Supramolecular polymerization of sulfated dendritic peptide amphiphiles into multivalent L-selectin binders

The synthesis of a sulfate-modified dendritic peptide amphiphile and its self-assembly into one-dimensional rod-like architectures in aqueous medium is reported. The influence of the ionic strength on the supramolecular polymerization was probed via circular dichroism spectroscopy and cryogenic transmission electron microscopy. Physiological salt concentrations efficiently screen the charges of the dendritic building block equipped with eight sulfate groups and trigger the formation of rigid supramolecular polymers. Since multivalent sulfated supramolecular structures mimic naturally occurring L-selectin ligands, the corresponding affinity was evaluated using a competitive SPR binding assay and benchmarked to an ethylene glycol-decorated supramolecular polymer.


General considerations Solvents and reagents
Unless stated otherwise, all solvents and reagents were obtained from commercial sources in at least pro analysi (p.a.) quality and were used without further purification. The absolutation of solvents was performed according to literature-known procedures [S1]. Ultrapure water was obtained from an Elga PURELAB flex 4 system by Veolia (Paris, France). The propargylated [G2]-dendron was synthesized in accordance to literature-known procedures [S2].

Reaction conditions
Air-and moisture-sensitive reactions were carried out under argon atmosphere with common Schlenk techniques and dry solvents. For this purpose, all glassware were previously ovendried or dried via a heat gun in vacuo at least three times. In addition, all necessary solvents and liquid starting materials were added via a septum and with disposable syringes, which were previously flushed with argon. Solids were added under a continuous counterflow of argon.

Chromatography
All flash-chromatographic purifications were carried out using silica gel with an average particle size of 35-70 μm and a pore size of 60 Å by Acros Organics (Geel, Belgium). A nitrogen pressure of 0.3-0.5 bar was applied. The eluents were freshly prepared of pro analysi grade solvents or distilled technical grade solvents. The analysis of the collected fractions was performed via TLC. The TLC-analyses were carried out on silica-coated aluminum sheets 60 F254 by Merck KGaA (Darmstadt, Germany) with fluorescence indicator. The analytes were detected by the following methods: UV absorption at a wavelength of 254 nm; vanillin-stain (solution prepared of 100 mL methanol with 1.0 g vanillin, 12 mL acetic acid, and 4mL conc.
sulfuric acid); ninhydrin stain (solution prepared of 1.5 g ninhydrin in 500 mL methanol and 15 mL acetic acid); KMnO4 stain (solution prepared of 6 g KMnO4, 40 g K2CO3 and 13 mg NaOH in 600 mL H2O. In case of staining agents, the plate is immersed shortly and the color reaction subsequently proceeds upon heating.

NMR spectroscopy
All 1 H and 13 C NMR spectra were recorded on a Bruker Avance II 400 spectrometer equipped with a 5 mm broadband observe probehead (z-gradient) using standard Bruker release pulse

Mass spectrometry
High resolution electrospray ionization mass spectra (ESI-HRMS) were recorded on an Agilent 6545 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, USA) with a LockSpray interface. Alternatively, ionization using field desorption was performed on a MAT 95 by Finnigan (San Jose, USA). MALDI samples were measured on an Autoflex maX MALDI-TOF/TOF device by Bruker. The corresponding matrices used are stated in the synthetic procedures.

CD
All circular dichroism spectra were recorded on a J-815 spectrometer from JASCO (Tokyo, Japan) and processed with Spectra Manager Version 2.12.00. The samples were measured using 110-QS cuvettes made of SUPRASIL® quartz glass by Hellma (Mühlheim, Germany) with a path length of 2 mm at 293°K, if not stated otherwise. The spectra were plotted in MS Excel® 365. For each solvent a blank spectrum was measured and subtracted from the raw data.

TEM
The transmission electron microscopy (TEM) images were recorded on a Tecnai T12 by FEI (Hillsboro, USA), which was equipped with a LaB6 cathode operating at 120 kV. The electron micrographs were recorded with a 4k × 4k CMOS by TVIPS (Oslo, Norway). The copper grids CF300-CU with a 3-4 nm thick carbon film from Electron Microscopy Sciences (Hatfield, USA) were glow discharged prior to use. For each analysis, 5 μL (10-100 μM aqueous solution) of the test item was adsorbed on the grid for 2 min. The grids were then stained for 1 min with 2.0 wt % uranyl acetate from Polysciences (Warminster, USA). The generated droplet was S5 removed with a Whatman® grade 4 filter paper tip from GE Healthcare Bio Sciences (Uppsalla, Sweden).

Cryo TEM
Droplets of the sample solution (5 µL) were applied on hydrophilized holey carbon filmed grids

L-Selectin binding assay
The L-selectin binding assay was performed in a similar manner as described in [S3]. The binding of soluble L-selectin binders was studied via a competitive SPR-based inhibition assay performed on a BIAcore X device (GE Healthcare, Freiburg, Germany) as previously explained in detail [S4-S6]. In brief, the assay relies on the L-selectin binding to an artificial ligand, which is composed of sulfated tyrosine and the tetrasaccharide sialyl Lewis X multimerized on a polyacrylamide backbone and attached via biotin to a streptavidin-coated gold chip (GE Healthcare, Freiburg, Germany). Protein A-coated gold nanoparticles (15 nm, Aurion, Wageningen, Netherlands) were loaded with an L-selectin/Fc chimera (R&D Systems, Minneapolis, USA), and the binding signal to the artificial ligand was recorded. Samples without inhibitors were set as a 100% binding control. In turn, binding signals of the L-selectin-loaded gold nanoparticles preincubated with different concentrations of the potential inhibitors were taken, which yielded the respective dose-response curves. The IC50 values were determined by fitting the relative binding response data with a sigmoidal dose-response curve.

BTAF3AhxEG2NH2, S9
The polypeptide was synthesized using a custom solid-support from Rapp Polymere O-

S12
Freshly prepared stock solutions of the reactants were prepared prior to the reaction.

Following concentrations were provided:
A S12 20 mg/mL in DMF B Propargylated dendron 20 mg/mL in DMF C TBTA 25 mM in DMF D CuSO4×5H2O 50 mM in H2O E NaAsc 100 mM in H2O Solutions A (2.0 µmol, 1 equiv), B (2.2 equiv), C (1.6 equiv), and D (2.0 equiv) were dispensed and diluted with a mixture of TFE/DMF 1:1 to reach a final concentration of 1 mg/mL, calculated for the functional monomer. The mixture was purged with argon and subjected to two freezepump-thaw cycles. Subsequently, solution E (4 equiv) was introduced and the reaction mixture was once more subjected to a freeze-pump-thaw cycle. The reaction proceeded over 3 d at 50 °C. The solvents were removed in vacuo and the residing product taken up in DMF and subjected to SEC on S-X1 (DMF). The DMF was removed in vacuo and the residing film rehydrated with H2O to yield the corresponding conjugate after lyophilization.  Figure S5: 1 H NMR spectrum of I (600 MHz, DMSO-d6, 294 K). S16 Figure S6: 13 C NMR spectrum of I (151 MHz, DMSO-d6, 294 K).