Vir-gene-inducing activities of hydroxycinnamic acid amides in Agrobacterium tumefaciens

Article abstract of DOI:10.1016/S0031-9422(98)00209-X

Expression of Agrobacterium tumefaciens virulence genes and transformation of dicots by this organism are dependent upon host plant phenolic compounds. Several alkylsyringamides have recently been shown to be powerful inducers of these vir-genes. These synthetic amides, and especially ethylsyringamide, are much stronger inducers than syringic acid. In this work, four alkylamides derived from ferulic or sinapic acids were synthesized by a dicyclohexylcarbodiimide method and tested for their potential to induce vir-gene expression on A. tumefaciens strains harbouring virB::lacZ or virE::lacZ fusion plasmids. Their effectiveness was compared to that of ethylsyringamide and tyraminylferulamide, a naturally occurring amide in plants. Whatever the amine moiety of the amide (ethylamine, propylamine, tyramine or β-alanine ethyl ester) conjugation of the acid functional group clearly diminished the toxicity to the bacteria of the respective acid at high concentration and thereby increased the vir-inducing potential. However, none of the inducers tested exhibited higher activity than acetosyringone, the reference compound for vir-gene induction, with the exception of ethylsyringamide at concentrations above 1 mM. When tested on Agrobacterium tumefaciens strain A348(pSM243cd), ethylferulamide and ethylsinapamide were more efficient than the corresponding phenolic acids but only above 100 μM.

Full text of DOI:10.1016/S0031-9422(98)00209-X

Phytochemistry Vol. 49, No. 6, pp. 1537±1548, 1998
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0031-9422/98/$ - see front matter
PII: S0031-9422(98)00209-X
VIR-GENE-INDUCING ACTIVITIES OF
HYDROXYCINNAMIC ACID AMIDES IN
AGROBACTERIUM TUMEFACIENS
KARINE BERTHELOT, DAVID BURET, BRIGITTE GUERIN, DIDIER DELAY,
JONATHAN NEGREL and FRANCIS M. DELMOTTE*
Glycobiologie, Centre de Biophysique Moleculaire, CNRS UPR 4301, aliated to the University of
Orleans, rue Charles Sadron, 45071 Orleans cedex 2, France; Laboratoire de Phytoparasitologie
INRA-CNRS, INRA BV 1540, 21034 Dijon cedex, France
(Received in revised form 16 February 1998)
Key Word IndexÐAgrobacterium tumefaciens; vir-genes; phenolic amide; syringic acid; sina-
pic acid; ferulic acid; ethylamine; tyramine.
AbstractÐExpression of Agrobacterium tumefaciens virulence genes and transformation of dicots by this
organism are dependent upon host plant phenolic compounds. Several alkylsyringamides have recently
been shown to be powerful inducers of these vir-genes. These synthetic amides, and especially ethylsyringa-
mide, are much stronger inducers than syringic acid. In this work, four alkylamides derived from ferulic or
sinapic acids were synthesized by a dicyclohexylcarbodiimide method and tested for their potential to
induce vir-gene expression on A. tumefaciens strains harbouring virB::lacZ or virE::lacZ fusion plasmids.
Their e€ectiveness was compared to that of ethylsyringamide and tyraminylferulamide, a naturally occur-
ring amide in plants. Whatever the amine moiety of the amide (ethylamine, propylamine, tyramine or b-
alanine ethyl ester) conjugation of the acid functional group clearly diminished the toxicity to the bacteria
of the respective acid at high concentration and thereby increased the vir-inducing potential. However,
none of the inducers tested exhibited higher activity than acetosyringone, the reference compound for vir-
gene induction, with the exception of ethylsyringamide at concentrations above 1 mM. When tested on
Agrobacterium tumefaciens strain A348(pSM243cd), ethylferulamide and ethylsinapamide were more e-
cient than the corresponding phenolic acids but only above 100 mM. # 1998 Elsevier Science Ltd. All
rights reserved
INTRODUCTION
phenolic compounds, such as acetosyringone and
hydroxyacetosyringone, which are inducers of the
virulence (vir) genes mediating T-DNA transfer (for
reviews [3, 4]). Some plant species, including mono-
cots, produce phenols but are recalcitrant to trans-
formation, possibly because of the absence of
speci®c vir-gene inducers [5]. The lignin precursors
in dicots, coniferyl alcohol and sinapyl alcohol are
strong vir-inducers [6, 7]. Two phenolic esters, (i)
the methyl ester of syringic acid which occurs in
various grapevine-derived wound exudates and (ii)
ethyl ferulate isolated from Triticum monococcum,
have been described as being more potent vir-indu-
Agrobacterium tumefaciens is a Gram-negative soil
bacterium which causes crown gall disease, a tumor-
ous growth of many dicotyledonous plants as well
as some monocotyledonous species. By its ability to
transfer genetic material to higher dicot plants,
Agrobacterium tumefaciens turned out to be a very
useful vector for genetic engineering, since it is
possible to replace some of the T-DNA genes (part
of the tumor-inducing plasmid of Agrobacterium)
with genes of agronomic interest (for recent reviews,
see [1, 2]). However this technique is ultimately
dependent on the ability of the bacterium to sense cers than acetosyringone [8, 9]. In addition, numer-
ous plant phenolic compounds are glycosylated
and potent vir-inducers [5, 10±13]. Recent studies
have also shown that several synthetic alkyla-
mides can activate the virulence system of
Agrobacterium [14, 15]. Interestingly these alkyla-
the environment: pH, sugars and particularly plant
*Author to whom correspondence should be addressed.
Tel: 33 (0)2 38-25-55-93; Fax: 33 (0)2 38-69-00-94;
E-mail: delmotte@cnrs-orleans.fr
1537

1538
K. BERTHELOT et al.
mides, and especially ethylsyringamide, are much di€erent phenolic acids tested was obtained with
more powerful inducers than the corresponding sinapic acid, but remained nevertheless lower than
acid, syringic acid [14, 15]. In an attempt to general- the response obtained in the presence of acetosyrin-
ize this result to hydroxycinnamic acids, we under- gone (Fig. 1B and D; Fig. 3A and C).
took the chemical synthesis of ethylsinapamide,
propylsinapamide,
and ethylferulamide. We report here their vir-indu-
cing activity on Agrobacterium tumefaciens strains
carrying a virB::lacZ and virE::lacZ gene fusion
plasmids. Their activity is compared with that of
Action of tyraminylferulamide and tyraminylsinapa-
mide on vir-gene induction
carbethoxyethylensinapamide,
The ®nding that the introduction of an amide
group into syringic acid strongly enhances its vir-
gene-inducing activity prompted us to test the ac-
tyraminylferulamide,
a natural hydroxycinnamic
tivity of tyraminylferulamide (10),
a naturally
amide which is of widespread occurrence in higher
plants and which is known to occur in Solanaceous
plants both in wound-healing [16, 17] and in crown-
gall tissues [18].
occurring phenolic amide in plants and of its ana-
logue, tyraminylsinapamide (11). 10 was a weak vir-
gene inducer and became more ecient than ferulic
acid only at concentrations above 500 mM. It exhib-
ited however a low toxicity at high concentration
on both strains studied (Fig. 1A and C). 11 began
to activate vir-genes at 100 mM and turned out to
be also less toxic at 1 mM than sinapic acid or acet-
osyringone (Fig. 1B and D). Both amides are there-
fore potentially useful vir-gene inducers if used at
concentrations above 500 mM.
RESULTS AND DISCUSSION
Alkylamides were synthesized according to
a
modi®ed carbodiimide original procedure [19], con-
sisting of the coupling of alkylamine onto the car-
boxyl group of hydroxycinnamic acids previously
activated by N-hydroxysuccinimide in the presence
of dicyclohexylcarbodiimide. This method, pre-
viously used for the synthesis of various amides de-
rived from syringic acid [14], allowed yields for the
preparation of sinapyl and ferulyl amides between
47 and 71%. Analyses of these amides by liquid
chromatography (HPLC) showed they were homo-
geneous and the hydrophobic character of these
new derivatives demonstrated by their retention
times (see Experimental section). The virulence-
inducing activity of the amides was tested on two
Agrobacterium tumefaciens strains A348(pSM243cd)
(locus virB) and A348(pSM358) (locus virE) carry-
ing the b-galactosidase reporter gene of E. coli
under the control of virB and virE promoters,
respectively [20]. This enzymatic activity was
measured by a sensitive spectro¯uorimetric method
using 4-methylumbelliferyl b-D-galactopyranoside as
substrate [11, 12, 14]. The vir-inducing activity of
sinapic acid (1), ferulic acid (5), syringic acid (7),
and acetosyringone (9), was also tested, over a wide
range of concentrations between 0.5 mM and 5 mM.
Acetosyringone was the ®rst vir-gene inducer ident-
i®ed by Stachel and co-workers [21] and is now
classically used as the reference compound for vir-
Action of ethylferulamide and ethylsyringamide on
vir-gene induction
Ethylferulamide (6) was a better inducer than
ferulic acid at concentrations above 300 mM
(Fig. 2A and C). The di€erence in vir-inducing ac-
tivity on strain A348(pSM243cd) was comparable
with that observed between syringic acid and ethyl-
syringamide (8) (Fig. 2A and B). 6 and 8 had di€er-
ent behaviour however on strain A348(pSM358). In
the concentration range tested, the dose-response
curve of ethylsyringamide was always above the
curve of syringic acid (Fig. 2D), whereas the induc-
tion level obtained in the presence of ethylferula-
mide exceeded the level reached with ferulic acid
only above 100 mM (Fig. 2C). The activity of ethyl-
syringamide (8) on strain A348(pSM243cd) was 19-
fold higher than that of syringic acid at the concen-
tration of 1 mM. 8 began to activate vir-genes at
150 mM, and showed a less toxic e€ect than aceto-
syringone at 5 mM. These results con®rm previous
data obtained after 6 hr of incubation [14].
Action of alkylsinapamides on vir-gene induction
Ethylsinapamide (2), propylsinapamide (3), and
gene inductions. Phenolic acids 1, 5 and 7 were less carbethoxyethylensinapamide (4) did not exhibit
active than acetosyringone, con®rming previous higher vir-inducing activities than either sinapic acid
results [22] (Figs 1±3). Free phenolic compounds, (1) or acetosyringone (9). The following order:
and especially phenolic acids, are known to become 9>1>2>3>4 was observed on the strain
toxic to Agrobacterium tumefaciens at high concen- A348(pSM358) (Fig. 3C and D) but at the concen-
tration (above 500 mM) [11, 12, 14, 23]. The strain tration of 100 mM, the same products exhibited a
A348(pSM358) exhibited a greater vir-gene-inducing di€erent induction activity on the other strain tested
sensitivity to phenolic acids than the strain A348(pSM243cd): 9>2>1>3>4 (Fig. 2A and 2B),
A348(pSM243cd) (Fig. 1C and D; Fig. 2C and D; indicating that the introduction of an amide func-
Fig. 3C). This result con®rms that the response to tion does not systematically enhance the activity of
signal molecules can vary according to the strain sinapic acid. Moreover, if we compare the action of
used [22, 24]. The best induction response of the ethylferulamide and ethylsinapamide on vir-gene in-

vir-Gene inducing activities of phenolic amides
duction, we can conclude that the O-methyl group
1539
EXPERIMENTAL
in R1 (Table 1) is not strictly required for optimal
induction. Melchers and co-workers [6] established
that at least one methoxyl and the para-hydroxyl
group were essential for the perception of phenolics,
but it seems that the substitution in R2 is the criti-
cal determinant. In addition Hess and co-workers
hypothesized [25] that phenolic compounds interact
chemically with their receptor in a hydrophobic
pocket. Alkylamides possess hydrophobic proper-
ties, but the most hydrophobic molecules, such as
carbethoxyethylensinapamide and tyraminylamides,
were not the most active. In our previous work, we
also observed that carbethoxyethylensyringamide
was a relatively poor inducer in comparison with
ethylsyringamide [14].
Bacterial strains and culture conditions
Agrobacterium tumefaciens, merodiploid strains
A348 (pSM358) and A348 (pSM243cd) were used
to assess vir-inducing activities. They are derived
from strain C58, harboring the octopine-Ti plasmid
(pTiA6) and another plasmid carrying virE::lacZ
and virB::lacZ fusions respectively [20]. Bacteria
were maintained at 258 on MG/L medium [30] sup-
1
plemented with 100 mg l
1
carbenicillin and
kanamycin (Sigma, L'Isle d'Abeau
100 mg l
Chesnes, France).
vir-Gene induction assays and b-galactosidase ac-
tivity measurement. Three-day old colonies of Agro-
bacterium tumefaciens strains were suspended and
cultivated for 24 hr in Murashige and Skoog med-
ium (MS) [31] supplemented with appropriate anti-
biotics at 288 using a rotary shaker (170 rpm). After
cultivation, bacteria were harvested by centrifu-
gation at 1,700 g for 10 min and resuspended in
fresh MS medium with antibiotics at approximately
2 Â108 bacteria per ml. Then, 2 ml culture were
transferred in sterilized test tubes and 63 ml of indu-
cer stock solns prepared in dimethylsulfoxide
(DMSO, Merck, Nogent-sur-Marne, France) were
added to give ®nal concns of 0.5, 1.58, 5, 15.8, 50,
158, 500, 1580 and 5000 mM. Controls without
inducer and with DMSO were also included. Bac-
teria were incubated with various inducers for 12 hr
at 258 on a rotary shaker (250 rpm). The bacterial
suspension was shaken vigorously using a Vortex
shaker and 1 ml of induced bacteria was removed
and centrifuged. The pellet was resuspended in
1.5 ml of 1 mM MgCl2, 0.1 M Na-Pi bu€er, pH 7.3.
After homogeneization, this vol. was divided into
aliquots of 500 ml in order to perform the exper-
iment in triplicate. Each 500 ml was then shaken
with 20 ml of 0.05% SDS and 20 ml CHCl3 for
In conclusion, our results con®rm that conju-
gation with ethylamine, can increase the vir-gene
inducing activity of ferulic and sinapic acids but
only on some Agrobacterium strains. The ®nding
that tyraminylferulamide is less active than ethylfer-
ulamide or ferulic acid seems to rule out the possi-
bility that hydroxycinnamic acid amides of
tyramine could play an important role in vir-gene
induction in vivo, although these amides are known
to be synthesized very early in wound-healing
tissues [16] and to be associated, like many vir-
genes inducing phenolics [7], to the formation of lig-
nin or lignin-like compounds [17]. Our results bring
new information about the structure-activity re-
lationships of phenolic compounds which may
induce vir-gene expression. These data should prove
useful to study the properties of the putative recep-
tor (Pbp39) which has been puri®ed from bacterial
proteins using an ethylsyringamide matrix [26] and
its interactions with the other receptors or proteins
known to be involved in the sensing of plant signal
molecules by Agrobacterium [22, 27±29].
Table 1. Description of di€erent phenolic derivatives showing the substitution at R1 and R2
R1
R2
Usual name
1
2
3
4
OCH3
OCH3
OCH3
OCH3
H
CH = CHCO2H
Sinapic acid
CH = CHCONHCH2CH3
CH = CHCONHCH2CH2CH3
CH = CHCONHCH2CH2CO2CH2CH3
CH = CHCO2H
Ethylsinapamide
Propylsinapamide
Carbethoxyethylensinapamide
Ferulic acid
5
6
7
8
9
10
11
H
CHCONHCH2CH3
CO2H
CONHCH2CH3
COCH3
CH = CHCONHCH2CH2(C6H4)OH
CH = CHCONHCH2CH2(C6H4)OH
Ethylferulamide
Syringic acid
Ethylsyringamide
Acetosyringone
N-tyraminylferulamide
N-tyraminylsinapamide
OCH3
OCH3
OCH3
H
OCH3

1540
K. BERTHELOT et al.
10 sec and incubated at 258 for 11 min to permeabi- of 0.1 M Na-Pi bu€er at pH 7.3) were added. After
lize bacterial membranes. Then 100 ml of 4-methy- 11 min incubation at 258, the reaction was stopped
lumbelliferyl b-D-galactopyranoside (Sigma, 3 mg by adding 500 ml of 1 M Na2CO3. The bacteria
dissolved in 200 ml of DMSO and diluted in 15 ml were centrifuged and the ¯uorescence intensity of
Fig. 1A and BÐCaption opposite

vir-Gene inducing activities of phenolic amides
1541
Fig. 1. The virulence activity of tyraminylferulamide (10, q) and tyraminylsinapamide (11, t).
Following 12 hr incubations at 258 and pH 5.6, b-galactosidase activity in Agrobacterium tumefaciens
strains A348(pSM243cd) and A348(pSM358), respectively carrying virB::lacZ (A and B) and virE::lacZ
(C and D), is expressed in ¯uorescence arbitrary units. To assist in comparing curves, the activity of
acetosyringone (9, w), ferulic acid (5, Q) and sinapic acid (1, T) are presented.
100 ml of the supernatant was measured on a ¯uor- General methods for chemistry and chemicals
escence microtiter plate reader (Titertek Fluoroskan
Mp: uncorr., Leitz hot-plate microscope; IR: KBr
pellets, Perkin-Elmer 16 PC FT-IR spectrometer;
II, Labsystems, lEx = 355 nm, lEm = 460 nm).

1542
K. BERTHELOT et al.
1H NMR: 300 MHz, AM 300 WB Bruker spectro- (19:1)]; TLC: silica gel Merck 60 F254using EtOAc-
photometer; MS: Lasermat 2000-MALDI TOF MeOH (17:3) as solvent and spots were detected by
spectrometer with an a-cyano-4-hydroxycinnamic ¯uorescence and by successive treatments with NH3
1
acid matrix [Sigma, 1 mg ml
in MeCN-EtOH and I2. UV/visible and ¯uorescence spectra were
Fig. 2A and BÐCaption opposite

vir-Gene inducing activities of phenolic amides
1543
Fig. 2. The virulence activity of ethylferulamide (6, q) and ethylsyringamide (8, r). Following 12 hr in-
cubations at 258 and pH 5.6, b-galactosidase activity in Agrobacterium tumefaciens strains
A348(pSM243cd) and A348(pSM358), carrying virB::lacZ (A and B) and virE::lacZ (C and D) respect-
ively, is expressed in ¯uorescence arbitrary units. The activity of acetosyringone (9, w), ferulic acid (5,
Q) and syringic acid (7, W ) are also presented.

1544
K. BERTHELOT et al.
recorded respectively with an Uvikon 860 spectro- at pH 7.4; 0.05 M Na2CO3 bu€er at pH 9.5.
photometer and a Shimadzu RF-5000 spectro¯uori- Phenolic acids, N-hydroxysuccinimide and amines
meter. Spectra were carried out in di€erent bu€ers: were purchased from Sigma. Ethylene glycol
0.1 M NaOAc bu€er at pH 5.2; 0.1 M Na-Pi bu€er dimethyl ether, triethylamine and acetosyringone
Fig. 3A and BÐCaption opposite

vir-Gene inducing activities of phenolic amides
1545
Fig. 3. The virulence activity of ethylsinapamide (2, t), propylsinapamide (3, Â) and carbethoxyethy-
lensynapamide (4, q). Following 12 hr incubations at 258 and pH 5.6, b-galactosidase activity in
Agrobacterium tumefaciens strains A348(pSM243cd) and A348(pSM358), respectively carrying
virB::lacZ (A and B) and virE::lacZ (C and D), is expressed in ¯uorescence arbitrary units. The activity
of acetosyringone (9, w) and synapic acid (1, T) are also presented in ®gures A and C.

1546
K. BERTHELOT et al.
were from Aldrich (Sigma). b-Alanine ethyl ester
hydrochloride was from Bachem (Bubendorf,
Switzerland). Silica Gel 60 (0.04±0.06 mm, 230±400
mesh) and other chemical reagents were purchased
from Merck.
l
l
maxEm = 454 nm; (pH 9.5)
maxEm = 501 nm; FTIR nmax cm 1: 3358 (NH),
lmaxEx = 368 nm,
2924 (CH), 1658 (CO amide I), 1594 (NH amide
II), 1512, 1300, 1210 and 1114 (CO methoxy), 982
1
and 830 (CH arom.); H NMR (DMSO-d6): d 8.71
(1H, s, OH), 7.89 (1H, t, J30, NH), 7.30 (1H, d,
J = 15.5 Hz, H-8), 6.83 (2H, s, H-2 and H-6), 6.43
(1H, d, J = 15.5 Hz, H-7), 3.78 (6H, s, 2 ÂOCH3),
3.17 (2H, quint., J = 6.5 Hz, CH2), 1.05 (3H, t,
J = 7 Hz, CH3); MS: m/z (rel. int.): 290 [M + K]+
(12), 274 [M + Na]+ (14), 251 [M]+ (30), 207
[M NHEt]+ (32), 179 [M CONHEt]+ (9), 72
[CONHEt]+ (12), 44 [NHEt]+ (17), 39 [K]+ (100),
23 [Na]+ (50).
Propylsinapamide (3). Crystals from EtOAc±
MeOH (17:3), (yield: 47%); Rf 0.72; mp 196±1988.
(Found: C, 62.82; H, 7.31; N, 5.19. C14H19NO4
requires: C, 63.40; H, 7.17; N, 5.28%). UV
(pH 5.2): lmax nm (log E): 312 (3.76); (pH 7.4): lmax
nm (log E): 312 (3.71); (pH 9.5): lmax nm (log E):
368 (3.83); ¯uorescence (pH 5.2) lmaxEx = 312 nm,
General procedure for synthesis of alkylamides 2, 3,
4, 6 and 8
Synthesis was carried out by a two-step pro-
cedure: activation of phenolic acid by the dicyclo-
hexylcarbodiimide method [19] followed by
coupling of alkylamine. N-hydroxysuccinimide
(NHS, 1.1 equiv) and N,N'-dicyclohexylcarbodii-
mide (DCC, 1.5 equiv) were added at room temp.
to a stirred soln of phenolic acid (5.0 g, 1 equiv) in
ethylene glycol dimethyl ether (90 ml). The soln was
stirred at room temp. for 30 min in the dark. Under
these conditions, the phenolic acid was quantitat-
ively transformed into the NHS ester of phenolic
acid. The abundant white ppt of N,N'-dicyclohexy-
lurea produced during the reaction was ®ltered o€
and the ®ltrate of NHS ester was directly coupled
to the alkylamine with free amino group (2.1
equiv). The soln was stirred at room temp. for 2 hr
in the dark. After ®ltration of liberated NHS, the
product is then half-conc under red. pres. and preci-
pitated. The dried product was dissolved in CHCl3
(120 ml), and washed, ®rst with a cold soln of 10%
KHSO4 (40 ml) and secondly with saturated aq.
NaCl (40 ml). For propylsynapamide H2SO4 was
put in place of KHSO4. Finally, the organic phase
was dried on anhydrous Na2SO4 and evaporated to
dryness. Products were crystallized in EtOAc±
MeOH (17:3) at room temp.
l
l
l
maxEm = 449 nm; (pH 7.4)
maxEm = 455 nm; (pH 9.5)
l
maxEx = 312 nm,
l
maxEx = 368 nm,
maxEm =505 nm; FTIR nmax cm 1: 3358 (NH),
2966 (CH), 1660 (CO amide I), 1594 (NH amide
II), 1512, 1322, 1210 and 1114 (CO methoxy), 984
1
and 800 (CH arom.); H NMR (DMSO-d6): d 8.71
(1H, s, OH), 7.89 (1H, s, broad, NH), 7.30 (1H, d,
J = 15.5 Hz, H-8), 6.83 (2H, s, H-2 and H-6), 6.46
(1H, d, J = 15.5 Hz, H-7), 3.78 (6H, s, 2 ÂOCH3),
3.11 (2H, q, J = 6 Hz, NHCH2), 1.45 (2H, sext.,
J = 7 Hz, CH2CH3), 0.86 (3H, t, J = 7.5 Hz, CH3);
MS: m/z (rel. int.): 304 [M + K]+ (5), 288
[M + Na]+ (9), 265 [M]+ (26), 207 [M NHPr]+
(11), 179 [M CONHPr]+ (3), 86 [CONHPr]+ (4),
58 [NHPr]+ (4), 39 [K]+ (100), 23 [Na]+ (19).
Carbethoxyethylensinapamide (4). Crystals from
EtOAc±MeOH (17:3), (yield: 56%); Rf 0.77;
mp 205±2078. (Found: C, 57.75; H, 6.65; N, 4.97.
C16H21NO6 requires: C, 59.44; H, 6.50; N, 4.33%).
UV (pH 5.2): lmax nm (log E): 312 (3.72); (pH 7.4):
lmax nm (log E): 316 (3.70); (pH 9.5): lmax nm
(log E): 372 (3.62); ¯uorescence (pH 5.2)
N-succinimidyl sinapate. Crystals from ethylene
glycol dimethyl ether; Rf 0.59, mp 179±1818. UV
(pH 5.2): lmax nm (log E): 340 (3.72); (pH 7.4): lmax
nm (log E): 337 (3.70); (pH 9.5): lmax nm (log E):
382 (3.62); ¯uorescence (pH 5.2) lmaxEx = 340 nm,
l
l
l
maxEm = 476 nm; (pH 7.4)
maxEm = 471 nm; (pH 9.5)
l
maxEx = 337 nm,
l
maxEx = 382 nm,
maxEm = 496 nm. FTIR nmax cm 1: 3394 (OH),
2942 and 2848 (CH), 1732 (CO ester), 1512, 1300,
1210 and 1114 (CO methoxy), 982 and 830 (CH
arom.). 1H NMR (DMSO-d6): d 9.20 (1H, s, OH),
7.84 (1H, d, J = 16 Hz, H-8), 7.18 (2H, s, H-2 and
H-6), 6.84 (1H, d, J = 16 Hz, H-7), 3.81 (6H, s, 2x
OCH3), 2.84 (4H, s, CH2CH2). MS: m/z (rel. int.):
223 [M NHS]+ (32), 207 [M ONHS]+ (100),
179 [M COONHS]+ (18), 142 [COONHS]+ (4),
98 [NHS]+ (4).
Ethylsinapamide (2). Crystals from EtOAc±
MeOH (17:3), (yield: 68%); Rf 0.66; mp 164±1668.
(Found: C, 61.73; H, 6.74; N, 5.48. C13H17NO4
requires: C, 62.15; H, 6.77; N, 5.58%). UV
(pH 5.2): lmax nm (log E): 312 (4.20); (pH 7.4): lmax
nm (log E): 312 (4.11); (pH 9.5): lmax nm (log E):
l
l
l
maxEx = 312 nm,
maxEx = 316 nm,
l
maxEm = 442 nm; (pH 7.4)
lmaxEm = 448 nm; (pH 9.5)
maxEx = 372 nm, lmaxEm = 505 nm; FTIR nmax
cm 1: 3394 (NH), 2962 (CH), 1736 (CO ester), 1660
(CO amide I), 1618 (NH amide II), 1512, 1262,
1104 (CO methoxy), 1028, 800 (CH arom.); 1H
NMR (DMSO-d6): d 8.73 (1H, s, OH), 7.98 (1H, t,
J30, NH), 7.31 (1H, d, J = 15.5 Hz, H-8), 6.83
(2H, s, H-2 and H-6), 6.46 (1H, d, J = 15.5 Hz, H-
7), 4.05 (2H, q, J = 7 Hz, OCH2CH3), 3.78 (6H, s,
2 ÂOCH3), 3.38 (2H, q, J = 6.5 Hz, NHCH2), 2.39
(2H, t, J = 6 Hz, CH2CO), 1.18 (3H, t, J = 7 Hz,
CH3); MS: m/z (rel. int.): 362 [M + K]+ (41), 346
[M + Na]+ (35), 323 [M]+ (47), 207 [M bAla-
368 (4.26); ¯uorescence (pH 5.2) lmaxEx = 312 nm, OEt]+ (100), 116 [bAla-OEt]+ (9), 39 [K]+ (53), 23
lmaxEm = 446 nm; (pH 7.4)
l
maxEx = 312 nm, [Na]+ (6).

vir-Gene inducing activities of phenolic amides
1547
Ethylferulamide (6). Crystals from EtOAc± (100), 328 [M Me]+ (8), 207 [M tyraminyl]+
(30).
HPLC. Analyses were carried out using an iso-
cratic mobile phase (MeCN±HOAc±H2O, 7:1:42)
MeOH (17:3), (yield: 71%); Rf 0.64; mp 159±1618.
(Found: C, 64.98; H, 6.88; N, 6.97. C12H15NO3
requires: C, 65.16; H, 6.79; N, 6.33%). UV
(pH 5.2): lmax nm (log E): 291 (4.01); (pH 7.4): lmax
nm (log E): 308 (4.05); (pH 9.5): lmax nm (log E):
354 (4.21); ¯uorescence (pH 5.2) lmaxEx = 291 nm,
1
with a ¯ow of 1 ml min and detection at A280
.
The sample (1 mg) was dissolved in mobile phase
solution (1 ml) and 10 ml of the solution was
injected onto an ODS Spheri 5 m analytical column
(4.6Â 220 mm; Spectra Physics) and a RP18 guard
column (7 mm Â2 cm). Retention times in min for 1
16.3, 2 24.7, 3 49.2, 4 58.1, 5 15.9, 6 21.4, 7 7.4, 8
10.5, 9 15.6, 10 85.7, 11 101.6.
l
l
l
maxEm = 432 nm; (pH 7.4)
maxEm = 436 nm; (pH 9.5)
l
maxEx = 308 nm,
l
maxEx = 354 nm,
maxEm = 487 nm; FTIR nmax cm 1: 3394 (OH),
3334 (NH), 2932 (CH), 1658 (CO amide I), 1586
(NH amide II), 1514, 1282, 1210, 1158, 1126 (CO
methoxy), 1032, 978, 822 (CH arom.); 1H NMR
(DMSO-d6): d 9.35 (1H, s, OH), 7.90 (1H, t, J30,
NH), 7.29 (1H, d, J = 15.5 Hz, H-8), 7.10 (1H, s,
H-2), 6.97 (1H, d, J = 8 Hz, H-6), 6.78 (1H, d,
J = 8 Hz, H-5), 6.40 (1H, d, J = 15.5 Hz, H-7),
3.79 (3H, s, OCH3), 3.17 (2H, quint., J = 6.5 Hz,
NHCH2), 1.05 (3H, t, J = 7 Hz, CH3); MS: m/z
(rel. int.): 265 [M + NHEt]+ (18), 260 [M + K]+
(8), 244 [M + Na]+ (12), 221 [M]+ (28), 177
[M NHEt]+ (10), 149 [M CONHEt]+ (2), 39
[K]+ (100), 23 [Na]+ (54).
AcknowledgementsÐWe would like to give special
thanks to Prof. E. W. Nester (University of
Washington, Seattle, WA, U.S.A.) who kindly pro-
vided us the Agrobacterium tumefaciens strains
A348(pSM243cd) and A348(pSM358). We thank
Mr H. Labbe for providing NMR spectra. Dr S.
Hawkins (INRA, Ardon) is acknowledged for criti-
cally reading through the manuscript. This research
was partly supported by grants from AIP/INRA
96/P 00030 Biologie du developpement. K.
Berthelot received fellowships from MENESRIP
Ethylsyringamide (8). Synthesis and character-
istics were previously described in Ref. [14].
(Ministere
de
l'Education
Nationale,
de
l'Enseignement Superieur et de la Recherche, et de
l'Insertion Professionnelle).
Tyraminylferulamide (10). Synthesis of 10 was
previously described [32, 33] and some chemical
characteristics of plant derived crystals of 10 were
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2 ÂOCH3), 3.33 (2H, q, J = 6.5 Hz, H-8'), 2.64
(2H, t, J = 7.5 Hz, H-7'); MS: m/z (rel. int.): 382
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