Determination of substituents distribution in carboxymethylpullulans by NMR spectroscopy

The distribution of carboxymethyl substituents in the alpha-(1 --> 6)-linke d maltotriosyl repeating units of a carboxymethylpullulan (CMP) series was investigated by high resolution NMR spectroscopy on very short oligomers (D Pn = 1.2-1.5) obtained by acid hydrolysis. A series of 2D NMR experiments o n parent pullulan, hydrolysed pullulan and CMP was used to assign the proto n and carbon chemical shifts of CMP acid hydrolysates. The degree of substi tution (DS) and the relative distribution of -CH2COONa groups at OH-2, OH-3 , OH-4 and OH-6 of glucose residues (DSi) were determined from H-1 NMR meas urements. From a set of CMP samples, widely different in degree of substitu tion, it was observed that the substitution at C-2 is predominant and decre ases according to the order C-2 > C-3 > C-6 > C-4. Taking into account the availability of each OH group in the parent pullulan, an order of relative reactivity of hydroxyl groups is defined according to the relation: R-i = D Si/n(i), where n(i) is the number of free OH groups in a maltotriose unit ( MTU) for a given site C-i, the reactivity order was found to be OH-2 > OH-4 > OH-6 > OH-3. (C) 2000 Elsevier Science Ltd. All rights reserved.