Effect of introducing amino acids into phenazine-1-carboxylic acid on phloem mobility

Article abstract of DOI:10.1080/14786419.2020.1716347

To develop new phenazine carboxylic acid derivatives with better phloem mobility, five novel 7-amino acid substituted phenazine-1-carboxylic acids were synthesised by introducing amino acids into PCA at the 7-position. The phloem mobility experiments in R

Full text of DOI:10.1080/14786419.2020.1716347

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Effect of introducing amino acids into phenazine-1-
carboxylic acid on phloem mobility
Yongtong Xiong, Xiang Zhu, Jinyu Hu, Yunping Wang, Xiaoying Du, Junkai Li &
Qinglai Wu
To cite this article: Yongtong Xiong, Xiang Zhu, Jinyu Hu, Yunping Wang, Xiaoying Du, Junkai Li
& Qinglai Wu (2020): Effect of introducing amino acids into phenazine-1-carboxylic acid on phloem
mobility, Natural Product Research, DOI: 10.1080/14786419.2020.1716347
To link to this article: https://doi.org/10.1080/14786419.2020.1716347
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1716347
Effect of introducing amino acids into phenazine-
1-carboxylic acid on phloem mobility
Yongtong Xionga, Xiang Zhua,b, Jinyu Hua, Yunping Wanga, Xiaoying Dua,b
Junkai Lia,b and Qinglai Wua,b
,
b
aSchool of Agriculture, Yangtze University, Jingzhou, China; Institute of Pesticides, Yangtze
University, Jingzhou, China
ABSTRACT
ARTICLE HISTORY
Received 13 November 2019
Accepted 6 January 2020
To develop new phenazine carboxylic acid derivatives with better
phloem mobility, five novel 7-amino acid substituted phenazine-1-
carboxylic acids were synthesised by introducing amino acids into
PCA at the 7-position. The phloem mobility experiments in Ricinus
communis seedlings showed that retaining the carboxyl group of
PCA and conjugating amino acids to its phenazine ring can also
endow PCA with phloem mobility. Comparing our previous research,
we found the amino acids substituted at 7-position on phenazine
ring of PCA could clearly enhance the phloem mobility of PCA than
that of amino acids conjugated with carboxyl group. Especially, the
phloem transport concentration of the compound 7-L-isoleucine sub-
stituted PCA (7d) was 21 times higher than PCA-L-isoleucine conju-
gate (8d). These data suggest that the introduction of amino acids
at different structural sites on the phenazine ring could effectively
enhance the phloem mobility of PCA and it is worth a further study.
KEYWORDS
Phenazine-1-carboxylic acid;
amino acid; synthesis;
phloem mobility
1. Introduction
It is estimated that only a very small part of pesticides (less than 0.1%) actually
reaches the sites of action, while the larger proportion is useless and causes serious
environmental pollution (Wang and Liu 2007). As environmental and health issues
become more serious, more attention is paid to reducing pesticide use and improving
CONTACT Xiaoying Du
cjdxdxy@163.com; Junkai Li
junkaili@sina.com; Qinglai Wu
wql106@163.com
Supplementary Information for this article can be accessed at https://doi.org/10.1080/14786419.2020.1716347.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

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Y. XIONG ET AL.
Figure 1. The structures of PCA and its derivatives.
the pesticide efficiency (Pimentel et al. 1993; Mehrazar et al. 2015). Fungicides with
phloem mobility have obvious advantages in controlling root pathogens and vascular
bundle pathogens, improving the effective utilisation rate of pesticides and changing
the traditional application methods (Edgington 1981; Chollet et al. 2004). In order to
obtain fungicides molecules with phloem mobility, an effective strategy is to change
the physicochemical properties of the molecules (Chollet et al. 2005). Another strategy
is to introduce endogenous small molecule such as a sugar or an amino acid that can
be transported within plants into the non-phloem mobile molecules (Wu et al. 2012;
Xie et al. 2016; Sheng et al. 2018).
Phenazine-l-carboxylic acid (PCA, Figure 1) is an antibiotic secreted by Pseudomonas
sp. M18 (Ge et al. 2004; Shao et al. 2010), which is a broad-spectrum and highly effect-
ive fungicide that inhibits plant pathogenic fungi and promotes plant growth
(Thomashow and Weller 1988; Lee et al. 2003). At present, PCA has registered a new
microbial source fungicide on rice in China and has been widely promoted. However,
PCA does not have phloem mobility (Niu et al. 2017; Yu et al. 2018).
In our previous work, in order to improve the phloem mobility of PCA, a series of PCA-
amino acid ester conjugates were designed and synthesised. Unfortunately, all conjugates
did not have phloem mobility (Niu et al. 2016). Soon after, our research group synthesised
a series of PCA-amino acid conjugates (Yu et al. 2018). Among them, most conjugates
exhibited phloem mobility. In addition, we found that different steric conformational
PCA-amino acid conjugates differ in phloem mobility (Zhu et al. 2019). Furthermore,
our work on the structural modification of PCA is based on the carboxyl group of PCA
(Xiong et al. 2017; Lu et al. 2018; Han et al. 2019; Qin et al. 2019; Zhu et al. 2019).
Based on our previous research work, we suspect that the introduction of amino
acids from the phenazine ring of PCA can also improve the phloem mobility of PCA.
Five novel 7-amino acid substituted phenazine-1-carboxylic acids were designed and
synthesised. In addition, the experiments in Ricinus communis seedlings were
performed to verify whether the compounds had phloem mobility after the amino
acids were conjugated to the phenazine ring.

NATURAL PRODUCT RESEARCH
3
2. Results and discussion
2.1. Chemical synthesis
The synthetic route of the intermediate and 7-amino acid substituted phenazine-
1-carboxylic acids 7a–7e is shown in Scheme 1. Intermediate 1 was obtained by
an Ullmann reaction in yield of 75%, followed by reduction with sodium borohy-
dride to give the intermediate 2 in yield of 81%. The pure compounds obtained in
the above two steps can be obtained by a simple post-treatment. Intermediate 2
was subjected to acid chloride reaction to obtain intermediate 3 in yield of 98%,
which was then esterified with methanol to obtain intermediate 4 in yield of 92%.
Intermediate 5 was obtained by bromination of intermediate 4 in yield of 73%.
Then, a nucleophilic reaction with the hydrochloride of the amino acid ester got
the intermediate 6 in yields ranging from 76% to 88%. Finally, the target com-
pounds 7a–7e was obtained by hydrolysis of the corresponding intermediate 6 in
good yields ranging from 74% to 82%. The structures of the target compounds
1
7a–7e were confirmed by H, 13C nuclear magnetic resonance (NMR) spectra and
high-resolution mass spectra (HRMS). (Analysis data of all the synthesised com-
pounds are available in supplementary information).
Scheme 1. Synthetic route of target compounds 7a–7e. Reagents and conditions: (A) cuprous
chloride, copper powder, N-ethylmorpholine, 2,3-butanediol, 70 ꢀC, 15 h; (B) Sodium borohydride,
sodium ethoxide, ethanol, reflux; (C) oxalyl chloride, CH2Cl2, reflux, 8 h; (D) methanol, 0 ꢀC, 6 h; (E)
N-bromosuccinimide, dibenzoyl peroxide, CCl4, reflux, 4 h; (F) N,N-diisopropylethylamine, 60 ꢀC, 4 h;
(G) lithium hydroxide, 1,4-dioxane/H2O (v/v ¼ 1:1), room temperature, 5 h.

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Y. XIONG ET AL.
Figure 2. Phloem mobility of 7a–7e and PCA. Phloem sap was collected at a 1 h interval for 5 h.
Each datapoint is the mean of 12 seedlings SE (n ¼ 3).
Figure 3. Compounds 7a–7e are different amino acids conjugated to the 7-position of
the phenazine ring of PCA. Compounds 8a–8e are different amino acids conjugated to
the carboxyl group of PCA.
2.2. Phloem mobility in Ricinus communis seedlings
Phloem mobility of 7a–7e and PCA were measured by the castor bean system which
is widely employed to study the phloem mobility of xenobiotics (Rocher et al. 2009;
Sheng et al. 2018). The cotyledons of Ricinus communis were incubated with each

NATURAL PRODUCT RESEARCH
5
compound of 0.2 mmol/L for 2 h. Then, phloem sap was collected and analysed using
Ultra High Performance Liquid Chromatography-Mass Spectrometer (UHPLC-MS).
As shown in Figure 2, the cotyledons incubated with PCA, the fungicide was not
detected in phloem sap even after 5 h. In contrast, when the cotyledons were incu-
bated with compounds 7a–7e, these compounds were clearly detected in phloem sap.
This confirmed our hypothesis that retaining the carboxyl group of PCA to conjugate
an amino acid to its phenazine ring can also endue PCA with phloem mobility.
Time-course experiments were performed within 5 h to evaluate the phloem
mobility of compounds 7a–7e over time. Figure 2 shows that compounds 7a–7e
were not detected at 1 h. The concentrations of compounds 7a, 7d and 7e in the
phloem sap increased with test time. Among them, compound 7d was found
to have the highest concentration in phloem sap of 22.63 1.39 mmol/L at 5 h,
indicating that it had the best phloem mobility. The phloem sap concentration
of compound 7c was 10.09 0.15 mmol/L at 3 h, approximately three times
higher than that at 2 h.
In our previous work, our group conjugated 12 amino acids to the carboxyl group
of PCA (Yu et al. 2018), five of which are identical to the amino acids conjugated to
the phenazine ring in this study (Figure 3). Therefore, we put the two results
together for comparison. As shown in supplementary information Figure S1, all PCA-
amino acid conjugates have phloem mobility. In addition, it can be clearly seen in
supplementary information Figure S1 that there is a large difference in the phloem
mobility of amino acid conjugated at different locations of PCA. Among them, the
concentration in the phloem sap of the compound 7-L-isoleucine substituted PCA
(7d) was 21 times higher than PCA-L-isoleucine conjugate (8d). For most com-
pounds, the phloem mobility of the same amino acid conjugated to the 7-position
of the phenazine ring of PCA is significantly better than that of the amino acid con-
jugated to the carboxyl group of PCA.
2.3. Prediction of phloem mobility using the Kleier model
The Kleier model was widely used to predict whether have the phloem mobility of xeno-
biotics based on the physicochemical properties (log Kow and pKa) (Hsu et al. 1995; Hsu
and Kleier 1996; Kleier and Hsu 1996). The experimental data results of many xenobiotics
were consistent with the theoretical predictions of the Kleier model. Thus, the physico-
chemical properties of the compounds 7a–7e and PCA were listed in supplementary
information Table S1. Based on their physicochemical properties, we marked the com-
pounds on the predicted phloem mobility may in supplementary information Figure S2.
As shown in Figure S2, all compounds 7a–7e and PCA were predicted to have
possibly mobile. Consistent with the results of the castor bean system, all compounds
have phloem mobility in addition to PCA. In addition, Xu et al. found that the mobility
of glycine-fipronil in castor seedlings is mediated by the amino acid carrier system
(Xie et al. 2016). While compounds 7a–7e are amino acid-PCA conjugates similar to
glycinergic-fipronil conjugate, which may also involve the participation of amino acid
carriers. The vector-mediated mechanism of these amino acid-PCA conjugates requires
further investigation.

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Y. XIONG ET AL.
3. Conclusions
Five novel 7-amino acid substituted phenazine-1-carboxylic acids 7a–7e were designed
and synthesised by amino acid substitution in C-7 position of phenazine ring. The
phloem mobility test results showed that all of the synthesised target compounds
7a–7e have phloem mobility. In addition, we compared the PCA-amino acid
conjugates of the previously reported amino acids conjugated to the carboxyl group
of PCA and found that most amino acid is conjugated to the phenazine ring for better
phloem mobility. Furthermore, the results of the phloem sap experimental data are
consistent with the theoretical predictions of the Kleier model. Overall, this study
provides an example of the conjugating of amino acids to different locations of
existing fungicide molecules, which can also endue it with phloem mobility. In order
to obtain compounds with better phloem mobility, it is the focus of our future research
on the effects of the same amino acid conjugated to different positions of PCA.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was funded by the National Key R&D Program of China (2018YFD0200500) and
National Natural Science Foundation of China (NO. 31672069)
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