1. PROBING PROTEIN STRUCTURE AND INTERACTIONS USING FUNCTIONALIZED NAPHTHALIMIDES L. Kelly, B. Abraham, M. Mullan Department of Chemistry and Biochemistry, University of Maryland, Baltimore County FIGURE 1. Synthetic Structural Probes iNTRODUCTION. Biomolecular interactions, particularly those involving nucleic acids and proteins with each other or with small molecules, are ubiquitous and critical to maintaining structure and function. Work in our laboratory has focused on using water soluble 1,8-naphthalimides and 1,4,5,8-naphthalene diimides as protein structural probes. The compounds initiate a number of photochemical reactions. It is of interest to develop small molecules that will cleave, crosslink, or photoaffinity label proteins and DNA. In this study, the fundamental reactions with amino acids and proteins are studied to understand the mechanisms of protein modification. 15  s EXPERIMENTAL RESULTS. Naphthalene imides and diimides are synthesized from commercially available aromatic anhydrides and primary amines. To date, the anionic, cationic, and polyamine derivatives shown in Figure 1 have been synthesized. Laser Flash Photolysis Studies. Transient spectroscopy was used to identify the specific amino acids that are oxidized by the naphthalimide excited states. The transient spectrum shown in Figure 2 illustrates that the oxidation of both tyrosine and tryptophan is mediated by the 1,8-naphthalimide triplet excited states (eq 1). When the experiment is conducted in an air-saturated aqueous solution, the spectra of the amino acid radicals are cleanly observed. 0.5  s FIGURE 2. Transient absorption spectrum of (II) (10  M) measured in an air-saturated aqueous solution of (a) 2.25 mM tyrosine and (b) tryptophan. The spectrum observed at long times (15  s) shows similar spectral features as the known spectrum of the tyrosyl and tryptophan radicals ((c) and (d), respectively (From ref. 1)). 15  s (a) (b) (c) (d) The rate constants for triplet-mediated electron transfer were measured using individual amino acids. Tyrosine and tryptophan were found to be the only amino acids that could be oxidized by the triplet state of compound (II). The native proteins, bovine serum albumin (BSA) and lysozyme also quenched the triplet state. Radicals were observed as lysozyme quenching products. The rate constants for the reaction shown in eq 1 were determined from the bimolecular quenching plots shown in Figure 3. The rate constants and radical (cage escape) yields are summarized in Table 1 (ref. 2). Amino Acid k q (x10 -9 M -1 s -1 ) Radical YieldTryptophan 2.60 ± 0.16 0.52 ± 0.04 Tyrosine 0.89 ± 0.03 0.33 ± 0.05 BSA 0.42 ± 0.02 Not observed Lysozyme 0.76 ± 0.01 0.22 ± 0.01 FIGURE 3. Bimolecular quenching plots for reaction of the triplet excited states of compound (II) with tryptophan (red) and tyrosine (blue). Inset: Triplet- state decay with added (0 – 50  M) tryptophan. TABLE 1. Summary of bimolecular rate constants for and radical yields from the reaction of 3 II* with amino acids and proteins. Photochemistry of Naphthalene Diimides. The redox properties of the naphthalene imides and diimides are known. 1,4,5,8-naphthalene diimides ((III) and (IV)) have one-electron reduction potentials that are ca. 0.5 V more positive than their 1,8-naphthalimide counterparts (I and II). Thus, it was of interest to compare their photoreactivity with amino acids and proteins. The fluorescence emission spectra of compounds III and IV were compared. These are shown in Figure 4. As seen from the figure, the relative intensity of compound IV (and other carboxylated naphthalene diimides) is significantly smaller than that of compound III. The quantum yields are indicated on the figure. FIGURE 4. Fluorescence emission spectra of aqueous buffered solutions of compounds III and IV. The solutions were optically matched at the excitation wavelength of 382 nm. The absolute fluorescence quantum yields are given. (IV) (  = 0.017) (III) (  = 0.0009) The data shown in Figure 4, along with other experiments (ref. 3) indicate that the singlet state of compound III is rapidly quenched via intramolecular electron transfer (Scheme I). By analogy to published work on phthalimides, we propose that, following intramolecular electron transfer, homolytic bond cleavage produces CO 2 and the carbon-centered radical. Preliminary evidence (Figure 5) suggests that this carbon-centered radical may covalently attach to a protein. FIGURE 5. HPLC chromatograph showing separation of (III) from the protein BSA. Upon irradiation (  > 320 nm; 0 – 20 min as indicated) of (III) in the presence of BSA, the peak at ca. 15 minutes shows the UV absorption spectrum of (III). Irradiation of (III), followed by mixing with a solution of BSA, did not indicate attachment of the naphthalene diimide to the protein. Prospects as Protein Structural Probes. Compounds (I) – (IV) have shown diverse photochemical reactivity with proteins. As shown in Figures 2 and 3, the naphthalene imide (II) predominantly reacts with proteins and amino acids via one- electron amino acid oxidation in the absence of oxygen. Gel electrophoresis confirms that oxidative cross-linking is the major protein modification product (Figure 6). The reaction is believed to occur following oxidation of either tyrosine or tryptophan, deprotonation to product the neutral radical, and radical-radical recombination. FIGURE 6. Separation of protein photoproducts using gel electrophoresis (20% SDS/PAGE, post-stained with coomassie blue). Solutions contain 80  M of lysozyme and 50  M of (II). In all cases, lanes 1 and 2 contain only lysozyme (no (II)); samples run in lanes 1 and 3 have been kept in the dark. Lane 2 has been exposed to light for the entire duration of photolysis. Lanes 4, 5, 6, 7 represent lysozyme samples that have been irradiated for 0.5, 1, 2, and 3 hours respectively, under nitrogen-saturated conditions. 14 kD 6.5 kD 27 kD Photoaffinity Labels. Benzophenone-ligand (drug) conjugates have been used to probe protein-ligand interaction sites. Production of the n-p* excited state, followed by hydrogen atom abstraction and radical recombination, gives rise to a covalent adduct between the protein and benzophenone. This is shown in Figure 7. Protein degradation and fragment analysis is then used to identify the ligand-protein interaction site. Unfortunately, benzophenone has a very small molar extinction coefficient above 300 nm and requires a high concentration to produce a reasonable absorption cross-section. FIGURE 7. Benzophenone-initiated protein photoaffinity labeling. Preliminary evidence (Figure 5) suggests that carboxyalkyl-functionalized 1,4,5,8- naphthalene diimide derivatives may be well-suited for photoaffinity labeling reagents. These compounds have molar extinction coefficients in excess of 20,000 M -1 cm -1. The proposed mechanism for protein photoaffinity labeling is given in Scheme III. CONCLUSION. We have shown that naphthalene imides and diimides can be light- activated to initiate a variety of protein modification event. Photoinduced cleavage, cross-linking, and affinity-labeling are useful tools to probe the structure and dynamics of proteins. The development of new photoaffinity labeling agents with large extinction coefficients offers the possibility of using these reagents at  M concentrations. SCHEME III. ACKNOWLEDGMENTS AND REFERENCES. This work was supported by NSF Grant CHE-9984874 (1)Wagenknecht, H. A.; Stempt, E. D.A.; Barton, J. K. Biochemistry 2000, 39, 5483- 5491. (2) Abraham, B.; Kelly, L. A. J. Phys. Chem. B 2003, 107, 12534 – 12541. (3) Abraham, B.; McMasters, S.; Mullan, M. A.; Kelly, L. A. J. Am. Chem. Soc. 2004, 126, 4293-4300.