Synthesis and activity of HCO-Met-Leu-Phe-OMe analogues containing β-alanine or taurine at the central position

Article abstract of DOI:10.1016/S0014-827X(03)00165-4

New synthetic analogues of the chemotactic N-formyltripeptide HCO-Met-Leu-Phe-OMe have been synthesized. The reported new models, namely Boc-Met-β-Ala-Phe-OMe (1), HCO-Met-β-Ala-Phe-OMe (2), Boc-Met-Tau-Phe-OMe (3), HCO-Met-Tau-Phe-OMe (4) and HCl·Met-Tau

Full text of DOI:10.1016/S0014-827X(03)00165-4

Il Farmaco 58 (2003) 1121ꢀ1130
/
www.elsevier.com/locate/farmac
Synthesis and activity of HCOꢀ
/
Metꢀ
/
Leuꢀ
/
Pheꢀ
/
OMe analogues
containing b-alanine or taurine at the central position
Cesare Giordano a, Gino Lucente a,b,*, Marianna Nalli b, Giampiero Pagani Zecchini a,b
,
Mario Paglialunga Paradisi a,b, Katia Varani c, Susanna Spisani d
a Istituto di Chimica Biomolecolare del CNR, Sezione di Roma c/o, Dipartimento di Studi Farmaceutici, Universita` degli Studi di Roma ‘La Sapienza’,
P.le A. Moro 5, 00185 Rome, Italy
b Dipartimento di Studi Farmaceutici, Universita` degli Studi di Roma ‘La Sapienza’, P.le A. Moro 5, 00185 Rome, Italy
c Dipartimento di Medicina Clinica e Sperimentale, Unita` di Farmacologia, Via Fossato di Mortara 19, Universita` di Ferrara, 44100 Ferrara, Italy
d Dipartimento di Biochimica e Biologia Molecolare, Via L. Borsari 46, Universita` di Ferrara, 44100 Ferrara, Italy
Received 14 March 2003; accepted 24 May 2003
Abstract
New synthetic analogues of the chemotactic N-formyltripeptide HCOꢀ
new models, namely BocꢀMetꢀb-AlaꢀPheꢀOMe (1), HCOꢀMetꢀb-Alaꢀ
MetꢀTauꢀPheꢀOMe (4) and HCl×MetꢀTauꢀPheꢀOMe (5), are characterized by the presence at the central position of a residue of
b-alanine or 2-aminoethanesulfonic acid (taurine) replacing the native -leucine. Whereas tripeptides 1 and 2 have been found quite
/
Metꢀ
/
Leuꢀ
/
Pheꢀ/OMe have been synthesized. The reported
/
/
/
/
/
/
/
Pheꢀ
/
OMe (2), Bocꢀ
/
MetꢀTauꢀPheꢀOMe (3), HCOꢀ
/
/
/
/
/
/
/
/
/
/
/
L
inactive as chemoattractants, all the three models containing the Tau residue exhibit a remarkable activity. Superoxide anion
production and lysozyme release have been also evaluated and the biological results are discussed together with the conformational
preferences of the examined models.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: b-Alanine; Chemotactic peptides; Human neutrophils; Taurine
1. Introduction
amino acids) or of 2-aminoethanesulfonic acids. Here
we report the first results concerning the most simple
models incorporating at the central position the achiral
residues of b-alanine (b-Ala) and of its sulfonic acid
analogue taurine (Tau).
In a previous article [1] we reported synthesis and
activity of analogues of the chemotactic tripeptide N-
formyl-
ter (fMLF-OMe) characterized by the presence at the
central position of achiral v-amino acid residues H2NÃ
(CH2)n ÃCOOH replacing the central -leucine. Models
L-methionyl-L-leucyl-L-phenylalanine methyl es-
In recent years studies on oligopeptides incorporating
b-Ala [2ꢀ
/
8] or Tau [9ꢀ17] residues have received
/
/
considerable attention in view of the tendency of these
models to adopt preferential secondary structures as
well as of the stability towards enzymatic degradation
/
L
incorporating g-aminobutyric, d-aminovaleric and o-
aminocaproic acid residues showed good and selective
antagonistic activity.
[18ꢀ21]. Furthermore, it is interesting to note here that
/
taurine, in addition to a variety of biological functions
[22,23], has well defined interactions with the activity of
neutrophils, the phagocytic cells on which the fMLF-
OMe receptors are located. Taurine is, in fact, a
recognized modulator of the ‘respiratory burst’ of
neutrophils and this property suggests that Tau deriva-
tives and analogues may be advantageous for the
development of antiinflammatory agents [24,25].
As a prosecution of these studies we started recently a
research program aimed at defining synthesis and
properties of fMLF-OMe analogues incorporating re-
sidues of achiral and chiral 3-aminopropanoic acids (b-
* Corresponding author.
E-mail address: gino.lucente@uniroma1.it (G. Lucente).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00165-4

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C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ1130
/
2. Chemistry
3. Biological results
The synthesis of Bocꢀ
BocꢀMetꢀTauꢀPheꢀOMe (3) and of the corresponding
N-formyl analogues HCOꢀMetꢀb-AlaꢀPheꢀOMe (2)
and HCOꢀMetꢀTauꢀPheꢀOMe (4) has been per-
formed according to the Scheme 1. Compounds 1 and
3 were prepared in solution by coupling BocꢀMetꢀOH
with HCl×b-AlaꢀPheꢀOMe (see Scheme 1) or HBr×
TauꢀPheꢀOMe [26] using isobutyl chloroformate for
carboxyactivation (mixed anhydride method). Acidoly-
sis of Bocꢀb-AlaꢀPheꢀOMe and 3, performed with
thionyl chloride in methanol, afforded HCl×b-Alaꢀ
PheꢀOMe and HCl×MetꢀTauꢀPheꢀOMe (5). A direct
transformation of the N-Boc derivatives 1 and 3 into the
corresponding N-formyl analogues 2 and 4 was per-
formed by following the procedure of Lajoie and Kraus
[27]. Thus, treatment of 1 and 3 with formic acid,
followed by ethyl 2-ethoxy-1,2-dihydro-1-quinolinecar-
boxylate (EEDQ), gave the formyl derivatives 2 and 4.
/
Metꢀ
/
b-Alaꢀ
/
Pheꢀ
/
OMe (1) and
The agonistic activity of the new ligands has been
determined on human neutrophils and compared with
that of the standard tripeptide fMLF-OMe; directed
migration (chemotaxis), superoxide anion production
and lysozyme release have been measured.
/
/
/
/
/
/
/
/
/
/
/
/
While the tripeptides 1 and 2 containing the central b-
Ala residue have been found quite inactive as chemoat-
tractants, both the corresponding analogues 3 and 4, in
which the Tau residue replaces the b-Ala, exhibit a
remarkable chemotactic activity, comparable to that
shown by the reference tripeptide fMLF-OMe (Fig. 1).
Furthermore, the Tau containing pseudotripeptide 5,
possessing a free N-terminal amino group, shows an
activity which is intermediate between that observed in
the case of the corresponding N-formyl derivative 4 and
N-Boc protected model 3. Concerning the superoxide
anion production and the lysozyme release, all the
examined compounds, regardless the nature of the
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
Scheme 1.

C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ
/
1130
1123
Fig. 1. Chemotactic activity of peptides 1ꢀ5.
/
central residue and the substituent at the N-terminal
amino group, are practically inactive, with the exception
of weak activity as secretagogue agents observed at high
concentration for the b-Ala containing N-formyl tripep-
tide 2 (data not shown).
The antagonistic activity was determined by measur-
ing the ability of the new analogues to inhibit the above
cited functions stimulated by an optimal dose of fMLF-
OMe. In the case of the chemotaxis, the b-Ala contain-
ing derivatives 1 and 2, which have been found inactive
as chemoattractants, have been examined. The influence
of increasing concentration of these two compounds on
the chemotaxis induced by 10 nM fMLF-OMe is shown
in Fig. 2A. A weak inhibition is observed for both the
models and the Boc derivative 1 is, as expected, the most
efficient antagonist.
the new analogues 3 and 4 and by the reference
tripeptide fMLF-OMe. In Table 1 the chemotactic
activity, superoxide anion production, lysozyme release,
and binding analysis of the new analogues 3 and 4 are
reported and compared with those exhibited by the
parent fMLF-OMe. The order of potency in [3H]-fMLF
displacement assays for the test peptides is: fMLF-
OMeꢀ
/
3ꢀ4. Thus, fMLF-OMe is the most potent
/
peptide, with its affinity in the nanomolar range (60
nM), while 3 and 4 display affinities in the micromolar
range (15 000 and 20 000 nM, respectively).
The above reported results clearly show that the
replacement, in the molecule of protypical ligand
fMLF-OMe, of the central leucine with a residue of b-
Ala affords the tripeptide 2 which is devoid of activity.
On the contrary, an analogous structural alteration of
the fMLF-OMe molecule, performed by introducing a
Tau residue in place of the native Leu, leads to the N-
formyl pseudotripeptide 4 which exhibits a chemotactic
activity comparable to that of the fMLF-OMe. Further-
more, models 3 and 5, which contain a bulky N-
acylating group and a free N-terminal amino group,
respectively, are both chemoattractants with activities
only slightly lower than those shown by fMLF-OMe
and by the corresponding N-formyl derivative 4 (see
Fig. 1). These latter findings are quite unexpected since
the N-deacylation of chemotactic N-formyltripeptides
affords inactive derivatives [28] and the introduction of
the bulky t-butyloxycarbonyl substituent at the Met NH
is associated with antagonistic activity [28,29].
The antagonism towards the superoxide anion pro-
duction exhibited by compounds 1, 2, and 5 is reported
in Fig. 2B; the N-deprotected derivative 5 exerts a
statistically significant reduction (P B0.05) on the
/
activity induced by 1 mM fMLF-OMe starting from
10ꢁ8 M and the inhibition progressively increases with
the concentration up to 44%; a weaker antagonist
activity is exhibited by 1 and 2. As shown in Fig. 2C,
only a weak inhibitory action on the lysozyme release is
observed for compounds 1, 2, and 5; in this case, the
formyl derivative 2 is the most efficient antagonist.
Receptor binding experiments have been carried out
on derivatives 3 and 4. Fig. 3 shows the [3H]-fMLF
displacements caused by increasing concentrations of

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C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ1130
/
Fig. 2. Antagonistic effect of b-Ala derivatives 1 and 2 and Tau-containing hydrochloride 5 on chemotaxis activated by 10 nM fMLF-OMe (A), on
superoxide anion production (B) and on release of granule enzymes (C) triggered by 1 mM fMLF-OMe.
undertaken to ascertain the involvement of the NH
groups in intramolecular H-bonds. In Fig. 4 is reported
the chemical shift dependence of the NH resonances as a
function of DMSO-d6 concentration in CDCl3 solution
(10 mM).
The results of these titration experiments clearly show
that in all the four models 1ꢀ4 the NH of the central
/
residue (i.e. b-Ala and Tau) presents a pronounced
solvent inaccessibility and in particular, in the case of
the N-Boc protected model 1, the b-Ala NH is
practically unaffected by the increase of the DMSO-d6
concentration (Ddꢂ
NH groups of the external Met and Phe residues of 1ꢀ
with the exception of the two BocꢀNH groups of 1 and
/0.05 ppm). On the contrary, the
/4,
/
3, interact efficiently with the solvent and this is
particularly evident in the case of the Phe residue of
the Tau containing tripeptides 3 and 4 (Dd values of
1.42 and 1.40 ppm, respectively). As far as the rather low
solvent accessibility of the two BocꢀNH groups of 1 and
/
3 is concerned, this is only in part attributable to the
participation to intramolecular H-bondings; in fact, as
previously observed [30], the bulky t-butyloxycarbonyl
group significantly hinders the interaction of the NH
with the solvent. In accordance with this effect and as
can be seen in Table 2, the Met formamido NH groups
of 2 and 4 exhibit Dd values (0.88 and 0.91 ppm,
respectively) higher than those of the corresponding Boc
protected NH groups (0.41 and 0.46 ppm, respectively).
The presence in the examined tripeptides of an
intramolecularly H-bonded NH group is also revealed
by the examination of the IR spectra in the NH
stretching region. In the spectrum of 1 two bands are
observed at 3348 and 3430 cmꢁ1, corresponding to the
H-bonded and free NH groups, respectively. The ratio
between the intensity of the two absorptions was found
Fig. 3. Competition curves of specific [3H]-fMLF binding to human
neutrophils by the test compounds. Curves are representative of a
single experiment taken from a series of three independent experi-
ments. Non specific binding was determined in the presence of 100 mM
fMLF.
4. Conformational studies
In order to investigate the role of the backbone
conformation on the remarkable biological activity
exhibited by pseudotripeptides 3 and 4, containing the
Tau central residue, as compared to the inactivity
observed in the case of the corresponding peptides 1
1
and 2, incorporating the b-Ala, an H NMR study was

C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ
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1130
1125
Table 1
Chemotactic activity, superoxide anion production, lysozyme release, and binding analysis of fMLF-OMe, 3, and 4
Peptide
Chemotaxis C.I.
Superoxide production O2ꢁ nmole
% Lysozyme release
Receptor binding IC50 (nM)
609
15 0009
20 0009
fMLF-OMe
1.209
/
0.08
(10ꢁ9 M)
529
/3
529
/
3
/
4
(10ꢁ6 M)
(10ꢁ6 M)
3
0.9390.07
(10ꢁ9 M)
1.0690.08
(10ꢁ9 M)
Efficacy data of chemotaxis are expressed as chemotactic index (C.I.), superoxide anion production is expressed as net nanomole of O2ꢁ/1ꢃ
cells/5 min, and lysozyme release is expressed in% (9SEM). In parentheses the concentration of the maximum agonistic activity. All data are
represented by the mean of six independent experiments performed in duplicate. IC50 values represent the mean9
/
2.290.1
(10ꢁ5 M)
10.290.3
(10ꢁ5M)
/
1090.5
(10ꢁ5 M)
1490.6
(10ꢁ5M)
/
/
150
4
/
/
/
/130
/
106
/
/SEM of three independent
determinations performed in duplicate.
practically concentration independent over the range
tetrahedral hybridization of the sulfur atom, the high
10.0ꢀ/1.0 mM. Thus, peptide self-association occurs, if
polar character and the acidꢀ/base property of the
any, at limited extent under these experimental condi-
tions and the band at 3348 cmꢁ1 can be assigned to the
intramolecularly H-bonded b-Ala NH group. The IR
spectrum of 3 shows a very close outcome in the NH
stretching region: two bands are observed at 3346 and
3433 cmꢁ1, and the band at lower frequency can be
assigned to an intramolecularly bonded Tau NH.
sulfonamide junction [14ꢀ16,31].
/
Concerning the above reported binding studies, the
results are in accordance with the weak activity in
relation to O2ꢁ production and lysosomal enzyme
release but are not consistent with the remarkable and
selective chemotactic activity exhibited by pseudopep-
tides 3 and 4. However, an analogous behaviour has
already been observed in the case of fMLF-OMe
analogues containing unnatural residues such as 4-
amino-tetrahydrothiopyran-4-carboxylic acid (Thp). In
these cases, full agonists such as fMLF-OMe, are
effective in displacing the labelled peptide from its
binding sites, while selective chemoattractants, as
[Thp1]fMLF-OMe or 3 and 4, are much less efficacious
[32]. This different behaviour can be rationalized on the
basis of the existence of at least two different functional
5. Discussion and conclusion
Although the above reported conformational studies
can be interpreted in terms of different foldings, they
suggest the presence of a significant population of the
tripeptide molecules in a conformation extended at the
N- and C-terminal residues and the involvement of the
central Tau and b-Ala NH groups in an intraresidue H-
receptor subtypes or isoforms [33ꢀ35]; low doses of a
/
full agonist (or a ‘pure’ chemoattractant) are required to
interact with a high-affinity receptor subtype that
activates transduction pathway responsible for chemo-
tactic response, while the increase of the full agonist
concentration allows binding with the low-affinity
subtype, able to activate the trasduction pathways
responsible for O2ꢁ production and lysozyme release.
As a consequence a peptide selective for chemotaxis,
which preferentially interacts with the high-affinity
subtype is not efficient in effectively displacing the full
agonist [3H]-fMLF, whose dose is sufficiently high to
interact also with the low-affinity receptor subtype.
a
bond (C6 conformation); the JNHÃC H values observed
for Met and Phe residues, as reported in Table 2, are in
accordance with this hypothesis. In particular, the
a
higher JNHÃC H values found for the C-terminal Phe
residue of the pseudopeptides 3 and 4 as compared with
1 and 2, indicate that the mixture of conformers which
populate the CHCl3 solution should be, in this case,
more rich of structures adopting an extended C-terminal
portion. However, the participation of the central NH to
H-bond interaction with the N-terminal carbonyl group
(C7 conformation) cannot be ruled out.
By taking into account the above discussed common
conformational features shown by the b-Ala and the
Tau containing models, this difference does not seem
prevalent to explain the different chemotactic responses.
Thus, although more b-sulfonamido pseudopeptides
should be examined to clarify the picture, the different
activity exhibited by the two N-formyltripeptides 2 and
4, as well as the unexpected activity of 3 and 5, seem
strictly connected with the intrinsic structural and
6. Experimental
6.1. Chemistry
Melting points were determined with a Buchi oil bath
¨
apparatus and are uncorrected. Optical rotations were
taken at 20 8C with a Schmidt-Haensch Polartronic D
polarimeter (1 dm cell, c 1.0 in CHCl3). IR spectra (KBr
chemical properties of the SO2Ã
/
NH as compared with
the usual COÃNH, with particular reference to the
/

1126
C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ1130
/
Fig. 4. Delineation of hydrogen-bonded NH groups in tripeptides 1ꢀ
DMSO-d6 concentration (% v/v) in CDCl3 solution. Peptide concentration 10 mM. (A) Bocꢀ
PheꢀOMe (2), (C) BocꢀMetꢀTauꢀPheꢀOMe (3), (D) HCOꢀMetꢀTauꢀPheꢀOMe (4).
/
4. Chemical shift dependence of the NH resonances as a function of the
/
Metꢀb-AlaꢀPheꢀOMe (1), (B) HCOꢀMetꢀb-Alaꢀ
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
disks) were recorded employing a Perkinꢀ
/
Elmer FT-IR
pared as described in the literature [36,26]. Bocꢀ
OH (Fluka Chemie, AG, Switzerland), Bocꢀb-Alaꢀ
(Fluka Chemie), HCl×PheꢀOMe (Fluka Chemie), and
fMLF-OH (Sigma, USA) were employed without pur-
ification. Elemental analyses were performed in the
laboratories of the Servizio Microanalisi del CNR,
Area della Ricerca di Roma, Montelibretti, Italy and
/
Metꢀ
/
Spectrum 1000 spectrometer. 1H NMR spectra were
determined in CDCl3 solution (unless otherwise speci-
fied) with a Bruker AM 200 spectrometer using Me4Si as
internal standard. Thin-layer and preparative layer
chromatographies were performed on silica gel Merck
60 F254 plates. The drying agent was sodium sulfate.
/
/OH
/
/
Parent fMLF-OMe and HBr×
/
Tauꢀ
/
Pheꢀ/OMe were pre-
were within 90.4% theoretical values. The abbrevia-
/

C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ
/
1130
1127
Table 2
hydrochloride and NMM in dry CH2Cl2 (3.6 ml) were
added as described for Bocꢀb-AlaꢀPheꢀOMe. Usual
work up afforded a residue (0.485 g), which was purified
by preparative layer chromatography [CHCl3ꢀMeOH
(95:5)] to give the pure title compound 1 (0.381 g, 79%)
as a foam. M.p. 105ꢀ106 8C (EtOAc). [a]Dꢂ 628. IR:
3356, 3319, 2949, 1736, 1686, 1647, 1526, 1168 cmꢁ1. 1H
NMR d (ppm): 1.44 [9H, s, C(CH3)3], 1.78ꢀ2.17 (2H, m,
Met b-CH2), 2.11 (3H, s, SÃCH3), 2.34 (2H, m, b-Ala a-
CH2), 2.55 (2H, t, Jꢂ7.3 Hz, Met g-CH2), 3.06 and 3.16
(2H, A and B of an ABX, Jꢂ7.8, 5.5, and 13.8 Hz, Phe
Solvent accessibility of peptide NH groups: differences (Dd, ppm)
between NH chemical shift values observed in CDCl3 solution
containing (CD3)2SO (10%) and in neat CDCl3; see also Fig. 4
/
/
/
/
Compound
Met NH
b-Ala NH
Phe NH
/
/
ꢄ
/
1
2
0.41 (7.3)
0.88 (7.4)
0.05
0.19
0.72 (7.3)
0.84 (7.8)
/
Tau NH
/
3
4
0.46 (7.9)
0.91 (8.1)
0.20
0.29
1.42 (8.8)
1.40 (9.1)
/
/
a
In parentheses the JNHÃC H values (Hz) for Met and Phe residues.
b-CH2), 3.30 and 3.78 (2H, 2 m, b-Ala b-CH2), 3.76
(3H,s, COOCH3), 4.17 (1H, m, Met a-CH), 4.84 (1H, m,
tions used are as follows: b-Ala, 3-aminopropanoic acid;
Boc, tert-butyloxycarbonyl; DMF, N,N-dimethylfor-
mamide; DMSO, dimethyl sulfoxide; EEDQ, ethyl 2-
Phe a-CH), 5.27 (1H, d, Jꢂ
/
7.3 Hz, Met NH), 6.58 (1H,
d, Jꢂ
/
7.3 Hz, Phe NH), 7.10 (1H, br, b-Ala NH), 7.16-
7.40 (5H, m, aromatic). Anal. (C, H, N) for
ethoxy-1,2-dihydro-1-quinolinecarboxylate;
Krebs-Ringer-phosphate containing 0.1% w/v
cose; NMM, N-methylmorpholine.
KRPG,
-glu-
C23H35N3O6S.
D
6.1.3. HCOꢀ
/
Metꢀ
/
b-Alaꢀ
/
PheꢀOMe (2)
/
The Boc-tripeptide 1 (0.169 g, 0.35 mmol) was
dissolved in formic acid (2.1 ml) and the mixture stirred
at room temperature for 1 day. After removal of the
excess of formic acid in vacuo, the residue was dissolved
in 2.1 ml of dry DMF. EEDQ 97% (0.107 g, 0.42 mmol)
was added and the solution stirred at room temperature
for 24 h. Evaporation under reduced pressure afforded a
residue which was dissolved in dry chloroform and the
product was precipitated by n-hexane. Washing with
dry ether afforded the pure formylpeptide 2 (0.133 g,
6.1.1. Boc-b-Alaꢀ
Isobutyl chloroformate 98% (0.13 ml, 1 mmol) was
added at ꢁ15 8C to a stirred solution of Bocꢀb-Alaꢀ
/
Pheꢀ
/
OMe
/
/
/
OH (0.189 g, 1 mmol) and NMM (0.13 ml, 1.2 mmol) in
dry CH2Cl2 (5 ml). The temperature was maintained at
ꢁ
/
15 8C for 10 min, and HCl×
mmol), NMM (0.11 ml, 1 mmol) and dry CH2Cl2 (3.6
ml) were then added. The mixture was stirred at ꢁ15 8C
/
PheꢀOMe (0.216 g, 1
/
/
for 15 min and then allowed to warm to room
temperature. Dry DMF (20 drops) was added and
stirring was continued 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 under reduced
pressure to give pure oily title compound (0.35 g, 100%),
which slowly crystallized at room temperature. M.p.
93%) as a white powder. M.p. 123 8C. [a]Dꢂ
/
ꢄ668. IR:
/
3293, 3084, 2951, 2919, 1739, 1646, 1538, 1443, 1384,
1
1223 cmꢁ1. H NMR d (ppm): 1.85ꢀ
b-CH2), 2.13 (3H, s, SÃCH3), 2.36 (2H, m, b-Ala a-
CH2), 2.56 (2H, m, Met g-CH2), 3.08 and 3.14 (2H, A
and B of an ABX, Jꢂ7.9, 5.5, and 13.5 Hz, Phe b-CH2),
/
2.20 (2H, m, Met
/
/
3.34 and 3.72 (2H, 2 m, b-Ala b-CH2), 3.77 (3H, s,
COOCH3), 4.56 (1H, m, Met a-CH), 4.85 (1H, m, Phe
91ꢀ
1683, 1650, 1524 cmꢁ1. H NMR d (ppm): [1.43 9H, s,
C(CH3)3], 2.37 (2H, t, Jꢂ6 Hz, b-Ala a-CH2), 3.07 and
3.15 (2H, A and B of an ABX, Jꢂ6.3, 5.7 and 13.8 Hz,
/
92 8C. [a]Dꢂ
/
ꢄ
/
518. IR: 3369, 3317, 2984, 1740,
1
a-CH), 6.75 (1H, d, Jꢂ
Jꢂ7.4 Hz, Met NH), 7.14ꢀ
b-Ala NH), 8.17 (1H, s, HÃ
C19H27N3O5S.
/
7.8 Hz, Phe NH), 6.91 (1H, d,
/
/
/7.38 (6H, m, aromatic and
/
/
CO). Anal. (C, H, N) for
Phe b-CH2), 3.36 (2H, m, b-Ala b-CH2), 3.73 (3H, s,
COOCH3), 4.87 (1H, m, Phe a-CH), 5.10 (1H, br, b-Ala
NH), 6.11 (1H, d, Jꢂ
/
7.3 Hz, Phe NH), 7.05ꢀ7.33 (5H,
/
6.1.4. Bocꢀ
/
Metꢀ
/
Tauꢀ
/
Pheꢀ
/
OMe (3)
m, aromatic). Anal. (C, H, N) for C18H26N2O5.
BocꢀMetꢀOH (0.272 g, 1.09 mmol) was activated
/
/
with isobutyl chloroformate 98% (0.14 ml, 1.09 mmol)
and NMM (0.14 ml, 1.3 mmol) in dry tetrahydrofuran
(8 ml) as described for compound 1. Addition of a
6.1.2. Bocꢀ
Thionyl chloride (0.076 ml, 1.05 mmol) was added to
a solution of Bocꢀb-AlaꢀPheꢀOMe (0.35 g, 1 mmol) in
dry methanol (1 ml), cooled at ꢁ15 8C. After stirring at
15 8C for 30 min and at 45 8C for 2.5 h, the solution
/
Metꢀ
/
b-Alaꢀ
/
Pheꢀ
/
OMe (1)
/
/
/
solution of HBr×
/
Tauꢀ
/
PheꢀOMe [26] (0.41 g, 1.09
/
/
mmol) and NMM (0.12 ml, 1.09 mmol) in dry tetra-
hydrofuran (5 ml) and usual work up afforded an oily
residue which was crystallized from diethyl ether-n-
ꢁ
/
was evaporated under vacuum to give intermediate
hydrochloride as a foam. This salt was used without
hexane (0.484 g, 84%). M.p. 119ꢀ
IR: 3343, 2985, 1738, 1661, 1521, 1344, 1149 cmꢁ1. H
NMR d (ppm): 1.43 [9H, s, C(CH3)3], 1.80ꢀ2.18 (2H, m,
Met b-CH2), 2.11 (3H, s, SÃCH3), 2.55 (2H, t, Jꢂ7.3
Hz, Met g-CH2), 2.87 (2H, m, Tau a-CH2), 2.99 and
/
121 8C. [a]Dꢂ
/
ꢁ
/
138.
1
further purification. Bocꢀ
/
MetꢀOH (0.249 g, 1 mmol)
/
was activated with isobutyl chloroformate 98% (0.133
ml, 1 mmol) and NMM (0.13 ml, 1.2 mmol) in dry
CH2Cl2 (5 ml) and equivalent amounts of the above
/
/
/

1128
C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ1130
/
3.19 (2H, A and B of an ABX, Jꢂ/5, 8, and 13.9 Hz, Phe
6.2.2. Cell preparation
b-CH2), 3.55 (2H, m, Tau b-CH2), 3.78 (3H,s,
Cells were obtained from the blood of healthy
subjects, and human peripheral blood neutrophils were
purified using the standard techniques of dextran
(Pharmacia, Uppsala, Sweden) sedimentation, centrifu-
gation on Ficoll-Paque (Pharmacia), and hypotonic lysis
of contaminating red cells. Cells were washed twice and
resuspended in KRPG, pH 7.4, at a final concentration
COOCH3), 4.20 (1H, m, Met a-CH), 4.43 (1H, m, Phe
a-CH), 5.18 (1H, d, Jꢂ
Jꢂ8.8 Hz, Phe NH), 7.10 (1H, br, Tau NH), 7.21ꢀ
(5H, m, aromatic). Anal. (C, H, N) for C22H35N3O7S2.
/
7.9 Hz, Met NH), 6.92 (1H, d,
/
/
7.43
6.1.5. HCOꢀ
/
Metꢀ
/
Tauꢀ
/
PheꢀOMe (4)
/
of 50ꢃ
/
106 cells/ml and kept at room temperature until
100% pure and 99% viable,
as determined using the Trypan blue exclusion test.
The Boc-derivative 3 (0.263 g, 0.5 mmol) was
dissolved in formic acid (3 ml) and the mixture stirred
at room temperature for 1 day. After removal of the
excess of formic acid in vacuo, the residue was dissolved
in 3 ml of dry DMF. EEDQ 97% (0.153 g, 0.6 mmol)
was added and the solution stirred at room temperature
for 24 h. Evaporation under reduced pressure afforded a
residue which was purified by preparative layer chro-
used. Neutrophils were 98ꢀ
/
6.2.3. Random locomotion
Random locomotion was performed with 48-well
microchemotaxis chamber (Bio Probe, Milan, Italy)
and migration into the filter was evaluated by the
method of leading-front [37]. The actual control random
matography [CHCl3ꢀ/MeOH (98:2)] to give the pure title
compound 4 (0.2 g, 88%) as a foam. [a]Dꢂ
/
ꢁ
/
198. IR
movement is 35 mm9
/
3 SE of 10 separate experiments
(CHCl3): 3340, 1743, 1674, 1339, 1145 cmꢁ1. H NMR
(CDCl3): d (ppm): 1.85ꢀ2.18 (2H, m, Met b-CH2), 2.11
(3H, s, SÃCH3), 2.55 (2H, t, Jꢂ 7.3 Met g-CH2), 2.89
(2H, m, Tau a-CH2), 2.99 and 3.19 (2H, A and B of an
ABX, Jꢂ4.9, 8.8, and 13.9 Hz, Phe b-CH2), 3.51 (2H,
m, Tau b-CH2), 3.80 (3H, s, COOCH3), 4.42 (1H, m,
Phe a-CH), 4.62 (1H, m, Met a-CH), 6.18 (1H, d, Jꢂ
9.1 Hz, Phe NH), 6.92 (1H, d, Jꢂ8.1 Hz, Met NH),
7.20ꢀ7.40 (6H, m, aromatic and Tau NH), 8.12 (1H, s,
HÃCO). Anal. (C, H, N) for C18H27N3O6S2.
1
performed in duplicate.
/
/
/
6.2.4. 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 10ꢁ12 to 10ꢁ5 M.
Data were expressed in terms of chemotactic index,
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. Standard errors are
/
/
/
/
6.1.6. HCl×
Thionyl chloride (0.044 ml, 0.60 mmol) was added to
a solution of BocꢀMetꢀTauꢀPheꢀOMe (3) (0.3 g, 0.57
mmol) in dry methanol (3 ml), cooled at ꢁ15 8C. After
stirring at ꢁ15 8C for 30 min and at 45 8C for 2.5 h, the
/
Metꢀ
/
Tauꢀ
/
Pheꢀ
/
OMe (5)
/
/
/
/
/
in the 0.02ꢀ0.09 chemotactic index range.
/
/
solution was evaporated under vacuum to give the
hydrochloride 5 as a foam in quantitative yield.
6.2.5. Superoxide anion (O2ꢁ) production
[a]Dꢂ
1437, 1361, 1144 cmꢁ1
(ppm): 2.01 (2H, m, Met b-CH2), 2.06 (3H, s, SÃ
2.51 (2H, t, Jꢂ 7.1 Hz, Met g-CH2), 2.85ꢀ3.06 (4H, m,
/
ꢄ
/
108. IR (CHCl3): 3035, 1738, 1710, 1680,
1H NMR (DMSO-d6): d
CH3),
The superoxide anion was measured by the super-
oxide dismutase-inhibitable reduction of ferricyto-
chrome c (Sigma, USA) modified for microplate-based
assays. Tests were carried out in a final volume of 200 ml
.
/
/
/
Phe b-CH2 and Tau a-CH2), 3.22 and 3.37 (2H, 2 m,
Tau b-CH2), 3.64 (3H, s, COOCH3), 3.81 (1H, poorly
containing 4ꢃ
/
105 neutrophils, 100 nmole cytochrome c
and KRPG. At zero time different amounts (10ꢁ9ꢀ
/
8ꢃ
/
resolved t, Met a-CH), 4.17 (1H, m, Phe a-CH), 7.21ꢀ
/
10ꢁ5 M) of each peptide were added and the plates were
incubated into a microplate reader (Ceres 900, Bio-TeK
Instruments, Inc.) with the compartment temperature
set at 37 8C. Absorbance was recorded at wavelenghts of
550 and 468 nm. The difference in absorbance at the two
wavelenghts was used to calculate nmole of O2ꢁ
produced using an absorptivity for cytochrome c of
18.5 mMꢁ1 cmꢁ1. Neutrophils were incubated with 5
mg/ml cytochalasin B (Sigma) for 5 min prior to
activation by peptides. Results were expressed as net
7.33 (5H, m, aromatic), 8.08 (1H, d, Jꢂ6.6 Hz, Phe
/
NH), 8.41 (2H, s, Met NH2), 8.80 (1H, poorly resolved t,
Tau NH). Anal. (C, H, N) for C17H28ClN3O5S2.
6.2. Biological assays
6.2.1. Peptides
Stock solutions of fMLF-OMe and peptide analo-
gues, 10ꢁ2 M, were prepared in DMSO and diluted in
KRPG, pH 7.4, before use. At the concentration used,
DMSO did not interfere with any of the biological
assays performed.
nmole of O2ꢁ per 1ꢃ
mean of six separate experiments performed in dupli-
cate. Standard errors are in 0.1ꢀ
4 nmole O2ꢁ range.
/
106 cells per 5 min and are the
/

C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ
/
1130
1129
6.2.6. Enzyme assay
The release of neutrophil granule enzymes was
evaluated by determination of lysozyme activity, mod-
radioactivity was counted on Top Count (efficiency
57%) with Micro-Scint-20 (30 ml in 96-well plates).
The cold drug concentrations displacing 50% of
labelled ligand (IC50) were obtained by computer
analysis of displacement curves. All data were analysed
using the non-linear regression curve fitting computer
program Graph Pad Prism (Graph Pad, San Diego,
CA). All the values obtained are the mean of three
independent experiments performed in duplicate.
ified for microplate-based assays. Cells, 3ꢃ
106/well,
/
were first incubated in triplicate wells of microplates
with 5 mg/ml cytochalasin B at 37 8C for 15 min and then
in the presence of each peptide in a final concentration
of 10ꢁ9ꢀ
were then centrifuged at 400ꢃ
/
8ꢃ
/
10ꢁ5 M for a further 15 min. The plates
g 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 microplate
reader at 465 nm. Enzyme release was expressed as a net
percentage of total enzyme content released by 0.1%
Acknowledgements
This work was supported in part by the Italian
‘Ministero dell’Istruzione, dell’Universita` e della Ricer-
ca’. We are grateful to Banca del Sangue of Ferrara for
providing fresh blood.
Triton X-100. Total enzyme activity was 859
/
1 mg/1ꢃ
/
107 cells/min. The values are the mean of five separate
experiments done in duplicate. Standard errors are in
the range 1ꢀ6%.
/
6.2.7. Antagonist assay
References
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-OMe. Antagonist activity data (% of activity)
were obtained by comparing the chemotactic index,
nmole of O2ꢁ or percentage of lysozyme release in the
absence (100%) and in the presence of the derivative.
[1] G. Pagani Zecchini, E. Morera, M. Nalli, M. Paglialunga
Paradisi, G. Lucente, S. Spisani, Synthesis and activity on human
neutrophil functions of fMLF-OMe analogs containing alkyl
spacers at the central position, Farmaco 56 (2001) 851ꢀ858.
/
[2] S. Cerrini, E. Gavuzzo, G. Lucente, F. Pinnen, G. Zanotti, Ten-
membered cyclotripeptides. Synthesis and crystal structure of
cyclo(-b-alanyl-
L
-phenylalanyl-
13.
L-prolyl-), Int. J. Peptide Protein
Res. 34 (1989) 6ꢀ
/
Chemotactic index of 10 nM fMLF-OMe was 1.159
0.10 SE. O2ꢁ generation produced by 1 nM fMLF-OMe
was 4392 nmol/1ꢃ
106 cells/5 min. Enzyme activity
triggered by 1 mM fMLF-OMe was 5795%/3ꢃ
106
cells/min.
/
[3] S. Cerrini, E. Gavuzzo, G. Lucente, G. Luisi, F. Pinnen, L.
Radics, Synthesis and conformation of cyclo(-MebAla-Phe-Pro-)
and cyclo(-MebAla-Phe-DPro-), Int. J. Peptide Protein Res. 38
/
/
/
/
(1991) 289ꢀ297.
/
[4] V. Pavone, B. Di Blasio, A. Lombardi, C. Isernia, C. Pedone, E.
Benedetti, G. Valle, M. Crisma, C. Toniolo, R. Kishore, b-
Alanine and b-bends. X-Ray diffraction structures of three linear
Derivatives were added to neutrophils 10 min before
the incubation step for cellular functionality. Each value
represents an average of six separate experiments
performed in duplicate. Standard errors are within
10% of the mean value.
oligopeptides, J. Chem. Soc., Perkin Trans. 2 (1992) 1233ꢀ
[5] W.F. DeGrado, J.P. Schneider, Y. Hamuro, The twists and turns
of b-peptides, J. Peptide Res. 54 (1999) 206ꢀ217 (and references
cited therein).
/1237.
/
[6] V. Pavone, A. Lombardi, G. D’Auria, M. Saviano, F. Nastri, L.
Paolillo, B. Di Blasio, C. Pedone, b-Alanine containing peptides:
a novel molecular tool for the design of g-turns, Biopolymers 32
6.2.8. Statistical analysis
The nonparametric Wilcoxon test was used in the
statistical evaluation of differences between groups.
(1992) 173ꢀ183.
/
[7] V. Pavone, A. Lombardi, M. Saviano, F. Nastri, R. Fattorusso,
O. Maglio, C. Isernia, L. Paolillo, C. Pedone, b-Alanine contain-
ing cyclic peptides with turned structure: the pseudo type II b-
6.2.9. Receptor binding assay
turn. VI, Biopolymers 34 (1994) 1517ꢀ1526.
/
Binding assays were carried out essentially according
to Spisani et al. [38]. In competition experiments were
carried out to determine the IC50 values, 6 nM [3H]-
[8] A.K. Thakur, R. Kishore, Influence of hydrophobic interactions
on the conformational adaptability of the b-Ala residue, J.
Peptide Res. 57 (2001) 455ꢀ461 (and references cited therein).
/
[9] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, G. Pochetti, D.
Rossi, Structure and conformation of peptides containing the
fMLF (specific activityꢂ
Products, Du Pont de Nemours, Milan, Italy) was
incubated with 100 ml of human neutrophils (5ꢃ
106)
/
71.5 Ci/mmol, NEN Research
sulphonamide junction. I. N-acetyl-tauryl-
L
-penylalanine methyl
324.
/
ester, Int. J. Peptide Protein Res. 34 (1989) 319ꢀ
/
at different concentrations of the test compounds at
37 8C for 15 min. Non specific binding was measured in
the presence of 100 mM fMLF, and was about 20% of
total binding. Bound and free radioactivity was sepa-
rated by filtering the assay mixture through Whatman
GF/C glass-filters using a Micro-Mate 196 Cell Har-
vester (Packard Instrument Company). The filter-bound
[10] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, F. Pinnen, G.
Pochetti, D. Rossi, Structure and conformation of peptides
containing the sulphonamide junction. II. Synthesis and confor-
mation of cyclo(-MeTau-Phe-DPro-), Int. J. Peptide Protein Res.
34 (1989) 471ꢀ479.
/
[11] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, F. Pinnen, G.
Pochetti, D. Rossi, Structure and conformation of peptides
containing the sulphonamide junction. III. Synthesis, crystal

1130
C. Giordano et al. / Il Farmaco 58 (2003) 1121ꢀ1130
/
and molecular structure of a taurine containing peptidic oxa-
cyclol, Int. J. Peptide Protein Res. 37 (1996) 167ꢀ173.
[12] A. Calcagni, E. Gavuzzo, F. Mazza, F. Pinnen, G. Pochetti, D.
Rossi, Structure and conformation of peptides containing the
sulphonamide junction. IV. Synthesis and crystal conformation of
[26] J. Diaz, C. Cazaubon, H. Demarne, J.-P. Gagnol, R. Guegan, Y.
Muneaux, P. Perreaut, J.-P. Richaud, M. Vedel, R. Roncucci,
New analogs of somatostatin with unexpected effects in vivo on
insulin basal secretion in the rat (1), Eur J. Med. Chem.-Chim.
/
Ther. 20 (1985) 219ꢀ
[27] G. Lajoie, J.-L. Kraus, Rapid and efficient method for the N-
formylation of C-blocked peptides, Peptides 5 (1984) 653ꢀ654.
/
227.
N-benzoyl-
L
-phenylalanyl-tauryl-L-leucine methyl ester, Gazzetta
Chim. Ital. 122 (1992) 17ꢀ
/23.
/
[13] S. Paik, E.H. White, Synthesis of b-aminosulfonopeptides acti-
vated through selective N-nitration of a taurine amide unit,
[28] R.J. Freer, A.R. Day, J.A. Radding, E. Schiffmann, S. Aswani-
kumar, H.J. Showell, E.L. Becker, Further studies on the
structural requirements for synthetic peptide chemoattractants,
Tetrahedron Lett. 37 (1996) 4663ꢀ4666.
/
[14] A. Calcagni, D. Rossi, M. Paglialunga Paradisi, G. Lucente, G.
Luisi, E. Gavuzzo, F. Mazza, G. Pochetti, M. Paci, Peptides
containing the sulphonamide junction: Synthesis, structure and
conformation of Z-Tau-Pro-Phe-NHiPr, Biopolymers 41 (1997)
Biochemistry 19 (1980) 2404ꢀ2410.
/
[29] C.K. Derian, H.F. Solomon, J.D. Higgins, III, M.J. Beblavy, R.J.
Santulli, G.J. Bridger, M.C. Pike, D.J. Kroon, A.J. Fischman,
Selective inhibition of N-formylpeptide-induced neutrophil acti-
vation by carbamate-modified peptide analogues, Biochemistry 35
555ꢀ567.
/
[15] C. Gennari, M. Gude, D. Potenza, U. Piarulli, Hydrogen-bonding
donor/acceptor scales in b-sulfonamidopeptides, Chem. Eur. J. 4
(1996) 1265ꢀ1269.
/
[30] M. Aschi, G. Lucente, F. Mazza, A. Mollica, E. Morera, M.
Nalli, M. Paglialunga Paradisi, Peptide backbone folding induced
by the Ca-tetrasubstituted cyclic a-amino acids 4-amino-1,2-
dithiolane-4-carboxylic Acid (Adt) and 1-aminocyclopentane-1-
carboxylic acid (Ac5c). A joint computational and experimental
(1998) 1924ꢀ1930.
/
[16] A. Calcagni, E. Gavuzzo, G. Lucente, F. Mazza, E. Morera, M.
Paglialunga Paradisi, D. Rossi, Peptides containing the sulpho-
namide junction. 2. Structure and conformation of Z-Tau-Pro-
DPhe-NHiPr, Biopolymers 54 (2000) 379ꢀ
[17] A. Calcagni, P.G. Ciattini, A. Di Stefano, S. Dupre`, G. Luisi, F.
Pinnen, D. Rossi, A. Spirito, c(SO2ÃNH) transition state
isosteres of peptides. Synthesis and bioactivity of sulfonamido
pseudopeptides related to carnosine, Farmaco 54 (1999) 673ꢀ677.
[18] D. Seebach, J.L. Matthews, Peptides: a surprise at every turn,
Chem. Commun. (1997) 2015ꢀ2022 (and references cited therein).
[19] K. Gademann, T. Hintermann, J.V. Schreiber, b-Peptides: twist-
ing and turning, Curr. Med. Chem. 6 (1999) 905ꢀ925 (and
references cited therein).
/387.
study, Org. Biomol. Chem. 1 (2003) 1980ꢀ1988.
[31] T. Ohwada, I. Okamoto, K. Shudo, K. Shudo, K. Yamaguchi,
/
/
Intrinsic pyramidal nitrogen of N-sulfonylamides, Tetrahedron
Lett. 39 (1998) 7877ꢀ7880.
/
/
[32] 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. Signall. 12 (2000) 391ꢀ398.
/
[33] A. Dalpiaz, M.E. Ferretti, G. Vertuani, S. Traniello, A. Scatturin,
S. Spisani, C- and N-terminal residue effect on peptide derivatives
antagonism toward the formyl-peptide receptor, Eur. J. Pharma-
[20] H.N. Gopi, G. Ravindra, P.P. Pal, P. Pattanaik, H. Balaram, P.
Balaram, Proteolytic stability of b-peptide bonds probed using
quenched fluorescent substrates incorporating a hemoglobin
col. 436 (2002) 187ꢀ196.
/
cleavage site, FEBS Lett. 535 (2003) 175ꢀ178.
/
[34] A. Dalpiaz, S. Spisani, C. Biondi, E. Fasbbri, M. Nalli, M.E.
Ferretti, Studies on human neutrophil biological functions by
means of formylpeptide receptor agonists and antagonists, Curr.
[21] D.B.A. de Bont, K.M. Sliedregt-Bol, L.J.F. Hofmeyer, R.M.J.
Liskamp, Increased stability of peptidosulfonamide peptidomi-
metics towards protease catalyzed degradation, Bioorg. Med.
Drug Targets*/Immune Endocrine Metabolic Disorders 3 (2003)
Chem. 7 (1999) 1043ꢀ
[22] J.B. Lombardini, M.S. Mozaffari, S.W. Schaffer, Taurine, Amino
Acids 19 (2000) 497ꢀ738.
/1047.
33ꢀ42.
/
[35] Y. Le, Y. Yang, Y. Cui, H. Yazawa, W. Gong, C. Qiu, J.M.
Wang, Receptors for chemotactic formyl peptides as pharmaco-
logical targets, Int. Immunopharmacol. 2 (2002) 113.
/
[23] J.B. Lombardini, S.W. Schaffer, Special issue: taurine: discovered
185 years ago and still intrigues the scientific community, Amino
[36] G. Vertuani, S. Spisani, M. Boggian, S. Traniello, A. Scatturin,
Conformational studies of synthetic tripeptide chemoattractants,
Acids 23 (2002) 343ꢀ
/458.
[24] J. Muhling, M. Fuchs, C. Fleck, A. Sablotzki, M. Krull, M.G.
¨
¨
Int. J. Peptide Protein Res. 29 (1987) 525ꢀ532.
[37] S.H. Zigmond, J.G. Hirsch, Leukocyte locomotion and chemo-
/
Dehne, J. Gonter, S. Weiss, J. Engel, G. Hempelmann, Effects of
arginine, -alanyl- -glutamine or taurine on neutrophil (PMN)
free amino acid profiles and immune functions in vitro, Amino
Acids 22 (2002) 39ꢀ53.
L
L
taxis. New methods for evaluation and demonstration of cell-
derived chemotactic factor, J. Exp. Med. 137 (1973) 387ꢀ410.
/
/
[38] S. Spisani, A. Breveglieri, E. Fabbri, G. Vertuani, O. Rizzuti, G.
Cavicchioni, Modifications of the amide bond at position 3 in
fMLP analogs select neutrophil functions, Peptide Res. 9 (1996)
[25] L. Della Corte, R.R. Crichton, G. Duburs, K. Nolan, K.F.
Tipton, G. Tirzitis, R.J. Ward, The use of taurine analogues to
investigate taurine functions and their potential therapeutic
279ꢀ282.
/
applications, Amino Acids 23 (2002) 367ꢀ379.
/