Synthesis and properties of chemotactic peptide analogs. II. HCO-Met-Leu-Phe-OMe analogs containing cyclic alpha,alpha-disubstituted amino acids as Met and Phe mimicking residues.

As a part of a research program aimed at studying structure activity relationship in the field of chemotactic peptides, modified analogs of the potent chemoattractant HCO-Met-Leu-Phe-OH (fMLP) of the general formula HCO-Xaa-Leu-Yaa-OMe are examined. 4-Aminotetrahydrothiopyran-4-carboxylic acid (Thp) and 2-aminoindane-2-carboxylic acid (Ain) have been chosen as achiral, conformationally restricted amino acids suitable to mimick the external Met and Phe residues of fMLP-OMe. Studies on a first model, namely [Ain3]fMLP-OMe 1, have already been reported (12). Here the two remaining analogs [Thp1, Ain3] 2 and [Thp1] 3 have been synthesized. The conformation in the crystal of the disubstituted analog 2 has been determined and compared with those adopted by the parent fMLP-OMe and by previously studied models. The backbone conformation of 2 is characterized by helical folding centred at each of the three residues with the central Leu presenting helical handedness opposite to those of the two adjacent achiral residues. This conformation presents strong similarities with that adopted in the crystal by fMLP-OMe and resembles the conformation of fMLP bound to immunoglobulin (Bence-Jones dimer). The conformationally restricted analogs 2 and 3 are more active than the parent in the stimulation of directed mobility of human neutrophils but are practically inactive in the superoxide production. Crystals of 2 are orthorhombic, s.g. P2(1)2(1)2(1), with a = 21.934 (8), b = 10.856 (2), c = 10.380 (2) A. The structure has been refined to R = 0.071 for 2301 independent reflections with I greater than 1.5 sigma.     

Conformational behaviour of A cyclolinopeptide a analogue: Two-dimensional NMR study of cyclo(Pro1-Pro-Phe-Phe-Ac6c-IIe-ala-Val8)

The cyclic octapeptide cyclo[-Pro¹-Pro-Phe-Phe-Ac₆c-Ile-ala-Val⁸-] [C₈-Ac₆c], containing the Pro¹-Pro-Phe-Phe sequence, followed by a bulky helicogenic C^α,α-dialkylated glycine residue Ac₆c (1-aminocyclohexane-1-carboxylic acid), and a _D-Ala residue at position 7 has been synthesized. This cyclic peptide is a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A (CLA). It has been designed with the aim of studying the role that the Ac₆c and _D-Ala residues play on the conformational behaviour of the whole molecule and their influence on the conformation of the Pro¹-Pro-Phe-Phe sequence when compared with cyclolinopeptide A. C₈Ac₆c has been investigated in chloroform and acetonitrile solutions by 2D NMR techniques. Only one set of sharp signals is observed in both solvents. This evidence strongly supports the hypothesis that only one conformational state exists in the chosen solvents. The interpretation of the experimental data points to the existence for C₈-Ac₆c of a very rigid structure stabilized by intramolecular hydrogen bonds. The measured NOE effects allow the calculation of internuclear distances, which have been used as restraints in molecular dynamic calculations. The proposed conformation of the molecule shows that the Pro-Pro-Phe segment retains the conformation observed in natural CLA both in solution and in the solid state and that the Ac₆c residue indeed reinforces the ring rigidity not permitting the formation of any appropriate cavity in which inorganic cations could be complexed.     

Modified chemotactic peptides: Synthesis,conformation, and biological activity of for-Thp-Leu-ΔzPhe-OMe

For-Thp-Leu-Δ^zPhe-OMe ( 2 ), an analogue of the chemotactic tripeptide For-Met-Leu-Phe-OMe, containing 4-aminotetrahydrothiopyran-4-carboxylic acid (Thp) and (Z)-2,3-didehydrophenylalanine (Δ^zPhe) as achiral, conformationally restricted mimics of Met and Phe, respectively, has been synthesized. In the crystal the new formyltripeptide adopts a type I β-turn conformation stabilized by a weak H bond between the formylic oxygen and the Δ^zPhe NH. ¹H-nmr analysis based on NH solvent accessibility and nuclear Overhauser effect experiments suggests that the β-turn is not preferred in CDCl₃ solution where a γ-turn, centered at the Thp residue, prevails. The biological activity of 2 has been determined on human neutrophils and compared to that of previously studied analogues. The tripeptide 2 is practically unable to elicit superoxide anion production and lysozyme release, while slight, but not statistically significant activity was induced in chemotaxis. The role of the orientation of the aromatic ring with respect to the backbone adjacent atoms is discussed. © 1994 John Wiley & Sons, Inc.     

Preferred conformation of peptides rich in Ac8c,a medium-ring alicyclic Cα,α-disubstituted glycine

A complete series of terminally blocked, monodispersed homo-oligopeptides (to the pentamer level) from the sterically demanding, medium-ring alicyclic C^α,α-disubstituted glycine 1-aminocyclooctane-1-carb oxylic acid (Ac₈c), and two Ala/Ac₈c tripeptides, were synthesized by solution methods and fully characterized. The preferred conformation of all the oligopeptides was determined in deuterochloroform solution by IR absorption and ¹H-NMR. The molecular structures of the amino acid derivative Z-Ac₈c-OH, the dipeptide pBrBz- (Ac₈c)₂-OH and the tripeptide pBrBz-(Ac₈c)₃-OtBu were assessed in the crystal state by X-ray diffraction. Conformational energy computations were performed on the monopeptide Ac-Ac₈c-NHMe. Taken together, the results obtained strongly support the view that the Ac₈c residue is an effective β-turn and helix former. A comparison is also made with the conformational preferences of α-aminoisobutyric acid, the prototype of C^{α, α}-disubstituted glycines, and of the other members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3, 5–7) investigated so far. The implications for the use of the Ac₈c residue in peptide conformational design are considered.     

Conformational characterization of peptides rich in the cycloaliphatic Cα,α-disubstituted glycine 1-amino-cyclononane-1-carboxylic acid

A series of N- and C-protected, monodispersed homo-oligopeptides (to the pentamer level) from the cycloaliphatic C^α,α,-dialkylated glycine 1-aminocyclononane-1-carboxylic acid (Ac₉c) and two Ala/Ac₉c tripeptides have been synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and ¹H-NMR. The molecular structures of the amino acid derivatives mClAc-Ac₉c-OH and Z-Ac₉c-OtBu, the dipeptide pBrBz-(Ac₉c)₂-OtBu, the tetrapeptide Z-(Ac₉c)₄-OtBu, and the pentapeptide Z-( Ac₉c)₅-OtBu were determined in the crystal state by X-ray diffraction. Based on this information, the average geometry and the preferred conformation for the cyclononyl moiety of the Ac₉c residue have been assessed. The backbone conformational data are strongly in favour of the conclusion that the Ac₉c residue is a strong β-turn and helix former. A comparison with the structural propensity of α-aminoisobutyric acid, the prototype of C^α,α-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3−8) is made and the implications for the use of the Ac₉c residue in conformationally constrained analogues of bioactive peptides are briefly examined. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci. 3: 367–382 No. of Figures: 10. No. of Tables: 6. No. of References: 62     

Synthesis,conformation, and biological activity of two fMLP-OMe analogues containing the new 2-[2′-(methylthio) ethyl] methionine residue

The new C^α-tetrasubstituted α-amino acid residue 2-[2′-(methylthio) ethyl]methionine (Dmt) has been introduced into the reference chemotactic tripeptide HCO-Met-Leu-Phe-OMe (fMLP-OMe) in place of the leucine or methionine, respectively. The biological activity of the new analogues [Dmt²] fMLP-OMe (2) and [Dmt¹] fMLP-OMe (3) has been determined; whereas 2 is active toward human neutrophils, stimulating directed migration, superoxide anion generation, and lysozyme release, 3 results practically inactive in all tested assays. A conformational analysis on 2 and 3 has been performed in solution by using ir absorption and ¹H-nmr. The conformation of 2 was also examined in the crystal by x-ray diffraction methods. Both 2 and 3 adopt fully extended conformation in correspondence with the Dmt residue. Biological and conformational results are discussed and compared with related previously studied models. © 1997 John Wiley & Sons, Inc. Biopoly 42: 415–426, 1997     

Contrasting solution conformations of peptides containing α,α-dialkylated residues with linear and cyclic side chains

The conformational properties of α,α-dialkylated amino acid residues possessing acyclic (diethylglycine, Deg: di-n-propylglycine, Dpg; di-n-butylglycine, Dbg) and cyclic (1-amino-cycloalkane-1-carboxylic acid, Ac_nc) side chains have been compared in solution. The five peptides studied by nmr and CD spectroscopy are Boc-Ala-Xxx-Ala-OMe, where Xxx = Deg(I). Dpg (II), Dbg (III), Ac₆c (IV), and Ac₇c (V). Delineation of solvent-shielded NH groups have been achieved by solvent and temperature dependence of NH chemical shifts in CDCl₃ and (CD₃)₂SO and by paramagnetic radical induced line broadening in pepiide III. In the Dxg peptides the order of solvent exposure of NH groups is Ala(1) > Ala(3) > Dxg(2), whereas in the Ac_nc peptides the order of solvent exposure of NH groups is Ala(1) > Ac_nc(2) > Ala(3). The nmr results suggest that Ac_nc peptides adopt folded β-turn conformations with Ala(1) and Ac_nc(2) occupying i + 1 and i + 2 positions. In contrast, the Dxg peptides favor extended C₅ conformations. The conformational differences in the two series are clearly borne out in CD studies. The solution conformations of peptides I-III are distinctly different from the β-turn structure observed in crystals. Low temperature nmr spectra recorded immediately after dissolution of crystals of peptide II provide evidence for a structural transition. Introduction of an additional hydrogen-bonding function in Boc-Ala-Dpg-Ala-NHMe (VI) results in a stabilization of a consecutive β-turn or incipient 3₁₀-helix in solution. © 1995 John Wiley & Sons, Inc.     

Studies on fMLP-receptor interaction and signal transduction pathway by means of fMLP-OMe selective analogues

For-Thp-Leu-Ain-OMe ([Thp(1), Ain(3)] fMLP-OMe) (2), for-Met-delta(z)Leu-Phe-OMe ([delta(z)Leu(2)] fMLP-OMe) (3), for-Thp-Leu-Phe-OMe ([Thp(1)] fMLP-OMe) (4), and for-Met-Leu-Ain-OMe ([Ain(3)] fMLP-OMe) (5) are for-Met-Leu-Phe-OMe (fMLP-OMe) (1) analogues which discriminate between different responses of human neutrophils. Peptides 3 and 5, similar to fMLP-OMe, enhance neutrophil cyclic AMP (cAMP) as well as calcium levels, while analogues 2 and 4, which evoke only chemotaxis, do not alter the concentration of these intracellular messengers. When we tested the peptides' ability to displace [3H]-fMLP from its binding sites, the following order of potency was observed: analogue 1 > 3 > 5 > 2 > 4. A particularly low activity at the receptor level characterized analogues 2 and 4. Their low effectiveness was not improved by the addition of cytochalasin B, by different incubation temperatures, or by the absence of endogenous guanine nucleotides, conditions known to influence fMLP receptor fate and functionality. We speculate that, in certain conditions, the fMLP receptor may undergo conformational changes that impede the binding of pure chemoattractants.     

Conformational Characterization of the 1-Aminocyclobutane-1-carboxylic Acid Residue in Model Peptides

A series of N- and C-protected, monodispersed homo-oligopeptides (to the dodecamer level) from the small-ring alicyclic C^α,α-dialkylated glycine 1-aminocyclobutane-1-carboxylic acid (Ac₄c) and two Ala/Ac₄c tripeptides were synthesized by solution methods and fully characterized. The conformational preferences of all the model peptides were determined in deuterochloroform solution by FT-IR absorption and ¹H-NMR. The molecular structures of the amino acid derivatives Z-Ac₄c-OH and Z₂-Ac₄c-OH, the tripeptides Z-(Ac₄c)₃-OtBu, Z-Ac₄c-(L -Ala)₂-OMe and Z-L -Ala-Ac₄c-L -Ala-OMe, and the tetrapeptide Z-(Ac₄c)₄-OtBu were determined in the crystal state by X-ray diffraction. The average geometry of the cyclobutyl moiety of the Ac₄c residue was assessed and the τ(N–C^α–C′) bond angle was found to be significantly expanded from the regular tetrahedral value. The conformational data are strongly in favour of the conclusion that the Ac₄c residue is an effective β-turn and helix former. A comparison with the structural propensities of α-aminoisobutyric acid, the prototype of C^α,α-dialkylated glycines, and the other extensively investigated members of the family of 1-aminocycloalkane-1-carboxylic acids (Ac_nc, with n=3, 5–8) is made and the implications for the use of the Ac₄c residue in conformationally constrained peptide analogues are briefly examined. © 1997 European Peptide Society and John Wiley & Sons, Ltd     

δ-Selective Opioid Peptides Containing a Single Aromatic Residue in the Message Domain: An NMR Conformational Analysis

The sequence of deltorphin I, a δ-selective opioid agonist, has been systematically modified by inserting conformationally constrained C^α,α disubstituted apolar residues in the third position. As expected, substitution of Phe with Ac₆c, Ac₅c and Ac₃c yields analogues with decreasing but sizeable affinity. Surprisingly, substitution with Aib yields an analogue with almost the same binding affinity of the parent compound but with a greatly increased selectivity. This is the first case of a potent and very selective opioid peptide containing a single aromatic residue in the message domain, that is, only Tyr¹. Here we report a detailed conformational analysis of [Aib³]deltorphin I and [Ac₆c³]deltorphin I in DMSO at room temperature and in a DMSO/water cryomixture at low temperature, based on NMR spectroscopy and energy calculations. The peptides are highly structured in both solvents, as indicated by the exceptional finding of a nearly zero temperature coefficient of Val⁵ NH resonance. NMR data cannot be explained on the basis of a single structure but it was possible to interpret all NMR data on the basis of a few structural families. The conformational averaging was analysed by means of an original computer program that yields qualitative and quantitative composition of the mixture. Comparison of the preferred solution conformations with two rigid δ-selective agonists shows that the shapes of [Aib³]deltorphin I and [Ac₆c³]deltorphin I are consistent with those of rigid agonists and that the message domain of opioid peptides can be defined only in conformational terms.     

Modified chemotactic peptides: Synthesis and activity of an azaTic-containing fMLP-OMe analogue

Summary The synthesis and the biological activity of a pseudopeptide analogue of the chemotacticN-formyltripeptide fMLP-OMe, containing the aza Tic (3,4-dihydro-2(1H)-phthalazinecarboxylic acid) residue replacing the native phenylalanine, is described. Whereas pseudopeptides containing lineara-azaammo acids are currently studied, data on the new group of analogues containing cyclic-aza residues capable of limiting the rotameric distribution of the side chains (topological control) are just emerging in the literature. At our best knowledge, the here described [azaTic³]fMLP-OMe represents the first example of the introduction of this new type of-aza residue into a natural bioactive peptide.     

Crystal structure of a tripeptide containing aminocyclododecane carboxylic acid: a supramolecular twisted parallel β‐sheet in crystals

The crystal structure of a tripeptide Boc‐Leu‐Val‐Ac₁₂c‐OMe ( 1 ) is determined, which incorporates a bulky 1‐aminocyclododecane‐1‐carboxylic acid (Ac₁₂c) side chain. The peptide adopts a semi‐extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C^α,α‐dialkylated residue Ac₁₂c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel β‐sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac₁₂c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti‐parallel β‐sheet in crystals, twisted or helical β‐sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted β‐sheet assembly in crystals. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Helical formation of a 13-residue C-peptide analogue of ribonuclease A in sodium dodecyl sulfate solution

The conformation of a 13-residue C-peptide analogue of ribonuclease A——in surfactant solutions was studied by CD. The CD spectrum of the peptide in excess NaDodSO₄ solution was typical for a helical conformation; the spectrum appeared to be virtually independent of pH (2.5–6) and temperature (3–25°C). Analysis of the CD data indicated a helicity of about 65–70% with no α-sheet and β-turn; this corresponded to 8 or 9 residues in the helical form or slightly more than two turns of α-helix. This compares with an average of about one turn of α-helix for the C-peptide analogue in water at pH 4.7 and 7°C. The conformation of the peptide in cationic surfactant, dodecyl ammonium chloride, and nonionic surfactant, dodecyl heptaoxyethylene ether, solution resembled that in water. We concluded that the C-peptide analogue can develop a maximum helicity close to the corresponding segment in ribonuclease A in hydrophobic environment provided by the clustering of NaDodSO₄ molecules to the cationic side groups of the peptide, except that the end effects may destabilize two or three residues each at both ends of the helix. Thus, in the interior of a protein molecule this hydrophobic effect may overshadow the charged-group effect than can be explained by the helix dipole model for the helical segments on the exterior of the protein molecule.     

Development of potent anti-infective agents from Silurana tropicalis: Conformational analysis of the amphipathic,alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7

Peptide XT-7 (GLLGP⁵LLKIA¹⁰AKVGS¹⁵NLL.NH₂) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC₅₀ = 140 µM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC₅₀ > 500 µM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d₃)-H₂O mixed solvent system, XT-7 is characterised by a right handed α-helical conformation between residues Leu³ and Leu¹⁷ whereas [G4K]XT-7 adopts a more restricted α-helical conformation between residues Leu⁶ and Leu¹⁷. A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu³ and Leu¹⁷. However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H₂O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly⁴ → Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.     

Structural versatility of peptides from Cα,αdialkylated glycines: Linear Ac3c homo-oligopeptides

The crystal-state molecular structures of five linear Ac₃c homo-oligopeptides to the tetramer were determined by x-ray diffraction. The oligomers are H-(Ac₃c)₂-OMe, Fmoc-(Ac₃c)₂-OMe MeOH, Ac-(Ac₃c)₂-OMe, pBrBz-(Ac₃c)₃-OMe · H₂O, and t-Boc-(Ac₃c)₄-OMe · 2H₂O. The results indicate the propensity of the tri- and tetrapeptides to fold into type I β-bends and distorted 3₁₀-helices, respectively, in partial contrast to Aib, Ac₅c, and Ac₆c homo-peptides of comparable main-chain length, where regular type III β-bends and 3₁₀-helical structures were found. When the influence of the constraints produced by the intramolecular H bonds of the C₁₀-type is absent, other less common structural features may be observed. The average geometry of the cyclopropyl group of the Ac₃c residue is found to be asymmetric and the N? C^α? C′ bond angle significantly expanded from the regular tetrahedral value.     

Development of helix-stabilized antimicrobial peptides composed of lysine and hydrophobic α,α-disubstituted α-amino acid residues

Lysine-based amphipathic nonapeptides, including homochiral peptides [Ac-(l-Lys-l-Lys-Xaa)₃-NH₂ (Xaa = Gly, Ala, Aib, Ac₅c, or Ac₆c) and Ac-(d-Lys-d-Lys-Aib)₃-NH₂], a heterochiral peptide [Ac-(l-Lys-d-Lys-Aib)₃-NH₂], and a racemic mixture of diastereomeric peptides [Ac-(rac-Lys-rac-Lys-Aib)₃-NH₂] were designed and synthesized to investigate the relationship between their preferred secondary structures and their antimicrobial activity. Peptide 5, [Ac-(l-Lys-l-Lys-Ac₆c)₃-NH₂] formed a stable α-helical structure and exhibited strong activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa).     

Effects of five-membered ring amino acid incorporation into peptides for coiled coil formation

A five-membered ring amino acid (Ac₅c), the peptides of which exhibit a preference for helical secondary structures, was introduced into peptides for the purpose of designing coiled coil peptides with high binding affinities. We prepared five types of peptides containing Ac₅c with different numbers or at different positions. The incorporation of Ac₅c into peptides enhanced their α-helicities; however, in contrast to our expectations, it did not result in stable coiled coil formation. The structures of side chains in hydrophobic amino acids, not α-helicities appeared to be important for stable hydrophobic interactions between peptides. Although we were unable to develop coiled coil peptides with high binding affinities, the present results will be useful for designing novel coiled coil peptides.     

Crystal state conformation of three model monomer units for the β-bend ribbon structure

The molecular and crystal structures of three compounds, representing the repeating units of the β-bend ribbon (an approximate 3₁₀-helix, with an intramolecular hydrogen-bonding donor every two residues), have been determined by x-ray diffraction. They are Boc-Aib-Hib-NHBzl, Z-Aib-Hib-NHBzl, and Z-L -Hyp-Aib-NHMe (Aib, α-aminoisobutyric acid; Bzl, benzyl; Boc, t-butyloxycarbonyl; Hyp, hydroxyproline Hib, α-hydroxyisobutyric acid; Z, benzyloxycarbonyl). The two former compounds are folded in a β-bend conformation: type III (III′) for Boc-Aib-Hib-NHBzl, while type II (II′) for the Z analogue. Conversely, the structure of Z-L -Hyp-Aib-NHMe, although not far from a type II β-bend, is partially open.     

A comparative NMR and molecular dynamics study of the conformations of bradykinin B1 and B2, B2, and B1-specific receptor antagonists B-9430, B-9436, and B-9858

Extensive proton magnetic resonance experiments were carried out on three bradykinin peptide antagonists B-9430, B-9436, and B-9858 in aqueous solutions as well as in sodium dodecylsulphate micelles (B-9430 and B-9436) and CD₃OH/H₂O (60%/40%) mixtures for B-9858. All three peptides showed no observable secondary structure in aqueous solution. However, in their respective structure-inducing solvents, B-9430 (B₁ and B₂ receptor antagonist) and B-9436 (a B₂ receptor antagonist) exhibit a type II β-turn involving residues 2–5, and B-9430 also exhibits a type II′ β-turn involving residues 6–9 (in sodium dodecylsulfate micellar solutions), whereas B-9858, a B₁-specific receptor antagonist, exhibits only a type II β-turn involving residues 2–5 (in CD₃OH/H₂O solutions). Simulated annealing calculations on B-9858 confirm the experimental conclusions based on the nmr data. In addition, simulated annealing of the (2S, 3aS, 7aS)-octahydroindole-2-carboxylic acid (Oic residue), which is present in two of the three decapeptides studied, show that the one-chair conformation of the six-membered ring predominates, in agreement with the experimental data. The activities of these peptides are compared with their secondary structures and the specific receptor activity appears to depend on the presence of specific amino acid residues, such as N-(2-indanyl)glycine (Nig) and D[α-(2-indanyl)glycine] (D-Igl) as well as on elements of secondary structure. © 1997 John Wiley & Sons, Inc. Biopoly 42: 521–535, 1997     

Design,synthesis and conformational analysis of hGM-CSF[13-31]-Gly-Pro-Gly-[103-116]

On the basis of the X-ray structure and results from structure--activity relationship studies, the following GM–CSF analogue was designed and synthesized by solid-phase methodology: hGM–CSF[13-31]-Gly-Pro-Gly-[103–116]-NH₂. This analogue was constructed to comprise helices A and D of the native hGM–CSF, covalently linked in an antiparallel orientation by the tripeptide spacer Gly-Pro-Gly, which is known as a turn-inducing sequence. The conformational analysis of the analogue by CD spectroscopy revealed an essentially random structure in water, while α-helix formation was observed upon addition of TFE. In 40% TFE the helix content was ∼45%. By two-dimensional NMR experiments in 1:1 water/trifluoroethanol mixture two helical sequences were identified comprising the segments corresponding to helix A and helix D. In addition to medium-range NOESY connectivities, a long-range cross-peak was found involving the leucine residues at positions 13 and 35. Based on the experimentally derived data (54 NOEs), the structure was refined by restrained molecular dynamics simulations over 120 ps at various temperatures. A representative conformation derived from the computer simulation is mainly characterized by two helical segments connected by a loop region. The overall three-dimensional structure of the analogue is comparable to the X-ray structure of hGM–CSF in that helices A and D are oriented in an antiparallel fashion, forming a two α-helix bundle. Nevertheless, there are small differences in the topology of the helices between the solution structure of the designed analogue and the X-ray structure of hGM–CSF. The possible implications of these conformational features at the effects of biological activity are discussed. © 1997 European Peptide Society and John Wiley & Sons, Ltd. J. Pep. Sci.3: 323–335 No. of Figures: 10. No. of Tables: 5. No. of References: 46