We have prepared two peptides based on the hydrophobic core (Lys-Leu-Val-Phe-Phe) of amyloid beta-protein (Abeta) that contain alpha,alpha-disubstituted amino acids at alternating positions, but differ in the positioning of the oligolysine chain (AMY-1, C-terminus; AMY-2, N-terminus). We have studied the effects of AMY-1 and AMY-2 on the aggregation of Abeta and find that, at stoichiometric concentrations, both peptides completely stop Abeta fibril growth. Equimolar mixtures of AMY-1 and Abeta form only globular aggregates as imaged by scanning force microscopy and transmission electron microscopy. These samples show no signs of protofibrillar or fibrillar material even after prolonged periods of time (4.5 months). Also, 10 mol % of AMY-1 prevents Abeta self-assembly for long periods of time; aged samples (4.5 months) show only a few protofibrillar or fibrillar aggregates. Circular dichroism spectroscopy of equimolar mixtures of AMY-1 and Abeta show that the secondary structure of the mixture changes over time and progresses to a predominantly beta-sheet structure, which is consistent with the design of these inhibitors preferring a sheet-like conformation. Changing the position of the charged tail on the peptide, AMY-2 interacts with Abeta differently in that equimolar mixtures form large ( approximately 1 mum) globular aggregates which do not progress to fibrils, but precipitate out of solution. The differences in the aggregation mediated by the two peptides is discussed in terms of a model where the inhibitors act as cosurfactants that interfere with the native ability of Abeta to self-assemble by disrupting hydrophobic interactions either at the C-terminus or N-terminus of Abeta.
Both amide-to-ester and amide-to-E-olefin backbone amide mutation methods were employed to perturb the same H-bond (formed by the NH of F23 and the CO of R14) in the pin WW domain.Comparison of the thermodynamic folding energies of the ester mutant and the E-olefin mutant, accounting for the transfer free energy differences measured on relevant model compounds, yielded an estimated value of 0.3 kcal/mol for the O-O repulsion term (ΔG O-Orep ) in a β-sheet context. The value of ΔG O-Orep enabled us to calculate the intrinsic F23-R14 H-bond free energy to be 1.3 kcal/ mol.There is growing interest in deciphering the contributions of backbone-backbone hydrogen bonding to protein folding energetics. 1 Since backbone hydrogen bonds are formed between main chain amides (Fig 1A), they can be perturbed by replacing the amide bond of interest in a protein with an iso-structural moiety which has reduced or lacks H-bonding capacity. Currently, the most convenient approach to perturb backbone H-bonding is to replace amides with esters ( Fig 1A). 2 An amide-to-ester (A-to-E) mutation eliminates the H-bond donor (N-H) and weakens the H-bond acceptor (C=O). A-to-E mutations are conservative in that the trans conformation of the linkage is maintained, as well as the ϕ, ψ dihedral angle preference of the flanking substructure. One concern with this approach is the possible electrostatic repulsions introduced between the O replacing the NH and carbonyl oxygen of the acceptor amide ( Fig 1A; red line). The magnitude of this O-O repulsion is unclear, which complicates the extraction of H-bond energies from A-to-E perturbation thermodynamic data. 1d, 2c, d It has been proposed that an amide-to-E-olefin (A-to-O) mutation is the ideal peptide bond perturbation. 3 An A-to-O mutation in a protein eliminates one H-bond donor (NH) and one Hbond acceptor (CO) without introducing electrostatic repulsions. However, this strategy has rarely been realized due to the difficulties associated with stereospecific synthesis of alkenecontaining isosteres and incorporating them into proteins. Recently, our group has reported a convenient protocol for the preparation of the Phe-Phe E-olefin dipeptide isostere and its incorporation into proteins. 4 Herein, we report perturbation of the Phe22-Phe23 amide bond in the Pin WW domain employing both A-to-O and A-to-E mutations ( Fig 1B). An energetic comparison of the ester mutant and E-olefin mutant enables us to quantify the repulsive O-O interaction introduced by A-to-E mutations and to establish the H-bond energy.The Pin WW domain, the ligand binding domain of the human Pin 1 protein, is one of the smallest and best-studied β-sheet proteins. 2c, 5, 6 It is a 34-residue polypeptide that folds into a twisted 3-stranded anti-parallel β-sheet structure (Fig 1B). Mutational studies show that the jkelly@scripps.edu. Pin WW domain is highly tolerant to side chain mutations at nearly every position. 6 Taking advantage of this fact, we carried out this study with the Val22Phe, Tyr23Phe variant of the PIN ...
Amyloid beta (Abeta) peptide amyloidogenesis, involving the formation of numerous distinct quaternary structures, appears to cause Alzheimer's disease. However, the precise identification of the toxic structure(s) and the neurotoxicity mechanism(s) remains elusive. Mutating the Abeta 1-40 Phe19-Phe20 backbone amide bond to an isostructural E-olefin bond enables formation of spherical aggregates to the exclusion of detectable amyloid fibrils. Herein, the fibrillization and toxicity of amide-to-ester mutants of Abeta 1-40 at the 19-20 position and surrounding backbone amide bonds are compared to the fibrillization and toxicity of the 19-20 E-olefin Abeta analogue and wild type Abeta. Whereas isostructural amide-to-E-olefin mutations eliminate both the H-bond donor and acceptor capabilities, isostructural amide-to-ester mutations eliminate the donor while retaining the ester carbonyl as a weakened acceptor. None of the amide-to-ester Abeta 1-40 mutants prevent fibrillization; in fact several exhibit hastened amyloidogenesis. The 18-19 amide-to-ester substitution is the only backbone mutation within the hydrophobic core region of the fibril (residues 17-21) that significantly slows fibrillization. Despite forming different morphologies, the 19-20 E-olefin mutant, the 18-19 amide-to-ester mutant, and WT Abeta 1-40 fibrils all exhibit similar toxicities when applied to PC12 cells at 18 h into the aggregation reactions, as assessed by MTT metabolic activity measurements. This result suggests that a common but low abundance aggregate morphology, that is accessible to these Abeta analogues, mediates toxicity, or that several different aggregate morphologies are similarly toxic.
Herein, we report a stereospecific E-olefin dipeptide isostere synthesis that can be used to make gram quantities of the Phe-Phe isostere desired for eliminating a specific backbone H-bond donor and acceptor in the Alzheimer's disease related Abeta peptide. The Phe19-Phe20 E-olefin analogue of Abeta(1-40) was prepared by solid-phase peptide synthesis and was subjected to amyloidogenesis conditions. This analogue can aggregate into spherical morphologies but does not progress on to form protofibrils or fibrils as is the case for the all-amide sequence, providing insight into the structural requirements for amyloidogenesis.
The preparation of sterically hindered and polyfunctional C(alpha,alpha)-disubstituted alpha-amino acids (alpha alpha AAs) via alkylation of ethyl nitroacetate and transformation into derivatives ready for incorporation into peptides are described. Treatment of ethyl nitroacetate with N,N-diisopropylethylamine (DIEA) in the presence of a catalytic amount of tetraalkylammonium salt, followed by the addition of an activated alkyl halide or Michael acceptor, gives the doubly C-alkylated product in good to excellent yields. Selective nitro reduction with Zn in acetic acid or hydrogen over Raney Ni gives the corresponding amino ester that, upon saponification, can be protected with the fluorenylmethyloxycarbonyl (Fmoc) group. The first synthesis of an orthogonally protected, tetrafunctional C(alpha,alpha)-disubstituted analogue of aspartic acid, 2,2-bis(tert-butylcarboxymethyl)glycine (Bcmg), is described. Also, the sterically demanding C(alpha,alpha)-dibenzylglycine (Dbg) has been incorporated into a peptide using solid-phase synthesis. It was found that once sterically congested Dbg is at the peptide N-terminus, further chain extension becomes very difficult using uronium or phosphonium salts (PyAOP, PyAOP/HOAt, HATU). However, preformed amino acid symmetrical anhydride couples to N-terminal Dbg in almost quantitative yield in nonpolar solvent (dichloroethane-DMF, 9:1).
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