The structure of (adeninylpropy1)cobalamin (AdePrCbl), a coenzyme B12 ((5'-deoxyadenosyl)cobalamin) analogue in which the ribose moiety of the adenosyl group has been replaced by a propylene chain, has been determined by X-ray diffraction methods. AdePrCbl crystallizes in the orthorhombic space group P212,21, with 2 = 4, a = 23.868 (9) A, b = 21.024 (7) A, c = 16.047 (4) A, and V = 8053.07 A3. The final R value is 0.100 based on 6621 observed reflections. The general conformations of the corrin ring, benzimidazole, phosphate, and ribose in AdePrCbl are very similar to those of (5'-deoxyadenosy1)cobalamin and methylcobalamin except for the amide side chains, which show some variability in the orientations of their amide groups. The adenine ring in AdePrCbl lies over the D ring of the corrin system, rotated about 120' clockwise from its position in coenzyme BI2. The ten water molecules in the crystal structure of AdePrCbl are well located and show no evidence of disorder. Complete 'H and I3C NMR assignments of AdePrCbl have been made by using the following two-dimensional NMR methods: homonuclear Hartmann-Hahn spectroscopy (HOHAHA), rotating frame Overhauser enhancement spectroscopy (ROESY), 'H-detected heteronuclear multiple-quantum-coherence (HMQC) spectroscopy, and 'H-detected multiple-bond heteronuclear multiplequantum-coherence spectroscopy (HMBC). In addition to the adenine orientation found in the crystal structure, a second orientation, in which the adenine lies over the B ring of the corrin, is suggested by 'H NOES and by a comparison of the 'H and I3C shifts of AdePrCbl to those of coenzyme B12. Our results suggest that alkyladenine groups in cobalamins may have a highly fluxional character permitting several orientations of the adenine. Previous studies have shown that binding to the B12-dependent enzymes ribonucleotide reductase and diol dehydrase is tighter for (adeninylpenty1)cobalamin than for coenzyme BI2 and the other (adeninylalky1)cobalamins. On the basis of our studies, we conclude that the flexibility of the alkyl chain, exhibited by the fluxional character of the alkyladenine group, and the orientation of the adenine ring could be responsible for the increased affinity of this analogue for the enzyme. Differences in the orientation of the adenine and the fluxional character of the alkyladenine group, in addition to corrin ring flexibility, may also be useful in explaining the changes in the circular dichroism spectra of (adeniny1alkyl)cobalamins upon binding to ethanolamine ammonia-lyase.

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The three cyanocobalaminmonocarboxylic acid isomers known to be produced by the mild acid hydrolysis of the b-, d-, and e-propionamide side chains of vitamin B12 have been unambiguously assigned by modern 2D NMR methods. Previously, structural assignments had been made by less definitive NMR methods, and both X-ray and neutron diffraction studies had failed to locate unambiguously the position of the carboxyl group. The b and e isomers were structurally assigned in this study, on the basis of the assignment of the 13C NMR signal of the carboxyl group from HMBC (1H-detected heteronuclear multiple-bond correlation) spectra. The carboxyl group resonances exhibited the greatest changes in chemical shift between the protonated (pH 2) and deprotonated (pH greater than 7) forms of the acids. The d isomer was assigned by difference. Since the HMBC experiments required the assignments of side-chain CH2 signals, homonuclear Hartmann-Hahn, 2D homonuclear correlation, 2D nuclear Overhauser effect, 1H-detected heteronuclear multiple quantum coherence, and HMBC spectroscopies were used to assign completely the 1H and 13C NMR spectra of the b and e isomers at pH approximately 7. By comparison with the 13C NMR spectra of the b and e isomers, nearly one-fourth of the resonances of the 13C NMR spectrum of vitamin B12 have been reassigned. The sites of incorporation of 13C-labeled precursors in B12 biosynthesis found in previous studies have been verified by a comparison of 13C assignments. The results of studies using cobalamins modified at the b-, d-, and e-propionamide side chains in which the incorrect structural assignments were used (before 1980), particularly studies of B12-dependent enzymes, require reinterpretation using the correct structural assignments.

A contracted ring degradation product, WYE-120318 (compound 2), was discovered during the development phase for methylnaltrexone bromide (compound 1) drug substance. The compound was isolated by high-performance liquid chromatography fractionation, and its structure was determined by spectroscopic data analyses. WYE-120318 is formed from methylnaltrexone through a benzyl-benzilic acid type rearrangement reaction to yield an α-hydroxy-cyclopentanecarboxylic acid substructure. The proposed structure and the formation mechanism are confirmed by the synthesis of WYE-120318 from methylnaltrexone (compound 1). A similar benzyl-benzilic acid type rearrangement reaction can be envisioned as the biological origin of remisporine A (compound 3), a naturally occurring cyclopentadienyl compound that autocatalytically dimerizes to remisporine B (compound 4). The structure of remisporine A was deduced from its dimer 4. Coniothyione (compound 5) can be considered as the first example of a stable natural product bearing the remisporine A skeleton. However, the regiochemistry of the chlorosubstitution in the coniothyrione structure needs to be revised to compound 6 on the basis of the nuclear magnetic resonance data and biogenesis analysis.