Chemical structure of hydrolysates of cereulide and their time course profile

Article abstract of DOI:10.1016/j.bmcl.2020.127050

Hydrolysates of an emetic toxin cereulide were found in the broth of Bacillus cereus. The ester cleaved depsipeptides of cereulide were synthesized using liquid phase fragment condensation method starting from commercially available amino acids. The chemical structure of hydrolysates was verified tetradepsipeptide L-O-Val-L-Val-D-O-Leu-D-Ala and dodecadepsipeptide (D-O-Leu-D-Ala-L-O-Val-L-Val)₃ using LC-TOFMS. Quantitative analysis of cereulide in the broth revealed production of cereulide in the stationary phase and decomposition in the death phase. The increase in tetradepsipeptide continued after the stationary phase until decomposition occurred.

Full text of DOI:10.1016/j.bmcl.2020.127050

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chemical structure of hydrolysates of cereulide and their time course profile
⁎
Toshihito Nakaa,c, , Yuka Takakia, Yoshihide Hattorib, Hiroshi Takenakab, Yoichiro Ohtab,
Mitsunori Kirihatab, Shinji Tanimoric
a Toyo College of Food Technology, 4-23-2 Minami-hanayashiki, Kawanishi, Hyogo 666-0026, Japan
b Research Center for Boron Neutron Capture Therapy, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan
c Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Nakaku, Sakai, Osaka 599-8531, Japan
A R T I C L E I N F O
A B S T R A C T
InChIKeys:
Hydrolysates of an emetic toxin cereulide were found in the broth of Bacillus cereus. The ester cleaved depsi-
peptides of cereulide were synthesized using liquid phase fragment condensation method starting from com-
mercially available amino acids. The chemical structure of hydrolysates was verified tetradepsipeptide L-O-Val-L-
Val-D-O-Leu-D-Ala and dodecadepsipeptide (D-O-Leu-D-Ala-L-O-Val-L-Val)3 using LC-TOFMS. Quantitative ana-
lysis of cereulide in the broth revealed production of cereulide in the stationary phase and decomposition in the
death phase. The increase in tetradepsipeptide continued after the stationary phase until decomposition oc-
curred.
JWWAHGUHYLWQCQ-UHZBFKKDSA-N
AMAQROXGXMKUMP-STQMWFEESA-N
LVRFTAZAXQPQHI-RXMQYKEDSA-N
UIKJRDSCEYGECG-UHFFFAOYSA-N
CLQJEHJUWLIROW-TZMCWYRMSA-N
GSKIIPFDRMHDQF-ZDEZRRDYSA-N
BTRVMPHKISKDEI-ZRNYENFQSA-N
RWZITXLYZQEETE-YJMBLLCNSA-N
OMWPVXFSTIXLAR-IOZPFHIPSA-N
OSAZBLNZXXOTDX-RBAFHZQWSA-N
KFODNABJTDQUSL-VONWAMCDSA-N
AKOCOTUURHEJAS-UHZBFKKDSA-N
JPZIQQNBGQBNLI-ABLOACMZSA-N
QWEAKSDNSASMLT-IEBWSBKVSA-N
VYRGEWOWDSEHHM-VXGBXAGGSA-N
IKRVOHUYMWAWHK-ZDEZRRDYSA-N
XITAEGDCQXMLLB-ZRNYENFQSA-N
DKXIZVUCDYCGAF-YJMBLLCNSA-N
OTUOOLRJEZOJMC-IOZPFHIPSA-N
BNCBKZOVEHEPJM-RBAFHZQWSA-N
OQQJUSHWYNGULW-VONWAMCDSA-N
OFVZGTATHGPOEX-ABLOACMZSA-N
ZIXZMLRXRSHDEV-UHZBFKKDSA-N
UGYHZMLKMRHTCW-KBXIAJHMSA-N
JYRIQQDZDIGAOX-KBXIAJHMSA-N
NGEWQZIDQIYUNV-BYPYZUCNSA-N
MACYIGNLDGZWAB-NSHDSACASA-N
KGDBXHCULXVTTK-GJZGRUSLSA-N
WZWGQLMLJPIYOG-PMACEKPBSA-N
Keywords:
Bacillus cereus
Emetic toxin
Chemical biology
Hydrolysis
Time course profile
Bacillus cereus is a bacterium that lives in soil. It forms heat-resistant
food poisoning. Bacillus cereus can cause two types of food poisoning: an
emetic type and a diarrhea type.1 B. cereus strains that cause the emetic
spores, so it can grow after being cooked in contaminated food, causing
⁎ Corresponding author at: Toyo College of Food Technology, 4-23-2 Minami-hanayashiki, Kawanishi-shi, Hyogo 666-0026, Japan.
E-mail address: toshihito@toshoku.ac.jp (T. Naka).
https://doi.org/10.1016/j.bmcl.2020.127050
Received 13 December 2019; Received in revised form 10 February 2020; Accepted 18 February 2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:ToshihitoNaka,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2020.127050

T. Naka, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
type of food poisoning produce cereulide.2 Cereulide is resistant to heat
even at 126 °C and does not decompose during food processing.3–5 In
addition, cereulide has been reported to be resistant to enzymes such as
peptidase and pepsin.3–10
Intens.
x10
TSB24H2_1-06_01_11045.d: EIC 1170.7000 +All MS
4
2
3
Cereulide is a K+ ionophore that damages the cell membrane po-
tential,11 resulting in expansion of mitochondria in the cell and for-
mation of vacuoles in the protoplasm of sensitive cells, causing apop-
tosis.12–14 Cereulide causes vacuolation in HEp-2 cells, which are from a
human laryngeal cancer cell line,10,15 and this effect is used to de-
termine toxicity. Cereulide has homologues and these compounds also
cause HEp-2 vacuolation.16–19
1
0
Intens.
TSB24H2_1-06_01_11045.d: EIC 1188.7000 +
3000
2000
1000
Incubation at room temperature (21
1 °C, Helsinki) is suitable
for producing cereulide, and the production at 8 °C or 40 °C is only a
few percent that at room temperature.20 Cereulide is produced in the
stationary phase that occurs after the logarithm phase, and the con-
centration forms a plateau.20–22 Therefore, a few reports exist on cer-
eulide production, but no study has reported on decompose of cereulide
after the stationary phase.
2
0
Intens.
TSB24H2_1-06_01_11045.d: EIC 787.4000 +
4
x10
The spontaneous degradation of toxins in bacterial colony is im-
portant for food hygiene analysis. Only small amounts of food poi-
soning toxins produced by bacteria can be obtained from nature.
Therefore, development of a general method for synthesizing standards
is needed. The present report describes the synthesis and structural
determination of the cereulide hydrolysates found in culture broth. The
chemical structure was determined using HPLC-TOFMS. Quantitative
analysis using synthetic compounds as standards revealed the time
course profile of cereulide production and degradation.
1.0
0.5
0.0
Intens.
TSB24H2_1-06_01_11045.d: EIC 403.2000 +
1
5
x10
1.5
1.0
0.5
0.0
B. cereus strain No. JCM 17690 (derived from R. Gilbert F4810/72)
was used in this study. Five milliliters of trypticase soy broth (TSB) was
inoculated and incubated at 35 °C with shaking at 180 rpm for 24 h. The
culture broth was extracted with methanol and analyzed using an LC-
TOFMS instrument equipped with a C18 column without solid phase
treatment. Elution involved a gradient program with 0.1% aqueous
formic acid solution and 0.1% methanolic formic acid solution as the
mobile phase.
0
10
20
30
Time [min]
Fig. 1. The LC-MS chromatogram of broth methanol extracts. The broth was
cultured in 5 mL TSB for 24 h at 35 °C and 180 rpm. Chromatograms: 1st line:
m/z 1170.7 (peak 3 represents cereulide); 2nd line: m/z 1188.7 (peak 2 re-
presents dodecadepsipeptide); 3rd line: m/z 787.4 (no peak identified); 4th line:
m/z 403.2 (peak 1 represents tetradepsipeptides).
Cereulide was confirmed by comparison with its previously syn-
thesized reference standard23 via retention time of 36.0 min (Fig. 1) and
mass spectrum (Fig. 2). Analysis of NH4+ adduct ions indicated elution
of linear dodecadepsipeptide, which is the primary hydrolysate of
cereulide at 30.3 min. In addition, the H+ adduct ion of tetra-
depsipeptide, a secondary hydrolysate, was confirmed at a retention
time of 20.4 min. Side chains of the amino acids and hydroxy acids in
the cereulide structure contain only hydrocarbons. Hydrolysis increases
the polarity and so hydrolysates elute in short times in reversed-phase
chromatography. The octadepsipeptide should elute between 20.4 and
30.3 min but was not found in the culture broth.
Cereulide (1) is a cyclic depsipeptide, cyclo (L-O-Val-L-Val-D-O-Leu-
D-Ala)3, which contains three tetradepsipeptide repeat units (Fig. 3). In
general, the ester bonds are easier to cleave than the amide bonds.
Therefore, the hydrolysates found in the culture broth were presumed
to be dodecadepsipeptides (L-O-Val-L-Val-D-O-Leu-D-Ala)3 (2) or (D-O-
Leu-D-Ala-L-O-Val-L-Val)3 (3), and tetradepsipeptides L-O-Val-L-Val-D-O-
Leu-D-Ala (4) or D-O-Leu-D-Ala-L-O-Val-L-Val (5). The compounds were
synthesized to confirm their chemical structure.
Four depsipeptides were synthesized from commercially available
amino acids, condensing agents, and protective reagents using a pre-
viously reported liquid-phase fragment condensation method. The
synthetic intermediates tetradepsipeptide (15 and 16) and octadepsi-
peptidic alcohol (18) were used as prepared (Scheme 1).19
Further extension of the depsipeptide chain gave dodecadepsipep-
tide (2). Cleavage of the benzyl group in compound 16 gave tetra-
depsipeptide 4 in 51% overall yield in 7 steps (Scheme 2). Moreover,
tetradepsipeptidic acid 15 and octadepsipeptidic alcohol 18 were
coupled using p-toluoyl chloride as an acylation reagent to produce
dodecadepsipeptide 19 in 98% yield. Removing the tert-butyldi-
methylsilyl (TBDMS) group with 70% hydrofluoric acid/pyridine and
Fig. 2. Mass spectrum of peak 3 {Cereulide, [M + H]+ 1153.6833 (Exact mass
1153.6859), [M + NH4]+ 1170.7098, [M + K]+ 1191.6396} from Fig. 1.
2

T. Naka, et al.
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O
O
O
O
O
NH
O
NH
O
O
O
O
NH
O
O
O
O
O
HN
NH
NH
O
O
O
Cereulide (1)
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
HO
HO
O
O
O
O
O
2
O
O
OH
OH
O
O
O
O
O
O
O
O
NH
NH
NH
NH
NH
NH
O
3
O
O
O
O
O
O
O
O
O
O
O
O
NH
NH
NH
NH
HO
HO
O
O
5
OH
OH
O
O
O
O
4
Fig. 3. The chemical structure of cereulide and its hydrolysates.
cleavage of the benzyl group in compound 19 gave dodecadepsipeptide
2 in 12 steps with 19% overall yield.
occurred at 30.3 min in the chromatogram with m/z 1188.7 corre-
+
sponding to the mass of the NH4 adduct ion of dodecadepsipeptide
Subsequently, dodecadepsipeptide (3) and tetradepsipeptide (5)
were synthesized via a similar reaction scheme. The hydroxy group in
dipeptide 11 was protected with the TBDMS group using tert-butyldi-
methylsilyl chloride to afford compound 21, followed by cleavage of
the benzyl ester to obtain dipeptidic acid 22 (Scheme 3). Then, di-
peptidic acid 22 and dipeptidic alcohol 8 were condensed using p-to-
luoyl chloride to afford tetradepsipeptide 23. Hydrogenation of com-
pound 23 over 10% Pd/C eliminated the TBDMS and benzyl groups
simultaneously, giving tetradepsipeptide (5) in 78% overall yield in 6
steps. A small amount of HCl remaining in the 10% Pd/C catalyst re-
moved the TBDMS group; only the benzyl group was removed by hy-
drogen reduction using 20% Pd(OH)2 catalyst to obtain tetra-
depsipeptidic acid 24 in 80% yield. Removing the TBDMS group of
compound 23 using 70% HF/py gave tetradepsipeptidic alcohol 25 in
good yield.
(Fig. 4, 1st line). No broth compound was found at the retention time
for (L-O-Val-L-Val-D-O-Leu-D-Ala)3 (2), which was 31.3 min (Fig. 4, 2nd
line). Retention time of dodecadepsipeptide (D-O-Leu-D-Ala-L-O-Val-L-
Val)3 (3) was similar to that of the natural constituent (Fig. 4, 3rd line).
The spectrum of the mixed solution of broth and compound 3 contained
only one peak that did not separate. The composition of dodecadepsi-
peptide is C64H104N6O19, and the exact mass of its H+ adduct ion is
1171.6965. The measured mass of the natural product was 1171.7003,
with a measurement error of 3.2 ppm, and the error of the synthetic
compound 3 was 0.1 ppm, which showed high agreement. Therefore,
peak 2, as Fig. 2 shows, was determined to be compound 3.
The composition of tetradepsipeptide is C19H34N2O7, and the exact
mass of its H+ adduct ion is 403.2444. The natural depsipeptide peak
eluted with a retention time of 20.5 min with m/z 403.2 (Fig. 5a, 1st
line). The tetradepsipeptide L-O-Val-L-Val-D-O-Leu-D-Ala (4) eluted at
the same retention time of 20.5 min (Fig. 5a, 2nd line). The measured
mass of the natural product was 403.2441, and that of synthetic com-
pound 4 was 403.2440, both of which were within 0.5 ppm of the
theoretical value. In the culture medium, a small peak eluted at
18.5 min (Fig. 5b, 1st line). The tetradepsipeptide D-O-Leu-D-Ala-L-O-
Val-L-Val (5) eluted at the same time of 18.5 min (Fig. 5b, 3rd line). The
peak in the culture broth was below the quantification limits. The mass
of the synthetic compound was 403.2435 and the mass of the natural
compound was 403.2414, both of which were within 3 ppm of the
theoretical value.
Coupling of tetradepsipeptidic acid 24 and tetradepsipeptidic al-
cohol 25 using p-toluoyl chloride gave octadepsipeptide 26 and re-
moving the TBDMS group in compound 26 gave octadepsipeptidic al-
cohol 27 (Scheme 4). Coupling of tetradepsipeptidic acid 24 with
octadepsipeptidic alcohol 27 under conditions identical to those used
for synthesis of compound 26 provided dodecadepsipeptide 28 in 66%
yield. Removal of the TBDMS and benzyl protecting groups gave do-
decadepsipeptide 3 in 12% overall yield in 12 steps.
The chemical structures of the natural depsipeptides were con-
firmed with reversed-phase HPLC-TOFMS using synthetic standards. A
comparison of retention times showed that natural component peak
Recovery rate was examined by adding cereulide to the culture
3

T. Naka, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Scheme 1. Synthesis of octadepsipeptidic alcohol
(18)19 Reagents and conditions: (a) EDCI (N-ethyl-N’-
(3-dimethylaminopropylcarbodiimide), HOBt (N-hy-
droxybenzotriazole), DIPEA (N,N-diisopropylethyla-
mine), CH3CN, r.t.; (b) tert-butyldimethylchlorosilane
(TBDMSCl), imidazole, N,N-dimethylformamide
(DMF), 50 °C; (c) H2, 10% Pd/C, CH3OH, r.t.; (d) p-
toluoyl chloride, Et3N, DMAP (4-(N,N-dimethyla-
mino)pyridine), toluene, r.t. ; (e) 70% HF/pyridine,
tetrahydrofuran (THF), −10 °C to r.t.
broth. The TSB broth cultured at 35 °C for 2 weeks in a shaking in-
cubator (180 rpm) was sterilized through a 0.22-μm membrane filter.
Methanol (200 μL) containing 36 μg cereulide was added to 10 mL of
culture broth, and shaken at 40 °C for 20 min. The sample solutions
were ice-cooled, and quantitative analysis was performed using LC-MS.
The unused TSB, acting as a negative control, showed a good recovery
rate of 93.3% (Table 1). The recovery rate of the untreated broth cul-
tured for 2 weeks was 1.6%, while recovery of the boiled broth was
92.4%. Since cereulide hydrolysis activity in the culture broth was
inactivated by boiling, the cereulide was thought to be hydrolyzed by
esterase secreted outside of the cells.
The hydrolysates found in the culture broth were dodecadepsipep-
tide (3) ester cleaved between Val-O-Leu, and tetradepsipeptide (4)
cleaved between Ala-O-Val. The substrate specificity of the esterase was
not high enough to recognize two points. It was expected that the
subsequent ester degradation between Val-O-Leu was slow, leaving a
large amount of tetradepsipeptide (31) in the culture broth and only
trace of tetradepsipeptide (32) were observed due to the rapid
Scheme 2. Synthesis of tetradepsipeptide (4) and dodecadepsipeptide (2). Reagents and conditions: (a) H2, 10% Pd/C, CH3OH, r.t.; (b) p-toluoyl chloride, Et3N,
DMAP, toluene, r.t.; (c) 70% HF/pyridine, THF, −10 °C to r.t.
4

T. Naka, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Scheme 3. Synthesis of tetradepsipeptide (5). Reagents and conditions: (a) TBDMSCl, imidazole, DMF, 50 °C; (b) H2, 10% Pd/C, CH3OH, r.t.; (c) p-toluoyl chloride,
Et3N, DMAP, toluene, r.t.; (d) H2, 20% Pd(OH)2/C, ethyl acetate, r.t.; (e) 70% HF/pyridine, THF, −10 °C to r.t.
degradation between Ala-O-Val. In addition, it was not found in octa-
depsipeptide and other depsipeptide, they were speculated to degrade
rapidly to tetradepsipeptide. The dipeptide that is a hydrolysate of
tetradepsipeptide was not found.
concentration profile of cereulide and hydrolysates with culture time
was produced (Fig. 6). Bacillus cereus was cultured in TSB in a shaking
incubator at 35 °C. The viable count after the lag phase indicated that
the logarithm phase was up to 12 h and then gradually decreased to the
death phase via the stationary phase. Few viable bacteria were found at
Using the synthetic compounds as standards for LC-MS analysis, a
Scheme 4. Synthesis of dodecadepsipeptide (3). Reagents and conditions: (a) p-toluoyl chloride, Et3N, DMAP, toluene, r.t.; (b) 70% HF/pyridine, THF, −10 °C to r.t.;
(c) H2, 20% Pd(OH)2/C, ethyl acetate, r.t.
5

T. Naka, et al.
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Fig. 4. The LC-MS chromatograms of dodecadepsipeptides with m/z 1188.7. 1st line: an extract of 5 mL TSB cultured for 24 h at 35 °C and 180 rpm; 2nd line:
dodecadepsipeptide (L-O-Val-L-Val-D-O-Leu-D-Ala)3 (2) 10 μg/mL methanol; 3rd line: dodecadepsipeptide (D-O-Leu-D-Ala-L-O-Val-L-Val)3 (3) 10 μg/mL methanol.
Fig. 5. The LC-MS chromatogram of tetradepsipep-
tides: (a) mass chromatogram m/z 403.2, (b) en-
larged y-axis view of the natural product in (a). 1st
line: an extract of 5 mL TSB cultured for 24 h at 35 °C
and 180 rpm; 2nd line: tetradepsipeptide L-O-Val-L-
Val-D-O-Leu-D-Ala (4) 10 μg/mL methanol; 3rd line:
tetradepsipeptide D-O-Leu-D-Ala-L-O-Val-L-Val (5) 10
μg/mL methanol.
70 h.
Table 1
Cereulide was produced in the stationary phase, which is consistent
Recovery of cereulide.
with previous reports.20–22 Cereulide decreased after a maximum con-
centration of 0.23 μM was achieved at 39 h, and traces were found after
70 h. Dodecadepsipeptide 30 was found at 14 h, with the maximum
concentration of 0.02 µM occurring at 32 h, and the compound dis-
appeared in about 60 h. This trend was similar to that of cereulide.
Tetradepsipeptide 31 increased (up to 2.31 μM at 57 h) as the cereulide
amount decreased. Although the concentration in the broth was mon-
itored, no molar equivalents between cereulide and hydrolysates were
found. Future studies will monitor the concentrations of hydroxy acids
and amino acids in the broth over time.
a
Matrix
Recovery
μg
%
b
Negative control
33.6
0.4
0.2
93.3
1.6
1.2
0.1
0.6
Culture broth for 2 weeks
0.6
0.0
Boiled culture broth for 2 weeks
33.3
92.4
a
A previously reported synthesized cereulide was used.23 Added 36 μg per
10 mL broth; values represent mean
standard deviation of 3 measurements.
b
Unused fresh TSB.
6

T. Naka, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Fig. 6. The concentration variation of cereulide and its hydrolysate depsipeptides in culture broth. A previously reported synthesized cereulide was used.23
influence the work reported in this paper.
Table 2
Vacuole response on HEp-2 cells.
Acknowledgments
Compound
Vacuole formation (nM)a
Cereulide (1)b
3.24
0.66
331
419
We thank Dr. Fujihiko Matsunaga and Dr. Sakiko Inatsu (Toyo
College of Food Technology) for technical assistance in the bacterial
cultures, and Mr. Toru Takahashi and Ms. Aya Okiura (Toyo Institute of
Food Technology) for technical assistance in recording mass spectra.
(L-O-Val-L-Val-D-O-Leu-D-Ala)3 (2)
(D-O-Leu-D-Ala-L-O-Val-L-Val)3 (3)
L-O-Val-L-Val-D-O-Leu-D-Ala (4)
D-O-Leu-D-Ala-L-O-Val-L-Val (5)
1,630
1,482
> 10,000
> 10,000
a
b
Data are presented as mean concentration
standard deviation (n = 6).
Appendix A. Supplementary data
A previously reported synthesized cereulide was used.23
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127050. These data include MOL files
and InChiKeys of the most important compounds described in this ar-
ticle.
The vacuole response of the hydrolysates was examined using the
HEp-2 cells. The two dodecadepsipeptides 2 and 3 were weaker than
cereulide but vacuole positive. The two negative tetradepsipeptides 4
and 5 appeared to lose toxicity due to their small molecular size
(Table 2).
References
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of a Bacillus cereus strain were found and their chemical structures
elucidated by synthesis of authentic samples. The depsipeptides in-
cluded the tetradepsipeptide L-O-Val-L-Val-D-O-Leu-D-Ala and dodeca-
depsipeptide (D-O-Leu-D-Ala-L-O-Val-L-Val)3. The recovery rate in the
broth with added cereulide suggested enzymatic degradation of cer-
eulide. The two depsipeptides appeared to remain in the culture broth
because of their slow ester cleavage between L-Val-D-O-Leu.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
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