Synthesis of a highly substituted N-6-linked immobilized NAD(+) derivativeusing a rapid solid-phase modular approach: Suitability for use with the kinetic locking-on tactic for bioaffinity purification of NAD(+)-dependent dehydrogenases

This study is concerned with further development of the kinetic locking-on strategy for bioaffinity purification of NAD(+)-dependent dehydrogenases. S pecifically, the synthesis of highly substituted N-6-linked immobilized NAD (+) derivatives is described using a rapid solid-phase modular approach. Ot her modifications of the N-6-linked immobilized NAD(+) derivative include s ubstitution of the hydrophobic diaminohexane spacer arm with polar spacer a rms (9 and 19.5 Angstrom) in an attempt to minimize nonbiospecific interact ions. Analysis of the N-6-linked NAD(+) derivatives confirm (i) retention o f cofactor activity upon immobilization (up to 97%); (ii) high total substi tution 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 approa ch 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, hydr oxylamine, and D-phenylalanine, respectively, as locking-on ligands. Surpri singly, two of these test NAD(+)-dependent dehydrogenases (lactate and alco hol 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 dehy drogenase showed no affinity for the S-6-linked immobilized NAD(+) derivati ve, but was locked-on strongly to the N-6-linked immobilized derivative. Th at 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(+) de rivative in the presence of D-phenylalanine. This differential locking-on o f NAD(+)-dependent dehydrogenases to N-6-linked and S-6-. linked immobilize d NAD(+) derivatives cannot be explained in terms of final accessible subst itutions levels, but suggests fundamental differences in affinity of the th ree test enzymes for NAD(+) immobilized via N-6-linkage as compared to thio l-linkage. (C) 2000 Academic Press.