Phenylboronic acid-salicylhydroxamic acid biconjugates. 1. A novel boronicacid complex for protein immobilization

A chemical affinity system exhibiting antibody-like properties is described . The system exploits bioconjugates with appended phenylboronic acid (PBA) moieties and a support-bound phenylboronic acid complexing reagent derived from salicylhydroxamic acid (SHA) for protein immobilization on a chromatog raphic support. The structure of the PBA . SHA complex was characterized by B-11 NMR and mass spectrometry and compared with complexes derived from mo del compounds. Protein modification reagents were synthesized from 3-aminop henylboronic acid and utilized to prepare bioconjugates from alkaline phosp hatase (AP) and horseradish peroxidase (HRP). AP obtained from one source a fforded PEA bioconjugates exhibiting significant loss of enzymatic activity , whereas AP obtained from a second source afforded PEA bioconjugates exhib iting only a modest loss of enzymatic activity. Conversely, HRP afforded PE A bioconjugates exhibiting no loss of enzymatic activity. SHA-modified Seph arose was prepared by reaction of methyl 4- [(6-aminohexanoylamino)methyl] salicylate with CNBr-activated Sepharose 4B, followed by treatment with aqu eous alkaline hydroxylamine. PBA-AP and PBA-HRP conjugates were efficiently immobilized on SHA-Sepharose at pH 8.3. PBA-AP conjugates were retained af ter washing with acidic buffers at pH 6.7, 4.2, and 2.5, whereas PBA-HRP co njugates were retained after washing with buffer at pH 6.7, but were eluted to some extent at and below pH 4.2. The results are interpreted in terms o f multivalent interactions involving boronic acid complex formation between the enzyme bioconjugates and immobilized complexing reagent.