B12-retro-riboswitches: guanosyl-induced constitutional switching of B12 coenzymes.

Complete B12 derivatives are natural "molecular switches" as a result of the coordinative switch ("base on" or "base off") of the natural nucleotide base. Certain predesigned B12-nucleotide conjugates were shown recently to behave as "retro riboswitches", in which the nucleotide environment modified the equilibrium between these two isomeric B12 structures. In contrast, the "reverse" situation has been discovered in natural B12 riboswitches, in which the binding of coenzyme B12 induces a conformational switch in the RNA species. The first (predesigned) B12-retro-riboswitches were DNA conjugates of methylcobalamin. We describe herein two representative B12-retro-riboswitches, in which an appended (RNA) nucleotide is used to destabilize the base-on form and induce the base-on to base-off switch. Through use of heterogeneous solid-phase synthetic methods, Co(beta)-cyanocobalamin-(3''-->2')-2''-methoxyguaninyl-3''-ate was prepared first as the crucial covalent RNA conjugate of vitamin B12. This cyanocorrinoid opened the door to two organometallic B12-nucleotide conjugates, which were made by electrosynthetic means: the cyanocorrinoid was cleanly methylated or adenosylated at the cobalt center to furnish covalent RNA conjugates of the organometallic B12 cofactors methylcobalamin and coenzyme B12, respectively. At room temperature, aqueous solutions of both of these organometallic RNA-B12 conjugates exhibited properties indicative of significant weakening of the axial (Co--N) bond (of their base-on forms) and of an enhanced formation of the base-off species. The base-on to base-off switch was studied by UV/Vis and NMR spectroscopic studies, which showed that the switch was very temperature-dependent and was accentuated with increasing temperatures. Thermodynamic data of the two organometallic RNA-B12 conjugates revealed an important contribution of entropic effects to the observed base-on to base-off switch. The two organometallic RNA-B12 conjugates thus acted as B12-retro-riboswitches and allowed the observation of a temperature-dependent reverse switch in the B12 cofactor moiety, induced by the appended nucleotide moiety. This behavior may be of interest in the "RNA-world" hypothesis, in which (simple) B12 derivatives are thought to act as possible catalytic enhancers ("cofactors") in RNA-based "B12 ribozymes".

Enhancing the methyl-donor activity of methylcobalamin by covalent attachment of DNA.

The preparation of a covalent DNA conjugate of vitamin B12 by means of heterogeneous solid-phase synthesis is reported. The cyano-corrinoid made available, dipotassium Co(beta)-cyanocobalamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(2)-4), was cleanly methylated at the Co center by electrosynthetic means. Aqueous solutions of the resulting organometallic DNA-B12 conjugate K(2)-5 exhibited spectroscopic properties indicative of significant weakening of the axial (Co-N) bond, together with a 25-times higher basicity relative to Co(beta)-methylcobalamin (2). Methyl-transfer equilibria of pH-neutral aqueous solutions of K(2)-5 and cob(I)alamin (K-7) on one side, and of cob(I)alamin-(3''-->2'),(3''-->5')-bis-2''-deoxythymidyl-3''-ate (K(3)-8) and methylcobalamin (2) on the other, were studied at room temperature (Scheme 3). The NMR-derived data provided an equilibrium constant of ca. 0.3. Activation of K(2)-5 for abstraction of its Co-bound Me group by a nucleophile (such as cob(I)alamin) was, thus, indicated.

Homocoenzyme B12 and bishomocoenzyme B12: covalent structural mimics for homolyzed, enzyme-bound coenzyme B12.

Efficient electrochemical syntheses of "homocoenzyme B(12)" (2, Co(beta)-(5'-deoxy-5'-adenosyl-methyl)-cob(III)alamin) and "bishomocoenzyme B(12)" (3, Co(beta)-[2-(5'-deoxy-5'-adenosyl)-ethyl]-cob(III)alamin) are reported here. These syntheses have provided crystalline samples of 2 and 3 in 94 and 77 % yield, respectively. In addition, in-depth investigations of the structures of 2 and 3 in solution were carried out and a high-resolution crystal structure of 2 was obtained. The two homologues of coenzyme B(12) (2 and 3) are suggested to function as covalent structural mimics of the hypothetical enzyme-bound "activated" (that is, "stretched" or even homolytically cleaved) states of the B(12) cofactor. From crude molecular models, the crucial distances from the corrin-bound cobalt center to the C5' atom of the (homo)adenosine moieties in 2 and 3 were estimated to be about 3.0 and 4.4 A, respectively. These values are roughly the same as those found in the two "activated" forms of coenzyme B(12) in the crystal structure of glutamate mutase. Indeed, in the crystal structure of 2, the cobalt center was observed to be at a distance of 2.99 A from the C5' atom of the homoadenosine moiety and the latter was found to be present in the unusual syn conformation. In solution, the organometallic moieties of 2 and 3 were shown to be rather flexible and to be considerably more dynamic than the equivalent group in coenzyme B(12). The homoadenosine moiety of 2 was indicated to occur in both the syn and the anti conformations.