Synthesis and activity on human neutrophil functions of fMLF-OMe analogs containing alkyl spacers at the central position.

Article abstract of DOI:10.1016/S0014-827X(01)01140-5

We report here the synthesis and activity of new analogs of the N-formyl and N-tert-butyloxycarbonyl (Boc) derivatives of the tripeptide Met-Leu-Phe-OMe containing an achiral omega-amino acid residue replacing the hydrophobic central leucine. The tripeptides HCO-Met-NH-(CH2)n-CO-Phe-OMe and Boc-Met-NH-(CH2)n-CO-Phe-OMe (n = 3-5) containing the central homomorphic residue of 5-aminopentanoic acid (delta-aminovaleric acid; delta-Ava; n = 4) and the two non-homomorphic residues of 4-aminobutanoic acid (gamma-aminobutyric acid; gamma-Abu; n = 3) and 6-aminohexanoic acid (epsilon-aminocaproic acid; epsilon-Aca; n = 5) have been examined. The activity as agonists and antagonists in chemotaxis, lysozyme release, and superoxide anion production of the new analogs has been determined. The N-Boc derivatives 2a and 2b, incorporating the gamma-Abu and the delta-Ava residues, show good and selective antagonist activity on superoxide anion production.

Full text of DOI:10.1016/S0014-827X(01)01140-5

www.elsevier.com/locate/farmac
Il Farmaco 56 (2001) 851–858
Synthesis and activity on human neutrophil functions of
fMLF-OMe analogs containing alkyl spacers at the central
position
Giampiero Pagani Zecchini a, Enrico Morera a, Marianna Nalli a,
Mario Paglialunga Paradisi a, Gino Lucente a,*, Susanna Spisani b
a Dipartimento di Studi Farmaceutici e Centro di Studio per la Chimica del Farmaco del CNR, Uni6ersita` degli Studi di Roma ‘La Sapienza’,
P.le A. Moro 5, 00185 Rome, Italy
b Dipartimento di Biochimica e Biologia Molecolare, Via L. Borsari 46, Uni6ersita` di Ferrara, 44100 Ferrara, Italy
Accepted 13 June 2001
Abstract
We report here the synthesis and activity of new analogs of the N-formyl and N-tert-butyloxycarbonyl (Boc) derivatives of the
tripeptide Met-Leu-Phe-OMe containing an achiral v-amino acid residue replacing the hydrophobic central leucine. The
tripeptides HCO-Met-NH-(CH2)n-CO-Phe-OMe and Boc-Met-NH-(CH2)n-CO-Phe-OMe (n=3–5) containing the central homo-
morphic residue of 5-aminopentanoic acid (d-aminovaleric acid; d-Ava; n=4) and the two non-homomorphic residues of
4-aminobutanoic acid (g-aminobutyric acid; g-Abu; n=3) and 6-aminohexanoic acid (o-aminocaproic acid; o-Aca; n=5) have
been examined. The activity as agonists and antagonists in chemotaxis, lysozyme release, and superoxide anion production of the
new analogs has been determined. The N-Boc derivatives 2a and 2b, incorporating the g-Abu and the d-Ava residues, show good
and selective antagonist activity on superoxide anion production. © 2001 Elsevier Science S.A. All rights reserved.
Keywords: Alkyl spacers; v-Amino acids; Chemotactic peptides; Human neutrophils
ubstituted glycine residues, bearing two linear side
chains (Dxx) [1–3] or a cyclo-aliphatic ring system
(Acnc) [4–8], represents a particularly useful modifica-
tion strategy.
The occurrence of two main features appears relevant
in this approach: (i) restriction of the available range of
backbone conformations and induction of predictable
conformations on the basis of the typology of the
substituents at the Ca-carbon atom; (ii) possibility to
optimize the interaction of the central residue with the
corresponding receptor area by modulating side-chain
bulkiness and overall hydrophobicity.
By synthesizing the above-mentioned type of tripep-
tide models, possessing the general formula HCO-Met-
Xaa-Phe-OMe (with Xaa=Dxx or Acnc), it was
possible to stabilize both extended and folded backbone
conformations and demonstrate that neutrophil recep-
tors can efficiently interact with both types of ligands
[6]. The operation of the induced-fit mechanism as well
as flexibility at the central position of the tripeptide
1. Introduction
Chemotactic N-formyl peptides are involved in the
defense mechanism against bacterial infections through
binding with specific receptors located on the neu-
trophil membranes. In addition to the cell-directed
migration (chemotaxis), the peptide receptor interaction
gives rise to a series of biochemical events including
production of superoxide anions and lysosomal enzyme
release. The N-formyl tripeptide HCO-Met-Leu-Phe-
OH (fMLF) and its methyl ester (fMLF-OMe) repre-
sent the reference models on which a variety of
chemical modifications have been performed in order
to gain information on the structure–activity
relationships.
An examination of the literature reveals that replace-
ment of the hydrophobic central leucine with Ca,a-dis-
* Corresponding author.
E-mail address: gino.lucente@uniroma1.it (G. Lucente).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01140-5

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G. Pagani Zecchini et al. / Il Farmaco 56 (2001) 851–858
tripeptide backbone fragment (e.g. n=4 for a dipep-
tide; n=7 for a tripeptide) and, on this basis, the
spacer is considered homomorphic [11].
In the present paper we report the synthesis and
biological activity of the tripeptides Boc-Met-NH-
(CH2)n-CO-Phe-OMe (2a–c) and HCO-Met-NH-
(CH2)n-CO-Phe-OMe (3a–c) (n=3–5) containing the
central homomorphic residue of d-aminovaleric acid
(d-Ava; n=4) and the two non-homomorphic residues
of g-aminobutyric acid (g-Abu; n=3) and o-
aminocaproic acid (o-Aca; n=5).
2. Chemistry
The synthesis of the tripeptides 2a–c and 3a–c was
performed according to Scheme 1.
Scheme 1.
molecule should thus represent factors relevant for the
optimization of recognition and binding by the biologi-
cal target.
In the light of the findings reported above it seemed
interesting to incorporate, at the central position of the
fMLF-OMe-based chemotactic analogs, residues of v-
amino acids H2Nꢀ(CH2)nꢀCO2H characterized by an
achiral bridge made up of a certain number of methyl-
ene units inserted between the amino and the carboxylic
group. These residues are largely adopted as useful
spacers for designing peptide analogs possessing differ-
ent degrees of hydrophobicity and enhanced flexibility
as compared with the natural counterparts [9,10]. De-
pending upon the number of methylene units, the v-
amino acid residue may correspond to a dipeptide or
The peptides 1a–c and 2a–c were prepared in solu-
tion via standard coupling reactions which involved
isobutyl chloroformate for carboxyactivation (mixed
anhydride method) and the tert-butyloxycarbonyl (Boc)
group for amino protection. Boc-deprotection of com-
pounds 1a–c was performed via acidolysis with thionyl
chloride in methanol. Treatment of 2a–c with formic
acid followed by ethyl 2-ethoxy-1,2-dihydro-1-quino-
linecarboxylaxe (EEDQ) gave the formyl derivatives
3a–c.
Table 1
Physical, analytical, and spectral data of Boc-protected dipeptides 1a–c
Comp.
Yield (%)
M.p. (°C), crystallization solvent a
[h]D
Formula (MW) b
1a
1b
1c
83
100
96
76–77, EA–H
103–104, EA–H
75–76, EA–H
+47°
+51°
+57°
C19H28N2O5 (364.44)
C20H30N2O5 (378.47)
C
21H32N2O5 (392.49)
1H NMR (l)
Phe
v-Xaa
v-CH2
3.10
3.07
3.09
c
a-CH
4.84
4.90
4.89
NH
6.65
6.11
6.03
a-CH2
2.20
2.20
NH
4.84
4.68
4.63
1a
1b
1c
2.17
IR (cm−1
)
1a
1b
1c
3342, 3287, 2981, 1750, 1679, 1643, 1530
3354, 3326, 2965, 1753, 1739, 1681, 1651, 1532
3350, 2929, 1743, 1678, 1646, 1524
a EA=ethyl acetate; H=n-hexane.
b Analytical results of C, H, N were within 90.4% of the calculated values.
c Superimposed on the Phe b-CH2 signal.

G. Pagani Zecchini et al. / Il Farmaco 56 (2001) 851–858
853
Table 2
Physical, analytical, and spectral data of Boc-protected tripeptides 2a–c
Comp.
Yield (%)
M.p. (°C), crystallization solvent a
[h]D
Formula (MW) b
2a
2b
2c
76
72
73
116–117, EA
120–121, EA
133–136, EA–H
+43°
+32°
+40°
C24H37N3O6S (495.64)
C
25H39N3O6S (509.68)
C26H41N3O6S (523.70)
1H NMR (l)
Met
v-Xaa
Phe
a-CH
4.20
4.25
4.25
NH
5.41
5.46
5.43
a-CH2
2.17
2.20
v-CH2
NH
7.09
a-CH
4.84
4.89
4.88
NH
7.05
6.72
6.32
c
2a
2b
2c
3.13
3.12 c and 3.37 6.85
2.17
3.20 c
6.71
IR (cm−1
)
2a
2b
2c
3323, 3310, 2965, 1747, 1685, 1644, 1525
3309, 2927, 1726, 1683, 1641, 1543, 1524
3329, 3310, 2960, 1711, 1688, 1647, 1524
a EA=ethyl acetate; H=n-hexane.
b Analytical results of C, H, N were within 90.4% of the calculated values.
c Superimposed on the Phe b-CH2 signal.
Table 3
Physical, analytical, and spectral data of formyltripeptides 3a–c
Comp.
Yield (%)
M.p. (°C)
[h]D
Formula (MW) a
3a
3b
3c
95
92
78
106–107
133–134
125–127
+41°
+27°
+42°
C20H29N3O5S (423.54)
C
C
21H31N3O5S (437.57)
22H33N3O5S (451.60)
1H NMR (l)
HCO
Met
v-Xaa
Phe
a-CH
4.64
4.67
4.72
NH b
7.08
6.90–7.35
6.92–7.35
a-CH2
2.17
2.17
v-CH2
NH b
6.90–7.36
6.90–7.36
6.92–7.35
a-CH
4.82
4.86
4.86
NH
6.91
6.65
6.48
3a
3b
3c
8.14
8.15
8.15
3.18 c
3.11 c and 3.34
3.25 c
2.13
IR (cm−1
)
3a
3b
3c
3291, 3086, 2950, 2921, 1746, 1640, 1547
3282, 3093, 1737, 1639, 1561, 1556, 1449
3287, 3086, 2940, 2923, 1755, 1640, 1549
a Analytical results of C, H, N were within 90.4% of the calculated values.
b Superimposed on aromatic.
c Superimposed on the b-CH2 signal.
Physical, analytical, and spectral data of the three
All Boc-derivatives were found to be unable to in-
duce chemotaxis and to trigger superoxide anion pro-
duction. Concerning the lysozyme release the
derivatives 2a–c show a slight activity, never statisti-
groups of peptides reported in Scheme 1 are summa-
rized in Tables 1–3.
cally significant, in the concentration range 10−10
–
10−5 M (not shown). The formyltripeptides 3a–c,
although unable to elicit superoxide anion production,
did show significant activity as chemoattractants (Fig.
1A) and segretagogue agents (Fig. 1B) even if with a
lesser potency than the standard tripeptide. In particu-
lar, 3a, containing the g-Abu residue, is the most active
formylpeptide reaching the maximum activity (ca. 0.80
chemotactic index; 7% decrease) at a concentration of
10−7 M, whereas the o-Aca derivative 3c is the least
active agonist (ca. 0.30 chemotactic index; 60% decrease
3. Biological results
The agonist activity of formyltripeptides 3a–c and
the corresponding Boc-protected tripeptides 2a–c was
determined on human neutrophils in three in vitro
assays: directed migration (chemotaxis), superoxide an-
ion production, and lysozyme release. Their activity
was compared to that of the standard tripeptide fMLF-
OMe.

854
G. Pagani Zecchini et al. / Il Farmaco 56 (2001) 851–858
at 10−7 M). An intermediate activity was observed for
3b (ca. 0.60 chemotactic index; 32% decrease at 10−7
M). On the contrary, the maximum value of the
lysozyme release activity is shown by 3c at 10−7 M (ca.
30% lysozyme release).
OMe based analogs leads to agonists which, although
less potent than the parent tripeptide, are able to dis-
criminate the different biological responses associated
with the stimulation of human formylpeptide receptors
or receptor subtypes. All the three new analogs are, in
fact, active as chemoattractants and degranulating
agents, but practically inactive as superoxide anion
producers. Furthermore, the finding that 3c, the least-
active analog as a chemotactic agent, is found to be the
most active secretagogue and that 3a, the least-active
secretagogue, is the most active as a chemoattractant,
underlines the receptor functional heterogeneity and the
complex control systems of the signal transduction
mechanisms [12–15].
The selectivity shown by the N-formyltripeptide ago-
nists 3a–c is not maintained in the N-tert-butyloxycar-
bonyl derivatives 2a–c which inhibit, although with
different intensity, all the three examined biological
functions. While only a slight inhibition has been found
on chemotaxis and lysozyme release (Fig. 2A and C), a
marked inhibitory activity, well superior to that associ-
ated with the N-Boc tripeptide 4 containing a central
leucine residue, is observed for the two new analogs 2a
and 2b on superoxide anion production. In this case,
the most efficient inhibitor 2a is obtained when the
shortest spacer (i.e. g-Abu) has been introduced and the
inhibition reaches values which are comparable to those
shown by N-Boc-MLF-OMe (4) in the chemotactic
activity (Fig. 2A). High and selective affinity for the
receptor subtype responsible for O−2 production is then
associated with the new N-Boc analogs 2a and 2b
incorporating alkyl spacers with three (g-Abu) or four
methylene groups (d-Ava). It should also be noted that
The antagonist activity of the Boc-tripeptides 2a–c
was determined by measuring the ability to inhibit the
above-cited human neutrophil responses stimulated by
the optimal dose of fMLF and compared to that shown
by Boc-Met-Leu-Phe-OMe (4). The influence of in-
creasing concentration of the examined derivatives on
chemotaxis induced by 10 nM fMLF is shown in Fig.
2A. A dose-dependent inhibition of the chemotactic
activity can be observed for all the three compounds
and the inhibition becomes statistically significant (PB
0.05) at the concentration of 10−6 M for 2b and 2c and
at 10−10 M for 2a, indicating that 2a is the most
efficient analog. The influence on superoxide anion
production is reported in Fig. 2B; compounds 2a and
2b exert a statistically significant reduction (PB0.05)
on the activity induced by 1 mM fMLF starting from
10−9 M and the inhibition progressively increases with
the concentration up to ca. 60 and 45% for 2a and 2b,
respectively; a weaker antagonist activity is exhibited by
2c. As shown in Fig. 2C, only a weak inhibitory action
on the lysozyme release is observed for the three new
compounds.
4. Discussion and conclusions
The results reported above indicate that the introduc-
tion of an alkyl spacer at the central position of fMLF-
Fig. 1. Biological activity of formyltripeptides 3a–c towards human neutrophils: (A) chemotactic activity; (B) release of neutrophil granule
enzymes evaluated by determining lysozyme activity.

G. Pagani Zecchini et al. / Il Farmaco 56 (2001) 851–858
855
Fig. 2. Effect of Boc derivatives 2a–c and 4 on chemotaxis activated by 10 nM fMLF (A), on superoxide anion production (B), and on release
of neutrophil granule enzymes (C) triggered by 1 mM fMLF.
the most active antagonist 2a contains the shortest alkyl
spacer g-Abu as it is found in the case of the most
active agonist 3a.
5. Experimental
5.1. Chemistry
In conclusion, the strategy adopted here, based on
the replacement of the central leucine with achiral
residues of v-amino acids in chemotactic formyltripep-
tides, leads to moderately active and selective agonists
and, in the case of the N-Boc derivatives, to new
efficient antagonists of superoxide anion production.
By taking into account the considerable interest associ-
ated with the development of high-affinity ligands for
the human neutrophil receptor, in view of their poten-
tial use as diagnostic and therapeutical agents [16,17],
the results reported here indicate that proper structural
modifications at the central position of N-Boc-MLF-
OMe based ligands can lead to potent and selective
antagonists. Further studies are in progress in our
laboratories to better define this point.
Melting points were determined with a Kofler hot-
stage apparatus and are uncorrected. Optical rotations
were taken at 20 °C with a Schmidt–Haensch Polar-
tronic D polarimeter (1 dm cell, c 1.0 in CHCl3). IR
spectra (KBr disks) were recorded employing a Perkin–
1
Elmer 983 spectrophotometer. H NMR spectra were
determined in CDCl3 solution with a Bruker AM 200
spectrometer using Me4Si as internal standard. TLC
and PLC were performed on Merck 60 F254 silica gel
plates. The drying agent was sodium sulfate. Parent
fMLF-OMe, Boc-Met-Leu-Phe-OMe, Boc-d-Ava-OH,
and Boc-o-Aca-OH were prepared as described in the
literature [18,19]. Boc-Met-OH (Fluka Chemie AG,
Switzerland), HCl·Phe-OMe (Fluka Chemie AG,

856
G. Pagani Zecchini et al. / Il Farmaco 56 (2001) 851–858
Switzerland), Boc-g-Abu-OH (Sigma, USA), and
fMLF-OH (Sigma, USA) were employed without
purification. Elemental analyses were performed in the
laboratories of the Servizio Microanalisi del CNR,
Area della Ricerca di Roma, Montelibretti, Italy. The
abbreviations used are as follows: g-Abu, g-aminobu-
tyric acid (4-aminobutanoic acid); o-Aca, o-amino-
caproic acid (6-aminohexanoic acid); d-Ava, d-amino-
valeric acid (5-aminopentanoic acid); Boc, tert-butyl-
oxycarbonyl; EEDQ, ethyl 2-ethoxy-1,2-dihydro-
1-quinolinecarboxylate; KRPG, Krebs–Ringer-phos-
tion was stirred at r.t. for 24 h. Evaporation under
reduced pressure afforded an oily residue, in the case of
3a and 3b, which was dissolved in dry chloroform, and
the product was precipitated by n-hexane. Washing
with dry ether afforded the pure title compounds 3a
and 3b. The solid residue, obtained by evaporation of
the reaction mixture containing 3c, was filtered and
washed with dry ether.
5.2. Biological assays
phate containing 0.1% w/v
D
-glucose (pH 7.4); PLC,
5.2.1. Peptides
preparative layer chromatography.
Stock solutions of fMLF-OMe and peptide analogs,
10−2 M, were prepared in dimethyl sulfoxide and di-
luted in KRPG, pH 7.4, before use. At the concentra-
tion used, dimethyl sulfoxide did not interfere with any
of the biological assays performed.
5.1.1. General procedure for the synthesis of dipeptides
1a–c
Isobutyl chloroformate 95% (0.14 ml, 1 mmol) was
added at −15 °C to a stirred solution of Boc-v-Xaa-
OH (1 mmol) and N-methylmorpholine (0.13 ml, 1.2
mmol) in dry CH2Cl2 (4.8 ml). The temperature was
kept at −15 °C for 10 min, and then HCl·Phe-OMe
(0.216 g, 1 mmol), N-methylmorpholine (0.11 ml, 1
mmol), and dry CH2Cl2 (3.6 ml) were added. The
mixture was stirred at −15 °C for 15 min and then at
room temperature (r.t.) for 1 day. Ethyl acetate was
added in excess and the organic layer washed with 2 N
HCl, brine, saturated aqueous NaHCO3, and brine.
The organic phase was dried and evaporated to give a
pure solid residue in the case of 1b and 1c. Crude 1a
was purified by PLC [CH2Cl2–EtOAc (8:2)].
5.2.2. Cell preparation
Cells were obtained from the blood of healthy sub-
jects, and human peripheral blood neutrophils were
purified employing the standard techniques of dextran
(Pharmacia, Uppsala, Sweden) sedimentation, centrifu-
gation on Ficoll–Paque (Pharmacia), and hypotonic
lysis of contaminating red cells. The cells were washed
twice and resuspended in KRPG, pH 7.4, at a final
concentration of 50×106 cells/ml and kept at r.t. until
used. Neutrophils were 98–100% pure and 99% viable,
as determined by the Trypan blue exclusion test.
5.2.3. Random locomotion
5.1.2. General procedure for the synthesis of tripeptides
2a–c
Random locomotion was performed with 48-well mi-
crochemotaxis chamber (Bio Probe, Milan, Italy) and
the migration into the filter was evaluated by the
method of leading-front [20]. The actual control ran-
dom movement is 35 mm93 SE of ten separate experi-
ments performed in duplicate.
Thionyl chloride (0.076 ml, 1.05 mmol) was added to
a solution of Boc-v-Xaa-Phe-OMe (1a–c) (1 mmol) in
dry methanol (1 ml), cooled at −15 °C. After stirring
at −15 °C for 30 min and at 45 °C for 4 h, the
solution was evaporated under vacuum to give HCl·H-
v-Xaa-Phe-OMe as a foam. This salt was used without
further purification. Boc-Met-OH (0.249 g, 1 mmol)
was activated with isobutyl chloroformate 95% (0.14
ml, 1 mmol) and N-methylmorpholine (0.13 ml, 1.2
mmol) in dry CH2Cl2 (4.8 ml) as described above for
Boc-v-Xaa-OH. Addition of a solution of HCl·H-v-
Xaa-Phe-OMe and N-methylmorpholine (0.11 ml, 1
mmol) in dry CH2Cl2 (3.6 ml) and usual work-up
afforded an oily residue which was purified by PLC
[CH2Cl2–EtOAc (1:1)].
5.2.4. Chemotaxis
In order to study the potential chemotactic activity,
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 (Orha Behringwerke, Germany)
and used at concentrations ranging from 10−12 to 10−5
M. Data were expressed in terms of chemotactic index
(CI), which is the ratio: (migration toward test attrac-
tant minus migration toward the buffer)/migration to-
ward the buffer; the values are the mean of six separate
experiments performed in duplicate. Standard errors
are in the 0.02–0.09 chemotactic index range.
5.1.3. General procedure for the synthesis of
formylpeptides 3a–c
The N-Boc-protected peptide (1 mmol) was dissolved
in formic acid (6 ml) and the mixture was stirred at r.t.
for 24 h. After removal of the excess of formic acid in
vacuo, the residue was dissolved in dry chloroform (6
ml) and EEDQ 97% (1.2 mmol) was added. The solu-
5.2.5. Superoxide anion (O−2 ) production
The superoxide anion was measured by the superox-
ide dismutase-inhibitable reduction of ferricytochrome
c (Sigma, USA) modified for microplate-based assays.

G. Pagani Zecchini et al. / Il Farmaco 56 (2001) 851–858
857
Tests were carried out in a final volume of 200 ml
containing 4×105 neutrophils, 100 nmol cytochrome c
and KRPG. At zero time different amounts (10−10
5.2.8. Statistical analysis
The non-parametric Wilcoxon test was used in the
statistical evaluation of differences between groups.
–
5×10−5 M) of each peptide were added and the plates
were incubated into a microplate reader (Ceres 900,
Bio-TeK Instruments, Inc.) with the compartment tem-
perature set at 37 °C. Absorbance was recorded at
wavelengths of 550 and 468 nm. Difference in ab-
sorbance at the two wavelengths was used to calculate
nanomoles of O−2 produced using an absorptivity for
cytochrome c of 18.5 mM−1 cm−1. Neutrophils were
incubated with 5 mg/ml cytochalasin B (Sigma) for 5
min prior to activation by peptides. Results are ex-
pressed as net nanomoles of O−2 /1×106 cells/5 min and
are the mean of six separate experiments done in dupli-
cate. Standard errors are in the 0.1–4 nmol O−2 range.
Acknowledgements
This work was supported in part by the Ministero
dell’Universita` e della Ricerca Scientifica e Tecnologica
(MURST). We are grateful to Banca del Sangue of
Ferrara for providing fresh blood.
References
[1] A.R. Dentino, P.A. Raj, K.K. Bhandary, M.E. Wilson, M.J.
Levine, Role of peptide backbone conformation on biological
activity of chemotactic peptide, J. Biol. Chem. 266 (1991)
18460–18468.
[2] I. Torrini, M. Paglialunga Paradisi, G. Pagani Zecchini, G.
Lucente, E. Gavuzzo, F. Mazza, G. Pochetti, S. Traniello, S.
Spisani, Synthesis, conformation, and biological activity of two
fMLP-OMe analogues containing the new 2-[2%-(methylthio)-
ethyl]methionine residue, Biopolymers 42 (1997) 415–426.
[3] C.W. Tornøe, H. Sengeløv, M. Meldal, Solid-phase synthesis of
chemotactic peptides using a-azido acids, J. Peptide Sci. 6 (2000)
314–320.
5.2.6. Enzyme assay
The release of neutrophil granule enzymes was evalu-
ated by determining the lysozyme activity, modified for
microplate-based assays. Cells, 3×106 per well, were
first incubated in triplicate wells of microplates with 5
mg/ml cytochalasin B at 37 °C for 15 min and then in
the presence of each peptide in a final concentration of
10−10–2×10−5 M for a further 15 min. The plates
were then centrifuged at 400g for 5 min and the
lysozyme was quantified nephelometrically by the rate
of lysis of cell wall suspension of Micrococcus lysodeik-
ticus. The reaction rate was measured using a mi-
croplate reader at 465 nm. Enzyme release was
expressed as a net percentage of the total enzyme
content released by 0.1% Triton X-100. Total enzyme
activity was 8591 mg/1×107 cells/min. The values are
the mean of five separate experiments done in dupli-
cate. Standard errors are in the 1–6% range.
[4] C. Toniolo, M. Crisma, G. Valle, G.M. Bonora, S. Polinelli,
E.L. Becker, R.J. Freer, Sudhanand, R.B. Rao, P. Balaram, M.
Sukumar, Conformationally restricted formyl methionyl tripep-
tide chemoattractants:
a three-dimensional structure–activity
study of analogs incorporating a Ca,a-dialkylated glycine at
position 2, Peptide Res. 2 (1989) 275–281.
[5] G. Cavicchioni, A. Breveglieri, M. Boggian, G. Vertuani, E.
Reali, S. Spisani, The importance of the peptide bond at position
2 in HCO-Met-Leu-Phe-OMe analogues as shown by studies on
human neutrophils, J. Peptide Sci. 2 (1996) 135–140.
[6] S. Prasad, R.B. Rao, H. Bergstrand, B. Lundquist, E.L. Becker,
P. Balaram, Conformation–activity correlations for chemotactic
tripeptide analogs incorporating dialkyl residues with linear and
cyclic alkyl side chains at position 2, Int. J. Peptide Protein Res.
48 (1996) 312–318.
[7] I. Torrini, G. Pagani Zecchini, M. Paglialunga Paradisi, G.
Lucente, G. Mastropietro, E. Gavuzzo, F. Mazza, G. Pochetti,
S. Traniello, S. Spisani, Modified chemotactic peptides: synthe-
sis, conformation, and activity of HCO-Thp-Ac6c-Phe-OMe,
Biopolymers 39 (1996) 327–337.
[8] M. Gatos, F. Formaggio, M. Crisma, G. Valle, C. Toniolo,
G.M. Bonora, M. Saviano, R. Iacovino, V. Menchise, S.
Galdiero, C. Pedone, E. Benedetti, Conformational characteriza-
tion of peptides rich in the cycloaliphatic Ca,a-disubstituted
glycine 1-amino-cyclononane-1-carboxylic acid, J. Peptide Sci. 3
(1997) 367–382.
[9] A. Banerjee, A. Pramanik, S. Bhattacharjya, P. Balaram, Omega
amino acids in peptide design: incorporation into helices, Bio-
polymers 39 (1996) 769–777.
[10] C. Galoppini, S. Meini, M. Tancredi, A. Di Fenza, A. Triolo, L.
Quartara, C.A. Maggi, F. Formaggio, C. Toniolo, S. Mazzucco,
A. Papini, P. Rovero, A new class of pseudopeptide antagonists
of the kinin B1-receptor containing alkyl spacers, J. Med. Chem.
42 (1999) 409–414.
5.2.7. Antagonist assay
Antagonist activity was determined by measuring the
ability of a derivative to inhibit chemotaxis, superoxide
anion production or granule enzyme release as induced
by fMLF. Antagonist activity data (percentage of activ-
ity) were obtained by comparing the chemotactic index,
nanomoles of O−2 or percentage of lysozyme release in
the absence (100%) and in the presence of the deriva-
tive. Chemotactic index of 10 nM fMLF was 1.159
0.10 SE. O−2 generation produced by 1 mM fMLF was
4392 nmol/1×106 cells/5 min. Enzyme activity trig-
gered by 1 mM fMLF was 5795%/3×106 cells/min.
Derivatives were added to neutrophils 10 min before
the incubation step for cellular functionality. Each
value represents an average of six separate experiments
done in duplicate. Standard errors are within 10% of
the mean value.
[11] A. Banerjee, P. Balaram, Stereochemistry of peptides and
polypeptides containing omega amino acids, Curr. Sci. 73 (1997)
1067–1077.

858
G. Pagani Zecchini et al. / Il Farmaco 56 (2001) 851–858
[12] P. Gierschik, D. Sidiropoulos, K.H. Jakobs, Two distinct Gi-
proteins mediate formyl peptide receptor signal transduction in
human leukemia (HL-60) cells, J. Biol. Chem. 264 (1989) 21470–
21473.
[13] S. Spisani, M.C. Pareschi, M. Buzzi, M.L. Colamussi, C. Biondi,
S. Traniello, G. Pagani Zecchini, M. Paglialunga Paradisi, I.
Torrini, M.E. Ferretti, Effect of cyclic AMP level reduction on
human neutrophil responses to formylated peptides, Cell. Signal.
8 (1996) 269–277.
[14] E. Fabbri, S. Spisani, C. Biondi, L. Barbin, M.L. Colamussi, A.
Cariani, S. Traniello, I. Torrini, M.E. Ferretti, Two For-Met-
Leu-Phe-OMe analogues trigger selective neutrophil responses. A
differential effect on cytosolic free Ca2+, Biochim. Biophys.
Acta 1359 (1997) 233–240.
[15] E. Fabbri, S. Spisani, L. Barbin, C. Biondi, M. Buzzi, S.
Traniello, G. Pagani Zecchini, M.E. Ferretti, Studies on fMLP-
receptor interaction and signal transduction pathway by means
of fMLP-OMe selective analogues, Cell. Signal. 12 (2000) 391–
398.
[16] D.J. Rose, K.P. Maresca, T. Nicholson, A. Davison, A.G. Jones,
J. Babich, A. Fischman, W. Graham, J.R.D. DeBord, J. Zubieta,
Synthesis and characterization of organohydrazino complexes of
technetium, rhenium and molybdenum with the {M(h1-
HxNNR)(h2-HyNNR)} core and their relationship to radiola-
beled organohydrazine-derivatized chemotactic peptides with
diagnostic applications, Inorg. Chem. 37 (1998) 2701–2716.
[17] T.W. Kuijpers, R.S. Weening, D. Roos, Clinical and laboratory
work-up of patients with neutrophil shortage or dysfunction, J.
Immunol. Methods 232 (1999) 211–229.
[18] G. Vertuani, S. Spisani, M. Boggian, S. Traniello, A. Scatturin,
Conformational studies of synthetic tripeptide chemoattractants,
Int. J. Peptide Protein Res. 29 (1987) 525–532.
[19] E.C. Jorgensen, G.C. Windridge, T.C. Lee, Angiotensin II
analogs. III. Synthesis and biological evaluation of some des-as-
partyl-angiotensins, J. Med. Chem. 13 (1970) 352–356.
[20] S.H. Zigmond, J.G. Hirsch, Leukocyte locomotion and chemo-
taxis. New methods for evaluation and demonstration of cell-
derived chemotactic factor, J. Exp. Med. 137 (1973) 387–410.