2 methylpyridine borane complex

The generation 2.0 PAMAM dendrimer with a cystamine core (647829, Sigma Aldrich) was conjugated to three different glycans via reductive amination. 20 equivalents of LS-Tetrasaccharide d (LSTd, Elicityl) and D-(+)-Galactose (G0750 Sigma Aldrich) per dendrimer were dissolved in Dimethylsulphoxide (DMSO) and acetic acid (8:2). Per dendrimer, 160 equivalents 2-Methylpyridine borane complex (65421 Sigma Aldrich) was added to a total volume of 200 µL. The reaction was incubated at 65 °C for 2 h with frequent vortexing. The reaction products were purified over disposable PD10 desalting columns (GE17-0851-01 GE Healthcare) in 50 mM Ammonium Formate pH 4.4 (NH₄HCO₃).
The presence of α2-3 sialic acid was validated using an ELISA-type assay using Maackia Amurensis Lectin I (MAL-I) (Vector Laboratories, Peterborough, UK). Briefly, NUNC maxisorb plates (RosKilde) were coated overnight at 4 °C with 5 µM of the products. The wells were subsequently blocked for 2 h at room temperature with carbo-free blocking buffer (Vector, SP5040). Incubation with the biotinylated MAL-I and peroxidase-labelled streptavidin (Sigma-Aldrich) allowed spectrophotometric quantification of the binding with 3,3′,5,5′-tetramethylbenzidine (TMB, Sigma-Aldrich) at 450 nm on the iMark^TM Microplate Absorbance Reader (Bio-RAD).

Methanol (gradient quality) was from Romil Ltd. (Cambridge, UK). Acetone, water, hexane, chloroform, and dichloromethane used in extractions were all HPLC grade from Fisher Scientific (Hampton, NH, USA). Acetonitrile and water for instrumental analyses were Optima LC–MS grade (Fisher Scientific). Trifluoroacetic acid (TFA) (≥99.0%), formic acid (for LC–MS LiChropur, 97.5–98.5%), 2-methylpyridine borane complex (2PBC) (95%), sodium cyanoborohydride (reagent grade, 95%), 2-methoxyethanol (ReagentPlus, ≥99.0%), 1-butanol (ACS reagent, ≥99.4%), Girard′s reagent T (GRT, (hydrazinocarbonylmethyl)trimethylammonium chloride, LiChropur, 99.0–101.0%), and (2-aminoethyl)trimethylammonium chloride hydrochloride (AETMA) (99%) were from Sigma–Aldrich (St. Louis, MO, USA).

Rapid PNGase F was purchased from New England BioLabs (Frankfurt am Main, Germany). Acetonitrile, 1-butanol, ethanol, acetic acid, 1-propanol, and 96-well filter plates were obtained from Merck (Darmstadt, Germany). Formic acid, 2-methylpyridine borane complex, and 2-anthranilic acid were acquired from Sigma-Aldrich (Munich, Germany); 96-well Strata SI-1 SPE plates were from Phenomenex (Aschaffenburg, Germany). Monoclonal antibodies and Fc-fusion proteins were procured from in-house development at Novartis. Reversed-phase chromatographic columns were from Phenomenex (Luna Omega 1.6 μm C18; 150 × 2.1 mm; Luna Omega Polar 1.6 μm C18; 150 × 2.1 mm) and Agilent (Zorbax RRHD Bonus RP 1.8 μm C14; 150 × 2.1 mm) and the HILIC column was purchased from Waters (ACQUITY UPLC Glycan BEH Amide 1.7 μm, 150 × 2.1 mm).

The glycodendrimers were synthesized by conjugation of a generation 2.0 PAMAM dendrimer with cystamine core (Sigma-Aldrich) via reductive amination of the free amino moieties. Approximately 20 equivalents per dendrimer of 3′-Sialyl-N-acetyllactosamine (for the transcriptomic analysis only), LS-Tetrasaccharide d (LSTd, in all other experiments; both from Elicityl) or D-(+)-Galactose (negative control, Sigma-Aldrich) were dissolved in Dimethylsulphoxide and acetic acid (8:2) to generate the α2-3sia and control dendrimer, respectively. Per dendrimer 160 equivalents of the 2-Methylpyridine borane complex (Sigma-Aldrich) were added in a total volume of 200 μl and incubated for 2 hours at 65°C with frequent vortexing. Disposable PD10 desalting columns (GE Healthcare) in 50 mM Ammonium Formate pH 4.4 (NH₄HCO₃) were used to purify the dendrimers. Subsequent lyophilization cycles were used to retrieve the glycodendrimers, followed by validation using LC-MS.