Foldamer-tetra-maleimidopropionyl-G0-PAMAM (TMP) and -bis-maleimido-butane (BMP) conjugates studied.

<p>ACHC stands for trans-2-aminocyclohexanecarboxylic acid. In the structure of <b>7</b>, the H14 helical conformation adopted by the foldamer segments in water is indicated (red), whereas the flexible linker is in an arbitrary conformation (green).</p

Screening for the foldameric recognition segments.

<p>Informative regions of the STD spectra (<b>A</b>–<b>F</b> for <b>1</b>–<b>6</b>, respectively) in the presence of Aβ(1–42) oligomers. The spectra were recorded in 20 mM phosphate buffer at pH 7.4, the total concentration of the Aβ(1–42) was 72 μM, and the ligands were applied at 2 mM. The secondary structure type is indicated to the right of the structures. ‘H’ stands for helix, and the numbers show the size of the H-bonded pseudocycles stabilizing the helices.</p

Affinity precipitation as detected by TEM.

<p><b>(A)</b> TEM image of the 72 µM Aβ oligomer. (<b>B</b>) TEM image of the 72 µM Aβ oligomer upon addition of 36 µM <b>7</b>.</p

Aβ oligomers did not precipitate out of the solution at 1 µM.

<p>Aβ concentrations determined by ELISA in supernatants obtained from samples containing 1 µM Aβ oligomer mixed with 0, 0.2, 0.5 and 1.0 equivalents of <b>7</b> and centrifuged at 15000×g (room temperature, 3 h).</p

NMR assignments and long-range NOE interactions.

<p>Data are displayed for the foldamer segments and the maleimide diastereomers for <b>7</b>, <b>8</b>, <b>9</b> and <b>10</b> in panels <b>A</b>, <b>B</b>, <b>C</b> and <b>D</b>, respectively. Crosspeaks in the overlaid TOCSY (red) and ROESY (blue) spectra prove the H14 structure of the foldamers. The long-range NOEs supporting the helical conformation were observed in aqueous medium. The addition of the thiol group to the maleimido moiety generates an additional stereogenic center. The NMR resonances of the Cys-maleimide linker region are split and their integrals indicate that the addition is not stereoselective; <i>R</i> and <i>S</i> configurations can be found in equimolar ratio (<i>S</i> and <i>R</i> maleimide diastereomers are signed with black and blue, respectively). Since this undetermined configuration moiety is in the flexible part of the molecule, the effect of the chiral center does not propagate further toward either the foldamer part or the PAMAM template.</p

Lower tendency of the LMW Aβ oligomer fraction to affinity precipitation.

<p>(<b>A</b>) SEC analysis of pure Aβ oligomers (black) and mixtures of 50 µM Aβ oligomers with <b>7</b> in 0.25 (green) and 1.0 (blue) equivalents. The samples were injected on the SEC column after filtration. The control SEC chromatogram of <b>7</b> was recorded (red) and it exhibited anomalously longer retention time due to its compact geometrical arrangement. (<b>B</b>) LC-MS results on the LMW fraction taken at 19 min. Both Aβ and <b>7</b> were identified in the chromatogram in a comparable amount. (<b>C</b>) LC-MS results on the HMW precipitate, which confirmed the heterocomplex nature of the product.</p

Ligand 7 protects against Aβ-induced LTP impairment.

<p>(<b>A</b>) The oligomeric Aβ(1–42) sample applied at 720 nM hinders synaptic potentiation. (<b>B</b>) Compound <b>7</b> applied at 950 nM prevents the LTP impairment caused by Aβ(1–42) oligomers. The control substance <b>11</b> has no effect against Aβ(1–42) oligomers. (<b>C</b>) Neither <b>7</b> nor <b>11</b> alone exerted any effect on LTP in the absence of Aβ oligomers. (<b>D</b>) The summarized results observed 180 min after LTP (**P<0.01, ***P<0.001 versus control). Statistical analysis was carried out by using two-tailed Student's t-test, n = 6 or 7 slices per group. Data are presented as means ±SEM. (<b>E</b>) Divalent <b>8</b> applied at 950 nM did not exhibit statistically significant effect against Aβ(1–42) oligomers. Arrows indicate LTP induction, EPSP stands for excitatory postsynaptic potential.</p

Tight binding between ligand 7 and the Aβ oligomers as determined by ITC.

<p>(<b>A</b>) ITC enthalpogram for the titration of the 72 μM Aβ oligomer with <b>7</b> up to 0.55 equivalents (triangles, left scale). Data was fitted with the two independent site model (black). The corresponding z-average diameters (squares) measured by using DLS are displayed on the right vertical scale. (<b>B</b>) ITC enthalpograms obtained for <b>1</b> (diamonds) and <b>8</b> (circles) at 72 μM Aβ oligomer concentration.</p

Design principles of the foldamer-dendrimer conjugates.

<p>Foldamers based on unnatural β-amino acid building blocks (R: proteinogenic side chains) fold into short helices (<b>A</b>). Foldamers exhibiting weak binding to the target can be identified by using NMR spectroscopic methods (<b>B</b>). Chemoselective ligation of the synthetic recognition segments with flexible linkers yields amplified affinity to the target (<b>C</b>). Blue spheres are schematic representation of the Aβ oligomers.</p

Selective binding to the LMW Aβ oligomers in the nM range.

<p>Size exclusion chromatographic separation of the HMW and LMW fractions of the Aβ oligomeric sample (top panel). Concentration-dependent dot blot experiments performed with the HMW (<b>A</b>) and the LMW fractions (<b>B</b>). The ligand loadings were 10 µg aliquots of <b>7</b>, and the sequence specific antibody BAM10 (1∶500) was utilized. Lanes within the panels are parallel experiments.</p