Dipeptides Containing Pyrene and Modified Photochemically Reactive Tyrosine: Noncovalent and Covalent Binding to Polynucleotides

Abstract

Dipeptides 1 and 2 were synthesized from unnatural amino acids containing pyrene as a fluorescent label and polynucleotide binding unit, and modified tyrosine as a photochemically reactive unit. Photophysical properties of the peptides were investigated by steady-state and time-resolved fluorescence. Both peptides are fluorescent (Φf = 0.3–0.4) and do not show a tendency to form pyrene excimers in the concentration range < 10−5 M, which is important for their application in the fluorescent labeling of polynucleotides. Furthermore, both peptides are photochemically reactive and undergo deamination delivering quinone methides (QMs) (ΦR = 0.01–0.02), as indicated from the preparative photomethanolysis study of the corresponding N-Boc protected derivatives 7 and 8. Both peptides form stable complexes with polynucleotides (log Ka > 6) by noncovalent interactions and similar affinities, binding to minor grooves, preferably to the AT reach regions. Peptide 2 with a longer spacer between the fluorophore and the photo-activable unit undergoes a more efficient deamination reaction, based on the comparison with the N-Boc protected derivatives. Upon light excitation of the complex 2·oligoAT10, the photo-generation of QM initiates the alkylation, which results in the fluorescent labeling of the oligonucleotide. This study demonstrated, as a proof of principle, that small molecules can combine dual forms of fluorescent labeling of polynucleotides, whereby initial addition of the dye rapidly forms a reversible high-affinity noncovalent complex with ds-DNA/RNA, which can be, upon irradiation by light, converted to the irreversible (covalent) form. Such a dual labeling ability of a dye could have many applications in biomedicinal sciences.