Differences arising in human neutrophil activation passing from N-formyl to N-acetyl-oligopeptides

Article abstract of DOI:10.1006/bioo.2000.1175

N-formyl- and N-acetyl-peptides were synthesized and compared in order to understand which features can best elicit biological responses. The behavior of N-formyl-peptides confirms the previously found sequential obligations in the residues, while acetyl-

Full text of DOI:10.1006/bioo.2000.1175

Bioorganic Chemistry 28, 252–259 (2000)
doi:10.1006/bioo.2000.1175, available online at http://www.idealibrary.com on
Differences Arising in Human Neutrophil Activation Passing from
N-Formyl to N-Acetyl-oligopeptides
Susanna Spisani* and Giorgio Cavicchioni†,1
*Department of Biochemistry and Molecular Biology, and
†Department of Pharmaceutical Sciences, University of Ferrara, 44100 Ferrara, Italy
Received December 15, 1999
N-formyl- and N-acetyl-peptides were synthesized and compared in order to understand
which features can best elicit biological responses. The behavior of N-formyl-peptides confirms
the previously found sequential obligations in the residues, while acetyl-derivatives do not
seem suitable for an efficacious stimulation of human neutrophils.
᭧ 2000 Academic Press
Key Words: human neutrophils; formyl-peptides; acetyl-peptides; chemotactic activity; super-
oxide anion production; lysozyme release.
INTRODUCTION
Regulation of the level of circulating neutrophils is very important because they
control microbial infection by removing and killing pathogens. Neutrophils detect
invading agents by means of signal molecules called chemoattractants, generated by
bacterial infection and tissue damage (1); they express specific G-protein coupled
receptors (2) for chemoattractants, which enable them to sense invading foreign
particles and to approach the site of infection by triggering cytoskeletal reorganization
and cell shape change (3,4).
N-formyl-Met-Leu-Phe-OH (fMLP), which derives from bacterial sources or dis-
rupted mitochondria, is the reference chemotactic peptide together with its synthetic
methylester derivative for-Met-Leu-Phe-OMe (fMLP-OMe).
Neutrophil chemotaxis toward the site of invasion is activated by low concentrations
of the ligand (5); in contrast, its high concentration activates an array of other
responses, including the release of hydrolytic enzymes from the granules into the
extracellular fluid, and the triggering of a “respiratory burst,” which is characterized
by the generation of superoxide anion (OϪ2 ) (6).
Although it seems well-established that the formyl as amino-terminal blocking
group is crucial for both binding and biological activity (7), a number of studies have
1 To whom correspondence and reprint requests should be addressed. Fax: ϩ39-532-291296. E-mail:
g5z@dns.unife.it.
252
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BIOLOGICAL ACTIVITY OF FORMYL- AND ACETYL-PEPTIDES
253
nevertheless been performed on acetyl-oligopeptides, but the picture is still unclear.
While it seems ascertained that acetyl-Met-Leu-Phe-OMe (Ac-MLP-OMe) is devoid
of any activity, biological responses have been found for acetyl-peptides containing
a varied and/or longer sequence: e.g., acetyl-peptides containing five or more residues
elicit a potent chemotactic activity for human neutrophils (8,9).
These observations prompted us to try and give a final answer to the question
whether a biological response is present (and if so, which) when the sequence in the
prototype peptide is changed, and/or when the formyl group is replaced by an acetyl
group in short peptides.
For this purpose, formyl- and acetyl-tri and tetra-peptides were synthetized to test
their efficiency in triggering biological responses in human neutrophils. Chemotaxis,
superoxide anion production, and lysozyme release of Ac-Met-Leu-Phe-OMe 1, for-
Met-Phe-Leu-OMe 2, Ac-Met-Phe-Leu-OMe 3, for-Met-Phe-Leu-Val-OMe 4, Ac-
Met-Phe-Leu-Val-OMe 5, for-Met-Leu-Phe-Val-OMe 6, and Ac-Met-Leu-Phe-Val-
OMe 7 were compared with those observed for the reference peptide fMLP-OMe.
These sequences have been chosen because they are present in the N-terminal
sequence of the calpain small subunit and exhibit chemotactic activity (8).
MATERIALS AND METHODS
Chemistry
The 1H-NMR spectra were recorded in deuterated chloroform (CDCl3) and dimeth-
ylsulphoxide (DMSO-d6) on a Bruker AC200 spectrometer at 200 MHz. Chemical
shifts are expressed as ␦ (ppm) related to the TMS signal.
Optical rotations were determined in MeOH at 20ЊC with a Perkin–Elmer Model
241 polarimeter.
Melting points were determined on a Reichert–Kofler block and are uncorrected.
Thin layer chromatography was performed on precoated silica gel F254 (Merck)
with the solvent system: methylene choloride/toluene/methanol 17/1/2.
Satisfactory microanalyses were obtained for all compounds, analytical results
being within Ϯ0.4% of the theoretical values.
Amino acids hydrochlorides as well as their Boc and acetyl derivatives were
purchased from Fluka. Removal of the Boc group was performed by treatment with
a 1:1 mixture of trifluoroacetic acid (TFA)-CHCl3. Peptide coupling was achieved
(i) by the racemization-free mixed-anhydride method with isobutylchloroformate
(IBCF) and triethylamine (TEA) (10), and (ii) by the 1-hydroxy-benzotriazole
(HOBt)–N-(3-dimethylaminopropyl)-NЈ-ethylcarbodiimide hydrochloride (EDC)
method (11), while the formyl group was introduced according the N-ethoxycarbonyl-
2-ethoxy-1,2-dihydroquinoline (EEDQ) method (12).
Purification of all the final products were achieved by reverse phase HPLC
analysis on a Waters Delta Prep 3000 and revelation with UV spectrometer Waters
484 at 220 nm using, as stationary less polar phase a Delta Pack C 18-300 A column
(30 mm ϫ 30 cm, particles 15 ␮m) with a proper eluting system.
Ac-Met-Leu-Phe-OMe 1
The peptide was synthesized following standard procedures in solution (Fig. 1).
1
Solid (mp.185–187ЊC; Rf 0.53; [␣] ϭ Ϫ15.5Њ, c ϭ 1). H-NMR (CDCl3): 0.88 and

254
SPISANI AND CAVICCHIONI
FIG. 1. Scheme of synthesis of compounds 1, 2, and 3. All the abbreviations are reported under
Materials and Methods.
0.91 (6H; 2CH3; 2d; J ϭ 5.22 Hz); 1.35–1.60 (2H; CH2; m); 1.90–2.05 (3H; CH2ϩCH;
m); 2.00 (3H; CH3CO; s); 2.08 (3H; SCH3; s); 2.49 (2H; CH2; m); 3.06 and 3.14
(2H; CH2; 2dd; AB of ABX; JAB ϭ 14.41 Hz; JAX ϭ 5.60 Hz; JBX ϭ 6.27 Hz); 3.70
(3H; OCH3; s); 4.45–4.90 (3H; 3CH; m); 6.72 (1H; NH; d; J ϭ 8.14 Hz); 6.93 (1H;
NH; d; J ϭ 7.83 Hz); 7.07–7.13 (3H; C6H5ϩNH; m); 7.23–7.28 (3H; C6H5; m).
For-Met-Phe-Leu-OMe 2
The peptide was synthesized following standard procedures in solution (Fig. 1).
1
Solid (mp. 176–178ЊC; Rf 0.49; [␣] ϭ Ϫ70.3Њ, c ϭ 1). H-NMR (CDCl3/DMSO-d6
1:1): 0.84 and 0.87 (6H; 2CH3; 2d; J ϭ 7.08 Hz); 1.45–1.95 (5H; 2CH2ϩCH; m);
1.97 (3H; SCH3; s); 2.31 (2H; CH2; t; J ϭ 7.68 Hz); 2.80 and 3.05 (2H; CH2; 2dd;
AB of ABX; JAB ϭ 13.02 Hz; JAX ϭ 4.38 Hz; JBX ϭ 8.59 Hz); 3.60 (3H; OCH3;
s); 4.25–4.45 (2H; 2CH; m); 4.55 (1H; CH; m); 6.72 (1H; NH; d; J ϭ 8.14 Hz);
7.17–7.13 (5H; C6H5; s); 7.97 (1H; HCO; s); 8.02–8.16 (2H; 2NH; m).
Ac-Met-Phe-Leu-OMe 3
The peptide was synthesized following standard procedures in solution (Fig. 1).
1
Solid (mp.168–170ЊC; Rf 0.45; [␣] ϭ Ϫ42.7Њ, c ϭ 1). H-NMR (CDCl3): 0.87 and
0.90 (6H; 2CH3; 2d; J ϭ 5.62 Hz); 1.45–1.65 (2H; CH2; m); 1.80–2.02 (3H; CH2ϩCH;
m); 1.96 (3H; CH3CO; s); 2.05 (3H; SCH3; s); 2.47 (2H; CH2; m); 3.06 (2H; CH2;
m); 3.69 (3H; OCH3; s); 4.43–4.89 (3H; 3CH; m); 6.62 (1H; NH; d broad); 6.72
(1H; NH; d broad); 6.95 (1H; NH; d broad); 7.10–7.40 (5H; C6H5; m).
For-Met-Phe-Leu-Val-OMe 4
The peptide was synthesized following standard procedures in solution (Fig. 2).
1
Solid (mp.215–218ЊC; Rf 0.65; [␣] ϭ Ϫ9.1Њ, c ϭ 1). H-NMR (CDCl3/DMSO-d6
1:1): 0.85–0.87 (12H; 4CH3; 2d); 1.42–1.53 (2H; CH2; m); 1.54–1.90 (3H; CH2ϩCH;
m); 1.96–2.09 (1H; CH; m); 2.03 (3H; SCH3; s); 2.29–2.36 (2H; CH2; m); 2.81 and
3.02 (2H; CH2; 2dd); 3.61 (3H; OCH3; s); 4.18 (1H; CH; m); 4.35 (1H; CH; m);
4.53 (1H; CH; m); 7.17 (5H; C6H5; m); 7.69 (1H; NH; d; J ϭ 8.27 Hz); 7.83 (1H;
NH; d; J ϭ 6.99 Hz); 7.85 (1H; HCO; s); 7.90 (1H; NH; d; Jϭ8.17 Hz); 8.07 (1H;
NH; d; J ϭ 8.31 Hz).

BIOLOGICAL ACTIVITY OF FORMYL- AND ACETYL-PEPTIDES
255
FIG. 2. Scheme of synthesis of compounds 4, 5, 6, and 7. All the abbreviations are reported under
Materials and Methods.
Ac-Met-Phe-Leu-Val-OMe 5
The peptide was synthesized following standard procedures in solution (Fig. 2).
1
Solid (mp.Ͼ220ЊC; Rf 0.56; [␣] ϭ Ϫ27.1Њ, c ϭ 1). H-NMR (DMSO); 0.89–0.96
(12H; 4CH3; 2d); 1.47–1.85 (3H; CH2ϩCH; m); 1.92 (3H; SCH3; s); 2.07 (3H; CH3;
s); 2.15 (1H; CH; m); 2.49 (2H; CH2; t; J ϭ 7.0 Hz); 3.12 (2H; CH2; AB of ABX;
JAB ϭ 6.50 Hz; JAX ϭ 3.17 Hz; JBB ϭ 2.54 Hz); 3.74 (3H; OCH3; s); 4.41–4.58
(3H; 3CH; m); 4.75 (1H; CH; m); 6.31 (1H; NH; d; J ϭ 6.63 Hz); 6.95 (1H; NH;
d; J ϭ 10.00 Hz); 7.02 (1H; NH; d; J ϭ 9.12 Hz); 7.10–7.40 (7H; C6H5ϩNH; m).
For-Met-Leu-Phe-Val-OMe 6
The peptide was synthesized following standard procedures in solution (Fig. 2).
Solid (mp.218–220ЊC; Rf 0.61; [␣] ϭ Ϫ48.7Њ, c ϭ 1). 1H-NMR (CDCl3): 0.85–0.88
(12H; 4CH3; 2d); 1.42–1.60 (2H; CH2; m); 1.90–2.20 (5H; 2CH2ϩCH; m); 2.04
(3H; SCH3; s); 2.47 (2H; CH2; m); 3.05 (2H; CH2; d; J ϭ 6.82 Hz); 3.70 (3H; OCH3;
s); 4.44 (1H; CH; m); 4.71 (1H; CH; m); 4.96 (1H; CH; m); 7.18 (1H; NH; d broad);
7.21 (5H; C6H5; m); 7.37 (1H; NH; d broad); 7.57 (1H; NH; d broad); 7.79 (1H;NH;
d broad); 8.21 (1H; HCO; s).
Ac-Met-Leu-Phe-Val-OMe 7
The peptide was synthesized following standard procedures in solution (Fig. 2).
1
Solid (mp.Ͼ220ЊC; Rf 0.69; [␣] ϭ Ϫ51.4Њ, c ϭ 1). H-NMR (CDCl3); 0.83–0.89
(12H; 4CH3; 2d); 1.48 (2H; CH2; m); 1.93–2.17 (4H; CH2ϩ2CH; m); 2.03 (3H;
SCH3; s); 2.08 (3H; CH3; s); 2.49 (2H; CH2; m); 3.08 (2H; CH2; m); 3.70 (3H; OCH3;
s); 4.42 (2H; 2CH; m); 4.61–4.80 (2H; 2CH; m); 6.59 (1H; NH; d; J ϭ 7.03 Hz);
6.68 (1H; NH; d; J ϭ 7.14 Hz); 7.00 (1H; NH; d; J ϭ 7.78 Hz); 7.10–7.40 (6H;
C6H5ϩNH; m).
Biological Essay (13)
Human neutrophils were purified employing the standard techniques of dextran
sedimentation of heparinized blood, followed by centrifugation on Ficoll–Paque and

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SPISANI AND CAVICCHIONI
hypotonic lysis of red cells. The cells were washed twice and resuspended in Krebs–
Ringer phosphate containing 0.1% w/v glucose (KRPG), pH 7.4. The percentage of
neutrophils was 98–100% pure and Ն99% viable as determined by Tripan blue
exclusion test.
Random locomotion was performed with a 48-well microchemotaxis chamber and
the migration into the filter was evaluated by the method of leading-front. The
actual control random movement is 32 ␮m Ϯ 3 SE of 10 separate experiments done
in duplicate.
Chemotaxis was studied by adding each peptide to the lower compartment of the
chemotaxis chamber. Peptides were diluted from a stock solution (10Ϫ2 M in DMSO)
with KRPG containing 1 mg/ml of bovine serum albumin and used at concentrations
ranging from 10Ϫ12 to 10Ϫ5 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.
Superoxide anion production was measured by the superoxide dismutase-inhibitable
reduction of ferricytochrome c modified for microplate based assays. The tests were
carried out in a final volume of 200 ␮l containing 4ϫ10Ϫ5 neutrophils, 100 nmol
cytochrome c, and KRPG. At zero time, different amounts (10Ϫ8–5ϫ10Ϫ5 M) of each
peptide were added and the plates were incubated into a microplate reader (Ceres
900, Bio-Tek Instruments, Inc.) with the compartment T set at 37ЊC. Absorbance was
recorded at wavelenghts of 550 and 468 nm. Differences in absorbance at the two
wavelenghts were used to calculate nanomoles of OϪ2 produced, using molar extinction
coefficient for cytochrome c of 15.5 mMϪ1 cmϪ1. Neutrophils were preincubated with
5 ␮g/ml cytochalasin B for 5 min prior to activation by peptides.
Granule enzyme assay. Release of neutrophil granule enzymes was evaluated by
determining lysozyme activity modified for microplate-based assays. Cells were incu-
bated in microplates wells in the presence of each peptide in a final concentration of
10Ϫ8–5 ϫ10Ϫ5 M for 15 min at 37ЊC. The plates were then centrifuged for 5 min at
400g and the lysozyme was quantified nephelometrically by the rate of lysis of cell
wall suspension of Micrococcus lysodeikticus. Neutrophils were preincubated with 5
␮g/ml cytochalasin B for 15 min at 37ЊC 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 Ϯ 1 mg/liter ϫ107 cells/min.
Statistical analysis. The nonparametric Wilcoxon test was used in the statistical
evaluation of differences between groups.
RESULTS
Chemotaxis is shown in Fig. 3. Blocking the N-terminal amino group by an acetyl
group in tripeptides 1 and 3 leads to a loss of the chemotactic activity; the same
behavior is seen for derivative 2, which carries the formyl group. A more elaborate
pattern emerges from the evaluation of tetrapeptides. Compounds 4, 6, and 7 show
statistically significant activities as chemoattractants. In particular, compound 6 is
more efficient and potent than the parent peptide at a concentration of 10Ϫ11 M. The
corresponding acetylated 7 and formylated 4, where the Phe residue is shifted at
position 2, evidence a reduced efficiency (activity peak only at the concentration of

BIOLOGICAL ACTIVITY OF FORMYL- AND ACETYL-PEPTIDES
257
FIG. 3. Chemotactic activity of fMLP-OMe and its analogues toward human neutrophils. The points
are the means of five separate experiments done in duplicate. SE are in the 0.02–0.09% chemotactic
index range.
10Ϫ7 M) and less potency (about 35%) than fMLP-OMe. The corresponding acetyl-
petide 5, conversely, is totally devoid of any activity.
Superoxide anion production is shown in Fig. 4. In acetyl-tripeptide derivatives 1
and 3 the response is totally absent, while the acetylated tetrapeptides 5 and 7 show
their maximal potency only at 10Ϫ5 M, thus exhibiting an efficacy ten times less than
fMLP-OMe. In particular, the potency of 7 is as high as the control, while compound
5 shows an activity about one-third of the control. All the formylated peptides,
however, are able to activate OϪ2 production with an order of potency at 10Ϫ5 M of:
fMLP-OMe Ͼ 6 Ͼ 4 Ͼ 2.
Hydrolytic enzyme release is statistically not significant in acetyl-tripeptides 1 and
3, while formyl-tripeptide 2 reaches the value of the control only at the concentration
of 10Ϫ5 M. Acetylated tetrapeptides seem to be sensitive to the sequence of the chain
residues: in fact, compound 5 reaches its maximum only at a concentration of 10Ϫ5
M, while compound 7 exhibits a similar pattern of activity to the reference at all the
concentrations tested. Formyl-tetrapeptides 4 and 6 possess a more potent degranulat-
ing activity than fMLP-OMe at 10Ϫ6 and 10Ϫ7 M, respectively.

258
SPISANI AND CAVICCHIONI
FIG. 4. Superoxide anion production of fMLP-OMe and its analogues toward human neutrophils.
The points are the means of five separate experiments done in duplicate. SE are in the 0.1–4 nmol
O2Ϫ range.
CONCLUSIONS
Chemotaxis. Acetylation is unable to elicit chemotactic activity on small peptides.
Our results, furthermore, suggest that an elongation of the peptidic chain in the Met-
Leu-Phe sequence could afford a biological response, as previously referred (9).
As concerns formylated compounds, an alteration of the residue sequence strongly
influences both the efficacy and potency of the derivatives.
Superoxide anion production is absent in acetylated tripeptides, but can be elicited
from acetyl-tetrapeptides exclusively at concentrations higher than the control. All
the formylated peptides are instead able to elicit a good biological response.
Lysozyme release. The behavior shown by acetylated tripeptides indicates that they
are devoid of activity in this function too. Formyl-tetrapeptides are always efficacious
FIG. 5. Release of neutrophil granule enzymes evaluated by determining lysozyme activity induced
by fMLP-OMe and its analogues. The points are the means of five separate experiments done in duplicate.
SE are in the 1–6% range.

BIOLOGICAL ACTIVITY OF FORMYL- AND ACETYL-PEPTIDES
259
as secretagogue agents, while the corresponding acetyl-tetrapeptides seem to be sensi-
tive to the sequence of the residues.
We can conclude that (i) acetyl-oligopeptides do not represent a useful tool for
finding ligands able to fully activate human neutrophil responses, (ii) formyl-oligopep-
tides always, in some manner, elicit a sensitivity to the residue sequence in tripeptides,
while tetrapeptides do not evidence this type of sensitivity.
Our results agree with both our previous studies (14,15) and present knowledge
of the features of the amino-terminal pocket (7,16).
ACKNOWLEDGMENTS
This work is part of a 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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