An investigation of the syntheses of cellulose and agarose derivatives containing sulphate, N-(6-aminohexyl)-2-naphthalenesulphonamide and carboxyl groups for the purification of proteins : a thesis presented in partial fulfilment for the requirements for the degree of Master of Science in Chemistry at Massey University
The syntheses of three different cellulose and agarose derivatives were investigated, namely, cellulose sulphate, N-(6- aminohexyll-2-naphthalenesulphonamide (2-ANS) cellulose and cellulose and agarose with multiple carboxyl groups.
In the case of cellulose sulphate, an attempt was made to find a sulphating reagent and conditions for a commercially convenient method of preparing a cellulose derivative with a sulphate substitution level of 3.5 meq/g. This synthesis was found to require the control of at least one of the following factors: (a) water present in the system,
(b) the quantity of sulphating reagent and (c) temperature. The stability of the sulphated cellulose in 0.08M sodium hydroxide at 83°C over 28 days was also evaluated. It was found that the sulphate substitution level decreased linearly over 4 weeks at the rate of 1%
Two routes of preparing 2-ANS-cellulose derivative were studied, namely, (1) the coupling of 2-ANS to epoxide activated cel lulose and
(2) the coupling of 2-naphthalene sulphonyl chloride (2-NSCl) to diaminohexyl (DAH)-cellulose. Both methods of synthesis were found to be equally feasible. However, the former method required the prior multi-step preparation of 2-ANS, while the latter method was carried out stepwise on the cellulose matrix. The excess reagents were readily washed away before the next step was undertaken. Also, the preparation of 2-NSCI from sodium 2-naphthalene sulphonate was quantitative. The capacity of these 2-ANS-cellulose derivatives for bovine serum albumin (BSA) was also investigated. The products prepared by method 1 showed a much lower capacity (0.05 - 0.38 gBSA/g) for BSA than those prepared by method 2 (0.49 - 0.78 gBSA/g).
The syntheses of cellulose and agarose derivatives containing alpha (A)- and beta (B)-citrylhexamethylenediamine (CM,D), aspartic acid (Asp) and 6-aminohexylaspartate (Asp-AH) groups were investigated using both epoxide and 1,1'-carbonyldiimidazole (CDl) activation procedures. The use of these products for the purification of bovine lactoferrin (Lf) was assessed. The nature of the binding action of Lf to the CM,D-matrices was also studied. It was found that (a) high CM,D substitution level on the matrix, (b) high porosity of the matrix and (c) the removal of additional cationic properties from the matrix by replacing the basic nitrogen linkage resulting from the epoxide activation by a non-basic urethane linkage resulting from CDI activation, led to an increase in the strength of Lf binding to the derivative. The results also suggested that the Lf binding was predominantly ionic in nature. Finally, it was found that Lf purification on A-CM,D-agarose gave a product of higher purity than that on Asp-agarose and Asp-AH-agarose.