Chemical structure of posttranslational modification with A farnesyl group on tryptophan

Article abstract of DOI:10.1271/bbb.80006

Bacillus subtilis and related bacilli produce a posttranslationally modified oligopeptide, the ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing. The ComX_RO-C-2 pheromone from strain RO-C-2 must be modified with a farnesyl group on the Trp residue, but the precise structure is not known. Here we report the precise nature of posttranslational farnesylation of ComX_RO-C-2 pheromone on the Trp residue, resulting in the formation of a tricyclic structure. The ComX ₁₆₈ pheromone, produced by the standard laboratory strain used in the study of B. subtilis, is also posttranslationally farnesylated according to phylogenetic resemblance.

Full text of DOI:10.1271/bbb.80006

Biosci. Biotechnol. Biochem., 72 (3), 914–918, 2008
Communication
Chemical Structure of Posttranslational Modification
with A Farnesyl Group on Tryptophan
y
Masahiro OKADA,1; Hisao YAMAGUCHI,1 Isao SATO,1 Fumitada TSUJI,1
y
1;
David DUBNAU,2 and Youji SAKAGAMI
1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
2Public Health Research Institute, 225 Warren Street, Newark, NJ 07103, USA
Received January 7, 2008; Accepted February 3, 2008; Online Publication, March 7, 2008
[doi:10.1271/bbb.80006]
Bacillus subtilis and related bacilli produce a post-
translationally modified oligopeptide, the ComX pher-
omone, that stimulates natural genetic competence
controlled by quorum sensing. The ComXRO-C-2 pher-
omone from strain RO-C-2 must be modified with a
farnesyl group on the Trp residue, but the precise
structure is not known. Here we report the precise
nature of posttranslational farnesylation of ComXRO-C-2
pheromone on the Trp residue, resulting in the for-
mation of a tricyclic structure. The ComX168 phero-
mone, produced by the standard laboratory strain used
in the study of B. subtilis, is also posttranslationally
farnesylated according to phylogenetic resemblance.
The ComX168 and ComXRO-C-2 pheromones, produced
by strains 168 and RO-C-2 respectively, are modified
with a farnesyl group, because the increase in molecular
weight matches the farnesyl modification detected by
MS analyses.5,6) Given the role of the modification in
each ComX variant, the structure of the farnesyl
modification might be similar to that of geranylation
but proof of the precise structure is unavailable. Since
each ComX pheromone shows group-specific bioactiv-
ity, determination of the nature of the modifications is
necessary in order to correlate group-specific structures
and activities, but sufficient preparation of purified
natural ComX pheromone has proven to be difficult
because the concentration of secreted pheromone in the
medium was low and the ComX pheromone is highly
unstable.5,10,12,13) Hence, we considered doing a syn-
thesis of a possible ComX peptide possessing the
farnesyl modified Trp residue and a comparison with
the synthetic peptide and culture broth containing the
natural ComX pheromone. Here we report the precise
structure of the ComXRO-C-2 pheromone, providing the
first example of the chemical structure of a naturally
occurring farnesyl group on a Trp residue.
We prepared a modified Trp residue with a farnesyl
group based on synthesis of the ComXRO-E-2 phero-
mone, which possesses a geranyl modified Trp residue
(Scheme 1A).10,11) C-Farnesylation of tricyclic com-
pound 1 using farnesyl bromide and NaH in THF
afforded diastereomixture of 2 in 73% yield and 1:1.6
(ꢀ-farnesyl:ꢁ-farnesyl) diastereomeric ratio. The yield
and the ratio of the desired ꢀ-farnesyl compound were
lower than those of C-geranylation, but employment of
C-farnesylation in NMP greatly diminished the ratio, to
1:9. After reductive deprotection of a Bz group chemo-
selectively, the diastereomixture was easily separated
by column chromatography using a solvent system of
Key words: Bacillus; ComX; posttranslational modifica-
tion; quorum sensing; tryptophan
In quorum sensing, bacteria regulate their gene
expression by secreting specific extracellular signaling
molecules.1,2) Bacillus subtilis and related bacilli pro-
duce a posttranslationally modified oligopeptide that
stimulates natural genetic competence,3,4) termed the
ComX pheromone.5) Natural isolates of bacilli exhibit
striking polymorphism in the amino acid sequence of
ComX, but each possesses an invariant Trp residue.6,7)
Previous studies have indicated that the Trp residue is
modified with an isoprenoid, which is essential for
pheromone activity, to form the mature ComX pher-
omone.6,8,9) The ComXRO-E-2 pheromone from B. sub-
tilis strain RO-E-2 was recently found to have a unique
modified Trp residue with a geranyl group at the 3
position of its indole ring, resulting in the formation of a
tricyclic structure.10,11) Successively, the ComXRO-H-1
pheromone from B. mojavensis strain RO-H-1 has also
been confirmed to have the geranyl modified Trp residue
in the same manner as the ComXRO-E-2 pheromone.12)
y
To whom correspondence should be addressed. Masahiro OKADA, present address: Graduate School of Science, Tohoku University, Aoba-ku,
Sendai 980-8578, Japan; Tel: +81-22-795-6555; Fax: +81-22-795-6557; E-mail: okada-org@mail.tains.tohoku.ac.jp; Youji SAKAGAMI,
Tel: +81-52-789-4116; Fax: +81-52-789-4118; E-mail: ysaka@agr.nagoya-u.ac.jp
Abbreviations: Clt, 2-chlorotrityl; Far, farnesyl; Pbf, 2,2,4,6,7-pentamethyl-dihydrobenzofurane-5-sulfonyl; Pp, 2-phenyl-2-propyl

Structure of Posttranslational Farnesylation on Trp
915
A
farnesyl bromide,
R
R
NaH,
DIBAL-H
THF, 0 °C, 5 h,
CO Me
73%
2
THF,
CO2Me –78 °C,
2 h, 37%
N
N
CO2Me
N
N
N
N
H
H
Bz
Bz
3
1
2 (α:β = 1:1.6)
R =
1) Fmoc-OSu, dioxane,
NaHCO3, H2O, rt, 1.5 h
1) LiOH, THF, MeOH,
H2O, rt, 30 min
R
Fmoc-Trp*(Far) 5
2) catecholborane, THF,
0 °C, 2 h
3) piperidine, CH3CN,
rt, 30 min (80% in 3 steps)
2) Fmoc-OSu, NaHCO3,
dioxane, H2O, rt,
1.5 h, 81% (in 2 steps)
N
N
CO2Me
H
H
H
Trp*(Far)-OMe 4
Asp(Pp)-Gly-Clt resin
B
C
Boc-Orn
H
N
H
5, HATU, HOAt, DIPEA, NMP, rt, 2 h
Fmoc-Trp*(Far)-Asp(Pp)-Gly-Clt resin
SPPS with peptide synthesizer
Glu(Pp)-Trp*(Far)-Asp(Pp)-Gly-Clt resin
N
Fmoc
Fmoc
NTf
6,
Pyr, MeOH, rt, 3 h, 96%
H
H
N
N
Fmoc
Fmoc
1) 8, HATU, HOAt, DIPEA, NMP, rt, 2 h
2) piperidine, CH3CN, rt, 1 h
N
3) TFA/HFIP/CH3CN (6/10/84), 0 °C, 24 h
Boc
HO2C
N
H
CO2H
Boc-Arg(Fmoc)2
7
HN
H
HO
O
O
1) BnBr, NaHCO3, DMF, 0 °C, 14 h,
79%
2) TFA, CH2Cl2, 0 °C, 1 h
3) Fmoc-Thr, HBTU, HOBt, DIPEA,
CH2Cl2, rt, 2 h, 85% (in 2 steps)
4) Pd/C, H2, THF, EtOAc, rt, 7 h, 48%
H
H
N
N
N
H2N
N
H
N
CO2H
H
O
O
O
CO2H
NH
ComXRO-C-2 peptide 9
(ComXRO-C-2 pheromone)
Fmoc-Thr-Arg(Fmoc)2
8
HN
NH2
Scheme 1. Synthesis of ComXRO-C-2 Peptide 9 (the ComXRO-C-2 Pheromone).
chloroform and methanol to afford optically active 3.
Fmoc protection, chemoselective double-bond reduction
with catecholborane, and Fmoc deprotection gave
the ComXRO-C-2 pheromone according to the synthesis
of Fmoc-Arg(Boc)2 (Scheme 1B).14,15) Treatment of
Boc-Orn with the guanidinylation reagent 6 in MeOH
1
2
ꢀ
*
*
Trp (Far)-OMe 4. The resulting 4 had only a cis
configuration between the newly added H-2 proton and
the farnesyl group based on NOE analysis. Hydrolysis
and Fmoc protection gave Fmoc-Trpꢀ(Far) 5 as two
conformational isomers of the amide bond in approx-
imately 1:1 ratio.
afforded the desired Boc-Arg(Fmoc)2 7. The dipep-
tide, Fmoc-Thr-Arg(Fmoc)2 8, was prepared via
condensation with Thr derivative in four steps.
After the formation of peptide bonds with a combi-
nation of peptide synthesizer and manual synthesis, as
previously reported,10–13,16) both 2-chlorotrityl (Clt)
resin and protecting groups such as a 2-phenyl-2-propyl
(Pp) group were cleaved under mild acidic conditions by
treatment with 6% TFA at 0 ꢁC for 24 h (Scheme 1C).
After HPLC purification, the structure of ComXRO-C-2
Fmoc-Agr(Pbf) is generally employed in solid-phase
peptide synthesis, but deprotection of the Pbf group did
not proceed with 7% TFA at 0 ꢁC for 24 h, the same
condition as the final deprotections used in the synthesis
of the ComXRO-H-1 pheromone.12) Hence we prepared
Boc-Arg(Fmoc)2 7 for solid-phase peptide synthesis of
3
*
peptide 9 was confirmed by ESI-HRMS.
We compared the natural ComXRO-C-2 pheromone in
1
Chemical data of Trpꢀ(Far)-OMe 4: 1H-NMR (400 MHz, CDCl3): ꢂ ¼ 1:56 (s, 3H), 1.58 (s, 3H), 1.60 (s, 3H), 1.68 (s, 3H), 1.94–2.07 (m, 8H),
2.34–2.80 (m, 4H), 3.34 (s, 3H), 3.88 (dd, 1H, J ¼ 7:4, 7.7 Hz), 4.81 (s, 1H), 5.06–5.11 (m, 2H), 4.81 (s, 1H), 5.15 (m, 1H), 6.54 (dd, 1H,
J ¼ 0:8, 7.5 Hz), 6.69 (dd, 1H, J ¼ 0:8, 7.4 Hz), 6.98–7.03 (m, 2H); 13C NMR (100 MHz, CDCl3) ꢂ 16.0, 16.3, 17.7, 25.7, 26.5, 26.8, 36.5, 39.7,
39.9, 41.1, 52.8, 57.7, 60.1, 82.5, 109.5, 118.8, 119.6, 123.8, 124.1, 124.3, 128.1, 131.3, 133.4, 135.1, 138.1, 149.4, 174.4; ESI-HRMS m=z
([M þ H]þ) Calcd. for C27H36N2O2: 421.2850, Found 421.2847.
*
2
3
Chemical data of Boc-Arg(Fmoc)2 7: 1H-NMR (400 MHz, CD3OD): ꢂ ¼ 1:40 (s, 9H), 1.75–1.82 (m, 3H), 1.95 (m, 1H), 3.55 (m, 1H), 4.23 (m,
1H), 4.27–4.37 (m, 4H), 4.56 (d, 1H, J ¼ 6:8 Hz), 7.32 (t, 4H, J ¼ 7:4 Hz), 7.38–7.43 (m, 4H), 7.66 (d, 2H, J ¼ 7:4 Hz), 7.72 (d, 2H,
J ¼ 7:4 Hz), 7.85 (d, 4H, J ¼ 7:4 Hz).
*
*
HRMS data of ComXRO-C-2 peptide 9; ESI-HRMS m=z ([M þ 2H]2þ) Calcd. for C48H68N8O10: 458.2524, Found 458.2525.

916
M. OKADA et al.
763 (−Far)
A
B
22.7 min
m/z = 967.5
Natural pheromone
Natural pheromone
22.7 min
m/z = 967.5
ComXRO-C-2 peptide 9
241
334
893(b5)
387(b3)
370
258(b2)
10
20
Time (min)
30
ComXRO-C-2 peptide 9
763(−Far)
100
50
C
−Far
(763)
−Gly
Thr−Arg−Glu−Trp*−Asp
b2 b3 b5
241
(258) (387)
370
(893)
334
258(b2)
387(b3)
893(b5)
ComXRO-C-2 peptide 9
0
0.1
1
10
100
200
500
1000
Mass (m/z)
Concentration (nM)
Fig. 1. Analyses of the Natural ComXRO-C-2 Pheromone and the Synthetic ComXRO-C-2 Peptide 9.
A, MALDI-TOF-MS/MS analyses of the natural ComXRO-C-2 pheromone and the synthetic ComXRO-C-2 peptide 9. MALDI-TOF-MS/MS
were acquired using an 4700 Proteomics Analyzer (Applied Biosystems, Foster City, CA). Squares represent the precursor ions at m=z 967.5. B,
LC/ESI-MS analyses of the natural ComXRO-C-2 pheromone and the synthetic ComXRO-C-2 peptide 9. LC/ESI-MS were acquired using an
API2000 LC/ESI-MS System (Applied Biosystems, Foster City, CA) in Q1MI scan mode (m=z value 967.5). A toriacontyl column
(250 ꢂ 4:6 mm ID, Develosil C30-UG-5, Nomura Chemical, Tokyo) was attached to a HPLC system and the sample solutions were eluted at a
flow rate of 1.0 ml/min with 25% CH3CN in 0.1% aqueous ammonium acetate for 2 min and then a gradient of 25–55% CH3CN in 0.1% aqueous
ammonium acetate for 30 min. C, Concentration-response curve using synthetic ComXRO-C-2 peptide 9. The curve represents the means of
duplicate samples.
the culture broth with the synthetic ComXRO-C-2 peptide
9 by MALDI-TOF-MS. They had the same molecular
composition possessing the expected ion at m=z 967.5.
The MS/MS spectrum of the natural ComXRO-C-2
pheromone was identical to that of the synthetic
ComXRO-C-2 peptide 9 by analysis of the precursor
ion at m=z 967.5 (Fig. 1A). Furthermore, the synthetic
ComXRO-C-2 peptide 9 showed the same retention time
as the natural pheromone by LC-ESI-MS monitoring at
m=z 967.5 (Fig. 1B). We then investigated the biological
activity of the synthetic ComXRO-C-2 peptide 9 for
B. subtilis strain RO-C-2 using the B. subtilis tester
strain, which responds to the ComXRO-C-2 pheromone,
by monitoring the expression of an srfA-lacZ fusion.6) ꢁ-
Galactosidase assay was carried out by measurement
of the intensities of absorbance at 420 nm by a standard
method with ONPG, as previously reported (Fig. 1C).
Peptide 9 showed potent biological activity, with an
EC50 value of 2 nM. This value was reasonable for
comparing the observed biological activity of the culture
broth with the equivalent concentration of the natural
ComXRO-C-2 pheromone in the culture broth calculated
by the intensity of LC-ESI-MS. Additionally, it has been
found that the precisely-modified Trp residue is essential
to biological activity,16) and that synthetic ComXRO-C-2
peptide 9 has biological activity approximately equal
to other natural ComX pheromones, such as the
ComXRO-E-2 pheromone.5,10–12) Thus the structure of
the ComXRO-C-2 pheromone was confirmed to be
synthetic ComXRO-C-2 peptide 9, and the posttransla-
tional modification was farnesylation on the Trp residue
at the 3 position of its indole ring, resulting in the
formation of a tricyclic structure.
We have prepared six recombinant mature ComX
variants using an Escherichia coli expression system.6)
These previous results, together with the present find-
ings, reveal that posttranslational isoprenoidal modifi-
cation in the ComX pheromones can be classified into
two types, with geranyl and farnesyl modifications
respectively (Table 1). For instance, the ComX168
pheromone, produced by the standard laboratory strain
used in the study of B. subtilis, is also modified with
a farnesyl group as the ComXRO-C-2 pheromone. This
similarity corresponds to the phylogenetic resemblance

Structure of Posttranslational Farnesylation on Trp
917
Table 1. Posttranslational Modifications of ComX Variants
Bacillus Strain
Sequence
Chemical Structure of Wꢀ
168
ADPITRQWꢀGD
TREWꢀDG
HN
H
RO-C-2
N
Farnesyl
O
RO-E-2
RO-H-1
RS-B-1
RO-B-2
GIFWꢀEQ
MLDWꢀKY
HN
H
MMDWꢀHY
N
Geranyl
YTNGNWꢀVPS
O
of the modifying enzymes (ComQ) and the ComX
precursors of the two strains.6,7) Although the precise
modification of the ComXNAF4 pheromone from
B. natto, which we were unable to produce from
E. coli, is unknown, our chemical synthetic method-
ology can potentially clarify the structure of this
pheromone in the near future.
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Acknowledgments
The work done at Nagoya University was supported
by a Grant-in-Aid for Scientific Research (S) (no.
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Sports, Science, and Technology of Japan, and that done
in Newark was supported by NIH grant GM057720.

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