This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. Specifically, the synthesis of highly substituted N-6-linked immobilized NAD(+) derivatives is described using a rapid solid-phase modular approach. Other modifications of the N-6-linked immobilized NAD(+) derivative include substitution of the hydrophobic diaminohexane spacer arm with polar spacer arms (9 and 19.5 Angstrom) in an attempt to minimize nonbiospecific interactions. Analysis of the N-6-linked NAD(+) derivatives confirm (i) retention of cofactor activity upon immobilization (up to 97%); (ii) high total substitution levels and high percentage accessibility levels when compared to S-6-linked immobilized NAD(+) derivatives (also synthesized with polar spacer arms); (iii) short production times when compared to the preassembly approach to synthesis. Model locking-on bioaffinity chromatographic studies were carried out with bovine heart L-lactate dehydrogenase (L-LDH, EC 1.1.1.27), bakers yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) and Sporosarcinia sp. L-phenylalanine dehydrogenase (L-PheDH, EC 1.4.1.20), using oxalate, hydroxylamine, and D-phenylalanine, respectively, as locking-on ligands. Surprisingly, two of these test NAD(+)-dependent dehydrogenases (lactate and alcohol dehydrogenase) were found to have a greater affinity for the more lowly substituted S-6-linked immobilized cofactor derivatives than for the new N-6-linked derivatives. In contrast, the NAD(+)-dependent phenylalanine dehydrogenase showed no affinity for the S-6-linked immobilized NAD(+) derivative, but was locked-on strongly to the N-6-linked immobilized derivative. That this locking-on is biospecific is confirmed by the observation that the enzyme failed to lock-on to an analogous N-6- linked immobilized NADP(+) derivative in the presence of D-phenylalanine. This differential locking-on of NAD(+)-dependent dehydrogenases to N-6-linked and S-6-. linked immobilized NAD(+) derivatives cannot be explained in terms of final accessible substitutions levels, but suggests fundamental differences in affinity of the three test enzymes for NAD(+) immobilized via N-6-linkage as compared to thiol-linkage.