Determining the occurrence of a 3(10)-helix and an alpha-helix in two different segments of a lipopeptaibol antibiotic using TOAC, a nitroxide spin-labeled C(alpha)-tetrasubstituted alpha-aminoacid

Trichogin GA IV is a 11-residue lipopeptaibol antibiotic exhibiting membrane modifying properties. We synthesized step-by-step by solution methods three trichogin analogues, each with a double Aib (alpha-aminoisobutyric acid)-->TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) replacement. The strict similarity in the conformational propensities of Aib and TOAC allowed us to exploit these analogues in a detailed investigation of the conformation of this lipopeptaibol in different organic solvents and in a membrane-mimetic environment using in particular the double spin labeling ESR technique. We conclude that the secondary structure in solution remains essentially unchanged if compared to that previously found in the crystal state for trichogin. More specifically, the N-terminal region of the peptide folds in a 3(10)-helix, while the central and C-terminal regions are mainly alpha-helical. An additional, significant proof for the modest plasticity of the trichogin structure was obtained by an X-ray diffraction analysis of the nOct-[TOAC4,8, Leu-OMe11] analogue. For the three analogues permeability measurements revealed membrane-modifying properties comparable to those of natural trichogin.     

Conformation and membrane activity of an analogue of the peptaibol antibiotic trichogin GA IV with a lipophilic amino acid at the N-terminus

We have synthesized by solution-phase methods two analogues of the 11-residue lipopeptaibol antibiotic trichogin GA IV in which the N-terminal n-octanoyl group is replaced either by an N-acetylated 2-amino-2-methyl-l -undecanoic acid or by an N-acetylated α-aminoisobutyric acid. CD, FTIR absorption, and NMR analyses unequivocally show that the main structural features of trichogin GA IV are preserved in these analogues. Since only the peptide containing the lipophilic chain exhibits membrane-modifying properties, these results strongly support the view that moving the long acyl moiety from the N^α-blocking group to the side chain of the N-terminal extra-residue does not affect the conformational properties or the membrane activity of trichogin GA IV. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib residues on conformation and bioactivity

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by L-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to L-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.     

Synthesis, conformational analysis, and spectroscopic characterization of peptides based on Daf, the first rigid transition-metal receptor, cyclic C(alpha,alpha)-disubstituted glycine

Three series of terminally protected model oligopeptides to the nonamer level, based on 9-amino-4,5-diazafluorene-9-carboxylic acid, the first rigid bipyridine-type C(alpha,alpha)-disubstituted glycine, and either Gly, L-Ala, or Aib residues were synthesized by solution methods and fully characterized. The molecular structures of two derivatives and one tripeptide were determined in the crystal state by x-ray diffraction. Moreover, the solution preferred conformations of these peptides were assessed by Fourier transform infrared absorption and (1)H-NMR techniques. A comparison with the known structural tendencies of the strictly related C(alpha,alpha)-disubstituted glycyl residues 1-aminocyclopentane-1-carboxylic acid and 9-aminofluorene-9-carboxylic acid is made, and the implications for the use of the 9-amino-4,5-diazafluorene-9-carboxylic acid residue in conformationally constrained analogs of bioactive peptides are briefly examined. A spectroscopic (uv absorption, fluorescence, CD) characterization of this novel heteroaromatic C(alpha,alpha)-disubstituted glycine is also reported. Finally, preliminary conformational data and membrane activity measurements are discussed for an analog of the lipopeptaibol antibiotic [L-Leu(11)-OMe] trichogin GA IV in which a 9-amino-4,5-diazafluorene-9-carboxylic acid residue was synthetically incorporated in position 1 (replacing the original Aib residue).     

Orientation and immersion depth of a helical lipopeptaibol in membranes using TOAC as an ESR probe

Trichogin GA IV is a lipopeptaibol antibiotic characterized by the sequence nOct–Aib¹–Gly–Leu–Aib⁴–Gly–Gly–Leu–Aib⁸–Gly–Ile–Lol (nOct: n‐octanoyl; Aib: α‐aminoisobutyric acid; Lol, leucinol), which exhibits membrane‐modifying properties. We synthesized step‐by‐step by solution methods three trichogin analogues, each with a single Aib → 2,2,6,6‐tetramethylpiperidin‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) substitution. The similarity in the conformational propensities of the C^α‐tetrasubstituted α‐amino acids Aib and TOAC allowed us to exploit these analogues to investigate the orientation and therefore the mechanism of action of trichogin in the membranes by the electron spin resonance (ESR) technique. A conformational analysis by Fourier transform ir absorption and CD in different organic solvents and in a membrane‐mimetic environment indicated that the conformation of the natural lipopeptaibol remains almost unchanged in the three analogues. Moreover, for all of the analogues permeability measurements revealed membrane‐modifying properties comparable to those of trichogin. Our ESR investigation demonstrated that, in liposomes based on phosphatidylcholine, trichogin lays parallel to the membrane surface with its hydrophobic face oriented toward the membrane interior. These results suggest that trichogin might modify membrane permeability via a carpet‐like mechanism, at least in liposomes and in the absence of a transmembrane potential. © 1999 John Wiley & Sons, Inc. Biopoly 50: 239–253, 1999     

Ion transport across a phospholipid membrane mediated by the peptide trichogin GA IV

Trichogin GA IV is a special member of a class of peptaibols that are linear peptide antibiotics of fungal origin, characterised by the presence of a variable number of alpha-aminoisobutyric acid residues, an acyl group at the N-terminus and a 1,2-amino alcohol at the C-terminus. Most of the peptaibols display ion-channel-forming or at least membrane-modifying properties. The 11-residue-long trichogin GA IV is not only one of shortest peptaibols, but it is also unique for its n-octanoyl group instead of the more common found acetyl group at the N-terminus. For the first time we have found that this lipopeptaibol is able to enhance conduction of monovalent cations through membranes of large unilamellar vesicles (LUVs). The influence of the [Leu-OMe]trichogin GA IV analogue (TRI) on ion permeation was studied under a variety of conditions (lipid composition, lipid-to-peptide ratio and a transmembrane potential). Parallel experiments were performed with the 16-residue long, channel-forming peptaibol, zervamicin (ZER). For the two peptides, the permeability between K(+) and Na(+) was found to be different. In addition, the ion diffusion rate dependencies on the peptide concentration are observed to be different. This might indicate that a different number of aggregated molecules are involved in the rate-limiting step, i.e. 3-4 (TRI) and 4-7 (ZER). In the presence of TRI, dissipation of the transmembrane potential, Delta psi, was observed with a rate to be dependent on the magnitude of both initial Delta psi and peptide concentration. Both peptides were activated by a cis-positive but not by cis-negative Delta psi. Under identical conditions the ion-conducting efficiency of zervamicin was 100-200 times higher than that of trichogin. Our results show that, unlike for zervamicin, the membrane-modifying activity of trichogin is not associated with a channel mechanism.     

Synthesis, conformation, and bioactivity of novel analogues of the antiviral lipopeptide halovir A.

We synthesized by solution-phase methods three analogues, [L-Leu(6)-OMe], [L-(alphaMe)Leu(3), L-Leu(6)-OMe], and [L-(alphaMe)Val(4), L-Leu(6)-OMe] of halovir A. The [L-Leu(6)-OMe] analogue is known to be biologically equipotent to its naturally occurring, antiviral, lipopentapeptide amide parent compound. The preferred conformations of the L-(alphaMe)Leu- and L-(alphaMe)Val-containing analogues, with a potentially reinforced helicity, were compared with those of [L-Leu(6)-OMe] halovir A and the natural peptide itself by use of a combination of FT-IR absorption and NMR techniques. Measurements of the antiviral activities against herpes simplex virus type-1 (HSV-1) of halovir A and its three analogues were also carried out. Interestingly, the [L-(alphaMe)Val(4), L-Leu(6)-OMe] analogue exhibits the most significant activity in reducing HSV-1 infectivity, notably higher than that of halovir A itself.     

Trichogin: a paradigm for lipopeptaibols.

Lipopeptaibols are members of a novel family of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of turn/helix inducing alpha-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. Using solution methods, the prototypical lipopeptaibol trichogin GA IV and a large series of appropriately designed analogues were synthesized, which allow: (i) determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity, and (ii) exploitation of a number of physico-chemical techniques aimed at assessing the trichogin preferred conformation under a variety of conditions and at investigating its mechanism of interaction with the phospholipid membranes.     

Probing membrane permeabilization by the antibiotic lipopeptaibol trichogin GA IV in a tethered bilayer lipid membrane

The lipopeptaibol trichogin GA IV (TCG) can be incorporated in the lipid bilayer moiety of a mercury-supported tethered bilayer lipid membrane (tBLM) at a non-physiological transmembrane potential of about -240mV, negative on the trans side of the bilayer. Once incorporated in the tBLM, TCG is stable over the range of physiological transmembrane potentials and permeabilizes the membrane at transmembrane potentials negative of -80÷-90mV. The chronocoulometric behavior is consistent with a kinetics of nucleation and growth of bundles of TCG building blocks with ion-channel properties. The TCG building blocks also permeabilize the lipid bilayer, albeit at more negative transmembrane potentials, and can be tentatively regarded as dimers of aligned TCG helical monomers. The cyclic voltammograms of tBLMs incorporating TCG point to a voltage-gated behavior of the TCG channel, similar to that exhibited by the peptaibol alamethicin.     

Location and aggregation of the spin-labeled peptide trichogin GA IV in a phospholipid membrane as revealed by pulsed EPR

The lipopeptaibol trichogin GA IV is a 10 amino acid-long residue and alpha-aminoisobutyric acid-rich antibiotic peptide of fungal origin. TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled analogs of this membrane active peptide were investigated in hydrated bilayers of dipalmitoylphosphatidylcholine by electron spin echo envelope modulation (ESEEM) spectroscopy and pulsed electron-electron double resonance (PELDOR). Since, the ESEEM of the spin label appears to be strongly dependent on the presence of water molecules penetrated into the membrane, this phenomenon was used to study the location of this peptide in the membrane. This was achieved by comparing the ESEEM spectra for peptides labeled at different positions along the amino acid sequence with spectra known for lipids with spin labels at different positions along the hydrocarbon chain. To increase the ESEEM amplitude and to distinguish the hydrogen nuclei of water from lipid protons, membranes were hydrated with deuterated water. The PELDOR spectroscopy technique was chosen to study peptide aggregation and to determine the mutual distance distribution of the spin-labeled peptides in the membrane. The location of the peptide in the membrane and its aggregation state were found to be dependent on the peptide concentration. At a low peptide/lipid molar ratio (less than 1:100) the nonaggregated peptide chain of the trichogin molecules lie parallel to the membrane surface, with TOAC at the 4th residue located near the 9th-11th carbon positions of the sn-2 lipid chain. Increasing this ratio up to 1:20 leads to a change in peptide orientation, with the N-terminus of the peptide buried deeper into membrane. Under these conditions peptide aggregates are formed with a mean aggregate number of about N = 2. The aggregates are further characterized by a broad range of intermolecular distances (1.5-4 nm) between the labels at the N-terminal residues. The major population exhibits a distance of approximately 2.5 nm, which is of the same order as the length of the helical peptide. We suggest that the constituting monomers of the dimer are antiparallel oriented.     

Aggregation and water-membrane partition as major determinants of the activity of the antibiotic peptide trichogin GA IV

Water-membrane partition and aggregation behavior are fundamental aspects of the biological activity of antibiotic peptides, natural compounds causing the death of pathogenic organisms by perturbing the permeability of their membranes. A synthetic fluorescent analog of the natural lipopeptaibol trichogin GA IV was used to study its interaction with model membranes. Time-resolved fluorescence data show that in water, an equilibrium between monomers and small aggregates is present, the two species having different affinity for membranes. Therefore, association curves are strongly dependent on peptide concentration. A similar heterogeneity is present in the membrane phase, which strongly suggests the occurrence of a monomer-aggregate equilibrium in this case, too. The relative population of each species was determined and a strong correlation between the concentration of membrane-bound aggregates and membrane leakage was found, thereby suggesting that liposome perturbation is due to peptide aggregates only. Light-scattering measurements demonstrate that leakage is not due to liposome micellization. Moreover, experiments with markers of different sizes show that molecules with a diameter of approximately 4 nm are released only to a minor extent. Overall, these results suggest that, within the concentration range explored, pore formation by peptide aggregates is the most likely mechanism of action for trichogin in membranes.     

The antimicrobial peptide trichogin and its interaction with phospholipid membranes.

The interaction of the antimicrobial peptide trichogin GA IV with phospholipid bilayers has been studied. A series of analogs of trichogin was synthesized in which the nitroxide spin label, 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC), replaced one of the three alpha-aminoisobutyric acid (Aib) residues in the sequence. These modified peptides were used to assess the location of different residues of the peptide in a phospholipid bilayer composed of egg phosphatidylcholine containing 0.4 mol% of a fluorescently labelled phospholipid. We demonstrate that the substitution of Aib residues with TOAC does not alter the manner in which the peptide affects membrane curvature or induces vesicle leakage. The proximity of the nitroxide group on the peptide to the 4,4-difluoro-4-bora-3a,4a-diaza-S-indacene (BODIPY) fluorophore attached to the phospholipid was estimated from the extent of quenching of the fluorescence. By this criterion it was concluded that the peptide penetrates into the bilayer and that Aib4 is the most deeply inserted of the Aib residues. The results suggest that the helix axis of the peptide is oriented along the plane of the membrane. All of the peptides were shown to raise the bilayer to the hexagonal phase transition temperature of dipalmitoleoylphosphatidylethanolamine, indicating that they promote positive membrane curvature. This is a property observed with peptides that do not penetrate deeply into the bilayer or are oriented along the bilayer normal. We also demonstrate trichogin-promoted leakage of the aqueous contents of liposomes. These results indicate that the peptides cause bilayer destabilization. The extent of leakage induced by trichogin is very sensitive to the peptide to lipid ratio over a narrow range.     

Peptide-membrane binding is not enough to explain bioactivity: A case study

Membrane-active peptides are a promising class of antimicrobial and anticancer therapeutics. For this reason, their molecular mechanisms of action are currently actively investigated. By exploiting Electron Paramagnetic Resonance, we study the membrane interaction of two spin-labeled analogs of the antimicrobial and cytotoxic peptide trichogin GA IV (Tri), with opposite bioactivity: Tri(Api⁸), able to selectively kill cancer cells, and Tri(Leu⁴), which is completely nontoxic. In our attempt to determine the molecular basis of their different biological activity, we investigate peptide impact on the lateral organization of lipid membranes, peptide localization and oligomerization, in the zwitter-ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane We show that, despite their divergent bioactivity, both peptide analogs (i) are membrane-bound, (ii) display a weak tendency to oligomerization, and (iii) do not induce significant lipid rearrangement. Conversely, literature data show that the parent peptide trichogin, which is cytotoxic without any selectivity, is strongly prone to dimerization and affects the reorganization of POPC membranes. Its dimers are involved in the rotation around the peptide helix, as observed at cryogenic temperatures in the millisecond timescale. Since this latter behavior is not observed for the inactive Tri(Leu⁴), we propose that for short-length peptides as trichogin oligomerization and molecular motions are crucial for bioactivity, and membrane binding alone is not enough to predict or explain it. We envisage that small changes in the peptide sequence that affect only their ability to oligomerize, or their molecular motions inside the membrane, can tune the peptide activity on membranes of different compositions.     

(alphaMe)Aun: a highly lipophilic, chiral, Calpha-tetrasubstituted alpha-amino acid. Incorporation into model peptides and preferred conformation.

Using a chemo-enzymatic approach we prepared the highly lipophilic, chiral, Calpha-methylated alpha-amino acid (alphaMe)Aun. Two series of terminally protected model peptides containing either D-(alphaMe)Aun in combination with Aib or L-(alphaMe)Aun in combination with Gly were synthesized using solution methods and fully characterized. A detailed solution conformational analysis, based on FT-IR absorption, 1H NMR and CD techniques, allowed us to determine the preferred conformation of this amino acid and the relationship between chirality at its alpha-carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that D-(alphaMe)Aun favors the formation of the left-handed 3(10)-helical conformation.     

Self-assembling and membrane modifying properties of a lipopeptaibol studied by CW-ESR and PELDOR spectroscopies.

Trichogin GA IV is a short lipopeptaibol antibiotic that is capable of enhancing the transport of small cations through the phospholipid double layer of the membrane. The antibiotic activity of the undecapeptide is thought to be based on either its self-assembling or membrane-modifying property. The chemical equilibrium between self-aggregated and non-aggregated molecular states was studied by CW-ESR spectroscopy using solutions of TOAC nitroxide spin-labelled trichogin analogues in an apolar solvent to mimic the membrane bound state. At room temperature the two different sets of signals observed in the spectrum were attributed to the presence of both monomers and aggregates in the sample. The ESR spectra of the monomeric and aggregated forms were separated and the dependence of the fraction of monomeric peptide molecules on concentration was obtained over the range 5 x 10(-6) to 7 x 10(-4) M. A two-step aggregation mechanism is proposed: dimerization of peptide molecules followed by aggregation of dimers to assemblies of four peptide molecules per aggregate. The equilibrium constants were estimated for both steps. In addition, the lower lifetime limit was determined for dimers and tetramers. It is shown that when the peptide concentration exceeds 10(-5) M. the major part of the peptide molecules in solution has the form of tetrameric aggregates. Independently, the PELDOR technique was used to investigate the concentration dependence of the parameters of the dipole-dipole interaction between spin labels in frozen (77 K] glassy solutions of aggregates of mono-labelled TOAC analogues. The number of molecules in aggregates as well as the frequency and amplitude of PELDOR signal oscillations were found to be concentration independent in the range 5 x 10(-4) to 8 x 10(-3) M. In the frozen glassy solution state, the number of peptide molecules per aggregate was determined to be close to four, which is in agreement with the value obtained for spin-labelled trichogin at room temperature. The present data provide experimental evidence in favour of a self-assembling rather than a membrane-modifying ion conduction mechanism.     

Receptor selectivity of enkephalin analogs carrying artificial address peptides

The synthetic [Leu]enkephalin analogs YGGFLGP(KS'S'S')2-OMe (S' represents Sar residue, ENK-S'8), YGGFLGP(KPPP)2-OMe (ENK-P8), and YGGFLGP(KA'LA')2-OMe (A' represents 2-aminoisobutyric acid (Aib), ENK-A'8) were designed to investigate the role of membrane affinity in opioid receptor binding. CD measurement in ethanol revealed that ENK-S'8 took a random coil conformation, while ENK-P8 and ENK-A'8 partly adopted a 3(1)- and alpha-helical conformation, respectively. ENK-A'8 was shown to be distributed to lipid bilayer membrane due to the amphiphilic alpha-helical structure, while ENK-S'8 and ENK-P8 stayed in aqueous phase. Opioid receptor affinities ENK-S'8 and ENK-P8 were similar to [Leu]enkephalinamide, indicating that the connection of hydrophilic peptide segments to the C-terminal of [Leu]enkephalinamide did not affect the receptor affinity. On the other hand, delta- and mu-receptor affinities of ENK-A'8 were about 1/20 and 1/6 times, respectively, those of [Leu]enkephalinamide. Therefore, ENK-A'8 has higher selectivity for mu-receptor than [Leu]enkephalinamide. These results are explained in terms of the membrane compartment concept proposed by Schwyzer.     

Mechanism of membrane activity of the antibiotic trichogin GA IV: a two-state transition controlled by peptide concentration

Synthetic fluorescent analogs of the natural lipopeptide trichogin GA IV were used to investigate the peptide position and orientation in model membranes. A translocation assay based on Forster energy transfer indicates that trichogin is associated to both the outer and inner leaflet of the membrane, even at low concentration, when it is not active. Fluorescence quenching measurements, performed by using water soluble quenchers and quenchers positioned in the membrane at different depths, indicate that at low membrane-bound peptide/lipid ratios trichogin lies close to the region of polar headgroups. By increasing peptide concentration until membrane leakage takes place, a cooperative transition occurs and a significant fraction of the peptide becomes deeply buried into the bilayer. Remarkably, this change in peptide position is strictly coupled with peptide aggregation. Therefore, the mechanism of trichogin action can be envisaged as based on a two-state transition controlled by peptide concentration. One state is the monomeric, surface bound and inactive peptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity.     

Analogs of the antimicrobial peptide trichogin having opposite membrane properties.

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n-octanoyl-Aib-Gly-Leu-Aib-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe; ST, n-octanoyl-Aib-Ser-Leu-Aib-Ser-Ser-Leu-Aib-Ser-Ile-Leu-OMe; BT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe; and DT, n-octanoyl-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-Aib-Ser(tBu)-Leu-Aib-Ser(tBu)-Ser(tBu)-Leu-Aib-Ser(tBu)-Ile-Leu-OMe. The trichogin GA IV differs from GT only in the nature of the C-terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head-to-tail dimer of BT. We show that BT and DT lower the bilayer-to-hexagonal phase transition temperature (T(H)) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide-promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi-elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.     

4‐Cyano‐α‐methyl‐l‐phenylalanine as a Spectroscopic Marker for the Investigation of PeptaibioticMembrane Interactions

Two analogs of the ten‐amino acid residue, membrane‐active lipopeptaibiotic trichogin GA IV, mono‐labeled with 4‐cyano‐α‐methyl‐L ‐phenylalanine, a potentially useful fluorescence and IR absorption probe of the local microenvironment, were synthesized by the solid‐phase methodology and conformationally characterized. The single modification was incorporated either at the N‐terminus (position 1) or near the C‐terminus (position 8) of the peptide main chain. In both cases, the replaced amino acid was the equally helicogenic α‐aminoisobutyric acid (Aib) residue. We performed a solution conformational analysis by use of FT‐IR absorption, CD, and 2D‐NMR spectroscopies. The results indicate that both labeled analogs essentially maintain the overall helical propensity of the naturally occurring lipopeptaibiotic. Peptide? membrane interactions were assessed by fluorescence and ATR‐IR absorption techniques. Analogies and differences between the two peptides were highlighted. Taken together, our data confirm literature results that some of the spectroscopic parameters of the 4‐cyanobenzyl chromophore are sensitive markers of the local microenvironment.     

3H-[1,2,4]-Triazolo[5,1-i]purin-5-amine derivatives as adenosine A2A antagonists

A novel series of 3-substituted-8-aryl-[1,2,4]-triazolo[5,1-i]purin-5-amine analogs related to Sch 58261 was synthesized in order to identify potent adenosine A(2A) receptor antagonists with improved selectivity over the A(1) receptor, physiochemical properties, and pharmacokinetic profiles as compared to those of Sch 58261. As a result of structural modifications, numerous analogs with excellent in vitro binding affinities and selectivities were identified. Moreover, compound 27 displayed both superior in vitro and highly promising in vivo profiles.