Evaluating the Role of Chromophore Side Group Identity in Mediating Solution-Phase Rotational Motion Kelly P. Greenough and G. J. Blanchard* Michigan State UniVersity, Department of Chemistry, East Lansing, Michigan 48824-1322 ReceiVed: August 22, 2006; In Final Form: NoVember 4, 2006 We report on the rotational diffusion dynamics of the chromophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) in a series of protic and polar aprotic solvents, as a function of the identity of the side group appended to the chromophore amine functionality. The central issue we address is whether or not the side groups play a role in mediating the anisotropic reorientation dynamics of the chromophore. To understand the motional properties of the chromophores in detail, we use both one-photon and two-photon excited fluorescence anisotropy decay measurements, and from these complementary excitation methods, we extract two of the Cartesian components of the rotational diffusion constant, D. The experimental data indicate that, regardless of the functionality of the pendant side group, the reorienting moieties exhibit ratios of D z /D x in the range 1.8-2.0. There is a small but discernible difference between the substituted chromophores. For all of the substituted NBD chromophores, dielectric friction plays a discernible role in determining their reorientation dynamics. Introduction The motion of a molecule in solution is controlled by a number of factors, ranging from its size, shape, and polarity to the nature of its interactions with its immediate environment. It is these interactions that play a critical role in mediating processes such as chemical reaction kinetics, for example. As a result of a broad effort over the past decade or more, a series of chromophores has been identified that are capable of providing information on their local environment with both their time- and frequency-domain responses. Molecules such as pyrene are known to exhibit solvent polarity-dependent fluo- rescence spectral profiles, and the fluorescence lifetime of this molecule depends sensitively on the amount of oxygen present. 1 Other chromophores have been developed that are tethered to specific structures, allowing them to locate in comparatively well-defined regions of heterogeneous systems. The so-called lock-and-key approach to the examination of crystallization phenomena makes use of chromophores that have as pendant side groups the crystallizing entity. 2-4 This approach to the optical interrogation of molecular-scale phenomena has proven to be useful to a broad range of biological and chemical investigations. One chromophore that has been used widely for the study of biological and biomimetic systems is 7-nitrobenz-2-oxa-1,3- diazole, NBD. 5-12 The reason for the popularity of this chromophore is that its fluorescence spectral profile and lifetime both depend sensitively on the polarity of its immediate environment. In addition, the chromophore NBD is amenable to synthetic substitution or modification of its amine side group, rendering it useful for localization within heterogeneous systems. A variety of sterols, lipids, and other compounds are available with a pendant NBD chromophore. A recurring question in these studies is the role that the chromophore side group plays in mediating its dynamics. In an effort to address this issue, we have undertaken a study of the reorientation and fluorescence lifetime of NBD possessing several different side groups in a series of polar protic and aprotic solvents. Our data indicate that the reorientation dynamics of the NBD chromophore depend on the identity of the chromophore side group to a limited extent and that we can account for the experimental data in the context of a combination of frictional and dielectric interactions between the dipolar chromophore and its immediate environment. Experimental Section Materials. The fluorescent probes (Chart 1) succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino) hexanoate (SNBD) and 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino hexanoic acid (NBDHA) were obtained from Molecular Probes, Inc., and used without further purification. The probe 4-benzylamino-7- nitrobenzofurazan (BBD) was purchased from Sigma-Aldrich and was also used as received. The solvents methanol, 1-pro- panol, 1-butanol, 1-pentanol, dimethyl sulfoxide (DMSO), N,N- dimethylformamide (DMF), and acetonitrile (ACN) were pur- chased from Sigma-Aldrich in their highest purity available and * Author to whom correspondence should be addressed. Email: blanchard@chemistry.msu.edu. CHART 1: Structures of the Substituted NBD Chromophores Used in This Work 558 J. Phys. Chem. A 2007, 111, 558-566 10.1021/jp065423n CCC: $37.00 © 2007 American Chemical Society Published on Web 12/21/2006