Design, Synthesis, and Structural Analysis of Turn Modified cyclo-(αβ³αβ²α)₂ Peptide Derivatives toward Crystalline Hexagon-Shaped Cationic Nanochannel Assemblies

Abstract

Synthesis and characterization of peptides 3-17.General methods: (S)-Fmoc-β 3 -homoleucine-OH and (R)-Fmoc-2-aminodecanoic acid were obtained from Senn Chemicals AG. Solvents and chemicals were used as received from their supplier. Solvents were stored over 4 Å molecular sieves. Solvents for column chromatography and extractions were of technical grade and distilled prior to use. THF was distilled over LiAlH 4 , CH 2 Cl 2 over CaH 2 and MeOH over NaBH 4 prior to use. All reactions were performed at room temperature under inert atmosphere. Flash chromatography was performed on Screening Devices silica gel 60 (0.04-0.063 mm). TLC analysis was conducted on DC-alufolien (Merck, Kieselgel 60, F254) with detection by UV-absorption at 254 nm or by spraying with a solution of ninhydrin (3 g/L) in EtOH/AcOH (20:1 v/v), or KMnO 4 solution (20 g in 1% aq K 2 CO 3 ), followed by charring at ~ 150 °C. 1 H and 13 C NMR spectra were recorded with a Bruker DMX-600 spectrometer (600/150 MHz) or with a Bruker AV-400liq spectrometer (400/100 MHz). Chemical shifts (δ) are given in ppm relative to TMS (0.00 ppm) or CD 3 OH (3.31 ppm) as internal standard. CD spectra were recorded at 298 K on a Jasco J-715 spectropolarimeter using 0.2 cm path length quartz cells. The CD spectra are an average of four scans, collected at 0.1 nm intervals between 190 and 250 nm with scanning speed 50 nm/min. The peptides were prepared at concentrations 0.1 mM in MeOH. Ellipticity is reported as mean residue ellipticity [θ], with approximate errors of ±10% at 220 nm. High resolution mass spectra were recorded by direct injection (2 μL of a 2 μM solution in water/acetonitrile; 50:50 v/v and 0.1% formic acid) on a mass spectrometer (Thermo Finnigan LTQ Orbitrap) equipped with an electro spray ion source in positive mode (source voltage 3.5 kV, sheath gas flow 10, capillary temperature 250˚C) with resolution R = 60000 at m/z 400 (mass range m/z = 150-2000) and dioctylphthalate (m/z = 391.28428) as a "lock mass". The high resolution mass spectrometer was calibrated prior to measurements with a calibration mixture (Thermo Finnigan). LC/MS analyses were performed on a LCQ Adventage Max (Thermo Finnigan) equipped with a Gemini C18 column (Phenomenex). The applied buffers were A: H 2 O, B: MeCN and C: 1.0% aq. TFA. HPLC purifications were performed with a Gilson GX-281 automated HPLC system, equipped with a preparative Gemini C18 column (150 × 21.20 mm, 5μ). The applied buffers were: A: 0.2% aq. TFA, B: MeCN. Loading of the HMPB-MBHA-resin:The HMPB-MBHA-resin (theoretical loading = 1.2 mmol/g, 2 mmol, 1.67 g) was suspended in 1,2-dichloroethane (10 mL) and concentrated thrice. Then a solution of the first amino acid (5 eq, 10 mmol), DIC (5 eq, 10 mmol, 1.54 mL) and DMAP (0.01 eq, 20 μmol, 3 mg) in dry DCM/DMF (50 mL, 10:1 v/v) was added. The mixture was shaken for 3 hours and then drained, washed subsequently with DCM, NMP, DCM and Et 2 O. The resin was dried before determination of the loading. The loading procedure was repeated when the loading of the resin wa...

Inhibiting and Reversing Amyloid‐β Peptide (1–40) Fibril Formation with Gramicidin S and Engineered Analogues

“…Synthesis of gramicidin S and analogues : The synthesis, characterization, and crystallographic structural studies of gramicidin S ( 1 ) and GS analogues 4 – 8 were previously reported in the literature 34–36. GS analogues 2 and 3 were synthesized with an adaptation of a previously reported method 30.…”

“…Synthesis of gramicidin S and analogues: The synthesis, characterization, and crystallographic structural studies of gramicidin S (1) and GS analogues 4-8 were previously reported in the literature. [34][35][36] GS analogues 2 and 3 were synthesized with an adaptation of a previously reported method. [30] All peptides were synthesized by using a standard 9-fluorenylmethoxycarbonyl (Fmoc) protection peptide synthesis protocol, involving the assembly of the protected linear decapeptides on a solid support, mild acidic cleavage, and macrocyclization in solution by using PyBOP/ HOBt/DiPEA in DMF at high dilution.…”

“…33, 34 Structural studies of native GS ( 1 ) and the analogues 4 – 8 (see Figures 5 and 9) with 1 H and 13 C NMR spectroscopy were described in recent papers 29. 34–36 The dispersion of the amide proton signals in the 1 H NMR spectrum indicated that there are no multiple conformations in solution, and the observed chemical shifts, coupling constants, and nuclear Overhauser effects demonstrated that this conformation corresponds to a stable cyclic β‐hairpin configuration. The 1 H and 13 C NMR spectra for analogues 2 and 3 (see Figure 7) showed multiple signals per nucleus, which indicates that they exist in solution as multiple interchanging conformations, and although they have similar structures to gramicidin S, we cannot be certain that they adopt β‐hairpin conformations.…”

Developments in optics and performance at BL13-XALOC, the macromolecular crystallography beamline at the Alba Synchrotron

“…From the start of user operation until March 2014, 60 research groups have tested 8363 crystals and collected 3281 datasets (defining one dataset as having 90 or more image frames), which has allowed the users to solve a large variety of structures that include integral membrane proteins or large-unit-cell crystals. As a result, the scientific production has started in earnest with the first structures being deposited in the Protein Data Bank and the first papers being published in peer-reviewed journals (Bacarizo & Camara-Artigas, 2013;Gallego et al, 2013;Gallego del Sol & Marina, 2013;Otero et al, 2013).…”

Anti-amyloidogenic Heterocyclic Peptides

“…The structure of GS identified by crystallography [129] was shown to dock into the β-hairpin-like structure of Aβ(18-42) with its hydrophobic and hydrophilic residues interacting, respectively, with the apolar hydrophobic and polar hydrophilic interfaces within the amyloidogenic fibrillar tubes [106]. A family of mixed (αβαβα) 2 cyclopeptides was designed based on GS and shown to form β-hairpin-like structures [131] that fit the channel interface of Aβ better than GS, probably because of the increased flexibility [106]. Further ThT assays and docking simulations performed on the GS derivatives illustrated the importance of hydrophilic and aromatic residues for interactions with the fibrils.…”