Synthetic GFP Chromophore and Control of Excited-State Proton Transfer in DNA: An Alternative Concept for Fluorescent DNA Labels with Large Apparent Stokes’ Shifts

Abstract

Synthetic GFP-like chromophores bearing an ortho-phenol group instead of the para-phenol group of natural GFP can mimic the hydrogen bonding network in the protein by an intramolecular hydrogen bond to the imidazolone group. This hydrogen bond influences the excited state of the model by proton transfer (ESPT). The corresponding GFP model chromophore 1 was synthesized with an additional azide functionality that can be used to ligate it to acetylene-modified biomolecules by Cu(I)-catalyzed cycloaddition. The chromophore 1 was incorporated as a synthetic modification into oligonucleotides using a postsynthetic methodology and characterized within the DNA environment by optical spectroscopy. In order to elucidate the effect of DNA on ESPT a second GFP chromophore 2 was synthesized carrying a methyl group that prevents ESPT processes. It became evident that DNA is able to provide an artificial environment for the GFP chromophore that controls the photophysical property in such a way that nearly solely the ESPT-driven, red-shifted fluorescence is occurring. The apparent Stokes’ shift is larger than 200 nm (9000 cm^-¹). Moreover, the comparison with the methylated chromophore 2 in DNA elucidates that DNA increases the fluorescence intensity of both GFP models presumably by restricting the conformational flexibility. Although the observable quantum yields are too low to consider the fluorophore for any bioanalytical application, the combination of both effects, red-shifted ESPT-controlled fluorescence with large apparent Stokes’ shifts and increase of intensity by restricting of internal conversion, provides an important concept for the design of fluorescent labels for DNA and RNA.

Key words

cyanine - fluorescence - green fluorescent protein - oligonucleotide - proton transfer

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