Effects of synthetic alkamides on Arabidopsis fatty acid amide hydrolase activity and plant development

Article abstract of DOI:10.1016/j.phytochem.2014.11.011

Alkamides and N-acylethanolamines (NAEs) are bioactive, amide-linked lipids that influence plant development. Alkamides are restricted to several families of higher plants and some fungi, whereas NAEs are widespread signaling molecules in both plants and animals. Fatty acid amide hydrolase (FAAH) has been described as a key contributor to NAE hydrolysis; however, no enzyme has been associated with alkamide degradation in plants. Herein reported is synthesis of 12 compounds structurally similar to a naturally occurring alkamide (N-isobutyl-(2E,6Z,8E)decatrienamide or affinin) with different acyl compositions more similar to plant NAEs and various amino alkyl head groups. These "hybrid" synthetic alkamides were tested for activity toward recombinant Arabidopsis FAAH and for their effects on plant development (i.e., cotyledon expansion and primary root length). A substantial increase in FAAH activity was discovered toward NAEs in vitro in the presence of some of these synthetic alkamides, such as N-ethyllauroylamide (4). This "enhancement" effect was found to be due, at least in part, to relief from product inhibition of FAAH by ethanolamine, and not due to an alteration in the oligomerization state of the FAAH enzyme. For several of these alkamides, an inhibition of seedling growth was observed with greater results in FAAH knockouts and less in FAAH over-expressing plants, suggesting that these alkamides could be hydrolyzed by FAAH in planta. The tight regulation of NAE levels in vivo appears to be important for proper seedling establishment, and as such, some of these synthetic alkamides may be useful pharmacological tools to manipulate the effects of NAEs in situ.

Full text of DOI:10.1016/j.phytochem.2014.11.011

Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Effects of synthetic alkamides on Arabidopsis fatty acid amide hydrolase
activity and plant development
a,d,e
Lionel Faure a, Ronaldo Cavazos b, Bibi Rafeiza Khan c, Robby A. Petros b, , Peter Koulen
,
⇑
Elison B. Blancaflor a,c, Kent D. Chapman a,
⇑
a Center for Plant Lipid Research, Department of Biological Sciences, University of North Texas, Denton, TX 76203, USA
b Department of Chemistry, University of North Texas, Denton, TX 76203, USA
c The Samuel Roberts Noble Foundation, Plant Biology Division, Ardmore, OK 73401, USA
d Vision Research Center, Department of Ophthalmology, School of Medicine, University of Missouri – Kansas City, Kansas City, MO, USA
e Department of Basic Medical Science, School of Medicine, University of Missouri – Kansas City, Kansas City, MO, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Alkamides and N-acylethanolamines (NAEs) are bioactive, amide-linked lipids that influence plant devel-
opment. Alkamides are restricted to several families of higher plants and some fungi, whereas NAEs are
widespread signaling molecules in both plants and animals. Fatty acid amide hydrolase (FAAH) has been
described as a key contributor to NAE hydrolysis; however, no enzyme has been associated with alkamide
degradation in plants. Herein reported is synthesis of 12 compounds structurally similar to a naturally
occurring alkamide (N-isobutyl-(2E,6Z,8E)decatrienamide or affinin) with different acyl compositions
more similar to plant NAEs and various amino alkyl head groups. These ‘‘hybrid’’ synthetic alkamides
were tested for activity toward recombinant Arabidopsis FAAH and for their effects on plant development
(i.e., cotyledon expansion and primary root length). A substantial increase in FAAH activity was discov-
ered toward NAEs in vitro in the presence of some of these synthetic alkamides, such as N-ethyllauroyla-
mide (4). This ‘‘enhancement’’ effect was found to be due, at least in part, to relief from product inhibition
of FAAH by ethanolamine, and not due to an alteration in the oligomerization state of the FAAH enzyme.
For several of these alkamides, an inhibition of seedling growth was observed with greater results in
FAAH knockouts and less in FAAH over-expressing plants, suggesting that these alkamides could be
hydrolyzed by FAAH in planta. The tight regulation of NAE levels in vivo appears to be important for
proper seedling establishment, and as such, some of these synthetic alkamides may be useful pharmaco-
logical tools to manipulate the effects of NAEs in situ.
Received 25 April 2014
Received in revised form 28 October 2014
Available online xxxx
Keywords:
Arabidopsis thaliana
Lipids
Fatty acid amide hydrolase
Alkamide
N-Acylethanolamine
Affinin
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
but are not limited to butyl, isopropyl, or isobutyl moieties
(Boonen et al., 2012; López-Bucio et al., 2006).
Alkamides and N-acylethanolamines (NAEs) are small, amide-
containing lipids (Chapman et al., 1998; López-Bucio et al., 2006).
The compositions of alkamides and NAEs vary in chain length
and the number of double bonds in their acyl groups (Boonen
et al., 2012; Chapman, 2004; Chapman et al., 1998, 1999). The
structural diversity of alkamides is greater than NAEs due to the
composition of the amino alkyl head group, which can include
NAEs occur widely and are used as signaling substances in ani-
mals, plants and some microorganisms (Blancaflor et al., 2014;
Coulon et al., 2012; Okamoto et al., 2007). In plants, N-palmitoy-
lethanolamine (NAE 16:0), N-oleoylethanolamine (NAE 18:1), N-
linoleoylethanolamine (NAE 18:2) and N-linolenoylethanolamine
(NAE 18:3) are generally the most abundant NAE types
(Blancaflor et al., 2014; Chapman et al., 1999) reflecting both the
common fatty acids found in membranes and their biosynthetic
origin. Alkamides, on the other hand, appear to be more restricted
in their distribution; they have been found in about 10 plant fam-
ilies and some fungi (Boonen et al., 2012; López-Bucio et al., 2006;
Ramírez-Chávez et al., 2004) but have not been reported in animal
systems. While NAEs are derived from membrane phospholipid
precursors, alkamides in plants are likely made from amino acid
⇑
Corresponding authors at: Department of Chemistry, University of North Texas,
Denton, TX 76203, USA. Tel.: +1 (940) 369 8238 (R.A. Petros). Center for Plant Lipid
Research, University of North Texas, Department of Biological Sciences, 1155 Union
Circle, #305220, Denton, TX 76203-5017, USA. Tel.: +1 (940) 565 2969/4771;
fax: +1 (940) 565 4136 (K.D. Chapman).
E-mail addresses: Robby.Petros@unt.edu (R.A. Petros), chapman@unt.edu (K.D.
Chapman).
http://dx.doi.org/10.1016/j.phytochem.2014.11.011
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
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L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
precursor (Cortez-Espinosa et al., 2011). Despite these structural
and biosynthetic differences, NAEs and alkamides exhibit some
similar biological effects when applied to plants suggesting there
may be some overlap in metabolism and/or targets of both groups
of compounds.
Different alkamide moieties have been reported in plants
(Boonen et al., 2012; López-Bucio et al., 2006; Ramírez-Chávez
et al., 2004; Hajdu et al., 2014). The most common naturally-occur-
ring alkamide has an acyl group containing a 2E double bond and
an isobutyl head group, identified as N-isobutyl-(2E,6Z,8E)decatri-
enamide, also called affinin (Ramírez-Chávez et al., 2004). Different
plants such as Echinacea purpurea or Echinacea angustifolia can
accumulate high levels of alkamides in certain organs, and extracts
have been used for many years for their purported therapeutic
benefits (López-Bucio et al., 2006). Recently several alkamide com-
pounds such as N-benzyl-(9Z,12Z)octadecadienamide were shown
et al., 2010). N-(2-Hydroxyethyl)dodecanamide (also referred to
as N-lauroylethanolamine or NAE 12:0) has been widely used to
study the effects of NAEs especially on plant root development
(Chapman, 2004; Coulon et al., 2012). Although the precise mech-
anism remains unknown, NAEs, in part, appear to interact with the
abscisic acid signaling pathway to mediate their negative effects on
seedling growth (Keereetaweep et al., 2013; Teaster et al., 2007).
Besides their medicinal properties, alkamides are also known
for their insecticidal effects (Ramírez-Chávez et al., 2004). Recently,
a new function for alkamides has been ascribed to affinin, and a
reduced form of this lipid (N-isobutyl-(2E)decanamide and N-iso-
butyldecanamide), for the modulation of Arabidopsis seedling
growth (Ramírez-Chávez et al., 2004). Application of exogenous
affinin at levels below 28
lM increased the primary root length,
whereas affinin concentrations above 28
l
M inhibited root devel-
opment (Ramírez-Chávez et al., 2004). The effects of alkamides
on plant development may operate through the cytokinin-signal-
ing pathway to control the activity of the plant meristem and dif-
ferentiation processes (López-Bucio et al., 2007). In addition,
genetic evidence suggests that alkamides modulate lateral root for-
mation via interaction with the jasmonic acid signaling pathway
(Méndez-Bravo et al., 2011).
Thus, NAEs and alkamides share some similarities and differ-
ences in structure, distribution and their influence on plant devel-
opment (López-Bucio et al., 2006, 2007). The studies of the effects
of N-decanoylethanolamide (NAE 10:0) on root development
to inhibit FAAH activity (IC50 = 4 lM) and/or to interfere with the
cellular uptake of N-(2-hydroxyethyl)-(5Z,8Z,11Z,14Z)eicosatetrae-
namide (also named anandamide) the most widely recognized NAE
in the mammalian endocannabinoid system (Hajdu et al., 2014).
NAEs have been associated with several functions in plants such
as seedling establishment (Blancaflor et al., 2003; Chapman, 2004;
Chapman et al., 1999; Kilaru et al., 2007; Wang et al., 2006), flow-
ering (Teaster et al., 2012), responses to pathogens (Kang et al.,
2008; Tripathy et al., 1999), inhibition of phospholipase (Austin-
Brown and Chapman, 2002) and lipoxygenase (Keereetaweep
Fig. 1. Chemical Structure of ‘‘hybrid’’ alkamides. Three different categories of alkamide like compounds: (A–D) acyl chain with 12:0 (number of carbon atom:number of
double bonds); (E–H) 16:0; (I–L) 18:2; (M) 10:3; (N) 10:0.
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L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
3
compared with different alkamides demonstrated similarities in
the biological activities between NAEs and alkamides (López-
Bucio et al., 2007). Since both types of lipids share a similar acyla-
mide structures, and in some instances exhibit similar negative
effects on seedling growth, it has been suggested that alkamides
and NAEs may act through common signaling mechanisms
(López-Bucio et al., 2007), but this remains to be determined.
Enzymes capable of metabolizing the different alkamides and
potentially terminate their different functions have not been
reported in planta. On the other hand, different enzymes are known
to metabolize NAEs including lipoxygenases (Keereetaweep et al.,
2013; Kilaru et al., 2011; Shrestha et al., 2002), N-acylethanol-
amine-hydrolyzing acid amidase (Ueda et al., 2010) and fatty acid
amide hydrolases (FAAHs) (Cravatt et al., 1996; Shrestha et al.,
2003; Wei et al., 2006). FAAHs have been described as key enzymes
terminating the effects of NAEs by hydrolyzing these lipids to their
corresponding free fatty acids (FFA) and ethanolamine.
In animals, different structural analogs of the endocannabinoid,
NAE 20:4 or the sleep-inducing primary amide, oleamide, with
alkyl ‘‘head-groups’’ similar to plant alkamides were tested for their
Fig. 2. Characterization of purified FAAH protein. (A) SDS–PAGE and Western blot of enriched FAAH from E. coli on a 12.5% polyacrylamide gel or PVDF membrane
(respectively). (B) Migration of purified FAAH protein on a Superdex-200 gel filtration. Molecular mass standards are indicated in kilodaltons. (C) SDS–PAGE (left panel) and
Western-blot (right panel) of fractions a, b and c. (D) Enzymatic activity in l
mol minÀ1 mg of proteinÀ1 of the different fractions (a, b and c), reaction carried out in 50 mM Bis-
Tris propane–HCl (pH 9.0) at 30 °C, 30 min, in a final volume of 0.15 ml. Data points represent means S.D. of triplicate assays. Vo, void volume. Proteins were separated by
SDS–PAGE (10% resolving gels) and visualized in gels by Coomassie-blue staining. Western blot was probed with mouse monoclonal antibodies. The recombinant proteins
expressing the HIS tag at the C-terminus were detected by chemiluminescence using a 1-to-2000 dilution of mouse monoclonal anti-HIS antibodies (ABGENT San Diego, CA)
and a solution of 1-to-4000 dilution of goat anti-mouse IgG conjugated to a peroxidase (Bio-Rad).
Fig. 3. TLC analysis of the lipid composition of the amidohydrolase assay with FAAH protein and different alkamide substrates (1–12). Reactions were initiated by the
addition of purified FAAH protein (0.9 lg) with 50 mM Bis-Tris propane–HCl, (pH 9.0), at 30 °C for 30 min, 120 rpm, and 100 lM of different alkamides or NAE 12:0 in a final
volume of 0.45 ml. Lipids were extracted and then separated by TLC (hexane/Et2O/AcOH, 80:20:2, v/v/v). Lipids were visualized by spraying the plate with a solution of
primuline and exposure under a UV lamp (Testet et al., 2005). Position of the origin, free lauric acid formed by hydrolysis of NAE 12:0 and free fatty acid (FFA) 12:0 standard
are indicated (arrows).
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L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
competitive effects on the mammalian FAAH activities (Boger et al.,
2000; Lang et al., 1999; Vandevoorde et al., 2003). None of these
alkamide-like compounds were described as good substrates for
rat FAAH protein. Here this concept was expanded by synthesizing
a broad range of NAE-alkamide ‘‘hybrid’’ compounds (1–12), and
their impact tested on FAAH activity and plant growth. Specifically,
a group of alkamides with different acyl chains (12:0, 16:0 and
18:2) and different head groups (isobutylamine, propylamine, iso-
propylamine and ethylamine) was synthesized. The synthetic alka-
mides (Fig. 1) could be hydrolyzed by FAAH in vitro, although with
lower efficiency compared to NAEs. The N-dodecyl series (1–4)
actually enhanced the Arabidopsis FAAH activity in vitro. None of
the synthetic ‘‘hybrid’’ compounds were as potent as the ‘‘native’’
NAE (12:0) or alkamide (affinin) at inhibiting seedling root elonga-
tion. However, for the N-dodecyl series (1–4), there were reductions
in cotyledon size that appeared to follow patterns of either natural
NAE in a manner that was consistent through the action of endog-
enous FAAH activity (e.g., N-ethyldodecanamide), or with natural
alkamide (e.g., affinin). Taken together, these results provide
insights about the potential interaction of FAAH with alkamide
metabolism, and provide a novel set of compounds that may be use-
ful for manipulating endogenous NAE levels in vivo through their
inhibition or enhancement of FAAH activity.
2. Results
2.1. Synthetic ‘‘hybrid’’ alkamides
A series of synthetic alkamides were designed with common
acyl chains found in plants (NAEs-12:0 (lauric), 16:0 (palmitic),
and 18:2 (linoleic)) and each of four different amino alkyl
head-groups, found in naturally-occurring alkamides (i.e., ethyl-,
propyl-, isopropyl- and isobutyl-amine) (Fig. 1). Details of the syn-
thesis and characterization of the compounds are described in the
experimental procedures and in the supplemental documents.
Stock solutions (10 mM in DMSO) were prepared and diluted to
the desired concentration for enzyme or growth assays. For
Fig. 4. Representative gas chromatography–MS analysis of the lipid composition of the amidohydrolase assay with FAAH protein and different N-acyl-amide substrates or
NAE 12:0. Reactions were initiated by the addition of FAAH protein (0.3 g) with 50 mM of Bis-Tris propane–HCl, (pH9.0), at 30 °C for 30 min, 120 rpm and 100 M of
different alkamide substrates in a final volume of 150 l. Lipids were extracted and analyzed by GC–MS. (A–E) Total ion chromatograms of extracted lipids from the reactions
l
l
l
with, NAE 12:0, (3), (4), (8), and (12). (F–J) mass spectra of NAE 12:0, (3), (4), (8), and (12), respectively.
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L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
5
simplicity, the different alkamides are referred to by a number
from 1 to 12, in addition to their suggested names in SciFinderR
and IUPAC designations also are provided in Fig. 1 for reference.
(Fig. 2B) (denoted a, b and c) were analyzed by SDS–PAGE and
Western-blotting (Fig. 2C). The amidohydrolase activity (AHase)
(l
mol minÀ1 mg of proteinÀ1) of each fraction was measured by
following the hydrolysis of [1-14C]NAE 12:0 to its respective
[1-14C]FFA (Fig. 2D). Fraction ‘‘c’’ was assigned to the imidazole
used to elute the protein from the Ni–NTA column because it con-
tained no protein and exhibited no AHase activity, (Fig. 2C and D).
Fraction ‘‘a’’ contained a high molecular mass protein complex
(around 700 kDa, Fig. 2B), assigned to FAAH protein by SDS–PAGE
and Western blotting analysis (Fig. 2C). FAAH enzymatic assays of
this fraction confirmed robust AHase activity (Fig. 2D). Together,
these results indicate that Arabidopsis FAAH formed an oligomeric
complex similar to purified rat FAAH protein (Patricelli et al.,
1998). The low molecular mass found in fraction ‘‘b’’ (30 kDa)
may have been a truncated form of FAAH, as it retained some
low AHase activity (Fig. 2D); however, it did not bind the monoclo-
nal antibody to the C-terminal His-tag in Western-blotting
(Fig. 2C).
2.2. Protein purification and amidohydrolase assays
Enzymatic assays were performed with recombinant FAAH pro-
tein (UniProt # Q7XJJ7, At5g64440p) purified to near-homogeneity
by affinity chromatography using a Ni-column (QiagenÒ) and by
gel filtration with a Superdex 200 column. Protein purity after elu-
tion from the Ni–NTA column was determined by SDS–PAGE and
Western blotting using an anti-HIS tag monoclonal antibody
(Fig. 2A). Bands observed in both SDS–PAGE and Western blotting
were consistent with the molecular mass calculated for FAAH pro-
tein (i.e., 70 kDa). Results of the purified FAAH fraction by gel filtra-
tion analysis (Fig. 2B) using a Superdex 200 column (GE health care
life sciences) displayed the presence of three major regions of
absorbance (280 nm). Different fractions from the gel filtration
Fig. 5. Amidohydrolase activity (
Reactions were initiated by the addition of purified FAAH protein (0.3
proceeded at 30 °C, 30 min, 120 rpm shaking in a final volume of 0.15 ml. Data points represent means S.D. of triplicate assays. (A–D) 100
l
mol minÀ1 mgÀ1 of protein) of purified FAAH with different [1-14C]NAEs in presence (or absence) of different alkamides compounds (1–12).
g) with 50 mM Bis-Tris propane–HCl, (pH 9.0), and 100 M of different radioactive NAEs. Reaction
M of [1-14C]NAE 12:0 was used
M of [1-14C]NAE 18:2; (M) 100 M of [1-14C]NAE 16:0. p-Value of <0.05 is indicated by * as determined by
l
l
l
as substrate; (E–H) 100
l
M of [1-14C]NAE 16:0; (I–L) 100
l
l
Student’s t test.
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2.3. Effect of synthetic alkamides on FAAH
One goal of this work was to determine if any of the newly-syn-
thesized alkamides (1–12) would be hydrolyzed by the recombi-
nant FAAH proteins. With prolonged reaction times and high
protein concentrations, all the alkamides were hydrolyzed to some
degree. However, using conditions previously reported for the
characterization of NAE hydrolytic activity in plants (Faure et al.,
2014; Shrestha et al., 2003), it appeared that the newly-synthe-
sized alkamides were relatively poor substrates for recombinant
FAAH protein relative to NAEs (Fig. 3). Primuline staining of acyl
lipids on the TLC plates (White et al., 1998) provided a visual rep-
resentation of free fatty acid (FFA) formation from the various alka-
mides, but was not intended to be quantitative in these assays.
Most of the alkamides appeared to be hydrolyzed by purified FAAH
although formation of FFA from the N-12:0 series (1–4) was diffi-
cult to detect by TLC (Fig. 3).
Fig. 7. Amidohydrolase activity (
protein with different [1-14C]N-acylethanolamines (NAEs) in presence of 100
N-isopropyllauroylamide (3) or N-ethyllauroylamide (4). Reactions were initiated
by the addition of purified FAAH (0.3 g) in 50 mM Bis-Tris propane–HCl (pH 9.0)
l
mol minÀ1 mg of proteinÀ1) of purified FAAH
M of
l
l
To confirm the hydrolysis of the alkamides to FFA, GC–MS anal-
ysis was also performed of the lipids in the reaction mixtures after
30 min incubation with purified FAAH protein confirming the for-
mation of FFA (e.g., lauric acid) from the alkamides (Fig. 4 and Sup-
plemental Fig. S1). When analyzed by GC–MS, more FFA was
released from NAE than from any of the alkamides (i.e., FFA 12:0
from NAE 12:0 at relative ion counts of 1.5E+07 was higher by an
order of magnitude than FFA recovered from the alkamides,
Fig. 4, Supplemental Fig. S1), although this should be interpreted
with some caution due to differences in chromatography and ion-
ization properties among the different alkamides and NAEs. Total
ion chromatograms suggested that amidohydrolase activity was
higher toward alkamides with longer acyl chain lengths
(18:2 > 16:0 > 12:0, Fig. 4). Alkamides with alpha or beta methyla-
tion in the amino alkyl head group (isobutylamine, isopropyl-
amine) (1, 5, 9, 3, 7, 11) were generally poorer substrates than
linear analogs (ethylamine, propylamine) (2, 6, 10, 4, 8, 12)
(Fig. 4; Supplemental Fig. S1). Similarly, affinin with its branched
isobutyl group showed little hydrolysis by FAAH under these
experimental conditions (Supplemental Fig. S1).
and different radiolabeled NAEs (12:0, 16:0 and 20:4) in a final volume of 0.15 ml.
Reactions proceeded at 30 °C, 30 min, with shaking (120 rpm). Data points
represent means S.D. of triplicate assays. Plots were generated with SigmaPlot
software version 12.0. p-Value of <0.05 is indicated by * as determined by Student’s
t test. ns, not significant.
synthetic alkamide compounds competed poorly with their corre-
sponding NAEs. Overall and somewhat surprisingly, alkamides
with shorter acyl chains actually induced an increase in AHase
activity with significant increases measured for the N-isopro-
pyllauroylamide (3) and N-ethyllauroylamide (4) (Fig. 5C and D,
respectively), while those with longer acyl chains (e.g., 18:2)
tended to decrease in Arabidopsis FAAH activity (Fig. 5I–L). The
naturally occurring affinin exhibited no significant effects on NAE
hydrolysis by FAAH (Fig. 5M). These results confirmed that
although FAAH was able to hydrolyze the synthetic alkamides,
they were considerably less suitable substrates when compared
to NAEs. Of particular interest was the apparent enhancement of
NAE hydrolysis by FAAH that was conferred by some of the shorter
chain alkamides (Figs. 5–8).
To more directly compare the FAAH-mediated hydrolysis of the
synthetic alkamides and affinin with NAE, equimolar concentra-
tions of alkamides and radiolabeled NAEs were tested with purified
enzyme (Fig. 5). For each assay, the acyl chain of the [1-14C]NAE
substrate was matched with corresponding alkyl group of
the alkamide being tested (e.g., [1-14C]NAE 12:0 was used with
the N-12:0-alkamide series; 1–4). Of twelve compounds tested,
Enhancement of FAAH activity by (3), by way of example, was
observed in radiometric scans following the conversion of radiola-
beled NAE 12:0–12:0 free fatty acid (Fig. 6), where contributions
from non-enzymatic processes were ruled out using heat-dena-
tured controls (Fig. 6B). Similar enhancements in FAAH activity
were observed for other NAE substrates including [1-14C]N-palmi-
toylethanolamine (NAE 16:0), or [1-14C]anandamide (NAE 20:4)
only one showed
N-isobutylpalmitoylamide (5) (Fig. 5E), suggesting that these
a significant inhibition of FAAH activity,
when the hydrolysis was conducted in the presence of 100
(3) or (4) (Fig. 7). An approximately threefold enhancement was
lM of
Fig. 6. Radiochromatograms of the total [1-14C]lipid components of following FAAH reactions with 100
isopropyllauroylamide (3). Reactions were initiated by the addition of purified FAAH protein (0.3 g) with 50 mM of Bis-Tris propane–HCl (pH 9.0), and 100
12:0 in a final volume of 0.15 ml. Reaction proceeded at 30 °C, for 30 min with shaking (120 rpm). (A) Assay with 0.01% of DSMO (solvent control); (B) assay with heat
denatured FAAH protein (5 min at 100 °C) and 100 M of (3); (C) assays with 100 M of (3). Total lipid content has been extracted has described by Shrestha et al. (2006).
l
M of [1-14C]NAE 12:0 in presence (or absence) of 100
lM of N-
l
l
M of [1-14C]NAE
l
l
Chromatograms were obtained by radiometric scanning of the TLC plate with a BioScan instrument.
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Fig. 8. Kinetic characterization of purified recombinant AtFAAH protein in presence of 100
l
M of N-isopropyllauroylamide (3) or N-ethyllauroylamide (4). (A) Initial
velocities were measured at increasing concentrations of [1-14C]NAE 12:0. Reactions were initiated by the addition of purified protein (0.3
l
g) with 50 mM Bis-Tris propane–
HCl (pH 9.0), at 30 °C in a final volume of 0.15 ml. (B) Represent the apparent kinetic parameters of the enzymes estimated by transformation of these original data (i.e.,
double-reciprocal plots). Data points represent means S.D. of triplicate assays. Plots were generated with Prism software version 3.0 (GraphPad Software, San Diego) and
data were fitted to a nonlinear regression (curve fit) using one site binding (hyperbola) equation with an R2 between 0.96 and 0.97.
observed in assays containing compounds (3) or (4) for each of the
radiolabelled NAE tested except for the [1-14C]anandamide for
which only (4) was able to significantly increase FAAH activity
(Fig. 7). Anandamide (NAE 20:4) is not an endogenous NAE in
higher plants, so perhaps the minimal modulation of Arabidopsis
FAAH activity toward anandamide is not altogether surprising.
ments were made at increasing concentrations of NAE 12:0.
Apparent kinetic parameters of the FAAH-catalyzed enzymatic
reaction were calculated and are summarized in Fig. 8B. Values
for kinetic parameters of the recombinant FAAH protein (Kmapp
and Vmapapx) obtained without alkamides were similar to Vapp and
Kampp determined elsewhere (0.23
l
mol minÀ1 mgÀ1 and 17m.a6x lM)
(Shrestha et al., 2003). There was a significant increase in the
app
V
with either (3) or (4) (Fig. 8B; t-test, confidence level 95%).
2.4. N-Isopropyllauroylamide (3) and N-ethyllauroylamide (4) and
FAAH activity
max
Only modest differences in the Kampp were observed in the presence
of either alkamide compound (Fig. 8). The catalytic efficiency (kcat
/
Km) of the FAAH protein increased by a factor of ꢀ2 in presence of
either (3) or (4) indicating that the increase of the enzyme activity
in presence of these two alkamides appeared to occur through an
increase in turnover rate of the recombinant FAAH enzyme. Similar
enzymatic assays were performed with free amino alkyl head
groups alone (ethylamine, propylamine, isopropylamine and
To more accurately characterize the enhancement effect of the
alkamide compounds on FAAH activity, the kinetic parameters of
the purified, recombinant Arabidopsis FAAH enzyme were mea-
sured in the presence of 100 lM of (3) or (4) at increasing concen-
trations of [1-14C]NAE 12:0 (Fig. 8). Arabidopsis FAAH exhibited
typical Michaelis–Menten kinetics when initial velocity measure-
Fig. 9. Effect of ethanolamine or ethylamine on FAAH activity and protection from inhibition by 100
were initiated by the addition of purified FAAH protein (0.3 g) in 50 mM Bis-Tris propane–HCl (pH 9.0), at 30 °C for 30 min, with shaking (120 rpm), and 100
[1-14C]NAE 12:0 in a final volume of 0.15 ml. (A) With different concentrations of ethylamine or ethanolamine. (B) Plus 100
M of (3) or (4). Solvent control corresponds to
lM of N-isopropyllauroylamide (3) or N-ethyllauroylamide (4). Reactions
l
lM of
l
0.01% (v/v) of DMSO. Data points represent means S.D. of triplicate assays. Plots were generated with SigmaPlot software version 12.0. p-Value of <0.05 and <0.03 are
indicated by * and **, respectively, as determined by Student’s t test. ns, not significant.
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L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
isobutylamine), with no effect on FAAH activity being observed.
Further, gel filtration separations conducted in the presence of
the synthetic alkamides showed no influence of the alkamides on
the oligomerization state of FAAH (Supplemental Fig. S2).
et al., 2003). Because inhibition of FAAH was substantial at 1 mM
ethanolamine (at pH 9), and because both (3) and (4) relieved this
inhibition, it was concluded that enhancement of FAAH activity
by these alkamides is mediated by a protection from product inhi-
bition, and not through a pH effect on the enzyme.
Recently, two N-phenoxyacylethanolamines were described that
exhibited similar enhancement effects toward both rat and Arabid-
opsis FAAH, which appeared to be through a relief from product
inhibition by free ethanolamine (Faure et al., 2014). Here these alka-
mide compounds were tested to establish if they might have a sim-
ilar function. There was a dose-dependent inhibition in the AHase
activity of FAAH at increasing ethanolamine concentrations
(Fig. 9A). By sharp contrast, there was no reduction in hydrolase
activity in presence of ethylamine (Fig. 9A), a molecule structurally
similar to ethanolamine but lacking the hydroxyl group. Both (3)
and (4) relieved ethanolamine inhibition up to a concentration of
about 1 mM ethanolamine and increased FAAH activity up to 10
and 50 mM ethanolamine for (3) and (4), respectively (Fig. 9B). No
changes in solution pH were observed (pH 9) up to 1 mM ethyla-
mine or ethanolamine. While the pH of the reactions did rise to
10 at 100 mM ethanolamine, this was not appreciably higher than
the pH optimum of 9 for the Arabidopsis FAAH enzyme (Shrestha
2.5. Comparison of effects of the synthetic alkamides, NAE 12:0 and
affinin on plant growth
Quantitative measurements of seedling growth (cotyledon
expansion and primary root length) were recorded in the presence
of these different alkamides (Figs. 10–12). Overall, cotyledon area
was reduced in seedlings grown in two different concentrations
of the synthetic alkamides (20 and 50 lM, Fig. 10). Within the N-
12:0 series (1–4), three compounds, (2), (3), and (4), showed
greater growth-inhibition in faah1-knockouts and less inhibition
in FAAH1-overexpressing seedlings compared to wild-type (WT,
Col(0); Fig. 10), suggesting that these alkamide compounds may
be partially metabolized by the endogenous FAAH protein in vivo.
WT seedlings treated with two different concentrations of affinin
(20 and 50 lM) also showed significant reduction in cotyledon
Fig. 10. NAE sensitivity of Arabidopsis seedlings in presence of different concentrations of new synthetic alkamides (1–12), NAE 12:0 or affinin. (A–J) Cotyledon size of 10 day
old Arabidopsis seedlings treated with increasing concentrations of NAE 12:0 or different alkamides (WT, KO1, OE1, black, dark gray, light gray bars, respectively). (K)
Representative images of 10 day old WT cotyledon seedlings treated with 50 lM of different alkamides, NAE 12:0 or affinin. The area of each cotyledon was measured with
the software ImageJ. p-Value of <0.05,<0.03,<0.001 are indicated by *, **, ***, respectively, as determined by Student’s t test. ND, not determined. WT, wild type (Col-0); KO1,
faah1 KO; or OE1, FAAH1 over-expressor.
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L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
9
development compared to untreated seedlings, but FAAH appeared
3. Discussion
to have no influence on growth inhibition by this naturally
occurring alkamide (Fig. 10), consistent with activity assays
in vitro. These results suggest that neither affinin nor N-iso-
butyllauroylamide (1) are likely to be hydrolyzed by Arabidopsis
FAAH in vivo.
Compared to the N-12:0 series (1–4), most of the N-18:2-alka-
mide compounds (9–12) were not as effective at inhibiting seed-
ling growth (Fig. 10G–J). Only the N-ethyllinoleoylamide (12) at a
The new synthetic alkamides used here are hybrid molecules,
sharing some structural similarities with NAEs (Blancaflor et al.,
2014) and also with the naturally occurring alkamides (Ramírez-
Chávez et al., 2004) (Fig. 1). Because of the broad substrate selec-
tivity of FAAH, it was not surprising that the different synthetic
alkamides functioned as substrates for Arabidopsis FAAH (Fig. 3).
Hydrolysis of the alkamides visualized by primuline on TLC plates
was not intended to be a quantitative assay, but demonstrated that
these alkamides could be converted to free fatty acids, and this was
further confirmed by GC–MS (Fig. 4 and Supplemental Fig. S1). It
concentration of 50 lM showed some statistically significant inhi-
bition of cotyledon expansion particularly in the faah1 knockouts
(Fig. 10). Independent of plant genotype, growth inhibition (root
or cotyledon) was not observed for the N-16:0-series of alkamide
compounds (5–8) (data not shown). There were only modest (t-
test, confidence level 95%), or in some cases no, inhibitory effects
of these synthetic alkamides on seedling root length for the N-12
series (1–4) (Fig. 11) or the N-18:2 series (9–12) (Fig. 12) in com-
parison to the notable growth inhibition by NAE 12:0 (Blancaflor
et al., 2003; Wang et al., 2006) or affinin (Ramírez-Chávez et al.,
appeared that synthetic alkamides with an
a- or b-methylation
(isopropyl and isobutyl) were not hydrolyzed as well as the com-
pounds with linear alkyl ‘‘head’’ groups (ethyl and propyl) (Supple-
mental Fig. S1). Interestingly, these same
a- or b-methylated
compounds also were resistant to hydrolysis by rat FAAH (data
not shown) consistent with previous reports with branched ana-
logs of N-palmitoylethanolamine or oleamide (Boger et al., 2000;
Vandevoorde et al., 2003).
Perhaps most intriguing was the increase of FAAH activity
toward NAEs that was observed in the presence of (3) or (4)
(Figs. 5–8). These synthetic alkamides were rather poor substrates
for FAAH themselves, but it was unexpected that they would
2004). However, addition of NAE 12:0 (30
12:0 or N-18:2-alkamides compounds (1–4; 9–12) (30
l
M) with any of the N-
M)
l
increased the severity of the growth inhibition (cotyledons and pri-
mary root) indicating a cumulative inhibition effect of these alka-
mide compounds in plant development (data not shown).
Fig. 11. Effect of different concentrations of the N-12:0-amide series (1–4), NAE 12:0 or affinin on the primary root length. (A–F) Root lengths of seedlings treated with
increasing concentration of NAE 12:0 or different alkamides (WT, KO1, OE1, black, dark gray, light gray bars, respectively). (G) Representative images of 10 days WT seedlings
treated with 50 lM of different alkamides or NAE 12:0. The length of each primary root was measured with the software ImageJ. p-Value of <0.05, <0.005, <0.001 are indicated
by *, **, ***, respectively, as determined by Student’s t test. WT, wild type (Col-0); KO1, faah1 KO; or OE1, FAAH1 over-expressor. Scale bar 1 cm.
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L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
enhance significantly the FAAH activity toward NAEs (Figs. 5–7).
Both compounds appeared to increase the Vmax and turnover rate
of the enzyme (Fig. 8). The enhanced NAE hydrolase activity was
explained by the observation that the alkamides provided protec-
tion from product inhibition by free ethanolamine (Fig. 9), as
opposed to an influence by these compounds on the newly-
revealed oligomeric state of Arabidopsis FAAH (Fig. 2, Supplemen-
tal Fig. S2). The product inhibition by ethanolamine was recently
identified in studies where N-phenoxyacylethanolamines showed
a similar enhancement effect on both Arabidopsis and rat FAAH
(Faure et al., 2014). The structures of the alkamides synthesized
here were quite different from the N-phenoxyacylethanolamines
(i.e., NAE containing a phenol group in acyl chain (Faure et al.,
2014); so the similar effect on FAAH was still somewhat surprising.
One difference was that neither (3) nor (4) increased the activity of
purified rat FAAH (data not shown), suggesting a specific feature of
Arabidopsis FAAH protein toward these new hybrid alkamides. The
precise mechanism by which (3) and (4) enhance FAAH activity
will require additional structural insights. These alkamides may
bind to a specific site on the FAAH protein and block binding of eth-
anolamine directly. Or, they may act to alter the overall conforma-
tion of FAAH so as to prevent this ethanolamine feedback
inhibition. It is also possible that these compounds could alter sub-
strate availability as a general detergent effect. Nonetheless, the
protection from feedback regulation of FAAH by these compounds
has revealed a previously unappreciated property of FAAH with
respect to its inhibition by ethanolamine. The specificity of this
inhibition becomes evident by the comparison with ethylamine,
lacking the OH group, which had no effect on FAAH activity
(Fig. 9). This negative feedback regulation suggests that free etha-
nolamine in tissues may influence the NAE signaling pathway in
plant systems. It is worth noting that unlike animal systems, etha-
nolamine is formed from serine decarboxylation in plant tissues,
and free ethanolamine levels in Arabidopsis leaf tissues are
reported to be about 250 nmol/g fresh weight (Rontein et al.,
2003). Still, it remains to be determined whether free ethanol-
amine is an important regulatory feature of NAE metabolism in
planta.
The naturally occurring affinin was not hydrolyzed appreciably
by FAAH under conditions used here (Fig. 3 and Supplemental
Fig. S1). Moreover, inhibition of NAE 12:0 hydrolysis could not be
observed when 100 lM of affinin was added in the presence of
purified FAAH protein (Fig. 5M). This suggests that affinin may
be metabolized by enzymes other than FAAH in planta, or that it
may be difficult to identify conditions for hydrolysis of affinin by
FAAH in vitro. N-decanoyl-homoserine lactone (C10-HL), a quo-
rum-sensing molecule produced by bacteria, was shown to be a
substrate for FAAH, and acts similarly to affinin in plant tissues
(Ortíz-Castro et al., 2008). Growth assays in the presence of
50 lM of exogenous affinin to the plant media showed an overall
decrease in cotyledon size and root elongation (Ramírez-Chávez
et al., 2004). However, unlike C10-HL (Ortíz-Castro et al., 2008),
no tolerance or sensitivity toward affinin was observed with FAAH
overexpressing- or faah1 knockout-seedlings, respectively (Figs. 10
and 11). It may be that additional metabolic pathways other than
FAAH are involved in the metabolism of alkamides in plants, indi-
cating the need for further studies in this area. Notably, affinin
showed a trend towards increasing FAAH’s capacity to metabolize
NAE 12:0 when added at equimolar concentrations, although this
increase was not significant at p < 0.05 (Fig. 5). However, affinin’s
structural similarity to (1), suggests that affinin might influence
FAAH activity directly in plants, but this will also require further
investigation.
Because of the structural similarities to both NAEs and natu-
rally-occurring alkamides, it was anticipated that the new syn-
thetic compounds reported here would negatively influence
seedling growth in a manner similar to the parent compounds. Fur-
ther, Arabidopsis plants were used with either a T-DNA disruption
in FAAH (faah knockout) or overexpressing FAAH to test whether
sensitivities to the synthetic alkamides would suggest their
Fig. 12. Effect of different concentrations of the N-18:2-alkamide series (9–12) on the primary root length. (A–D) Root lengths of seedlings treated with increasing
concentration of the different N-18:2-amide compounds (WT, KO1, OE1, black, dark gray, light gray bars, respectively). (E) Representative images of 10 days WT seedlings
treated with 50 lM of different N-18:2-amide series compounds. The length of each primary root was measured with the software ImageJ. p-Value of <0.05,<0.005,<0.001 are
indicated by *, **, ***, respectively, as determined by Student’s t test. WT, wild type (Col-0); KO1, faah1 KO; or OE1, FAAH1 over-expressor. Scale bar 1 cm.
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L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
11
hydrolysis by FAAH in planta in these different genotypes, similar
to NAEs (Wang et al., 2006). Indeed, the synthetic alkamide com-
pounds inhibited plant growth development in a manner similar
to NAE 12:0 treatment (Blancaflor et al., 2003), although the effects
of the alkamides were not as pronounced as NAE 12:0 (Figs. 10–12).
It is presumed that endogenous FAAH could mediate the tolerance
of seedlings to several of these alkamides. For example, faah1-KO
by first producing the fatty acid chloride (Hillard et al., 1995) and
purifying by TLC as described elsewhere (Shrestha et al., 2002).
4.2. Synthesis of alkamides
Synthesis of compounds was performed using published meth-
ods (Vandevoorde et al., 2003). All solvents were purified and dried
under standard methods. Organic extracts were dried using anhy-
drous MgSO4. NMR spectra were recorded on a Varian 400 MHz
(1H) and 500 MHz (13C) NMR spectrometers. Chemical shifts were
recorded as d values against tetramethylsilane as an internal
standard.
seedlings treated with micromolar amounts (20–50 lM) of the
N-12:0 alkamides displayed a more severe inhibition of growth
with respect to both root length and cotyledon area when com-
pared to WT, an effect that was not seen for (1); whereas, the
FAAH-OE seedlings showed a better tolerance for this series of
compounds (Figs. 10–12). Consistent with their structural similar-
ities, (1) showed growth inhibition effects very similar to affinin on
seedling development (Ramírez-Chávez et al., 2004), and these
inhibitory effects were independent of the FAAH transcript or
enzyme activity levels (Fig. 11). It will be interesting to test this
new synthetic alkamide with mutants like drr1 or dhm1 that are
tolerant or hypersensitive to affinin and decanamide
(Morquecho-Contreras et al., 2010; Pelagio-Flores et al., 2013) or
to determine if (1) will influence cytokinin or jasmonate signaling,
pathways that interact with natural alkamides (López-Bucio et al.,
2007; Méndez-Bravo et al., 2011) but not with NAEs.
Typical synthetic procedure: to a 2-neck dry round-bottom
flask, dry CH2Cl2 (50 mL was added, followed by addition of corre-
sponding acyl chloride (1 M equivalent)). The reaction vessel was
cooled to 0 °C followed by drop-wise addition of 10 M equivalents
of the corresponding amine. The reaction mixture was allowed stir
at room temperature for 1 h. Extraction of synthetic alkamides was
performed using two successive aqueous extractions (50 mL) of 5%
Na2CO3, followed by 1 M HCl and brine. The organic phase was
dried over MgSO4. Evaporation of solvent provided a white solid
product or a viscous yellow liquid depending on the fatty acyl ser-
ies. High resolution mass spectra (HRMS) were obtained for each
compound on a Thermo Scientific Hybrid MALDI-LTQ Orbitrap XL
mass spectrometer in positive ion mode with 2,5-dihydroxyben-
zoic acid (DHB) as a MALDI matrix. Ions corresponding to [M+H]+
and [M+Na]+ were identified for each compound and observed
masses differed from expected masses between 0.0001 and
0.0006. HRMS data for observed and calculated [M+H]+ are pro-
vided below for each compound after NMR information.
3.1. Concluding remarks
Recently, additional, naturally-occurring alkamides have been
identified in extracts of Lepidium and Heliopsis species with effects
on the cellular uptake and/or the hydrolysis of anandamide by
FAAH, continuing to point to cross-over between plant alkamides
and endocannabinoid signaling in mammals (Hajdu et al., 2014).
It will be of interest to test the effects of these new potent alka-
mides like N-benzyl-(9Z,12Z)octadecadienamide on plant develop-
ment, perhaps with the different FAAH mutants. Taken together
the synthetic alkamides described here have several unusual prop-
erties in plant systems: some, like (1) act similarly to natural alka-
mides, others, like (4) or (12) act in a manner more similar to NAEs
and appear to be hydrolyzed by FAAH in planta, others, like the N-
16:0 series (5–8) have very little activity in vitro or in vivo, and still
others, like (3) enhance FAAH activity in vitro by preventing feed-
back inhibition of ethanolamine. As such, these synthetic alka-
mides represent a range of potentially useful pharmacological
tools for the further dissection of NAE and alkamide functions in
plants, or in the biochemical characterization of the FAAH protein
and represent potential novel compounds for regulating cellular
physiology, growth and lipid signaling. Similarly, it may be of
interest to evaluate the effects on endocannabinoid signaling in
mammals of these synthetic ‘‘hybrid’’ alkamides.
4.2.1. N-(2-Methylpropyl)dodecanamide or N-isobutyllauroylamide
(1)
White solid. 60.4% yield. 1H NMR (CDCl3, 400 MHz), d (ppm):
5.43 (bs, 1H, NH), 3.10 (t, 1H, –CH2), 2.17 (t, 2H, –CH2), 1.76 (mp,
2H, –CH2), 1.63 (mp, 2H, –CH2), 1.29–1.25 (overlapping signals,
16H, –CH2), 0.92–0.90 (overlapping signals, 9H, –CH3). HRMS:
[M+H]+ of m/z 256.2638; calculated mass: 256.2640 for C16H33NO.
4.2.2. N-Propyldodecanamide or N-propyllauroylamide (2)
White solid. 81.7% yield. 1H NMR (CDCl3, 400 MHz), d (ppm):
5.42 (bs, 1H, NH), 3.22 (q, 1H, –CH2), 2.15 (t, 2H, –CH2), 1.62 (mp,
2H, –CH2), 1.52 (mp, 2H, –CH2), 1.29–1.25 (overlapping signals,
16H, –CH2), 0.94–0.86 (overlapping signals, 6H, –CH3). HRMS:
[M+H]+ of m/z 242.2482; calculated mass: 242.2484 for C15H31NO.
4.2.3. N-(1-Methylethyl)dodecanamide or N-isopropyllauroylamide
(3)
4. Experimental
White solid. 64.1% yield. 1H NMR (CDCl3, 400 MHz), d (ppm):
5.227 (bs, 1H, NH), 4.091 (mp, 1H, –CH), 2.114 (t, 2H, –CH2),
1.608 (mp, 2H, –CH2), 1.277–1.252 (overlapping signals, 16H,
–CH2), 1.46 (d, 6H, –CH3), 0.877 (mp, 3H, –CH3). 13C NMR (CDCl3,
500 MHz) d (ppm): 172.6, 41.3, 37.2, 29.8–29.5 (6 overlapping sig-
nals), 26.1, 23.0, 22.9, 14.3. HRMS:[M+H]+ of m/z 242.2481; calcu-
lated mass: 242.2484 for C15H31NO.
4.1. Materials
[1-14C] Lauric acid was from Amersham Biosciences, [1-14C] pal-
mitic acid was purchased from NEN (New England Nuclear, Boston,
MA), and [1-14C] arachidonic acid was purchased from PerkinElmer
Life Sciences. Ethanolamine, anandamide, isopropyl-b-D-thiogalac-
topyranoside (IPTG), Triton-X100, ethylamine, propylamine, isobu-
tylamine, isopropylamine and acyl chlorides, primuline dye
(yellow 59) were from Sigma–Aldrich Chemical Co. (St. Louis). N-
4.2.4. N-Ethyldodecanamide or N-ethlyllauroylamide (4)
White solid. 77.6% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.403 (bs, 1H, NH), 3.230 (mp, 2H, –CH2), 2.137 (t, 2H, –CH2), 1.613
(mp, 2H, –CH2), 1.286–1.246 (overlapping signals, 16H, –CH2), 1.30
(d, 3H, –CH3), 0.873 (mp, 3H, –CH3). 13C NMR (in CDCl3, 500 MHz) d
(ppm): 173.5, 37.1, 34.5, 32.1, 29.9–29.6 (6 overlapping signals),
26.1, 23.0, 15.1, 14.3. HRMS:[M+H]+ of m/z 228.2324; calculated
mass: 228.2327 for C14H29NO.
dodecyl-b-D-maltoside (DDM) was from Calbiochem (LA Jolla,
CA). Silica Gel G (60 Å)-coated glass plates for thin-layer chroma-
tography (TLC, 10 Â 20 cm or 20 Â 20 cm, 0.25 mm thickness) were
from Whatman (Clifton, NJ). Different N-[1-14C] acylethanolamines
(and non-radiolabeled NAEs) were synthesized from ethanolamine
and corresponding [1-14C] fatty acids (and non-radiolabeled FFAs)
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4.2.5. N-(2-Methylpropyl)hexadecanamide or
(overlapping signals, 4H, –CH2), 1.61 (mp, 2H, –CH2), 1.35–1.29
N-isobutylpalmitoylamide (5)
(overlapping signals, 14H, –CH2), 1.12 (t, 3H, –CH3), 0.87 (t, 3H,
–CH3). HRMS: [M+H]+ of m/z 308.2949; calculated mass:
308.2953 for C20H37NO.
White solid. 91.4% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.453 (bs, 1H, NH), 3.076 (mp, 2H, –CH2), 2.162 (t, 2H, –CH2), 1.753
(mp, 1H, –CH), 1.637 (mp, 2H, –CH2), 1.246 (overlapping signals,
24H, –CH2), 0.951–0.857 (overlapping signals, 9H, –CH3). 13C
NMR (in CDCl3, 500 MHz) d (ppm): 173.5, 47.1, 37.2, 32.2, 29.9–
29.6 (9 overlapping signals), 28.8, 26.2, 23.0, 20.4, 14.4. HRMS:
[M+H]+ of m/z 312.3263; calculated mass: 312.3266 for C20H41NO.
4.3. Plant materials and growth
Ten mg of different Arabidopsis thaliana seeds (faah1-KO, FAAH-
OE, Col(0); Wang et al., 2006) were surface sterilized and then
stratified in the dark for 2 days at 4 °C prior to sowing in solid
MS (Murashige and Skoog) medium containing different concen-
trations of NAE 12:0 or alkamides (Teaster et al., 2007). Growth
4.2.6. N-Propylhexadecanamide or N-propylpalmitoylamide (6)
White solid. 82.2% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.42 (bs, 1H, NH), 3.20 (mp, 2H, –CH2), 2.15 (t, 2H, –CH2), 1.62 (mp,
2H, –CH2), 1.50 (mp, 2H, –CH2), 1.28–1.25 (overlapping signals,
24H, –CH2), 0.94–0.86 (overlapping signals, 9H, –CH3). HRMS:
[M+H]+ of m/z 298.3107; calculated mass: 298.3110 for C19H39NO.
of seedlings was in 16 h-light/8 h-dark cycle (60 l )
mol mÀ2 sÀ1
for 11 days at 20 °C. Images of primary roots and cotyledons were
captured using a Nikon DX camera. Root lengths and cotyledon
areas were measured using ImageJ software (1.4.3.67 version).
Statistical tests (Student’s t-test) were conducted by using the
Microsoft Excel 2010 software.
4.2.7. N-(1-Methylethyl)hexadecanamide or
N-isopropylpalmitoylamide (7)
White solid. 62.2% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.20 (bs, 1H, NH), 4.09 (mp, 1H, –CH), 2.11 (t, 2H, –CH2), 1.61 (mp,
2H, –CH2), 1.28–1.25 (overlapping signals, 24H, –CH2), 1.15 (d, 6H,
–CH3), 0.88 (mp, 3H, –CH3). HRMS: [M+H]+ of m/z 298.3106; calcu-
lated mass: 298.3110 for C19H39NO.
4.4. Protein expression and purification
The recombinant plasmid FAAH1-pTrcHis2 (At5g64440, Uni-
prot # Q7XJJ7) was constructed as described in Shrestha et al.
(2003). Arabidopsis-FAAH proteins (FAAH) were expressed and
purified in a 50 mM Bis-Tris propane–HCl (pH 9), Triton X-100
(1% v/v) using an a QiQexpressÒ NI–NTA Fast Start (QiagenÒ) col-
umn as described elsewhere (Faure et al., 2014; Kim et al., 2013).
An FPLCÒ system (Amersham Pharmacia Biotech) was used for
all gel filtration chromatography separations. The purified protein
from the Ni-column was concentrated by filtration–centrifugation
using a Centricon YM-30 (Millipore, Bedford, MA) device to a final
4.2.8. N-Ethylhexadecanamide or N-ethylpalmitoylamide (8)
White solid. 43.5% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.39 (bs, 1H, NH), 3.29 (mp, 2H, –CH2), 2.14 (t, 2H, –CH2), 1.61 (mp,
2H, –CH2), 1.28–1.25 (overlapping signals, 24H, –CH2), 1.13 (t, 3H,
–CH3), 0.88 (t, 3H, –CH3). HRMS: [M+H]+ of m/z 284.2949; calcu-
lated mass: 284.2953 for C18H37NO.
volume of 200 ll and then loaded onto a Superdex 200 gel filtra-
4.2.9. N-(2-Methylproyl)-(9Z,12Z)octadecadienamide or
N-isobutyllinoleamide (9)
tion column (GE health care life sciences). The column was equili-
brated with 50 mM Bis-Tris propane–HCl, (pH 9), 0.1 M NaCl,
0.2 mM DDM and the eluted proteins were monitored by UV absor-
bance at 280 nm. Fractions were collected and assayed for enzyme
activity and analyzed by SDS–PAGE and Western blotting to con-
firm the location of the eluted FAAH protein (Faure et al., 2014).
Molecular mass calibration of the column was done using blue
dextran (669 kDa), b-amylase (200 kDa), alcohol dehydrogenase
(150 kDa), albumin (66 kDa) and carbonic anhydrase (29 kDa).
Yellow oil. 78.1% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.40–5.28 (overlapping signals, 5H, NH and C@CAH), 3.08 (t, 2H,
–CH2), 2.77 (t, 2H, –CH2), 2.17 (t, 2H, –CH2), 2.06–2.04 (overlapping
signals, 4H, –CH2), 1.76 (mp, 1H, –CH), 1.63 (mp, 2H, –CH2), 1.35–
1.31 (overlapping signals, 14H, –CH2), 0.92–0.90 (overlapping sig-
nals, 9H, –CH3). HRMS: [M+H]+ of m/z 336.3262; calculated
mass:336.3266 for C22H41NO.
4.2.10. N-Propyl-(9Z,12Z)octadecadienamide or N-propyllinoleamide
(10)
4.5. FAAH assays
Yellow oil. 73.9% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.52 (bs, 1H, NH), 5.40–5.28 (overlapping signals, 4H, C@CAH)
3.19 (mp, 2H, –CH2), 2.75 (t, 2H, –CH2), 2.14 (t, 2H, –CH2), 2.02
(overlapping signals, 4H, –CH2), 1.61 (mp, 2H, –CH2), 1.50 (mp,
2H, –CH2), 1.35–1.29 (overlapping signals, 14) [M+H]+ of m/z
322.3107; calculated mass: 322.3110 for C21H39NO.
The NAE amidohydrolase assays were conducted as previously
described (Kim et al., 2013) with few modifications. The reactions
were conducted for 30 min at 30 °C, in BTP buffer (150 ll, 50 mM
Bis-Tris propane–HCl, pH 9.0), with different concentrations of cold
or radiolabelled NAEs, and different concentrations of alkamides at
varying concentrations of purified protein (see legend of Figs. for
details in reaction compositions). The reactions were terminated
by adding hot iPrOH (2 ml, 70 °C). The lipids were extracted and
the distribution of lipids was evaluated by radiometric scanning
of TLC plates as described (Shrestha et al., 2006). For the non-radio-
active assays, lipids were located by UV of the TLC plate after
spraying a solution of primuline as described (Testet et al., 2005)
or they were analyzed by GC–MS. Extracted lipid after enzymatic
reactions were evaporated under N2, and derivatized with BSTFA
4.2.11. N-(1-Methylethyl)-(9Z,12Z)octadecadienamide or
N-isopropyllinoleamide (11)
Yellow oil. 80.1% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.40–5.28 (overlapping signals, 5H, NH and C@CAH), 4.06 (mp,
1H, –CH), 2.75 (t, 2H, –CH2), 2.11 (t, 2H, –CH2), 2.04 (overlapping
signals, 4H, –CH2), 1.60 (mp, 2H, –CH2), 1.35–1.29 (overlapping sig-
nals, 14H, –CH2), 1.14–1.12 (d, 6H, –CH3), 0.87 (t, 3H, –CH3). HRMS:
[M+H]+ of m/z 322.3104; calculated mass: 322.3110 for C21H39NO.
(50
TMS-ether derivatives were dried under N2 and suspended in hex-
ane 50 l) for injection. Lipid analyses were performed using a GC–
ll, Fisher Scientific, Houston, TX, USA) for 30 min at 55 °C.
4.2.12. N-Ethyl-(9Z,12Z)octadecadienamide or N-ethyllinoleamide
(12)
l
MS model Agilent GC 7890A/MSD 5975C system and a capillary
HP-5 MS column (30 m  0.250 mm, 0.25-mm coating thickness;
Agilent Technologies) in full mass scan mode as previously
described (Keereetaweep et al., 2013). Identification of the
FFAs or alkamides was based on identification of characteristic
Yellow oil. 59.5% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
Yellow oil. 73.9% yield. 1H NMR (in CDCl3, 400 MHz), d (ppm):
5.53 (bs, 1H, NH), 5.40–5.28 (overlapping signals, 4H, C@CAH)
3.27 (mp, 2H, –CH2), 2.75 (t, 2H, –CH2), 2.13 (t, 2H, –CH2), 2.02
Please cite this article in press as: Faure, L., et al. Effects of synthetic alkamides on Arabidopsis fatty acid amide hydrolase activity and plant development.
Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.11.011

L. Faure et al. / Phytochemistry xxx (2014) xxx–xxx
13
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molecular and fragment ions in comparison with known standards.
To test ethanolamine inhibition, assays containing purified FAAH
protein (0.3 lg) were first incubated with 100 lM of the different
alkamides and then with different concentrations of ethanolamine
or ethylamine (0–100 mM) as described (Faure et al., 2014). Assays
were conducted in the presence of detergent to facilitate solubility
of lipophilic compounds for enzyme assays (Shrestha et al., 2003).
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Acknowledgments
KDC is grateful to the National Science Foundation for providing
individual research and development leave to assist in the prepara-
tion of this manuscript by agreement under the Intergovernmental
Personnel Act. This research was supported by the United States
Department of Energy, Office of Science, Basic Energy Sciences
(DE-FG02-12ER15647). We thank Jantana Keereetaweep for excel-
lent assistance with GC–MS, and Dr. Jorge Molina Torres, Laborato-
rio de Fitobioquímica Deptartamento de Biotecnología
y
Bioquímica, CINVESTAV-IPN, Irapuato, México for the generous gift
of affinin. Drew Sturtevant performed high-resolution mass mea-
surements of the synthetic compounds on
a ThermoFisher
MALDI-Orbitrap XL mass spectrometer purchased, in part, with a
grant from the Hoblitzelle Foundation.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2
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Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.11.011