Nanovesicles displaying functional linear and branched oligomannose self-assembled from sequence-defined Janus glycodendrimers

Abstract

Cell surfaces are often decorated with glycoconjugates that contain linear and more complex symmetrically and asymmetrically branched carbohydrates essential for cellular recognition and communication processes. Mannose is one of the fundamental building blocks of glycans in many biological membranes. Moreover, oligomannoses are commonly found on the surface of pathogens such as bacteria and viruses as both glycolipids and glycoproteins. However, their mechanism of action is not well understood, even though this is of great potential interest for translational medicine. Sequence-defined amphiphilic Janus glycodendrimers containing simple mono- and disaccharides that mimic glycolipids are known to self-assemble into glycodendrimersomes, which in turn resemble the surface of a cell by encoding carbohydrate activity via supramolecular multivalency. The synthetic challenge of preparing Janus glycodendrimers containing more complex linear and branched glycans has so far prevented access to more realistic cell mimics. However, the present work reports the use of an isothiocyanate-amine "click"-like reaction between isothiocyanate-containing sequence-defined amphiphilic Janus dendrimers and either linear or branched oligosaccharides containing up to six monosaccharide units attached to a hydrophobic amino-pentyl linker, a construct not expected to assemble into glycodendrimersomes. Unexpectedly, these oligoMan-containing dendrimers, which have their hydrophobic linker connected via a thiourea group to the amphiphilic part of Janus glycodendrimers, self-organize into nanoscale glycodendrimersomes. Specifically, the mannose-binding lectins that best agglutinate glycodendrimersomes are those displaying hexamannose. Lamellar "raft-like" nanomorphologies on the surface of glycodendrimersomes, self-organized from these sequence-defined glycans, endow these membrane mimics with high biological activity.

Keywords: automated glycan assembly; cell membrane mimics; isothiocyanate–amine coupling.

Fig. 1.

Synthetic route for preparation of sugars containing triethylene glycol azido and amino linkers (A). Synthetic route for sugar containing the hydrophobic N-pentyl amino linkers (B). Automated glycan assembly of oligoMan containing N-pentyl amino linkers (C). Reagents and conditions: (i) BF₃·Et₂O, CH₂Cl₂, 0 °C to 23 °C, 15 h, 80%; (ii) NaN₃, DMF, 80 °C, 15 h, 74%; (iii) MeONa, methanol, pH = 10, 23 °C, 7 h, 95%; (iv), H₂, Pd/C, methanol, 23 °C, 15 h, 95%; (v) H₂NNH₂·HOAc, DMF, then CCl₃CN, DBU, CH₂Cl₂, 79%; (vi) TMSOTf, CH₂Cl₂, 39%; (vii) MeONa, methanol, then H₂, Pd/C, 82%.

Fig. 2.

Synthesis of ITC functionalized JD. Reagent and conditions: (i) NaN₃, DMF, 15 h, 85 °C; (ii) SOCl₂, benzyltriethylammonium chloride (BTEAC), 65 °C, 2 h, 93% for 2 steps; (iii) K₂CO₃, DMF, 80 °C, 48 h, 94%; (iv) H₂, Pd/C, methanol, 55 °C, 15 h, 100%; (v) K₂CO₃,18-C-6, DMF, 80 °C, 15 h, 90%; (vi) KOH, ethanol, 90 °C, 2 h, then 2 M HCl, 100%; (vii) CDMT, NMM, THF, 12 h, 93%; (viii) DCC, DPTS, CH₂Cl₂, 23 °C, 24 h, 80%; (ix) 2 M HCl, methanol, 23 °C, 2 h, 98%, (x) DCC, DPTS, CH₂Cl₂, 23 °C, 24 h, 93%; (xi) H₂, Pd/C, methanol, 12 h, 81%; (xii) CS₂, NEt₃, THF, 0 °C, then TsCl, 23 °C, 95%.

Fig. 3.

Sequence-defined JGDs obtained by conventional click reaction via CuAAC (20) (A) and by ITC–amine “click”-like reaction (B), reported here.

Fig. 4.

Oligosaccharides with pentyl-amino linker used for the preparation of JGDs via ITC–amine “click” reaction. Man and Lac were prepared by solution phase synthesis. Man, Gal, Glc, GlcNAc, and oligoMan were prepared by AGA.

Fig. 5.

Library of sequence-defined amphiphilic JGDs containing monosaccharide (Man, Gal, Glc, and GlcNAc), disaccharide (Lac), and oligoMan. An amphiphilic JD, JD-3 (8), which was coassembled with JGDs, is presented in the top row, left side.

Fig. 6.

GDSs self-assembled from JGDs containing Gal and Lac prepared by ITC–amine “click” reaction with pentyl (C5) or triethylene glycol (3EO) linkers and their agglutination with human galectin-1 (Gal-1) and galectin-8S (Gal-8S).

Fig. 7.

Agglutination assays of GDSs from amphiphilic JGDs containing oligoMan with ConA. (A–C) Agglutination assays of GDS from JGDs containing Man (A), diMan (B), and triMan (C). The GDSs were prepared by self-assembly of the JGDs (100%) or coassembled with JD-3 (20 and 50 mol % of JGDs). (D and E) Agglutination assays of GDS from of JGDs containing (α1–2) oligoMan (D), (α1–6) oligoMan (E), and comparison of three JGDs containing isomeric linear or branched trimannose (F). The GDSs were prepared by coassembled with JD-3 (20 mol % of JGDs).

Fig. 8.

Surface topography of GDS self-organized from Lac-containing sequence-defined JGDs including JGD(5/1_Lac^C5) (A–C) with a hydrophobic C5 linker and JGD(5/1_Lac^3EO) (D–F) with a hydrophilic 3EO linker deposited on mica. AFM height images (A and D), phase images (B and E) with inserted FFT, and the corresponding height profiles (C and F) of GDSs on mica measured at ambient humidity (40%). Arrows in A and D indicate the directions for analysis of the height profiles in C and F.