Simple and scalable synthesis of 5- and 6-carboxytetramethylrhodamines (TAMRAs) is reported. Acylation of 3-dimethylaminophenol with 1,2,4-benzenetricarboxylic anhydride afforded a mixture of 4-dimethylamino-2-hydroxy-2',4'(5')-dicarboxybenzophenones, which can be easily separated into individual compounds upon recrystallization from methanol and acetic acid. Individual benzophenones were reacted with 3-dimethylaminophenol to give 5- or 6-carboxytetramethylrhodamines. The dyes were converted into hydroxyprolinol-based phosphoramidite reagents suitable for oligonucleotide synthesis. 5- and 6-TAMRA isomers on oligonucleotides showed similar absorption and emission spectra. Fluorescence quantum yield of the dyes correlates with the presence of dG nucleosides in the adjacent region of oligonucleotide sequence. Several energy transfer primers containing on their 5'-termini (6-FAM)dT(n)(6-TAMRA) dye system (n = 0, 2, 4, 6, 8, 10, 12, 14) were prepared, and their spectral properties were studied.

The Diels–Alder reaction of 3‐methoxyestra‐1,3,5(10),14,16‐pentaen‐17‐yl acetate (1) with nitroethylene gives 3‐methoxy‐16‐nitro‐14,17‐ethenoestra‐1,3,5(10)‐trien‐17β‐yl acetate (2). Cycloadduct 2 can be reduced with TiCl3, giving oxazine 3 as a result of sigmatropic rearrangement, whereas the treatment of 2 with Lewis acids (TiCl4, SnCl4) leads to a derivative of the cyclic hydroximic acid 10; the latter is viewed as a product of intramolecular rearrangement. Cycloadduct 2 suffers a weak base‐induced cleavage of the C(16)–C(17) bond, releasing the nitrile oxide intermediate, which can be trapped by a dipolarophile or reduced with triphenylphosphane. Thus, the trapping of the nitrile oxide with propargyl alcohol gives the isoxazole 12 in 50 % yield, and the reduction of the nitrile oxide with triphenylphosphane gives nitrile 9 in 94 % yield. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

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