Mechanism of activation of hydroxyl‐containing polymers with N‐hydroxysuccinimide and carbodiimides: Reason for leakage

Abstract The carbodiimide‐mediated reaction of N‐hydroxysuccinimide with carboxyl groups immobilized to hydroxyl‐containing polymers (such as Sepharose or Trisacryl) leads to N‐hydroxysuccinimide ester and N‐hydroxysuccinimide derivatives of β‐alanine which react subsequently with the hydroxyl group of the polymer via ester and carbamate bonds. These derivatives are formed upon interaction of dicyclohexyl carbodiimide with three equivalents of N‐hydroxysuccinimide followed by a Lossen rearrangement. The amount of β‐alanine thus coupled is very high compared to the number of carboxyl groups present on the resin. The β‐alanine bound through the ester bond comprises about 90% of the β‐alanine bound. Alkaline treatment of the ester bonded β‐alanine containing polymers (prior to coupling of amino‐containing ligands) causes a rearrangement yielding β‐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 β‐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 (e.g. nitrophenyl). Upon coupling with amino‐containing ligands, these esters yield resins bearing chemically stable bonds.