Structural Diversity of Peptoids: Tube-Like Structures of Macrocycles

Peptoids, or poly-N-substituted glycines, are characterised by broad structural diversity. Compared to peptides, they are less restricted in rotation and lack backbone-derived H bonding. Nevertheless, certain side chains force the peptoid backbone into distinct conformations. Designable secondary structures like helices or nanosheets arise from this knowledge. Herein, we report the copper-catalysed alkyne-azide cycloaddition (CuAAC) of macrocycles to form innovative tube-like tricyclic peptoids, giving access to host–guest chemistry or storage applications. Different linker systems make the single tubes tuneable in size and enable modifications within the gap. An azobenzene linker, which is reversibly switchable in conformation, was successfully incorporated and allowed for light-triggered changes of the entire tricyclic structure.


General experimental procedures
Solvents and reagents were purchased from commercial sources and used without further purification. Abbreviations are as follows: 1,1,1,3,3,3-hexafluoroisopropyl alcohol (HFIP), acetonitrile (ACN), Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (PyBOP), N,N'-diisopropylcarbodiimide (DIC), N,N'diisopropylethylamine (DIPEA), peptide grade dimethylformamide (pDMF), lithium diisopropylamide (LDA), tetrahydrofuran (THF). Reagents and products were weighted on SARTORIUS analytical scales, models LA310S and BP211D. Moisture and air-sensitive reactions were carried out under argon according to the common SCHLENK technique [1]. Liquids were transferred via plastic syringes and V2A-steel needles. Powdered solids were added against an argon flow. Reactions at 0 °C were cooled with an ice/water bath. Purification by column chromatography was performed according to STILL [2]. Silica gel 60 (MERCK, 0.04-0.063 mm) served as a stationary phase. Solvents for elution were purchased in an analytical-grade purity and used mixtures are reported as volume ratios (v/v). Freeze-drying of aqueous solutions was performed on a lyophilisator from CHRIST model Alpha 1-2 LD plus, equipped with a VACUUBRAND RZ 2.5 pump.

Solid-phase synthesis
Solid-phase synthesis of linear peptoids was performed by the published submonomer technique [3,4] in 6 mL plastic-fritted syringes (MULTISYNTHEC GmbH), closed with a plastic cap. As a solid support, a 2-chlorotrityl chloride resin (CARBOLUTION, 1.60 mmol/g loading density) was used. Reaction steps were performed on a KS 501 digital circular shaker (IKA-LABORTECHNIK) at room temperature. Yields were calculated according to the resin loading value.

S3
General Procedure (GP1) for the synthesis of cyclic peptoids [3,5] For the synthesis of linear precursors in a fritted syringe, a 2-chlorotrityl chloride resin (300 mg, 0.480 mmol, 1.60 mmol/mg loading density, 100-200 mesh, 1.00 equiv.) was swollen in 3.00 mL of methylene chloride for 2 h. After filtration, a freshly prepared solution of bromoacetic acid (2.59 mmol, 5.40 equiv.) and N,N'-diisopropylethylamine (DIPEA, 2.59 mmol, 5.40 equiv.) in 2.5 mL of DCM was added and shaken for 1 h at 21 °C. The resin was extensively washed with peptide grade N,N'-dimethyl-formamide (pDMF). For the following substitution reaction, a solution of the corresponding amine (3.98 mmol, 8.30 equiv.) in 2.5 mL of pDMF was added to the resin and shaken for 30 min at room temperature (overnight in case of aniline). Following extensive washing with pDMF, a solution of bromoacetic acid (4.80 mmol, 10.0 equiv.) and N,N'diisopropylcarbodiimide (DIC, 4.80 mmol, 10.0 equiv.) in 2.5 mL pDMF were added and shaken for 30 min at room temperature (2 h in the case of aniline). The acetylation and substitution steps were alternated repeatedly until the desired peptoid length was achieved. For cleavage, a solution of 33% hexafluoroisopropanol in DCM was added and the mixture was shaken overnight. The solvent was removed under reduced pressure and the residue was resolved in 10.0 mL of acetonitrile/water (1:1). The mixture was lyophilized overnight to gain a colorless powder.
Without any purification, the full amount of the linear precursor was resolved in 100 mL of dry methylene chloride and degassed with argon. For the cyclization following a protocol by KIRSHENBAUM [5], benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 1.44 mmol, 3.00 equiv.) and DIPEA (2.88 mmol, 6.00 equiv.) were added. The mixture was stirred at room temperature overnight. The solvent was removed under reduced pressure and purification was performed via preparative reverse phase HPLC.

General Procedure (GP2) for the CuAAC of two peptoids [6]
Under inert conditions, both peptoids (1.00 equiv. each) were dissolved in dry methylene chloride. Afterwards, 2,6-lutidine (6.00 equiv.) was added to the solution and stirred for 5 min. Then, tetrakis(acetonitrile)copper(I) hexafluorophosphate (Cu(CH3CN)4PF6, 1.00 equiv.) was added and the mixture was stirred for 3 days. The solvent was removed under reduced pressure and the product was purified via preparative reverse phase HPLC.

General Procedure (GP3) for the CuAAC of a peptoid and a linker [6]
Under inert conditions, the peptoid (1.00 equiv.) and the linker (5.00-10.0 equiv.) were dissolved in dry methylene chloride. Afterwards, 2,6-lutidine (8.00 equiv.) was added to the solution and stirred for 5 min. Then, Cu(CH3CN)4PF6 (1.00 equiv.) was added and the mixture was stirred for 3 days. The solvent was removed under reduced pressure and the product was purified via preparative reverse phase HPLC.

Synthesis of small molecules 3-Azidopropan-1-amine (17) [7]:
3-Chloropropylamine hydrochloride (6.00 g, 46.0 mmol, 1.00 equiv.) was dissolved in 100 mL of water. Sodium azide (9.00 g, 138 mmol, 3.00 equiv.) was added and the mixture was stirred for 18 h at 21 °C . The solvent was reduced to 2/3 of its volume. The remaining mixture was cooled to 0 °C and 100 mL of diethyl ether were added. Potassium hydroxide (6.00 g, 107 mmol, 2.30 equiv.) was added in portions and the organic layer was separated. The aqueous layer was extracted twice with diethyl ether, the organic layers were combined, dried over Na2SO4 and the solvent was removed under reduced pressure. The product was obtained as a colorless liquid in 82% yield (3.78 g, 37.8 mmol).

Switch experiments
For switch experiments via NMR, the sample was dissolved in deuterated acetonitrile and stored under exclusion of light. Measurements were performed on a BRUKER 500 spectrometer. 1 H signals were recorded with 500 MHz. After the measurement, the sample was radiated for 30 min with UV light and an additional spectrum was accommodated.    For switch experiments via UV-VIS, a 20 μM solution of each sample was prepared and stored under exclusion of light. Measurements were performed on a LAMBDA 750 spectrometer from PERKINELMER. The spectrum was recorded in the range of 200 nm to 800 nm wavelength. After the measurement, the sample was radiated with UV light (365 nm) for 30 sec and the measurement was repeated. Reversibility was recorded after irradiation with UV light (365 nm) for 1 min first and subsequent irradiation with visible light (460 nm) for 1 min.