Cu-catalyzed click conjugation of cobalamin to a BODIPY-based fluorophore: A versatile tool to explore the cellular biology of vitamin B.

The Cu-catalyzed click conjugation of an azide-functionalized vitamin B (cobalamin) and an alkyne-labeled 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) led to the formation of a highly stable fluorescent BODIPY-labeled vitamin B (λ/λ = 495/508 nm). The formation of what has been identified as an iodine adduct of the conjugate was also observed as a side-product during this reaction and could be removed using HPLC. BODIPY-labeled vitamin B was characterized by NMR and HR-ESI-MS. In vitro studies on wild-type human fibroblasts indicated that BODIPY-labeled vitamin B could internalize in a manner similar to that of untagged vitamin B. ATP-binding cassette sub-family D member 4 (ABCD4) is a lysosomal localized transporter required to export vitamin B from the lysosomal lumen to the cytosol. Mutations in this transporter result in the accumulation of vitamin B in lysosomes. In human fibroblasts harbouring a mutation in ABCD4, BODIPY-labeled vitamin B accumulated in the lumen of lysosomes. Our data suggests the potential use of BODIPY-labeled vitamin B to investigate the intracellular behavior of the vitamin in the context of disorders related to the abnormal cellular utilization of the vitamin. Moreover, results presented here demonstrate that click chemistry could be exploited for the conjugation of vitamin B to various other fluorophores.