Zinc Substitution of Cobalt in Vitamin B12: Zincobyric acid and Zincobalamin as Luminescent Structural B12‐Mimics

Abstract Replacing the central cobalt ion of vitamin B12 by other metals has been a long‐held aspiration within the B12‐field. Herein, we describe the synthesis from hydrogenobyric acid of zincobyric acid (Znby) and zincobalamin (Znbl), the Zn‐analogues of the natural cobalt‐corrins cobyric acid and vitamin B12, respectively. The solution structures of Znby and Znbl were studied by NMR‐spectroscopy. Single crystals of Znby were produced, providing the first X‐ray crystallographic structure of a zinc corrin. The structures of Znby and of computationally generated Znbl were found to resemble the corresponding CoII‐corrins, making such Zn‐corrins potentially useful for investigations of B12‐dependent processes. The singlet excited state of Znby had a short life‐time, limited by rapid intersystem crossing to the triplet state. Znby allowed the unprecedented observation of a corrin triplet (E T=190 kJ mol−1) and was found to be an excellent photo‐sensitizer for 1O2 (ΦΔ=0.70).


Synthesis, purification and spectral analysis of zincobyric acid
All operations were performed with protection from light.
In a 5 mL glass vial 2.0 mg (2.3 µmol) crystalline hydrogenobyric acid (Hby) [2] were dissolved in 2.3 mL 0.5 M Zn(OAc) 2 pH 6 and stirred for 80 min at RT. The reaction solution was diluted with H 2 O to 15 mL and loaded on a Sep-Pak® C18 Classic Cartridge. The adsorbed Znby was washed with 20 mL H 2 O followed by 20 mL 100 mM NaBF 4 aq. pH 6 and 20 mL H 2 O. The Znby was eluted with 3 mL 100 µM NaBF 4 in MeOH and the solvents were evaporated in Ar-stream at RT. The solid Znby was dissolved in 50µL H 2 O and ~200 µL MeCN. The Znby was crystallized by S3 slow addition of ~1 mL MeCN, storage at RT for 3 h, and at 5±3°C overnight. Additional 2 mL MeCN were added and the suspension was stored for further 24h at 5±3°C. The mother liquor was separated, the crystals were washed with 2 x 1 mL MeCN and dried in HV for 6 h. 1.3 mg (1.4 µmol, 61%) crystalline Znby was obtained as orange crystals. Further 0.5 mg (0.5 µmol, 22%; total: 1.8 mg, 1.9 µmol, 83 %) of Znby were obtained from the mother liquor.   Figure S2).

Synthesis, purification and spectral analysis of zincobalamin
All operations were performed with protection from light.

Computational structure calculations of zincobyric acid
As previous studies showed that the conformations of the amide side chains may differ from the crystal structure, and are susceptible to intramolecular H-bonding, the previously published optimized gas phase structure of Hby [2] was taken as a starting structure to obtain consistent results. The two inner hydrogen atoms were removed and replaced by Zn 2+ and the resulting structure on Znby(4) was geometry optimized using Density Functional Theory. To avoid further issues with intramolecular hydrogen bonding, the X-ray crystal structure of Co(II)-heptamethyl-cobyrinate perchlorate (Co II -cobester, Cbin II ) [7] was taken as an alternative starting structure and modified by replacing Co(II) by Zn(II), giving Znby(4) Cob . Subsequent structure optimization revealed the geometry of the corrin ligand was very similar to the one of Znby(4). To assess the effect of coordination of the 5 th ligand in Znby on the structure of the corrin ring and the coordination geometry of Zn, three models were investigated: (i) Znby Cob , where the coordination on the -face is modelled with an acetate ligand, (ii) Znby() Cob , where an acetate ligand is coordinated on the -face, and (iii) Znby(4) Cob , where Zn is only 4-fold coordinated and the ligand on the -face is omitted. To determine the lowest energy conformation of the acetate ligand, a dihedral scan was performed for Znby Cob and Znby() Cob , where the structures were allowed to fully relax and only the dihedral angle under study was fixed. In both cases, the acetate ligand prefers and east-west orientation. However, several low energy conformations could be determined in both cases. For structural comparison, the core structures of Znby Cob and Hby, were aligned on the respective corrin ring atoms.
The Znbl structure was obtained by modification of a previously optimized gas phase structure of Cbl II derived from the X-ray crystal structure of an antivitamin B 12 . [8] All calculations were performed with the quantum chemical suite Turbomole. [9] A def2-TZVP basis set was used for all atoms. [10] In order to speed up calculation time the resolution-of-identify (RI) technique was utilized. [11] To critically assess the performance of density functionals, both, the BP86 [12] and the PBE [13] , density functional were used for structure optimizations together with empirical dispersion corrections of the D3 type with Becke-Johnson damping (BJ). [14] However, structural differences were found to be minimal. All structures were visualized with PyMol. [15] Table S5. Calculated structural parameters in Å and in ° of the optimized structure of Znbl and Cbl II and comparison with experimental single crystal X-ray data of Cbl II [16] .