Texas Red-X and rhodamine Red-X, new derivatives of sulforhodamine 101 and lissamine rhodamine B with improved labeling and fluorescence properties

Texas Red sulfonyl chloride (TR-SC) and Lissamine rhodamine B sulfonyl chloride (LRB-SC) are popular dyes often used to prepare red fluorescent conjugates that are useful second labels in combination with fluorescein. Unfortunately, being sulfonyl chloride derivatives, both are unstable to moisture during storage and prone to hydrolysis in the conjugation reaction. Their instability causes the percentage of reactive dye to vary from lot to lot and requires use of low temperatures and a relatively high pH to optimize conjugation efficiency. Succinimidyl esters of the aminohexanoic acid sulfonamides of both dyes have been prepared, which are designated Texas Red-X succinimidyl ester (TR-X-SE) and Rhodamine Red-X succinimidyl ester, respectively. Their spectral properties are similar to those of their sulfonyl chloride analogs; moreover, incorporation of the succinimidyl ester at the end of the aliphatic chain spacer facilitates conjugation, decreases precipitation of proteins during conjugation and storage, and usually increases the fluorescence yield of the conjugate. Comparison of the rate of hydrolysis of TR-SC with that of TR-X-SE shows that, while the former was completely hydrolyzed within 5 min by exposure to water, TR-X-SE retains most of its reactivity for more than an hour. The reactivity of both new derivatives is high between pH 7.5 and 8.5, allowing conjugation of proteins that do not tolerate the high pH required for reaction with sulfonyl chlorides. In addition, Texas Red maleimides and haloacetamides containing spacer groups were prepared for labeling sulfhydryl groups. A Texas Red-X derivative of phalloidin has also been prepared, and its use for labeling F-actin has been characterized.