DOI: 10.1002/chem.200801104 Water-Soluble Nile Blue Derivatives: Syntheses and Photophysical Properties Jiney Jose, Yuichiro Ueno, and Kevin Burgess* [a] Introduction Nile Blue, A, is a fluorescent probe that has been known for over 110 years. [1, 2] In polar media its absorption and emis- sion maxima shift to the red, which is indicative of stabilized charge separation in the excited state; consequently, this dye has been used to monitor events that depend upon solvent polarity. [3–5] It has also been used for fluorescence resonance energy transfer (FRET) studies. [6, 7] Nile Blue tends to have a higher affinity for cancerous cells than healthy ones [8] and it is a photosensitizer for oxygen; [9, 10] these two properties together can be useful in photodynamic therapy. [11] Howev- er, two properties of Nile Blue in aqueous media are limit- ing for many applications, specifically 1) low solubility and 2) low quantum yield. Some work has been published on Nile Blue derivatives with improved water solubilities. [12–15] The aggregation of in- herently flat, lipophilic aromatic dyes is disfavored when they are functionalized with water-solubilizing substituents and their quantum yields can improve as a result. Conse- quently, Nile Blue derivatives with hydrophilic groups can have improved solubilities and fluorescence outputs. Deriva- tives B and C are the most interesting probes to arise from these studies. [12] Their quantum efficiencies are improved by as much as a factor of ten, however, they have no carboxylic acid handle for attachment to biomolecules, and the sharp- ness of their emissions broadens as the solvent is changed from methanol to water, which is perhaps indicative of ag- gregation. Other work involved the incorporation of groups that offer only incrementally enhanced water solubilities, [13] lack of quantum yield data, [14] and/or no experimental proce- dures for the syntheses. [14, 15] This paper reports the syntheses of 2-hydroxy Nile Blue derivatives 1 and 2 and compares their fluorescence proper- ties with those of B and C. For both probes, a 2-hydroxy substituent was incorporated to enhance water solubility and other hydrophilic groups are situated on both ends of the molecule to reduce the potential for aggregation. Not all Abstract: Four water-soluble 2-hy- droxy-Nile Blue derivatives, 1a, 1b, 2a, and 2b, were prepared by condensation reactions performed under relatively mild conditions (90 8C, N,N-dimethyl- formamide with no added acid). These fluorescent probes had more favorable fluorescence characteristics than two known water-soluble Nile Blue deriva- tives. Specifically, they were superior to the known dyes with respect to their quantum yields in aqueous media and the sharpness of their fluorescence emissions. Concentration-dependant UV absorption and fluorescence emis- sion studies indicated that the dyes did not aggregate in aqueous solution at concentrations of less than 1–4 mm. The new water-soluble materials 1a, 1b, 2a, and 2b emit in a desirable region of the fluorescence spectrum (l = 670– 675 nm). Overall they are potentially interesting for labeling biomolecules in aqueous environments. Keywords: biochemical markers · dyes/pigments · fluorescence · Nile Blue · water-soluble groups [a] J. Jose, Y. Ueno, K. Burgess Department of Chemistry Texas A&M University, Box 30012 College Station, TX, 77841 (USA) Fax: (+ 1) 979 845-8839 E-mail : burgess@tamu.edu Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200801104.  2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Chem. Eur. J. 2009, 15, 418 – 423 418