Photooxidation of Amino Acids and Proteins Mediated by Novel 1,8-Naphthalimide Derivatives

Abstract

The ground- and excited-state interactions of N-(2-ethanoic acid)-1,8-naphthalimide (NI-ala), along with tryptophan and tyrosine-substituted 1,8-naphthalimide derivatives (NI-trp and NI-tyr), with amino acids and native proteins have been studied in aqueous buffered solution. The singlet excited state of NI-ala reacts with the aromatic amino acids, tryptophan, tyrosine, and histidine with a rate constant at or exceeding the diffusion-controlled limit. Reactivity with bovine serum albumin (BSA) and lysozyme was also demonstrated. However, in all cases, no long-lived redox products were observed from the singlet state quenching. The reactivity of the triplet excited state of NI-ala with the individual amino acids and proteins was studied using laser flash photolysis. Triplet-state reactivity was demonstrated with tryptophan and tyrosine, along with BSA and lysozyme proteins. In the case of tryptophan, tyrosine, and lysozyme, long-lived redox products were observed and quantified. Neutral tryptophan and tyrosyl radicals were observed by transient spectroscopy as quenching products. Upon photolysis of all three 1,8-naphthalimide derivatives, both cleavage and cross-linking of the lysozyme are observed. These studies have identified the potential oxidative targets in native proteins and demonstrated the utility of our naphthalimide derivatives to photomodify native proteins.

Photochemical Reduction of Cytochrome c by a 1,4,5,8‐Naphthalenediimide Radical Anion¶

“…These results suggest that the protein acts as the electron donor in Step 3, a process that could be mediated by oxidizable amino acid residues in the protein. This conclusion is supported by the reports of Abraham and Kelly ( 27 ), who observed photoinduced ET from bovine serum albumin and lysozyme to excited state 1,8‐naphthalimide derivatives. They identified tryptophan, tyrosine, histidine and lysine as the donor amino acids.…”

Enzyme Modification and Oxidative Cross‐linking Using Carboxylate‐, Phenol‐ and Catechol‐Conjugated 1,8‐Naphthalimides

“…The spectral shapes of the three compounds are identical. However, the fluorescence intensities of NI‐Tyr and NI‐Dopa are more than 100 times smaller than that of NI‐Ala (ɸ f = 0.34 ± 0.02) . The fluorescence quantum yields were found to be 0.0059 ± 0.0014 and 0.0027 ± 0.0012 for NI‐Tyr and NI‐Dopa , respectively.…”

“…indicate that conjugation of tyrosine or dopa to 1,8‐naphthalimides introduces a singlet‐state (S 1 ) deactivation pathway that is not present in NI‐Ala . We have previously shown that tyrosine quenches the singlet state of NI‐Ala at diffusion‐controlled rates . The rate constant for quenching by lysozyme is almost two orders of magnitude larger than that expected for a diffusion‐controlled process.…”

“…We have previously reported the triplet‐mediated lysozyme oxidation using NI‐Ala and NI‐Tyr . The report demonstrated that irradiation of NI‐Ala or NI‐Tyr in the presence of lysozyme produced dimers.…”

“…UV (10 m m phosphate buffer pH 7.0) λ max ( ε ): 344 nm (12 600 ± 200 m −1 cm −1 ), 274 nm (4200 ± 100 m −1 cm −1 ) and 234 nm (27 300 ± 400 m −1 cm −1 ) . 1 H NMR (DMSO‐d 6 ): δ 8.53 (dd, 4H, naphth), 7.92 (t, 2H, naphth), 6.83 (d, 2H, phen), 6.44 (d, 2H, phen), 5.78 (t, H, CH) and 2.42 (m, 2H, CH 2 ).…”