Conversion of Pyridylamino Sugar Chains to Corresponding Reducing Sugar Chains

 Lyophilized pyridylamino saccharide (>1 nmol).

 Put on the bottom of a test tube.

 Add 1 mL of the 0.1% Acetic acid solution.

 Mix well.

 Add Palladium black (3 mg).

 Reduce with hydrogen gas at an atmospheric pressure and at room temperature for 3 h.

 Filtrate by Milex filter (0.22 μM).

 Evaporate to dryness.

 Add 0.2 mL of anhydrous hydrazine.

 Heated in a sealed tube at 70°C for 2 min.

 Remove excess hydrazine in vacuo.

 Gel filtration on a Bio-Gel P-2 column (1.2 × 90 cm; 10 mM acetic acid).

 Concentrate Ninhydrin positive fractions and lyophilized.

 1-amino-1-deoxy derivative (about 1 nmol/ 5 μL of water) is put on the bottom of a test tube.

 Add 8 μL of saturated hexamethylenetetramine aqueous solution.

 Add 1.5 μL of 50% acetic acid aqueous solution (pH of the mixed reaction solution is 4.5).

 Heat in a sealed tube at 100°C for 45 min.

 Add 100 μL of water.

 Concentrate to about 20 μL with a Speed Vac concentrator to remove the volatile reagents.

 Add 200 μL of water.

 Apply onto a TOYOPAK ODS cartridge.

 Wash with 600 μL of water.

 Collect eluate and washings.

 Add 0.6 g of Dowex 50W X2 (H⁺).

 Pour into a small glass column.

 Wash with 3 column volumes of water.

 Collect eluate and washings, and concentrated, and freeze-dried.