Limitations of N-hydroxysuccinimide esters in affinity chromatography and protein immobilization.

The carbodiimide-mediated reaction of N-hydroxysuccinimide with carboxyl groups immobilized to hydroxyl-containing polymers (such as Sepharose or Trisacryl) leads to an undesirable side reaction in high yields. The product of this reaction interferes with the application of such columns for further affinity-based purification. In addition to the desired N-hydroxysuccinimide ester, a bis(N-hydroxysuccinimide)derivative of beta-alanine [namely, N- [(succinimidooxy)carbonyl]-beta-alanine N-hydroxysuccinimide ester] is probably produced that reacts subsequently with the hydroxyl group of the polymer via ester and carbamate bonds. These beta-alanine derivatives are formed upon interaction of dicyclohexylcarbodiimide with 3 equiv of N-hydroxysuccinimide followed by a Lossen rearrangement. The amount of beta-alanine thus coupled is very high compared to the number of carboxyl groups present on the resin. The beta-alanine bound through the ester bond comprises about 90% of the beta-alanine bound. Alkaline treatment of the ester-bonded beta-alanine-containing polymers (prior to coupling of amino-containing ligands) causes a rearrangement yielding beta-alanine with a free carboxyl group coupled through a stable carbamate linkage. After coupling of amino-containing ligands, the above-described rearrangement cannot occur, and the beta-alanine-linked ligand leaks from the polymer via hydrolysis of the ester bond. The newly formed carboxyl groups (derived from the rearrangement) can be used to prepare active esters. In view of the above, we developed methods for the preparation of nitrophenyl esters as well as N-hydroxysuccinimide esters free of unstable beta-alanine derivatives on polymers containing hydroxyl groups. Upon coupling with amino-containing ligands, these esters yield resins bearing chemically stable bonds.