Design and synthesis of multivalent α-1,2-trimannose-linked bioerodible microparticles for applications in immune response studies of Leishmania major infection

Leishmaniasis, a neglected tropical disease, currently infects approximately 12 million people worldwide with 1 to 2 million new cases each year in predominately underdeveloped countries. The treatment of the disease is severely underdeveloped due to the ability of the Leishmania pathogen to evade and abate immune responses. In an effort to develop anti-leishmaniasis vaccines and adjuvants, novel carbohydrate-based probes were made to study the mechanisms of immune modulation. In this study, a new bioerodible polyanhydride microparticle was designed and conjugated with a glycodendrimer molecular probe. This molecular probe incorporates a pathogen-like multivalent display of α-1,2-trimannose, for which a more efficient synthesis was designed, with a tethered fluorophore. Further attachment of the glycodendrimer to a biocompatible, surface eroding microparticle allows for targeted uptake and internalization of the pathogen-associated oligosaccharide by phagocytic immune cells. The α-1,2-trimannose-linked bioerodible microparticles were found to be safe after administration into the footpad of mice and demonstrated a similar response to α-1,2-trimannose-coated latex beads during L. major footpad infection. Furthermore, the bioerodible microparticles allowed for investigation of the role of pathogen-associated oligosaccharides for recognition by pathogen-recognition receptors during L. major-induced leishmaniasis.


General experimental
The following procedures are reported on the largest scale reactions, regardless of yields. All non-aqueous reactions were conducted under argon atmosphere using standard Schlenk techniques for the exclusion of moisture and air. For all room temperature reactions, the ambient temperature was between 20-23 °C. All solvents were dried using 4 Å or 3 Å molecular sieves, or by a solvent drying system with basic alumina columns unless otherwise noted. Analytical thin layer chromatography was performed on Sorbtech silica gel 250 μm, using UV light as the visualizing agent and a solution of bromocresol green, cerium ammonium molybdate (CAM), KMnO 4 , ninhydrin, p-anisaldehyde, or vanillin , as developing agents and then heat. Column chromatography was performed using Sorbtech 60 Å (230 × 400 mesh) or by Teledyne RediSep ISCO columns.
Compounds characterized by 1 H and 13 C spectra were recorded on 400 MHz, 500 MHz or 600 MHz Varian spectrometers. All chemical shifts are referenced to TMS or residual non-deuterated solvent (CDCl 3 : 1 H NMR = 7.26 ppm, 13  (q) and multiplet (m)], coupling constants [Hz], integration, assignment). Carbon spectra were recorded with proton decoupling and the chemical shifts are reported in ppm (C) relative to TMS. Perfluorinated carbon signals on all Cbz-F containing compounds were not reported due to available NMR methods and low signal due to C-F splitting in 13 C NMR. All melting points are uncorrected.
The reaction was left to stir for 1 h, then suspended in CH 2 Cl 2 (300 mL). The organic layer was washed with cold saturated NaHCO 3 (500 mL), dried over MgSO 4, and concentrated under reduced pressure.
The hemiacetal was resuspended in CH 2 Cl 2 (100 mL) and cooled to 0 °C.

Trimannose-linked bioerodible microparticles (2)
A polyanhydride copolymer composed of 20:80 CPH/SA was synthesized via melt polycondensation as previously described. [12] The resulting polymer was dissolved at 10 mg/mL in methylene chloride. The polymer solution was spray dried using a mini spray dryer (B-290, Büchi, Switzerland). The resulting particle morphology