Structural basis for recognition of N-formyl peptides as pathogen-associated molecular patterns.

The formyl peptide receptor 1 (FPR1) is primarily responsible for detection of short peptides bearing N-formylated methionine (fMet) that are characteristic of protein synthesis in bacteria and mitochondria. As a result, FPR1 is critical to phagocyte migration and activation in bacterial infection, tissue injury and inflammation. How FPR1 distinguishes between formyl peptides and non-formyl peptides remains elusive. Here we report cryo-EM structures of human FPR1-Gi protein complex bound to S. aureus-derived peptide fMet-Ile-Phe-Leu (fMIFL) and E. coli-derived peptide fMet-Leu-Phe (fMLF). Both structures of FPR1 adopt an active conformation and exhibit a binding pocket containing the R2015.38XXXR2055.42 (RGIIR) motif for formyl group interaction and receptor activation. This motif works together with D1063.33 for hydrogen bond formation with the N-formyl group and with fMet, a model supported by MD simulation and functional assays of mutant receptors with key residues for recognition substituted by alanine. The cryo-EM model of agonist-bound FPR1 provides a structural basis for recognition of bacteria-derived chemotactic peptides with potential applications in developing FPR1-targeting agents.

A subset of five human mitochondrial formyl peptides mimics bacterial peptides and functionally deactivates human neutrophils.

BACKGROUND: Trauma causes inflammation by releasing mitochondria that act as Danger-Associated Molecular Patterns (DAMPs). Trauma also increases susceptibility to infection. Human mitochondria contain 13 N-formyl peptides (mtFPs). We studied whether mtFPs released into plasma by clinical injury induce neutrophil (PMN) inflammatory responses, whether their potency reflects their similarity to bacterial FPs and how their presence at clinically relevant concentration affects PMN function. METHODS: N-terminal sequences of the 13 mtFPs were synthesized. Changes in human PMN cytosolic Ca concentration ([Ca]i) and chemotactic responses to mtFPs were studied. Sequence similarity of mtFPs to the canonical bacterial peptide f-Met-Leu-Phe (fMLF/fMLP) was studied using the BLOcks SUbstitution Matrix 62 (BLOSUM 62) system. The presence of mtFPs in plasma of trauma patients was assayed by Enzyme-linked immunosorbent assay (ELISA). The effects of the most potent mtFP (ND6) on PMN signaling and function were then studied at ambient clinical concentrations by serial exposure of native PMN to ND6, chemokines and leukotrienes. RESULTS: Five mtFPs (ND6, ND3, ND4, ND5, and Cox 1) induced [Ca]i flux and chemotaxis in descending order of potency. Evolutionary similarity to fMLF predicted [Ca]i flux and chemotactic potency linearly (R = 0.97, R = 0.95). Chemoattractant potency was also linearly related to [Ca]i flux induction (R = 0.92). Active mtFPs appear to circulate in significant amounts immediately after trauma and persist through the first week. The most active mtFP, ND6, suppresses responses to physiologic alveolar chemoattractants (CXCL-1, leukotriene B4) as well as to fMLF where CXCL-1 and leukotriene B4 do not suppress N-formyl peptide receptor (FPR)-1 responses to mtFPs. Prior FPR-1 inhibition rescues PMN from heterologous suppression of CXCR-1 and BLT-1 by mtFPs. CONCLUSION: The data suggest mtFPs released by injured tissue may attract PMN to trauma sites while suppressing PMN responses to other chemoattractants. Inhibition of mtFP-FPR1 interactions might increase PMN recruitment to lung bacterial inoculation after trauma. These findings suggest new paradigms for preventing infections after trauma. LEVEL OF EVIDENCE: Therapeutic, Level IV.

Bioactive Steroids with Methyl Ester Group in the Side Chain from a Reef Soft Coral Sinularia brassica Cultured in a Tank.

A continuing chemical investigation of the ethyl acetate (EtOAc) extract of a reef soft coral Sinularia brassica, which was cultured in a tank, afforded four new steroids with methyl ester groups, sinubrasones A-D (1-4) for the first time. In particular, 1 possesses a ß-D-xylopyranose. The structures of the new compounds were elucidated on the basis of spectroscopic analyses. The cytotoxicities of compounds 1-4 against the proliferation of a limited panel of cancer cell lines were assayed. The anti-inflammatory activities of these new compounds 1-4 were also evaluated by measuring their ability to suppress superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced human neutrophils. Compounds 2 and 3 were shown to exhibit significant cytotoxicity, and compounds 3 and 4 were also found to display attracting anti-inflammatory activities.

Cysteinyl-glycine reduces mucosal proinflammatory cytokine response to fMLP in a parenterally-fed piglet model.

BACKGROUND: PepT1 transports dietary and bacterial peptides in the gut. We hypothesized that cysteinyl-glycine would ameliorate the inflammatory effect of a bacterial peptide, formyl-methionyl-leucyl-phenylalanine (fMLP), in both sow-fed and parenterally-fed piglets. METHODS: An intestinal perfusion experiment was performed in piglets (N = 12) that were sow-reared or provided with parenteral nutrition (PN) for 4 d. In each piglet, five segments of isolated intestine were perfused with five treatments including cysteine and glycine, cysteinyl-glycine, fMLP, free cysteine and glycine with fMLP, or cysteinyl-glycine with fMLP. Mucosal cytokine responses and intestinal morphology was assessed in each gut segment. RESULTS: PN piglets had lower mucosal IL-10 by approximately 20% (P < 0.01). Cysteinyl-glycine lowered TNF-α response to fMLP in PN-fed animals and IFN-γ response to fMLP in both groups (P < 0.05). The free cysteine and glycine treatment reduced TNF-α in sow-fed animals (P < 0.05). fMLP affected villus height in parenterally (P < 0.05), but not sow-fed animals. CONCLUSION: Parenteral feeding conferred a susceptibility to mucosal damage by fMLP. The dipeptide was more effective at attenuating the inflammatory response to a bacterial peptide than free amino acids. This may be due to competitive inhibition of fMLP transport or a greater efficiency of transport of dipeptides.

Improving dipolar recoupling for site-specific structural and dynamics studies in biosolids NMR: windowed RN-symmetry sequences.

Experimental characterization of one-bond heteronuclear dipolar couplings is essential for structural and dynamics characterization of molecules by solid-state NMR. Accurate measurement of heteronuclear dipolar tensor parameters in magic-angle spinning NMR requires that the recoupling sequences efficiently reintroduce the desired heteronuclear dipolar coupling term, fully suppress other interactions (such as chemical shift anisotropy and homonuclear dipolar couplings), and be insensitive to experimental imperfections, such as radio frequency (rf) field mismatch. In this study, we demonstrate that the introduction of window delays into the basic elements of a phase-alternating R-symmetry (PARS) sequence results in a greatly improved protocol, termed windowed PARS (wPARS), which yields clean dipolar lineshapes that are unaffected by other spin interactions and are largely insensitive to experimental imperfections. Higher dipolar scaling factors can be attained in this technique with respect to PARS, which is particularly useful for the measurement of relatively small dipolar couplings. The advantages of wPARS are verified experimentally on model molecules N-acetyl-valine (NAV) and a tripeptide Met-Leu-Phe (MLF). The incorporation of wPARS into 3D heteronuclear or homonuclear correlation experiments permits accurate site-specific determination of dipolar tensors in proteins, as demonstrated on dynein light chain 8 (LC8). Through 3D wPARS recoupling based spectroscopy we have determined both backbone and side chain dipolar tensors in LC8 in a residue-resolved manner. We discuss these in the context of conformational dynamics of LC8. We have addressed the effect of paramagnetic relaxant Cu(ii)-EDTA doping on the dipolar coupling parameters in LC8 and observed no significant differences with respect to the neat sample permitting fast data collection. Our results indicate that wPARS is advantageous with respect to the windowless version of the sequence and is applicable to a broad range of systems including but not limited to biomolecules.

De novo chemoattractants form supramolecular hydrogels for immunomodulating neutrophils in vivo.

Most immunomodulatory materials (e.g., vaccine adjuvants such as alum) modulate adaptive immunity, and yet little effort has focused on developing materials to regulate innate immunity, which get mentioned only when inflammation affects the biocompatibility of biomaterials. Traditionally considered as short-lived effector cells from innate immunity primarily for the clearance of invading microorganisms without specificity, neutrophils exhibit a key role in launching and shaping the immune response. Here we show that the incorporation of unnatural amino acids into a well-known chemoattractant-N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-offers a facile approach to create a de novo, multifunctional chemoattractant that self-assembles to form supramolecular nanofibrils and hydrogels. This de novo chemoattractant not only exhibits preserved cross-species chemoattractant activity to human and murine neutrophils, but also effectively resists proteolysis. Thus, its hydrogel, in vivo, releases the chemoattractant and attracts neutrophils to the desired location in a sustainable manner. As a novel and general approach to generate a new class of biomaterials for modulating innate immunity, this work offers a prolonged acute inflammation model for developing various new applications.

Liposomes formulated with fMLP-modified cholesterol for enhancing drug concentration at inflammatory sites.

Improving efficacy of inflammation treatment by increasing drug delivery to the inflammatory sites is a challenging endeavor. N-formyl-methionyl-leucyl-phenylalanine (fMLP), the first discovered leukocyte chemotaxis peptide, is composed of formyl methionine, leucine and phenylalanine. It conjugates with formyl peptide receptors on the target cells with high receptor expression on the surface such as macrophages. With this in mind, we developed a novel fMLP-modified liposome (fMLP-LIP) for enhancing drug delivery to the inflammatory sites and resolving the systemic reaction issue with conventional anti-inflammatory drugs. Being a more stable and cheaper liposomal component than phospholipids, cholesterol (CHO) has been thoroughly investigated as an alternative anchor. In this study, fMLP was covalently conjugated with CHO with polyethylene glycol link to prepare the liposomes, cellular uptake of liposomes by differentiated human U937 cells was examined and cellular uptake experiment in vitro was employed to optimize fMLP-LIP prescription and investigate the uptake mechanism. An in vivo inflammatory model was established to evaluate the targeting performance of fMLP-LIP to inflammatory site. The in vitro and in vivo findings indicate that the fMLP ligands playing an important role in increasing drug delivery to inflammatory sites and fMLP-LIP as a promising anti-inflammatory drug carrier.

Characterization of the shear stress regulation of CD18 surface expression by HL60-derived neutrophil-like cells.

Shear stress-induced cleavage of cell surface CD18 integrins is reported to be part of an anti-inflammatory control mechanism that minimizes neutrophil activity in the blood under physiologic conditions. The cysteine protease, cathepsin B (catB), has been implicated in this mechanoregulatory mechanism, but its molecular dynamics remain to be elucidated. Moreover, attempts to do so using molecular approaches are hindered by the limited ex vivo life span of primary neutrophils. As an alternative, we explored the potential use of HL60-derived neutrophilic cells as a transfectable culture model that exhibits a shear-induced CD18 cleavage response comparable to primary neutrophils. HL60 cells were differentiated into neutrophil-like cells (dHL60-NCs) and exposed to laminar shear stress ([Formula: see text] for 10 min). Based on cytometric analyses, sheared cells cleaved CD18 and CD11a, but not CD11b, integrins. Treatment of cells with E64 or doxycycline prior to and during shear exposure inhibited CD18, but only attenuated CD11a, cleavage. Neither aprotinin nor pepstatin affected shear-induced CD18 or CD11a cleavage. Notably, dHL60-NCs expressed minimal catB. Thus, multiple cysteine proteases in addition to catB may cleave CD18 on sheared leukocytes. In fact, our findings indicate that multiple non-cysteine proteases also participate in the shear-related cleavage of CD11/CD18 heterodimers. Finally, shear-induced cleavage of CD18 and CD11a by dHL60-NCs was inhibited by fMLP concentrations of at least [Formula: see text]. Collectively, our findings indicate that shear-induced CD11/CD18 cleavage is phenotypic of neutrophilic cells, including those derived from HL60 cells. Moreover, our results verify shear stress as a key anti-inflammatory stimulus for neutrophils under physiologic conditions.

Effects of synthetic peptides on the inflammatory response and their therapeutic potential.

Recently, interest in small peptide molecules as potential drug candidates has revived. In this review, two series of synthetic peptides and their selective effects on the inflammatory response have been described, focusing on the intracellular pathways involved and on their therapeutic potential. A series of F(D)LF(D)LF analogs has been synthesized, including either N- t-Boc or different N-ureido substituents. The free acid derivatives as they are good candidates as antiinflammatory drugs are able to antagonize the multiple neutrophil functions evoked by N-formyl-L-methionyl-L-leucyl-Lphenylalanine (fMLF), i.e. chemotaxis, superoxide anion production and lysozyme release. Their methyl-ester derivatives are ineffective. The second series of peptides derives from the endogenous protein kinase C (PKC) inhibitor PKI55, a 55-amino acid protein, whose synthesis is induced by PKC activation, so that a feedback loop of inhibition is established. In vitro experiments showed that PKI55 inhibits recombinant PKC isoforms α, ß1, ß2, γ, δ, ζ, ; to identify the minimal amino acid sequence of PKI55 protein maintaining the inhibitory effects on PKC, peptides derived from both C- and N-terminal sequences have been synthesized. The N-terminal peptides 5 (MLYKLHDVCRQLWFSC), 8 (CRQLWFSC) and 9 (CRQLW), that in human neutrophils retain the inhibitory activity on PKC, decrease the chemotaxis, and, in mice, display anti-inflammatory and analgesic action, after both central and peripheral administration of very low doses. Furthermore, the peptide 5 shows neuroprotective activity in a model of cerebral ischemia in vitro, favouring the recovery of synaptic function. These findings suggest interesting possible therapeutic applications for these peptides.

Intramolecular 1H-13C distance measurement in uniformly 13C, 15N labeled peptides by solid-state NMR.

A (1)H-(13)C frequency-selective REDOR (FS-REDOR) experiment is developed for measuring intramolecular (1)H-(13)C distances in uniformly (13)C, (15)N-labeled molecules. Theory and simulations show that the experiment removes the interfering homonuclear (1)H-(1)H, (13)C-(13)C and heteronuclear (1)H-(15)N, (13)C-(15)N dipolar interactions while retaining the desired heteronuclear (1)H-(13)C dipolar interaction. Our results indicate that this technique, combined with the numerical fitting, can be used to measure a (1)H-(13)C distance up to 5Å. We also demonstrate that the measured intramolecular (1)H-(13)C distances are useful to determine dihedral angles in proteins.

Absolute peptide quantification by lutetium labeling and nanoHPLC-ICPMS with isotope dilution analysis.

The need of analytical methods for absolute quantitative protein analysis spurred research on new developments in recent years. In this work, a novel approach was developed for accurate absolute peptide quantification based on metal labeling with lutetium diethylenetriamine pentaacetic acid (Lu-DTPA) and nanoflow high-performance liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry (nanoHPLC-ICP-IDMS). In a two-step procedure peptides were derivatized at amino groups with diethylenetriamine pentaacetic anhydride (DTPAA) followed by chelation of lutetium. Electrospray ionization mass spectrometry (ESI MS) of the reaction product demonstrated highly specific peptide labeling. Under optimized nanoHPLC conditions the labeled peptides were baseline-separated, and the excess labeling reagent did not interfere. A 176Lu-labeled spike was continuously added to the column effluent for quantification by ICP-IDMS. The recovery of a Lu-DTPA-labeled standard peptide was close to 100% indicating high labeling efficiency and accurate absolute quantification. The precision of the entire method was 4.9%. The detection limit for Lu-DTPA-tagged peptides was 179 amol demonstrating that lutetium-specific peptide quantification was by 4 orders of magnitude more sensitive than detection by natural sulfur atoms present in cysteine or methionine residues. Furthermore, the application to peptides in insulin tryptic digest allowed the identification of interfering reagents decreasing the labeling efficiency. An additional advantage of this novel approach is the analysis of peptides, which do not naturally feature ICPMS-detectable elements.

Observation of a low-temperature, dynamically driven structural transition in a polypeptide by solid-state NMR spectroscopy.

At reduced temperatures, proteins and other biomolecules are generally found to exhibit dynamic as well as structural transitions. This includes a so-called protein glass transition that is universally observed in systems cooled between 200 and 230 K, and which is generally attributed to interactions between hydrating solvent molecules and protein side chains. However, there is also experimental and theoretical evidence for a low-temperature transition in the intrinsic dynamics of the protein itself, absent any solvent. Here, we use low-temperature solid-state NMR to examine site-specific fluctuations in atomic structure and dynamics in the absence of solvents. In particular, we employ magic angle spinning NMR to examine a structural phase transition associated with dynamic processes in a solvent-free polypeptide, N-f-MLF-OH, lattice at temperatures as low as 90 K. This transition is characterized by the appearance of an extra set of lines in 1D (15)N spectra as well as additional cross peaks in 2D (13)C-(13)C and (13)C-(15)N spectra. Interestingly, the gradual, temperature-dependent appearance of the new spectral component is not accompanied by the line broadening typical of dynamic transitions. A direct comparison between the spectra of N-f-MLF-OH and the analog N-f-MLF-OMe, which does not display this transition, indicates a correlation of the structural transition to the temperature dependent motion of the aromatic phenylalanine side chain. Several quantitative solid state NMR experiments were employed to provide site-specific measurements of structural and motional features of the observed transition.

Synthesis and bioactivity of chemotactic tetrapeptides: fMLF-OMe analogues incorporating spacer aminoacids at the lateral positions.

A small library of N-For and N-Boc tetrapeptidic analogues of the chemotactic tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe), obtained by incorporating three different spacer aminoacids (Gly, betaAla and Pro) between the native residues of Met and Leu (N-For- and N-Boc-Met-Xaa-Leu-Phe-OMe; Xaa2 series) and Leu and Phe (N-For- and N-Boc-Met-Leu-Xaa-Phe-OMe; Xaa3 series), have been synthesized and examined for their biological activity as agonists and antagonists. Chemotaxis, lysozyme release and superoxide anion production have been measured. All the N-For analogues maintain good to moderate chemotactic activity with the betaAla3 15 model reaching the maximum value. All the N-Boc tetrapeptides are efficient chemotactic antagonists. Conversely, with the exception of the moderate antagonistic activity exhibited by the N-Boc Xaa2 models against lysozyme release, all the other N-Boc analogues do not show significant activity against both superoxide anion and lysozyme release.

The main functions and structural modifications of tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) as a chemotactic factor.

Gram negative bacteria-derived and synthetic N-formyl peptides play a key role in host defense as chemotactic factors for phagocytic leukocytes. The first compound to be identified was N-formylmethionyl-leucyl-phenylalanine (fMLP) which contains highly potent leukocyte chemoattractant. Natural fMLP was subsequently purified and identified in supernatants of gram negative bacteria. Recently, much more attention has been focused on the human formyl peptide receptor (FPR) and its variant formyl peptide receptor-like 1 (FPRL1) and formyl peptide receptor-like 2 (FPRL2). Chemotactic factors such as fMLP interact with their specific cell surface receptors, which results in multiple biological responses through a G protein-coupled signal pathway. In this review, the functions and structural modifications of fMLP are discussed in view of future drug development.

Study of synthetic peptides derived from the PKI55 protein, a protein kinase C modulator, in human neutrophils stimulated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH.

Elucidation of the involvement of protein kinase C subtypes in several diseases is an important challenge for the future development of new drug targets. We previously identified the PKI55 protein, which acts as a protein kinase C modulator, establishing a feedback loop of inhibition. The PKI55 protein is able to penetrate the cell membrane of activated human T-lymphocytes and to inhibit the activity of alpha, beta(1) and beta(2) protein kinase C isoforms. The present study aimed to identify the minimal amino acid sequence of PKI55 that is able to inhibit the enzyme activity of protein kinase C. Peptides derived from both C- and N-terminal sequences were synthesized and initially assayed in rat brain protein kinase C to identify which part of the entire protein maintained the in vitro effects described for PKI55, and then the active peptides were tested on the isoforms alpha, beta(1), beta(2), gamma, delta, epsilon and zeta to identify their specific inhibition properties. Specific protein kinase C isoforms have been associated with the activation of specific signal transduction pathways involved in inflammatory responses. Thus, the potential therapeutic role of the selected peptides has been studied in polymorphonuclear leukocytes activated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH to evaluate their ability to modulate chemotaxis, superoxide anion production and lysozyme release. These studies have shown that only chemotactic function is significantly inhibited by these peptides, whereas superoxide anion production and lysozyme release remain unaffected. Western blotting experiments also demonstrated a selective reduction in the levels of the protein kinase C beta(1) isoform, which was previously demonstrated to be associated with the polymorphonuclear leukocyte chemotactic response.

Optimizing size and copy number for PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarrier uptake by macrophages.

Curing HIV-1 infection has remained elusive because of low and fluctuating drug levels arising from poor absorption, the development of viral reservoirs and sanctuary sites, toxicity, and patient nonadherence. The present study addresses the issue of insufficient drug exposure in macrophages. Viral reservoir sites such as macrophages are believed to be responsible for the viral rebound effect observed upon the discontinuation of anti-HIV drug therapy. In our proposed model, a drug can be covalently attached to a nanocarrier in order to facilitate the delivery of therapeutic agents to the site(s) of infection. As an initial step, we propose the covalent attachment of several copies of N-formyl-Met-Leu-Phe (fMLF), a known chemo-attractant for macrophages. In this article, one or more copies of fMLF were conjugated to multifunctional commercially available or novel, peptide-based PEG nanocarriers in which the structure was varied by appending PEGs with average molecular weights of 5, 20, and 40 kDa. U937 cell-specific binding and cellular uptake were analyzed. The results of uptake studies indicate that (i) uptake is energy dependent and mediated by a fMLF receptor, (ii) appending only 2 copies of the targeting ligand to the multifunctional nanocarrier appears sufficient for binding in vitro, and (iii) of the three configurations studied, the nanocarrier with a molecular weight of about 20 kDa, corresponding to a size of 20-60 nm, demonstrated the highest uptake. The results of the current studies demonstrate the feasibility of targeting macrophages and the suitability of using these synthetically versatile peptide--backbone PEG nanocarriers. The convenience, flexibility and possible limitations of this nanocarrier approach are discussed.

Anti-formyl peptide antibodies.

Antibodies that selectively bind to N-formylmethionyl leucyl phenylalanine (fMLF, also known as fMLP) have been generated. These antibodies bound to fMLF with higher affinity than to non-formylated peptide MLF: the differences in the binding energies between fMLF and MLF were 1.4->2.1 kcal/mol.

Synthesis, conformation and biological activity of centrally modified pseudopeptidic analogues of For-Met-Leu-Phe-OMe.

For-Met-betaAlapsi[CSNH]-Phe-OMe (3), For-Met-betaAlapsi[CH2NH]-Phe-OMe (5), For-Met-NH-pC6H4-SO(2-Phe-OMe 8a), For-Met-NH-mCH4-SO2-Phe-OMe (8b) and the corresponding N-Boc precursors (2, 4, 7a, b) have been synthesized and their activity towards human neutrophils has been evaluated in comparison with that shown by the reference tripeptide For-Met-Leu-Phe-OMe (fMLF-OMe). Chemotaxis, lysozyme release and superoxide anion production have been measured. (1)H NMR titration experiments and IR spectra have been discussed in order to ascertain the preferred solution conformation adopted by the tripeptide 3 with particular reference to the presence of a folded conformation centred at the centrally positioned thionated beta-residue.

Hybrid alpha/beta-peptides: for-Met-Leu-Phe-OMe analogues containing geminally disubstituted beta2,2- and beta 3,3-amino acids at the central position.

The two fMLF-OMe analogues For-Met-beta(3)hAc(6)c-Phe-OMe (6) and For-Met-beta(2)hAc(6)c-Phe-OMe (12) and their corresponding N-Boc derivatives 5 and 11 have been synthesized and their biological activity towards human neutrophils evaluated. The N-formyl peptides 6 and 12 exhibit good activity as chemoattractans and 12 is highly active in superoxide anion production. The preferred solution conformation of the two N-formyl derivatives has been discussed.

Biological activity of for-Met-Leu-Phe-OMe analogs: relevant substitutions specifically trigger killing mechanisms in human neutrophils.

Two analogs of the prototypical peptide for-Met-Leu-Phe-OMe (fMLP-OMe), for-Gln-Tyr-Phe-OMe (1) and for-Gln-Tyr-Tyr-OMe (2), carrying unusual hydrophilic residues, were synthesized in order to investigate whether they provoked specific biological responses, as well as intracellular calcium mobilization, in human neutrophils. Whereas neither compound stimulates chemotaxis, both are able to elicit lysosomal enzyme production. However compound 1 is able to trigger copious superoxide anion production while compound 2 only elicits minor superoxide anion production. In binding experiments on formylpeptide receptors, the newly synthesized compounds for-Gln-Tyr-Phe-OMe (1) and for-Gln-Tyr-Tyr-OMe (2) showed affinity values in the micromolar range. These derivatives demonstrate inability to find a positive contribute from single substitutions. A very important result of this research is the evidence of the ability of the formyl group alone to trigger the primary target of the human neutrophil activity, i.e. killing mechanisms, by activating the specific receptor conformation.