The geranyl-modified tryptophan residue is crucial for ComX RO-E-2 pheromone biological activity

Article abstract of DOI:10.1016/j.bmcl.2011.04.123

The ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in gram-positive bacteria, Bacillus. We have reported the structure of the ComX_RO-E-2 pheromone, which is produced by the RO-E-2 strain of Bacillus subtilis. ComX_RO-E-2 analogs with substituted amino acids and isoprenoid modified tryptophan residues (e.g., prenyl, geranyl, and farnesyl), were synthesized and examined for biological activity. These results indicate that Phe-Trp*(Ger)-NH₂ is the minimum pharmacophore of the ComX _RO-E-2 pheromone. Furthermore, the length of the isoprenoid moiety (i.e., modification style), and the presence of double bonds, are crucial for biological activity. The modification style of the ComX pheromone is more important than the peptide sequence with respect to biological activity.

Full text of DOI:10.1016/j.bmcl.2011.04.123

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4041–4044
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The geranyl-modified tryptophan residue is crucial for ComXRO-E-2
pheromone biological activity
Fumitada Tsuji a, Ko Kobayashi a, Masahiro Okada b, Hisao Yamaguchi a, Makoto Ojika a, Youji Sakagami a,
⇑
a Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, Aichi 464-8601, Japan
b Graduate school of Bioscience and Biotechnology, Chubu University, Kasugai, Aichi 487-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which
stimulates natural genetic competence in gram-positive bacteria, Bacillus. We have reported the structure
of the ComXRO-E-2 pheromone, which is produced by the RO-E-2 strain of Bacillus subtilis. ComXRO-E-2 ana-
logs with substituted amino acids and isoprenoid modified tryptophan residues (e.g., prenyl, geranyl, and
farnesyl), were synthesized and examined for biological activity. These results indicate that Phe-
Trp⁄(Ger)-NH2 is the minimum pharmacophore of the ComXRO-E-2 pheromone. Furthermore, the length
of the isoprenoid moiety (i.e., modification style), and the presence of double bonds, are crucial for bio-
logical activity. The modification style of the ComX pheromone is more important than the peptide
sequence with respect to biological activity.
Received 15 January 2011
Revised 20 April 2011
Accepted 27 April 2011
Available online 12 May 2011
Keywords:
Bacillus subtilis
ComX pheromone
Posttranslational modification
Quorum sensing
Ó 2011 Elsevier Ltd. All rights reserved.
Structure–activity relationships
Quorum sensing is a well known phenomenon in which bacte-
ria regulate their behavior, dependent on cell density.1 Bacteria se-
crete specific extracellular signaling molecules and utilize such
molecules to sense their cell density. Bacilli secrete the ComX pher-
omone, which stimulates natural genetic competence controlled
by quorum sensing.2 In the presence of the ComX pheromone, a
membrane-located receptor (histidine kinase ComP) autophosph-
orylates and subsequently donates a phosphate via a two-compo-
nent system ultimately activating competence gene expression.3,4
Recent efforts from our group have demonstrated that the
ComXRO-E-2 pheromone (1), from the B. subtilis strain RO-E-2, is a
hexapeptide containing a geranyl-modified tryptophan residue.5
This novel functional group modification results in the formation
(e.g., Bacillus anthracis). Their respective quorum sensing phero-
mones most likely share similar structures with those of the ComX
pheromone, although studies on their quorum sensing systems
have not been well established to date. We hope that this study
will provide the foundation necessary for the further development
of small molecules to be used in controlling these bacteria by the
quorum sensing system.
We have reported that the tricyclic structure and stereochemi-
cal configuration of the modified tryptophan residue is necessary
for pheromone function. In addition, we have explored the struc-
ture–activity relationships using an alanine scan and with trun-
cated peptides.7–9
of a tricyclic structure (Fig. 1). Our group has also reported the
6
structure of the farnesylated pheromone, ComXRO-C-2
.
We have
been interested in defining the structure–activity relationships of
ComXRO-E-2 analogs, with the ultimate goal being the identification
of a simple synthetic molecule that could control bacterial growth.
These structure–activity relationships provide new insight into the
interactions between the endogenous pheromone and its respec-
tive receptor. The ComX pheromone bears a unique structure and
the ComP receptor belongs to the histidine kinase receptor-protein
family. This constitutes a receptor class in which ligand–receptor
interactions have not been adequately investigated. Several viru-
lent bacteria are known to be taxonomically similar to B. subtilis
Figure 1. Chemical structure of ComXRO-E-2 pheromone. Bold Trp⁄(Ger) represents
the modified tryptophan residue with geranyl group in the ComXRO-E-2 pheromone
(bold lines).
⇑
Corresponding author. Tel: +81 52 789 4116; fax: +81 52 789 4118.
E-mail address: ysaka@agr.nagoya-u.ac.jp (Y. Sakagami).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.123

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F. Tsuji et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4041–4044
Here, we disclose the active subunit of the ComXRO-E-2 phero-
on the tripeptide. Next, we synthesized the corresponding dipep-
tides to determine the minimum bioactive structure-subunit (i.e.,
the pharmacophore). We attempted to synthesize a C-terminal
blocked dipeptide, [4-5]ComXRO-E-2-OMe, because peptides con-
taining a modified tryptophan residue at the C- or N-terminus
led to low biological activity in previous studies.9 Efforts to synthe-
size this dipeptide methyl ester were unfruitful, but [4-5]ComXRO-
mone and highlight the importance of the geranyl side chain pen-
dant on the modified tryptophan residue. Various ComXRO-E-2
analogs were synthesized using solid-phase synthetic tech-
niques.5,7–10 Biological activities of these peptides, as well as the
ComXRO-E-2 pheromone, were investigated using b-galactosidase
assays.3,5,7,8,10,11 These data were used to construct the respective
dose–response curves. The biological activities of these peptides
were represented by three values normalized to the activity of
the ComXRO-E-2 pheromone (1) (Table 1). The first and second val-
ues correspond to the concentrations of each peptide required to
elicit the same b-galactosidase activity as that observed with the
ComXRO-E-2 pheromone at EC25 and EC50, respectively. The third
is the maximum activity as a percentage of that obtained with
the ComXRO-E-2 pheromone.
The structure–activity relationships of the tripeptides was
investigated in comparison with [3-5]ComXRO-E-2 (2), the smallest
peptide known to exhibit biological activity as demonstrated in
previous work.9 [E5A][3-5]ComXRO-E-2 (3) showed slightly higher
biological activity than tripeptide 2, and dose-dependent activity
was observed with [E5G][3-5]ComXRO-E-2 (4) (Table 1). These re-
sults indicate that the C-terminal amino acid residue of the tripep-
tide can be replaced by other amino acids. In previous work,
alanine scan experiments with the original ComXRO-E-2 hexapep-
tide pheromone have demonstrated that the third phenylalanine
is exchangeable with alanine.9 However, the replacement of the
N-terminal phenylalanine residue with alanine (tripeptide 5) in
the tripeptide 2, significantly attenuates biological activity. Among
the tripeptides, a fixed C-terminal alanine, tyrosine and isoleucine
exchange (tripeptide 7 and 9) was tolerated, but other amino acid
replacements led to very low activities (6, 8, 10, 11, 12; Table 1). A
structure–activity relationships as a function of aromaticity and/or
steric bulk among the N-terminal amino acid residues of these trip-
eptides was not observed. These results indicate that the phenylal-
anine residue is not exchangeable with another amino acid residue
(diketopiperazine 13) was readily obtained. To prevent forma-
E-2
tion of the diketopiperazine, the N-terminus was functionalized
as the acetyl amide 14. Unfortunately, the dipeptides 13 and 14
did not elicit biological activity. Gratifyingly, bioactivity was ob-
served with [4-5]ComXRO-E-2-NH2 (15), with activity comparable
to that of 4. Moreover, Bz-Trp⁄(Ger)-NH2 (16) showed no biological
activity. Therefore, it can be posited that the minimal bioactive
subunit of the ComXRO-E-2 pheromone is Phe-Trp⁄(Ger)-NH2.
The effect of modifying the tryptophan side chain was also
studied. [Pre]ComXRO-E-2 (17) and [Far]ComXRO-E-2 (18) were pre-
pared, which contain prenyl and farnesyl side chains, respectively
(Table 2). The peptide 17 lost all activity and 18 lost most activity.
Previous studies have demonstrated that the tricyclic structure and
the stereochemical configuration of the modified tryptophan
moiety are important for biological activity.7,8 Despite this, [Pre]-
ComXRO-E-2 (17) and [Far]ComXRO-E-2 (18), retaining the typical
tricyclic structure and stereochemical configuration of the parent
tryptophan residue, led to reduced biological activities. These
results indicate that the geranyl side chain of the tryptophan
residue is an important factor in expressing biological activity in
ComXRO-E-2. These results are also in accord with the finding that
the RO-E-2 tester strain exhibits cross activities with other gerany-
lated pheromones, but little activity in the presence of ComXRO-C-2
and ComX168 pheromone, both of which have farnesyl modified
tryptophan groups.3
Next, we hydrogenated the Fmoc-geranylated tryptophan with
Rhodium on Alumina and obtained two Fmoc-amino acids with
saturated/partially saturated side chains. Using these compounds,
Table 1
Biological activities of ComXRO-E-2 and ComXRO-E-2 analogs

F. Tsuji et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4041–4044
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Table 2
Chemical structures and biological activities of ComXRO-E-2 and ComXRO-E-2 analogs
we synthesized two peptides [H2Ger]ComXRO-E-2 (19) and [H4Ger]-
ComXRO-E-2 (20), incorporating side chains with the same carbon
skeleton as ComXRO-E-2 (Table 2). [H4Ger]ComXRO-E-2 (20) was
obtained as a mixture of epimers that were inseparable by HPLC.
Therefore, [H4Ger]ComXRO-E-2 (20) was tested as a mixture of
epimers. [H2Ger]ComXRO-E-2 (19) retained partial activity, but
[H4Ger]ComXRO-E-2 (20) led to a reduced response. These results
indicate that the double bonds of the geranyl group, as well as
the length, are important for biological activity.
isoprenylation of cysteine, are known to distinguish the length
of the isoprenoidal chain.15 The mechanism of chain length dis-
tinction has been termed the ‘ruler hypothesis’. This hypothesis
is supported by X-ray crystallographic analysis of FTase and
GGTase-I and enables one to account for the substrate specificity
of the respective enzymes. Therefore, further analysis of the X-ray
crystallographic data of ComP, as well as FTase and GGTase-I,
could provide insight into the mechanism of isoprenoidal chain
length distinction. Perhaps ComP can be classified as a new type
of receptor that distinguishes the isoprenoidal chain length, but
not the peptide sequence. Because, receptors that have a signal
transduction system (two-component system) are distributed
widely throughout bacteria, fungi and plants, it is possible that
ComP exists as a novel receptor type in various organisms.
According to our results from the structure–activity relationship
studies of the ComXRO-E-2 pheromone, the structure of the labeled
ligand has been optimized to facilitate investigation of the molec-
ular interactions between the ComX pheromone and the ComP
receptor.
The quorum sensing pheromone of gram-negative bacteria, a so
called autoinducer, is an acylhomoserine lactone, and the species
specificity of the pheromones is dependent on the acyl group
length.16–18 Gram-positive bacteria, Bacillus, may also use the pep-
tide pheromone side chain length with respect to species specific-
ity. B. subtilis and related Bacilli may be unique gram-positive
bacteria, because not only the modification, but also the receptors
were evolved for the acquisition of species-specificity. In gram-
negative bacteria, an autoinducer (acylhomoserine lactone) is a
lead compound in the development of a novel type of drug for
use in controlling bacterial growth.19–21 This work provides the
foundation necessary to aid in similar trials of virulent gram-
positive bacteria in the near future.
Tremerogen A-10 is a dodecapeptide mating pheromone of
Tremella mesenterica, which has an S-farnesyl modified cysteine
methyl ester at the C-terminus.12,13 Earlier structure–activity rela-
tionship studies of tremerogen A-10 analogs have indicated that
the longer isoprenoid chain generates the greatest biological
activity, and that the alkyl chain is replaceable with an isoprenoid
side chain.14 These results suggest that the farnesyl moiety is not
crucial in eliciting biological activity with tremerogen A-10.
In contrast, the geranyl moiety is critical for the observed bio-
logical activity associated with the ComX pheromone as men-
tioned above. Furthermore, the peptide chain length is crucial in
tremerogen A-10, but it is not necessary in inducing biological
activity with the ComX pheromone.13,14 Despite the similar post-
translational modifications between these two classes of isopre-
noidal peptides, it is interesting that the structural requirements
necessary for biological activity is completely different. This could
imply that the interactions between the peptide containing the
isoprenoidal tryptophan and its corresponding receptor are
different from that of the peptide containing the isoprenoidal cys-
teine. It can be suggested that the ComX receptor (ComP), which
is a membrane-located histidine kinase, distinguishes the length
of the isoprenoid chain. FTase (farnesyltransferase) and GGTase-I
(geranylgeranyltransferase type I), enzymes that catalyze the

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F. Tsuji et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4041–4044
hexafluorophosphate (HATU) and 1-hydroxy-7-azabenzotriazole (HOAt) in
Acknowledgements
CH2Cl2, N,N-diisopropylethylamine (DIPEA) under nitrogen. The Fmoc-
protected modified tryptophan residues containing the tetrahydrogeranyl
group and the dihydrogeranyl group were prepared by hydrogenation using
Rhodium–Alumina as the catalyst under an atmosphere of hydrogen gas. The
Fmoc-protected tryptophan residue bearing a geranyl group was used as a
starting material in this transformation. The structures of all modified
tryptophan residues were confirmed by 1H NMR spectroscopy. Other
residues were purchased from commercial sources (Watanabe chemical,
Nova biochem). Peptide bond formation was accomplished with a peptide
synthesizer (Applied Biosystems 433A) except for the modified tryptophan
coupling. After which, each crude peptide was purified by HPLC and the
resulting product structures were confirmed by MS/MS analysis.
This work was supported by a Grant-in-Aid for JSPS Fellows (no.
21 2118) for F.T. and a Grant-in-Aid for Scientific Research (S) (no.
18101009) for Y.S. from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.123.
11. Biological activity was investigated as previously reported using the B. subtilis
tester strain in the expression of a srfA-lacZ fusion, which responds to added
ComXRO-E-2 pheromone.3,5,7,8 Briefly, the strain was cultured overnight and
subsequently diluted 100-fold. The culture was added to a sample solution and
incubated at 37 °C for 5 h at 150 rpm, then b-galactosidase activity was
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acid
with
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium