Engineering biology and chemical approaches to the construction of vitamin B12 analogues and antivitamins B12 as probes and therapeutic agents.

Vitamins are indispensable cofactors that expand the chemical capabilities of enzymes beyond the inherent limitations of amino acid side chains. Among them, vitamin B₁₂ is particularly remarkable due to its exceptional structural complexity, the presence of a cobalt-centered corrin ring, and its exclusive biosynthetic origin in prokaryotes. This review explores the biosynthesis, transport, and biological significance of B₁₂, with an emphasis on the growing toolbox of synthetic analogues designed for research and therapeutic use. Recent advances in synthetic biology have enabled the complete heterologous expression of the aerobic B₁₂ biosynthesis pathway in Escherichia coli, facilitating the high-yield production of biosynthetic intermediates and cobalt-free B₁₂-precursors. These intermediates serve as platforms for the generation of metbalamins, metal-substituted cobalamin analogues incorporating rhodium, nickel, zinc, and other transition metals. In parallel, novel organo-antimetabolites and fluorescently labelled derivatives have been developed to probe B₁₂-dependent enzymes, trace vitamin transport in living systems, and selectively disrupt microbial or disease-linked metabolism. These synthetic analogues function as versatile tools for imaging, mechanistic dissection, and metabolic inhibition and more specifically in the case of molecules that counteract the physiological effects of vitamin B₁₂ in animal systems hold potential as antivitamins B₁₂. Collectively, they offer powerful new approaches to study nutrient trafficking, engineer cofactor interactions, and develop targeted antimicrobial or anticancer strategies. The review concludes by discussing future directions in applying engineering biology and chemical synthesis to further diversify and exploit the functional potential of the cobalamin scaffold.