15] Synthesis of pyrimidine 14C-labeled thiamine

Abstract

This chapter examines the synthesis of pyrimidine 14 C-labeled thiamine. Thiamine labeled in its thiazole moiety either with 14 C or 35 S is commercially available. The labeled thiamine so formed must be further purified by paper chromatography. The crude thiamine is dissolved in a small amount of water, streaked on a Whatman No. 3 paper, and chromatographed for 16 hours (by using descending solvent system of isobutanol:acetic acid:water, 4: 1: 1). The thiamine can be located under ultraviolet (UV) light, being seen as a quenching band. It can be located also by converting it to thiochrome by spraying a narrow strip of the paper with alkaline potassium ferricyanide; in this case, it will be seen as a bright blue fluorescent spot under UV light (365 nm). The relevant area on the paper is cut out and eluted with dilute HCl. Determination of the radioactivity of an aliquot and assay for thiamine content (thiochrome method) will give the specific activity of the solution. A further amount of thiamine can be obtained from the mother liquor from the crystallization of the thiamine.