Synthesis and biological evaluation of new active For-Met-Leu-Phe-OMe analogues containing para-substituted Phe residues

Article abstract of DOI:10.1002/psc.2414

In the present study, we report synthesis and biological evaluation of the N-Boc-protected tripeptides 4a-l and N-For protected tripeptides 5a-l as new For-Met-Leu-Phe-OMe (fMLF-OMe) analogues. All the new ligands are characterized by the C-terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a-l and the antagonism of 4a-l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined on human neutrophils. No synthesized compounds has higher activity than the standard fMLF-OMe tripeptide to stimulate chemotaxis, although compounds 5a and 5c with -CH₃ and -C(CH₃)₃, respectively, in position 4 on the aromatic ring, are better than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i, containing -F and -I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed.

Full text of DOI:10.1002/psc.2414

Research Article
Received: 12 December 2011
Revised: 28 February 2012
Accepted: 14 March 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2414
Synthesis and biological evaluation of
new active For-Met-Leu-Phe-OMe
analogues containing para-substituted
Phe residues
Adriano Mollica,a* Federica Feliciani,a Azzurra Stefanucci,a
Roberto Costante,a Gino Lucente,b Francesco Pinnen,a
Daniela Notaristefanoc and Susanna Spisanic
In the present study, we report synthesis and biological evaluation of the N-Boc-protected tripeptides 4a–l and N-For
protected tripeptides 5a–l as new For-Met-Leu-Phe-OMe (fMLF-OMe) analogues. All the new ligands are characterized by
the C-terminal Phe residue variously substituted at position 4 of the aromatic ring. The agonism of 5a–l and the antagonism
of 4a–l (chemotaxis, superoxide anion production, lysozyme release as well as receptor binding affinity) have been examined
on human neutrophils. No synthesized compounds has higher activity than the standard fMLF-OMe tripeptide to stimulate
chemotaxis, although compounds 5a and 5c with -CH3 and -C(CH3)3, respectively, in position 4 on the aromatic ring, are better
than the standard tripeptide to stimulate the production of superoxide anion, in higher concentration. Compounds 4f and 4i,
containing -F and -I in position 4, respectively, on the aromatic ring of phenylalanine, exhibit significant chemotactic
antagonism. The influence of the different substitution at the position 4 on the aromatic ring of phenylalanine is discussed.
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: biological activity; chemotaxis; receptor binding; human neutrophils; fMLF-OMe analogues; peptidomimetics
relationships concerning N-formylpeptides are still scarcely
understood and continue to be the object of active investigation
Introduction
The chemotactic agent N-formyl-L-methionyl-L-leucyl-L-phenylal-
anine (For-Met-Leu-Phe; fMLF) can be isolated from bacterial
suspensions and can also be found in disrupted mitochondria
[1,2]. This small N-acyl tripeptide is the prototype and the most
active component of a family of N-formyl-methionyl peptides
that act as molecular switches capable to trigger the directional
migration (chemotaxis) towards the infection sites of human
neutrophils, the phagocytic cells specialized in host defense
against invading pathogens. The induced chemotactic response
is mediated by the interaction of the N-formylpeptides with
specific G-protein-linked receptors (high-affinity formylpeptide
receptor, FPR and its low-affinity subtype, FPRL-1) located on
neutrophil cell membrane [3–5]. The FPRs activation initiates
the migration of neutrophils along the increasing concentration
gradient of chemoattractant agents and is followed by the
production of bacterial killing cytotoxic agents triggered by the
interaction of the ligand with low-affinity FPRL-1 receptors.
Relevant and extensively studied among these functions are the
catalyzed conversion of molecular oxygen to superoxide anion
(OÀ2 ) (respiratory burst) [6] and the release of lysosomal enzymes.
However, because of the complexity of the intracellular signaling
pathways [7] and the peculiar characteristics of the involved
receptors, which are rapidly desensitized and internalized soon
after the interaction with the ligand, several aspects concerning
the involved biochemical mechanisms and the structure–activity
[8–12].
Since the Schiffmann original discovery of the chemoattractant
activity of N-formylpeptides [13], the tripeptide fMLF and its
methyl ester For-Met-Leu-Phe-OMe (fMLF-OMe) have been
adopted as reference model for structure–activity relationships
and conformational studies [8]. Great attention has been
dedicated to the role exerted by the N-formyl group and the
Met residue at the first position. Results clearly indicate that
these two N-terminal elements are required for optimum activity
although different amino acids, with linear or cyclic aliphatic side
chains, can suitably replace the central leucine. Comparable less
attention has been dedicated to the C-terminal third residue.
*
Correspondence to: Adriano Mollica, Dipartimento di Scienze del Farmaco,
Università di Chieti-Pescara “G. d’Annunzio”, Via dei Vestini 31, 66100 Chieti,
Italy. E-mail: a.mollica@unich.it
a Dipartimento di Scienze del Farmaco, Università di Chieti-Pescara “G. d’Annunzio”,
Via dei Vestini 31, 66100 Chieti, Italy
b Istituto di Chimica Biomolecolare (CNR) c/o Dipartimento di Chimica e
Tecnologie del Farmaco, “Sapienza”, Università di Roma, P.le A.Moro,
00185 Roma, Italy
c
Dipartimento di Biochimica e Biologia Molecolare, Università di Ferrara, Via L.
Borsari 46, 44121 Ferrara, Italy
J. Pept. Sci. (2012)
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

MOLLICA ET AL.
Early studies performed on the fMLF-OH analogue For-Nle-Leu-
Phe(4-Cl)-OH suggested that the receptor area interacting with
the aromatic ring should be spatially restricted [14]. More
recently, a series of fMLF-OMe analogues with hydrophilic
residues replacing the Phe at position 3 has been examined,
and a good interaction with the receptor subtype stimulating
the chemotactic migration has been evidenced [15]. However, a
systematic replacement of the native residue at position 3 with
Phe analogues, characterized by different substituents on the
aromatic ring, is not thus far available. This approach should
give valuable information on the features of the hydrophobic
receptor pocket as well as on the possibility to obtain ligands
capable to select the various neutrophil biological functions.
Here, we report results concerning synthesis and biological
evaluation of a series of fMLF-OMe analogues in which the para
hydrogen atom of the C-terminal Phe aromatic ring is replaced
by a series of substituents possessing different steric and
electronic properties (Figure 1). The biological activity (chemo-
taxis, superoxide anion production and lysozyme release) has
been determined on human neutrophils and compared with
that of the reference ligand fMLF-OMe. The N-formyl tripeptides
5a–l were examined as agonists, whereas the corresponding
N-Boc derivatives 4a–l, obtained as intermediates during the
synthesis of 5a–l, were tested for their activity as antagonists
towards the biological functions stimulated by fMLF-OMe.
Antagonistic activity at the FPRs is the object of active research
due to the great potential as diagnostic and therapeutic agents
of this type of ligands [16,17]. The different biochemical behavior
of N-formyl and N-Boc derivatives – agonism versus antagonism –
is well known and only in part related to the different size of
the groups. The key factor resides in the type of the bond joining
the N-terminal protecting group to the backbone, which is
urethanic in the Boc as opposed to amidic in the For group. In
following this observation, several potent chemotactic antago-
nists, characterized by urethanic or ureic protecting group at
the amino terminus, have been synthesized [18–22].
Experimental Procedures
All the starting materials and reagents were purchased from
Sigma-Aldrich (Sigma-Aldrich Corp., Saint Louis, MO, USA).
1H NMR spectra were determined in CDCl3 solution with a Varian
300 MHz (Varian Medical Systems, Inc., Palo Alto, CA, USA), and
chemical shifts were referred to TMS. The mass spectra were
performed on a TOF-ESI spectrometer. Thin layer chromatography
was performed on silica gel Merck (Merck & Co., Inc., Whitehouse
Station, NJ, USA) 60 F254 plates. Experimental detail and character-
ization of all new compounds can be found in the supporting
information.
Synthesis
The synthesis of the intermediate products 1a-l and final pro-
ducts 4a-l and 5a-l is reported in Schemes 1, 2, 3 respectively.
The Boc-protected tripeptides 4a–l were synthesized starting
from Boc-Met-Leu-OMe. The dipeptide methyl ester was hydro-
lysed to the intermediate Boc-Met-Leu-OH and then coupled with
the different HCl.H-Phe(4-R)-OMe (1a–l) by using the usual EDC/
HOBt procedure (Scheme 2). Aminoesters 1a–l were obtained in a
single step starting from the commercially available N-protected
aminoacids Boc-Phe(4-R) -OH by treatment with SOCl2/MeOH as
reported in Scheme 1. The N-Boc tripeptides 4a–l were then di-
rectly converted into the corresponding N-formyl derivatives
according to the procedure of Lajoie-Kraus [23]. Thus, treatment
with formic acid, followed by ethyl 2-ethoxy-1,2-dihydro-1-
quinolinecarboxylate (EEDQ), gave the derivatives 5a–l (Scheme 3).
All products were synthesized in solution by using the EDC/
HOBt/DIPEA coupling method. The corresponding active esters
of additives such as N-hydroxy derivatives are less reactive than
O-acylisourea. However, these additive increases the efficiency
of carbodiimide-mediated reactions. First of all, they suppress
the formation of N-acylurea. The beneficial effect of HOBt is
attributed to its capacity to protonate O-acylisourea, thus
preventing the intramolecular reaction from occurring and
shifting the reaction to form the corresponding active esters
and thereby decreasing the degree of racemization in numerous
cases [24].
R'
R'
S
a: CH3
b: C6H5
c: tBu
O
H
d: CF3
e: CN
f: F
g: Cl
h: Br
i: I
OMe
N
R
N
N
H
H
O
O
4 a-l: R = tBuOCO-
5 a-l: R = HCO-
The Na-terminal Boc-protected peptides were all deprotected
by a mixture of TFA in DCM 1 : 1 at r.t. The intermediate TFA salts
were used for the following reaction without further purifications.
Intermediate products 3, 5 were purified by precipitation in
l: NO2
fMLF-OMe: R = HCO- ; R' = H
Figure 1. Relevant structures discussed in the text.
R:
R
R
1a: CH3
1b: C6H5
1c: tBu
1d: CF3
1e: CN
a
OH
OMe
HCl. H2N
Boc
N
1f: F
H
1g: Cl
1h: Br
1i: I
O
O
1 a-l
1l: NO2
Scheme 1. Synthesis of compounds 1a–l: (a) SOCl2, MeOH.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. (2012)

CHEMOTACTIC PEPTIDES fMLF-OMe ANALOGUES
S
S
S
O
O
a
H
b
OH
H
N
Boc
N
N
Boc
OMe
N
Boc
H
OH
N
H
H
O
O
O
2
3
R
R:
S
4a: CH3
O
4b: C6H5
4c: tBu
4d: CF3
4e: CN
4f: F
c
H
N
OMe
Boc
N
N
H
H
O
O
4g: Cl
4h: Br
4i: I
4l: NO2
4 a-l
Scheme 2. Synthesis of compounds 4a–l: (a) HCl.H-Leu-OMe, DIPEA, HCl.EDC, HOBt, CH2Cl2; (b) NaOH 1N, MeOH; (c) HCl.H-Phe(4-R)-OMe, EDC,
HOBt.H2O, DIPEA, CH2Cl2.
Scheme 3. Synthesis of compounds 5a–l: (a) HCOOH; (b) CHCl3, EEDQ.
EtOAc, the filtrate was washed with 3 Â 50 ml of NaHCO3 s.s.,
3 Â 50 ml of 5% citric acid, 3 Â 50 ml of deionized water, dried
under vacuum, followed by trituration with warm diethyl ether
and used in the next step without further purification. All
Boc-protected products 1a–l, 2a–l, 3a–l were purified by silica
gel column chromatography. All the final products 4a–l and
5a–l were purified by RP-HPLC with C18 semipreparative column
using a linear gradient of H2O/acetonitrile as eluent from 5%
acetonitrile to 90% acetonitrile in 45 min. All the intermediate
salts were used in the next step without further purifications.
The purity of the final products, determined by NMR analysis
and by analytical RP-HPLC (C18-bonded 4.6 Â 150 mm) at a flow
rate of 1 ml/min on a Waters binary pump 1525 (Waters Corpora-
tion, Milford, MA, USA) using an isocratic elution of 20% CH3CN/
H2O 0.1% TFA, monitored with a Waters 2996 Photodiode Array
Detector, was found to be >95%.
methanol (10 ml), and the resulting solution was stirred at r.t. for
3 h. The oily residue obtained after evaporation under reduced
pressure was triturated with Et2O to give the desired product as
chloridrate salt, used for the subsequent reaction.
Coupling reaction
To an ice-cooled mixture containing N-protected amino acid or
peptide (0.7 mmol) in CH2Cl2 (20 ml), EDC.HCl (0.8 mmol), HOBt
(0.8 mmol), DIPEA (2.3 mmol) and the required C-protected amino
acid (0.7 mmol) were added. The reaction mixture was allowed to
warm at r.t. overnight and evaporated under reduced pressure.
The residue was then dissolved in EtOAc and washed with three
portions of 5% citric acid, NaHCO3 s.s., brine. The organic phase
was dried on Na2SO4, and the solvent evaporated under reduced
pressure to give the desired product.
Methyl ester hydrolysis
General Synthetic Procedures
To an ice-cooled solution of methyl ester (5.3 mmol) in methanol
(60 ml), 1N NaOH (16 ml) was added dropwise. The mixture was
stirred at 0 ꢀC for 15 min and then at room temperature
overnight. The solvent was evaporated under reduced pressure,
water (15 ml) was added and the solution extracted with diethyl
Esterification of Boc-protected amino acids
Thionyl chloride (1.6mmol) was added dropwise at 0ꢀC to a stirred
solution of Boc-protected amino acid (0.8mmol) in anhydrous
J. Pept. Sci. (2012)
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

MOLLICA ET AL.
ether. The aqueous phase was then acidified with 5% citric acid
and extracted with AcOEt (3 Â 20 ml). The organic phase was
dried on Na2SO4 and evaporated under reduced pressure to give
the desired compound.
Neutrophils were incubated with 5 mg/ml cytochalasin B (Sigma)
for 5 min prior to activation by peptides. Results were expressed
as net nmoles of OÀ2 /1Â 106 cells/5min and are the mean of six
separate experiments performed in duplicate.
Tripeptide formylation
Enzyme assay
The N-Boc-tripeptide (0.2 mmol) was dissolved in formic acid
(5 ml) and the mixture was stirred at room temperature for 24 h.
After removal of the excess of formic acid under reduced
pressure, the residue was dissolved in 5 ml of dry chloroform.
EEDQ (0.2 mmol) was added, and the solution was stirred at room
temperature for 24 h. Evaporation under reduced pressure
afforded a crude residue, which was purified by silica gel chroma-
tography to give the pure desired compound.
The release of neutrophil granule enzymes was evaluated by
determination of lysozyme activity, modified for microplate-based
assays. Cells, 3Â 106/well, were first incubated in triplicate wells
of microplates with 5 mg/ml cytochalasin B at 37ꢀC for 15 min and
then in the presence of each peptide at a final concentration of
10À10–5 Â 10À5 M for a further 15 min. The plates were then
centrifuged at 400Â g for 5 min, and the lysozyme was quantified
nephelometrically by the rate of lysis of cell wall suspension of
Micrococcus lysodeikticus. The reaction rate was measured using a
microplate reader at 465 nm. Enzyme release was expressed as
a net percentage of total enzyme content released by 0.1% Triton
X-100. Total enzyme activity was 85 Æ 1 mg/1 Â 107 cells/min. The
values are the mean of five separate experiments performed
in duplicate.
Biological Activity Studies
Cell preparation
Cells were obtained from the blood of healthy subjects, and human
peripheral blood neutrophils were purified by using the standard
techniques of dextran (Pharmacia, Uppsala, Sweden) sedimenta-
tion, centrifugation on Ficoll-Paque (Pharmacia), and hypotonic
lysis of contaminating red cells. Cells were washed twice and
resuspended in Krebs–Ringer phosphate containing 0.1% w/v
glucose (KRPG), pH 7.4, at final concentration of 50 Â 106 cells/ml
and kept at room temperature until used. Neutrophils were
98–100% viable, as determined using the Trypan Blue exclusion test.
The study was approved by the local Ethics Committee, and
informed consent was obtained from all participants.
Binding assays
In a final volume of 0.2 ml, 5.10À7 cells/ml were incubated in the
3
presence of 6 nM H-fMet-Leu-Phe and different concentrations
of displacing agents. The incubation was carried out at 37 ꢀC for
15 min, and the bound ligand was separated from the free ligand
by rapid filtration of the mixture through Whatman GF/B glass
fiber filters. Radioactivity of the filters was then measured in a
Beckman LS 1801 spectrometer (Beckman Coulter Inc., Brea, CA,
USA). Reported values refer to specific binding, defined as the
total amount of 3H-fMLF bound minus the nonspecific. Non-
specific is defined as the amount of binding not inhibited by
10-mM unlabeled fMLF.
Random locomotion
Random locomotion was performed with 48-well microchemotaxis
chamber (Bio Probe, Milan, Italy), and migration into the filter was
evaluated by the leading-front method [25]. The actual control
random movement is 35 Æ 3 mm SE of 10 separate experiments
performed in duplicate.
Antagonist activity
Antagonist activity was determined by measuring the peptide’s
ability to inhibit chemotaxis, OÀ2 production or granule enzyme
release as activated by fMLF-OMe. Antagonist’s activity data
(percentage of activity) were obtained by comparing the chemo-
tactic index, nanomoles of OÀ2 or the percentage of lysozyme
release in the absence (100%) and in the presence of analogues.
Derivatives were added to neutrophils 10 min before the incuba-
tion step for cellular functionality. Each value represents an
average of six separate experiments performed in duplicate.
Chemotaxis
Each peptide was added to the lower compartment of the
chemotaxis chamber. Peptides were diluted from a stock solution
with KRPG containing 1 mg/ml of bovine serum albumin (BSA;
Orha Behringwerke, Germany) and used at concentrations ranging
from 10À12 to 10À5 M. Data were expressed in terms of chemotactic
index (C.I.), which is the ratio (migration toward test attractant
minus migration toward the buffer/migration toward the buffer);
the values are the mean of six separate experiments performed
in duplicate.
Statistical analysis
The nonparametric Wicoxon test was used in the statistical evalua-
tion of differences between groups. Differences were considered to
be statistically significant at P ≤ 0.05.
Superoxide anion (OÀ2 ) production
This anion was measured by the superoxide dismutase-inhibitable
reduction of ferricytochrome c (Sigma, St. Louis, MO, USA) modified
for microplate-based assays. Tests were carried out in a final
volume of 200 mL containing 4 Â 105 neutrophils, 100-nmol
cytochrome c (Sigma) and KRPG. At zero time, different amounts
(10À10–5 Â 10À5 M) of each peptide were added, and the plates
were incubated into a microplate reader (Ceres 900, Bio-TeK
Instruments, Inc.; Winooski, VT, USA) with the compartment
temperature set at 37 ꢀC. Absorbance was recorded at 550 and
468 nm. The difference in absorbance at the two wavelengths
was used to calculate nmoles of OÀ2 produced using a molar
Results and Discussion
Agonism
Biological activity of the new analogues 4a–l and 5a–l has been
tested on human neutrophils and compared with that shown
by fMLF-OMe used as standard ligand. Chemotactic activity
data of the new compounds 5a–l are reported in Figures 2A
and 3A, and are expressed in terms of chemotactic index (C.I.).
The resulting dose–response curves, as usually found for
extinction coefficient for cytochrome c of 18.5 mmolÀ1 cmÀ1
.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. (2012)

CHEMOTACTIC PEPTIDES fMLF-OMe ANALOGUES
Figure 2. Biological activities of N-For tripeptides 5a-e and fMLF-OMe. (A) chemotactic activity; (B) superoxide anion production; (C) release of
neutrophil granule enzymes evaluated by determining the lysozyme activity.
Figure 3. Biological activities of N-For tripeptides 5f–l and fMLF-OMe. (A) chemotactic activity; (B) superoxide anion production; (C) release of
neutrophil granule enzymes evaluated by determining the lysozyme activity.
chemoattractants, increase to reach a peak and then decrease
rapidly as the concentration of the ligand increases. As shown
in the Figures 2A–3A, the new ligands 5a–l do not reach the
efficacy of the standard, which shows a C.I value of 1.10 at a
concentration of 10À9 M. It is however remarkable that the
two tripeptides 5d and 5e, characterized by Phe(4-CF3) and
Phe(4-CN) residue at position 3 of the backbone, exhibit the C.I.
maximum at a concentration sensibly lower (10À11 and 10À10 M,
respectively) than that required by fMLF-OMe, with a value that,
in the case of 5d, is slightly higher than that exhibited by the
standard at the same concentration (C.I. at 10À11 M: 0.88 and
0.80 for 5d and fMLF-OMe, respectively). Thus, both 5d and 5e, al-
though less efficacious than the parent, are both the most potent
of series, being maximally effective at the concentration of 10À10
and 10À11 M, respectively. A high activity is also shown by the
Phe(4-Cl) containing tripeptide 5g which, with a C.I. peak of 0.98
J. Pept. Sci. (2012)
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

MOLLICA ET AL.
at 10À7 M, although moderately potent, is the most efficacious
among all the here-reported derivatives. As for the other three
halogen derivatives, the best effect is exerted by the Phe(4-Br)
substituent in 5h (C.I. maximum = 0.82 at 10À8 M) . The Phe(4-I)
5i is slightly less favorable than 5h but is clearly better than that
of the Phe(4-F) residue in 5f (C.I. peak 0.65 at 10À8 M). Notably,
the effect induced by the electronegative and bulky substituent
-NO2 in 5l is comparable to that of the bromine (5h) and sensibly
lower to that shown by fluorine in 5f; thus, this latter substituent
remains the less favorable among the tested electronegative
substituents. Finally, the three ligands 5a–c, containing methyl,
phenyl or t-butyl groups show low activity with the worst value
exhibited by the t-butyl containing analogue 5c.
Figures 2B and 3B report the capacity of the new ligands to
stimulate superoxide anion production. With the only exception
of the fluorine containing analogue 5f, whose peak of activity
appears at the same concentration of the standard (10À6 M), all
the analogues, regardless of the substituents at the Phe aromatic
ring, show peaks grouped at a higher concentration (around
10À5 M). Compounds 5a and 5c, containing -CH3 and -C(CH3)3
substituents, show, in this range of concentration, the highest
activity with peaks of 62 and 65 nmoles of OÀ2 , which are slightly
higher than the standard, thus exhibiting practically comparable
potency. Among the ligands 5e–l, containing electron with-
drawing substituents, the best results are shown by 5i and 5l,
which are characterized by the presence of -NO2 and -I groups
Figure 4. Effect of N-Boc-protected tripeptides 4a–e on the neutrophil activities triggered by fMLF-OMe. (A) chemotactic activity; (B) superoxide anion
production; (C) release of neutrophil granule enzymes evaluated by determining the lysozyme activity.
Figure 5. Effect of N-Boc-protected tripeptides 4f–l on the neutrophil activities triggered by fMLF-OMe. (A) chemotactic activity; (B) superoxide anion
production; (C) release of neutrophil granule enzymes evaluated by determining the lysozyme activity.
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. (2012)

CHEMOTACTIC PEPTIDES fMLF-OMe ANALOGUES
at the Phe para position, respectively. Notably, these latter
substituents are less effective than the alkyl -CH3 and -C(CH3)3
groups in 5a and 5c.
electron withdrawing substituents at the phenylalanine para
position.
With the consideration of the entire group of the analogues
here under study, it can be seen that most of them show a
selective ability to stimulate neutrophils. In fact, as general
feature, some of them are able to induce chemotaxis although
this property is generally accompanied by scarce superoxide
anion production or lysozyme release. These data are not surpris-
ing as it is well recognized that the transduction pathway
underlying the chemotactic response is different from those
responsible for superoxide anion production and lysozyme
release [26]. Distinct mechanisms are in fact involved in each of
the examined neutrophil responses, and a distinctive imprint of
signal protein activation, for a single set of cellular responses,
may exist. Moreover, it has been shown that each physiological
function shows different requirements for receptor occupancy.
As reported in the experimental section, the release of neutro-
phil granule enzymes was evaluated by determining the lysozyme
activity expressed as % of lysozyme release and is reported in
Figures 2C and 3C. The analogues 5e–l, containing electron with-
drawing substituents at the Phe residue, show good release
values (in the range 55–72% at a 10À5 M) for all the analogues of
this series except for 5h whose maximum (62%) appears at
10À7 M. Thus, as compared with the standard, 5h shows lower
efficacy and higher potency, whereas 5d has higher efficacy but
lower potency. Among the three nonhalogenated derivatives,
5a–c the C6H5- containing analogue 5b is almost inactive,
whereas the Me- and tBu- containing ligands (5a and 5c) show
good activity, comparable to that induced by the presence of
Figure 6. Competition curves of specific [3H] fMLF binding to human neutrophils by agonists 5a–e (A) and 5f–l (B). Curves are a representative exper-
iment taken from a series of three independent experiments. Nonspecific binding was determined in the presence of 10-mL fMLF-OMe.
Figure 7. Competition curves of specific [3H] fMLF binding to human neutrophils by antagonists 4a–e (A) and 4f–l (B). Plot is a representative
experiment replicate three times using different cell preparations. Nonspecific binding was determined in the presence of 10-mL fMLF-OMe.
J. Pept. Sci. (2012)
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci

MOLLICA ET AL.
In the case of the superoxide anion production, continuous
occupancy of almost 100% of the receptors to achieve and
maintain an optimal response is required, and this is not neces-
sary for the chemotactic response [20].
the examined compounds is more active than fMLF-OMe to
stimulate chemotaxis, thus confirming that the presence of a
shallow binding pocket scarcely adapts to accommodate para-
substituted aromatic rings. In conclusion, the here-described
fMLF-OMe analogues provide new information on structure–
activity relationship concerning chemotactic peptides and
formyl-peptide receptors, leading to molecules which, depending
upon the nature of acylating groups at the N-terminal position
and substitution in 4 position on the aromatic ring of phenylala-
nine, exhibit significant chemotactic agonism or antagonism with
selectivity towards the neutrophil biological functions. The differ-
ent behavior can be rationalized on the basis of the existence of
at least two different states and/or with different subtypes of FPRs
[22,23,25]. Low doses of a full agonist (or a ‘pure’ chemoattractant)
are required to interact with a high-affinity receptor subtype,
which activates the transduction pathway responsible for chemo-
tactic response, whereas an increase in agonist concentration
allows binding with the low-affinity subtype, able to activate the
transduction pathways responsible for OÀ2 production and lyso-
zyme release [28]. As a consequence, a peptide selective for killing
mechanisms, which preferentially interacts with the low-affinity
subtypes, is efficient in effectively displacing the full agonist
[3H]-fMLF [29] only at high concentration. A further investigation
using specific receptor inhibitors of FPR or FPRL1 should give use-
ful information and will be the object of incoming research. How-
ever, the here-reported results, obtained by using simple and
structurally strongly related fMLF-OMe analogues, represent an
interesting contribution toward the comprehension of the subtle
mechanisms involved in the different neutrophil biological func-
tions. Furthermore, the good versatility and good efficacy of the
here-reported new molecules are a promising characteristic partic-
ularly as concerns pharmacological application.
Antagonism
All Boc-protected tripeptides 4a–l have been tested to evaluate
the antagonist activity on FPR receptor. The antagonism was
determined by measuring the inhibition of the activity by deriva-
tives on human neutrophils stimulated by standard tripeptide
fMLF-OMe (Figures 4 and 5). A significant dose-dependent
inhibition of chemotactic index, which reaches the value 70% at
10À9 M concentration, is observed for compounds 4f and 4i, with
-F and -I in position 4, respectively, on the aromatic ring of
phenylalanine, whereas tripeptide 4c, with -C(CH3)3 in the same
position on the aromatic ring, shows the lower ability to inhibit
the activity of standard tripeptide fMLF-OMe (30%). None of the
Boc-protected tripeptides 4a–l show significant antagonism
towards both superoxide anion production and in lysozyme
release (Figures 4 and 5).
Binding
Competition binding assays were carried out to gain information
regarding receptor affinity of formylated (5a–l) and Boc-protected
analogues (4a–l). fMLF-OMe and Boc-MLF-OMe were used as
reference compounds, respectively, for agonists and antagonists.
For-Met-Leu-Phe-OMe showed the highest binding affinity, in
agreement with its overall activity in the displacing of [3H]-fMLF,
followed by analogues 5d > 5c > 5e ≥ 5a (Figure 6). As regards
the other compounds, the rank order of binding affinity is
generally similar to that obtained in chemotaxis experiments
with the exception of tripeptide 5c that shows a considerable
The analytical data of all synthesized products are provided in
the Supplemental Information section associated with this article.
receptor affinity, which is 44% of [3H] fMLF inhibition at 10À6
M
concentration; no significant chemotactic activity was revealed,
probably due to the presence of a bulky group (4-tBu) on the
Phe residue.
Acknowledgements
This work was supported by grants from Fondazione Cassa di
Risparmio di Ferrara. We are grateful to Banca del Sangue of
Ferrara for providing fresh blood.
All Boc derivatives 4a–l (antagonists) show lower receptor
affinity, Boc-MLF-OMe was used as standard [27] (Figure 7).
Compounds 4e and 4l have the best affinity, followed by 4c, 4d
and 4h. As expected, Boc-protected compounds 4a–l bind to
the formylpeptide receptors at concentration of 103 times higher
of their respective formylated compounds; this reflects the
obtained inhibition activity data (Figures 3 and 4). The presence
of Boc group, which is a key feature to exhert antagonism activity
at the formyl peptide receptors [16], also determines the
observed weak affinity of these compounds. The poor binding
shown by the Boc-MLF analogues are not surprising and agree
with the scarce data referred by Derian et al. [16]. The agonist
and antagonist modification seen in this series of Phe3 modified-
chemotactic-peptides in general suggests that considerable
changes at the aromatic ring of the Phe residues in position 3 are
unfavorable for binding affinity.
References
1 Carp H. Mitochondrial N-formylmethionyl proteins as chemoattractants
for neutrophils. J. Exp. Med. 1982; 85: 264–275.
2 Marasco WA, Phan SH, Krutzsch H, Showell HJ, Feltner DE, Nairn R,
Becker EL, Ward PA. Purification and identification of formyl-
methionyl-leucyl-phenylalanine as the major peptide neutrophil
chemotactic factor produced by Escherichia coli. J. Biol. Chem.
1984; 259: 5430–5439.
3 Seifert R, Wenzel-Seifert K. The human formyl peptide receptor as
model system for constitutively active G-protein-coupled receptors.
Life Sci. 2003; 73: 2263–2280.
4 Boulay F, Tardiff M, Brouchon L, Vignais P. Synthesis and use of a novel
N-formyl peptide derivative to isolate a human N-formyl peptide
receptor cDNA. Biochem. Biophys. Res. Comm. 1990; 168: 1103–1109.
5 Su SB, Gong W, Gao JL, Shen W, Murphy PM, Oppenheim JJ, Wang JM.
A seven-transmembrane, G protein-coupled receptor, FPRL1, mediates
the chemotactic activity of serum amyloid A for human phagocytic cells.
J. Exp. Med. 1999; 189: 395–402.
Conclusion
6 Baggiolini M, Boulay F, Badwey JA, Cornutte JT. Activation of neutrophil
leukocytes: chemoattractant receptors and respiratory burst. FASEB
1993; 7: 1004–1010.
7 Selvatici R, Falzarano S, Mollica A, Spisani S. Signal transduction
pathways triggered by selective formylpeptide analogues in human
neutrophils. Eur. J. Pharmacol. 2006; 534: 1–11.
In the present paper, we have examined the fMLF-OMe analogues
4a–l and 5a–l, which combine the presence of various substituents
at the para position of the Phe aromatic ring with two N-terminal
protecting groups, namely H-CO- and tBu-OCO-, which are
characterized by different size and chemical properties. None of
wileyonlinelibrary.com/journal/jpepsci
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
J. Pept. Sci. (2012)

CHEMOTACTIC PEPTIDES fMLF-OMe ANALOGUES
8 Rathore RS. Conformational investigations on analogs of inflamma-
tion response inducing chemotactic tripeptide fMLP. Pept. Sci. 2005;
80: 651–664.
9 Cavicchioni G, Fraulini A, Falzarano S, Spisani S. Structure-activity
relationship of for-L-Met L-Leu-L-Phe-OMe analogues in human
neutrophils. Bioorg. Chem. 2006; 34: 298–318.
10 Torino D, Mollica A, Pinnen F, Feliciani F, Spisani S, Lucente G. Novel
chemotactic For-Met-Leu-Phe-OMe (fMLF-OMe) analogues based on
Met residue replacement by 4-amino-proline scaffold: synthesis and
bioactivity. Bioorg. Med. Chem. 2009; 17: 251–259.
11 Mollica A, Paglialunga Paradisi M, Varani K, Spisani S, Lucente G.
Chemotactic peptides: fMLF-OMe analogues incorporating proline-
methionine chimeras as N-terminal residue. Bioorg. Med. Chem.
2006; 14: 2253–2265.
for synthetic peptide chemoattractants. Biochemistry 1980; 19:
2404–2410.
19 Higgins JD III, Bridger GJ, Derian CK, Beblavy MJ, Hernandez PE, Gaul
FE, Abrams MJ, Pike MC, Solomon HF. N-terminus urea-substituted
chemotactic peptides: new potent agonists and antagonists toward
the neutrophil fMLF receptor. J. Med. Chem. 1996; 39: 1013–1015.
20 Prossnitz ER, Ye RD. The N-formyl peptide receptor: a model for the
study of chemoattractant receptor structure and function. Pharmacol.
Ther. 1997; 74: 73–102.
21 Le Y, Oppenheim JJ, Wang JM. Pleiotropic roles of formyl peptide
receptors. Cytokine Growth Factor Rev. 2001; 12: 91–105.
22 Dalpiaz A, Spisani S, Biondi C, Fabbri E, Nalli M, Ferretti ME. Studies on
human neutrophil biological functions by means of formyl-peptide
receptor agonists and antagonists. Curr. Drug Targets Immune Endocr.
Metabol. Disord. 2003; 3: 33–42.
12 Giordano C, Lucente G, Mollica A, Nalli M, Pagani Zecchini G,
Paglialunga Paradisi M, Gavuzzo E, Mazza F, Spisani S. Hybrid a/b3-
peptides with proteinogenic side chains. Monosubstituted analogues
of the chemotactic tripeptide For-Met-Leu-Phe-OMe. J. Pept. Sci.
2004; 10: 510–523.
23 Lajoie G, Kraus JL. Rapid and efficient method for the N-formylation of
C-blocked peptides. Peptides 1984; 5: 653–654.
24 El-Faham A, Albericio F. Peptide coupling reagents, more than a letter
soup. Chem. Rev. 2011; 111: 6557–6602.
13 Schiffmann E, Corcoran BA, Whal SM. N-formylmethionyl peptides as
chemoattractants for leucocytes. Proc. Natl. Acad. Sci. (USA) 1975; 72:
1059–1062.
14 Day AR, Radding JA, Freer RJ, Showell HJ, Becker ER, Schiffmann E,
Corcoran B, Aswanikumar S, Pert C. Synthesis and binding charac-
teristics of an intrinsically radiolabeled chemotactic acyl tripeptide.
Na-formyl-norleucyl-leucyl-phenylalanine. FEBS Lett. 1977; 77:
291–294.
25 Dalpiaz A, Ferretti ME, Vertuani G, Traniello S, Scatturin A, Spisani S.
C- and N-terminal residue effect on peptide derivatives’ antagonism
toward the formyl-peptide receptor. Eur. J. Pharmacol. 2002; 436:
187–196.
26 Selvatici R, Falzarano S, Traniello S, Pagani Zecchini G, Spisani S. For-
mylpeptides trigger selective molecular pathways that are required
in the physiological functions of human neutrophils. Cell. Signal.
2003; 15: 377–383.
15 Spisani S, Turchetti M, Varani K, Falzarano S, Cavicchioni G. Hydrophilic
residues at position 3 highlight unforeseen features of the fMLP
receptor pocket. Eur. J. Pharmacol. 2003; 469: 13–19.
27 Cavicchioni G, Varani K, Niccoli S, Rizzuti O, Spisani S. An evaluation of
fMLP pocket dimensions and features using formyltetrapeptides. J.
Pept. Res. 1999; 54: 336–343.
16 Derian CK, Solomon HF, Higgins III JD, Beblavy MJ, Santulli RJ, Bridger GJ,
Pike MC, Kroon DJ, Fischman AJ. Selective inhibition of N-formylpeptide-
induced neutrophil activation by carbamate-modified peptide ana-
logues. Biochemistry 1996; 35: 1265–1269.
28 Pagani Zecchini G, Nalli M, Mollica A, Lucente G, Paglialunga Paradisi
M, Spisani S. Isopeptide bonds in chemotactic tripeptides. Synthesis
and activity of lysine-containing fMLF analogs. J. Pept. Res. 2002; 59:
283–291.
17 Le Y, Murphy PM, Wang JM. Formyl-peptide receptors revisited.
Trends Immunol. 2002; 11: 541–548.
18 Freer RJ, Day AR, Radding JA, Schiffmann E, Aswanikumar S, Showell
HJ, Becker EL. Further studies on the structural requirements
29 Cavicchioni G, Fraulini A, Turchetti M, Varani K, Falzarano S, Pavan B.
Biological activity of for-Met-Leu-Phe-OMe analogs: relevant substitu-
tions specifically trigger killing mechanisms in human neutrophils.
Eur. J. Pharmacol. 2005; 512: 1–8.
J. Pept. Sci. (2012)
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jpepsci