Synthesis and photophysical studies of new pyrenylamino acids Goreti Pereira, a Elisabete M. S. Castanheira, b,* Peter Schellenberg, b M. Belsley, b Paula M. T. Ferreira, a Luís S. Monteiro a a Centre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. b Centre of Physics, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal. 1. Introduction Most of fluorescence studies concerning peptides and proteins make use of the natural fluorescent amino acids, tyrosine, tryptophan and phenylalanine. However, the development of new fluorescent non-natural amino acids that can allow selective excitation and detection and act as site specific probes constitutes an important area of research in peptide chemistry. 1-3 Pyrene has unique photophysical properties, such as high fluorescence quantum yield, long excited-state lifetimes and the ability to form excimers when two pyrene moieties are in close proximity. 4,5 There are several reports describing the synthesis and applications of pyrenylalanine as a fluorescent probe in peptides and proteins. 6-8 Previously, we have reported the synthesis of several new pyrenylamino acid derivatives. 9,10 These compounds were prepared from dehydroamino acid derivatives using several types of reactions, namely Michael additions, substitution reactions and palladium catalyzed cross- couplings. The photophysical properties of some of these compounds showed their potential utility as fluorescence probes for biological systems. 9,10 Continuing our work concerning the synthesis of non-proteinogenic fluorescent amino acids, we decided to prepare three new pyrenylamino acids using as substrates β,β-disubstituted dehydroalanine derivatives and a strategy previously developed by us. 11,12 Recently, we described the synthesis of α-substituted-β,β- dibromoalanines from a β,β-dibromodehydroalanine by treatment with oxygen nucleophiles and primary amines. Furthermore, by reacting a β-bromo-β-triazolyldehydroalanine with primary amines it was possible to prepare several α- amino-β-iminoalanines. 12 These compounds afforded the α- aminoglycines when treated with silica in dichloromethane. 12 In this work, this methodology was applied to the synthesis of a α-(pyren-1-yl)methylamino-β,β-dibromoalanine, a α- (pyren-1-yl)methylamino-β-iminoalanine and a α-(pyren-1- yl)methylaminoglycine. The photophysical properties of these compounds were studied in several solvents of different polarity and time-resolved fluorescence studies were performed. 2. Results and discussion The methyl ester of the N-tert-butoxycarbonyl-β,β- dibromodehydroalanine (1) was treated with pyren-1- ylmethanamine hydrochloride in the presence of triethylamine (NEt 3 ) in methanol to give the α-addition product the methyl 3,3-dibromo-2-(tert-butoxycarbonylamino)-2-[(pyren-1-yl)- methylamino)]propanoate 2 in 86% yield (Scheme 1). Compound 1 was obtained from the methyl ester of N-tert- butoxycarbonyldehydroalanine by treatment with N- bromosuccinimide (NBS) followed by NEt 3 . In the 1 H NMR spectrum of compound 2 it is possible to observe the high chemical shift of the β-CH proton (6.32 ppm) due to the electron-withdrawing effect of the two bromine atoms. * Corresponding author. Tel.: +351-253604321; fax: +351-253604061; e-mail: ecoutinho@fisica.uminho.pt ARTICLE INFO ABSTRACT Article history: Received Received in revised form Accepted Available online Two new pyrenylamino acid derivatives were synthesized from β-bromodehydroalanine derivatives in good yields using addition and elimination reactions. The photophysical properties of the two new pyrenylamino acids were studied in several solvents. Steady-state and time-resolved fluorescence measurements revealed that the bipyrenylamino acid undergoes excimer formation, this process being solvent dependent. Rate constants for excimer formation and dissociation were calculated. The monopyrenylamino acid exhibits a photophysical behavior similar to that of pyrene, including the sensitivity to solvent polarity. The results point to a potential use of these new pyrenyl derivatives as fluorescent probes for peptides and proteins. Keywords: β-bromodehydroalanines Pyrenylamino acids Photophysical studies Fluorescent probes