Conversion of pyridylamino sugar chains to corresponding reducing sugar chains

Shin-ichi Nakakita

Introduction

Pyridylamino (PA) saccharides can be converted into the corresponding reducing sugar chains using a two-step process (Figure 1). The first step is its conversion into 1-amino-l-deoxy derivatives, and the second is its conversion into the corresponding reducing sugar chains using the Sommelet reaction (1, 2).

Protocol

In this chapter, the protocol for converting PA sugar chains into reducing sugar chains will be described.

Materials

1. Palladium (Wako Pure Chemical Industries, Ltd., Osaka, Japan)

2. Hexamethylenetetramine (Wako Pure Chemical Industries Ltd.)

3. Hydrazine anhydrous (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan)

Instruments

1. Speed Vac Concentrator (Thermo Fisher Scientific Inc., Waltham, MA)

2. Block incubator (Astec Co., Ltd., Fukuoka, Japan)

3. Rotary oil pump connected to cold trap (Yamato Scientific Co., Ltd., Tokyo, Japan)

4. Hydrogen Generator (GL-Science, Tokyo, Japan)

Methods

1.

Preparation of 1-amino-1-deoxy derivative from pyridylamino saccharide

a.: Lyophilized PA saccharide (>1 nmol).
b.: Put it in a test tube.
c.: Add 1 mL of 0.1% Acetic acid solution.
d.: Mix it well.
e.: Add Palladium black (3 mg) (see Note 1).
f.: Reduce it using hydrogen gas at atmospheric pressure and at room temperature for 3 h.
g.: Filtrate by Milex filter (0.22 μM).
h.: Evaporate to dryness.
i.: Add 0.2 mL of anhydrous hydrazine (see Note 2).
j.: Heat it in a sealed tube at 70°C for 2 min.
k.: Remove excess hydrazine in vacuo.
l.: Gel filtration on a Bio-Gel P-2 column (1.2 × 90 cm; 10 mM acetic acid).
m.: Concentrate Ninhydrin positive fractions and lyophilized.

2.

Conversion of 1-amino-l-deoxy derivative into reducing saccharide (Sommelet reaction)

a.: 1-Amino-1-deoxy derivative (about 1 nmol/5 μL of water) is put in a test tube.
b.: Add 8 μL of saturated hexamethylenetetramine aqueous solution.
c.: Add 1.5 μL of 50% acetic acid aqueous solution (pH of the mixed reaction solution is 4.5).
d.: Heat it in a sealed tube at 100°C for 45 min.
e.: Add 100 μL of water.
f.: Concentrate to about 20 μL with a Speed Vac concentrator to remove volatile reagents.
g.: Add 200 μL of water.
h.: Apply to a TOYOPAK ODS cartridge.
i.: Wash with 600 μL of water.
j.: Collect eluate and washings.
k.: Add 0.6 g of Dowex 50W X2 (H⁺).
l.: Pour it into a small glass column.
m.: Wash with 3 column volumes of water.
n.: Collect eluate and washings, concentrate, and freeze-dry.

Notes

1. Palladium black may ignite when dry; thus, store it in water after use.

2. Hydrazine anhydrous is a highly toxic toxin. Wear protective glasses and gloves when using it.

3. Discussion

This method clearly indicates that 1-amino-l-deoxy sugars can be converted into corresponding sugar chains with reducing power using the Sommelet reaction.

References

Takahashi C, Nakakita S, Hase S. Conversion of pyridylamino sugar chains to corresponding reducing sugar chains. J Biochem. 2003 Jul;134(1):51–5. [PubMed: 12944370] [CrossRef]

Sommelet M. Sur un mode de décomposition des halogénoalcoylates d'hexaméthylène – tétramine. Compt Rend. 1913;157:852–854.

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Bookshelf ID: NBK593933PMID: 37590670

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