Kvach Maksim V, Tsybulsky Dmitry A, Shmanai Vadim V, Prokhorenko Igor A, Stepanova Irina A, Korshun Vladimir A
Institute of Physical Organic Chemistry, Minsk, Belarus.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.
Curr Protoc Nucleic Acid Chem. 2013 Mar;Chapter 4:4.55.1-4.55.33. doi: 10.1002/0471142700.nc0455s52.
This unit describes the preparation of 5- and 6-carboxy derivatives of the xanthene fluorescent dyes fluorescein (FAM), 4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE), and tetramethylrhodamine (TAMRA) as individual isomers, and their conversion to non-nucleoside phosphoramidite reagents suitable for oligonucleotide labeling. The use of a cyclohexylcarbonyl (Chc) protecting group for blocking of phenolic hydroxyls facilitates the chromatographic separation of isomers of carboxy-FAM and carboxy-JOE as pentafluorophenyl esters. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride gave a mixture of 4-dimethylamino-2-hydroxy-2',4'(5')-dicarboxybenzophenones, easily separable into individual compounds upon fractional crystallization. Individual isomeric benzophenones are precursors of 5- or 6-carboxytetramethylrhodamines. The dyes were converted into 6-aminohexanol- (JOE), 4-trans-aminocyclohexanol- (FAM and JOE), and hydroxyprolinol-based (TAMRA) phosphoramidite reagents.
本单元描述了呫吨荧光染料荧光素（FAM）、4'，5'-二氯-2'，7'-二甲氧基荧光素（JOE）和四甲基罗丹明（TAMRA）的5-和6-羧基衍生物作为单个异构体的制备方法，以及它们转化为适用于寡核苷酸标记的非核苷亚磷酰胺试剂的过程。使用环己基羰基（Chc）保护基来封闭酚羟基有助于将羧基-FAM和羧基-JOE的异构体作为五氟苯基酯进行色谱分离。用1,2,4-苯三甲酸酐对3-二甲基氨基酚进行酰化反应，得到4-二甲基氨基-2-羟基-2'，4'（5'）-二羧基二苯甲酮的混合物，通过分步结晶可轻松分离成单个化合物。单个异构二苯甲酮是5-或6-羧基四甲基罗丹明的前体。这些染料被转化为基于6-氨基己醇-（JOE）、4-反式氨基环己醇-（FAM和JOE）和羟脯氨醇的亚磷酰胺试剂。
