for 1 min. The upper methanolic layer was removed and
10 m,
injected onto an UPLCTM BEH C18 column (1.7
ll) were added and the resulting emulsion re-centri-
ll
l
2.1 ꢁ 100 mm; Waters Acuity). The column was then eluted at
0.2 ml minꢀ1 with a mixture of 0.5% formic acid (solvent A) and
acetonitrile containing 0.5% formic acid (solvent B) using a linear
gradient of 5–95% solvent B over 9 min, followed by isocratic elu-
tion with 95% B for a further 2 min. The eluant was monitored by
diode array detection for UV–Vis-absorbing metabolites, with the
absorbance at 330 nm used for quantification after calibrating
the system with a flavonoid standard (quercetin 3-O-diglucoside).
The eluant was then analysed by MS after electrospray ionization
using a Micromass QTof spectrometer operating in positive ion
mode. Settings were sample cone voltage = 41 kV, capillary volt-
age = 2.55 kV, extraction cone voltage = 5 kV, source tempera-
ture = 100 °C and desolvation temperature = 180 °C. Metabolites
were identified from parent and fragment mass ions with reference
to published spectra (Cuyckens et al., 2003).
Nielsen, J.K., Olsen, C.E., Petersen, M.K., 1993. Acylated flavonol glycosides from
cabbage leaves. Phytochemistry 34, 539–544.
Ojima, I., 2004. Use of fluorine in the medicinal chemistry and chemical biology of
bioactive compounds: a case study on fluorinated taxane anticancer agents.
Chembiochem 5, 628–635.
Sawada, S., Suzuki, H., Ichimaida, F., Yamaguchi, M., Iwashita, T., Fukui, Y., Hemmi,
H., Nishino, T., Nakayama, T., 2005. UDP-Glucuronic acid: anthocyanin
glucuronosyltransferase from Red Daisy (Bellis perrenis) flowers. J. Biol. Chem.
280, 899–906.
Smart, B.E., 2001. Fluorine substituent effects (on bioactivity). J. Fluor. Chem. 109,
3–11.
St-Pierre, B., Laflamme, P., Alarco, A.M., De Luca, V., 1998. The terminal
O-acetyltransferase involved in vindoline biosynthesis defines a new class of
proteins responsible for coenzyme A-dependent acyl transfer. Plant J. 14, 703–
713.
Stochmal, A., Simonet, A.M., Macias, F.A., Oliviera, M.A., Abreu, J.M., Nash, R.,
Oleszek, W., 2001. Acylated apigenin glycosides from alfalfa (Medicago sativa L.)
var. Artal. Phytochem. 57, 1223–1226.
Acknowledgements
The authors acknowledge the Biotechnology and Biological
Sciences Research Council for their support of a studentship (OC)
and a research development fellowship (RE).
Suzuki, H., Nakayama, T., Yonekura-Sakakibara, K., Fukui, Y., Nakamura, N., Nakao,
M., Tanaka, Y., Yamaguchi, M., Kusumi, T., Nishino, T., 2001. Malonyl-CoA:
anthocyanin 5-O-glucoside-6000-O-malonyltransferase from scarlet sage (Salvia
References
Alluis, B., Dangles, O., 1999. Acylated flavone glucosides: synthesis, conformational
investigation, and complexation properties. Helv. Chim. Acta 82, 2201–2212.
Baker, D.C., Dougall, D.K., Glassgen, W.E., Johnson, S.C., Metzger, J.W., Rose, A., Seitz,
H.U., 1994. Effects of supplied cinnamic acids and biosynthetic intermediates on
the anthocyanins accumulated by wild carrot suspension-cultures. Plant Cell
Tissue Org. Cult. 39, 79–91.
splendens) flowers
– enzyme purification, gene cloning, expression and
characterization. J. Biol. Chem. 276, 49013–49019.
Walker, K., Croteau, R., 2000. Taxol biosynthesis: molecular cloning of
a
benzoyl-CoA: taxane
2 alpha-O-benzoyltransferase cDNA from Taxus and
functional expression in Escherichia coli. Proc. Natl. Acad. Sci. USA 97,
13591–13596.