Versatile synthesis of end-reactive polyrotaxanes applicable to fabrication of supramolecular biomaterials

Abstract

Cyclodextrin (CD)-threaded polyrotaxanes (PRXs) with reactive functional groups at the terminals of the axle polymers are attractive candidates for the design of supramolecular materials. Herein, we describe a novel and simple synthetic method for end-reactive PRXs using bis(2-amino-3-phenylpropyl) poly(ethylene glycol) (PEG-Ph-NH₂) as an axle polymer and commercially available 4-substituted benzoic acids as capping reagents. The terminal 2-amino-3-phenylpropyl groups of PEG-Ph-NH₂ block the dethreading of the α-CDs after capping with 4-substituted benzoic acids. By this method, two series of azide group-terminated polyrotaxanes (benzylazide: PRX-Bn-N_3, phenylazide: PRX-Ph-N_3,) were synthesized for functionalization via click reactions. The PRX-Bn-N₃ and PRX-Ph-N₃ reacted quickly and efficiently with p-(tert-butyl)phenylacetylene via copper-catalyzed click reactions. Additionally, the terminal azide groups of the PRX-Bn-N₃ could be modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules via a copper-free click reaction; this fluorescently labeled PRX was utilized for intracellular fluorescence imaging. The method of synthesizing end-reactive PRXs described herein is simple and versatile for the design of diverse functional PRXs and can be applied to the fabrication of PRX-based supramolecular biomaterials.

Keywords: azide group; biomaterials; click chemistry; cyclodextrin; polyrotaxane.

Scheme 1

Scheme for the end-capping of the PEG-NH₂ (1)/α-CD pseudopolyrotaxane with 4-(azidomethyl)benzoic acid (2a) (Reaction 1). Synthesis of end-reactive PRXs (4a–c) by the end-capping of the PEG-Ph-NH₂ (3)/α-CD pseudopolyrotaxane with 2a–c (Reaction 2).

Figure 1

SEC charts for crude products of reaction 1 (upper chart) and 2 (lower chart).

Figure 2

(A) SEC charts of α-CD, PEG-Ph-NH₂ (3), PRX-Bn-N₃ (4a), PRX-Ph-N₃ (4b), and PRX-Ph-Me (4c). (B) FTIR transmission spectra of α-CD, PEG-Ph-NH₂ (3), PRX-Bn-N₃ (4a), PRX-Ph-N₃ (4b), and PRX-Ph-Me (4c).

Scheme 2

End-group modification of 4a-c with model alkyne via copper-catalyzed click reaction.

Figure 3

(A) ¹H NMR spectra of the PRXs before (4a, 4b, 4c) and after copper-catalyzed click reactions with p-(tert-butyl)phenylacetylene for 60 min (5a, 5b, 5c). Spectra were recorded in DMSO-d₆. (B) Time-course of click reaction between the azide-terminated PRXs (4a: open circles, 4b: closed squares) and p-(tert-butyl)phenylacetylene.

Scheme 3

Scheme of the synthesis of water-soluble PRX (6) and the following end group modification with copper-free click reaction (7).

Figure 4

SEC charts of the HEE-PRX-Bn-N₃ (6) (A, B) and the HEE-PRX-DF488 (7) (C, D) monitored with fluorescence detector (excitation wavelength: 488 nm, emission wavelength: 515 nm) (A, C) and refractive index (RI) detector (B, D).

Figure 5

CLSM images of HeLa cells treated with HEE-PRX-DF488 (7) (500 μg/mL) for 26 h (scale bars: 20 μm). The acidic endosomes/lysosomes and nuclei are stained with LysoTracker Red DND-99 and Hoechst 33342, respectively.