Organometallic DNA–B₁₂ Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin‐Transport Proteins

Abstract

The synthesis and structural characterization of Co-(dN) -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN) -Cbl, which are organometallic DNA-B conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN) -Cbl, or radiolabeled vitamin B ( Co-CNCbl) and Co-(dN) -Cbl or Co-(dN) -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN) -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.

The structure of the rat vitamin B12 transporter TC and its complex with glutathionylcobalamin

“…Even though there is a substantial buried surface area between the α- and β-domain, Cbl is required for α- and β-domain association in Cbl transporters ( 22 , 23 ). Cbl binds sequentially to the β-domain of Cbl transporters and then the α-domain closes onto the β-domain–Cbl complex ( 22 , 23 , 24 , 35 ), which is consistent with PISA analysis. Previously it has been shown that the vitamin B 12 transporter TC is highly tolerant toward Cbl analogs that carry larger substitutions at the β-axial site including, for example, GSH ( Figure 2 , Figure 3 , Figure 4 ) and 18-, 25-, or 39-nt long oligonucleotides ( 35 , 40 ).…”

“…S4 : blue spectra 1–4), suggesting that TC can sequester and stabilize GSCbl. A similar observation has been made for oligonucleotide-substituted Cbls, which quickly undergo photolysis, but not when they are bound to TC ( 35 ). Once TC has been degraded in the lysosome, the bound Cbl form is liberated and processed by CblC for use as coenzyme.…”

Vitamin B12 and its binding proteins in milk from cow and buffalo in relation to bioavailability of B12

“…Anonymous human saliva was used as a source of native human HC ≈ 64 nM. Recombinant human HC (Furger et al, 2012;Mutti et al, 2017) was employed in the experiments, where a high concentration of the protein was required.…”

Vitamin B12b Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells

“…Bauer with coworkers found that the B 12 derivative nitrosylcobalamin in combination with interferon has a pronounced toxic effect on tumor cells [10]. Studies are in progress in an attempt to design drugs of directed action based on conjugates of B 12 with known antitumor drugs [11,12]. It has been shown earlier that cobalamins are capable of catalyzing the formation of reactive oxygen species in the presence of natural reducing agents, e.g., ascorbate and thiols, which leads to oxidative stress [13][14][15].…”