The importance of the methionine configuration in for-Met-Leu-Phe-OMe biological activities

Article abstract of DOI:10.1006/bioo.1999.1155

for-D-Met-L-Leu-L-Phe-OMe was synthesized in order to understand the importance of the methionine configuration on human neutrophil biological activity. Our results show that both chemotactic response and lysozyme release are lower than those of the parent fMLP-OMe, indicating that the specific receptor pocket possesses a well-located, restricted positive area that does not completely face the D-Met residue. The triggering of superoxide anion production does not seem to be sensitive to this variation of the configuration. (C) 2000 Academic Press.

Full text of DOI:10.1006/bioo.1999.1155

Bioorganic Chemistry 8, 39–44 (2000)
doi:10.1006/bioo.1999.1155, available online at http://www.idealibrary.com on
The Importance of the Methionine Configuration in
for-Met-Leu-Phe-OMe Biological Activities
Susanna Spisani* and Giorgio Cavicchioni1,†
*Department of Biochemistry and Molecular Biology and †Department of Pharmaceutical Sciences,
University of Ferrara, 44100, Ferrara, Italy
Received May 14, 1999
for-D-Met-L-Leu-L-Phe-OMe was synthesized in order to understand the importance of the
methionine configuration on human neutrophil biological activity. Our results show that both
chemotactic response and lysozyme release are lower than those of the parent fMLP-OMe,
indicating that the specific receptor pocket possesses a well-located, restricted positive area that
does not completely face the D-Met residue. The triggering of superoxide anion production
does not seem to be sensitive to this variation of the configuration.
© 2000 Academic Press
INTRODUCTION
N-formylpeptides are involved in the defense mechanisms against bacterial infec-
tions by binding to specific receptors (FPR), which involve heterotrimeric G-proteins
in the transduction pathway. Occupancy of the FPR results in neutrophil chemotaxis,
oxidase activation, or degranulation, depending on the peptide concentrations and
experimental conditions (1).
In the field of the structure–activity relationships concerning chemotactic N-formyl-
tripeptides, several studies have been made on the backbone modification of the
prototypical chemotactic ligand N-for-Met-Leu-Phe-OH (fMLP) and its for-Met-Leu-
Phe-OMe analogue (fMLP-OMe) (2–4) for their pharmaceutical relevance. Attention
was recently focused by us, as well as by other groups, to ascertain the features of the
receptor pocket where the first residue allocates. In particular, the substitutions at the
Met residue can be split in two categories: the first has the aim of defining the pocket
dimensions varying the side chain features (5), the second of defining the charge inter-
action conditions which influence both the intensity and position of the residue charge
(6,7). It has been ascertained that the side chain of the residue at position 1 penetrates
in a hydrophobic pocket of limited depth and capacity. A positively charged area has
been proposed that surrounds the electron-rich sulphur atom, which in turn must be in
a well-defined precise position (5,8).
1 To whom correspondence and reprint requests should be addressed. Fax:+39-532-291296.
E-mail:g5z@dns.unife.it.
39
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Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.

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As a continuation of our studies, we thought it would be interesting to ascertain the
influence of a configurational change in the Met residue on human biological responses.
We therefore synthesized the tripeptide for-D-Met-L-Leu-L-Phe-OMe 1, since only
scarce, marginal information is available (9); we tested this peptide on neutrophil
functions involved in the inflammatory response, such as directional locomotion
(chemotaxis), superoxide anion (O−2 ) production, and lysozyme release. The biologi-
cal data were compared with those of the parent compound fMLP-OMe
METHODS
Chemical Synthesis
The formylpeptide 1 was synthetized following standard procedures in solution
(Scheme 1), purified by HPLC, and characterized by TLC and 1H-NMR.
Fmoc-D-Met, Boc-L-Leu, and L-Phe-OMe hydrochloride residues were purchased
from Fluka. Removal of the t-Boc group was performed by treatment with a 1:1 mix-
ture of TFA-CHCl3. Peptide coupling was achieved (i) by the racemization-free
mixed-anhydride method with isobutylchloroformate (IBCF) and triethylamine
(TEA), and (ii) by the 1-hydroxy-7-azabenzotriazole (HOAt)-N-(3-dimethylamino-
propyl)-N′-ethylcarbodiimide hydrochloride (EDC) method. Piperidine (Pip) and
dimethylformamide (DMF) was used to remove the Fmoc group. The formyl group
was introduced at the level of tripeptide using EDC and HCOOH.
HCO-D-Met-L-Leu-L-Phe-OMe. M.p. 166–168∞C; [α] = –34.3°C, c = 1 in MeOH. 1H-
NMR (CDCl3) δ 8.13 (s, 1H, HCO), 7.29 (s, 5H, phenyl), 7.11 (d, J = 7.58 Hz, 1H, NH),
6.55 (d, J = 7.35 Hz, 1H, NH), 6.40 (d, J = 8,01 Hz, 1H, NH), 4.83 (m, 1H, CH), 4.62 (m,
1H, CH), 4.38 (m, 1H, CH), 3.72 (s, 3H, CH3), 3.05 and 3.17 (AB of ABX, 2dd, JAB
13.19 Hz, JAX = 6.63 Hz, JBX = 5.74 Hz, 2H, CH2), 2.46 and 2.59 (AB of ABX, 2dd, JAB
=
=
13.19 Hz, JAX = 6.56 Hz, JABX = 7.14, Hz, 1H, CH2), 2.11 (s, 3H, CH3), 1.42–1.62 (m, 5H,
2CH2 + CH), 0.89 and 0.92 (2d, J = 5.53 Hz, 6H, 2CH3).
BIOLOGICAL ASSAYS
Human neutrophils were purified employing the standard techniques of dextran sed-
imentation of heparinized blood, followed by centrifugation on Ficoll-Paque. The
SCHEME 1. Synthesis of compound 1.

METHIONINE CONFIGURATION IN FORMYLTRIPEPTIDES
41
cells were washed twice and resuspended in Krebs–Ringer–Phosphate containing
0.1% w/v Glucose (KRPG), pH 7.4, at a concentration of 50 × 106 cells/ml.
Chemotaxis was performed with a 48-well microchemotaxis chamber, and the
migration into the filter was evaluated by the leading-front method (10). Chemotaxis
was studied by adding each peptide to the lower compartment of the chemotaxis
chamber. Peptides were diluted from a stock solution (10−2 M in dimethylsulfoxide)
with KRPG containing 1 mg/ml of bovine serum albumin and used at concentrations
ranging from 10−12 to 10−5 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 actual control random movement is 32 µm
± 3 Standard Error (SE) of 10 separate experiments done in duplicate. The dose–response
curves are typical of chemoattractants that rise to a peak and then decline to zero as
the concentration of ligand is increased above its optimum value (11). A likely expla-
nation for this decrease is that the chemical gradient was diminished from the
increased rate of diffusion of that attractant, resulting in a greater degree of saturation
of the cells in the upper compartment of the chemotactic chamber (12).
Superoxide anion production was measured by the superoxide dismutase-inhibitable
reduction of ferricytochrome c (3) modified for microplate based assays. The tests
were carried out in a final volume of 200 µl containing 4 × 105 neutrophils, 100 nmol
cytochrome c, and KRPG. At zero time, different amounts (10−8–10−4 M) of each pep-
tide were added and the plates were incubated into a microplate reader (Ceres 900,
Bio-Tek instruments, INC) with the compartment T set at 37°C. Absorbance was
recorded at wavelenghts of 550 and 468 nm. Differences in absorbance at the two
wavelenghts were used to calculate nmoles of O−2 produced, using molar extinction
coefficient for cytochrome c of 15.5 mM−1 cm−1. Data are expressed as net nanomoles
of O2-production/1 × 106 neutrophils/5 min. Neutrophils were preincubated with 5
µg/ml cytochalasin B for 5 min prior to activation by peptides.
Granule enzyme assay. Release of neutrophil granule enzymes was evaluated by deter-
mining lysozyme activity (3) modified for microplate-based assays. Cells were incubated in
microplates wells in the presence of each peptide in a final concentration of 10−9–10−4 M for
15 min at 37°C. The plates were then centrifuged for 5 min at 400 g and the lysozyme was
quantified nephelometrically by the rate of lysis of cell wall suspension of Micrococcus
lysodeikticus. Neutrophils were preincubated with 5 µg/ml cytochalasin B for 15 min at
37°C prior to activation by peptides. Reaction rate was measured with a microplate reader
at 465 nm. Enzyme was expressed as net percentage of total enzyme content released by
0.1% Triton X-100. Total enzyme activity was 85 ± 1 mg/1 × 107 cells/min.
RESULTS AND DISCUSSION
As shown in Fig. 1, the introduction of the D-Met significantly influences the chemo-
tactic response, which is strongly reduced to an amount varying from a half to a third
of the prototype values at physiological concentrations (10−10–10−8 M). It induces about
0.7 C.I. at 10−10 M, evidencing an efficacy 10 times higher than fMLP-OMe.
In Fig. 2 the superoxide anion production is reported. Compound 1 has a behavior
similar to the parent fMLP-OMe only at high concentrations, showing its peak at
10−5 M. Its potency, however, is lower than fMLP-OMe at 10−6 M (23 and 44 nmol,
respectively).

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SPISANI AND CAVICCHIONI
FIG. 1. Chemotactic activity of fMLP-OMe (᭹) and its analogue (᭜) toward human neutrophils. The
points are the mean of five separate experiments done in duplicate. SE are in the 0.02–0.09% chemotactic
index range.
Lysozyme release of compound 1 is shown in Fig. 3, and the corresponding values
highlight its lower potency as secretagogue agent, when compared with the standard
peptide, at the optimal concentrations of 10−6–10−5 M.
CONCLUSION
The present results, obtained by studying the Met in D configuration, indicate on one
hand that the positive charge of the receptor pocket is confined to a precise, restricted
part of it, so that the sulphur atom of the D-Met cannot optimally interact with it; on
the other hand they stress the minor obligations required by the activation of the super-
oxide anion production.
From the data given here and those previously reported by us (13), we can affirm
that: (a) the activation of all the biological responses require only the “primary hook-
ing” between the protic amide bond at positions 2 of the ligand and the receptor pock-
et, while further bonds at positions 2 and 3, as well as the characteristic amino acid
FIG. 2. Superoxide anion production of fMLP-OMe (᭹) and its analogue (᭜) toward human neu-
trophils. The points are the mean of five separate experiments done in duplicate. SE are in the 0.1–4 nmol
O−2 range.

METHIONINE CONFIGURATION IN FORMYLTRIPEPTIDES
43
FIG. 3. Release of neutrophil granule enzymes evaluated by determining lysozyme activity induced
by fMLP-OMe (᭹) and its analogue(᭜). The points are the mean of five separate experiments done in
duplicate. SE are in 1–6% range.
features, can improve both the efficacy and potency of the responses; (b) the chemo-
tactic activity is strongly responsive to a complete ligand-receptor hooking, as well as
to the electronic features of the first residue and the steric features of the third one;
(c) these factors influence instead the efficacy alone of the superoxide anion production,
whereas a good potency is almost always present; (d) lysozyme release cannot so far
be characterized in an independent manner, because this biological response is some-
times related to the chemotactic response and sometimes to the superoxide anion
production.
ACKNOWLEDGMENTS
This work is part of research program supported by MURST (Research Funds 40% and 60%), CNR,
and AIRC. We are grateful to the Banca del Sangue of Ferrara for providing fresh blood and Mrs. Linda
Bruce, a qualified English teacher, for revision of the text.
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