Synthesis, Characterization, Solution Stability, and X-ray Crystal Structure of the Thiolatocobalamin γ-Glutamylcysteinylcobalamin, a Dipeptide Analogue of Glutathionylcobalamin:  Insights into the Enhanced Co−S Bond Stability of the Natural Product Glutathionylcobalamin

• Published in: Inorganic chemistry 2001-06, Vol.40 (12), p.2686-2692
• Main Authors: Suto, Robert K, Brasch, Nicola E, Anderson, Oren P, Finke, Richard G
• Format: Article
• Language: English
• Subjects: Cobalt - chemistry; Crystallography, X-Ray; Dipeptides - chemical synthesis; Dipeptides - chemistry; Glutamine - analogs & derivatives; Glutamine - chemical synthesis; Glutamine - chemistry; Glutathione - analogs & derivatives; Glutathione - chemistry; Molecular Conformation; Solutions; Sulfur - chemistry; Vitamin B 12 - analogs & derivatives; Vitamin B 12 - chemistry
• ISSN: 0020-1669; 1520-510X
• DOI: 10.1021/ic001365n

Glutathionylcobalamin (γ-glutamylcysteinylglycinylcobalamin; γ-GluCysGly-Cbl) is a natural product which functions as an intermediate in the biosynthesis of the active B12 coenzymes adenosylcobalamin and methylcobalamin. Of interest to the present studies is glutathionylcobalamin's unique stability in comparison to other thiolatocobalamins, notably the ≥6 × 104 fold less stable cysteinylcobalamin, Cys-Cbl. In order to determine which parts of the glutathione tripeptide contribute to the overall stability of glutathionylcobalamin, two cysteine-containing dipeptides, which are truncated versions of glutathione, were used to synthesize their corresponding cobalamins, specifically γ-glutamylcysteinylCbl (γ-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl). As with glutathionylCbl, the dipeptide γ-GluCys-Cbl forms a stable thiolatocobalamin. However and most interestingly, CysGly-Cbl is observed to be unstable much like Cys-Cbl. The results require that the extra stability of glutathionylcobalamin and its congeners, compared to cysteinylcobalamin and its analogues, must be derived from destabilization by the γ-NH3 + group in cysteinylcobalamin, or stabilization by the γ-NHC(O)− amide linkage in glutathionylcobalamin, or both. To probe any ground-state structural basis for the possible stabilization in γ-GluCys-containing cobalamins, γ-GluCys-Cbl was crystallized and yielded the first X-ray structural determination of a true thiolatocobalamin, and only the second structure of a cobalamin containing a Co−S bond, the first example being Randaccio and co-workers' 1999 structure of the thioketone complex, thioureacobalamin, (NH2)2CSCbl. Key features of the structure of γ-glutamylcysteinylcobalamin include (i) a normal Co−S bond length of 2.267(2) Å, (ii) a Co−N(axial) bond length of 2.049(6) Å, (iii) two alternate conformations of the γ-glutamylcysteinyl moiety, and (iv) folding of the corrin ring upward by 24.2°, the highest degree of folding yet observed for a cobalamin. These results do not show any strong stabilization (e.g., no shortened Co−S bond), although it is not clear for certain what the effect is (stabilizing or destabilizing) of the elongated Co−N(axial) bond; instead, the crystallographic results suggest that the metastable Cys-Cbl probably has a Co−S cleavage transition state that is stabilized (along with, possibly, any ground-state destabilization of the Co−S bond). Overall, the results strongly suggest that placing a positive charge on the