Electric dipole moments of the fluorescent probes Prodan and Laurdan: experimental and theoretical evaluations Cíntia C. Vequi-Suplicy, Kaline Coutinho & M. Teresa Lamy Biophysical Reviews ISSN 1867-2450 Volume 6 Number 1 Biophys Rev (2014) 6:63-74 DOI 10.1007/s12551-013-0129-8 1 23 Your article is protected by copyright and all rights are held exclusively by International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag Berlin Heidelberg. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self- archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Biophys Rev (2014) 6:63–74 DOI 10.1007/s12551-013-0129-8 REVIEW Electric dipole moments of the fluorescent probes Prodan and Laurdan: experimental and theoretical evaluations Cíntia C. Vequi-Suplicy & Kaline Coutinho & M. Teresa Lamy Received: 13 August 2013 /Accepted: 3 December 2013 /Published online: 14 January 2014 # International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag Berlin Heidelberg 2014 Abstract Several experimental and theoretical approaches Keywords Prodan . Laurdan . Optical absorption and can be used for a comprehensive understanding of solvent fluorescence . Electric dipole moment . Lippert–Mataga effects on the electronic structure of solutes. In this review, we equation . Quantum mechanics calculations revisit the influence of solvents on the electronic structure of the fluorescent probes Prodan and Laurdan, focusing on their electric dipole moments. These biologically used probes were Introduction synthesized to be sensitive to the environment polarity. However, their solvent-dependent electronic structures are The solvent effect on electronic properties of molecules is a still a matter of discussion in the literature. The absorption topic of great interest, both in the experimental (Reichardt and emission spectra of Prodan and Laurdan in different 2004;Lippert1955;Matagaetal.1956) and theoretical solvents indicate that the two probes have very similar elec- (Tomasi 2004; Tomasi et al. 2005; Miertus et al. 1981;Field tronic structures in both the ground and excited states. et al. 1990; Coutinho and Canuto 1997) areas. Solvents can Theoretical calculations confirm that their electronic ground produce significant changes in molecular properties, such as states are very much alike. In this review, we discuss the absorption and emission spectra, reactivity, electrochemistry, electric dipole moments of the ground and excited states solubility, among others (Reichardt 2004). Several experimen- calculated using the widely applied Lippert–Mataga equation, tal and theoretical approaches can be used for a comprehen- using both spherical and spheroid prolate cavities for the sive understanding of solvent effects. In this review, we revisit solute. The dimensions of the cavity were found to be crucial the influence of solvents on the electronic structure of the for the calculated dipole moments. These values are compared fluorescent probes Prodan and Laurdan, focusing on the elec- to those obtained by quantum mechanics calculations, consid- tric dipole moments of these fluorophores. ering Prodan in vacuum, in a polarizable continuum solvent, Prodan and Laurdan (Fig. 1)arelargelyusedinbiological and using a hybrid quantum mechanics–molecular mechanics relevant systems. They were synthesized to be sensitive to the methodology. Based on the theoretical approaches it is environment polarity, so their emission spectra shift about evident that the Prodan dipole moment can change even 120 nm from cyclohexane to water (Weber and Farris 1979; in the absence of solute–solvent-specific interactions, Parasassietal.1986; Catalan et al. 1991;Lakowicz2006). which is not taken into consideration with the experi- When inserted into membranes, their emission spectra are mental Lippert–Mataga method. Moreover, in water, for extremely dependent on the lipid bilayer phase (gel or fluid) electric dipole moment calculations, it is fundamental to con- (Zeng and Chong 1991; Rottenberg 1992; Ferretti et al. 1993; sider hydrogen-bonded molecules. Ambrosini et al. 1994, 2001;Allevaetal.1995;Belletal.1996; Bagatolli et al. 1997; De Vequi-Suplicy et al. 2006;Moyano et al. 2008;Lucioetal.2010; Vequi-Suplicy et al. 2013). Special Issue Advances in Biophysics in Latin America : : However, the mechanism that makes Prodan and Laurdan C. C. Vequi-Suplicy K. Coutinho M. T. Lamy (*) so sensitive to polarity changes, and to lipid gel–fluid phase Instituto de Física, Universidade de São Paulo, CP 66318, transition, is still under discussion. A number of polemic CEP 05315-970 São Paulo, SP, Brazil e-mail: [email protected] points are presented in the literature on the structure and URL: http://fig.if.usp.br/~mtlamy/ electronic distribution of the fluorophores in different Author's personal copy 64 Biophys Rev (2014) 6:63–74 Fig. 1 Prodan (top) and Laurdan H H H30 22 24 (bottom) structures and atom H 29 23 H8 H H1 12 numbers used in the text 27 21 H H 7 28 2 9 26 25 H11 N 20 6 4 17 3 H H34 31 10 18 H 33 H H 13 5 O 32 19 15 H H16 14 H H H H H H H H N H H H H H H O H H solvents, both in the ground and excited states (Balter et al. calculations of the ground state electric dipole moment will be 1988; Bunker et al. 1993;Mennuccietal.2008). For example, discussed, considering both the polarizable continuum model one very controversial point is the structure and electronic (PCM) (Miertus et al. 1981;Tomasietal.2005) and a more distribution of the excited state. At least two emission bands complex discrete model, with quantum mechanics/molecular are present in the emission spectra of Prodan or Laurdan. mechanics hybrid calculations (S-QM/MM) (Coutinho and These have been attributed to two excited electronic states: a Canuto 1997;Canutoetal.2000; Georg et al. 2006). locally excited state (LE), and an internal charge transfer (ICT) or a twisted ICT state (TICT) (Rollinson and Drickamer 1980; Nowak et al. 1986; Ilich and Prendergast Comparing the optical absorption and emission spectra 1989; Parusel et al. 1997, 1998, 2001; Viard et al. 1997; of Prodan and Laurdan Parusel 1998; Kozyra et al. 2003; Tomin et al. 2003; Tomin 2006; Tomin and Hubisz 2006; Novaira et al. 2007, 2008; To understand the electric structure of Prodan and Laurdan, it Adhikary et al. 2009;Morozovaetal.2009; Everett et al. is fundamental to analyze the optical absorption and emission 2010; ). In the latter state, the dimethylamine and the spectra of the fluorophores in different solvents. Figure 2 propanoyl groups would be rotated out of the naphthalene shows the absorption spectra of Prodan (black) and Laurdan ring due to the high charge separation. However, several (red) in solvents with different polarities: cyclohexane, chlo- authors do not agree with the above explanation (Balter roform, dichloromethane, acetonitrile, methanol and water1. et al. 1988; Catalan et al. 1991;Bunkeretal.1993; Samanta Considering that the word “polarity” is used in a very wide and Fessenden 2000;LoboandAbelt2003;Mennuccietal. way (Reichardt 2004), Table 1 shows the different parameters 2008;Roweetal.2008). for the solvents cited above, developed to assess the sample Another discussion in the literature concerns the dipole polarity: refraction index (n), electric dipole moment (μ), moment values of Prodan and Laurdan (Weber and Farris dielectric constant (ε), and the polarity scales, Δf (Lippert 1979; Nowak et al. 1986;Balteretal.1988; Balter et al. N 1955;Matagaetal.1956)andET (Reichardt 1994). 1988; Ilich and Prendergast 1989; Catalan et al. 1991; In all solvents used, Prodan and Laurdan can be seen to Bunker et al. 1993; Sun et al. 1997; Parusel et al. 1997, display very similar optical absorption spectra (Fig. 2), apart 1998, 2001; Parusel 1998; Kawski et al. 2000;Frischetal. from water, where Laurdan is not soluble. This is a strong 2004; Huang et al. 2006), and this is the focus of the present review. Here we consider the evaluation of electric dipole mo- 1 Several papers mention the wavelengths of the maximum absorption of ments for Prodan and Laurdan using two different approaches. the lower energy band of Prodan in different solvents (Weber and Farris The experimental approach is based on the largely used 1979;Heiseletal.1987;Balteretal.1988; Catalan et al. 1991; Kawski – 1999; Moyano et al. 2006). Possibly due to the broad absorption band, the solvatochromic shift method, with the Lippert Mataga equation cited positions of the maximum absorption of the lower energy band vary (Lippert 1955; Mataga et al. 1956),andonamodificationofthis from 354.9 to 361 nm in chloroform, and from 356.8 to 364 nm in water. equation, considering a different geometry for the solute cavity In some solvents, the Prodan optical absorption spectrum has been (Scholte 1949). This methodology assumes that the fluorophore published (Bunker et al. 1993; Parusel et al. 1998; Artukhov et al. 2007; Sun et al. 1997). To our knowledge, this is the first time that electric dipole moments, both for the ground and the excited absorption spectra of Laurdan are published and compared with Prodan states, are independent of the solvent. Conversely, theoretical in different solvents. Author's personal copy Biophys Rev (2014) 6:63–74 65 Fig. 2 Comparing Prodan (black line) and Laurdan (red line) absorption spectra in water (a), methanol (b), acetonitrile (c), dichloromethane (d), chloroform (e) and cyclohexane (f). [Prodan]=[Laurdan]=4 μM. Dashed lines at 280 and 350 nm are for guiding the eyes only indication that the two probes have very similar ground state energy (higher wavelength) absorption band (the latter also energies and geometries.