Solution conformational analysis of amphiphilic helical,synthetic analogs of the lipopeptaibol trichogin GAIV

The step-by-step synthesis by solution methods of the [Ser^2,5,6,9, Leu-OMe¹¹] analog of trichogin GA IV is described. The four Ser residues have been incorporated into the sequence as replacements of the naturally occurring Gly residues to increase the amphiphilicity of the 3D-structure of the lipopeptaibol. A detailed solution conformational analysis has been performed on this undecapeptide and its prototypical [Leu-OMe¹¹] trichogin GA IV analog using FTIR absorption and CD spectroscopies, and two-dimensional NMR under a variety of experimental conditions, including a membrane-mimetic environment. Both peptides adopt a mixed 3₁₀/α-helical structure, which in the micellar system was found to be less flexible for the Ser-containing analog. For both analogs permeability measurements revealed membrane-modifying properties comparable to those of the natural lipopeptaibol.     

(αMe)Aun: a highly lipophilic,chiral, Cα‐tetrasubstituted α‐amino acid. Incorporation into model peptides and preferred conformation

Abstract: Using a chemo‐enzymatic approach we prepared the highly lipophilic, chiral, C^α‐methylated α‐amino acid (αMe)Aun. Two series of terminally protected model peptides containing either d ‐(αMe)Aun in combination with Aib or l ‐(αMe)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 α‐carbon atom and screw sense of the helix that is formed. The results obtained strongly support the view that d ‐(αMe)Aun favors the formation of the left‐handed 3₁₀‐helical conformation.     

Amphipathic control of the 310‐/α‐helix equilibrium in synthetic peptides

Abstract: A series of short, amphipathic peptides incorporating 80% C^α,C^α‐disubstituted glycines has been prepared to investigate amphipathicity as a helix‐stabilizing effect. The peptides were designed to adopt 3₁₀‐ or α‐helices based on amphipathic design of the primary sequence. Characterization by circular dichroism spectroscopy in various media (1 : 1 acetonitrile/water; 9 : 1 acetonitrile/water; 9 : 1 acetonitrile/TFE; 25 mm SDS micelles in water) indicates that the peptides selectively adopt their designed conformation in micellar environments. We speculate that steric effects from ith and ith + 3 residues interactions may destabilize the 3₁₀‐helix in peptides containing amino acids with large side‐chains, as with 1‐aminocyclohexane‐1‐carboxylic acid (Ac₆c). This problem may be overcome by alternating large and small amino acids in the ith and ith + 3 residues, which are staggered in the 3₁₀‐helix.     

Peptides from chiral Cα,α-disubstituted glycines Synthesis and characterization,conformational energy computations and solution conformational analysis of Cα-methyl,Cα-isopropylglycine [(αMe)Val] derivatives and model peptides*

Conformational energy computations on Ac-l -(αMe)Val-NHMe indicate that turns and right-handed helical structures are particularly stable conformations for this chiral Cα-methyl, Cα-alkylglycyl residue. We have synthesized and characterized a variety of l -(αMe)Val derivatives and peptides (to the pentamer level). The results of the solution conformational analysis, performed using infrared absorption, ¹H nuclear magnetic resonance, and circular dichroism, are in general agreement with those obtained from the theoretical investigation, in the sense that the l -(αMe)Val residue turns out to be a strong β-turn and right-handed helix former. A comparison is also made with the conclusions extracted from published work on peptides rich in other Cα-methyl, Cα-alkylglycyl residues.     

Improved solid‐phase synthesis of α,α‐dialkylated amino acid‐rich peptides with antimicrobial activity*

Abstract: A homologous series of nonapeptides and their acetylated versions were successfully prepared using solid‐phase synthetic techniques. Each nonapeptide was rich in α,α‐dialkylated amino acids [one 4‐aminopiperidine‐4‐carboxylic acid (Api) and six α‐aminoisobutyric acid (Aib) residues] and also included lysines or lysine analogs (two residues). The incorporation of the protected dipeptide 9‐fluorenylmethyloxycarbonyl (Fmoc)‐Aib‐Aib‐OH improved the purity and overall yields of these de novo designed peptides. The helix preference of each nonapeptide was investigated in six different solvent environments, and each peptide's antimicrobial activity and cytotoxicity were studied. The 3₁₀‐helical, amphipathic design of these peptides was born out most prominently in the N‐terminally acetylated peptides. Most of the peptides exhibited modest activity against Escherichia coli and no activity against Staphylococcus aureus. The nonacetylated peptides (concentrations ≤100 μm ) and the acetylated peptides (concentrations ≤200 μm ) did not exhibit any significant cytotoxicity with normal (nonactivated) murine macrophages.     

Circular dichroism studies of helical oligopeptides: Can 310 and α-helical conformations be chiroptically distinguished?

Circular dichroism studies of seven helical oligopeptides containing α-aminoisobutyric acid (Aib) in methanol and trifluoroethanol (TFE) solutions are reported. Peptides ranging from 10 to 21 residues in length have been examined. In all cases distinct negative CD bands characteristic of helical peptides are obtained at 220 and 205 nm corresponding to the n-π* π-π* transitions, respectively. The ratio R = [θ]n-π*/[θ]π-π* is < 1.0 for all peptides studied. Using crystal structure and n.m.r. results for a 10 residue 310 helical peptide and literature values for an α-helical 11-residue peptide, it is shown that both helical conformations yield R values of ? 0.8 in alcoholic solvents. The CD data are considered in the light of ¹H n.m.r. studies on these oligopeptides. The results suggest that 310 and α-helical conformations cannot be distinguished by CD methods.     

New tools for the control of peptide conformation: the helicogenic Cα‐methyl,Cα‐cyclohexylglycine*

Abstract: The novel C^α‐tetrasubstituted α‐amino acid C^α‐methyl, C^α‐cyclohexylglycine was prepared by hydrogenation of its C^α‐methyl, C^α‐phenylglycine precursor. Terminally protected homodi‐, homotri‐, and homotetrapeptides from C^α‐methyl, C^α‐cyclohexylglycine and co‐oligopeptides to the pentamer level in combination with Gly or α‐aminoisobutyric acid residues were prepared by solution methods and fully characterized. The results of a conformational analysis, performed by use of Fourier transform infrared (FT‐IR) spectrophotomet absorption, ¹H NMR, and X‐ray diffraction techniques, support the contention that this C^α‐methylated, C^β‐trisubstituted aliphatic α‐amino acid is an effective β‐turn and 3₁₀‐helix inducer in tri‐ and longer peptides as its C^α‐methyl valine parent compound, but partially divergent from the corresponding aromatic C^α‐methyl, C^α‐diphenylmethylglycine residue, known to promote folded and fully extended structures to a significant extent in these oligomers.     

Synthesis,crystal structure and molecular conformation of Nα‐Boc‐l‐leucyl‐(Z)‐β‐(3‐pyridyl)‐α,β‐ dehydroalanyl‐l‐leucine methyl ester*

Abstract: A protected tridehydropeptide containing (Z)‐β‐(3‐pyridyl)‐α,β‐dehydroalanine (Δ^Z3Pal) residue, Boc‐Leu‐Δ^Z3Pal‐Leu‐OMe ( 1 ), was synthesized via Erlenmeyer azlactone method. X‐ray crystallographic analysis revealed that the peptide 1 adopts an extended conformation, which is similar to that of a Δ^ZPhe analog, Boc‐Leu‐Δ^ZPhe‐Leu‐OMe ( 2 ).     

The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

Facile synthesis of Nα‐protected‐l‐α,γ‐diaminobutyric acids mediated by polymer‐supported hypervalent iodine reagent in water

Abstract: Hofmann rearrangement of N^α‐Boc‐l ‐Gln‐OH mediated by a polymer‐supported hypervalent iodine reagent poly[(4‐diacetoxyiodo)styrene] (PSDIB) in water afforded N^α‐Boc‐l ‐α,γ‐diaminobutyric acid (Boc‐Dab‐OH, 1 ) in 87% yield. N^α‐Z‐derivative (Z‐Dab‐OH, 2 ) was prepared with PSDIB in 83% yield. Since the reaction of N^α‐Fmoc‐Gln‐OH by this procedure did not proceed because of the insolubility of Fmoc‐Gln‐OH in aqueous media, we synthesized Fmoc‐Dab(Boc)‐OH ( 5 ) from 2 in 54% yield. Polymyxin B heptapeptide (PMBH) which contains four Dab residues was successfully synthesized in a solution‐phase synthesis.     

A topographically and conformationally constrained,spin‐labeled, α‐amino acid: crystallographic characterization in peptides*

Abstract: 2,2,6,6‐Tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) is a topographically and conformationally restricted, nitroxide containing, C^α‐tetrasubstituted α‐amino acid. Here, we describe the molecular and crystal structures, as determined by X‐ray diffraction analyses, of a TOAC terminally protected derivative, the cyclic dipeptide c(TOAC)₂·1,1,1,3,3,3‐hexafluoropropan‐2‐ol (HFIP) solvate, and five TOAC‐containing, terminally protected, linear peptides ranging in length from tetra‐ to hepta‐peptides. Incipient and fully developed, regular or distorted 3₁₀‐helical structures are formed by the linear peptides. A detailed discussion on the average geometry and preferred conformation for the TOAC piperidine ring is also reported. The X‐ray diffraction structure of an intramolecularly cyclized side product resulting from a C‐activated TOAC residue has also been determined.     

(αMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides

Abstract: Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, C^α‐methylated α‐amino acid l ‐(αMe)Nva with a short, linear side‐chain. A set of terminally protected model peptides to the pentamer level containing either (αMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (αMe)Nva peptides were also synthesized using side‐chain hydrogenation of the corresponding C^α‐methyl, C^α‐allylglycine (Mag) peptides. A detailed solution and crystal‐state conformational analysis based on FT‐IR absorption, ¹H NMR and X‐ray diffraction techniques allowed us to define that: (i) (αMe)Nva is an effective β‐turn and 3₁₀‐helix former; and (ii) the relationship between (αMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. l ‐(αMe)Nva induces the preferential formation of right‐handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on α‐amino acids with a C^α‐methyl substituent and a C^α‐linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.     

Synthesis of [3α‐3H] 17α‐Hydroxy pregnenolone and [3α‐3H] Pregnenolone

For the first time, [3α‐³H] 17α‐hydroxy pregnenolone (1) was synthesized through a multiple step sequence. The presence of [3β‐³H] isomer in RP‐HPLC purified product was identified by tritium NMR. The [3β‐³H] isomer was then separated from [3α‐³H] 17α‐hydroxy pregnenolone with chiralPAK AD‐H column. [3α‐³H] pregnenolone (2) was synthesized from commercial available 5‐pregnen‐3,20‐dione in one step with an improved procedure.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

Ac10c: a medium‐ring,cycloaliphatic Cα,α‐disubstituted glycine. Incorporation into model peptides and preferred conformation

Abstract: Two complete series of N‐protected oligopeptide esters to the pentamer level from 1‐amino‐cyclodecane‐1‐carboxylic acid (Ac₁₀c), an α‐amino acid conformationally constrained through a medium‐ring C^α_i ? C^α_i cyclization, and either the l ‐Ala or Aib residue, along with the N‐protected Ac₁₀c monomer and homo‐dimer alkylamides, were synthesized using solution methods and fully characterized. The preferred conformation of these model peptides was assessed in deuterochloroform solution using FT‐IR absorption and ¹H NMR techniques. Furthermore, the molecular structures of two derivatives (Z‐Ac₁₀c‐OH and Fmoc‐Ac₁₀c‐OH) and two peptides (the dipeptide ester Z‐Ac₁₀c‐l ‐Phe‐OMe and the tripeptide ester Z‐Aib‐Ac₁₀c‐Aib‐OtBu) were determined in the crystal state using X‐ray diffraction. The experimental results support the view that β‐bends and 3₁₀‐helices are preferentially adopted by peptides rich in Ac₁₀c, the third largest cycloaliphatic C^α,α‐disubstituted glycine known. This investigation allowed us to complete a detailed conformational analysis of the whole 1‐amino‐cycloalkane‐1‐carboxylic acid (Ac_nc, with n = 3–12) series, which represents the prerequisite for our recent proposal of the ‘Ac_nc scan’ concept.     

Expedient synthesis of [18F]‐labeled α‐trifluoromethyl ketones

Several [¹⁸F]‐labeled α‐trifluoromethyl ketones have been synthesized. Reactions of 2,2‐difluoro‐1‐aryl‐1‐trimethylsiloxyethenes ( 1a–d ) with [¹⁸F]‐F₂ at low temperature produced [¹⁸F]‐labeled α‐trifluoromethyl ketones ( 2a–d ). Radio‐labeled products were isolated by purification with column chromatography in 22–28% yields, decay corrected (d.c.) in three runs per compound. Radiochemical purity was >99% with specific activities 15–20 GBq/mmol at the end of synthesis (EOS). The synthesis time was 35–40 min from the end of bombardment (EOB). This one‐step simple method is highly useful for the radiochemical synthesis of potential biologically active [¹⁸F]‐labeled α‐trifluoromethyl ketones for PET imaging. Copyright © 2003 John Wiley & Sons, Ltd.     

Peroxisome proliferator‐activated receptor‐γ coactivator‐1α in muscle links metabolism to inflammation

1. In higher eukaryotes, metabolism and immunity are tightly coupled. However, whereas in evolutionary terms a compromised immune response due to undernourishment has been the predominant problem, the inflammatory response to obesity and other lifestyle‐associated diseases has increased in relevance in Western societies in the past 100 years. 2. Traditionally, fat tissue has been considered as the major source of pro‐inflammatory secreted factors in these pathologies. However, in recent years the contribution of other tissues to disease‐causing chronic inflammation has been increasingly appreciated. 3. Peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) is one of the key regulatory factors in active skeletal muscle. Aberrant expression of PGC‐1α in inactive muscle fibres could be linked to a sedentary lifestyle, persistent systemic inflammation and a higher risk for many chronic diseases. Accordingly, modulation of PGC‐1α activity in skeletal muscle may have a broad range of therapeutic effects. Here, recent advances in the understanding of the role of muscle PGC‐1α in health and disease are reviewed.     

Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic α‐helices

Abstract: The effects of 2 molal Na₂SO₄ at neutral pH on hydrophobic and electrostatic interactions between amphipathic α‐helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl‐ L EE L KKK L EE L KKK L EE L ‐NH₂) and LEE (acetyl‐ L EE L EEE L EE L EEE L EE L ‐NH₂). In phosphate buffer at neutral pH, only LEK adopted a predominantly α‐helical conformation, attributable to glu^––lys⁺ interactions where a major contribution is evidently a hydrogen bond (Biochemistry 32 : 9668–9676). Despite the presence of lys⁺ in the e and g′ positions of the abcdefg heptad repeat, LEK exhibited mean‐residue ellipticities at 222 nm ([θ]₂₂₂) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na₂SO₄ at 25–75°C, the helical content of LEK increased, with the greatest increase observed at 75°C. The value of the ellipticity ratio R ([θ]₂₂₂/[θ]₂₀₈) of LEK in 2 molal Na₂SO₄ also increased, indicating a stronger interhelical association. At 50°C and 75°C, LEK remained predominantly α‐helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na₂SO₄, however, the peptide formed α‐helices that associated to form a coiled coil. At 50°C and 75°C, LEE became predominantly random coil but the remaining α‐helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix‐stabilizing and helix‐destabilizing electrostatic interactions in amphipathic α‐helices by Na₂SO₄.     

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.     

Structural versatility of peptides from Cα,α-disubstituted glycines: crystal-state conformational analysis of peptides from Cα-methylhomophenylalanine, (αMe)Hph

The molecular and crystal structures of the C^α-tetrasubstituted, δ-branched α-amino acid C^α-methyl-homophenylalanine, H-d -(αMe)Hph-OH, and three peptides (to the pentamer level), including the homotripeptide, have been determined by X-ray diffraction. The peptides are Z-l -(αMe)Hph-(l -Ala)₂-OMe, pBrBz-[d -(αMe)Hph]₃-OtBu and Ac-(Aib)₂-l -(αMe)Hph-(Aib)₂-OtBu. All the (αMe)Hph residues prefer φ,ψ torsion angles in the helical region of the conformational map. The two terminally blocked tripeptides adopt a β-bend conformation stabilized by a 1→4 C = O?H-N intramolecular H-bond. The terminally blocked pentapeptide is folded in a regular 3₁₀-helix. In general, the relationship between (αMe)Hph α-carbon chirality and helix handedness is the same as that exhibited by protein amino acids. A comparison is also made with the conclusions extracted from published work on peptides from other types of C^α-alkylated aromatic α-amino acids. © Munksgaard 1996.