Cleavage of aspartyl β-phenacyl esters by selenophenol under neutral conditions

Neutral selenophenol in DMF accomplished the removal of the β-phenacyl protecting group of aspartic acid in solution, on the peptide Boc-Asp(β-OPac)-Phe amide and on the resin peptide Boc-Trp(Nⁱⁿ-For)-Met-Asp(β-OPac)-Phe- (CCK 30–33) without α, β-rearrangement.     

Use of Fmoc-N-(2-hydroxy-4-methoxybenzyl) amino acids in peptide synthesis

The use of N, O-bisFmoc-N-(2-hydroxy-4-methoxybenzyl) amino acid derivatives in the synthesis of peptides with difficult sequences has already been described. With these amino acid derivatives the reversible protecting group 2-hydroxy-4-methoxybenzyl (Hmb) for the backbone amide bonds of peptide chains is introduced, and thus the aggregation due to hydrogen-bond interchain association is inhibited. This paper describes the synthesis and use of Fmoc-N-(2-hydroxy-4-methoxybenzyl)amino acid derivatives as an alternative means of introducing Hmb backbone protection. These new monoFmoc derivatives were obtained in higher yield than the bisFmoc derivatives. Coupling yields to the amino peptide resin were the same as those obtained with bisFmoc derivatives, under the TBTU/HOBt/DIEA conditions. We also compared different syntheses of a difficult peptide with the Fmoc approach [triple coupling, capping, use of chaotropic agents, backbone protection using monoFmoc (Hmb)Ala] and with optimized Boc chemistry. Both the backbone protection and optimized Boc chemistry approaches gave the desired product in excellent yield and purity. © Munksgaard 1997.     

Surface-induced conformational switching in amphiphilic peptide segments of apolipoproteins B and E and model peptides†

The conformational and surface-binding properties of a synthetic peptide corresponding to Tyr-apolipoprotein B-100(1000–1016) amide, SP-4, which was previously shown to mimic the focal accumulation pattern of LDL on the healing de-endothelialized rabbit aorta [Shih et al. (1990) Proc. Natl. Acad. Sci. USA 87 , 1436–1440], have been investigated. SP-4 behaves as an amphiphilic α-helical peptide at the air-water interface and bound to siliconized quartz slides. However, its N^α-acetylated analogue formed β-sheet structures at the air-water interface. Nonhomologous peptide models of SP-4 also exhibited mixed α-helical and β-sheet surface-binding behavior. Peptides corresponding to the cationic apolipoprotein (apo) B/E receptor binding regions of apoE (SP-2) and apoB (SP-11) were also studied. SP-2 behaved as an amphiphilic α helix, but, surprisingly, SP-11 formed surface-induced β-sheets. These results demonstrate that all of the peptides studied have surface-binding properties, and suggest further that either α-helical or β-sheet peptide structures may determine the binding of LDL to the arterial wall or the apoB/E receptor.     

Structure,solubility and reactivity of peptides

A conformational study of two protected peptide segments, (1–10 and 11–28), spanning the entire sequence of thymosin α₁, in solvents of different polarity and capability of forming hydrogen bonds, is reported. By using infrared absorption and circular dichroism techniques the occurrence of the random coil conformation, the self-associated β-structure, and the α-helix (the latter adopted only by the longer peptide) was established. The self-associated species of the two peptide segments were disrupted either by adding increasing amounts of hexamethylphosphoramide or by dilution. This structural transition was monitored by the disappearance of the amide-I C=O stretching band of strongly intermolecularly hydrogen-bonded molecules (near 1630 cm^-1) in the infrared absorption spectra. The tendency of these peptides to aggregate is paralleled by a decrease in their solubility. The conformational findings are discussed in terms of the solvent-dependent product yields obtained in the reaction of segment (1–10) with the N^α-deprotected (11–28) segment to give the fully protected thymosin α₁.     

Synthesis and solubility properties of peptide fragments of human hemoglobin α-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin α-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a <P_C > value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that β-sheet structure played an important role in the insolubility of peptides. When a peptide has a β-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

Conformation of a water-soluble β-sheet model peptide. A circular dichroism and Fourier-transform infrared spectroscopie study of double D-amino acid replacements

Among peptide secondary structures β-sheet domains have been much less intensively studied than α-helical conformations, mainly because of the lack of well characterized model peptides. In the present paper the secondary structure of a water-soluble de novo peptide consisting of 26 amino acids (DPKGDPKGVTVTVTVTVTGKGDPKPD-NH₂) and the corresponding double D-amino acid replacement set have been studied by circular dichroism and Fourier-transform infrared spectroscopy. The model peptide was found to be unstructured in aqueous solution at peptide concentrations < 10^?3 mol/L but to adopt a predominantly β-sheet structure in the presence of 15 mM sodium dodecyl sulfate or at apolar/water interfaces. Although the peptide is composed of amino acids with low helical propensity, it formed a single-stranded helical structure in aqueous trifluoroethanol. The D-amino acid replacement set was synthesized in order to study the conformational stability of the model peptide selectively in distinct regions. The data show that both the α-helix present in 50% trifluoroethanol as well as the β-sheet domain formed in the presence of sodium dodecyl sulfate or at apolar/water interfaces, are located in the region between Val⁹ and Thr¹⁸. Pairwise substitution of adjacent amino acids by their corresponding D-amino acids provides a pronounced β-sheet disturbance. These findings demonstrate that double D-amino acid replacements may be used to locate β-sheet domains in peptides.     

Novel Pentapeptide GLP‐1 (32–36) Amide Inhibits β‐Cell Apoptosis In Vitro and Improves Glucose Disposal in Streptozotocin‐Induced Diabetic Mice

We proposed that a pentapeptide, LVKGR amide, GLP‐1 (32–36) amide, derived from the gluco‐incretin hormone, glucagon‐like peptide‐1 (GLP‐1), might possess favorable actions against diabetes. Therefore, GLP‐1 (32–36) amide was synthesized and the effects of it were examined in INS‐1 cell and streptozotocin‐induced diabetic mice model. To determine the protective effects of GLP‐1 (32–36) amide on INS‐1 cell viability and apoptosis, cells were exposed to 1 μm streptozotocin (STZ) and GLP‐1 (32–36) amide for 24 h. Results showed that GLP‐1 (32–36) amide treatment decreased apoptosis rate and significantly retained cell viability compared with saline‐treated controls. Then, GLP‐1 (32–36) amide was administered intraperitoneally to streptozotocin‐induced diabetic mice with normal mice used as control. Body weight, energy intake, plasma glucose, and histopathology of the pancreas were assessed. Results showed that GLP‐1 (32–36) amide protected β‐cell viability and apoptosis against STZ‐induced toxicity, inhibited weight gain, and relieved symptoms of polydipsia. Moreover, GLP‐1 pentapeptide‐treated mice showed a slight trend toward reduced glucose excursions in intraperitoneal glucose tolerance test at the end of the experiment. GLP‐1 (32–36) amide exerted favorable protective actions in streptozotocin‐induced diabetic mice. The peptide curtailed weight gain and alleviates symptoms of polydipsia. These findings suggested the probable utility of GLP‐1 (32–36) amide, a peptide mimetic derived there from GLP‐1, for adjuvant treatment of diabetes.     

Efficient solution phase synthesis of [l-(β-mercapto-β, β-cyclopentamethylenepropionic acid)- 2-(O-ethyl-d-tyrosine)-4-valine-9-desglycine]arginine vasopressin

A convergent synthesis of the peptide [1-(β-mercapto-β,β-cyclopentamethylenepropionic acid)-2-(O-ethyl-d -tyrosine)-4-valine-9-desglycine]arginine vasopressin (1), based on the classical solution phase method, was developed. The molecule is assembled by a 3 + 4 coupling via the amide method; then the disulfide bridge is installed by iodine treatment of the bis-acetamidomethyl protected thiols, and the terminal arginine amide added by a 7 + 1 coupling. The method (see Scheme 1) has been used to prepare gram quantities of 1 in more than 98% purity and in 13% yield (based on tetrapeptide intermediate 13) after a single stage purification. The method appears to be particularly suitable for the large scale preparation of 1 and other vasopressin congeners. A novel, albeit low level, transfer of acetamidomethyl group from the sulfur of cysteine to the asparagine amide side-chain was detected following hydrogen chloride treatment of Boc-containing intermediates.     

Solution conformation of N-terminal fragments of trichosanthin small domain (TCS 182-200)

Three peptides, T14, T18 and TDK, derived from the N-terminus of trichosanthin small domain (TCS 182-200) have been investigated by circular dichroism. Secondary structure and structural transitions of the above peptides under different conditions were studied. Alcohol prompts a transition of the T18 peptide from a β-sheet to an α-helical structure. It also increases the α-helicities of T14 and TDK. The β-sheet of T18 peptide appears more hydrophobic than the α-helix of T14 or TDK. The effects of polypeptide sequence and solvent on secondary structure formation of these model peptides are discussed.     

Structure-activity relationship study: short antimicrobial peptides

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an α-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an α-helical structure correlated with the antimicrobial activity and a β-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of l -amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the β-sheet structure retained antifungal activity in the same condition.     

Crystal structure of a dipeptide Boc-Aib-Phe-OMe

In order to understand the effect of the restrictions posed by the Aib residue on peptide conformation we studied the crystal structure of a dipeptide tBoc-Aib-Phe-OMe. Crystals of this compound are triclinic, space group PI with a= 9.600(1) Å, b=10.262(1) Å, c= 10.799(1) Å, α= 98.43°(1), β=99.18°(1), °=98.87°(1), V= 1021.69(18) ų and Z=2. The structure was solved by direct methods and refined to an R-factor of 4.98%. The backbone conformational angles for the Aib residue in molecule A are in the left-handed helical region, while in molecule B they are in the right-handed helical region. The Phe residue in molecule A is in the right-handed helical conformation, while in molecule B it is in the β-region. The peptide units are trans and show significant deviation from planarity [(ω₁= 166.67(5)° and ω₂=–177.9(5)]. © Munksgaard 1997.     

Effects of N‐Methylated Amyloid‐β30–40 Peptides on the Fibrillation of Amyloid‐β1–40

Alzheimer's disease is a neurodegenerative disorder associated with amyloid‐β (Aβ) fibrillation. N‐Methylated amyloid‐β peptides are potent inhibitors of amyloid‐β fibrillation. We investigated the inhibitory effect of N‐Methylated Aβ_30–40 peptides on Aβ_1–40 fibrillation. N‐Methylated Aβ_30–40 peptides affected the fibrillation, and this effect was dependent on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. N‐Methylated Aβ_30–40 peptides were co‐aggregated with Aβ_1–40. Spectroscopic technique was adopted to investigate an origin of the observed dependence. Suppression of thioflavin T (ThT) fluorescence count was correlated with the dissociation constant K_d of monomer–dimer equilibrium of each N‐Methylated Aβ_30–40 peptide. Monomeric N‐Methylated peptides decreased ThT fluorescence count during Aβ_1–40 fibrillation. Secondary structure content was not largely different between Aβ_1–40 fibrils and co‐aggregates. These results suggested that N‐Methylated Aβ_30–40 peptides disrupted the regular β‐sheet structure of Aβ_1–40 fibrils and affected the ThT fluorescence count. The monomer–dimer equilibrium of N‐Methylated peptides was (partly) responsible for the observed dependence of their inhibitory effect on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. Our study provides a hint to design new N‐Methylated inhibitor peptides of fibrillation.     

Crystal structures of heterochiral peptides

Crystal structure of a heterochiral peptide. viz. Boc-Val¹-d -Ala²-Leu³-Ala⁴-OMe with a d chiral residue in the second position of a sequence, has been determined [a= 40.44(1), b= 4.887(5), c= 15.381(5) Å, β= 109.6(1)°, space group C2, Z= 4, R= 0.11]. The peptide is in a parallel β-sheet structure terminated by a distinct local bend. The structure is stabilised by N–H?O as well as C^α–H?O hydrogen bonds. The contiguous C^α-H?O hydrogen bond observed in this structure is an unique observation. © Munksgaard 1991.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Implementation of an FTIR calibration curve for fast and objective determination of changes in protein secondary structure during formulation development

The aim of this study was to develop a quick and objective method for the determination of changes in protein secondary structure by Fourier transform infrared spectroscopy (FTIR). Structural shifts from native regions (α-helix, intramolecular β-sheet) to aggregated strands (intermolecular β-sheet) were used to evaluate protein damage. FTIR spectra of 16 different proteins were recorded and quantified by peak fitting of the non-deconvolved and baseline corrected amide I bands. The resulting percentile secondary structures were correlated with the shape and intensity of the area normalized amide I bands using an interval partial least squares algorithm (iPLS). Structural elements were focused on the following regions: α-helix 1660–1650 cm⁻¹, intramolecular β-sheet 1695–1683 cm⁻¹ and 1644–1620 cm⁻¹, intermolecular β-sheet 1620–1595 cm⁻¹. Three calibration curves were created from the data sets. Calculated α-helix content ranged from 0% to 79.59%, intramolecular β-sheet from 10.64% to 63.89% and intermolecular β-sheet from 0.23% to 9.70%. The linear relationship between actual values (as determined by peak fitting) and calculated values was evaluated by correlation coefficient and root mean square error of calibration while cross-validation was performed to detect possible outliers. Results were verified by including two proteins as validation standards and comparing the calculated values to peak fitting and X-ray data.     

Crystal structure of tert.-butyloxycarbonyl-L-prolyl-D-alanyl-D-alanyl-N-methylamide Dimeric β-sheet formation

The crystal structure of the tripeptide t-Boc-L-Pro-D-Ala-D-Ala-NHCH₃, monohydrate, (C₁₇H₃₀N₄O₅·H₂O, molecular weight = 404.44) has been determined by single crystal X-ray diffraction. The crystals are mono-clinic, space group P2₁, a = 9.2585(4), b = 9.3541(5). c = 12.4529(4) Å, β= 96.449(3)°, Z = 2. The peptide units are in the trans and the tBoc-Pro bond in the cis orientation. The first and third peptide units show significant deviations from planarity (Δω=5.2° and Δω=3.7°, respectively). The backbone torsion angles are: φ¹, = -60°, ψ_1/= 143.3°, ω₁= -174.8°, φ₂= 148.4°, ψ₂= -143.1°, ω₂= -179.7°, φ₃= 151.4°, ψ₃= -151.9°, ω₃= -176.3°. The pyrrolidine ring of the proline residue adopts the C₂— C^γ conformation. The molecular packing gives rise to an antiparallel β-sheet structure formed of dimeric repeating units of the peptide. The surface of the dimeric β-sheet is hydrophobic. Water molecules are found systematically at the edges of the sheets interacting with the urethane oxygen and terminal amino groups. Surface catalysis of an L-Ala to D-Ala epimerization process by water molecules adsorbed on to an incipient β-sheet is suggested as a mechanism whereby crystals of the title peptide were obtained from a solution of tBoc-Pro-D-Ala-Ala-NHCH₃.     

ISOLATION AND PHYSICAL CHARACTERIZATION OF BOVINE LENS CRYSTALLINS

The soluble proteins from bovine lens homogenate were separated on Sepharose CL-6B (2 times 200 cm) in 0.05 m tris-NaHSO₃ pH 8.2 buffer containing 20 mM EDTA. Five sharp and defined fractions (HMα, α, βH, βL, γ) were obtained. Each crystallin fraction was further purified by rechromatography on the same column. Each protein fraction was pure as judged by ultracentrifugation and SDS-gel electrophoresis. The molecular weights of the five fractions were 3.04 times 10⁶, 5.83 times 10⁵, 1.58 times 10⁵, 4.59 times 10⁴, 2.14 times 10⁴ as determined from sedimentation coefficient and intrinsic viscosity data by Scheraga-Mandelkern equation, which was in close agreement with that obtained by gel filtration. The polypeptide composition of crystallins as determined by SDS-gel electrophoresis revealed one band for high molecular weight α (HMα) and α, three for βH, two for βL and one for γ. The gross CD patterns of crystallins were about the same in the peptide region (200 nm ? 250 nm) with a minimum centered at about 217 nm, indicative of α β-sheet structure in all crystallins. The [0] values at 217 nm ranged from –1700 to –3700 degrees cm² per decimole. The CD spectra of these crystallins in the aromatic region (250 nm ? 300 nm) were different, reflecting the different contributions of aromatic amino acids to the tertiary structure of crystallins.     

Electrostatic effects on the α-helix and β-strand formation of BPTI(16–36) studied by Monte Carlo simulated annealing

Abstract: The electrostatic effects on the secondary structure forming tendencies of a peptide fragment with residues 16–36 of bovine pancreatic trypsin inhibitor, BPTI( 16 - 34 ), are studied using Monte Carlo simulated annealing simulations. We consider three dielectric functions ε(r) of distance r: constant dielectric function (ε = 2; strong electrostatic interactions) and sigmoidal functions varying from ε (0) = 2 to ε(∞) = 47 (intermediate) and to ε(∞) = 78 (weak). Simulations with ε = 2 suggest that this peptide exhibits a significant propensity for β-strand formations in accordance with a β-sheet structure of the relevant segment in native BPTI. The tendency for α-helix formations becomes almost comparable with that of β-strands in the simulation with ε(∞) = 47, and there appears no appreciable conformational propensity for this case. Finally, the results with ε(∞) = 78 generate low-energy conformations with conspicuous α-helices. These findings suggest the possibility that the change in electrostatic interactions can be the key factor for the conformational transitions of peptides between α-helix and β-sheet that have recently been observed in experiments. These changes in electrostatic interactions can arise from those in various environmental factors such as conformations of the rest of the protein molecule and solvent conditions.     

CONFORMATIONAL CALCULATIONS ON GASTRIN C-TERMINAL TETRAPEPTIDE

Energy optimizations were performed on some typical conformations of the gastrin C-terminal peptide amide NAc-Trp-Met-Asp-Phe-NH₂. Two families of lowest energy conformations were found corresponding to: (a) α-helical structures; (b) conformations having β-structure at the level of Trp residue, and C₇-structure at the level of Asp residue. The two aromatic rings were folded on the peptide backbone and ca. 5 Å distant from each other (centre to centre). The last family, favoured by energy and population probability, can better account for conformational experimental results and biological activity observations.     

Conformational investigation of α, β-dehydropeptides

The crystal structure of Ac-Pro-ΔVal-NHCH₃ was examined to determine the influence of the α,β-dehydrovaline residue on the nature of peptide conformation. The peptide crystallizes from methanol-diethyl ether solution at 4° in needle-shaped form in orthorhombic space group P2₁2₁2₁ with a= 11.384(2) Å, b = 13.277(2) Å, c = 9.942(1) Å. V = 1502.7(4) ų Z = 4, D_m= 1.17 g cm^?3 and D_c=1.18 g cm^?3 The structure was solved by direct methods using SHELXS-86 and refined to an R value of 0.057 for 1922 observed reflections. The peptide is found to adopt a β-bend between the type I and the type III conformation with φ₁=?68.3(4)°, ψ₁=? 20.1(4)°, φ₂=?73.5(4)°= and Ψ₂=?14.1(4)°=. An intramolecular hydrogen bond between the carbonyl oxygen of ith residue and the NH of (i+ 3)th residue stabilizes the β-bend. An additional intermolecular N.,.O hydrogen bond joins molecules into infinite chains. In the literature described crystal structures of peptides having a single α,β-dehydroamino acid residue in the (i+ 2) position and forming a β-bend reveal a type II conformation.