Amphiphilic dendrons as supramolecular holdase chaperones

The aggregation of incompletely or incorrectly folded proteins is implicated in diseases including Alzheimer's, cataracts, and other maladies. Natural systems express protein chaperones to prevent or even reverse harmful protein aggregation. Synthetic chaperone-like systems have sought to mimic the action of their biological counterparts but typically require substantial optimization and high concentrations to be functional, or lack programmability that would enable the targeting of specific protein substrates. Here we report a series of amphiphilic dendrons that undergo assembly and inhibit the aggregation of fragment 16–22 amyloid β protein (Aβ16–22). We show that monodisperse dendrons with hydrophilic tetraethylene glycol chains and a hydrophobic core based on naphthyl and benzyl ethers undergo supramolecular assembly in aqueous solutions to form sphere-like particles. The solubility of these dendrons and their assemblies is tuned by varying the relative sizes of their hydrophilic and hydrophobic regions. Two water-soluble dendrons are discovered and shown, via fluorescence experiments with rhodamine 6G, to generate a hydrophobic environment. Furthermore, we demonstrate that sub-stoichiometric concentrations of these amphiphilic dendrons stabilize Aβ16–22 peptide with respect to aggregation, mimicking the activity of holdase chaperones. Our results highlight the potential of these amphiphilic molecules as the basis for a novel approach to artificial chaperones that may address many of the challenges associated with existing synthetic chaperone mimics.


Materials and Methods
Materials.Anhydrous solvents were used in all reactions except for the synthesis of 2 and were either prepared from commercial solvents using a Pure Process Technology Solvent Purification System (CH2Cl2 and THF) or purchased as anhydrous reagents directly from Thermo Fisher Scientific (CH2Cl2, THF and DMF).NaHCO3 used for esterification reactions was stored in a desiccator to prevent water adsorption.LiCl and LiAlH4 (2.4 M in THF) (both from Acros), NaHCO3 (from EMD Millipore), HCl (12 M aq.) (from Macron Fine Chemicals), DMSO-d6 (from Cambridge Isotope Laboratories), thionyl chloride (2 M in CH2Cl2) (from Sigma-Aldrich), tetraethylene glycol (4, from Combi-Blocks), 1,4-dihydroxy-2-naphthoic acid (6, from TCI Chemicals) and methyl 3,4dihydroxybenzoate (10, Biosynth) were used as received.All other chemicals were purchased from Thermo Fisher Scientific and used as received.Deionized water was used for all aqueous washes and solutions.
General Methods.All reactions (except for the synthesis for 2) were conducted under a nitrogen atmosphere using anhydrous solvents in glassware that was dried in an oven (120 °C) for at least 30 min and cooled under vacuum prior to use.Room temperature denotes ambient temperature in our laboratories, 23 ± 2 °C.
Column chromatography.Column chromatography was carried out using a Büchi Pure C-810 Flash Chromatography System using prepacked Büchi EcoFlex Silica columns as the stationary phase and the indicated solvents as mobile phase.Elution of desired products was monitored using absorption at 254 nm.Fractions were visualized by thin-layer chromatography on silica gel plates.
Nuclear magnetic resonance (NMR) spectroscopy.NMR spectra were recorded on a Bruker DPX-500 instrument at ambient temperature in DMSO-d6. 1 H and 13 C spectra were referenced to the signal arising from residual nondeuterated solvent.Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants, integration.
Mass spectrometry.High resolution mass spectrometry (HRMS) data were recorded were recorded on a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer in electrospray ionization mode.
Samples were prepared at a concentration of 10 µg/mL in a 50:50 mixture of MeCN and water + 0.1 % formic acid.

Synthesis of Aβ16-22 Peptide
Fragment 16-22 β amyloid peptide (Aβ16-22) was a kind gift from the Nilsson group at the University of Rochester, synthesized through solid-phase peptide synthesis and purified using analytical high-pressure liquid chromatography (HPLC). 6Lyophilized, purified peptide samples were dissolved in 60% MeCN in water with 1% trifluoroacetic acid and sonicated for 5 min.Peptide concentrations were analyzed via analytical HPLC using a reverse phase Phenomenex Gemini column (5 μm, C18, 110 Å, 250 × 4.6 mm, Phenomenex) on a Shimadzu LC-2010A for comparison to a concentration curve calibrated by amino acid analysis.The peptide solution was aliquoted into Eppendorf tubes, frozen, then lyophilized.Immediately prior to use, each lyophilized aliquot was dissolved in water to a final concentration of 500 μM.

Materials and Methods
UV-vis spectra were recorded on an Agilent Cary 3500 Multicell Peltier.Spectra were recorded with a scanning speed of 600 nm/min, average time of 0.100 s, a data interval of 1 nm, and spectral bandwidth of 2.00 nm.Solutions were measured in a capped quartz cuvette (Starna Cells) with a path length of either 1 mm or 10 mm, selected to ensure the maximum absorbance was between 0.1 and 3. Solutions in 10 mm cuvettes were stirred with a magnetic stirrer bar at 600 rpm.
Solutions of 1 and 3 were prepared from aqueous stock solutions (1000 µM and 250 µM, respectively) by dilution to the appropriate concentration with water.Solutions of 2 were prepared from a stock solution (1000 µM) in MeCN (spectrophotometric grade, Thermo Scientific) by dilution to the appropriate concentration with water and MeCN.
For full-spectrum studies, solutions were heated at a rate of 0.5 °C/min from 20 to 75 °C, held at 75 °C for 10 min, then cooled from 75 to 20 °C.Spectra were recorded from 700 to 200 nm at 20, 40, 60 and 75 °C upon both heating and cooling.
For discrete wavelength studies, solutions were heated at a rate of 0.5 °C/min from 20 to 75 °C, held at 75 °C for 10 min, then cooled from 75 to 20 °C.This heat-hold-cool cycle was repeated three times.The absorbance at 217, 249, 295, and 343 nm was recorded at 1 °C intervals.Full-wavelength spectra from 700 to 200 nm were recorded at 20 °C at the start and end of the experiment (i.e., before the first heating and after the third cooling).S8     Circularity is a measure of the ratio of the area of the particle divided by the area of a circle with an equivalent perimeter length.Area, perimeter, and circularity were determined from the AFM height data shown in Figure 3a-d.The more circular a particle, the closer to 1 this value will be.A circularity of 1 denotes a perfectly circular particle while a circularity of 0 denotes a highly irregular, non-circular particle.Broken grey line at circularity = 1 is a guide for the eye.

Materials and Methods
Transmission electron microscopy (TEM) measurements were performed on a Hitachi 7650 TEM working at 80 kV with an attached 11-megapixel Gatan Erlangshen digital camera for image capture.Images were recorded using Digital Micrograph software.
Solutions of 1 and 3 were prepared in the same way as for AFM (SI Section 4.1).For staining, 10 µL of the sample solution together with 10 µL of phosphotungstic acid (PTA) solution (2% aq., pH 6.5) were loaded onto 200 mesh carbon/formvar copper grids and incubated for 2 min.The excess solution was wicked off with filter paper and the grids were allowed to dry.

TEM Data
Figure S8.TEM of dendrons at 30 µM.TEM images of (a) 1 and (b) 3 after negative staining with phosphotungstic acid.
TEM images of 1 (Figure S8a) show poorly assembled morphologies with only a few particle-like structures.Given the amphilphilic nature of dendrons 1 and 3 and the significantly smaller hydrophobic region in 1 compared to 3, we hypothesize that the PTA stain may be disrupting the assembly of 1.

Materials and Methods
Fluorescence emission spectra were recorded at room temperature on a Spex Fluoromax-3 fluorometer (Jobin-Yvon Horiba) from 400 to 700 nm, with an excitation wavelength of 488 nm, slit widths of 2 nm, and integration time of

Figure S1 .
Figure S1.Assembly of 2 in aqueous MeCN.UV-vis spectra of 2 collected upon cooling solutions from 75 °C (red line) to 20 °C (blue line) at the indicated concentrations in (a-d) 10% MeCN in water and (e) 20% MeCN in water.Spectra were collected at 75, 60, 40, and 20 °C.Spectra in a and b collected in a cuvette with path length (l) of 10 mm; spectra in c-e were collected in a cuvette with l = 1 mm.(f) Magnified section of the spectrum in c.

Figure S2 .
Figure S2.Assembly of 3 in water.UV-vis spectra of 3 collected upon cooling aqueous solutions at the indicated concentrations from 75 °C (red line) to 20 °C (blue line).Spectra were collected at 75, 60, 40, and 20 °C.Spectra in a collected in a cuvette with path length (l) of 10 mm; spectra in b-d were collected in a cuvette with l = 1 mm.

Figure S3 .
Figure S3.Assembly of 1 in water.UV-vis spectra of 1 collected upon cooling aqueous solutions at the indicated concentrations from 75 °C (red line) to 20 °C (blue line).Spectra were collected at 75, 60, 40, and 20 °C.Spectra in a and b collected in a cuvette with path length (l) of 10 mm; spectra in c and d were collected in a cuvette with l = 1 mm.

Figure S6 .
Figure S6.AFM of 1 at 30 µM.AFM (a) height and (b) amplitude images of 1 spin-coated on a mica substrate prepared from a 30 µM solution in water that was heated to 75 °C and subsequently cooled to 25 °C (at 0.5 °C/min).Scale bars = 200 nm.Note that (a) is identical to Figure 3a in the main text.

Figure S7 .
Figure S7.Degree of circularity of 1 and 3. Circularity is a measure of the ratio of the area of the particle divided by the area of a circle with an equivalent perimeter length.Area, perimeter, and circularity were determined from the AFM height data shown in Figure3a-d.The more circular a particle, the closer to 1 this value will be.A circularity of 1 denotes a perfectly circular particle while a circularity of 0 denotes a highly irregular, non-circular particle.Broken grey line at circularity = 1 is a guide for the eye.

Figure S13 .
Figure S13.Samples of CR and Aβ16-22 after 26 h.(a) UV-vis spectra of the samples from Figure S12 after incubation at 25 °C for 26 h after sample preparation.Spectra were collected in a cuvette with path length (l) of 1 mm at 25 °C.(b) Photograph of the samples from a immediately after UV spectra were collected.Formation of a precipitate and a change in color are visible in cuvettes containing CR + Aβ16-22 and CR + Aβ16-22 + 1.