NMR analysis of Nile Blue (C. I. Basic Blue 12) and Thionine (C. I. 52000) in solution David Hazafy a , Marie-Virginie Salvia a , Andrew Mills a , Michael G. Hutchings c , Maxim P. Evstigneev b , John A. Parkinson a, * a WestCHEM Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, UK b Department of Physics, Sevastopol National Technical University, Sevastopol 99053, Crimea, Ukraine c School of Chemistry, University of Manchester, Oxford Road, Manchester M13 9PL, UK article info Article history: Received 7 April 2010 Received in revised form 29 July 2010 Accepted 30 July 2010 Available online 7 August 2010 Keywords: Nile Blue Thionine NMR spectroscopy Self-assembly Numerical analysis Molecular modelling abstract The dyes Nile Blue (C. I. Basic Blue 12) and Thionine (C. I. 52000) were examined in both ionic and neutral forms in different solvents using NMR and UVevisible spectroscopy to firmly establish the structures of the molecules and to assess the nature and extent of their aggregation. 1 H and 13 C NMR assignments and chemical shift data were used, together with nuclear Overhauser effect information, to propose a self- assembly structure. These data were supplemented with variable temperature, dilution and diffusion- based experimental results using 1 H NMR spectroscopy thereby enabling extended aggregate structures to be assessed in terms of the relative strength of self-association and the extent to which extended aggregates could form. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction C. I. Basic Blue 12 (Nile Blue, NB), 1, is a classical dye which, despite being known for more than 100 years [1] remains of current interest as the basis for the development of new dyes and stains. It belongs to a class of molecules whose basic framework is that of a benzophenoxazine, a class which also includes Nile Red, a phenoxazinone, here termed Red Nile Blue (RNB) and Meldola’s Blue (C. I. Basic Blue 6, C. I. 51175). These and related compounds are extensively used in scientific appli- cations that make use of their fluorescent and solvatochromic characteristics. For RNB such applications have recently included visualization of protein conformational changes for engineered proteins containing a 4Cys motif in a live cell setting using an arsenic-modified RNB derivative [2] whereas NB has seen application as a stain for Escherichia coli in flow cytometry [3], in a modified form as a chemodosimeter for Hg 2þ in biological media [4] and has also been used to monitor processes that depend on solvent polarity [5e7], in (F)RET [8,9] and as a photosensitizer for oxygen in photodynamic therapy applica- tions [10,11]. The property that can make these molecules attractive as stains and imaging agents is their high fluorescence quantum yield together with their solvatochromism. In polar environments, their fluorescence is reduced significantly and additionally, for molecules that would be of general use in identifying and binding to biomolecules, their aqueous solubility is generally very poor. Active research is taking place to define aqueous analogues of these benzophenoxazines and increasing success in producing these is being achieved [12]. Such mole- cules show higher fluorescence quantum yields than the parent molecules, a feature related to altered aggregation characteris- tics. As with many flat aromatic molecules, these phenoxazine- based molecules generally self-associate. Some detail of the manner by which this occurs has been reported and it is this aggregating property that is believed to be generally responsible for quenching the fluorescence response [13e15] and which is alleviated in polar solvents. When these molecules can be functionalized for enhanced aqueous solubility, self-assembly can be disrupted which logically leads to enhanced fluorescence [16]. Whilst many of the physical properties of these dyes have been studied, little has been reported of any fine detail on the manner in which this self-association occurs. Also in the case of * Corresponding author. Tel.: þ44 141 548 2820; fax: þ44 141 548 4822. E-mail address: john.parkinson@strath.ac.uk (J.A. Parkinson). Contents lists available at ScienceDirect Dyes and Pigments journal homepage: www.elsevier.com/locate/dyepig 0143-7208/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.dyepig.2010.07.014 Dyes and Pigments 88 (2011) 315e325