The Phenanthridine-modified Tyrosine Dipeptide: Synthesis and Non-covalent Binding to DNA and RNA

Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA. This work is licensed under a Creative Commons Attribution 4.0 International License

Non-Covalent Binding of Tripeptides-Containing Tryptophan to Polynucleotides and Photochemical Deamination of Modified Tyrosine to Quinone Methide Leading to Covalent Attachment

A series of tripeptides TrpTrpPhe (1), TrpTrpTyr (2), and TrpTrpTyr[CH2N(CH3)2] (3) were synthesized, and their photophysical properties and non-covalent binding to polynucleotides were investigated. Fluorescent Trp residues (quantum yield in aqueous solvent ΦF = 0.03–0.06), allowed for the fluorometric study of non-covalent binding to DNA and RNA. Moreover, high and similar affinities of 2×HCl and 3×HCl to all studied double stranded (ds)-polynucleotides were found (logKa = 6.0–6.8). However, the fluorescence spectral responses were strongly dependent on base pair composition: the GC-containing polynucleotides efficiently quenched Trp emission, at variance to AT- or AU-polynucleotides, which induced bisignate response. Namely, addition of AT(U) polynucleotides at excess over studied peptide induced the quenching (attributed to aggregation in the grooves of polynucleotides), whereas at excess of DNA/RNA over peptide the fluorescence increase of Trp was observed. The thermal denaturation and circular dichroism (CD) experiments supported peptides binding within the grooves of polynucleotides. The photogenerated quinone methide (QM) reacts with nucleophiles giving adducts, as demonstrated by the photomethanolysis (quantum yield ΦR = 0.11–0.13). Furthermore, we have demonstrated photoalkylation of AT oligonucleotides by QM, at variance to previous reports describing the highest reactivity of QMs with the GC reach regions of polynucleotides. Our investigations show a proof of principle that QM precursor can be imbedded into a peptide and used as a photochemical switch to enable alkylation of polynucleotides, enabling further applications in chemistry and biology

Photochemical Reactivity of Naphthol-Naphthalimide Conjugates and Their Biological Activity

Quinone methide precursors 1a–e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10−2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10−5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105–106 M−1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M−1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a–e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light

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

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 −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