Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design.

Anders Foller Larsen,Moa S Wranne,Christopher P Lawson,L Marcus Wilhelmsson,Henrik Gradén,Søren Preus,Blaise Dumat,Mattias Bood,Morten Grøtli

doi:10.1038/srep12653

Abstract

Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (εΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

Highlights

Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics
We recently reported the synthesis of the boronic pinacol ester (4, Table 1) in two synthetic steps from commercially available 6-chloro-7-iodo7-deazapurine[36]
From the compound screen performed in this study, we successfully identified two very promising compounds, 2-CNqA and 1-CNqA, which display improved fluorescence quantum yields and molar absorptivities with up to a 13-fold increase in brightness compared with quadracyclic adenine (qA)

Summary

Introduction

Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs. Fluorescence is a versatile and sensitive tool for investigating biological systems[1,2,3,4]. The use of fluorescence spectroscopy to study nucleic acids requires the introduction of synthetic modifications to the natural nucleosides which, like most biomolecules, are virtually non-fluorescent[6] These modifications typically involve tethering an external fluorophore to a native nucleoside via a long flexible linker[7,8]. For such experiments to become wide-spread and reliable there is a pressing need for FBAs with enhanced brightness and photostability, as well as with versatile fluorescence properties

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