Tetrazole derivatives and matrices as novel cobalamin coordinating compounds

Cobalamin (Cbl, vitamin B₁₂) consists of two moieties: (i) the corrin ring with the central Co-ion in the oxidation states Co^3+/2+/1+ and (ii) the nucleotide side chain. The lower position of the ring is typically occupied by the nucleotide base (Bzm), whereas the upper surface coordinates exchangeable ligands. We have found that amino-tetrazole can coordinate to H₂O · Cbl (Co³⁺) with K_d = 10⁻⁵-10⁻⁶ M. A specific group (presumably tetrazole, TZ) can be easily created in CNBr-activated Sepharose by treatment with . The prepared matrix (STZ) contained ≈10 mM of the active groups, which bound H₂O · corrinoids with K_d = 10⁻⁵-10⁻⁶ M. Stability of STZ-Cbl bonds gradually increased and reached K_d = 10⁻⁷ M over 10-20 h (20 °C, pH 6-7). This effect can be ascribed to partial displacement of Bzm and coordination of TZ to the lower position. The binding was most efficient at pH 4-7 and low ionic strength, yet, noticeable adsorption took place even at extreme conditions, pH 1-9 and I = 0-2 M. Reduced corrins (Co²⁺) also exhibited high affinity for STZ. The bound ligands could be eluted as H₂O · Cbl (pH 0), HO · Cbl (pH 14) or diCN · Cbl (pH 9-12, CN⁻). The adsorbent is applicable for one-step purification of corrins from a crude extract; separation of aquo- and diaquo-forms; specific capturing of H₂O · Cbl from a mixture containing organo-Cbls or protein-bound Cbl, analysis of peptide-Cbl dissociation kinetics, etc.