Synthesis and characterisation of pure isomers of iodoacetamidotetramethylrhodamine

Abstract

The isomeric benzoylnitrobenzoate esters 5 and 6, prepared by condensation of 4-nitrophthalic anhydride and 3-(dimethy1amino)phenoI followed by esterification, were separated by fractional crystallisation and their structures assigned by NOE difference spectroscopy. Reduction of the nitro group in each compound followed by acetylation and ester hydrolysis gave the isomeric acetamido acids 7 and 8, which were efficiently condensed with 3-(dimethylamino)phenol in the presence of trimethylsilyl polyphosphate to give the acetamidorhodamines 9 and 10, respectively. These compounds were converted by standard means into the pure 6and 5-(iodoacetamid0)tetramethylrhodamines 1 and 2. The visible spectroscopic properties of the compounds were examined and accurate extinction coefficients determined.Paper 4/02892I

Optimised synthesis of 6-iodoacetamidotetramethylrhodamine

“…1 However, aspects of experimental detail required modification to ensure successful execution of the synthesis. Thus isomerically-pure 4-nitrophthalic anhydride 3 was prepared from commercial mixed isomers of nitrophthalic acid with minor modifications of published procedures as previously described, 1 and condensed with 3-dimethylaminophenol 4. The crude product was esterified with methanol (catalysed by concentrated H 2 SO 4 ) and the mixture of isomers 5 and 6 (ratio ~4:6) was fractionally crystallised from methanol to give the pure 4-nitroisomer 5.…”

“…Two further crystallisations allowed isolation of pure 5. In contrast, repeated efforts to obtain the 5-nitro isomer 6 by crystallisation from ethanol, as previously described, 1 were unsuccessful. The two isomers could be resolved by thin-layer chromatography (using multiple elutions) but this was not a practicable means to separate useful quantities of the 5-nitro isomer.…”

“…Quantities of 9 and 2 were determined spectrophotometrically, with solutions diluted in EtOH−water (9:1) and based on ε 549 96,900 M -1 cm -1 as previously determined. 1 To keep all the information together, details of the full experimental protocol for Schemes 1 and 2 are given below, although parts of the procedures (mainly in the conversion of 5 to 7) do not differ significantly from steps previously described. 1 Note also that, although rhodamine compounds are shown here as their open-chain, fluorescent tautomers, they are named as their spiro-lactone forms, as in the previous work.…”

“…A solution of 4-nitrophthalic anhydride 3 (33 g, 171 mmol, prepared as previously described 1 ) and redistilled 3-(dimethylamino)phenol 4 (24.2 g, 176.4 mmol) in dry toluene (570 ml) was heated under reflux for 6 h and cooled. The solvent was evaporated and the residue was dissolved in CHCl 3 and washed with 2 M HCl.…”

“…Some time ago, one of us described the synthesis and characterisation of the pure 5-and 6-isomers of iodoacetamidotetramethylrhodamine (compounds 1 and 2 respectively), 1 that are useful for specific labelling of cysteine residues in proteins or peptides. These and other reactive rhodamine dyes are widely used in biological microscopy applications particularly because of their good fluorescence properties, specifically brightness and resistance to photobleaching.…”