Disproof of the Proposed Structures of Bradyoxetin, a Putative Bradyrhizobium japonicum Signaling Molecule, and HMCP, a Putative Ralstonia solanacearum Quorum-Sensing Molecule

Article abstract of DOI:10.1021/acs.jnatprod.0c01369

First, we revisited the reported NMR data of bradyoxetin, a putative cell density factor of Bradyrhizobium japonicum, and found some inconsistencies in the proposed structure. To elucidate the correct structure, we synthesized model oxetane compounds and confirmed that the NMR data of the synthetic compounds did not match those of the reported bradyoxetin. After reinterpreting the reported NMR data, we concluded that bradyoxetin must be chloramphenicol. Next, some derivatives of 2-hydroxy-4-((methylamino)(phenyl)methyl)cyclopentanone (HMCP), which is a putative quorum-sensing molecule of Ralstonia solanacearum, were synthesized. The NMR spectra of the synthesized compounds were completely different from those of the reported natural products. Based on theoretical studies, including the estimation of 1H and 13C NMR chemical shifts using density functional theory calculations, we confirmed the correctness of the structure of the synthesized compound. These results strongly suggest that the proposed structure of HMCP could be incorrect.

Full text of DOI:10.1021/acs.jnatprod.0c01369

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Article
Disproof of the Proposed Structures of Bradyoxetin, a Putative
Bradyrhizobium japonicum Signaling Molecule, and HMCP, a
Putative Ralstonia solanacearum Quorum-Sensing Molecule
Arata Yajima,* Ryo Katsuta, Mikaho Shimura, Ayaka Yoshihara, Tatsuo Saito, Ken Ishigami,
and Kenji Kai*
Cite This: J. Nat. Prod. 2021, 84, 495−502
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ABSTRACT: First, we revisited the reported NMR data of bradyoxetin,
a putative cell density factor of Bradyrhizobium japonicum, and found
some inconsistencies in the proposed structure. To elucidate the correct
structure, we synthesized model oxetane compounds and confirmed that
the NMR data of the synthetic compounds did not match those of the
reported bradyoxetin. After reinterpreting the reported NMR data, we
concluded that bradyoxetin must be chloramphenicol. Next, some
derivatives of 2-hydroxy-4-((methylamino)(phenyl)methyl)-
cyclopentanone (HMCP), which is a putative quorum-sensing molecule
of Ralstonia solanacearum, were synthesized. The NMR spectra of the
synthesized compounds were completely different from those of the reported natural products. Based on theoretical studies,
1
including the estimation of H and 13C NMR chemical shifts using density functional theory calculations, we confirmed the
correctness of the structure of the synthesized compound. These results strongly suggest that the proposed structure of HMCP could
be incorrect.
acteria, fungi, and other microorganisms communicate
Bwith each other using small molecules that play an
important role in cell-to-cell communication. Among the life
regulatory systems, information transfer between individual
microorganisms is one of the most important mechanisms for
promoting the survival of a species and its interactions with
other organisms. This built-up information network relies
mainly on the chemical signals between individual cells of the
same species. This information network is not limited to the
same species, but may be extended to different species or even
different kingdoms. One major class of bacterial signaling
molecules is known as the autoinducers or quorum-sensing
(QS) pheromones.1 Notably, QS is a cell density-responsive
transcriptional system that regulates a variety of life events,
such as pathogenicity control, biofilm formation, pigment and
antibiotic biosynthesis, and interbacterial competence acquis-
ition. N-Acylhomoserine lactones (AHLs, Figure 1), which
regulate a wide variety of bacterial activities, are known as one
of the major classes of QS signaling molecules, and most
Gram-negative bacteria produce AHL(s) to control an internal
QS circuit.2 As represented by AHLs, microbial signaling
molecules are often relatively simple in structure. This may be
because it is not evolutionarily advantageous to have a complex
biosynthetic system for a signaling molecule. Although they are
likely to be simple compounds, elucidating the structure of
microbial signaling molecules is often difficult because their
production levels are extremely low and several physical
properties, such as NMR spectra, may not be adequately
measured.3 Consequently, research on the identification of
microbial signaling molecules has not been sufficiently
advanced, and it is likely that there are still undiscovered
types of signaling molecules. However, structurally orphaned
(rare and very unusual) compounds, which are interesting
from the perspective of natural product chemistry, have been
reported. Herein, we discuss the structural disproof of two
structurally orphaned microbial signaling molecules, bradyox-
etin and 2-hydroxy-4-((methylamino)(phenyl)methyl)-
cyclopentanone (HMCP).
Bradyoxetin, a Putative Signaling Molecule of
Bradyrhizobium japonicum. Rhizobia and legumes form
nitrogen-fixing nodules in the roots and live together in a very
specific interaction between the bacteria and the plant. The
symbiosis involves several steps of exchanging chemical signals,
such as flavonoids from the plant and a Nod factor from the
bacteria with each other; the various genes are differentially
expressed in different ways.4 Differentiated rhizobia in the
Received: December 21, 2020
Published: January 29, 2021
© 2021 American Chemical Society and
American Society of Pharmacognosy
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1
Figure 2. Partial H NMR data of known phenyloxetane derivatives.
on the oxetane ring should show a coupling constant of
approximately 7 Hz, which could be mistaken for vicinal
coupling that would be caused by the strain of the four-
membered ring, whereas the coupling constant of those of the
bradyoxetin was reported to be 11.5 Hz. Because the NMR
spectra of bradyoxetin were recorded in CD3OD/D2O, the
possibility that these differences are caused by the use of
different solvents cannot be excluded. Furthermore, no
detailed NMR data of the trans-isomer of the phenyloxetane
derivative possessing an amino group at C-3, e.g., trans-5, were
available. To resolve these questions and verify the validity of
the structural determination of bradyoxetin, we planned to
synthesize analogues for NMR analysis.
Figure 1. Structures of AHLs and the proposed structure of
bradyoxetin.
nodules, referred to as bacteroids, reduce atmospheric nitrogen
to ammonium ions that can be available to the plant, and these
compounds are exchanged for energy sources from the plant.5
The AHLs produced by rhizobia are considered the most
important QS molecules involved in the symbiotic program of
the rhizobia. Both QS mechanisms and molecules have been
identified in many rhizobial species.6 For example, Rhizobium
leguminosarum produces AHL 1 (Figure 1) as a self-growth
inhibition signaling molecule;6a furthermore, we have reported
that the absolute configuration at the hydroxy-bearing C-3
plays an important role in the biological activity.7
Scheme 1 shows the synthesis of the phenyloxetane
derivatives cis-8 and trans-8. The known enamine 7 was
Scheme 1. Synthesis of Phenyloxetane (8)
The QS molecules discovered from rhizobia include some
structurally unique AHLs, such as cinnamoyl-HSL (2)6g and p-
coumaroyl-HSL (3).6i These unique aromatic classes of AHLs
have not been identified in any other bacteria. In 2001, Loh et
al. isolated a cell density factor (CDF), bradyoxetin (4), which
acts as an extracellular signaling molecule and accumulates as a
function of culture density from the rhizobia Bradyrhizobium
japonicum.8 It has been reported that bradyoxetin induces
NolA, which leads to nod gene suppression. The bradyoxetin
structure was proposed by extensive analyses of its NMR and
MS spectra. The proposed structure is unique and contains
two symmetrical oxetane rings and a free imine group.
However, there is no structurally similar natural product with
the features of bradyoxetin. Therefore, we comprehensively
examined the description given in Loh’s work,8 and several
questions arose. First, we thought that it was necessary to
verify whether the N-nonsubstituted imine group, which was
difficult to isolate because of its high reactivity and
susceptibility to hydrolysis, could exist stably as a natural
product. Next, some unusual points were found in the NMR
prepared from acryloyl chloride10 and was subjected to
photocycloaddition with benzaldehyde according to Bach’s
condition9 to afford cis-8 and trans-8 in a moderate yield.
However, the deprotection of the carboxybenzyl (Cbz) group
of 8 was unsuccessful, and we obtained undesired ring-opened
compounds under various reaction conditions. Although there
are several phenyloxetane compounds with nitrogen function-
ality at C-3 of its oxetane ring, no compound having a free
amino group (−NH2) has been reported. This fact might
reflect the high reactivity of oxetane, which makes the removal
of various substituents difficult. With the phenyloxetane
derivatives in hand, 1D and 2D NMR analyses of cis-8 and
trans-8 were conducted. Consequently, the NMR data for both
cis- and trans-isomers were similar to those of previously
1
data. For instance, in the H NMR spectra of known oxetane
compounds such as compounds 5 and 6, the chemical shifts of
the methylene protons α to the oxygen atom are reported to be
approximately 4−5 ppm,9 whereas the bradyoxetin signals are
reported to be 3.61 and 3.82 ppm, which is similar to general
ether derivatives (Figure 2). In addition, the geminal protons
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reported compounds. Even considering that the synthetic
products were protected by a Cbz group, the signals at the
oxetane site of the two products were significantly different
from those of the reported bradyoxetin, suggesting that the
proposed structure might be incorrect.
Next, we revisited the reported NMR data of bradyoxetin
and attempted to speculate on the correct structure. First, the
possibility that the nitrogen and oxygen atoms are reversed,
i.e., bradyoxetin is an azetidine derivative, was investigated.
However, the reported NMR data of similar azetidine
compounds are entirely different.11 Focusing on the reported
13C NMR spectrum of bradyoxetin, it is possible to find the
following structural information: bradyoxetin has a p-
disubstituted-phenyl group, a carbonyl-like carbon, and three
sequential carbon atoms possessing oxygen or nitrogen
substituents (9; Figure 3 and Table 1). Assuming that one
be explained unless we assumed that there was a nitro group as
X. Following this assumption, it is possible that the 166.4 ppm
signal can be a carbonyl carbon and an oxygen or nitrogen in
the side chain can be acylated. Although no proton nor carbon
1
signal corresponding to the acyl chain was recorded in the H
and 13C NMR spectra of bradyoxetin, some of its traces could
be found in the reported HSQC and HMBC spectra. For
instance, they observed a cross-peak between approximately
1
6.3 ppm of H and 66 ppm of 13C signals in the HSQC
spectrum, even though these signals were not recorded in the
1D NMR spectra. This phenomenon leads us to hypothesize
that the carbon atom of the acyl chain can be deuterated
because it is well known that the reduction of the signal
intensity by deuterium quadrupole relaxation or signal
broadening due to residual C−D coupling is often observed
in the 13C NMR spectrum of a deuterated compound.12 In the
process of estimating the bradyoxetin structure that satisfies
(1) a p-disubstituted-phenyl moiety, (2) a nitro group and a
sequential oxygen/nitrogen trisubstituted propyl chain, and
(3) an acyl group with deuterium-exchangeable proton(s), we
realize that chloramphenicol (10), a common antibiotic, can be
a candidate for that compound.
To confirm our hypothesis, we conducted an NMR study
1
using commercial chloramphenicol and found that the H and
13C NMR spectra were in good agreement with those of the
reported data for bradyoxetin (Table 1). The slight chemical
shift discrepancies might be caused by the concentration of the
sample or the pH of the solution. This phenomenon is often
observed when MeOH, a highly polar solvent, is used as the
solvent for NMR.13 Although signals H-11 (6.21 ppm) and C-
11 (67.1 ppm) were observed in the initial measurements, the
signal intensities of these peaks were reduced after allowing the
sample solution to stand for 2 weeks to promote the H−D
exchange at C-11 (Figures S1 and S2). Focusing on the MS
data, Loh et al. deduced the molecular formula of bradyoxetin
(C19H21N3O2) based on the observed peak at 324.0 ([M +
H]+, theoretical value 324.2). However, the MS peaks assumed
from the molecular formula of chloramphenicol
(C11H12Cl2N2O5) are 322.0, 324.0, and 326.0 ([M]+), the
major peaks, which are deduced from the isotopes of chlorine
atoms. The estimated MS data for chloramphenicol are
considered to be consistent with the reported data, although
the MS spectrum, including high-resolution MS data for
bradyoxetin, is not shown and therefore not comparable. It is
unclear why the results of the reported elemental analysis of
bradyoxetin do not agree with the calculated chloramphenicol
values. From our NMR studies and MS data, we concluded
that the compound reported as bradyoxetin must be
chloramphenicol. It should be noted that Loh et al. applied
chloramphenicol (50 μg/mL) to the culture medium of B.
japonicum, presumably to inhibit the growth of other
microorganisms.
Figure 3. Partial structure 9 based on bradyoxetin NMR data and the
structure of chloramphenicol (10).
Table 1. NMR Data of Chloramphenicol and Reported Data
of Bradyoxetin
δC
δH, mult. (J, Hz)
a
b
c
b
c
position chloramphenicol reported chloramphenicol
reported
1
2
71.5
58.2
71.3
58.5
5.17, m (2.8)
4.17, ddd, (2.8,
6.6, 7.2)
5.16 (2.9)
4.14 (2.9,
6.6, −)
3
61.9
62.2
3.64, dd (6.9,
11.4)
3.61 (7.2,
11.5)
3.83, dd (6.5,
11.4)
3.82 (6.6,
11.5)
4
150.5
128.2
124.4
148.2
167.2
67.1
151.5
128.2
124.0
148.5
166.4
5, 9
6, 8
7
10
11
7.63, d (8.7)
8.20, d (8.7)
7.65 (8.8)
8.18 (8.8)
HMCP, a Putative Ralstonia solanacearum Quorum-
Sensing Molecule. R. solanacearum is a Gram-negative β-
proteobacterium that causes a destructive disease called
“bacterial wilt” in more than 200 plant species over a broad
geographical range.14 This pathogen attacks a wide variety of
important crops, such as tomato, potato, and tobacco, as well
as other weeds, shrubs, and trees by invading the xylem vessels
of the plant roots. After the invasion, the bacterium diffuses
into the stem, where it grows vigorously and rapidly produces
extracellular polysaccharide (EPS), which blocks vascular
tissues and prevents water flow, thus blighting the invaded
6.21, s
a
b
According to reported bradyoxetin numbering. Recorded at 400
MHz (1H NMR) and 100 MHz (13C NMR) in CD3OD/D2O = 1:1.
c
Recorded at 500 MHz (1H NMR) and 125 MHz (13C NMR) in
CD3OD/D2O = 1:1.
of the substituents in the benzene ring was the carbon chain,
there must be some substituent attached to the other, which
Loh et al. assigned to the carbon of the imine. However, we
realized that the slightly shielded signal at 124.0 ppm could not
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plant without xanthochromia. The pathogen also produces
plant cell-wall-damaging enzymes, such as endoglucanase and
polygalacturonase, as well as ralfranones as secondary
metabolites, both of which are required for full bacterial
virulence.15 The production of these virulence and pathoge-
nicity molecules is controlled by an extensive network of
interactive signal transduction pathways, wherein PhcA, a
LysR-type transcriptional regulator, plays an important role. In
this system, the PhcA activity is regulated by the PhcS/PhcR
two-component system, and its key molecules have been
proven to be 3-hydroxylated-fatty acid methyl esters acting as
QS signaling molecules. In 1997, Denny et al. showed that
methyl 3-hydroxypalmitate (3-OH PAME, 11) was a phc QS
signaling molecule that triggers the expression of an EPS
biosynthetic gene in R. solanacearum AW1.16 Although 3-OH
PAME was the first QS signaling molecule of an endogenous
fatty acid derivative, another fatty acid derivative, 3-
hydroxymyristate (3-OH MAME, 12), was found in other R.
solanacearum strains, such as OE1-1, as the QS signaling
molecule in 2015.17
Figure 5 summarizes the synthesis of HMCP as described by
Umesha et al.19 Benzylmethylamine (14) was treated with n-
Figure 5. Synthetic procedure and 1H NMR data of HMCP reported
by Umesha. *It was not specified which of these two signals each
corresponded to.
BuLi at low temperatures, and the obtained intermediate was
then reacted with 2-hydroxy-4-methylcyclopentanone (15) to
obtain the desired HMCP as a colorless solid. Although the
synthetic procedure was not reported in detail, we noticed that
there were some suspicious features with this method. First,
the corresponding lithium amide should be formed first when
benzylmethylamine is treated with n-BuLi. It would be very
difficult to form a benzyl anion even if an excess amount of n-
BuLi is employed. Second, assuming that the benzyl anion can
be formed, it is impossible for the employed electrophile (15)
to react with the anion at its methyl group. Furthermore, if the
intended carbon−carbon bond formation occurs, the number
of carbon atoms in the product would not match those of the
proposed HMCP structure.
Figure 4. Structures of quorum-sensing molecules of R. solanacearum
and proposed structure for HMCP.
1
R. solanacearum also employs some AHLs as QS signaling
molecules.18 Although only two classes, fatty acid derivatives
and AHLs, had been identified as QS signaling molecules in R.
solanacearum, Umesha et al. found a new class in 2016.19
Spectroscopic analyses, such as 1H NMR and MS, revealed the
structure of the compound as HMCP (13). They claimed that
the structure of the compound was confirmed by chemical
synthesis. This is a surprising discovery in the history of QS
science because the reported HMCP structure does not share
any common structural features with that of the known QS
molecules of any microorganisms. The reported HMCP
structure has unique arrangements as a secondary amino
group at the benzylic position and α-hydroxy ketone in its five-
membered ring system. It is well known that some QS
signaling molecules bearing an amino group derived from an
amino acid (e.g., ComX pheromone20) or anthranilic acid (e.g.,
PQS21) or bearing five-membered ring systems, such as lactone
(e.g., AHL) or thiazole (e.g., IQS22), exist. However, the
reported HMCP structure is prominent from the perspective of
its structure. Furthermore, although HMCP has three
stereogenic centers, leading to eight possible stereoisomers,
no information about its relative or absolute configuration has
been reported. Owing to its high biological and chemical
importance, we were interested in synthesizing HMCP.
Next, we carefully checked the reported H NMR data of
HMCP. The reported assignments of each resonance are
summarized in Figure 5. Again, we found many unusual
descriptions in their claims. Therefore, we performed a
simulation on the 1H NMR spectrum of the reported
HMCP structure using commercial software (Table S1). We
found many differences between the reported and predicted
assignments. Unfortunately, because the 13C NMR spectrum of
the natural product has not been reported, it is impossible to
verify the data. Although the prediction of the chemical shifts
using computational methods is not always accurate, the
obtained shifts appeared to be highly reasonable. On the basis
of these results, we speculated that the reported structure of
the natural product might be incorrect. Thus, we decided to
synthesize the sample with the proposed HMCP structure to
conduct a direct comparison with their NMR spectra.
Scheme 2 summarizes the synthesis of the HMCP
derivatives. First, the commercially available cyclopent-3-ene
carboxylic acid was converted into ketone 17 via the known
amide 16.23 The reductive amination of the resulting keto
group of 17 with methylamine yielded the secondary amine 18.
Prior to the introduction of some oxygen functionalities into
the cyclopentenyl moiety of 18, it was necessary to protect the
amino group. We first selected an acetyl group (19) because
the Ac group could be tolerant toward various reaction
conditions. In the NMR spectra of 19, the rotational isomers
Before starting our synthetic study of HMCP, we carefully
checked the synthetic procedure reported by Umesha et al.
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a
synthesized 23a and 23b, although it was not possible to
determine which stereoisomers they were. Because the acetyl
groups of 23a and 23b could not be removed under standard
conditions, we could not obtain HMCP. Next, we employed
Fmoc, Alloc, and Teoc groups as the protecting groups of the
amino group of 18 because the protecting groups could be
removed under various reaction conditions. We synthesized
the corresponding N-protected HMCPs using the same
procedures as described above. Unfortunately, however, all
attempts to remove the protecting groups resulted in the
decomposition of the product under all examined reaction
conditions. These results indicate that the proposed structure
of HMCP (13) would be highly unstable toward basic, acidic,
or even neutral transition metal-catalyzed reaction conditions.
We assumed that the free amino group of HMCP may be
condensed with the acyloin moiety via Maillard-like reactions
to obtain a complex mixture.
Scheme 2. Synthesis of N-Acetyl HMCP
Although we could not prepare the pure sample of HMCP,
some N-protected HMCPs were obtained. Essentially, there
was no difference among the NMR spectra, except for the
signals originating from the substituted groups on the nitrogen
atom. This finding indicates that it is possible to compare the
NMR spectra of N-protected HMCPs with those of the
reported HMCP directly. To simplify the discussion, the
simplest compounds, N-acetyl HMCP (23a and 23b), were
selected. The observed NMR spectra were completely different
from those of the reported HMCP data and were in good
agreement with the expected values (Tables S2 and S3), except
for those of an additional acetyl group. However, it was still
possible that the differences might be attributed to the
difference in the configuration of the hydroxy-bearing C-10
because we prepared two out of four stereoisomers. To exclude
this possibility, we conducted a more reliable estimation of the
NMR spectra of the stereoisomers of N-acetyl HMCP by
means of density functional theory (DFT) calculations.24 The
observed NMR data of our synthesized 23a and 23b and
theoretical NMR data of four stereoisomers are shown in
Tables S2 and S3. The observed data are in good agreement
with the estimated data, supporting the correctness of the
structure of our synthetic compounds. Because there were
significant but very small differences between the estimated
data of the two stereoisomers, we concluded that the
a
Reagents and conditions: (a) MeNH2, Ti(Oi-Pr)4, EtOH, NaBH4, 0
°C (96%); (b) AcCl, Et3N, THF, 0 °C (90%); (c) OsO4, NMO, t-
BuOH, acetone, H2O (91%); (d) NaH, TESCl, THF, 0 °C (28% for
21a, 39% for 21b); (e) Dess-Martin periodinane, CH2Cl2, 0 °C (89%
for 22a, 89% for 22b); (f) HF-pyridine, MeCN, 0 °C (40% for 23a,
89% for 23b).
(approximately 3:1 in DMSO-d6) were observed, and the ratio
of the isomers was changed to approximately 2:1 at 90 °C. The
double bond of 19 was oxidized with OsO4 to afford the
corresponding diol 20. The facial selectivity of this oxidation
1
was estimated to be >97:3 by the H NMR spectrum, and the
1
disagreement between the H NMR spectrum of our sample
isomers were inseparable. The relative configuration of the
five-membered ring of the major isomer of 20 was deduced by
a NOESY experiment. It should be noted that compound 20 is
not a diastereomeric mixture relative to the cyclopentane ring.
The monoprotection of the hydroxy group of 20 as triethylsilyl
(TES) ether yielded 21a and 21b. Although the four
stereoisomers could be produced in this step (see Figure
S3), only two compounds were separated. There were two
possibilities: (1) two of the four isomers were selectively
produced, and (2) the separated 21a and 21b were both
diastereomeric mixtures that were indistinguishable by NMR
analysis. In any case, it was impossible to determine the relative
configuration of each compound. Finally, the Dess−Martin
oxidation of the remaining secondary alcohol of 21a and 21b
was followed by the removal of the TES group with HF-
pyridine to produce N-acetyl HMCP 23a and 23b,
respectively. Even if the relative configurations of compounds
21a and 21b were unknown, this subsequent two-step
conversion reaction narrows the number of possible structures
of N-acetyl HMCP to two (Figure S3). The 1H and 13C NMR
spectra provided good support for the structures of the
and that of the reported HMCP should not be caused by the
difference in their configuration. There are two significant
differences between the reported data of HMCP and our
synthetic compounds: (1) HMCP appears to have multiple
substituents on the benzene ring, whereas our compounds give
typical monosubstituted-benzene signals, and (2) the chemical
shifts and coupling patterns of the protons on the cyclopentane
ring do not match at all. Thus, we concluded that the reported
structure of the natural product is incorrect. Although we
conducted several bioassays using our synthesized N-acetyl
HMCP, no activity was observed against R. solanacearum.
We focused on two structurally unique microbial signaling
molecules, bradyoxetin and HMCP, and disproved their
assigned structures. First, we synthesized the model phenyl-
oxetane derivatives and found that the 1H NMR spectra of the
compounds were completely different from those in the data of
the reported natural bradyoxetin. Thus, we revisited the
reported NMR data of bradyoxetin. On the basis of NMR and
MS studies, we concluded that the compound reported as
bradyoxetin must be chloramphenicol. It was reported that
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Benzyl 2-Phenyloxetan-3-ylcarbamate (cis-8 and trans-8).
In a quartz tube, benzaldehyde (152 μL, 1.50 mmol) and 7 (532 mg,
3.00 mmol) were dissolved in MeCN (10 mL). This mixture was
irradiated for 19 h (λ = 365 nm; light source: Sen Lights Co.,
HL100CH-4). After concentration under reduced pressure, the
residue was chromatographed on SiO2 (hexane/EtOAc = 20:1) to
give a mixture of two isomers (5:1), cis-8 and trans-8 (167 mg, 39%).
Analytical samples were obtained by further chromatography
(CH2Cl2/acetone = 100:1).
MerR family proteins, such as NolA, activate their target genes
in the presence of some toxic compounds; thus, chloramphe-
nicol as an antibiotic may cause Loh’s observations. They
reported that the production of bradyoxetin was reduced under
Fe3+ replete culture conditions. This might be rationalized by
the chelation ability of chloramphenicol caused by two free
hydroxy groups and an amide nitrogen to Fe3+, which
prevented its extraction into the EtOAc phase. Even if the
observation of chloramphenicol was the result of an
experimental application from a reagent bottle, its signaling
molecule-like activity remains intriguing. Chloramphenicol was
isolated from Streptomyces sp. in 1948 by the Parke-Davis
group. Because there is no report of chloramphenicol being
identified from other microorganisms, Loh’s findings can
facilitate more important insights in microbiology if the
isolated chloramphenicol is a product of B. japonicum. Second,
we synthesized some HMCP derivatives, such as N-acetyl
HMCP, and found that the 1H NMR spectra of the
compounds were completely different from those reported
for natural HMCP. We carefully assessed the structural
adequacy of our synthetic compounds through computational
estimations of the NMR chemical shifts of the N-acetyl HMCP
stereoisomers. The significant difference in the NMR spectra
between the reported HMCP and our synthetic sample, as well
as theoretical data, strongly suggests that the assigned structure
of the natural product is incorrect, and the reported HMCP
synthesis procedure must be reconsidered. There is an urgent
need to reisolate the natural product from R. solanacearum, and
the correct structure of the active compound should be
determined using extensive NMR studies that include 13C
NMR and 2D experiments.
Determining the structure of natural organic compounds is
often a challenging task, and errors occasionally occur.25 The
causes might be a simple error in the interpretation of the
spectra, insufficient data due to the limited amount of
compound obtained, and, rarely, scientific fraud. The
elucidation of the structure of a microbial signaling molecule
is particularly difficult because its production is extremely low
and its physical properties, such as an NMR spectrum, may not
be sufficiently measured.26 If the structure of a compound
deduced by spectroscopic analyses is so unique that it has
never been found before, it would be a very exciting and
important discovery, but if the structure is rare and very
unusual, there is no doubt that it is necessary to collect all
sufficient data and reconsider.
Properties of cis-8: colorless oil: IR (film) νmax 3291, 1714 cm−1;
1H NMR (CD3OD, 400 MHz) δ 7.37−7.23 (8H, m), 7.08 (2H, m),
5.91 (1H, m), 5.03−5.00 (2H, m), 4.95 (1H, d, J = 12.8 Hz), 4.81
(1H, d, J = 12.8 Hz), 4.56 (1H, m); 13C NMR (CDCl3, 100 MHz) δ
155.2, 136.9, 136.0, 128.7, 128.5, 128.2, 128.0, 125.5, 86.5, 76.9, 66.8,
48.7; HRDARTMS m/z 284.1280 [M + H]+ (calcd for C17H18O3,
284.1281).
Properties of trans-8: colorless oil: IR (film) νmax 3314, 1683
1
cm−1; H NMR (CD3OD, 400 MHz) δ 7.44−7.25 (10H, m), 5.54
(1H, d, J = 5.5 Hz), 5.06 (1H, d. J = 12.3 Hz), 5.04 (1H, d. J = 12.3
Hz), 4.75 (1H, m), 4.62−4.55 (2H, m); 13C NMR (CDCl3, 100
MHz) δ 155.1, 139.8, 128.6, 128.4, 128.3, 128.2, 125.5, 90.3, 74.4,
67.1, 53.5; HRDARTMS m/z 284.1285 [M + H]+ (calcd for
C17H18O3, 284.1281).
Cyclopent-3-en-1-yl(phenyl)methanone (17). To a solution of
16 (5.00 g, 32.2 mmol) in THF (70 mL) was added dropwise
phenyllithium (1.6 M in n-dibutyl ether, 22.4 mL, 35.4 mmol) at −78
°C under Ar. After stirring for 1 h, the mixture was warmed to room
temperature (rt) and stirred for 1 h. The reaction mixture was
quenched with saturated aqueous NH4Cl and extracted with EtOAc.
The organic layer was washed with saturated aqueous NaHCO3, H2O,
and brine and dried with Na2SO4. After concentrating under reduced
pressure, the residue was chromatographed on SiO2 (hexane/EtOAc
= 10:1) to give 17 (4.97 g, 90%) as a colorless oil: IR (film) νmax
1756, 1697 cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.98 (2H, m), 7.56
(1H, m), 7.47 (2H, m), 5.69 (2H, s), 4.09 (1H, m), 2.76 (4H, m);
13C NMR (CDCl3, 100 MHz) δ 201.4, 136.4, 132.8, 128.9, 128.6,
44.1, 36.2; HRESIMS m/z 173.0947 [M + H]+ (calcd for C12H13O,
173.0961).
N-(Cyclopent-3-en-1-yl(phenyl)methyl)-N-methylacetamide
(19). To a solution of 17 (5.83 g, 33.9 mmol) in methylamine (2.0 M
in EtOH, 67.7 mL, 135 mmol) was added dropwise Ti(OiPr)4 (15.1
mL, 50.8 mmol) under Ar, and the solution was stirred for 5 h. After
cooling to 0 °C, NaBH4 (1.92 g, 50.8 mmol) was added, and the
mixture was stirred for 2 h. The reaction mixture was quenched with
H2O. The resulting inorganic precipitate was filtered off and was
washed with diethyl ether. The combined organic layer was washed
with 2 M HCl(aq) and 10% NaOH(aq) and dried with K2CO3.
Concentration under reduced pressure gave crude 18 (6.12 g, 96%) as
a brown oil. This compound was then used in the next step without
further purification. To a solution of crude 18 (1.28 g, 4.39 mmol) in
THF (6.4 mL) was added triethylamine (2.86 mL, 2.05 mmol) at 0
°C under Ar. After stirring for 30 min, acetyl chloride (732 μL, 10.3
mmol) was added. After stirring for 3 h, the reaction mixture was
quenched with saturated aqueous NH4Cl. The aqueous layer was
extracted with EtOAc, and the combined organic layer was washed
with H2O and brine and dried with Na2SO4. After concentration
under reduced pressure, the residue was chromatographed on SiO2
(hexane/EtOAc = 7:3) to give 19 (1.42 g, 90%) as a mixture of
EXPERIMENTAL SECTION
■
General Experimental Procedures. The IR spectra were
measured using a Jasco IR-4100 spectrometer. The H NMR spectra
1
were recorded on a Jeol JNM ECX400 (400 MHz) spectrometer
using CDCl3 at δ 7.26, CD3OD at δ 3.30, or TMS at δ 0.00 as the
internal standards. The 13C NMR spectra were recorded on a Jeol
JNM ECX400 (100 MHz) spectrometer using CDCl3 at δ 77.0 and
CD3OD at δ 49.0 as the internal standards. High-resolution MS data
were recorded on an Agilent 6530 Accurate-Mass Q-TOF (ESI) and
Jeol JMS-T100LP (DART) spectrometers. Column chromatography
was performed with Wakogel-C200 silica gel. Conformational
analyses, structural optimizations, and chemical shift calculations
were performed using Spartan’14 (version 1.1.8, Wavefunction, Inc.).
All of the air- or moisture-sensitive reactions were conducted under an
argon atmosphere. The dried solvents used for the reactions (THF
and CH2Cl2) were purchased from FUJIFILM Wako Pure Chemical
Corporation.
1
rotational isomers (3:1): colorless oil; IR (film) νmax 1630 cm−1; H
NMR (DMSO-d6, 400 MHz) δ 7.46−7.24 (5H, m), 5.77−5.62 (2H,
m), 5.53 (0.75H, d, J = 11.9 Hz), 4.69 (0.25H, d, J = 11.4 Hz), 3.26−
2.98 (1H, m), 2.68 (2.25H, s), 2.57 (0.75H, s), 2.46−2.26 (2H, m),
2.20 (0.75H, s), 2.15−2.01 (1H, m), 1.99 (2.25H, s), 1.90−1.72 (1H,
m); 13C NMR (CDCl3, 100 MHz) δ 170.8, 170.4, 139.6, 138.9, 129.7,
129.6, 129.5, 129.3, 128.64, 128.59, 128.4, 127.8, 127.4, 65.1, 59.4,
37.54, 37.46, 36.4, 35.9, 35.6, 30.0, 27.3, 22.31, 22.26; HRESIMS m/z
230.1535 [M + H]+ (calcd for C15H20NO, 230.1539).
N-((3,4-Dihydroxycyclopentyl)(phenyl)methyl)-N-methyl-
acetamide (20). To a solution of 19 (1.00 g, 1.05 μmol) in t-BuOH/
acetone/H2O (5:1:1, 14 mL) were added N-methylmorpholine-N-
500
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J. Nat. Prod. 2021, 84, 495−502

Journal of Natural Products
pubs.acs.org/jnp
Article
oxide (1.34 g, 11.4 μmol) and OsO4 (1% in t-BuOH, 1 mL), and the
solution was stirred for 12 h. The reaction mixture was filtered
through a Celite pad, and the filtrate was concentrated under reduced
pressure. The residue was diluted with EtOAc, and the resulting
solution was dried with Na2SO4. After concentration under reduced
pressure, the residue was chromatographed on SiO2 (hexane/EtOAc
= 1:1, EtOAc and MeOH) to give 20 (1.05 g, 91%) as a mixture of
rotational isomers (9:1). Properties of the major isomer: colorless oil;
N-Acetyl HMCP (23a and 23b). To a solution of 22a (20 mg,
53.3 μmol) in CH3CN (2 mL) was added HF-pyridine complex (150
μL, 207 μmol) at 0 °C. After stirring for 2 h, the mixture was
concentrated under reduced pressure. The residue was extracted with
diethyl ether. The organic layer was washed with saturated aqueous
NaHCO3, H2O, and brine and dried with Na2SO4. After
concentration under reduced pressure, the residue was chromato-
graphed on SiO2 (hexane/EtOAc = 1:1, CHCl3, CHCl3/MeOH =
8:1) to give 23a (5.6 mg, 40%) as a yellow oil. 23b (161 mg, 89%)
was synthesized from 22b (182 mg) in the same manner.
Properties of 23a: colorless oil; IR (film) νmax 3399, 1750, 1638
cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.37−7.28 (5H, m), 5.88 (1H,
d, J = 12.4 Hz), 4.41 (1H, dd, J = 8.3, 8.3 Hz), 3.07 (1H, m), 2.70
(3H, s), 2.49 (1H, dd, J = 8.3, 19.2 Hz), 2.19 (1H, m), 2.13 (3H, s),
2.06 (1H, m), 1.91 (1H, ddd, J = 8.3, 8.3, 12.3 Hz); 13C NMR
(CDCl3, 100 MHz) δ 216.3, 171.2, 137.6, 128.8, 128.5, 128.0, 73.1,
58.2, 39.7, 32.6, 30.1, 30.0, 22.1; HRESIMS m/z 262.1453 [M + H]+
(calcd for C15H20NO3, 262.1438).
1
IR (film) νmax 3344, 1613 cm−1; H NMR (CDCl3, 400 MHz) δ
7.28−7.15 (5H, m), 5.55 (1H, d, J = 12.4 Hz), 4.09 (2H, m), 3.42
(2H, br), 3.02 (1H, m), 2.57 (3H, s), 1.99 (3H, s), 1.78 (2H, m), 1.58
(1H, m), 1.36 (1H, m); 13C NMR (CDCl3, 100 MHz) δ 171.2, 138.6,
128.5, 128.4, 127.5, 73.7, 73.4, 60.0, 35.9, 34.5, 33.7, 30.1, 22.1;
HRESIMS m/z 264.1596 [M + H]+ (calcd for C15H22NO3,
264.1594).
N-((3-Hydroxy-4-((triethylsilyl)oxy)cyclopentyl)(phenyl)-
methyl)-N-methylacetamide (21a and 21b). To a solution of 20
(405 mg, 1.54 mmol) in THF (12 mL) was added sodium hydride
(60%, 111 mg, 2.78 mmol) at 0 °C under Ar. After stirring for 30 min,
chlorotriethylsilane (271 μL, 1.54 mmol) was added, and the mixture
was stirred for 5 h. The reaction mixture was quenched with saturated
aqueous NH4Cl, and the aqueous layer was extracted with CH2Cl2.
The organic layer was washed with H2O and brine and dried with
Na2SO4. After concentration under reduced pressure, the residue was
chromatographed on SiO2 (hexane/EtOAc = 10:1 to 1:1) to give 21a
(167 mg, 28%) and 21b (158 mg, 39%).
Less polar isomer 21a: colorless oil; IR (film) νmax 3460, 1644
cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.37−7.25 (5H, m), 5.65 (1H,
d, J = 11.9 Hz), 4.24 (1H, m), 4.00 (1H, m), 3.08 (1H, m), 2.75 (1H,
br), 2.67 (3H, s), 2.08 (3H, s), 1.86 (1H, m), 1.71 (1H, dd, J = 6.9,
7.8 Hz), 1.63 (1H, m), 1.30 (1H, m), 0.97 (9H, m), 0.64 (6H, m);
13C NMR (CDCl3, 100 MHz) δ 170.8, 138.8, 128.6, 128.4, 127.5,
74.1, 73.9, 59.8, 36.5, 35.2, 34.1, 30.0, 22.3, 6.7, 4.7; HRDARTMS m/
z 378.2466 [M + H]+ (calcd for C21H36NO3Si, 378.2459).
More polar isomer 21b: colorless oil; IR (film) νmax 3460, 1633
cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.35−7.24 (5H, m), 5.68 (1H,
d, J = 11.9 Hz), 4.20 (1H, m), 4.00 (1H, m), 3.10 (1H, m), 2.77 (1H,
br), 2.63 (3H, s), 2.07 (3H, s), 1.82 (2H, m), 1.50 (2H, m), 0.92 (9H,
t, J = 7.8 Hz), 0.56 (6H, q, J = 7.8 Hz); 13C NMR (CDCl3, 100 MHz)
δ 170.9, 139.2, 128.5, 128.4, 127.5, 74.4, 73.7, 59.8, 36.3, 34.8, 34.1,
30.1, 22.2, 6.6, 4.6; HRDARTMS m/z 378.2442 [M + H]+ (calcd for
C21H36NO3Si, 378.2459).
Properties of 23b: colorless oil; IR (film) νmax 3357, 1750, 1624
cm−1; 1H NMR (CDCl3, 400 MHz) δ 7.38−7.27 (5H, m), 5.88 (1H,
d, J = 11.9 Hz), 4.26 (1H, dd, J = 8.2, 8.2 Hz), 3.12 (1H, m), 2.68
(3H, s), 2.40 (1H, m), 2.31 (1H, m), 2.09 (3H, s), 1.98 (1H, m), 1.97
(1H, m); 13C NMR (CDCl3, 100 MHz) δ 216.1, 171.2, 137.3, 128.8,
128.6, 128.0, 72.8, 58.9, 38.3, 33.7, 30.7, 30.1, 22.2; HRDARTMS m/
z 262.1453 [M + H]+ (calcd for C15H20NO3, 262.1438).
Calculations. The conformational analyses of each stereoisomer
of HMCP and N-acetyl HMCP using the MMFF94 force field gave 7
to 11 conformers within 20 kJ/mol from the global minimum.
1
Geometry optimization of each conformer and H and 13C NMR
chemical shift calculations were both performed by DFT calculations
using the B3LYP/6-31G* level of theory. The calculated chemical
shifts of each compound were corrected based on the Boltzmann
distribution of the conformers.
Biofilm Formation Assay. To measure biofilm formation of R.
solanacearum, we used a standard PVC assay. Strain OE1-1 cells from
overnight bacterial cultures in CPG were diluted to an OD600 of 0.1 in
new medium. A sample (5 μL) of the cell suspension was seeded into
each well of a PVC 96-well microtiter plate (Thermo Fisher
Scientific) containing CPG media (95 μL) and compounds. Plates
were sealed with Breathe-Easy membrane (Sigma-Aldrich) and
incubated statically at 30 °C. After 24−30 h, each well was stained
with 25 μL of 1% crystal violet for 25 min at rt. PVC plates were
washed twice with Milli-Q water (200 μL), and the remaining liquid
was aspirated from the bottom of the plate. Adhering crystal violet
was dissolved in 95% EtOH (200 μL), transferred to a new
polystyrene plate, and measured as absorbance at 595 nm.
N-((3-Hydroxy-4-oxocyclopentyl)(phenyl)methyl)-N-
methylacetamide (22a and 22b). To a solution of 21a (173 mg,
458 μmol) in CH2Cl2 (11 mL) was added Dess-Martin periodinane
(607 mg, 1.43 mmol) at 0 °C. After stirring for 2 h, saturated aqueous
NaHCO3 and saturated aqueous Na2S2O3 were added, and the
aqueous layer was extracted with CH2Cl2. The combined organic
layer was washed with saturated aqueous NaHCO3, H2O, and brine
and dried with Na2SO4. After concentration under reduced pressure,
the residue was chromatographed on SiO2 (hexane/EtOAc = 3:1) to
give 22a (153 mg, 89%). 22b (161 mg, 89%) was synthesized from
21b (182 mg) in the same manner.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01369.
Properties of 22a: colorless oil; IR (film) νmax 1755, 1645 cm−1;
1H NMR (CDCl3, 400 MHz) δ 7.38−7.28 (5H, m), 5.84 (1H, d, J =
1
Supplemental figures and tables, copies of H and 13C
12.3 Hz), 4.26 (1H, dd, J = 6.9, 6.9 Hz), 3.09 (1H, m), 2.69 (3H, s),
2.47 (1H, dd, J = 8.7, 19.2 Hz), 2.13 (3H, s), 2.01−1.82 (3H, m),
0.97 (9H, t, J = 7.8 Hz), 0.65 (6H, q, J = 7.8 Hz); 13C NMR (CDCl3,
100 MHz) δ 214.5, 170.9, 137.8, 128.7, 128.6, 127.9, 73.5, 58.5, 40.1,
35.2, 30.7, 30.1, 22.0, 6.6, 4.7; HRDARTMS m/z 376.2302 [M + H]+
(calcd for C21H34NO3Si, 376.2303).
NMR spectra (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Properties of 22b: colorless oil; IR (film) νmax 1755, 1644 cm−1;
1H NMR (CDCl3, 400 MHz) δ 7.41−7.28 (5H, m), 5.83 (1H, d, J =
11.9 Hz), 4.09 (1H, dd, J = 5.5, 5.5 Hz), 3.18 (1H, m), 2.70 (3H, s),
2.39 (1H, dd, J = 8.2, 19.2 Hz), 2.17 (1H, dd, J = 8.2, 19.2 Hz), 2.08
(3H, s), 1.95 (1H, m), 1.74 (1H, m), 0.93 (9H, t, J = 7.8 Hz), 0.60
(6H, m); 13C NMR (CDCl3, 100 MHz) δ 214.2, 170.9, 137.7, 128.7,
128.5, 127.9, 73.4, 59.3, 39.0, 36.5, 31.7, 30.1, 22.2, 6.6, 4.7;
HRDARTMS m/z 376.2301 [M + H]+ (calcd for C21H34NO3Si,
376.2303).
Arata Yajima − Department of Chemistry for Life Sciences and
Agriculture, Faculty of Life Sciences, Tokyo University of
Agriculture, Tokyo 156-8502, Japan; orcid.org/0000-
0003-1167-6041; Email: ayaji@nodai.ac.jp
Kenji Kai − Graduate School of Life and Environmental
Sciences, Osaka Prefecture University, Osaka 599-8531,
Japan; orcid.org/0000-0002-4036-9959; Email: kai@
biochem.osakafu-u.ac.jp
501
https://dx.doi.org/10.1021/acs.jnatprod.0c01369
J. Nat. Prod. 2021, 84, 495−502

Journal of Natural Products
pubs.acs.org/jnp
Article
(10) Chanthamath, S.; Nguyen, D. T.; Shibatomi, K.; Iwasa, K. Org.
Lett. 2013, 15, 772−775.
Authors
Ryo Katsuta − Department of Chemistry for Life Sciences and
Agriculture, Faculty of Life Sciences, Tokyo University of
Agriculture, Tokyo 156-8502, Japan
Mikaho Shimura − Graduate School of Agriculture, Tokyo
University of Agriculture, Tokyo 156-8502, Japan
Ayaka Yoshihara − Graduate School of Life and
Environmental Sciences, Osaka Prefecture University, Osaka
599-8531, Japan
Tatsuo Saito − Department of Chemistry for Life Sciences and
Agriculture, Faculty of Life Sciences, Tokyo University of
Agriculture, Tokyo 156-8502, Japan; orcid.org/0000-
0001-7477-1674
Ken Ishigami − Department of Chemistry for Life Sciences and
Agriculture, Faculty of Life Sciences, Tokyo University of
Agriculture, Tokyo 156-8502, Japan
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01369
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. S. Hashimura for his help in the synthetic study.
We thank Professors T. Nukada and M. Onaka (Tokyo
University of Agriculture) for their helpful discussions. This
work was supported in part by JSPS KAKENHI to A.Y. and
K.K.
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