Bio gel p 2 column

Incubations of amylose V (0.6%wv⁻¹) and GtfB-ΔN (0.2 mg) were performed under the conditions described in “Substrate specificity of the L. reuteri NCC 2613 GtfB”. After incubation for 24 h at 37 °C, the reaction was stopped by transfer to 100 °C for 10 min. The polysaccharide was separated from trace amounts of small oligosaccharides (DP < 5) also present in the product mixture by size-exclusion chromatography on a Biogel P2 column (2.5 × 50 cm; Bio-Rad, Veenendaal, The Netherlands) using 10 mM NH₄HCO₃ as eluent at a flow rate of 48 ml h⁻¹.

The products of the radiolabelling XylT activity (as described above) were mixed with 0.5 ml 50% (v/v) Dowex 1x8 chloride form in water (Sigma-Aldrich, St. Louis, MO, US) and filtered through a 0.8 mL Pierce Centrifuge Tube (Thermo Scientific, Rockford, IL, US). The eluent was fractionated on a Bio-Gel P2 column (Bio-Rad, Hercules, CA, US) and fractions (1.8 ml) were collected. The radioactivity of each fraction (600 μl) was counted with a Tri-Carb 2100TR liquid scintillation counter (PerkinElmer, Waltham, CA, US) 4 mL of biodegradable aqueous scintillant (Amersham Biosciences, Pittsburgh, PA, US).

pNP-GalNAc (1; 41 mg, 0.12 mmol), GlcNAc (2; 106 mg, 0.48 mmol), and the Tyr470His mutant of the β-N-acetylhexosaminidase from T. flavus (1.4 U, 13.4 mg, 480 μL) were incubated in a mixture of acetonitrile 10% v/v) in McIlvaine buffer pH 5.0 (total reaction volume 2.4 mL) at 45 °C and 1000 rpm. The reaction was monitored by HPLC and TLC (propan-2-ol/H₂O/NH₄OH aq., 7/2/1, v/v/v). After 2 h, the conversion of pNP-GalNAc was almost complete and another batch of pNP-GalNAc was added (18 mg, 0.05 mmol). After 4 h, the reaction was stopped by boiling for 2 min, and, upon cooling down to r.t. (room temperature), the mixture was centrifuged (13,500 rpm; 10 min) and loaded onto a Biogel P-2 column (2.6 × 100 cm, Bio-Rad, USA) using water as a mobile phase at an elution rate of 8 mL/h. The fractions containing product were combined and lyophilized; the title compound 5 was obtained as a white solid (40 mg, 0.094 mmol, 58% isolated yield). HRMS (ESI⁺): found m/z 447.15845 (expected 447.15853 for [M + Na]⁺, C₁₆H₂₈O₁₁N₂Na). For the respective NMR and MS data, see Additional file 1: Table S1 and Figures S1, S5.

10 mm propargyl-NHS (Sigma) in DMSO (600 μl) was incubated with 10 mg/ml BSA (3 ml) in PBS at room temperature for 2 h. The BSA-alkyne generated was desalted on a PD10 column.
2 mg p-aminophenyl-phosphorylcholine was dissolved in 1.0 ml 0.1 m MES (pH 4.5) and added to an equal volume of 1 mg/ml azide-PEG₄-COOH (Iris Biotech) and 0.2 ml 10 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and incubated overnight at room temperature. The azide-PEG₄-phenyl-PCh was separated from the smaller precursors on a Biogel P2 column (15 ml, Bio-Rad) washed with PBS. Click chemistry between azide and alkyne was used to generate the BSA-PEG₄-PCh conjugate. The reaction was composed of 1.0 ml of 0.2 mm BSA-alkyne and 0.7 ml of 0.3 mm azido-PEG₄-phenyl-PCh, 2 μl of 500 mm fresh ascorbic acid, 50 μl of copper/Tris-[(1-benzyl-1H-1,2,3-triazol-4-yl) methyl]amine (TBTA) reagent (1 volume of 10 mm copper II/2 volumes of 50 mm TBTA, Sigma), and the reagents were incubated for 2 h with rolling. The BSA-PEG₄-PCh-containing fractions were collected after desalting on a PD10 column.

Frs. K‐B and K‐F were dissolved in 30 mL of 0.1 m NaOH, respectively, and the solution was incubated at 25 °C for 18 h. Then, the solution was neutralized with 1 m HCl, concentrated to a small volume, applied to a Bio‐Gel P‐2 column (2.5 × 100 cm) (Bio‐Rad, Tokyo, Japan), and eluted with water (0.25 mL·min⁻¹).