Characterization of the Excited States of 4-Amino-1,8-naphthalimides Towards an Understanding of Interactions with Proteins and Amino Acids

Abstract

Light is increasingly used in the development and design of new molecules in a variety of applications. Light is used as an initiator for chemical reactions, as a tool to excite photoactive molecules into a more reactive state, and as an energy source for imaging purposes. The use of photoactive molecules in biomedicine is of great interest due to the versatility and efficacy of the intended applications. One such application is the photochemical crosslinking of tissues, or ‘tissue welding,’ as a method of wound healing and an alternative to sutures. This is especially of interest for tissues which are unable to be easily treated with sutures, such as cartilaginous joint tissues in the knee. 4-Amino-1,8-naphthalimides were synthesized to evaluate them as photochemical crosslinkers and determine the efficiency of blue-light excitation. The 4-amino-1,8-naphthalimides were differentiated by the nature of the azacyclic or dimethyl 4-amino moiety and the imide substituent. The 4-amino substituents were chosen to determine the effect of ring size on the ability of the ring to enter a twisted intramolecular charge transfer state. Entering this state can change fundamental properties such as fluorescence quantum yield and excited state energies, as well as oxidation-reduction potentials, which would contribute to the reactivity for oxidative crosslinking. The photophysical properties of the 4-amino-1,8-naphthalimides were determined through absorption and steady-state fluorescence spectroscopy, as well as time-correlated single photon counting. Fluorescence quantum yields and fluorescence lifetimes were calculated, and photochemical reactivity with oxidizable amino acids and proteins were determined. Dynamic singlet-state quenching of fluorescence intensity was observed in the presence of L-tryptophan, and dynamic fluorescence intensity enhancement was observed in the presence of L-histidine or a glycine-tyrosine dipeptide. Dynamic singlet-state quenching of fluorescence lifetimes was observed in the presence of oxidizable amino acids. Static fluorescence enhancement was observed in the presence of protein (bovine serum albumin or lysozyme). The rate constants of dynamic quenching with L-tryptophan (~2-3.5 x 109 M-1 s-1) were up to one or two orders of magnitude greater than the rate constants for quenching with L-histidine (0.4-3.2 x 108 M-1 s-1) or a glycine-tyrosine dipeptide (0.55-1.1 x 109 M-1 s-1). Gel electrophoresis experiments showed that photoinduced oxidative crosslinking did not occur with either bovine serum albumin or lysozyme after multiple hours of blue-light irradiation of the ANIs.