Supramolecular Self‐Assembly of β3‐Peptides Mediated by Janus‐Type Recognition Units

Abstract To gain mechanistic insights, natural systems with biochemical relevance are inspiring for the creation of new biomimetics with unique properties and functions. Despite progress in rational design and protein engineering, folding and intramolecular organization of individual components into supramolecular structures remains challenging and requires controlled methods. Foldamers, such as β‐peptides, are structurally well defined with rigid conformations and suitable for the specific arrangement of recognition units. Herein, we show the molecular arrangement and aggregation of β3‐peptides into a hexameric helix bundle. For this purpose, β‐amino acid side chains were modified with cyanuric acid and triamino‐s‐triazine as complementary recognition units. The pre‐organization of the β3‐peptides leads these Janus molecule pairs into a hexameric arrangement and a defined rosette nanotube by stacking. The helical conformation of the subunits was indicated by circular dichroism spectroscopy, while the supramolecular arrangement was detected by dynamic light scattering and confirmed by high‐resolution electrospray ionization mass spectrometry (ESI‐HRMS).


GSP 1: Synthesis of diazoketone
Under inert gas atmosphere, a solution of the amino acid (1.00 eq) in anhydrous tetrahydrofuran (THF) (~10 mL/g amino acid) was cooled to -21 °C. After addition of triethylamine (NEt3, 1.10 eq) and isobutylchloroformate (1.10 eq) the reaction mixture was stirred 45 min at -21 °C to -15 °C. Then, the reaction mixture was warmed up to 0 °C and diazomethane [1] in dieethylether (Et2O, 0.6-0.7 M, 2.00 eq) was added under light exclusion. The solution was stirred first for 0.5 h at 0 °C, then 4-6 h at room temperature (RT) and quenched afterwards with AcOH (2.00 eq). Et2O (~10 mL/g amino acid) and sat. NaHCO3-solution (~10 mL/g amino acid) or a 6% aq. NaHCO3-solution [~10 mL/g per 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acid] was given and the layers were separated. The organic layer was washed with sat. NH4Cl-solution (~10 mL/g amino acid) and sat. NaCl-solution (~10 mL/g amino acid), dried over MgSO4 and the solvent was removed in vacuo. The product was immediately used for the next step or purified via flash column chromatography.

GSP 2: Synthesis of β 3 -amino acids
A solution of the diazoketone from GSP 1 (1.00 eq) in THF/H2O (9:1, v/v) was first cooled to 0 °C and then treated with silver benzoate (0.10 eq) excluding light and ultrasonicated for 1.5-4.5 h. The reaction was followed by TLC and diluted after full conversion with H2O (~10 mL/g amino acid). The pH was adjusted with 1 M aq. HCl to 2 and Et2O or ethyl acetate (EtOAc, ~10 mL/g amino acid) was added. The layers were separated and the aqueous layer was extracted with Et2O or EtOAc (3 × ~10 mL/g amino acid). The combined organic extracts were dried over MgSO4 and the solvent was removed under vacuum.
The crude product was purified by flash column chromatography.
The reaction solution was then adsorbed on silica gel and purified by column chromatography using the eluents DCM/MeOH 1 The moderate yield of 50% for the hydroxy-substituted product 7 was caused by classical competition between substitution and elimination reactions. The formation of the elimination product (23%) under the required conditions (pH = 9.5, 65 °C.) was unavoidable, which is why the reaction was monitored by thin layer chromatography (TLC) and stopped immediately after the starting material had completely converted. 2 The waste product of triphyenylphospine, triphenylphospine oxide, was difficult to completely separate under applied conditions.
(3) Coupling was realized by adding of an activated solution consisting of the amino acid (5.0 eq/3.0 eq), HOAt (5.0 eq/3.0 eq), HATU (4.5 eq/2.7 eq) and DIEA (10 eq) in DMF (450 µL per 10 µM resin) to the resin followed by microwave irradiation (25 W, 60 °C, 10 min). The coupling of Fmoc-ACHC-OH (9) was adapted from a procedure described by GELLMANN [6] : an activated solution of the amino acid (5.0 eq/3.0 eq), HATU (4.5 eq/2.7 eq), HOAt (5.0 eq/3.0 eq) and and incubated for 0.5-1 h at room temperature to reach full deprotection of protecting groups (here Boc). After cleavage, excess of TFA was removed by a gentle N2 stream. The product was precipitated in -21 °C cold Et2O (3 × 8 mL) and centrifuged for 30 min at 9000 rpm and -10 to 0 °C. The supernatant was discarded. The procedure of precipitation and centrifugation was repeated twice and the crude product was dried in vacuum. The crude peptide was dissolved in aqueous CH3CN and purified by HPLC followed by freeze-drying of the purified product and storage at -21 °C.    Concentration-and time-dependent CD measurements:

SUPPORTING INFORMATION
S13 CD spectra were performed from equimolar mixture of 1 and 2. Therefore, peptides 1 and 2 were mixed at different concentrations ranging from 50 to 500 µM, measured within 20 min and after overnight incubation at 4 °C. After CD measurements, UV spectrum of each sample was recorded for concentration-and time-dependent UV measurement (see Figure S3 C and D).

Electrospray Ionization (ESI) Mass Spectrometry
The data shown for the aggregates were recorded on a micrOTOF-Q II instrument from BRUKER DALTONIK (Bremen, Germany). An equimolar mixture of β 3 -CYA decapeptide 1 (25 µM) and β 3 -TAT decapeptide 2 (25 µM) in 5 mM ammonium acetate (pH = 7.1) was prepared, vortexed (10 s), centrifuged (10 s) and incubated overnight at 4 °C. Prior measurement, the sample was diluted to a final concentration of 20 µM (10 µM each peptide) and injected with a flow rate of 8 µL min -1 . The ESI source in the positive mode was operated at an ESI voltage of -4500 V for capillary and -500 V for end plate offset with nitrogen as nebulizer gas (5 L min −1 flow rate) and drying gas (0.7 bar backing pressure, 80 °C temperature).