Multivalent, Stabilized Mannose‐6‐Phosphates for the Targeted Delivery of Toll‐Like Receptor Ligands and Peptide Antigens

Abstract Mannose‐6‐phosphate (M6P) is recognized by the mannose‐6‐phosphate receptor and plays an important role in the transport of cargo to the endosomes, making it an attractive tool to improve endosomal trafficking of vaccines. We describe herein the assembly of peptide antigen conjugates carrying clusters of mannose‐6‐C‐phosphonates (M6Po). The M6Po's are stable M6P mimics that are resistant to cleavage of the phosphate group by endogenous phosphatases. Two different strategies for the incorporation of the M6Po clusters in the conjugate have been developed: the first relies on a “post‐assembly” click approach employing an M6Po bearing an alkyne functionality; the second hinges on an M6Po C‐glycoside amino acid building block that can be used in solid‐phase peptide synthesis. The generated conjugates were further equipped with a TLR7 ligand to stimulate dendritic cell (DC) maturation. While antigen presentation is hindered by the presence of the M6Po clusters, the incorporation of the M6Po clusters leads to increased activation of DCs, thus demonstrating their potential in improving vaccine adjuvanticity by intraendosomally active TLR ligands.


In vitro dendritic cell maturation assay
The test compounds were dissolved in DMSO at a concentration of 1 mM and sonicated in water bath for 15 minutes. Murine bone marrow-derived dendritic cells were seeded in 96-well plates at a density of 50.000 cells/well and incubated with titrated amounts of compounds. After 3 hours of incubation, the cells were washed once and incubated with fresh medium. After 16 hours, supernatant was harvested for ELISA analysis (Biolegend) to measure the amount of produced IL-12p40.

In vitro antigen presentation assay
The test compounds were dissolved in DMSO at a concentration of 1 mM and sonicated in water bath for 15 minutes. 50.000 D1 cells were seeded in 96-well flat bottom plates and incubated with the indicated test compounds. After 2 hours 50.000 B3Z or OTIIZ were added per well and incubated with the D1 cells overnight. The following day, B3Z or OTIIZ activation was detected by measurement of absorbance at 595 nm upon color conversion of chlorophenol red-β-D-galactopyranoside (Calbiochem) the lacZ reporter enzyme.

General experimental
All reagents were of commercial grade and used as received unless stated otherwise. Reaction solvents were of analytical grade and when used under anhydrous conditions stored over flame-dried 3Å molecular sieves. All moisture and oxygen sensitive reactions were performed under an argon atmosphere. Column chromatography was performed on silica gel (Screening Devices BV, 40-63 µm, 60 Å). For TLC analysis, pre-coated silica gel aluminum sheets (Merck, silica gel 60, F254) were used with detection by UV-absorption (254/366 nm) where applicable. Compounds were visualized on TLC by UV absorption (245 nm), or by staining with one of the following TLC stain solutions: (NH4)6Mo7O24•H2O (25 g/L), (NH4)4Ce(SO4)4•2H2O (10 g/L) and 10% H2SO4 in H2O; bromocresol (0.4 g/L) in EtOH; KMnO4 (7.5 g/L), K2CO3 (50 g/L) in H2O. Staining was followed by charring at ~150°C. 1 H, 13 C and 31 P NMR spectra were recorded on a Bruker AV-400 (400/100/162 MHz) spectrometer and all individual signals were assigned using 2D-NMR spectroscopy. Chemical shifts are given in ppm (δ) relative to TMS (0 ppm) in CDCl3 or via the solvent residual peak. Coupling constants (J) are given in Hz. LC-MS analysis were done on an Agilent Technologies 1260 Infinity system with a C18 Gemini 3 µm, C18, 110 Å, 50 x 4.6 mm column. Absorbance was measured at 214 nm and 256 nm and an Agilent Technologies 6120 Quadrupole mass spectrometer was used as detector. Peptides and conjugates were purified with a Gilson GX-281 preparative
The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The obtained intermediate (Rf = 0.14 (MeOH/DCM: 2/3 v/v) was dissolved in a mixture of AcOH/Milli-Q H2O (1/1, v/v, 25 mL) and heated to 90°C for 8 hours, after which it was cooled to room temperature. The reaction mixture was diluted with Milli-Q H2O and concentrated in vacuo. After co-evaporation with Milli-Q H2O (5x), the residue was dissolved in Milli-Q H2O, washed with DCM (4x) and concentrated in vacuo. Lyophilization yielded the title compound (0.50 g, 1.7 mmol, 81%) as a white solid. Rf

Di-tert-butyl methylphosphonate S6
To a solution of di-tert-butyl phosphite (26 g, 0.13 mol, 1.0 eq.) in THF (0.25 L) was added slowly a solution of n-butyllithium (1.6 M in hexane, 99 ml, 0.16 mol, 1.2 eq.) at -78°C under an argon atmosphere. After stirring for 1 h, a solution of iodomethane in THF (2 M, 85 ml, 0.17 mol, 1.3 eq.) was added and the reaction was allowed to warm up to room temperature overnight. Concentration in vacuo and purification by column chromatography (050% EtOAc in pentane) yielded the title compound as a slightly yellow liquid (21.7 g, 104 mmol, 80%). 1