Cu-catalyzed click conjugation of cobalamin to a BODIPY-based fluorophore: A versatile tool to explore the cellular biology of vitamin B12.

ABSTRACT

The Cu-catalyzed click conjugation of an azide-functionalized vitamin B12 (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 B12 (λex/λem = 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 B12 was characterized by NMR and HR-ESI-MS. In vitro studies on wild-type human fibroblasts indicated that BODIPY-labeled vitamin B12 could internalize in a manner similar to that of untagged vitamin B12. ATP-binding cassette sub-family D member 4 (ABCD4) is a lysosomal localized transporter required to export vitamin B12 from the lysosomal lumen to the cytosol. Mutations in this transporter result in the accumulation of vitamin B12 in lysosomes. In human fibroblasts harbouring a mutation in ABCD4, BODIPY-labeled vitamin B12 accumulated in the lumen of lysosomes. Our data suggests the potential use of BODIPY-labeled vitamin B12 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 B12 to various other fluorophores.