A formyl peptide substituted with a conformationally constrained phenylalanine residue evokes a selective immune response in human neutrophils

Article abstract of DOI:10.1016/j.bmc.2012.11.046

Formyl-Met-Leu-Phe-OH (fMLP) binds to formyl peptide receptors, FPR1 and FPR2, and evokes migration and superoxide anion production in human neutrophils. To obtain a more effective and selective ligand, fMLP analogs in which the Phe residue was substitute

Full text of DOI:10.1016/j.bmc.2012.11.046

Bioorganic & Medicinal Chemistry 21 (2013) 668–675
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A formyl peptide substituted with a conformationally constrained
phenylalanine residue evokes a selective immune response in human
neutrophils
Ryo Hayashi a, Masaya Miyazaki b,c, Satoshi Osada a, Hiroshi Kawasaki a, Ichiro Fujita d, Yuhei Hamasaki e,
Hiroaki Kodama a,
⇑
a Department of Chemistry, Graduate School of Science and Engineering, Saga University, 1 Honjo-machi, Saga 840-8502, Japan
b Department of Molecular and Material Sciences, Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Fukuoka 816-8580, Japan
c Measurement Solution Research Center, National Institute of Advanced Industrial Science and Technology, Saga 841-0052, Japan
d Faculty of Culture and Education, Saga University, Saga 840-8502, Japan
e Department of Pediatrics, Faculty of Medicine, Saga University, Saga 849-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Formyl-Met-Leu-Phe-OH (fMLP) binds to formyl peptide receptors, FPR1 and FPR2, and evokes migration
and superoxide anion production in human neutrophils. To obtain a more effective and selective ligand,
fMLP analogs in which the Phe residue was substituted with four isomers of cyclopropanephenylalanine
were synthesized. While Z-isomer peptides induced both migration and superoxide anion production, E-
isomer peptides elicited only chemotaxis. Homologous receptor desensitization experiments revealed
that E-isomer peptides bound to FPR2. Although a selective agonist of chemotaxis also binds to FPR2
without increasing intracellular calcium concentration, E-isomer peptide elevated the concentration to
the same level as fMLP. Understanding of mechanisms responsible for the selectivity of the reported
Received 10 October 2012
Revised 28 November 2012
Accepted 28 November 2012
Available online 7 December 2012
Keywords:
Formyl peptide
Cyclopropane phenylalanine
fMLP
Selective agonist
Formyl peptide receptors
selective agonists and
between receptor–ligand interactions and biological responses of human neutrophils.
Ó 2012 Elsevier Ltd. All rights reserved.
r
Phe-substituted analogs should prove useful for revealing the relationship
1. Introduction
peptides to the receptors causes multiple immune responses such
as superoxide production and chemotaxis, some agonists elicit a
selective response. For example, Torrini et al. identified formyl-
Thp-Leu-Ain-OMe ([Thp1,Ain3]fMLP-OMe, Fig. S2), characterized
Human neutrophils represent 50–60% of the total leukocytes in
circulation, and are highly specialized for their primary function,
that is, phagocytosis and destruction of microorganisms through
generation of superoxide anion and release of protease-containing
granules. Leukocyte recruitment to the sites of inflammation and
infection is dependent upon the presence of a gradient of locally
produced chemotactic factors. The bacterial peptide N-formyl-
methionyl-leucyl-phenylalanine (fMLP) is one such chemotactic
factor and is a highly potent leukocyte chemoattractant. It interacts
with receptors on the membranes of neutrophils, activating these
cells through G-protein-coupled pathways. Two functional fMLP
receptors in human neutrophils are characterized, namely formyl
peptide receptor (FPR1) and its subtype (FPR2) with high and
low affinities for fMLP, respectively.1
by the presence of an unusual
a-amino acid in which the native
Met and Phe external residues were substituted with 4-amino-tet-
rahydrothiopyran-4-carboxylic acid (Thp) and 2-aminoindane-2-
carboxylic acid (Ain), as a selective agonist which only induces
the chemotactic response in human neutrophils.2,3 As another
example of a selective agonist, Pagani Zecchini et al. found that for-
myl-Met-
the central Leu residue was replaced by (Z)-2,3-dihydroleucine
D D
ZLeu-Phe-OMe ([ ZLeu2]fMLP-OMe, Fig. S2), in which
(D
ZLeu), was only able to elicit superoxide anion production and
degranulation.4 These results seem to indicate that incorporation
of an amino acid surrogate with conformationally constrained side
chains is crucial for the design of highly selective and potent pep-
tide analogs with enhanced properties and has provided valuable
insights into how peptides interact with their receptors.5
The tripeptide fMLP and its methyl ester analog fMLP-OMe are
generally adopted as the reference models for evaluating the activ-
ity of newly synthesized analogs. While the binding of the formyl
Amino acids containing a cyclopropane skeleton are found in a
wide class of naturally occurring products, and are of interest
for peptide design because of their steric hindrance due to the
⇑
Corresponding author. Tel.: +81 952 28 8562; fax: +81 952 28 8548.
E-mail address: hiroaki@cc.saga-u.ac.jp (H. Kodama).
a,a-disubstitution. The presence of the tensioned three-membered
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.046

R. Hayashi et al. / Bioorg. Med. Chem. 21 (2013) 668–675
669
2.2.
r
EPhe-containing fMLP analogs only evoked chemotaxis in
human neutrophils
The biological activities of synthetic peptides were evaluated in
human neutrophils. fMLP was used as a control peptide. Figure 2A
shows the concentration dependence of chemotaxis in human neu-
E-isomer
Z-isomer
(2S, 3R)
(2R, 3S)
(+)- EPhe-OMe
(-)- EPhe-OMe
trophils toward
OMe. The chemotactic activity EC50 value of fMLP-OMe was
2.5 0.1 nM (Table 2). The other Phe-containing peptides also
rPhe-containing synthetic peptides 5–8 and fMLP-
1
2
r
showed chemotactic activity in human neutrophils but they were
about 2- to 8-fold lower as compared withfMLP (Fig. 2A and Table 2).
The chemotactic activities of peptides 6 and 8, in which the side
(2S, 3S)
(2R, 3R)
(+)- ZPhe-OMe
(-)- ZPhe-OMe
chains are
than that of the
Superoxide anion production in human neutrophils stimulated
Phe-containing peptides is shown in Figure 2B. fMLP-OMe in-
D-configuration, were approximately 3- and 2-fold higher
3
4
L
-configuration peptides 5 and 7, respectively.
Figure 1. Chemical structures of cyclopropane phenylalanine methyl esters ( Phe-
r
by
r
OMe).52,53
duced superoxide anion production with an EC50 value of
16 0.2 nM (Table 2). In contrast to chemotactic response, only
peptide 8, which contains the (ꢀ)-Z isomer, evoked superoxide an-
ion production with an EC50 of 250 80 nM. Peptide 7 containing
the (+)-Z isomer showed only 45% activity relative to fMLP at
ring introduces severe constrains in the proximal backbone torsion
angles. Cyclopropane phenylalanine ( Phe, Fig. 1) has been the
r
1.0 lM. On the other hand, E-isomer analogs 5 and 6 did not show
most widely studied. For example, all four stereoisomers were
definite activity although the peptides induced migration of hu-
man neutrophils. Thus, peptide 5 and 6 seemed to be selective ago-
nists of chemotaxis in human neutrophils.
incorporated into enkepharin1–3,6–9 and aspartame.4,10 In addition,
some peptides containing the
r
Phe residue were proven to be
much more resistant to enzymatic degradation by chymotryp-
sin.5,11 In the present study, to obtain a more effective and selective
ligand, fMLP analogs in which the Phe residue was substituted with
four stereoisomers of
fects of the peptides on chemotaxis and superoxide anion produc-
tion in human neutrophils were evaluated.
2.3. Both E-isomer and Z-isomer analogs induced intracellular
calcium mobilization in human neutrophils
r
Phe were synthesized (Table 1), and the ef-
[Thp1, Ain3]fMLP-OMe (Fig. S2), a selective agonist for chemo-
taxis,2 did not increase cytoplasmic calcium concentration,3,17
whereas [D
ZLeu2]fMLP-OMe (Fig. S2), which evoked superoxide
production and degranulation,4 increased the concentration.3,17,18
In addition, superoxide anion production by the addition of
2. Results and discussion
2.1. Synthesis
[D
ZLeu2]fMLP-OMe was reduced in the presence of a calcium ion
chelator.3 Likewise, superoxide anion production by human neutro-
phils stimulated by fMLP-OMe was also affected by the chelator.
These results suggested that an increase of cytoplasmic calcium con-
centration is required for NADPH oxidase in human neutrophils. In
contrast, substance P, transforming growth factor b1, and the mur-
ine S100 protein, only induced chemotactic responses and did not
influence calcium concentration.19,20 These results suggested that
intracellular calcium mobilization could be a good indicator for
investigating the mechanisms of selective agonists. Figure 3 shows
the maximum concentrations of intracellular calcium ion induced
Racemic
r
EPhe-OMe and
r
ZPhe-OMe were synthesized by a
diazoaddition method12 and malonate method,13,14 respectively.
The E-isomers (1 and 2) and the Z-isomers (3 and 4) were resolved
by formation of diastereomeric salts with brucine15 and O,O-dib-
enzoyltartaric acid,10 respectively (Fig. 1). The optical purities of
the stereoisomers were evaluated by comparison of reported [a]
D
values.16 The synthetic route and details are described in the Sup-
porting Information.
Peptide synthesis was performed by a conventional solution
phase method (Scheme 1). The coupling of Boc-Leu-OH with
Phe-OMe was achieved by a mixture anhydride (MA) method
to give dipeptide 9. After the Boc group was removed by the treat-
ment of trifluoroacetic acid (TFA), formyl-Met-OH was introduced
to dipeptide 9 utilizing 1-ethyl-3-(3-dimethylaminopropyl)carbo-
diimide (EDC) as a coupling reagent. Peptides 5, 6, 7 and 8 were
purified by silica gel chromatography.
by the addition of fMLP-OMe and
The cytoplasmic calcium concentrations induced by the stimulation
of Phe-containing peptides 5–8 were similar to that of fMLP-OMe
rPhe-containing peptides 5–8.
r
r
although the biological activities of peptides 5–8 were lower than
fMLP-OMe. Previous studies of receptor occupancy–response rela-
tionships demonstrated that whereas almost 100% of the receptors
needed to be occupied to achieve maximal superoxide anion pro-
duction, less than 1% receptor occupancy was required to generate
maximal actin polymerization,21,22 which represents an essential fa-
cet of neutrophil migration.23 If the selectivity of the E-isomer ana-
logs 5 and 6 was due to their low binding affinities to the receptors as
compared with the affinities of the Z-isomer analogs 7 and 8, the cal-
cium concentrations induced by peptides 5 and 6 may be lower than
that of fMLP-OMe and peptides 7 and 8. WKYMVm binds to both
FPR1 and FPR2, and elevated intracellular calcium concentration,
leading to chemotaxis, and endocytosis. In contrast, WKXMVm
(X = G or R) binds to only FPR2, and evoked chemotaxis, while cyto-
plasmic calcium concentration was not increased.24 Although the
mechanisms responsible for the selectivity of E-isomer peptides
are unclear, it is possible that peptides 5 and 6 might bind to a spe-
cific receptor, FPR1 or FPR2.
Table 1
Structures of fMLP-OMe and its derivatives containing cyclopropane phenylalanine
(rPhe)
Peptide
Sequence
fMLP-OMe
Formyl-Met-Leu-Phe-OMe
5
6
7
8
Formyl-Met-Leu-(+)-
EPhe-OMe
r
Formyl-Met-Leu-(ꢀ)-
r
EPhe-OMe
ZPhe-OMe
Formyl-Met-Leu-(+)-
r
Formyl-Met-Leu-(ꢀ)-
r
ZPhe-OMe

670
R. Hayashi et al. / Bioorg. Med. Chem. 21 (2013) 668–675
Met
Leu
Phe
Isomer of Phe
E-isomer
Z-isomer
Boc
Boc
H
OH
MA
H
OMe
OMe
(+)
(-)
(+)
(-)
9a
9b
9c
9d
TFA
formyl
formyl
OH
EDC-HOBt
OMe
5
6
7
8
Scheme 1. Synthesis of
rPhe-containing peptides 5–8.
fMLP-OMe
fMLP-
A
primes these cells for exaggerated superoxide anion production
after subsequent addition of an agonist,25–27 a process known as
neutrophil priming.28–30 Treatment with calcium ion ionophores
such as A23187 and ionomycin that do not bind to specific recep-
tors also causes neutrophil priming, suggesting that an increase of
cytoplasmic calcium ion leads to pre-activation of human neutro-
E(+)
Peptide 5, (+)-E
100
80
60
40
20
0
E(-)
Peptide 6, (-)-E
Z(+)
Peptide 7, (+)-Z
Z(-)
Peptide 8, (-)-Z
phils.
rPhe-containing fMLP-OMe analogs 5–8 increased intracel-
lular calcium concentration (Fig. 3) while only Z-isomers 7 and 8
initiated superoxide anion production in human neutrophils. Thus,
we thought that examination of the neutrophil priming activity of
the synthetic peptides could provide insight into the mechanisms
of the selective agonists. Figure 4 and Table 2 indicate the priming
effects of the synthetic peptides. Interestingly, enhanced superox-
ide anion production was observed only when the cells were pre-
treated with Z-isomer peptides 7 and 8. These results suggested
that Z-isomer peptides 7 and 8 might have high binding affinity
to a specific receptor that functions in superoxide anion production
in human neutrophils.
-11
-10
-9
-8
-7
-6
2.5. E-Isomer peptide binds to FPR2
Log peptide concentration (M)
When neutrophils encounter increasing concentration of a che-
moattractant, they gradually become non-responsive to further
stimulation by the same agonist.31 This process, known as homol-
ogous desensitization, is due to a decreased affinity of receptors for
G-proteins by association with arrestin family and internaliza-
tion.32 Thus, we performed receptor desensitization experiments
fMLP-OMe
fMLP-
B
E(+)
Peptide 5, (+)-E
E(-)
Peptide 6, (-)-E
Z(+)
Peptide 7, (+)-Z
to investigate
r
Phe-containing peptides-binding receptors.33–39
Z(-)
Peptide 8, (-)-Z
To measure such homologous desensitization in the case of FPRs,
we recorded intracellular calcium mobilization in response to
fMLP-OMe and
rPhe-containing peptides. The peptide concentra-
tions required for homologous receptor desensitization was deter-
mined (Fig. S3). As shown in Figure 5A and 5B, no significant
second rise of intracellular calcium concentration was observed
by stimulation with E- and Z-isomer peptide. These results indicate
that both peptides bind to FPRs. Similarly, intracellular calcium
concentration did not increased by addition of E-isomer peptide
in human neutrophils pre-stimulated with Z-isomer peptide
(Fig. 5D). Conversely, the second stimulation with Z-peptide raised
intracellular calcium concentration in human neutrophils pre-acti-
vated with E-isomer peptide (Fig. 5C). These results suggested that
E-isomer peptide bound to either FPR1 or FPR2 while Z-isomer
peptide interacted with both receptors. To verify the receptor
interacting with E-isomer peptide, desensitization experiments
were carried out utilizing the FPR2 selective agonist, MMK-1.35
The desensitization of FPR2 by MMK-1 inhibited E-isomer pep-
tide-induced intracellular calcium mobilization (Fig. 6A) although
the mobilization elicited by Z-isomer peptide was observed
(Fig. 6B). In addition, repression of an increase in the concentration
evoked by MMK-1 was observed in human neutrophils pretreated
with E- and Z-isomer peptide (Fig. 6C and 6D). These results
strongly suggest that E-isomer peptide associates with FPR2 while
Z-isomer peptide binds to both FPR1 and FPR2.
Log peptide concentration (M)
Figure 2. Chemotaxis (A) and superoxide production (B) of human neutrophils
stimulated by fMLP-OMe and its derivatives (cross, fMLP; open circle, peptide 5;
closed circle, peptide 6; open triangle, peptide 7; closed triangle, peptide 8). The
data expressed are the means of five separate experiments performed in triplicate.
2.4. Human neutrophils were primed by Z-isomer peptides, but
not E-isomer peptides
Exposure of human neutrophils to fMLP-OMe at concentrations
insufficient to cause superoxide anion production nevertheless

R. Hayashi et al. / Bioorg. Med. Chem. 21 (2013) 668–675
671
Table 2
Biological activities of fMLP-OMe and rPhe-containing peptides
Peptide
Isomer of
rPhe
Chemotaxis
Oꢀ2 Production
Relative activity (%)
Priming activitya (%)
EC50 (nM)
2.5 0.1
Relative activity (%)
EC50 (nM)
fMLP-OMe
100
18
53
12
26
16
2
100
—
—
—
6.4
5
6
7
8
(+)-E
(ꢀ)-E
(+)-Z
(ꢀ)-Z
14
4.7 0.2
21
9.1 2.4
2
10%b
10%b
10%b
114
114
175
200
6
250 80
a
Human neutrophils were preincubated with PBS or 0.1
lM of Phe-containing peptides for 5 min at 37 °C, followed by stimulated with 0.1
lM of fMLP-OMe. The priming
activity was estimated by comparison with superoxide anion production in human neutrophils pretreated with PBS.
b
The mean activity at 1.0 lM.
Jesaitis and co-worker investigated the binding site of fMLP in
FPR1 using receptor mutants40,41 and a photoaffinity labeled fMLP
peptide.42 They proposed that (1) the positioning of fMLP in the
binding pocket of FPR1 was parallel to the fifth transmembrane
(TM) helix, (2) the formyl group formed hydrogen bonds to
Asp106 and Arg 201, (3) the carboxyl group made an ion-pair with
Arg205, (4) Leu side chain was positioned around residues 83–85
(VRK), which is near the extracellular surface of the second TM he-
lix. The importance of Arg84 and Lys85 for the interaction between
fMLP and FPR1 was also reported by Radel et al.43 and Lala et al.44
The molecular modeling of FPR1-the photoaffinity labeled peptide
complex suggested that the Phe side chain bound to the pocket
consisting of the third, the fifth, the sixth, and the seventh TM heli-
ces of FPR1. We showed that FPR1 coupled with only Z-isomer pep-
tide while FPR2 recognized both E- and Z-isomer peptides. Thus, if
the
r
Phe also interacts with the pocket recognizing the Phe of
fMLP, the pocket of FPR1 might be smaller than that of FPR2.
3. Conclusions
5
6
7
8
Herein, we found that fMLP-OMe analogs in which the Phe resi-
due was substituted with the conformationally constrained amino
(+)-E
(-)-E
(+)-Z
(-)-Z
acid, cyclopropane phenylalanine (
biological response in human neutrophils. Among the four different
isomers of Phe studied (Fig. 1), the analogs replaced by E-isomer
Phe (peptides 5 and 6) showed only chemotactic activity while
the Z-isomer Phe-containing peptides 7 and 8 evoked both migra-
r
Phe, Fig. 1), elicited a selective
r
Figure 3. Maximum concentrations of intracellular calcium ion in human neutro-
phils stimulated by fMLP-OMe and its derivatives (0.10 M). The basal level of
intracellular calcium concentration was approximately 140 nM. The data expressed
are the means of five separate experiments performed in triplicate.
r
l
r
tion and superoxide anion production (Fig. 2 and Table 2). Homolo-
gous receptor desensitization experiments revealed that E-isomer
peptide interacted with FPR2 while Z-isomer peptide bound to both
FPR1 and FPR2. Some agonists such as [Thp1,Ain3]fMLP-OMe and
[D
ZLeu2]fMLP-OMe (Fig. S2), are known to specifically evoke biolog-
ical responses in human neutrophils, that is, [Thp1,Ain3]fMLP-OMe
and [D
ZLeu2]fMLP-OMe are selective agonists of migration and
superoxide anion production, respectively.45 Interestingly, while
pure chemoattractant [Thp1,Ain3]fMLP-OMe does not affect the
intracellular calcium concentration, peptides 5 and 6 caused intra-
cellular calcium mobilization as well as fMLP-OMe and Z - isomer
peptides 7 and 8. Fabbri et al. determined the binding affinities of
fMLP-OMe, [Thp1,Ain3]fMLP-OMe, and [ ZLeu2]fMLP-OMe utilizing
D
a competition assay with [3H]-fMLP in human neutrophils.46 The
binding affinity of [Thp1,Ain3]fMLP-OMe was about 50- and 600-
fold lower than that of [D
ZLeu2]fMLP-OMe and fMLP-OMe, respec-
tively. These results seem to suggest that the selectivity of
[Thp1,Ain3]fMLP-OMe was due to its significant low binding affinity
to receptors because less than 1% occupancy of FPRs was suggested
to be required for migration in human neutrophils. In contrast to
[Thp1,Ain3]fMLP-OMe, despite the fact that intracellular calcium
mobilization seems to be important for superoxide anion produc-
tion in human neutrophils, E-isomer peptides 5 and 6 were
selective agonists of migration while increasing intracellular cal-
cium concentrations. Understanding of mechanisms responsible
PBS
Peptide 6
Peptide 8
(-)-E
(-)-Z
Figure 4. Representative enhanced superoxide anion production in human neu-
trophils pre-incubated with Phe-containing fMLP-OMe analog. Human neutro-
phils were pre-incubated with PBS, peptide 6 or peptide 8 (0.1 M) for 5 min at
M) (closed
r
l
37 °C (open arrowhead) before stimulation by fMLP-OMe (1.0
l
arrowhead).

672
R. Hayashi et al. / Bioorg. Med. Chem. 21 (2013) 668–675
A
B
fMLP-OMe (1 µM)
40
40
30
20
10
fMLP-OMe (1 µM)
30
20
10
0
E-isomer peptide
Z-isomer peptide
(1 µM)
(1 µM)
0
0
50
100
150
200
0
50
100
150
200
Time (sec)
Time (sec)
D
C
Z-isomer peptide (1 µM)
E-isomer peptide (1 µM)
40
40
Z-isomer peptide
(1 µM)
30
20
10
0
30
20
10
0
E-isomer peptide
(1 µM)
0
50
100
150
200
0
100
200
300
Time (sec)
Time (sec)
Figure 5. Desensitization of intracellular calcium mobilization in human neutrophils triggered with fMLP-OMe, E-isomer peptide (the equal quantity mixture of peptides 5
and 6), and Z-isomer peptide (that of peptides 7 and 8) at the concentration indicated. Cells were stimulated with fMLP-OMe, followed by the addition of E- (A) and Z-isomer
peptide (B). Cells re-stimulated with Z- (C) and E-isomer peptide (D) after activation with E- and Z-isomer peptide, respectively. Curves show representative experiments.
Open triangles indicate the time point of the agonist addition.
for the selectivity of the reported selective agonists and
r
Phe-
chloroform–methanol (9:1); Rf4, chloroform–methanol–acetic acid
(95:5:1). Components on a TLC plate were colorized by carboniza-
tion with sulfuric acid, o-tolidine, iodine, and ninhydrin.
substituted analogs should prove useful for revealing the relation-
ship between receptor–ligand interactions and biological responses
of human neutrophils.
4.2. Peptide synthesis
4. Experimental
4.2.1. General procedure of Boc-Leu-
A solution of Boc-Leu-OH (72 mg, 0.30 mmol) and N-methyl-
morpholine (33.6 l, 0.30 mmol) in THF (1.0 ml) was cooled to
ꢀ15 °C. Isobutylchloroformate (40 l, 0.30 mmol) was added to
the solution slowly, and then the mixture was stirred for 10 min
at ꢀ15 °C. A solution of Phe-OMe salt (0.30 mmol) and triethyl-
amine (42 l, 0.30 mmol) in dichloromethane (3.0 ml) was added
to the anhydride mixture. The reaction was stirred for 15 min at
ꢀ15 °C, and then at room temperature for 5 h. After removing
the solvent using a rotary evaporator, the residue obtained was
dissolved in ethyl acetate (10 ml). The organic layer was washed
successively with water, 0.5 M sodium bicarbonate aqueous solu-
tion, and 5% potassium hydrogen sulfate aqueous solution. The
solution was dried over anhydrous sodium sulfate. The solvent
was evaporated, and the residue obtained was crystallized by the
addition of petroleum ether to give a white solid.
r
Phe-OMe (9)
4.1. Materials and methods
l
Amino acids, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrogen chloride (EDCꢁHCl), and N-hydroxybenzotriazole (HOBt)
were purchased from the Peptide Institute (Osaka, Japan). Other
chemicals for organic synthesis were obtained from Wako
Chemicals (Osaka, Japan) or Katayama Chemicals (Osaka, Japan),
and used without further purification. 1H NMR spectra were re-
corded with a JEOL-JMN GX 270 spectrophotometer (JEOL, Tokyo)
using tetramethylsilane as an internal standard, and signals were
assigned by 2D-correlation spectroscopy. Description of cyclopro-
pane protons is defined in Figure S1 Supplementary data. Optical
rotations were recorded with JEOL-JMS 300 (JEOL, Tokyo) at
25 °C. Thin-layer chromatography (TLC) was performed on silica
gel GF254 (Merck) with the following solvent systems (v/v); Rf2,
l
r
l

R. Hayashi et al. / Bioorg. Med. Chem. 21 (2013) 668–675
673
A
B
MMK-1(0.1 µM)
MMK-1(0.1 µM)
40
40
30
20
10
0
30
20
10
0
Z-isomer peptide
(1 µM)
E-isomer peptide
(1 µM)
0
50
100
150
200
0
50
100
150
200
Time (sec)
Time (sec)
C
D
Z-isomer peptide (1 µM)
40
40
E-isomer peptide (1 µM)
30
20
10
0
30
20
10
0
Z-isomer peptide
(1 µM)
E-isomer peptide
(1 µM)
MMK-1
(0.1 µM)
MMK-1
(0.1 µM)
0
50
100
150
200
0
80
160
240
Time (sec)
Time (sec)
Figure 6. Simultaneous desensitization of FPR2 by MMK-1 inhibited E-isomer peptide-induced intracellular calcium mobilization. The intracellular calcium mobilization
elicited by E- (A) and Z-isomer peptide (B) in human neutrophils pre-stimulated with the FPR2 selective agonist MMK-1. Cells were pretreated with E- (C) and Z-isomer
peptide (D) before stimulation with MMK-1. Curves show representative experiments. Open triangles indicate the time point of the agonist addition.
Boc-Leu-(+)-
r
EPhe-OMe (9a) yield, 72 mg (60%). ½a D
ꢂ
+22.0°, (c
3.73 (3H, s, methyl ester), 3.02 (1H, dd, Hx), 2.13 (1H, m,Ha), 1.72
(1H, m, Hb), 1.57 (9H, s, Boc group), 1.46 (1H, m, -protons of
0.20, methanol). TLC Rf2 = 0.79, TLC Rf4 = 0.610.44. 1H NMR (CDCl3);
d = 7.41–7.18 (5H, m, aromatic protons), 6.87 (1H, s, NH of (+)-
r
c
Leu), 1.24 (2H, m, b-protons of Leu), 0.82 (6H, dd, d-protons of
Leu). Found; C, 65.28; H, 7.89; N, 6.93. Calcd for C22H32N2O5; C,
65.32; H, 7.79; N, 6.93.
EPhe), 4.87 (1H, d, NH of Leu), 4.12 (1H, m,
a
-proton of Leu),
3.33 (3H, s, methyl ester), 2.86 (1H, dd, Hx), 2.21 (1H, m,Ha), 1.59
(1H, m, Hb), 1.57 (9H, s, Boc group), 1.49 (1H, m, -protons of
c
Boc-Leu-(ꢀ)-
r
ZPhe-OMe (9d) yield, 85 mg (70%). ½a D
ꢂ ꢀ88.5°, (c
Leu), 1.73 (2H, m, b-protons of Leu), 0.97 (6H, dd, d-protons of
Leu). Found; C, 64.88; H, 7.93; N, 6.87. Calcd for C22H32N2O5; C,
65.32; H, 7.79; N, 6.93.
0.20, methanol). TLC Rf2 = 0.77, TLC Rf4 = 0.610.42. 1H NMR (CDCl3);
d = 7.35–7.14 (5H, m, aromatic protons), 5.98 (1H, s, NH of (ꢀ)-
r
ZPhe), 4.68 (1H, d, NH of Leu), 3.90 (1H, m,
a-proton of Leu),
3.73 (3H, s, methyl ester), 2.99 (1H, dd, Hx), 2.18 (1H, m,Ha), 1.69
Boc-Leu-(ꢀ)-
r
EPhe-OMe (9b) yield, 82 mg (68%). ½a D
ꢂ
ꢀ35.3°, (c
0.20, methanol). TLC Rf2 = 0.87, TLC Rf4 = 0.610.61. 1H NMR (CDCl3);
d = 7.39–7.21 (5H, m, aromatic protons), 6.91 (1H, s, NH of (ꢀ)-
(1H, m, Hb), 1.57 (9H, s, Boc group), 1.47 (1H, m, -protons of
c
Leu), 1.25 (2H, m, b-protons of Leu), 0.80 (6H, dd, d-protons of
Leu). Found; C, 65.31; H, 7.96; N, 6.90. Calcd for C22H32N2O5; C,
65.32; H, 7.79; N, 6.93.
r
EPhe), 4.93 (1H, d, NH of Leu), 4.14 (1H, m,
a-proton of Leu),
3.33 (3H, s, methyl ester), 2.83 (1H, dd, Hx), 2.23 (1H, m,Ha), 1.60
(1H, m, Hb), 1.61 (9H, s, Boc group), 1.46 (1H, m, -protons of
c
Leu), 1.73 (2H, m, b-protons of Leu), 0.97 (6H, dd, d-protons of
Leu). Found; C, 64.82; H, 7.97; N, 6.93. Calcd for C22H32N2O5; C,
65.32; H, 7.79; N, 6.93.
4.2.2. General procedure of preparation of formyl-Met-Leu-
r
Phe-OMe (5, 6, 7, and 8)
Boc-protecting dipeptide 9 (81 mg, 0.20 mmol) was treated
Boc-Leu-(+)-
r
ZPhe-OMe (9c) yield, 75 mg (62%). ½a D
ꢂ
+54.5°, (c
with trifluoroacetic acid (1.0 ml) for 30 min in an ice-bath. Trifluo-
roacetic acid was evaporated using nitrogen gas flashing. Diethyl
ether was added to the residue obtained, resulting in white solid.
The solid was dissolved in dimethylformamide (0.50 ml), and then
0.20, methanol). TLC Rf2 = 0.75, TLC Rf4 = 0.610.40. 1H NMR (CDCl3);
d = 7.35–7.13 (5H, m, aromatic protons), 5.95 (1H, s, NH of (+)-
r
ZPhe), 4.67 (1H, d, NH of Leu), 3.85 (1H, m,
a-proton of Leu),

674
R. Hayashi et al. / Bioorg. Med. Chem. 21 (2013) 668–675
triethylamine (70
ll, 0.50 mmol) was added to the solution. For-
4.4. Chemotaxis
myl-Met-OH (35 mg, 0.20 mmol) and HOBt (32 mg, 0.24 mmol)
was added to the solution. After the mixture was cooled to 0 °C,
EDCꢁHCl (42 mg, 0.22 mmol) was added. The mixture was stirred
for 2 h in an ice-bath, and then for 10 h at room temperature.
The solvent was removed using a rotary evaporator in vacuo. The
residue obtained was dissolved in ethyl acetate (15 ml), and the or-
ganic layer was washed with water, 0.5 M sodium bicarbonate
aqueous solution, 5% potassium hydrogen sulfate, and brine. After
the solution was dried over anhydrous sodium sulfate, the solvent
was evaporated. Diethyl ether was added to the residue obtained,
resulting in white solid as the formyl peptide.
The chemotactic assay was performed in a micro chemotaxis
chamber.48 Cells were suspended in Krebs–Ringer phosphate buf-
fer containing 1 mM CaCl2, 5 mM D-glucose, and 0.1% bovine serum
albumin, at a concentration of 1.0 ꢃ 106 cells/ml. Peptide solutions
or buffer were placed in the lower wells. The polyvinylpyrrolidone-
free polycarbonate filter was assembled and incubation was car-
ried out at 37 °C for 60 min in humidified air.49 After incubation,
the filter was stained with Diff-Quick (Kokusai Siyaku, Kobe) solu-
tion and counted at 1000ꢃ magnification for 5 fields. The chemo-
tactic index was estimated as the migrated cell number. Data are
reported as the mean value standard error of three independent
experiments.
Formyl-Met-Leu-(+)-
r
EPhe-OMe (5) yield, 72 mg (77%). ½a D
ꢂ
+15.6°, (c 0.50, methanol). TLC Rf2 = 0.42, TLC Rf4 = 0.610.46. 1H
NMR (DMSO-d6); d = 8.78 (1H, s, NH of (+)-
NH of Met), 8.05 (1H, d, NH of Leu), 8.03 (1H, s, formyl group),
7.28–7.20 (5H, m, aromatic protons), 4.44 (1H, m, -proton of
Met), 4.31 (1H, m, -proton of Leu), 3.23 (3H, s, methyl ester),
2.76 (1H, dd, Hx), 2.44 (2H, t, -protons of Met), 2.09 (1H, m,Ha),
2.04 (3H, s, methyl protons of Met), 1.86 (2H, m, b-protons of
Met), 1.64 (1H, m, -protons of Leu), 1.50 (2H, m, b-protons of
r
EPhe), 8.27 (1H, d,
4.5. Superoxide anion production
a
a
Superoxide anion production was measured as superoxide dis-
mutase inhibitable reduction of ferricytochrome c.50,51 The reduc-
tion of the ferricytochrome c concentration was monitored with
a Shimadzu UV-3000 dual-wavelength spectrophotometer (Kyoto,
Japan) at 540–550 nM. A cell suspension (1 ꢃ 106 cells/ml), con-
c
c
Leu), 1.36 (1H, m, Hb), 0.90 (6H, dd, d-protons of Leu), Found; C,
59.57; H, 7.11; N, 9.07. Calcd for C23H33N3O5S; C, 59.59; H, 7.18;
N, 9.07.
taining 1 mM CaCl2, 5 mM D-glucose, and 100 mM ferricytochrome
c was preincubated at 37 °C for 5 min before the addition of vari-
ous concentrations of peptides. The superoxide anion release was
calculated based on a molar absorption coefficient of 19.1 ꢃ
103 Mꢀ1 cmꢀ1. Data are reported as the mean value standard er-
ror for at least three independent experiments.
Formyl-Met-Leu-(ꢀ)-
r
EPhe-OMe (6) yield, 72 mg (77%). ½a D
ꢂ
ꢀ116.8°, (c 0.50, methanol). TLC Rf2 = 0.43, TLC Rf4 = 0.610.48. 1H
NMR (DMSO-d6); d = 8.79 (1H, s, NH of (ꢀ)-
r
EPhe), 8.05 (1H, d,
NH of Met), 8.27 (1H, d, NH of Leu), 7.99 (1H, s, formyl group),
7.32–7.17 (5H, m, aromatic protons), 4.44 (1H, m, -proton of
Met), 4.31 (1H, m, -proton of Leu), 3.23 (3H, s, methyl ester),
2.72 (1H, dd, Hx), 2.43 (2H, t, -protons of Met), 2.07 (1H, m,Ha),
2.02 (3H, s, methyl protons of Met), 1.82 (2H, m, b-protons of
Met), 1.61 (1H, m, -protons of Leu), 1.50 (2H, m, b-protons of
a
a
4.6. Intracellular calcium mobilization
c
Intracellular calcium mobilization was measured by the Fura-2
method.50,51 Cells (2 ꢃ 106 cells/ml) were incubated with 4 mM
Fura-2 AM at 37 °C for 30 min, washed twice with PBS, and then
c
Leu), 1.37 (1H, m, Hb), 0.89 (6H, dd, d-protons of Leu), Found; C,
59.41; H, 7.19; N, 9.00. Calcd for C23H33N3O5S; C, 59.59; H, 7.18;
N, 9.07.
suspended in PBS containing 1 mM CaCl2 and 5 mM
D-glucose.
Changes in the fluorescence emission intensity at 490 nM (excita-
tions at 340 and 380 nM) were monitored with a Shimadzu RF-
5000 spectrofluorophotometer. The cells were lysed by the addi-
tion of Triton X-100 for the determination of the maximum fluo-
rescence, and the minimum fluorescence was then determined
by addition of ethylene glycol tetraacetic acid. The concentration
of intracellular calcium concentration was calculated using a Kd va-
lue of 224 nM for calcium ion. Data are reported as the mean
value standard error for three independent experiments.
Formyl-Met-Leu-(+)-
r
ZPhe-OMe (7) yield, 70 mg (76%). ½a D
ꢂ
+26.4°, (c 0.50, methanol). TLC Rf2 = 0.47, TLC Rf4 = 0.610.51. 1H
NMR (DMSO-d6); d = 8.13 (1H, s, NH of (+)-
NH of Met), 8.18 (1H, d, NH of Leu), 7.99 (1H, s, formyl group),
7.25–7.12 (5H, m, aromatic protons), 4.38–4.32 (1H, m, -proton
of Met), 4.24–4.15 (1H, m, -proton of Leu), 3.62 (3H, s, methyl es-
ter), 3.17 (1H, dd, Hx), 2.35 (2H, t, -protons of Met), 2.03 (3H, s,
methyl protons of Met), 1.81–1.59 (4H, m, b-protons of Met, Ha,
Hb), 1.45 (1H, m, -protons of Leu), 1.21 (2H, m, b-protons of
r
ZPhe), 7.70 (1H, d,
a
a
c
c
Leu), 0.77(6H, dd, d-protons of Leu), Found; C, 59.19; H, 7.13; N,
8.80. Calcd for C23H33N3O5S; C, 59.59; H, 7.18; N, 9.07.
Supplementary data
Formyl-Met-Leu-(ꢀ)-
r
ZPhe-OMe (8) yield, 70 mg (76%). ½a D
ꢂ
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.11.046.
ꢀ111.0°, (c 0.50, methanol). TLC Rf2 = 0.46, TLC Rf4 = 0.610.52. 1H
NMR (DMSO-d6); d = 8.29 (1H, s, NH of (ꢀ)-
r
ZPhe), 7.80 (1H, d,
NH of Met), 8.14 (1H, d, NH of Leu), 8.00 (1H, s, formyl group),
7.26–7.14 (5H, m, aromatic protons), 4.39 (1H, m, -proton of
Met), 4.16 (1H, m, -proton of Leu), 3.61 (3H, s, methyl ester),
3.01 (1H, dd, Hx), 2.40 (2H, t, -protons of Met), 2.03 (3H, s, methyl
protons of Met), 1.87–1.65 (4H, m, b-protons of Met, Ha, Hb), 1.19
(1H, m, -protons of Leu), 0.99–0.78 (2H, m, b-protons of Leu),
References and notes
a
1. Prossnitz, E. R.; Ye, R. D. Pharmacol. Ther. 1997, 74, 73.
2. Torrini, I.; Zecchini, G. P.; Paradisi, M. P.; Lucente, G.; Gavuzzo, E.; Mazza, F.;
Pochetti, G.; Spisani, S.; Giuliani, A. L. Int. J. Pept. Protein Res. 1991, 38, 495.
3. Fabbri, E.; Spisani, S.; Biondi, C.; Barbin, L.; Colamussi, M. L.; Cariani, A.;
Traniello, S.; Torrini, I.; Ferretti, M. E. Biochim. Biophys. Acta 1997, 1359, 233.
4. Zecchini, G. P.; Paradisi, M. P.; Torrini, I.; Lucente, G.; Gavuzzo, E.; Mazza, F.;
Pochetti, G.; Paci, M.; Sette, M.; Di Nola, A. Biopolymers 1993, 33, 437.
5. Hruby, V. J.; Li, G.; Haskell-Luevano, C.; Shenderovich, M. Biopolymers 1997, 43,
219.
a
c
c
0.69 (6H, dd, d-protons of Leu), Found; C, 59.50; H, 7.14; N, 9.03.
Calcd for C23H33N3O5S; C, 59.59; H, 7.18; N, 9.07.
6. Kimura, H.; Stammer, C. H.; Shimohigashi, Y.; Ren-Lin, C.; Stewart, J. Biochem.
Biophys. Res. Commun. 1983, 115, 112.
4.3. Isolation of human neutrophils
7. Mapelli, C.; Kimura, H.; Stammer, C. H. Int. J. Pept. Protein Res. 1986, 28, 347.
8. Shimohigashi, Y.; Costa, T.; Pfeiffer, A.; Herz, A.; Kimura, H.; Stammer, C. H.
FEBS Lett. 1987, 222, 71.
9. Shimohigashi, Y.; Takano, Y.; Kamiya, H.; Costa, T.; Herz, A.; Stammer, C. H.
FEBS Lett. 1988, 233, 289.
Human neutrophils were isolated from heparinized venous
blood obtained from healthy volunteers. Standard isolation tech-
niques involving Ficoll/Hypaque gradients were used,47 followed
by dextran sedimentation and hypotonic lysis to remove erythro-
cytes. Cells were suspended in PBS without calcium chloride.
10. Mapelli, C.; Stammer, C. H.; Lok, S.; Mierke, D. F.; Goodman, M. Int. J. Pept.
Protein Res. 1988, 32, 484.

R. Hayashi et al. / Bioorg. Med. Chem. 21 (2013) 668–675
675
11. Ogawa, T.; Yoshitomi, H.; Kodama, H.; Waki, M.; Stammer, C. H.; Shimohigashi,
Y. FEBS Lett. 1989, 250, 227.
34. Klein, C.; Paul, J. I.; Sauvé, K.; Schmidt, M. M.; Arcangeli, L.; Ransom, J.;
Trueheart, J.; Manfredi, J. P.; Broach, J. R.; Murphy, A. J. Nat. Biotechnol. 1998, 16,
1334.
35. Hu, J. Y.; Le, Y.; Gong, W.; Dunlop, N. M.; Gao, J. L.; Murphy, P. M.; Wang, J. M. J.
Leukoc. Biol. 2001, 70, 155.
36. Heit, B.; Tavener, S.; Raharjo, E.; Kubes, P. J. Cell Biol. 2002, 159, 91.
37. Fu, H.; Bylund, J.; Karlsson, A.; Pellmé, S.; Dahlgren, C. Immunology 2004, 112,
201.
38. Ernst, S.; Lange, C.; Wilbers, A.; Goebeler, V.; Gerke, V.; Rescher, U. J. Immunol.
2004, 172, 7669.
39. Karlsson, J.; Fu, H.; Boulay, F.; Bylund, J.; Dahlgren, C. Biochem. Pharmacol. 2006,
71, 1488.
40. Miettinen, H. M.; Mills, J. S.; Gripentrog, J. M.; Dratz, E. A.; Granger, B. L.;
Jesaitis, A. J. J. Immunol. 1997, 159, 4045.
41. Mills, J. S.; Miettinen, H. M.; Cummings, D.; Jesaitis, A. J. J. Biol. Chem. 2000, 275,
39012.
42. Mills, J. S.; Miettinen, H. M.; Barnidge, D.; Vlases, M. J.; Wimer-Mackin, S.;
Dratz, E. A.; Sunner, J.; Jesaitis, A. J. J. Biol. Chem. 1998, 273, 10428.
43. Radel, S. J.; Genco, R. J.; de Nardin, E. Infect. Immun. 1994, 62, 1726.
44. Lala, A.; Gwinn, M.; de Nardin, E. Eur. J. Biochem. 1999, 264, 495.
45. Selvatici, R.; Falzarano, S.; Mollica, A.; Spisani, S. Eur. J. Pharmacol. 2006, 534, 1.
46. Fabbri, E.; Spisani, S.; Barbin, L.; Biondi, C.; Buzzi, M.; Traniello, S.; Zecchini, G.
P.; Ferretti, M. E. Cell. Signal. 2000, 12, 391.
47. Böyum, A. Scand. J. Clin. Lab. Invest. Suppl. 1968, 97, 77.
48. Falk, W.; Goodwin, R. H.; Leonard, E. J. J. Immunol. Methods 1980, 33, 239.
49. Harvath, L.; Falk, W.; Leonard, E. J. J. Immunol. Methods 1980, 37, 39.
50. Miyazaki, M.; Kodama, H.; Fujita, I.; Hamasaki, Y.; Miyazaki, S.; Kondo, M. J.
Biochem. 1995, 117, 489.
12. Suzuki, M.; Gooch, E.; Stammer, C. Tetrahedron Lett. 1983, 24, 3839.
13. Mapelli, C.; Turocy, G.; Switzer, F. J. Org. Chem. 1989, 54, 145.
14. Ahmad, S.; Phillips, R. J. Med. Chem. 1992, 35, 1410.
15. Kimura, H.; Stammer, C. H. J. Org. Chem. 1983, 48, 2440.
16. Fernández, M.; de Frutos, M.; Marco, J. Tetrahedron Lett. 1989, 30, 3101.
17. Ferretti, M. E.; Spisani, S.; Pareschi, M. C.; Buzzi, M.; Cavallaro, R.; Traniello, S.;
Reali, E.; Torrini, I.; Paradisi, M. P.; Zecchini, G. P. Cell. Signal. 1994, 6, 91.
18. Ferretti, M. E.; Nalli, M.; Biondi, C.; Colamussi, M. L.; Pavan, B.; Traniello, S.;
Spisani, S. Cell. Signal. 2001, 13, 233.
19. Reibman, J.; Meixler, S.; Lee, T. C.; Gold, L. I.; Cronstein, B. N.; Haines, K. A.;
Kolasinski, S. L.; Weissmann, G. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6805.
20. Cornish, C. J.; Devery, J. M.; Poronnik, P.; Lackmann, M.; Cook, D. I.; Geczy, C. L.
J. Cell. Physiol. 1996, 166, 427.
21. Sklar, L. A.; Omann, G. M.; Painter, R. G. J. Cell Biol. 1985, 101, 1161.
22. Sklar, L. A.; Hyslop, P. A.; Oades, Z. G.; Omann, G. M.; Jesaitis, A. J.; Painter, R. G.;
Cochrane, C. G. J. Biol. Chem. 1985, 260, 11461.
23. Downey, G. P. Curr. Opin. Immunol. 1994, 6, 113.
24. Bae, Y.-S.; Song, J. Y.; Kim, Y.; He, R.; Ye, R. D.; Kwak, J.-Y.; Suh, P.-G.; Ryu, S. H.
Mol. Pharmacol. 2003, 64, 841.
25. Issekutz, A. C.; Lee, K. Y.; Biggar, W. D. Infect. Immun. 1979, 24, 295.
26. Karnad, A. B.; Hartshorn, K. L.; Wright, J.; Myers, J. B.; Schwartz, J. H.; Tauber, A.
I. Blood 1989, 74, 2519.
27. Bellavite, P.; Chirumbolo, S.; Lippi, G.; Guzzo, P.; Santonastaso, C. Cell Biochem.
Funct. 1993, 11, 93.
28. Hallett, M. B.; Lloyds, D. Immunol. Today 1995, 16, 264.
29. Condliffe, A. M.; Kitchen, E.; Chilvers, E. R. Clin. Sci. 1998, 94, 461.
30. Swain, S. D.; Rohn, T. T.; Quinn, M. T. Antioxid. Redox Signal. 2002, 4, 69.
31. Fu, H.; Karlsson, J.; Bylund, J.; Movitz, C.; Karlsson, A.; Dahlgren, C. J. Leukoc.
Biol. 2006, 79, 247.
51. Hayashi, R.; Osada, S.; Yoshiki, M.; Sugiyama, D.; Fujita, I.; Hamasaki, Y.;
Kodama, H. J. Biochem. 2006, 139, 981.
52. Stammer, C. H. Tetrahedron 1990, 46, 2231.
32. Ali, H.; Richardson, R. M.; Haribabu, B.; Snyderman, R. J. Biol. Chem. 1999, 274,
6027.
53. Cativiela, C.; Diaz-de-Villegas, M.; Jim nez, A. I.; L pez, P.; Marraud, M.; Oliveros,
L. Chirality 1999, 11, 583.
33. Tomhave, E. D.; Richardson, R. M.; Didsbury, J. R.; Menard, L.; Snyderman, R.;
Ali, H. J. Immunol. 1994, 153, 3267.