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.     

(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.     

Synthesis of enantiopure non‐natural α‐amino acids using tert‐butyl (2S)‐2‐[bis‐(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate as key‐intermediate:the first synthesis of(S)‐2‐amino‐oleic acid

Abstract: A general method for the synthesis of enantiopure non‐natural α‐amino acids is described. The key intermediate tert‐butyl (2S)‐2‐[bis(tert‐butoxycarbonyl)amino]‐5‐oxopentanoate was obtained from l ‐glutamic acid after suitable protection and selective reduction of the γ‐methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of δ,ε‐unsaturated α‐amino acids. This methodology was applied to the synthesis of (S)‐2‐amino‐oleic acid.     

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.     

(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.     

Studies on the conformational properties of CP‐1042−55, the hinge region of CP‐10, using circular dichroism and RP‐HPLC

Abstract: The conformational properties of CP‐10^42?55, a peptide corresponding to the hinge region of CP‐10, were investigated using circular dichroism spectroscopy and reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The circular dichroism studies indicated that CP‐10^42?55 formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mm sodium dodecyl sulfate, which comprised a mixture of α‐helix and β‐sheet. The effect of temperature on the conformation of CP‐10^42?55 was investigated between 5 and 40°C, with very small changes in the spectra being observed.RP‐HPLC was then used to investigate the effect of temperature on the conformation of CP‐10^42?55 in the presence of a hydrophobic surface. Using a C₁₈‐adsorbent, CP‐10^42?55 exhibited a conformational transition at 25°C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near‐planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25°C in theRP‐HPLC system most likely corresponds to the unfolding of an α‐helical and/or β‐sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment.     

Entrapping unusual folding characteristics of the β‐Ala residues in a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH3

Abstract: Crystal structure analysis of a model peptide: Boc‐β‐Ala‐Aib‐β‐Ala‐NHCH₃ (β‐Ala: 3‐amino propionic acid; Aib: α‐aminoisobutyric acid) revealed distinct conformational preferences for folded [φ≈136°, µ ≈ ?62°, ψ ≈100°] and semifolded [φ ≈ 83°, µ ≈ ?177°, ψ ≈ ?117°] structures of the N‐ and C‐terminus β‐Ala residues, respectively. The overall folded conformation is stabilized by unusual N_i···H‐N_i+1 and nonconventional C–H···O intramolecular hydrogen bonding interactions.     

Effects of single d‐amino acid substitutions on disruption of β‐sheet structure and hydrophobicity in cyclic 14‐residue antimicrobial peptide analogs related to gramicidin S

Abstract: Gramicidin S (GS) is a 10‐residue cyclic β‐sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure–activity studies of de novo‐designed 14‐residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single l ‐amino acid with its corresponding d ‐enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274 , 13181]. The diastereomer with a d ‐Lys substituted at position 4 caused the greatest improvement in specificity vs. other l to d substitutions within the cyclic 14‐residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted β‐sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275 , 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a d ‐ or l ‐amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the d ‐ and l ‐substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic d ‐amino acids d ‐Lys and d ‐Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the l ‐diastereomers, which demonstrated strong hemolysis. All of the l ‐substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the d ‐substituted peptides tended to improve based on the hydrophilicity of the residue. d ‐Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic d ‐amino acid substitutions had superior antimicrobial activity vs. the l ‐enantiomers although substitution of a hydrophobic d ‐amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted β‐sheet structure on antimicrobial activity.     

Synthesis of [14C]‐l‐tryptophan and [14C]‐5′‐hydroxy‐l‐tryptophan labeled in the carboxyl group

The synthesis of selectively ¹⁴C‐labeled l ‐tryptophan and its derivative 5‐hydroxy‐l ‐tryptophan using chemical and multienzymatic methods is reported. The mixture containing [1‐¹⁴C[‐dl ‐alanine, indole or 5‐hydroxyindole has been converted to [1‐¹⁴C]‐l ‐tryptophan or 5′‐hydroxy‐[1‐¹⁴C]‐l ‐tryptophan, respectively, in a one‐pot multienzymatic reaction using four enzymes: d ‐amino acid oxidase, catalase, glutamic‐pyruvic transaminase and tryptophanase. Copyright © 2003 John Wiley & Sons, Ltd.     

A combined extended and helical backbone for Boc‐(Ala‐Leu‐Ac7c‐)2‐OMe*

Abstract: The structure of the peptide Boc‐Ala‐Leu‐Ac₇c‐Ala‐Leu‐Ac₇c‐OMe (Ac₇c,1‐aminocycloheptane‐1‐carboxylic acid) is described in crystals. The presence of two Ac₇c residues was expected to stabilize a 3₁₀‐helical fold. Contrary to expectation the structural analysis revealed an unfolded amino terminus, with Ala(1) adopting an extended β‐conformation (φ = ?93°,ψ = 112°). Residues 2–5 form a 3₁₀‐helix, stabilized by three successive intramolecular hydrogen bonds. Notably, two NH groups Ala(1) and Ac₇c(3) do not form any hydrogen bonds in the crystal. Peptide assembly appears to be dominated by packing of the cycloheptane rings that stack against one another within the molecule and also throughout the crystal in columns.     

Conformational investigation of α,β‐dehydropeptides. X. Molecular and crystal structure of Ac‐ΔAla‐NMe2 compared with those of Ac‐l‐Ala‐NMe2, Ac‐dl‐Ala‐NMe2 and other dimethylamidesa

Abstract: A series of three homologous dimethyldiamides Ac‐ΔAla‐NMe₂, Ac‐l ‐Ala‐NMe₂ and Ac‐dl ‐Ala‐NMe₂ has been synthesized and the structures of these amides determined from single‐crystal X‐ray diffraction data. To learn more about the conformational preferences of compounds studied, the fully relaxed (φ–ψ) conformational energy maps in vacuo (AM1) of Ac‐ΔAla‐NMe₂ and Ac‐l ‐Ala‐NMe₂ were obtained, and the calculated minima reoptimized with the DFT/B3LYP/6–31G** method. The crystal‐state results have been compared with the literature data. Ac‐ΔAla‐NMe₂ and other α,β‐dehydroamino acid dimethyldiamides, Ac‐ΔXaa‐NMe₂ adopt the conservative conformation of the torsion angles φ, ψ = ～ ?45°, ～130°, which are located in the high‐energy region (region H) of Ramachandran diagram. Ac‐l ‐Ala‐NMe₂ and Ac‐dl ‐Ala‐NMe₂, as well as other saturated amino acid dimethylamides Ac‐l /dl ‐Xaa‐NMe₂, present common peptide structures, and no conformational preferences are observed. Molecular packing of the amides analysed reveals two general hydrogen‐bonded motifs. Dehydro and dl ‐species are paired into centrosymmetric dimers, and l ‐compounds form catemers. However, Ac‐ΔAla‐NMe₂ and Ac‐DL ‐Ala‐NMe₂ constitute exceptions: their molecules also link into catemers.     

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.     

NMR and CD conformational studies of the C‐terminal 16‐peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes

Abstract: The 16‐amino acid sequences of the C‐terminal helices of the homologous bacterial cytochromes c₅₅₁ from Pseudomonas aeruginosa and c₅₅₂ from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of α‐helices and 3₁₀‐helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c₅₅₁ and 20% for c₅₅₂, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2‐trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2‐trifluoroethanol. NMR spectra in phosphate buffer show the presence of both α‐ and 3₁₀‐helical structures. In water/2,2,2‐trifluoroethanol (50 : 50) α‐helices are predominant. CD temperature‐dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2‐trifluoroethanol (50 : 50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c₅₅₂ C‐terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.     

Synthesis of 5,5,6,6‐D4‐L‐lysine‐aflatoxin Bl for use as a mass spectrometric internal standard

Human exposure to the hepatocarcinogenic mycotoxin aflatoxin B_l results in modification of serum albumin lysine ε‐amino residues to form lysine‐aflatoxin adducts. A perdeuterated reference standard is now required to quantitatively measure this adduct in epidemiologic studies of liver cancer using isotopic dilution mass spectrometry. A convenient method for the preparation of D4‐L ‐lysine‐AFB_l using commercially available 5,5,6,6‐D4‐l ‐lysine is demonstrated for the first time. The application of two standard α‐amino protection methods is also reported that simplifies the production of natural isotopic abundance lysine‐AFB_l over the currently used method employing N_α‐acetyl‐l ‐lysine. t‐Boc‐N_α‐lysine was used to prepare lysine‐AFB_l; however, a preferred method for directing reaction of AFB_l‐dialdehyde to the ε‐amino group of 5,5,6,6‐D4‐l ‐lysine utilized cupric ions that were spontaneously removed during the reverse phase HPLC purification of D4‐lysine‐AFB_l using 1% HOAc. This strategy eliminates the need to otherwise synthesize and purify t‐Boc‐N_α‐ or N_α‐acetyl‐5,5,6,6‐D4‐lysine and then TFA or enzymatically deprotect overnight to obtain the target compound. Copyright © 2004 John Wiley & Sons, Ltd.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

A θ‐defensin composed exclusively of d‐amino acids is active against HIV‐1

Abstract: The ability of certain θ‐defensins, including retrocyclin‐1, to protect human cells from infection by HIV‐1 marks them as potentially useful molecules. θ‐Defensins composed of l ‐amino acids are likely to be unstable in environments that contain host and microbial proteases. This study compared the properties of two enantiomeric θ‐defensins, retrocyclin‐1, and RC‐112. Although these peptides have identical sequences, RC‐112 is composed exclusively of d ‐amino acids, whereas retrocyclin‐1 contains only l ‐amino acids. We compared the ability of these peptides to protect JC53‐BL human cells from infection by 30 primary HIV‐1 isolates. JC53‐BL cells are modified HeLa cells that express surface CD4, CXCR4, and CCR5. They also contain reporter cassettes that are driven by the HIV‐1 LTR, and express β‐galactosidase and luciferase. The HIV‐1 isolates varied in co‐receptor specificity and included subtypes A, B, C, D, CRF01‐AE, and G. RC‐112 was several fold more potent than retrocyclin‐1 across the entire HIV‐1 panel. Although RC‐112 bound immobilized gp120 and CD4 with lower affinity than did retrocyclin‐1, surface plasmon resonance experiments performed with 1 μg/mL of RC‐112 and retrocyclin‐1 revealed that both glycoproteins were bound to a similar extent. The superior antiviral performance of RC‐112 most likely reflected its resistance to degradation by surface‐associated or secreted proteases of the JC53‐BL target cells. θ‐Defensins composed exclusively of d ‐amino acids merit consideration as starting points for designing microbicides for topical application to the vagina or rectum.     

Synthesis and kinetics studies of N′‐(2‐(3,5‐disubstituted‐4H‐1,2,4‐triazol‐4‐yl)acetyl)‐6/7/8‐substituted‐2‐oxo‐2H‐chromen‐3‐carbohydrazide derivatives as potent antidiabetic agents

A novel series of N′‐(2‐(3,5‐disubstituted‐4H‐1,2,4‐triazol‐4‐yl)acetyl)‐6/7/8‐substituted‐2‐oxo‐2H‐chromen‐3‐carbohydrazides were synthesized and studied for their α‐glucosidase inhibition activity. Most of the synthesized compounds exhibited potential α‐glucosidase inhibition activity with IC₅₀ values ranging from 0.96 ± 0.02 to 32.86 ± 0.73 µg/ml. Among them, compounds 3e and 4e , having a methoxy group on the coumarin ring, proved to be the most potent ones, showing an enzyme inhibition activity with IC₅₀ = 0.96 ± 0.02 and 1.44 ± 0.06 µg/ml, respectively. The kinetic study through Lineweaver–Burk plots revealed that the inhibition mechanism of the most active compounds 3d, 3e, 4d , and 4e , on the α‐glucosidase activity, was found to be in the competitive mode.     

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.