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 Co3+/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 Kd = 10−5–10−6 M. A specific group (presumably tetrazole, TZ) can be easily created in CNBr-activated Sepharose by treatment with N3-. The prepared matrix (STZ) contained ≈10 mM of the active groups, which bound H2O · corrinoids with Kd = 10−5–10−6 M. Stability of STZ–Cbl bonds gradually increased and reached Kd = 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.

Graphical abstractWe have found that tetrazole derivatives can coordinate to H2O · cobalamin. A specific group (presumably tetrazole) can be easily created in CNBr-activated Sepharose by treatment with azide. The prepared matrix contained ≈10 mM of the active groups, which bound H2O · corrinoids with Kd = 10−5–10−6 M.Figure optionsDownload full-size imageDownload as PowerPoint slide