Modular and scalable synthesis of nematode pheromone ascarosides: Implications in eliciting plant defense response

Article abstract of DOI:10.1039/d0ob00652a

A highly efficient and modular synthesis of nematode pheromone ascarosides was developed, which highlights a 4-step scalable synthesis of the common intermediate 10 in 23percent yield from commercially available l-rhamnose by using orthoesterification/benzylation/orthoester rearrangement as the key step. Six diverse ascarosides were synthesized accordingly. Notably, biological investigations revealed that ascrNo.1 and ascrNo.18 treatment resulted in enhanced callose accumulation in Arabidopsis leaves. And ascrNo.18 also increased the expression of defense-related genes such as PR1, PDF1.2, LOX2 and AOS, which might contribute to the enhanced plant defense responses. This study not only allows a facile access to 1-O, 2-O, and 4-O substituted ascarosides, but also provides valuable insights into their biological activities in inducing plant defense response, as well as their mode of action.

Full text of DOI:10.1039/d0ob00652a

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Modular and scalable synthesis of nematode
pheromone ascarosides: implications in eliciting
Cite this: DOI: 10.1039/d0ob00652a
plant defense response†
Shuai Ning,‡a Lei Zhang,‡b Jinjin Ma,a Lan Chen,a Guangyao Zeng,a Chao Yang,b
a
Yingjun Zhou,a Xiaoli Guo*b and Xu Deng
*
A highly efficient and modular synthesis of nematode pheromone ascarosides was developed, which
highlights a 4-step scalable synthesis of the common intermediate 10 in 23% yield from commercially
available L-rhamnose by using orthoesterification/benzylation/orthoester rearrangement as the key step.
Six diverse ascarosides were synthesized accordingly. Notably, biological investigations revealed that
ascr#1 and ascr#18 treatment resulted in enhanced callose accumulation in Arabidopsis leaves. And
ascr#18 also increased the expression of defense-related genes such as PR1, PDF1.2, LOX2 and AOS,
which might contribute to the enhanced plant defense responses. This study not only allows a facile
access to 1-O, 2-O, and 4-O substituted ascarosides, but also provides valuable insights into their biologi-
cal activities in inducing plant defense response, as well as their mode of action.
Received 28th March 2020,
Accepted 16th June 2020
DOI: 10.1039/d0ob00652a
rsc.li/obc
phylum, including plant-parasitic nematodes (Fig. 1).7
Structurally, ascarosides are a family of glycosides of 3,6-
Introduction
Nematodes are ubiquitous and parasitize most plants and dideoxysugar L-ascaryloses with hydroxyl fatty acids of varying
animals. Plant-parasitic nematodes cause agricultural damage lengths attached at the penultimate (ω-1) or terminal (ω)
of more than $100 billion annually worldwide.1 The reco- carbon, which was labelled according to the small molecule
gnition of specific small molecular patterns has been shown to identifier (SMID) as promoted by Schroeder’s lab.8 Additional
play a central role in plant immune response.2,3 Plants possess modifications can occur at 2-O and 4-O of the sugar, includ-
pattern recognition receptors that serve to detect microbe- ing indole-3-carbonyl,9 4-hydroxybenzoyl,10,11 (E)-2-methyl-
associated molecular patterns (MAMPs), which thereby elicit 2-butenoyl,10,11 glucosyl,12 etc. Ascarosides serve essential
defense reactions known as MAMP-triggered immunity (MTI).4
Identification of MAMPs represents a sustainable crop disease
management approach. However, investigations on conserved
nematode-associated molecular patterns that are recognized
by plants remain scarce.5
Ascarosides represent an evolutionarily conserved family of
glycolipid pheromones derived from nematodes. Since the first
ascaroside ascr#1 was isolated and characterized from
C. elegans by Jeong et al. in 2005,6 more than 200 ascarosides
have been identified from over 20 different nematode species,
demonstrating their wide distribution in the nematode
aXiangya School of Pharmaceutical Science, Central South University, Changsha,
Hunan 410013, China. E-mail: dengxu3817@csu.edu.cn
bCollege of Plant Science and Technology, State Key Laboratory of Agricultural
Microbiology, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
E-mail: guoxi@mail.hzau.edu.cn
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0ob00652a
Fig. 1 Synthetic approaches towards ascarosides and the key
‡These authors contributed equally to this work.
intermediates.
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functions in chemical signalling of nematodes and regulate
development,6,13–15 lifespan,16,17 morphology18 and social
Results and discussion
behaviour,11,14,15,19–21 which are highly sex- and structure- Initially, we envisaged to avoid tedious anomeric protection/
dependent.7,22–24 Strikingly, ascarosides can be active at extre- deprotection by introducing the fatty acid side chain at the
mely low concentrations (1.0 fmol) and subtle modifications early stage. As shown in Scheme 1, the synthetic endeavours
in the chemical structure can exert significant impacts on commenced with Lewis acid-catalyzed glycosylation of ali-
their biological functions.10,11 Recently, Manosalva et al. phatic alcohol 2 with L-rhamnose derivative 1.32 Unexpectedly,
showed that ascr#18, the most abundant ascaroside in plant- this reaction was quite inefficient even after extensive screen-
parasitic nematodes, triggered plant hallmark defense ing and optimizations, which gave the corresponding coupling
responses, including activation of MAMP-triggered immunity, products as a 1 : 1 mixture of diastereoisomers. The desired
as well as salicylic acid- and jasmonic acid-mediated defense product 3 was obtained in only 20% yield upon removal of the
signalling pathways, which thereby increased resistance to acetyl group (Table S1†). We then proceeded to protect C2 and
pathogen infections in plants.25,26 This result suggests that C4 with distinct masking groups and remove the C3-oxygen
ascarosides represent
a
nematode-associated molecular group. Inspired by Mukhopadhyay and Field’s work which
pattern (NAMP). Additionally, ascr#18 secreted by plant-para- described a one-pot orthoester formation/benzylation/regio-
sitic nematodes can be metabolized by plants to shorter side- selective hydrolysis sequence of sugars to prepare partially pro-
chain ascarosides, which confer nematode repellence.27 These tected glycosyl acceptors,35 we applied the established protocol
results suggest that ascarosides not only elicit plant-defense to precursor 3 to afford the desired intermediate 4 in 68%
response by conventional pattern-triggered immunity, but can yield. Subsequent deoxygenation at C3 was not straight-
also be edited by plants to manipulate nematode chemical sig- forward. Initial nucleophilic reduction of 3-O-mesylate with
nalling, which demonstrates their potential utility to improve NaBH4 proved unsuccessful,32 and afforded the C2-deacety-
economic and environmental sustainability of agriculture. lated side product instead. The Barton–McCombie deoxygena-
However, their structure–activity relationships (SARs), as well tion reaction was proved to be effective for this transform-
as their mode of action, remain largely unexplored due to their ation.36 Specifically, activation of C3-OH with TCDI followed by
limited access.
radical reduction provided the desired C3-deoxygenated
To this end, several elegant synthetic strategies towards product 6 in 26.4% yield. The final oxidative cleavage of the
ascarosides have been developed (Fig. 1). For instance, Jeong double bond was also inefficient, only delivering 7 (ascr#1) in
et al., as well as several other groups, developed a synthetic 24% yield after saponification.
strategy of ascarosides featuring a 6-step synthesis of the key
Although ascarosides can be obtained via intermediate 6
2,4-di-O-benzoylascarylose in 36.5% yield from L-rhamnose.6,28–30 with fewer steps, the overall efficiency and scalability remains
Similarly, Martin et al. made an improvement in efficiency to far from satisfactory. Besides, modifications at the anomeric
51% overall yield in 6 steps from L-rhamnose.31 However, lack position require synthesis from the very early stage. To address
of versatility renders this route highly restricted in the regio- these problems, 2nd generation synthesis was further devel-
selective synthesis or modifications of 2′- or 4′-substituted
ascarosides. A significant synthetic advancement was achieved
by Zhang et al., which highlights a 9-step regioselective
manipulation of L-rhamnose to afford 4-O-TBDPS-2-O-Bz-ascar-
ylose in 55% overall yield. It thereby allowed the versatile syn-
thesis of several structurally diverse ascarosides.32 Besides,
Guo et al. developed a de novo asymmetric synthetic strategy
evolving through the Noyori reduction of an acylfuran and a
propargyl ketone, the Achmatowicz rearrangement and
diastereoselective palladium-catalyzed glycosylation and epoxi-
dation/ring-opening sequence.33,34 Since these processes
require more than 6 synthetic steps and tedious operation,
further improvements are still required. Herein, we developed
an efficient and modular synthetic route to a diverse set of
ascarosides with 1-tolylthio-2-O-acetyl-4-O-Bn-ascaroside 10
as a common intermediate, which can be easily scaled up in
4 steps, with 23% total yield from L-rhamnose by using
the orthoesterification/benzylation/orthoester rearrangement
sequence and the Barton–McCombie deoxygenation as the
key steps. Furthermore, the abilities of ascarosides in elicit-
ing defense responses, including callose deposition and
defense-associated gene expression in Arabidopsis, were also
studied.
Scheme 1 Synthesis of ascr#1.
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Scheme 2 Synthesis of the common intermediate 10.
Scheme 3 Synthesis of 1-O-substituted ascarosides.
oped. As shown in Scheme 2, a one-pot acetylation/thionyla-
tion/hydrolysis sequence of L-rhamnose delivered the TolS-acti-
vated rhamnose 8 in 69% overall yield,37 which was followed
by a one-pot orthoester formation/benzylation/regioselective
hydrolysis sequence to give intermediate 9 in 71% yield.38
Subsequent Barton–McCombie deoxygenation provided inter-
mediate 10 in 42% yield. Remarkably, the common precursor
10 can be easily prepared on a scale of more than 5 gram.
With enough amount of 10 in hand, we then proceeded to
attach it with different substituents to access diverse ascaro-
sides. For 1-O-substituted ascarosides, glycosylation of 10 with
freshly prepared (ω-1)-hydroxyacyl esters 11 upon treatment of
NIS in the presence of TMSOTf gave the coupling products 12
in 55–74% yields. Remarkably, only the desired α-anomer was
detected for all the substrates examined, which was attributed
to the anchimeric assistance of the 2-acetoxy group. This is the
first example of using a tolyl thioascaroside as the glycosyl
donor for the glycosylation reaction, wherein the efficiency is
comparable or slightly inferior to that of the previously
reported Lewis acid-activated glycosylation of 2,4-di-O-benzoyl
ascarylose,6,9 4-O-TBDPS-2-O-Bz ascarylose32 and their 1-O-tri-
chloroacetimidate derivatives via a two-step process31,32 or Pd-
catalyzed allylic glycosylation.33,34 Subsequent saponification
Scheme 4 Synthesis of 4-O-substituted ascarosides.
Perception of pathogen associated molecular patterns
with NaOH in 1,4-dioxane/H2O delivered 13, which was selec- (PAMPs) activates the first line of innate immunity to fend off
tively deprotected with catalytic hydrogenation to give the pathogen infection. Various defense responses in plant cells
desired ascarosides in moderate to good overall yields. By have been well characterized after pathogen recognition,
varying the fatty acid chain at the anomeric carbon, several including reactive oxygen species (ROS) production, callose
representative ascarosides 14a–14d, namely ascr#1, ascr#5, deposition, and immune gene activation.39–42 Although
ascr#10, and ascr#18, were synthesized accordingly ascr#18 has been shown to activate the defense signalling
(Scheme 3).
pathways26 and can be further metabolized into ascarosides
For 4-O-substituted ascarosides, a slightly modified syn- with a shorter side chain in plants,27 the immunity-related
thetic route was developed. As shown in Scheme 4, an acid- activity of other ascarosides is unclear. Whether callose depo-
mediated debenzylation of the common intermediate 10 gave sition and ROS production are influenced by ascarosides
the alcohol 15 in good yield, which was followed by installa- including ascr#18 is unknown. In order to investigate the
tion of an ester group (e.g. indole-3-carbonyl or senecioyl immune activation ability of different ascarosides, the bio-
group) at 4-O to give the corresponding esters 16a and 16b logical activities of several ascarosides in inducing plant
uneventfully. Subsequent glycosylation with the fatty acid 11e defense responses were further explored in Arabidopsis
delivered the coupling products 17a and 17b in 48% and 84% thaliana. As shown in Fig. 2, significant callose accumulation
yields respectively. The final selective hydrolysis of the acetyl in Arabidopsis leaves was detected upon treatment with
esters afforded the desired 4-O-substituted ascarosides 18a ascr#1 (14a/7, 1 μM) and ascr#18 (14c, 1 μM), but not with
(icas#3) and 18b (mbas#3) in 80% and 53% yields respectively. ascr#5 (14d) and ascr#10 (14b) (see Fig. S1A in the ESI†). This
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Fig. 2 Ascaroside treatment induces callose deposition in Arabidopsis
thaliana Col-0. (A) Ten-day-old seedlings were treated with 1 μM flg22
and 1 μM ascarosides (ascr#1 (14a/7), ascr#5 (14d) and ascr#18 (14c))
for 24 h and callose was detected by aniline blue staining. (B) Water
treatment serves as the control. ImageJ software was used to quantify
the number of callose deposits. Data are average SE (n ≥ 9). Different
letters denote significant differences by the one-way ANOVA test (p <
0.05). Bar = 200 μm.
Fig. 3 Immune gene expression in Arabidopsis thaliana Col-0 is
induced upon ascaroside treatment. Ten-day-old seedlings were treated
with ddH2O and 1 μM ascarosides for 60 min, and total RNA was
means that only specific side chains at the anomeric carbon
will trigger the optimal activity, which indicates that early
recognition by host PAMP receptors might be specific for
certain ascarosides. The stable activity obtained for ascr#18 is
consistent with the fact that ascr#18 is the most abundant
ascaroside excreted by plant-parasitic nematodes. However,
callose deposition is still considerably less pronounced with
ascaroside treatment compared with that triggered by the
positive control flg22. It is worth noting that no significant
ROS burst was detected (see Fig. S1B in the ESI†), which
might be rationalized that ROS burst is mediated by distinct
signalling pathways or the signal is too weak to be measured
since the ROS burst in the apoplast is often fast and
transient.
Using whole seedlings, we also examined the transcript
levels of genes involved in biosynthetic or responsive pathways
of the two major defense hormones, salicylic acid (SA) and jas-
monic acid (JA) (Fig. 3). qRT-PCR analysis revealed that
ascr#18 pretreatment increased the expression levels of the JA-
biosynthesis genes Lipoxygenase 2 (LOX2) and Allene Oxide
Synthase (AOS), and the JA responsive gene PDF1.2 in whole
seedlings, whereas neither ascr#1 nor ascr#5 shows any effect
under the conditions tested. In addition, the expression of the
SA-responsive marker PR1 is also induced by ascr#18,
suggesting the important role of ascarosides in the elicitation
of JA and SA-mediated plant defense responses in a structure-
dependent manner.26,43 These results provide a rationale for
the previous results that JA and SA are involved in plant-para-
sitic nematode-induced resistance.44 The fact that JA and SA
responsive pathways are activated upon ascaroside treatment
suggests potential application of ascarosides as plant immu-
nity inducers.
extracted for qRT-PCR analysis. Data represent the mean
SE of two
biological replicates. Different letters denote significant differences by
the one-way ANOVA test (n = 2, p < 0.05).
Conclusions
In summary, we developed an improved synthetic strategy to
enable efficient and modular synthesis of diverse ascarosides,
which features a 4-step scalable synthesis of the key intermedi-
ate 10 from commercially available L-rhamnose and uses a
tolyl thioascaroside as the glycosyl donor for the glycosylation
reaction. Furthermore, the biological activities of ascarosides
in eliciting plant defense response were also investigated. This
study not only allows a facile access to 1-O, 2-O, and 4-O substi-
tuted ascarosides, but also provides valuable insights into
their structure–activity relationships, as well as their mode of
action. Although variable immune responses present a chal-
lenge for future mechanistic study, further investigations for
the discovery of more potent elicitors and characterization of
the detailed mechanism as well as the receptors that are
responsible for plant immune response would be prominent
for better management of the pathogen.
Experimental
The procedures for the synthesis of the compounds mentioned
in this article and their characterization data are presented in
the ESI.†
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also grateful to the Analytical and Testing Centre of Central
South University for the access to their facilities.
Plant materials and growth conditions. The Arabidopsis
thaliana Col-0 accession was obtained from the Arabidopsis
Biological Resources Centre (ABRC). Seedlings were germi-
nated on half-strength Murashige and Skoog (MS) plates Notes and references
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