Practical Synthesis of Isomerically Pure 5- and 6-Carboxytetramethylrhodamines, Useful Dyes for DNA Probes

Abstract

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.

Solid-phase synthesis provides a modular, lysine-based platform for fluorescent discrimination of nitroxyl and biological thiols

“…Isomerically pure 5-carboxytetramethylrhodamine (5-CO 2 H-TAMRA, Scheme S3†)41 was first coupled to the Lys ε-position. Following reaction with iodoacetic acid N -hydroxysuccinimide ester ( S9 , Scheme S3†),42 the binding site was appended by stirring the resin with excess cyclam overnight in a dichloromethane–methanol solution.…”

“…Building on knowledge gained from designing CuDHX1, 27 we sought to achieve HNO selectivity for the first lysine conjugate, produced by employing Fmoc-Lys(Mtt)-OH as the linker, the yellow-emitting ( λ em ≈ 580 nm) rhodamine scaffold as the fluorescent moiety, and cyclam as the metal-binding site. Isomerically pure 5-carboxytetramethylrhodamine (5-CO 2 H-TAMRA, Scheme S3 † ) 41 was first coupled to the Lys ε-position. Following reaction with iodoacetic acid N -hydroxysuccinimide ester ( S9 , Scheme S3 † ), 42 the binding site was appended by stirring the resin with excess cyclam overnight in a dichloromethane–methanol solution.…”

Characterizing new fluorescent tools for studying 5-HT3 receptor pharmacology

“…1A) were synthesized accordingly (for detailed description of synthetic procedures, full spectroscopic characterization and purity assessment see Supplementary data). Briefly, fluorophores were either purchased (rhodamine 101 and acridone; Sigma Aldrich) or synthesized according to published procedures (Egawa et al, 2011;Holzhauser et al, 2010;Kvach et al, 2009), and coupled to the granisetron core via linkers. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 …”

Recent Progress in Small Spirocyclic, Xanthene-Based Fluorescent Probes

“…SiR-COOH, which SiR-Hoechst is based on, tends to exist in its spirocyclic form before binding to proteins, but can be converted to the fluorescent xanthene form after binding. These and related fluorophores can either exist as 5 -or 6 -carboxy-isomers and only recently the 4 -carboxy-isomers were synthesized by the same group, which will be addressed at a later point in the review [144][145][146][147][148]. Depending on the location of the carboxylic acid, the spectral properties might be identical, but their localization and cytotoxicity can be different.…”