Heavy vitamin B12 derivatives
The invention discussed in this application relates to vitamin B12-based compounds that are useful as quantitative standards, particularly for the assessment of vitamin B12 deficiency.
1. A process for preparing a heavy vitamin B12 having a mass shift of at least +7 over unlabelled vitamin B12, the process including:
providing 13 C-labelled 5,6-dimethylbenzimidazole having seven or more 13 C atoms and optionally one or more 2 H atoms,
providing cobinamide dicyanide, and
forming the heavy vitamin B12 from the 13 C-labelled 5,6-dimethylbenzimidazole and the cobinamide dicyanide in the presence of a bacterium,
wherein the heavy vitamin B12 comprises seven 13 C atoms located in the dimethylbenzimidazole moiety and is a compound of formula (I):
or a pharmaceutically acceptable salt, hydrate, solvate, or prodrug thereof,
wherein R is 5′-deoxyadenosyl, OH, H 2 O, CH 3 , or CN.
2. The process of claim 1 , wherein the seven 13 C atoms are located in the dimethylbenzimidazole moiety as follows:
3. The process of claim 1 , wherein the 13 C-labelled 5,6-dimethylbenzimidazole has eight 13 C atoms.
4. The process of claim 1 , wherein the 13 C-labelled 5,6-dimethylbenzimidazole has nine 13 C atoms.
5. The process of claim 1 , wherein the 13 C-labelled 5,6-dimethylbenzimidazole has one or more 2 H atoms.
6. The process of claim 1 , wherein the 13 C-labelled 5,6-dimethylbenzimidazole has two 2 H atoms.
7. The process of claim 1 , wherein the bacterium is Salmonella enterica ( S. enterica ).
8. The process of claim 7 , wherein the bacterium is S. enterica serovar Typhimurium ( S. typhimurium ).
9. The process of claim 8 , wherein the bacterium is strain PA09.
10. The process of claim 1 , wherein the process is carried out under aerobic conditions.
11. The process of claim 1 , wherein the process is carried out in the presence of ethanolamine as a carbon source.
12. The process of claim 1 , wherein the bacterium is grown aerobically in a minimal growth medium with ethanolamine provided as the sole carbon source and the 13C7-5,6-dimethylbenzimidazole and cobinamide dicyanide are provided in the growth medium.
13. The process of claim 1 , wherein the process comprises an initial step of preparing the 13 C-labelled 5,6-dimethylbenzimidazole.
14. The process of claim 13 , wherein the initial step comprises:
providing 13 C-labelled formic acid,
providing 13 C-labelled dimethylphenylenediamine having six 13 C atoms,
contacting the 13 C-labelled formic acid with the 13 C-labelled dimethylphenylenediamine,
thereby producing the 13 C-labelled 5,6-dimethylbenzimidazole.
15. The process of claim 14 , wherein the 13 C-labelled dimethylphenylenediamine has one or more 2 H atoms.
16. The process of claim 14 , wherein 13 C-labelled dimethylphenylenediamine has two 2 H atoms.
17. The process of claim 14 , which is carried out in the presence of heated hydrochloric acid.
18. The process of claim 1 , wherein the heavy vitamin B12 has a mass shift of from +7 to +10 over unlabelled vitamin B12.
19. The process of claim 1 , wherein the heavy vitamin B12 is heavy cyanocobalamin, heavy hydroxocobalamin, heavy deoxyadenosylcobalamin, heavy methylcobalamin or combinations thereof.