Chemical structure of cichorinotoxin, a cyclic lipodepsipeptide that is produced by Pseudomonas cichorii and causes varnish spots on lettuce

Article abstract of DOI:10.3762/bjoc.15.27

Pseudomonas cichorii, which causes varnish spots on lettuce and seriously damages lettuce production during the summer season in the highland areas of Japan (e.g., Nagano and Iwate prefectures) was isolated. The structure of a toxin produced by this organism was analyzed based on the detailed evaluation of its 2D NMR and FABMS spectra, and this compound has not been reported previously. We propose the name cichorinotoxin for this toxin. In conjunction with the D or L configurations of each amino acid, which were determined by Marfey’s method, we propose the structure of cichorinotoxin to be as follows: 3-hydroxydecanoyl-(Z)-dhThr¹-D-Pro²-D-Ala³-D-Ala⁴-D-Ala⁵-D-Val⁶-D-Ala⁷-(Z)-dhThr⁸-Ala⁹-Val¹⁰-D-Ile¹¹-Ser¹²-Ala¹³-Val¹⁴-Ala¹⁵-Val¹⁶-(Z)-dhThr¹⁷-D-alloThr¹⁸-Ala¹⁹-L-Dab²⁰-Ser²¹-Val²², and an ester linkage is present between D-alloThr¹⁸ and Val²² (dhThr: 2-aminobut-2-enoic acid; Dab: 2,4-diaminobutanoic acid). Thus, the toxin is a lipodepsipeptide with 22 amino acids. The mono- and tetraacetate derivatives and two alkaline hydrolysates, compounds A and B, were prepared. We discuss here the structure–activity relationships between the derivatives and their necrotic activities toward lettuce.

Full text of DOI:10.3762/bjoc.15.27

Chemical structure of cichorinotoxin, a cyclic
lipodepsipeptide that is produced by
Pseudomonas cichorii and causes
varnish spots on lettuce
Hidekazu Komatsu1, Takashi Shirakawa2, Takeo Uchiyama1 and Tsutomu Hoshino*1,§
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 299–309.
1Department of Applied Biological Chemistry, Faculty of Agriculture
and Graduate School of Science and Technology, Niigata University,
Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan and 2Department
of Planning and General Administration, Institute of Vegetable and
Floriculture Science, National Agriculture and Food Research
Organization, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8519, Japan
doi:10.3762/bjoc.15.27
Received: 28 September 2018
Accepted: 22 January 2019
Published: 01 February 2019
Associate Editor: N. Sewald
Email:
Tsutomu Hoshino* - hoshitsu@agr.niigata-u.ac.jp
© 2019 Komatsu et al.; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
§ Fax: +81-25-262-6854
Keywords:
cichorinotoxin; lipodepsipeptide; necrotic lesion of lettuce; phytotoxin;
Pseudomonas cichorii
Abstract
Pseudomonas cichorii, which causes varnish spots on lettuce and seriously damages lettuce production during the summer season
in the highland areas of Japan (e.g., Nagano and Iwate prefectures) was isolated. The structure of a toxin produced by this organism
was analyzed based on the detailed evaluation of its 2D NMR and FABMS spectra, and this compound has not been reported previ-
ously. We propose the name cichorinotoxin for this toxin. In conjunction with the D or L configurations of each amino acid, which
were determined by Marfey’s method, we propose the structure of cichorinotoxin to be as follows: 3-hydroxydecanoyl-(Z)-dhThr1-
D-Pro2-D-Ala3-D-Ala4-D-Ala5-D-Val6-D-Ala7-(Z)-dhThr8-Ala9-Val10-D-Ile11-Ser12-Ala13-Val14-Ala15-Val16-(Z)-dhThr17-D-
alloThr18-Ala19-L-Dab20-Ser21-Val22, and an ester linkage is present between D-alloThr18 and Val22 (dhThr: 2-aminobut-2-enoic
acid; Dab: 2,4-diaminobutanoic acid). Thus, the toxin is a lipodepsipeptide with 22 amino acids. The mono- and tetraacetate deriva-
tives and two alkaline hydrolysates, compounds A and B, were prepared. We discuss here the structure–activity relationships be-
tween the derivatives and their necrotic activities toward lettuce.
Introduction
Pseudomonas cichorii causes varnish spots on lettuce. Varnish and dicot species (e.g., Asteraceae, Solanaceae, Apiaceae, and
spots, also called midrib rot or bacterial rot [1-4], are dark Liliaceae; 54 species in 17 families) [5], but necrotic lesions
brown and can induce necrotic lesions [1-7]. P. cichorii, isolat- most commonly occur in lettuce plants and seriously hurt
ed by us, infects a wide range of host plants, including monocot lettuce production in the highland areas of Japan (e.g., Nagano
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Beilstein J. Org. Chem. 2019, 15, 299–309.
and Iwate prefectures) in the summer season [5]. Effective ways Supporting Information File 1, Figure S1) showed strong
of inhibiting the infection are of great interest, but the infection and broad absorption bands at 1535 cm−1 and 1660 cm−1, indi-
mechanism remains unknown. We isolated a phytotoxin from cating that cichorinotoxin has amide groups and that it is a
the bacterial bodies of P. cichorii isolate YM8705 to clarify its peptide. In addition, a band at 1740 cm−1 (medium) was also
mechanism of action. An injection of 750 pg of the isolated observed, suggesting that an ester bond is present. This toxin
toxin caused necrotic lesions on lettuce midrib [5]. Furthermore, was subjected to Edman degradation, but this technique provi-
this toxin was confirmed to have an elicitor-like effect on the ded no information about the composition of the amino acids,
production of the phytoalexin lettucenin A [6-8]. As described suggesting that the amino group at the N-terminus is modified
below, the toxin was determined to be a cyclic lipodepsipeptide or blocked. However, this toxin was positive for ninhydrin;
composed of a 3-hydroxydecanoic acid moiety and 22 amino thus, a basic amino functional group is likely present in
acid residues. Two types of cyclic lipodepsipeptide phytotoxins cichorinotoxin. FABMS (positive mode; matrix: m-nitrobenzyl
are well known: relatively lower molecular weight compounds alcohol, NBA) showed signals at m/z 2069 [M + H]+ and
composed of 9 amino acids, such as those found in syringo- m/z 2070 (isotope peak), and signals at m/z 2067 [M − H]+ and
statin [9], syringomycin [10,11] and syringotoxin [12], and m/z 2068 (isotope peak) were observed in the negative mode
higher molecular weight compounds consisting of 22 (Supporting Information File 1, Figure S2). This finding
or 25 amino acids, which have been found in syringopeptins demonstrates that the molecular ion of cichorinotoxin (M+) is
[13].
m/z 2068. To clarify the amino acid composition, cichorino-
toxin was hydrolyzed in 6 N HCl at 110 °C for 5 h, 10 h and
Herein, we report the structural determination of the toxin pro- 22 h, and analysis of the amino acids in the hydrolytic residues
duced by P. cichorii YM8705, which was determined mainly showed that Thr, Ser, Pro, Ala and Val were present in the
from detailed analyses of its 2D NMR spectra. Although the structure of this toxin. To determine the stereochemistry (either
structure is similar to that of syringopeptin 22A, composed of D or L) of each amino acid, the hydrolytic residues were reacted
22 amino acids, it is not identical, and this new phytotoxic com- with Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alani-
pound has not been reported previously; we propose the name namide, FDAA) [15] to generate diastereomers. The diastereo-
cichorinotoxin for this compound. Very recently, Huang et al. mers were then separated by HPLC with a C18 column using a
reported new phytotoxins, named cichopeptin A and B, from solvent system composed of CH3CN and triethylamine phos-
P. cichorii SF1-54 [14], which cause midrib rot disease in phate (pH 3.0, see Figures S5B–S5E, Supporting Information
lettuce much like cichorinotoxin. However, cichorinotoxin is File 1). Marfey’s method revealed the presence of 2,4-
structurally distinct from the cichopeptins. The structural differ- diaminobutanoic acid (Dab) and Ile in the toxin (Figures S5B
ences are as follows: four of the amino acid residues are differ- and S5D). Four diastereomers are present in both Thr and Ile
ent, and the lipophilic fatty acid also differs between the two (structures, see Figure S5A, Supporting Information File 1):
toxins. Moreover, one of 22 amino acids was not definitively L-allo-type; L-threo-type; D-threo-type; and D-allo-type. The
identified. Thus, cichopeptins A and B are different compounds four diastereomers (FDAA derivatives) of Thr were well sepa-
compared to the cichorinotoxin isolated by us. The structure of rated by HPLC (Supporting Information File 1, Figure S5C). In
cichopeptin proposed by Huang et al. was determined mainly by contrast, in the case of the Ile derivatives, separations between
MS/MS spectral analyses, and the stereochemistry (D, L) of the L-allo- and L-threo-types and between the D-allo- and
each of the amino acids was not determined. Herein, we present D-threo-types were unsuccessful, but separations between the
the almost complete assignments of all the 1H and 13C NMR L-allo- and D-allo-Ile and between the L-threo- and D-threo-Ile
signals of cichorinotoxin, and we determined the stereochemis- were achieved (Supporting Information File 1, Figure S5E).
try of 12 out of the 22 amino acids. In addition, we prepared Consequently, the HPLC analyses demonstrated that
chemically modified derivatives of cichorinotoxin and evalu- the Thr residue in the toxin was the D-allo-form with
ated the abilities of some derivatives to cause lettuce rot, in (2R,3R)-stereochemistry, and the Ile residue was either the
order to provide insight into which structural units have a D-allo-derivative, with (2R,3S)-stereochemistry, or the D-threo-
crucial role for causing the phytotoxic activity.
form, with (2R,3R)-stereochemistry. Table 1 summarizes
the composition and the stereochemistry of each amino
acid. As described below, NMR analysis of cichorinotoxin
confirmed that three units of dehydrothreonine (dhThr:
2-aminobut-2-enoic acid) were present in the toxin, and the
Z-geometry of the double bond was determined by NOESY
analysis (see Figure 6 and Supporting Information File 1, Figure
S10F).
Results and Discussion
Composition of cichorinotoxin:
3-hydroxydecanoic acid and 22 amino acids
The culture conditions of P. cichorii YM8705 and the isolation
procedure are described in the Experimental section. This
toxin was isolated as a white solid. Its IR spectrum (KBr tablet,
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Beilstein J. Org. Chem. 2019, 15, 299–309.
80 °C for 24 h. The reaction mixture was then analyzed by
GC–MS, which showed m/z 332 as the parent ion [M+], demon-
strating that two TMS molecules were attached (Supporting
Information File 1, Figure S3A). Commercially available
(±)-3-hydroxydecanoic acid was also derivatized with the above
mentioned TMS reagent (Supporting Information File 1, Figure
S3B). The two products showed the same GC retention time
and fragment ions in the MS, verifying that the fatty acid in
cichorinotoxin is 3-hydroxydecanoic acid. As described above,
a basic amino acid (positive in the ninhydrin test) is present in
cichorinotoxin. To confirm the presence of a free amino group,
cichorinotoxin (6.0 mg) was acetylated for 6 h at room tempera-
Table 1: The proportion of each enantiomer of each amino acid, which
were determined by an amino acid analyzer and by the peak areas of
the D- and L-amino acids as estimated by Marfey’s method.
Ala
Val
Pro
Ser D-alloThra Dabb
Ilec
D
L
7.65 4.33 1.49 0.68
1.21 1.42 0.82
1.0
–
–
0.91
–
–
0.92
The composition ratios of each amino acid presented here were in
good agreement with those of the overall structure of cichorinotoxin
shown in Figure 6. aD-alloThr indicates (2R, 3R)-stereochemistry.
bDab: 2,4-diaminobutanoic acid. cThe Ile residue is either the D-allo- or
the D-threo-type and not the L-allo- or L-threo-type.
Analyses of the fragment ions in the FABMS spectrum of ture with Ac2O (1.5 mL) in a mixed solvent (5 mL) composed
cichorinotoxin (Figure 1) showed the presence of the following of CH3CN/pH 9.7 Na2CO3/NaHCO3 buffer solution (1:1). The
peptide segment: Pro-Ala-Ala-Ala-Val-Ala-dhThr-Ala-Val FABMS spectrum (positive mode, NBA matrix, Supporting
(Figure 2). As described below, this toxin has a 3-hydroxyde- Information File 1, Figure S4A) showed a signal at m/z 2111 [M
canoic acid moiety as a fatty acid. Furthermore, the amino + H]+, indicating that the monoacetate was obtained; thus, the
group at the N-terminus is modified or blocked, as mentioned number of basic amino acids was limited to one. As shown in
above. Observation of a fragment ion of m/z 254 suggests that Table 1, Dab is the only basic amino acid present in the mole-
the 3-hydroxydecanoyl dehydrothreonine moiety (m/z 254) is cule. A prominent peak at m/z 2133 [M + Na: 2068 + 42 + 23]+
linked to the Pro residue (Figure 2). This segment was further was observed in addition to the signal at m/z 2111 [M + H]+. In
confirmed by detailed NMR analyses (Figure 3). To confirm the contrast, the reaction with Ac2O/Py showed a parent ion at m/z
presence of this fatty acid moiety, cichorinotoxin (2.0 mg) was 2259 in the FABMS spectrum (m-NBA matrix, Supporting
hydrolyzed at 120 °C for 90 min with 1.0 mL of 6 N aqueous Information File 1, Figure S4B), which suggested that the
HCl. EtOAc (1.5 mL) was added to the hydrolysate, and then tetraacetate was generated m/z [2068 + 42 × 4 + 23 = 2259], but
the fatty acid was extracted. The extract was dried over an- the molecular ion of the tetraacetate peak (m/z 2237 [M + H]+)
hydrous MgSO4 and concentrated in vacuo. A mixture (1.0 mL) did not appear in the FABMS spectrum (Supporting Informa-
of trimethylchlorosilane and N,O-bis(trimethylsilyl)acetamide tion File 1, Figure S4B). Consequently, these MS data of the
(10:1) was added to the residue, the solution was heated at mono- and tetraacetates, which were obtained under controlled
Figure 1: FABMS spectrum (positive mode, NBA matrix) of cichorinotoxin over the mass range of m/z 150─1000.
Figure 2: The segment identified based on the FABMS spectrum (peptide fragment b ions).
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Beilstein J. Org. Chem. 2019, 15, 299–309.
Figure 3: NMR analyses of the segment consisting of 3-hydroxydecanoyl-dehydrothreonin-proline (600 MHz, acetone-d6). DhThr1 and Pro2 indicate
that the first and second amino acids in the peptide backbone are dhThr and Pro, respectively.
reaction conditions, clearly indicated that one amino group S8–S12. The side chain of each amino acid (type of amino acid)
(Dab) and three OH groups are present in the toxin. As shown was mainly determined from the 1H,1H COSY and TOCSY
in Table 1, cichorinotoxin has three OH groups in its structure spectra. The NOESY spectrum provided important information
due to the presence of two serine residues and one threonine on the backbone of the amino acid sequence, as depicted in
residue. In addition, one 3-hydroxyfatty acid is present, as de- Figure 4. The presence of a dhThr moiety in cichorinotoxin was
scribed above (Figure 2). Thus, there are four OH groups in confirmed as follows (see dhThr1 in Figure 3). The methyl
cichorinotoxin. As mentioned below, the four acetate moieties signal (δH 1.82, d, J = 7.0 Hz, γCH3) had clear COSY cross
were determined to be at Dab, the two serine residues and the peaks with the olefinic proton at δH 5.89 (q, J = 7.0 Hz, βH),
β-hydroxyfatty acid based on NMR analyses. Thus, the OH which showed an HMQC cross peak with the carbon at δc 123.3
group on the Thr residue is blocked through an ester linkage (d). In the HMBC spectrum, the γCH3 moiety had clear correla-
(IR, 1740 cm–1).
tions with the double bond carbons (δc 132.8, s, αC and
δc 123.3, d, βC), and the βH exhibited clear HMBC correla-
tions with the αC and carbonyl carbon (δc 168.8, s, CO). These
NMR data clearly indicate the presence of a dhThr moiety
NMR analyses to determine the overall
structure of the monoacetate
The 1H NMR spectrum of cichorinotoxin (not the acetate deriv- (Figure 3), and the strong NOE between the γCH3 protons and
ative) in DMSO-d6 (600 MHz) was not sufficiently resolved to the αNH proton demonstrated that the double bond is in the
elucidate the structure (Supporting Information File 1, Figure Z-configuration. The αNH proton of dhThr showed a definitive
S6). However, the 1H NMR signals of the monoacetate were HMBC cross peak with the carbonyl carbon (δc 173.5, s, C-1)
well separated (600 MHz, acetone-d6, Supporting Information of the fatty acid (FA) and showed NOEs with H-2 (δH 2.64, m;
File 1, Figure S7). Therefore, the 1D and 2D NMR spectra of 2.72, m) of the FA, which exhibited HMBC correlations with
the monoacetate were analyzed to determine the overall struc- C-1, the alcoholic carbon (δc 69.3, d, C-3) and C-4 (δc 38.2, t)
ture. The 1H,1H COSY, TOCSY, NOESY, HMQC and HMBC of the FA, demonstrating that the 3-hydroxydecanoic acid (the
spectra are shown in Supporting Information File 1, Figures FA) is directly connected to the dhThr moiety via an amide
Figure 4: Key NMR observations used to construct the backbone sequence.
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bond. This structural unit (Figure 3) was further confirmed by porting Information File 1, Figure S12C) established that Dab20
the m/z 254 ion in the FABMS spectrum of natural cichorino- was acetylated; an HMBC cross peak between γCH2 of Dab20
toxin (Figure 1). We then determined the presence of three and COCH3 was observed.
dhThr residues in the cichorinotoxin (Figure 6) by analyzing the
NMR data; the βCH protons (olefinic protons) of the three Figure 6 depicts the key NMR data. Tables S1 and S2 (Support-
dhThr moieties are found at δH 6.37 (q, J = 7.0 Hz), δH 6.24 (q, ing Information File 1) summarize the 1H and 13C NMR data of
J = 7.0 Hz) and δH 5.89 (q, J = 7.0 Hz) in the 1H NMR spec- the monoacetate. Based on these data, cichorinotoxin contains a
trum of the monoacetate (Supporting Information File 1, Figure lipophilic fatty acid and 22 amino acids, and the peptide part is
S7). In the NMR spectra of the monoacetate, αH (δH 4.31) of cyclized through an ester linkage between two of the amino acid
Pro2 showed a clear HMBC cross peak with the carbonyl car- residues (Thr18 and Val22). Thus, cichorinotoxin is classified as
bon (CO, δc 168.8, s) of dhThr1, indicating that Pro is the 2nd a cyclic lipodepsipeptide.
amino acid residue, as shown in Figure 3. The five-membered
Pro skeleton was confirmed by the COSY and TOCSY spectra, To determine the stereochemistry of the amino acids (D or L),
as shown in Figure 3. The FA-dhThr-Pro fragment was further we tried to isolate partial fragments by acid hydrolysis. The
supported by the fragment ion at m/z 351 in the FABMS toxin was dissolved in 12 N HCl and reacted for 20 h at room
(Figure 1 and Figure 2). As described above, a basic amino acid temperature. The partial hydrolysates were subjected to
(positive to the ninhydrin test) is present; thus, the amino group reversed-phase HPLC (C18), which showed that 6 main peptide
was acetylated under basic conditions to afford the monoac- fragments were produced. These fragments were purified and
etate of cichorinotoxin (Figure 6). This basic amino acid was analyzed by FABMS. The FABMS spectrum of one of the frag-
determined to be 2,4(α,γ)-diaminobutanoic acid (Dab-20) by ments indicated that the segment had the structure shown in
detailed analyses of the NMR data. In the COSY and TOCSY Figure 5. This fragment was subjected to acid hydrolysis ac-
spectra of the acetate, the αNH proton (δH 8.61) was correlated cording to the method described above. Marfey’s method
with αCH (δH 3.76), βCH2 (δH 2.33; 2.08) and γCH2 (δH 3.21). revealed the following amino acid composition, including the
The γCH2 protons had a clear HMBC cross peak with the acetyl stereochemistry: Pro (D × 1), Ala (D × 4) and Val (D × 1).
carbonyl carbon (δc 170.5, s, COCH3), as shown in Figure None of the other generated peptide fragments were useful for
S12B (Supporting Information File 1). The spectrum shown in determining stereochemistry. Considering the D or L configura-
Figure S12B also indicated that the ester linkage (IR, 1740 tions (Table 1), we could propose the stereochemistry of 12 of
cm−1) is between βCH of Thr-18 (δH 5.23, m) and CO of Val- the amino acids (including the dhThr moieties), but the stereo-
22 (δc 170.5, s).
chemistries of the remaining 10 amino acids remain undeter-
mined (see Figure 6).
Detailed analyses of the 2D NMR data (1H,1H-COSY, TOCSY
and NOESY) and 1H,13C correlations (HMQC and HMBC)) of NMR data of the tetraacetate
the peptides and proteins [16-18], which are illustrated in To further confirm the structure of cichorinotoxin, the tetraac-
Figure 4, allowed us to construct the complete backbone amino etate was analyzed by NMR (600 MHz, acetone-d6). The
acid sequence of cichorinotoxin; the NOESY data are shown in majority of the 1H NMR signals of the tetraacetate (Figure
Figures S10A–S10F (Supporting Information File 1), and the S13A, Supporting Information File 1) were separated, which
HMBC data are shown in Figures S12A–S12C. Figures S10B was not the case in the spectrum of the monoacetate. The
and S10C highlight the NOE correlations of αNiH/αNi+1H. TOCSY, COSY and NOESY spectra of the tetraacetate are
Figures S10D and S10E mainly depict the NOE correlations of shown in Figures S13E–S13N (Supporting Information File 1).
αNiH/αCiH and αCiH/αNi+1H. Figure S10F mainly highlights The NOE data indicated that the amino acid sequence of the
the NOEs of βCiH/αNi+1H. Distinct NOEs were observed be- tetraacetate is as shown in Figure S14 (Supporting Information
tween the γNH of Dab-20 and COCH3, further verifying that File 1). The positions of the acetates were determined mainly
the γNH2 of Dab was acetylated. The HMBC spectrum (Sup- from the HMBC and COSY spectra (Figure 7). The β-CH2
Figure 5: Fragment obtained by acid hydrolysis and each amino acid was exclusively D-configuration (peptide fragment b ions).
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Beilstein J. Org. Chem. 2019, 15, 299–309.
Figure 6: The complete structure of cichorinotoxin monoacetate and the assignments of 1H NMR chemical shifts, which were determined by various
2D NMR techniques.
Figure 7: Determination of the positions of the acetyl groups in the tetraacetate.
protons of Ser12, Ser21 and Dab20 each had distinct HMBC residues were acetylated in the tetraacetate, and the amino
correlations with one of the acetyl carbonyl carbons, the signals group of Dab was acetylated in both the monoacetate and the
of which were confirmed by clear HMBC cross peaks for the tetraacetate. We found that the hydroxy group at C-3 of the FA
acetyl methyl protons (see Figures S13B–S13D in Supporting residue was acetylated despite the H-3 proton not having a clear
Information File 1). Thus, the hydroxy groups of the two Ser HMBC cross peak with the acetyl carbonyl carbon. The δH of
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Beilstein J. Org. Chem. 2019, 15, 299–309.
H-3 in the tetraacetate was shifted downfield by ca. 1.0 ppm J = 7.2 Hz, 1H for dhThr18) and 5.58 (q, J = 6.8 Hz, 1H for
(acetylation shift) relative to that in the monoacetate dhThr1). This finding indicated that an additional dhThr residue
(δH 4.19 → δH 5.37), confirming that the OH group of the FA was produced during the hydrolysis. Moreover, the NOE data
was acetylated. Consequently, the complete structure of indicated that the double bond in the newly formed dhThr was
cichorinotoxin tetraacetate is shown in Figure S14 (Supporting in the E-geometry (Figures S16C–16E), as shown in Figure 8.
Information File 1), and the fundamental scaffolds, composed The NOE data (Figure 8), which reveal the amino acid se-
of 3-hydroxydecanoic acid and the amino acid sequence back- quence, verified that Thr18, which is connected to CO2H of
bone, of the mono- and tetraacetates were identical. Tables S3 Val22 via an ester bond, was changed into dhThr and that the
and S4 (Supporting Information File 1) summarize the assign- backbone sequence from the fatty acid moiety to dhThr17 was
ments of 1H and 13C NMR signal data.
identical to that of parent cichorinotoxin, but the ester bond was
cleaved by the alkaline hydrolysis to yield the linear peptide, as
shown in Figure 8 and Figure S18 (Supporting Information
File 1).
Compounds A and B generated by alkaline
hydrolysis
This toxin is composed of a cyclic peptide formed by an ester
linkage between the OH of Thr18 and the CO2H of Val22. On the other hand, compound B contained three olefinic protons
Figure S15 (Supporting Information File 1) shows the primary (βCH of the dhThr residues) in its structure (Figure S17B), and
structures of cichopeptin A and B [14], syringopeptin 22-A and their signals appeared at δH 6.37 (q, J = 7.0 Hz, 1H for dhThr8);
22-B [13], corpeptin A and B [19], fuscopeptin [20] and 5.65 (q, J = 7.2 Hz, 1H for dhThr18); 5.59 (q, J = 7.0 Hz, 1H for
tolaasin I [21,22], which are typical lipodepsipeptides produced dhThr1). The chemical shifts of the two olefinic protons of
by Pseudomonas sp., and they are structural analogues of dhThr8 and dhThr1 were nearly the same as those of compound
cichorinotoxin. All of these peptides have a lipophilic fatty acid A. The NOESY spectrum, shown in Supporting Information
moiety connected to dhThr1-D-Pro2, and the amino acids File 1, Figure S17F, indicated that the double bonds of dhThr8
dhThr17 and alloThr18 are strictly conserved. Moreover, the and dhThr1 were in the Z-configuration, which is identical to
conserved alloThr moiety is involved in the cyclic ester linkage. unreacted cichorinotoxin, but the E-geometry was found for the
Therefore, the ester bond of natural cichorinotoxin was cleaved double bond of dhThr18, which is identical to that of compound
by alkaline hydrolysis (0.02 N KOH/MeOH) at room tempera- A. The amino acid sequence of compound B was determined
ture for 5 h to examine the role of the cyclic structure in the mainly from its NOESY spectrum (Supporting Information
ability to cause varnish spots on lettuce. Two products, A and File 1, Figures S17C, D and E). The fatty acid moiety was
B, were generated by the hydrolysis. The FABMS spectrum of retained, and the sequence from dhThr1 to Ala16 was the same
compound A indicated that the MW of A was identical to that as that of unreacted cichorinotoxin. As shown in Figure 9, the
of unreacted cichorinotoxin (Figure S16A, Supporting Informa- 17th residue was Thr-like amino acid, which was different from
tion File 1). As shown in Figure S16B, signals for four olefinic that (dhThr) in the parent cichorinotoxin and in compound A.
protons (βCH of dhThr) were observed in the 1H NMR spec- The subsequent amino acids in the sequence (18th to 22nd)
trum (600 MHz, DMSO-d6) δH 6.46 (q, J = 6.6 Hz, 1H for were identical to those in compound A. However, it is notable
dhThr17); 6.38 (q, J = 7.0 Hz, 1H for dhThr8); 5.78 (q, that the residue in the 17th position was not in fact genuine Thr
Figure 8: Partial structure of compound A as deduced from its NOE data; key NOEs are represented by double-headed arrows. The δH (ppm) are
given, and these signals were measured in DMSO-d6 (600 MHz). The double bond of dhThr18 was in the E-configuration.
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Figure 9: Partial structure of compound B (Val16 to Val22 residues). The chemical shifts (δH and δC (ppm)) are shown, and these values were ob-
tained in DMSO-d6 (600 MHz NMR instrument).
because the chemical shift of βCH was δC (ppm) 53.85, which the fatty acid, two serine residues (Ser12 and Ser21) and Dab20
is significantly upfield compared with the resonance of this car- are all acylated, had no activity. Ser12, which is present in the
bon in genuine Thr (approximately 67 ppm βCH-O function). linear portion of the peptide, is not conserved in the toxins listed
Furthermore, distinct NOEs were observed between the γCH in Supporting Information File 1, Figure S15; thus, it is likely
protons of Dab20 and the βCH protons of the 17th amino acid that the hydrophilic Ser21, embedded in the macrocycle of the
(Thr-like) and between the γCH protons of Dab20 and the γCH3 peptide, is crucial for the necrosis activity. The peptides in the
protons of the 17th Thr-like amino acid. These data unambigu- macrocycle, listed in Supporting Information File 1, Figure S15,
ously indicated that the Thr-like residue was linked with Dab consist of 5 or 8 amino acids, and of these, at least two of the
through the γ-nitrogen atom of Dab20, as shown in Figure 9.
hydrophilic amino acids are conserved; this could be the Dab-
Ser or the Dab-Dab sequence for example. The conversion of
Consequently, the overall structures of compounds A and B are the two hydrophilic amino acids into hydrophobic residues by
depicted in Figure S18 (Supporting Information File 1). acylation may have caused the complete loss of activity seen
Figure 10 shows the reaction mechanisms for generating com- with the tetraacetate (4). Acyclic compound A (a linear lipopep-
pounds A and B from cichorinotoxin by treatment with dilute tide) produced by alkaline treatment had no activity, supporting
KOH. The proton at α-position of D-alloThr18 was first elimi- that a macrocyclic peptide would be essential for biological ac-
nated by the base (OH− ion), followed by the release of Val22- tivity. The sequence Z-dhThr17-D-alloThr18 of cichorinotoxin
COO− ion and the production of dhThr. This process follows an was converted to Z-dhThr17-E-dhThr18 by alkaline hydrolysis.
E2-type elimination mechanism (see a Newman projection), This additional structural modification may have further de-
thus leading to compound A, which has an E-double bond for creased the activity. Compound B has a macrocycle composed
dhThr instead of a Z-double bond. Compound A was further of 4-amino acids, but it exhibited no necrotic activity. As seen
converted into compound B by nucleophilic attack of the free in the structures of all the toxins depicted in Figure S15 (Sup-
amino group of Dab20 on the C-3 of dhThr17.
porting Information File 1), the macrocycles are in terminal po-
sitions; thus, having the macrocycle at the termini may be re-
quired for the toxic activity. At the present stage, the crucial
structural units for the toxic activity cannot be specified and are
still uncertain. Further studies are required.
Necrotic lesion on lettuce by cichorinotoxin
derivatives (mono- and tetraacetates and
compounds A and B)
Figure S19A (Supporting Information File 1) shows the
bioassay method. Figure S19B indicates that the necrotic
lesions caused by the monoacetate (3) were somewhat less
severe, but the compound was still active; the tetraacetate (4)
showed almost no activity. For the monoacetate (3), the hydro-
philic NH2 of Dab20, which is one of the residues in the macro-
cycle of the peptide, is acetylated. The tetraacetate (4), in which
Conclusion
In summary, we succeeded in proposing the overall structure of
cichorinotoxin, 3-hydroxydecanoyl-(Z)-dhThr1-D-Pro2-D-Ala3-
D-Ala4-D-Ala5-D-Val6-D-Ala7-(Z)-dhThr8-Ala9-Val10-D-Ile11-
Ser12-Ala13-Val14-Ala15-Val16-(Z)-dhThr17-D-alloThr18-Ala19-
L-Dab20-Ser-21-Val22, in which an ester linkage is formed be-
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Beilstein J. Org. Chem. 2019, 15, 299–309.
Figure 10: Mechanism of the formation of compounds A and B from cichorinotoxin by alkaline hydrolysis.
tween D-alloThr18 and Val22. The configurations (D or L) of all are required for the toxic activity, but are not sufficient. Further
22 amino acids could not be determined in the present study. investigations are necessary to address the structure–activity
Authentic D-[1-13C]- and L-[1-13C]-Val were separately fed relationship.
into cultures of P. cichorii YM8705, and the cichorinotoxin
Experimental
thus obtained was analyzed by 13C NMR spectroscopy. Five
Val residues (four D-Val and one L-Val) are included in General analytical methods
cichorinotoxin (Figure 6 and Table 1). All the five 13C signals NMR spectra were recorded in acetone-d6 on a Bruker DMX
of CO- of Val residues appeared with almost the same signal in- 600 spectrometer; the chemical shifts (δ) are given in ppm rela-
tensities in each of the 13C NMR spectra, which were obtained tive to the residual solvent peaks (δH 2.04 and δC 29.80) as the
by the feedings of L- and D-Val (Figure S20B and S20C, Sup- internal references for the 1H and 13C NMR spectra, respective-
porting Information File 1). This finding definitively demon- ly. In the case of DMSO-d6 solutions, the chemical shifts are
strated that an epimerase (racemase) is involved in the biosyn- given in ppm relative to the solvent peaks (δH 2.49 and
thetic gene cluster. Differences in the 13C signal intensities were δC 39.50). The coupling constants, J, are given in Hz. FABMS
a little in the feeding experiments using L- or D-[1-13C]-Ala spectra were obtained on a JEOL SX 100 spectrometer using
(see Figure S20D and S20E, Supporting Information File 1). m-nitrobenzyl alcohol (NBA). A Shimadzu LC-10A HPLC
These results indicated that an epimerase is involved in the apparatus and a Shimadzu SPD-10AVP detector were used to
biosynthetic gene cluster of this strain. Thus, we failed to assign determine the D or L configuration of each amino acid.
the D or L configurations for all the amino acids by the feeding
experiments. However, we succeeded in proposing the com- Bioassay method
plete stereochemistry of the molecule by careful analysis of the The necrotic lesions were detected by the method given in
biosynthetic gene cluster (nonribosomal peptide synthetases, Figure S19A (Supporting Information File 1). The lesions were
NRPS). These results will be reported in due course. The usually detected after standing for 1 day. In the cases of the
present studies gave some information of which structural units mono- and tetraacetates and compounds A and B, the necrotic
307

Beilstein J. Org. Chem. 2019, 15, 299–309.
activities significantly decreased; thus, the samples were which were concentrated to dryness. The residues were evalu-
allowed to stand for 3 days to estimate the activities, as shown ated using an amino acid analyzer. Marfey’s method was em-
in Supporting Information File 1, Figure S19B.
ployed to determine the stereochemistry of each of the amino
acids. The dried hydrolysates obtained by heating for 10 h were
dissolved in 140 μL of H2O. To 25 μL of the solution were
Isolation of cichorinotoxin
Pseudomonas cichorii isolate YM8705 has been deposited in added 1% Marfey’s reagent ((1-fluoro-2,4-dinitro-5-fluoro-
NARO Genebank as MAFF730229 (NARO: National Agricul- phenyl)-L-alaninamide, 1.8 μM) dissolved in acetone and 10 μL
tural and Food Research Organization).
of 1 M NaHCO3 (10 μM). The solution was heated at 35 °C for
1 h, then 2 N aq HCl was added to quench the reaction, and then
P. cichorii YM8705 was cultured in YPD medium (polypep- it was concentrated to dryness. The residues were dissolved in
tone (10 g, Nippon Shinyaku Ltd), dry yeast extract (5 g, Oxoid DMSO and subjected to reversed-phase HPLC (C18) using a
Ltd.), NaCl (3 g) and mannitol (5 g, Wako Ltd.) in 1000 mL of mobile phase composed of 13% CH3CN/87% 50 mM triethyl-
H2O). Precultured seeds were grown at 28 °C for 24 h in amine phosphate. Amino acid compositions of natural
100 mL of YPD medium using a 500 mL Sakaguchi flask with cichorinotoxin (not Marfey’s derivatives) were analyzed by
reciprocal shaking. Next, 10 mL of the precultured seed culture Hitachi Keisoku Service Ltd (Tokyo).
was inoculated into 1000 mL of the YPD medium and cultured
at 28 °C for 72 h. The cells obtained from 6 L of culture were Partially hydrolyzed peptide fragments
collected by centrifugation and lyophilized. The dried cells Seven milliliters of 12 N HCl was added to 15 mg of cichorino-
were soaked in approximately 1.0 L of MeOH and stirred at toxin, and the reaction was allowed to stand at room tempera-
room temperature for 2 h. The cells were then removed by ture for 20 h. The hydrolysates were subjected to reversed-
filtration. The methanolic fraction, which contained the crude phase HPLC (the same ODS column) using MeOH/H2O
cichorinotoxin, was developed with a mixed solvent of (58:42) with 0.3% NH3 and afforded 6 major peaks. After
(CH3)2CO/MeOH (2:1) on a SiO2 TLC. The toxin was visual- purification, only one peak remained in the HPLC chro-
ized as a yellowish white spot by spraying either a BTB reagent matogram, and this compound was analyzed by FABMS. To
or deionized water. Crude cichorinotoxin was obtained by SiO2 clarify the stereochemistry of the amino acids present, Marfey’s
column chromatography eluting with the above solvent. Pure method was employed.
cichorinotoxin was obtained by reversed-phase HPLC (column:
Shiseido CAPCEL PAK C18 UG120; mobile phase: MeOH/ Hydrolysis of cichorinotoxin in a basic medium
1% aqueous NH3 = 87:13, detected at 254 nm) in a yield of The purified toxin (26 mg) was dissolved in 0.02 N KOH/
approximately 122 mg. Melting point: 181–184 °C.
MeOH and allowed to stand at ambient temperature for 5 h.
This reaction mixture was neutralized with 2 N aq HCl and then
Preparation of the monoacetate and the tetraacetate lyophilized. The residue was subjected to reversed-phase HPLC
This toxin (6.0 mg) was dissolved in 5 mL of a mixture (1:1) of (C18) eluting with MeOH/1% aq NH3 (71:29), and the eluate
CH3CN and 0.01 M carbonate buffer solution (pH 9.7), and was monitored at 254 nm. New peaks from compounds A
then 1.5 mL of Ac2O was added. The reaction was carried out at (earlier elution) and B (later elution) were observed in addition
room temperature for 6 h. Almost all of the toxin to the peak of cichorinotoxin. The isolated yields were as
had been consumed by SiO2 TLC analysis. Complete purifica- follows: 2.5 mg for compound A; 6.3 mg for compound B; and
tion was achieved by reversed-phase HPLC (MeOH/ 6.1 mg of recovered cichorinotoxin.
1% aq NH3 = 83:17), yielding 5.4 mg of the monoacetate.
Cichorinotoxin (8.0 mg) was dissolved in Py (3.0 mg), and Feeding experiments of D- and L-[1-13C]Val
1.5 mL of Ac2O was added. The reaction was continued for 1 h and -Ala
at ambient temperature, and the crude material was purified by P. cichorii YM8705 was grown in the culture medium
the same HPLC conditions as described for the monoacetate to composed of the following compositions: mannitol (10 g),
afford 6.8 mg of the tetraacetate.
Na2HPO4·12H2O (3 g), KH2PO4 (0.5 g), NaCl (3 g),
(NH4)2SO4 (1 g), MgSO4·7H2O (25 mg), CaCl2·2H2O (23 mg)
and dry yeast extract (0.1 g) dissolved in 1000 mL of H2O. To
the 500 mL Sakaguchi flask containing 100 mL medium,
Determination of the amino acid composition and of
the D and L configurations
To 1.0 mg of cichorinotoxin was added 1.0 mL of 6 N HCl, and 25–40 mg of the labelled Val and Ala was aseptically added
the mixture was frozen at −30 °C and degassed under a high and then grown at 28 °C for 120 h. The labelled cichorino-
vacuum pump. The glass vessel was sealed and heated at toxins were isolated by the same HPLC conditions as described
110 °C for 5 h, 10 h and 22 h to obtain the hydrolysates, each of above.
308

Beilstein J. Org. Chem. 2019, 15, 299–309.
11.Segre, A.; Bachmann, R. C.; Ballio, A.; Bossa, F.; Grgurina, I.;
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Pucci, P.; Scaloni, A.; Segre, A.; Simmaco, M. FEBS Lett. 1990, 269,
377–380. doi:10.1016/0014-5793(90)81197-v
Supporting Information
Supporting Information File 1
Analytical data (1H, 13C NMR, IR and MS), photographs of
bioassay methods.
13.Ballio, A.; Barra, D.; Bossa, F.; Collina, A.; Grgurina, I.; Marino, G.;
Moneti, G.; Paci, M.; Pucci, P.; Segre, A.; Simmaco, M. FEBS Lett.
1991, 291, 109–112. doi:10.1016/0014-5793(91)81115-o
14.Huang, C.-J.; Pauwelyn, E.; Ongena, M.; Debois, D.; Leclère, V.;
Jacques, P.; Bleyaert, P.; Höfte, M. Mol. Plant-Microbe Interact. 2015,
28, 1009–1022. doi:10.1094/mpmi-03-15-0061-r
[https://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-15-27-S1.pdf]
Acknowledgements
15.Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
doi:10.1007/bf02908688
Dr. Shinobu Fujimura in this department (Niigata University)
assisted in a preliminary work to clarify the amino acid compo-
sition. We also thank Prof. Fumio Sugawara, Tokyo University
of Science, for valuable comments on the structural determina-
tion. One of the authors (T. Shirakawa) appreciates the finan-
cial support from the Science and Technology Agency (Japan),
which allowed him to structurally characterize this toxin in this
department of Niigata University. Special thanks are given to
the late Prof. T. Uchiyama, who made a great contribution to
this investigation in an earlier research stage.
16.Wüthrich, K. NMR of Proteins and Nucleic Acids; John Wiley & Sons,
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ORCID® iDs
Tsutomu Hoshino - https://orcid.org/0000-0002-8484-2752
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