Conformational studies on β-bend containing a cis peptide unit

Conformational studies have been carried out on the X-cis-Pro tripeptide system (a system of three linked peptide units, in the trans-cis-trans configuration) using energy minimization techniques. For X, residues Gly, L-Ala, D-Ala and L-Pro have been used. The energy minima have been classified into different groups based upon the conformational similarity. There are 15, 20, 18 and 6 minima that are possible for the four cases respectively arid these fall into 11 different groups. A study of these minima shows that, (i) some minima contain hydrogen bonds - either 4→1 or 1→2 type, (ii) the low energy minima qualify themselves as bend conformations, (iii) cis′ and trans′ conformations are possible for the prolyl residue as also the C^γ-endo and C^γ-exo puckerings, and (iv) for Pro-cis-Pro, cis′ at the first prolyl residue is ruled out, due to the high energy. The available crystal structure data on proteins and peptides, containing cis-Pro segment have been examined with a view to find the minima that occur in solid state. The data from protein show that they fall under two groups. The conformation at X in X-cis-Pro is near extended when it is a non-glycyl residue. In both peptides and proteins there exists a preference for trans′ conformation at prolyl residue over cis′ when X is a non-glycyl residue. The minima obtained can be useful in modelling studies.     

Increased antibacterial activity of 15‐residue murine lactoferricin derivatives

Abstract: LFM W8 is a synthetic 15‐residue lactoferricin derivative (H₂N‐EKCLRWQWEMRKVGG‐COOH), corresponding to residues 16–30 of the mature murine lactoferrin protein except that the asparagine residue in position 8 of the native peptide is replaced with tryptophan. We have previously reported that the two tryptophan residues in positions 6 and 8 are of crucial importance for the antibacterial activity of many lactoferricin derivatives but, despite fulfilling this requirement, LFM W8 is inactive against Escherichia coli and Staphylococcus aureus. In order to solve this puzzle, a quantitative structure–antibacterial activity relationship study of synthetic LFM W8 derivatives was performed by replacing the glutamate residues in positions 1 and 9 with arginine or alanine, and the valine residue in position 13 with tyrosine. The results from the study were analyzed using multivariate data analysis. The derived mathematical model clustered the peptides into distinct groups which reflected their antibacterial activities, pointed out correlations between different structural parameters, highlighted the structural parameters that were important for antibacterial activity, and enabled us to predict the activity of a 15‐residue bovine lactoferricin derivative. The results showed that net charge and micelle affinity, as determined from the ratio of α‐helicity in sodium dodecyl sulfate micelles and in 1,1,1,3,3,3‐hexafluoro‐2‐propanol, were the most important structural parameters affecting antibacterial activity. The most active derivative, LFM R1,9 W8 Y13, displayed a minimal inhibitory concentration of 10 and 12 µm against E. coli and S. aureus, respectively. This represented more than 50‐fold and 40‐fold increases in antibacterial activity, respectively, compared with LFM W8.     

Preparation and properties of des-Tyr98 and des-Arg97-Tyr98 acylphosphatase (muscular isoenzyme)

Previous NMR reports indicated that Tyr⁹⁸, the C-terminal residue of the muscular form of acylphosphatase, is likely to be part of the enzyme's active site. In addition, there is evidence that an arginine residue participates to the catalyzed reaction, possibly as phosphate binding site. Among all Arg residues present in the muscular forms of acylphosphatase, four, i.e. Arg²³, Arg⁷⁴, Arg⁷⁷, and Arg⁹⁷, appear to be conserved in all species checked thus far. We prepared the des-Tyr⁹⁸ and des-Arg⁹⁷-Tyr⁹⁸ derivatives of the native acylphosphatase to investigate the properties of both modified enzymes. The enzyme lacking Tyr⁹⁸ was found to be catalytically less effective than the native one, whereas the des-Arg⁹⁷-Tyr⁹⁸ acylphosphatase was completely inactive. This evidence suggests that Arg⁹⁷ participates directly to the active site catalytic mechanism. Fluorescence and CD spectra revealed that the latter enzyme could have been undergone some conformational change that could account for the loss of activity; on the other hand, the one-dimensional NMR spectra of either native and des-Arg⁹⁷-Tyr⁹⁸ enzymes were strictly similar, thus demonstrating that the removal of the two C-terminal residues does not markedly affect the fold of the enzyme. The results reported are proof of a critical contribution of Arg⁹⁷ to the acylphosphatase active site; however, we cannot exclude that the function of this residue is merely to stabilize the active site conformation and dynamics.     

ROLE OF TYROSINE RESIDUES ON STRUCTURE-FUNCTION OF FRUCTOSE-1,6-BIPHOSPHATE ALDOLASE FROM CERATITIS CAPITATA

Tyrosine contributions to the structure-function relationship in the fructose-1, 6-biphospate aldolase from C. capitata have been investigated. There are three well defined groups of tyrosine residues with different roles in the structure of the insect aldolase. C-terminal tyrosine residues are essential for the maintenance of the catalytic conformation. Releasing of these residues by carboxypeptidase A treatment results in complex conformational changes according to CD studies. Another tyrosine residue group is located at the active site, and the substrate, fructose-1,6-biphosphate, protects it upon nitration. Chemical modification of this residue results in enzyme activity changes similar to those induced by carboxypeptidase digestion. Enzyme-substrate interaction results in a change of the microenvironment of at least three tyrosine residues per subunit with different accessibility for tetranitromethane.     

Conformation and structure of linear peptides with regularly alternating l- and d-residues: structure of the blocked hexapeptide tert-butyloxycarbonyl-(d-alloisoleucyl-l-isoleucyl)3 methyl ester monohydrate

Peptides with a regular sequence of enantiomeric residues (l and d ) along the chain have received considerable attention because of their accessibility to unique conformations and because they are model compounds for the naturally occurring peptide gramicidin A, which shows monovalent cation selective transmembrane transport. The solid-state structure of the linear hexapeptide t-Boc-(d -aIle-l -Ile)₃-OMe has been determined by X-ray diffraction techniques and refined to a final R factor of 0.068. The molecule shows a bent U-shaped conformation stabilized by three intramolecular H-bonds of the N—H?O=C type: a type II β-bend (4 → 1 bend or C₁₀ ring structure) with l -Ile² and d -aIle³ at positions 2 and 3 of the bend, an α-turn (5 → 1 bend or C₁₃ ring structure) and a 1 → 5 bend or C₁₇ ring structure. The first two 10-membered and 13-membered bends are enclosed in the latter 17-membered hydrogen-bonded ring structure. This structural motif is common to hepta- and octa-peptide cyclic molecules, showing that ring closure is not required to achieve a particular topology in the molecular design of specific bended conformations. © Munksgaard 1995.     

Insights from the Structure of an Active Form of Bacillus thuringiensis Cry5B

The crystal protein Cry5B, a pore-forming protein produced by the soil bacterium Bacillus thuringiensis, has been demonstrated to have excellent anthelmintic activity. While a previous structure of the three-domain core region of Cry5B(112–698) had been reported, this structure lacked a key N-terminal extension critical to function. Here we report the structure of Cry5B(27–698) containing this N-terminal extension. This new structure adopts a distinct quaternary structure compared to the previous Cry5B(112–698) structure, and also exhibits a change in the conformation of residues 112–140 involved in linking the N-terminal extension to the three-domain core by forming a random coil and an extended α-helix. A role for the N-terminal extension is suggested based on a computational model of the tetramer with the conformation of residues 112–140 in its alternate α-helix conformation. Finally, based on the Cry5B(27–698) structure, site-directed mutagenesis studies were performed on Tyr495, which revealed that having an aromatic group or bulky group at this residue 495 is important for Cry5B toxicity.     

Crystal structure and molecular conformation of achatin-I (H-Gly-d-Phe-Ala-Asp-OH), an endogenous neuropeptide containing a d-amino acid residue

In order to investigate the active conformation of achatin-I (H-Gly-d -Phe-Ala-Asp-OH), an endogenous neuropeptide from the Achatina fulica ganglia, its crystal structure and molecular conformation were analysed by the X-ray diffraction method. Crystals from methanol/dioxane are monoclinic, space group P2₁ with a=5.083(1), b= 9.125(1), c= 20.939(3) Å, β=94.73(1)° The structure was solved by direct methods and refined to R = 0.051 for 1714 independent reflections with Fo > σ(Fo). The molecule exists as a zwitterion with the Gly N-terminal end protonated and Asp β-carboxyl deprotonated; the C-terminal of Asp is in a neutral state. The molecule takes a kind of β turn structure with the d -Phe-Ala residues at the corner of the bend. This turn conformation is primarily formed by the strong intramolecular hydrogen bonds of NH(Gly)—O^δ1 (Asp) and NH(Asp)- O^δ1(Asp) pairs, thus forming a 15-membered ring structure. Judging from the published data concerning the structure-activity relationship, this turn conformation may reflect an important feature related to the neuroexcitatory activity of achatin-I.     

Conformational properties of hybrid peptides containing α‐ and ω‐amino acids*

Abstract: This review briefly surveys the conformational properties of guest ω‐amino acid residues when incorporated into host α‐peptide sequences. The results presented focus primarily on the use of β‐ and γ‐residues in αω sequences. The insertion of additional methylene groups into peptide backbones enhances the range of accessible conformations, introducing additional torsional variables. A nomenclature system, which permits ready comparisons between α‐peptides and hybrid sequences, is defined. Crystal structure determination of hybrid peptides, which adopt helical and β‐hairpin conformations permits the characterization of backbone conformational parameters for β‐ and γ‐residues inserted into regular α‐polypeptide structures. Substituted β‐ and γ‐residues are more limited in the range of accessible conformation than their unsubstituted counterparts. The achiral β,β‐disubstituted γ‐amino acid, gabapentin, is an example of a stereochemically constrained residue in which the torsion angles about the C^β–C^γ (θ₁) and C^α–C^β (θ₂) bonds are restricted to the gauche conformation. Hybrid sequences permit the design of novel hydrogen bonded rings in peptide structures.     

Empirical rules predicting conformation of cyclic tetrapeptides from primary structure

A useful set of empirical rules is put forward to predict the conformations of cyclic tetrapeptides and cyclic tetradepsipeptides on the basis of primary structure, briefly presented as follows: 1. A conformation allowing an intramolecular hydrogen bond (IMHB) of γ-turn is preferred, and an ester bond always adopts a trans form. 2. On a right-handed peptide ring, the carbonyl group acylating a D residue is oriented to the upper side of the main ring. 3. The carbonyl group acylating a d proline or an N-methyl-d -amino acid residue is oriented to the lower side of the ring, forming a cis bond. 4. The lddl configurational sequence adopts a cis-trans-cis-trans backbone with C_i symmetry. 5. A glycine residue behaves as a d residue in an l -peptide. Conformations of cyclotetrapeptides containing two glycine residues at diametric positions or containing an N-methyl-dehydroamino acid residue are predicted by use of appendices of rule 5. Almost all conformations of cyclic tetrapeptides are predicted by these rules. Energetical rationalization of the rules and prediction of possible new conformations are described. Conformations of cyclo (-l -Pro-l -Leu-d -Tyr(Me)-l -Ile-)(1) and cyclo (-l -Pro-d -Leu-d -Tyr(Me)-l -Ile)(2) are compared. Results of n.m.r. experiments showed that compound 1 adopts a unique cis-trans-trans-trans backbone with a γ-turn IMHB, and 2 has a cis-trans-cis-trans backbone with C_i symmetry. These observations confirmed the rules described above. Peptides 1 and 2 are the first diastereomeric peptides with trans (ld ) and cis (dd ) secondary amide bonds.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys³⁰⁷ and Staphylococcus aureus V8 protease at Glu³⁰², producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (～58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule     

Modulation of dimerization by residues distant from the interface in bovine neurophysin-II

The crystal structure of bovine neurophysin-II in its liganded state (Chen et al. [1991]. Proc. Natl. Acad. Sci. USA 88 , 4240-4244) indicates that the 1–6 sequence has a disordered conformation, lacks noncovalent contacts to other regions of the protein and is distant from the monomer-monomer interface. Cleavage of the 1–6 sequence by Staphylococcus protease V8 yielded a protein that, for the first time, crystallized in both liganded and unliganded states. Insights into the role of the 1–6 sequence in the unliganded state were obtained by NMR and related biophysical comparisons of the native and des-1–6 proteins. NMR spectra demonstrated that the environment and/or conformation of residues in the 1–6 sequence differed in liganded and unliganded states. Additionally, the unliganded des-1–6 protein exhibited a dimerization constant four to five times that of the native protein, potentially accounting for the observation that its peptide affinity was also increased. NMR studies further indicated that the increased dimerization constant of the des-1–6 protein correlated with the presence in the native protein of two isoenergetic forms of the monomer, in contrast to only a single form in the des-1–6 protein, as evidenced by signals from an internal dimerization-sensitive a-proton. Thus, the 1–6 sequence reduces the dimerization constant by stabilization of an alternative monomer conformation. A second product of Staphylococcus protease V8 digestion of the native protein was identified as the des-1–6 protein with an internal clip after binding site residue Glu-47, the clip presumably breaking the short 3, 10 helix that most directly connects the interface to the binding site. This product, although unable to bind peptide, retained the dimerization constant of the des-1–6 protein, suggesting a lack of importance of the helix in dimerization and contrasting with the effects of the 1–6 sequence. A model is proposed in which the 1–6 sequence stabilizes the second conformation of the unliganded monomer via interactions affecting the loop region that separates the two neurophysin domains and which has been shown to influence neurophysin self-association.     

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science 264 , 82–85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C^α torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (α_L) and that in family 2 is right-handed helical (α_R). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereo-chemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens. © Munksgaard 1995.     

New fMLF-OMe Analogues Containing Constrained Mimics of Phenylalanine Residue

In the context of a research program aimed at elucidating the HCO-Met-Leu-Phe-OMe (fMLF-OMe) structural features which control interactions with the receptor in correspondence with the C-terminal residue, four different analogues of the native ligand have been synthesized and evaluated. Compounds 1-3 possess the general formula HCO-Met-Leu-Xaa-OMe with Xaa = N-benzylglycine, N-benzylphenylalanine, and α,α-dibenzylglycine, respectively. In the analogue 4 the constraint at the C-terminus has been obtained by incorporating a 2-oxopiperazine ring, made up of two phenylalanine residues, to replace the native C-terminal Phe residue. The consequences of the chemical modifications on the activity of the new analogues are discussed.     

Conformational analysis of homochiral and heterochiral diprolines as β-turn-forming peptidomimetics: unsubstituted and substituted models

The effect of replacing one of the proline residues in either unsubstituted homochiral or heterochiral diproline segments with either a 2- or a 3-substituted prolyl residue on the allowed conformation of the diproline template has been examined. In heterochiral (l-d ) diprolines, placement of a 2-methyl-d -proline residue in the i+ 2 position and placement of either a cis- or trans-3-methyl-l -proline residue in the i+ 1 position results in substituted diproline peptides that adopt the same type II β-turn conformation as that identified experimentally for the unsubstituted diproline peptides. In contrast, placement of a cis-3-methyl-d -proline residue in the i+ 1 position of a homochiral (d-d ) diproline peptide seems to promote a different conformation than that seen in the unsubstituted case, whereas the trans-3-methyl-d -proline residue seems to provide a stabilizing influence for the predicted type VI'β-turn. The demonstrated ability of certain substituted diproline templates to adopt predictable conformations coupled with the development of asymmetric synthetic routes to both 2- and 3-substituted prolyl residues capable of mimicking a variety of side chains should make these templates useful tools in designing specific turn mimics of biologically active molecules.     

Orthogonal threading-through-β-sheet design of lung cancer EGFR extracellular domain-derived peptidic mimotopes binding to anti-EGFR antibody

Human epidermal growth factor receptor (EGFR) has been established as a therapeutic target of lung cancer and other diverse tumors. The antibody drug Cetuximab has been developed to target the third subdomain III (TSDIII) of EGFR extracellular domain (ECD) by competitively inhibiting epidermal growth factor binding. In this study, we performed systematic investigation on the crystal complex structure of EGFR ECD domain with Cetuximab to create a residue importance profile for the TSDIII subdomain, based on which a number of U-shaped, double-stranded linear peptides were derived and cyclized to orthogonally thread through most hotspot residues and many responsible residues within the TSDIII β-sheet plane; they represent mimotopes of the key antibody-recognition site of TSDIII subdomain. Computational analyses revealed that these linear peptides cannot spontaneously fold to the desired conformation in free state due to their intrinsic flexibility. Cell-free assays confirmed that the stapling can considerably improve the binding affinity of linear peptides to Cetuximab by up to 18-fold. The cOrt1 [3–18] cyclic peptide was measured to have the highest affinity in all designed linear and cyclic peptides.     

Design of peptides with αβ-dehydro residues: Synthesis,crystal structure and molecular conformation of N-Boc-L-Ile-ΔPhe-L-Trp-OCH3

The dehydro-peptide Boc-L-Ile-ΔPhe-L-Trp-OCH₃ was synthesized by the azlactone method in the solution phase. The peptide was crystallized from methanol in an orthorhombic space group P2₁2₁2₁ with a = 10.777(2), b= 11.224(2), c= 26.627(10) Å. The structure was determined by direct methods and refined to an R value of 0.069 for 3093 observed reflections [l≥ 2σ(l)].The peptide failed to adopt a folded conformation with backbone torsion angles: φ₁, = 90.8(8)°, ψ₁= -151.6(6)°, φ₂= 89.0(8)°, ψ₂= 15.9(9)°, φ₃= 165.7(7)°, ψ^T₃= -166.0(7)°. A general rule derived from earlier studies indicates that a three-peptide unit sequence with a ΔPhe at the (i+ 2) position adopts a β-turn II conformation. Because the branched β-carbon residues such as valine and isoleucine have strong conformational preferences, they combine with the ΔPhe residue differently to generate a unique set of conformations in such peptides. The presence of β-branched residues simultaneously at both (i+ 1) and (i+ 3) positions induces unfolded conformations in tetrapeptides, but a β-branched residue substituted only at (i+ 3) positron can not prevent the formation of a folded β-turn II conformation. On the other hand, the present structure shows that a β-branched residue substituted at the (i+ 1) position prevents the formation of a β-turn II conformation. These observations indicate that a β-branched residue at the (i+ 1) position prevents a folded conformation whereas it cannot generate the same degree of effect from the (i+ 3) position. This may be because of the trans disposition of the planar ΔPhe side-chain with respect to the C=O group in the residue. The molecules are packed in an anti-parallel manner to generate N₂-H₂…O₂ (-x,y-1/2, -z+ 3/2) and N^ε1₃-H^ε1₃…O₁(-x,y -1/2, -z+ 3/2) hydrogen bonds.     

Crystal and molecular structure of Boc-Phe-Val-OMe; comparison of the peptide conformation with its dehydro analogue

The crystal structure of the peptide Boc-Phe-Val-OMe determined by X-ray diffraction methods is reported in this paper. The crystals grown from aqueous methanol are orthorhombic, space group P 2₁2₁ 2₁, a= 11.843(2), b= 21.493(4), c= 26.676(4) A and V= 6790 ų. Data were collected on a CAD4 diffractometer using MoK_α radiation (λ= 0.7107 Å) up to Bragg angle θ=26°. The structure was solved by direct methods and refined by a least-squares procedure to an R value of 6.8% for 3288 observed reflections. There are three crystal-lographically independent peptide molecules in the asymmetric unit. All the three molecules exhibit extended conformation. The sidechain of the Val² residue shows two different conformations. The conformation of the peptide Boc-Phe-Val-OMe is compared with the conformation of Ac-ΔPhe-Val-OH. It is observed that while Boc-Phe-Val-OMe exhibits an extended conformation, Ac-ΔPhe-Val-OH shows a folded conformation. The results of this comparison highlight the conformation constraining property of the ΔPhe residue. Interestingly, even though Boc-Phe-Val-OMe and Ac-ΔPhe-Val-OH are conformation ally different, they exhibit similar packing patterns in the solid state. © Munksgaard 1995.     

Synthesis and binding potencies of cyclic hexapeptide analogs of somatostatin incorporating acidic and basic peptoid residues

The synthesis, binding affinity, and structure–activity relationships of compounds related to the cyclic hexapeptide, c[Pro⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰-Phe¹¹], L-363,301 (the numbering in the sequence refers to the position of the residue in native somatostatin) is reported. The Pro residue in this compound is replaced with the peptoid residues Nasp [N-(2-carboxyethyl) glycine], Ndab [N-(2-aminoethyl) glycine] and Nlys [N-(4-aminobutyl) glycine]. This series of compounds enables us to draw conclusions about the influence of positively or negatively charged residues in the bridging region on the binding affinity towards the isolated human somatostatin receptors. A loss of binding to the recombinant human somatostatin (hsst) receptors in the Nasp analog compared with L-363,301 and compared with the Ndab and Nlys analogs clearly demonstrates that the presence of an acidic residue in the bridging region is unfavorable for binding to the hsst receptors. Comparison between the Ndab analog and the Nlys analog suggests that the presence of a basic residue in the bridging region might be advantageous for binding to the hsst5 receptor provided that the residue bearing the basic group extends far enough to allow for interaction with the receptor, while the length of the basic peptoid residue does not influence binding to the hsst2 receptor. These results are useful for the design of hsst5 selective somatostatin analogs.     

Synthesis and properties of chemotactic peptide analogs I. Crystal structure and molecular conformation of HCO-Met-Leu-Ain-OMe

HCO-Met-Leu-Ain-OMe (2), an analog of the chemotactic peptide HCO-Met-Leu-Phe-OH, containing the conformationally blocked residue of the 2-aminoindane-2-carboxylic acid (Ain) has been synthesized and its crystal and molecular conformation has been determined. Crystals of 2 are monoclinic, space group P2₁, with a = 15.059(7), b = 18.548(7), c = 9.600(4)Å;β= 85.04(3). The structure has been solved by direct methods and refined to R = 0.069 for 2813 independent reflections with I > 2.5σ(I). Two independent molecules A and B have been found in the asymmetric unit of the crystal of 2. Their conformation can be described as extended at the Met and Leu residues, but folded at the C-terminal Ain residue. The helical folding is left- and right-handed in the A and B molecule, respectively. The crystal packing is characterized by ribbons of intermolecular hydrogen bonded molecules extended along the c direction. The constrained analog 2 is highly active in the superoxide production, thus indicating that a stabilization of a helical folding at the C-terminal region of chemotactic tripeptides maintains the activity. The orientation of the aromatic ring, with respect to its adjacent backbone atoms, does not seem critical for the activity.