Inhibition of β‐amyloid toxicity by short peptides containing N‐methyl amino acids

Abstract: Single N‐methyl amino acid‐containing peptides related to the central hydrophobic region β_16–20 (Lys‐Leu‐Val‐Phe‐Phe) of the β‐amyloid protein are able to reduce the cytotoxicity of natural β_1–42 in PC12 cell cultures. N‐methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t‐test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

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.     

Insights into the determinants of β‐sheet stability: 1H and 13C NMR conformational investigation of three‐stranded antiparallel β‐sheet‐forming peptides

Abstract: In a previous study we designed a 20‐residue peptide able to adopt a significant population of a three‐stranded antiparallel β‐sheet in aqueous solution (de Alba et al. [1999]Protein Sci. 8, 854–865). In order to better understand the factors contributing to β‐sheet folding and stability we designed and prepared nine variants of the parent peptide by substituting residues at selected positions in its strands. The ability of these peptides to form the target motif was assessed on the basis of NMR parameters, in particular NOE data and ¹³C_α conformational shifts. The populations of the target β‐sheet motif were lower in the variants than in the parent peptide. Comparative analysis of the conformational behavior of the peptides showed that, as expected, strand residues with low intrinsic β‐sheet propensities greatly disfavor β‐sheet folding and that, as already found in other β‐sheet models, specific cross‐strand side chain–side chain interactions contribute to β‐sheet stability. More interestingly, the performed analysis indicated that the destabilization effect of the unfavorable strand residues depends on their location at inner or edge strands, being larger at the latter. Moreover, in all the cases examined, favorable cross‐strand side chain–side chain interactions were not strong enough to counterbalance the disfavoring effect of a poor β‐sheet‐forming residue, such as Gly.     

A Meccano set approach of joining trpzip a water soluble β‐hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide

Abstract: A 16 residues long, water soluble, monomeric β‐hairpin peptide ‘trpzip’ (Cochran et al. Proc. Natl. Acad. Sci. U.S.A., 98 (2001), 5578), stabilized by tryptophan zipper has been linked via a tetraglycyl linker to a hydrophobic didehydrophenylalnine (ΔF) containing helical octapeptide. Circular dichroism studies of this 28 residues long peptide, ‘trpzipalpha’ (Ac‐GEWTWDDATKTWTWTE‐GGGG‐ΔFALΔFALΔFA‐NH₂) in water have revealed the presence of both the β‐hairpin and the helical conformations. This is the first instance where a ΔF containing peptide has been found to display a helical fold in water. The fluorescence emission wavelengths of tryptophan in Ac‐G‐W‐G‐NH₂, trpzip and trpzipalpha were 341.5, 332.8 and 332.6 nm, respectively. The fluorescence quantum yield of trpzip was 2.6‐fold higher than trpzipalpha suggesting that proximal interactions between the β‐hairpin and the helix caused the quenching of tryptophan fluorescence in the former by the ΔFs in the latter. The molar ellipticity of the far UV couplet characteristic of trpzip was reduced in trpzipalpha and the CD based thermal melting temperatures at 228 nm were 62 °C (trpzip) and 57 °C (trpzipalpha). A concentration‐dependent variable temperature CD study in water showed that in trpzipalpha, increasing temperature is detrimental to the β‐hairpin, but it augments the helical motif, perhaps by intermolecular oligomerization. Our results show that in water, trpzipalpha exhibits long‐range interactions between two different secondary structures. In contrast to trpzip, trpzipalpha has shown a greater tendency to oligomerize in water.     

From β‐N‐tert‐butyloxycarbonyl N‐carboxyanhydrides to β‐aminoacid derivatives

Abstract: β‐N‐tert‐butyloxycarbonyl‐N‐carboxyanhydrides are very reactive β‐amino acid derivatives. They react cleanly and smoothly with different nucleophiles like aminoesters, enolates, N‐methyl‐d ‐glucamine, amidoximes to afford in good to excellent yields peptides, β‐amino ketocompounds, β‐aminosugars and functionalized disubstituted 1,2,4‐oxadiazoles.     

Ginsenoside Rg3 promotes beta‐amyloid peptide degradation by enhancing gene expression of neprilysin

Objectives It has been hypothesized that the accumulation of beta‐amyloid peptide (Aβ) in the brain is a triggering event leading to the pathological cascade of Alzheimer's disease. The steady‐state levels of Aβ are determined by the metabolic balance between anabolic and catabolic activity and the dysregulation of this activity leads to Alzheimer's disease. Recent evidence has shown that neprilysin (NEP) is the rate‐limiting enzyme in the Aβ degradation in the brain. Ginseng, the root of Panax ginseng C.A. Meyer, is widely used as a tonic for the prevention and treatment of age‐related disorders in China. We aimed to investigate the basis of this use. Methods In this study, we investigated the effect of ginsenoside Rg3, one of the major active components of ginseng, on the metabolism of Aβ40 and Aβ42 in SK‐N‐SH cells transfected with Swedish mutant β‐amyloid precursor protein (SweAPP). Results The ELISA result showed that Rg3 significantly reduced the levels of Aβ40 and Aβ42, 19.65 ± 6.05%, 23.61 ± 6.74%, respectively (P < 0.01). The Western blot analysis showed that Rg3 reduced the levels of Aβ40 and Aβ42 through enhancing NEP gene expression, and real‐time PCR assay showed that 50 μM Rg3 could significantly enhance NEP gene expression (2.9 fold at 48 h). Conclusions Our findings suggest that the Rg3 compound of ginseng may be useful for treating patients suffering with Alzheimer's disease.     

(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.     

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.     

Novel 19F‐MRS β‐galactosidase reporter molecules incorporated nitrogen mustard analogues

In this study, we propose a novel molecular platform‐integrated fluorinated antitumor nitrogen mustards for ¹⁹F‐MRS assay of β‐galactosidase (β‐gal) activity. Following this idea, we have designed, synthesized, and characterized 2‐fluoro‐4‐[bis(2′‐chloroethyl)amino]phenyl β‐D‐galactopyranoside 5 , 2‐fluoro‐4‐{bis[2′‐O‐(β‐D‐galactopyranosyl)ethyl]amino}phenyl β‐D‐galactopyranoside 8 , 2‐fluoro‐4‐{bis[[1″‐(β‐D‐galactopyranosyl)‐1″, 2″, 3″‐triazol‐4″‐yl]methyl] amino}phenyl β‐D‐galactopyranoside 14 and 2‐fluoro‐4‐{bis[[1″‐(β‐D‐glucopyranosyl)‐1″, 2″, 3″‐triazol‐4″‐yl]methyl]amino}phenyl β‐D‐galactopyranoside 15 through glycosylation and click reaction strategies, and their structures were confirmed by NMR and HRMS or elemental analysis data. Among them, 2‐fluoro‐4‐[bis(2′‐chloroethyl)amino]phenyl β‐D‐galacto‐pyranoside 5 was found very sensitive to β‐gal (E801A) in PBS at 37°C with big Δδ_F response. Here, we demonstrated the feasibility of this platform for assessing β‐gal activity in solution, and in vitro with lacZ‐transfected human MCF7 breast and PC3 prostate tumor cells, by the characterization of β‐gal‐responsive ¹⁹F‐chemical shift changes Δδ_F and hydrolytic kinetics.     

β‐turn formation by a six‐residue linear peptide in solution

Abstract: A model peptide AAGDYY‐NH₂ (B1), which is found to adopt a β‐turn conformation in the TEM‐1 β‐lactamase inhibitor protein (BLIP) in the TEM‐1/BLIP co‐crystal, was synthesized to elucidate the mechanism of its β‐turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT‐IR and ¹H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT‐IR spectroscopy confirmed the formation of a β‐turn in solution by the model peptide. The dihedral angles [(φ₃, ?₃) (φ₄, ?₄)] of [(?52°, ?32°) (?38°, ?44°)] of Gly‐Asp fragment in the model peptide are consistent with those of a type III β‐turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of β‐turn formation and stability.     

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.     

Convenient and efficient synthesis of Boc‐/Z‐/Fmoc‐β‐amino acids employingN‐protected α‐amino acid fluorides

Abstract: A new and efficient method for the synthesis ofN^α‐Fmoc‐/Boc‐/Z‐β‐amino acids using the two‐step Arndt‐Eistert approach is described. Fmoc‐/Boc‐/Z‐α‐Amino acid fluorides were used for the acylation of diazomethane synthesizing Fmoc‐/Boc‐/Z‐α‐aminodiazoketones as crystalline solids with good yield and purity. They were then converted to the corresponding β‐amino acids using PhCOOAg/dioxane/H₂O.     

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.     

Chiral N‐Phosphonyl Imine Chemistry: Asymmetric Synthesis of α‐Alkyl β‐Amino Ketones by Reacting Phosphonyl Imines with Ketone‐Derived Enolates

A series of new chiral syn‐α‐branched β‐amino ketones has been synthesized by reacting chiral phosphonyl imines with ketone‐derived enolates. The N‐protection group on imine auxiliary was found to be crucial to the asymmetric induction. The absolute stereochemistry has been unambiguously determined by converting a product to a known sample.     

‘100 years of peptide synthesis’: ligation methods for peptide and protein synthesis with applications to β‐peptide assemblies*

Abstract: A brief survey of the history of peptide chemistry from Theodore Curtius to Emil Fischer to Bruce Merrifield is first presented. The discovery and development of peptide ligation, i.e. of actual chemical synthesis of proteins are described. In the main chapter, ‘ Synthesis of Proteins by Chemical Ligation ’ a detailed discussion of the principles, reactivities and mechanisms involved in the various coupling strategies now applied (ligation, chemical ligation, native chemical ligation) is given. These include coupling sites with cysteine and methionine (as well as the seleno analogs), histidine, glycine and pseudo‐prolines, ‘unrestricted’ amino‐acid residues (using the Staudinger reaction), as well as solid‐phase segment coupling by thioligation of unprotected peptides. In another section, ‘ Synthesis of β‐peptides by Thioligation ’, couplings involving β²‐ and β³‐peptides are described (with experimental details).     

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.     

Conformational analysis of endomorphin‐1 by molecular dynamics methods

Abstract: Endomorphin‐1 (EM1, H‐Tyr‐Pro‐Trp‐Phe‐NH₂) is a highly potent and selective agonist for the μ‐opioid receptor. A conformational analysis of this tetrapeptide was carried out by simulated annealing and molecular dynamics methods. EM1 was modeled in the neutral (NH₂‐) and cationic (NH‐) forms of the N‐terminal amino group. The results of NMR measurements were utilized to perform simulations with restrained cis and trans Tyr¹‐Pro² peptide bonds. Preferred conformational regions in the Φ₂–Ψ₂, Φ₃–Ψ₃ and Φ₄–Ψ₄ Ramachandran plots were identified. The g(+), g(?) and trans rotamer populations of the side‐chains of the Tyr¹, Trp³ and Phe⁴ residues were determined in χ₁ space. The distances between the N‐terminal N atom and the other backbone N and O atoms, and the distances between the centers of the aromatic side‐chain rings and the Pro² ring were measured. The preferred secondary structures were determined as different types of β‐turns and γ‐turns. In the conformers of trans‐EM1, an inverse γ‐turn can be formed in the N‐terminal region, but in the conformers of cis‐EM1 the N‐terminal inverse γ‐turn is absent. Regular and inverse γ‐turns were observed in the C‐terminal region in both isomers. These β‐ and γ‐turns were stabilized by intramolecular H‐bonds and bifurcated H‐bonds.     

Identification of Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐amino acid‐OH as new impurities in Fmoc‐protected amino acid derivatives*

Abstract: During the manufacture of a proprietary peptide drug substance a new impurity appeared unexpectedly. Investigation of its chemical structure established the impurity as a β‐Ala insertion mutant of the mother peptide. The source of the β‐Ala was identified as contamination of the Fmoc‐Ala‐OH raw material with Fmoc‐β‐Ala‐Ala‐OH. Further studies also demonstrated the presence of β‐Ala in other Fmoc‐amino acids, particularly in Fmoc‐Arg(Pbf)‐OH. In this case, it was due to the presence of both Fmoc‐β‐Ala‐OH and Fmoc‐β‐Ala‐Arg(Pbf)‐OH. It is concluded that β‐Ala contamination of Fmoc‐amino acid derivatives is a general and hitherto unrecognized problem to suppliers of Fmoc‐amino acid derivatives. The β‐Ala is often present as Fmoc‐β‐Ala‐OH and/or as a dipeptide, Fmoc‐β‐Ala‐amino acid‐OH. In collaboration with the suppliers, new specifications were introduced, recognizing the presence of β‐Ala‐related impurities in the raw materials and limiting them to acceptable levels. The implementation of these measures has essentially eliminated β‐Ala contamination as a problem in the manufacture of the drug substance.     

Synthesis of 3,7,8‐15N3‐N1‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin

3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was synthesized from the oxidation of 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine with 2 equivalents of Ir(IV) in pH 4.5 potassium phosphate buffer. The synthesis of 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine started with bromination of 1,7,NH₂‐¹⁵N₃‐2′‐deoxyguanosine. The resulting 1,7,NH₂‐¹⁵N₃‐8‐bromo‐7,8‐dihydro‐2′‐deoxyguanosine reacted with sodium benzyloxide to afford 1,7,NH₂‐¹⁵N₃‐8‐benzyloxy‐7,8‐dihydro‐2′‐deoxyguanosine. Subsequent catalytic transfer hydrogenation of 1,7,NH₂‐¹⁵N₃‐8‐benzyloxy‐7,8‐dihydro‐2′‐deoxyguanosine with cyclohexene and 10% Pd/C yielded 1,7,NH₂‐¹⁵N₃‐8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine. Purification of 3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was first carried out on a C18 column and the product was further purified on a graphite column. ESI‐MS was used to confirm the identity and to determine the isotopic purity of all the labeled compounds. The isotopic purity of 3,7,8‐¹⁵N₃‐N¹‐(β‐D‐erythro‐pentofuranosyl)‐5‐guanidinohydantoin was 99.4 atom% based on LC‐MS measurements. Copyright © 2003 John Wiley & Sons, Ltd.     

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.