Access to organometallic arylcobaltcorrins through radical synthesis: 4-ethylphenylcobalamin, a potential "antivitamin B(12)"

Abstract

Locked B(12): 4-Ethylphenylcobalamin, a novel organometallic arylcobalamin, has been synthesized in a radical reaction. This vitamin B(12) antimetabolite features a strong Co-C bond, and represents a "locked" form of vitamin B(12) . It may be used in animal studies to induce functional vitamin B(12) deficiency artificially to help clarify still controversial issues related to the pathophysiology of vitamin B(12) deficiency.

Figure 1

Two projections of a model of the structure of EtPhCbl derived from x-ray crystal structure analysis

Figure 2

Interaction of EtPhCbl with human Cbl-binding protein intrinsic factor (IF). (A) Binding of EtPhCbl to IF measured via competition with the fluorescent B₁₂-derivative CBC (k_+CBC = 64 · 10⁶ M⁻¹s⁻¹): IF was rapidly mixed with either CBC or a solution of CBC and EtPhCbl. An arrow indicates effect of competition between CBC and EtPhCbl, consistent with k_+EtPhCbl = 79 · 10⁶ M⁻¹s⁻¹. (B) Dissociation of EtPhCbl from IF: The pre-incubated complex of IF (1.0 μM) and either EtPhCbl or CNCbl (1.3 μM) was mixed with the ‘chasing’ B₁₂-derivative CBC (1.3 μM). From the increase of the fluorescence the dissociation rate constants of the complexes were calculated: k_−EtPhCbl ≈ 1.5 · 10⁻⁶ s⁻¹ and k_−CNCbl ≈ 6 · 10⁻⁷ s⁻¹ (see Supp. Info for further details).

Figure 3

A EtPhCbl is bound to CblC as a ‘base-off’ form, as seen in the characteristic spectral changes and hypsochromic shift of the absorbance maxima; B. EtPhCbl is processed very slowly by CblC (in the presence of glutathione, open circles), whereas MeCbl is rapidly demethylated (closed circles) – see Suppl. Info for exptl. Details.

Scheme 1

Left. Structural formulas of some relevant cobalamins: vitamin B₁₂ (R = CN, cyano-cobalamin, CNCbl), coenzyme B₁₂ (R = 5′-deoxyadenosyl, 5′-deoxyadenosyl-cobalamin, AdoCbl), methyl-cobalamin (R = methyl, MeCbl), aquo-cobalamin (R = H₂O, H₂OCbl, a cation), 4-ethylphenyl-cobalamin (R = 4-ethylphenyl, EtPhCbl). Right. Symbols of exemplary cobalamins and their biosynthetic conversions in mammals a) by the enzyme CblC, b) by adenosyltransferase and c) by methionine synthase. EtPhCbl is resistant to conversion by CblC into cob(II)alamin.

Scheme 2

Outline of the synthesis of EtPhCbl from H₂OCbl and 4-ethylphenyl diazonium tetrafluoroborate.

Scheme 3

Outline of the proposed mechanism of the formation of EtPhCbl via a hypothetical one-electron reduction of 4-ethylphenyl diazonium tetrafluoroborate by cob(II)alamin with loss of dinitrogen, and followed by recombination of cob(II)alamin with the 4-ethylphenyl radical.