Effect of NH2 rotation on the fluorescence of 2-aminopurine in solution

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Abstract

Since the introduction of 2-aminopurine (2AP) in 1969 as a fluorescent analogue of adenine, its intense fluorescence in aqueous solution and the subsequent reduction of this intensity in DNA has been a powerful tool for studies of structural changes in DNA. Herein, we show that the unusual intense fluorescence of 2AP in water is attributed to the formation of a closed complex between one water molecule and 2AP in the excited state. This configuration restricts the rotation of the 2-NH2 group which subsequently lowers the nonradiative decay rate. We supported this finding by attaching heavy masses to the amino group, dimethyl (2-(N(Me)2)) and diethyl (2-(N(Et)2)). By examining the fluorescence behavior in dioxane (an apolar, aprotic solvent), the lighter NH2 group can rotate in the excited state more freely which enhances the nonradiative loss of fluorescence. On the other hand, this rotation slows down sharply in the two heavy-group derivatives, leading to a restoration of the fluorescence intensity and lifetime very close to that of 2AP in water. Depletion of fluorescence was observed in the 2AP derivatives in water and is attributed to the population of a twisted intramolecular charge transfer (TICT) state due to the strong electron donating power of the NR2 groups, an effect that is absent for the parent 2AP.

Graphical abstract

The unusual intense fluorescence of 2-aminopurine (2AP) in aqueous solution is attributed to the restricted rotation of the 2-NH2 group by forming a closed complex between one water molecule and 2AP, reducing the loss of fluorescence. This mechanism is proved by attaching heavy masses to the amino group which reduces the rotational effect.

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Introduction

The potential use of 2-aminopurine (2AP) as a fluorescent analogue of adenine in DNA research was first suggested in 1969 by Stryer and co-workers [1]. The authors characterized the fluorescence of 2AP and reported a quantum yield of 0.68 in aqueous solution at pH 7.0 and a fluorescence maximum at ~370 nm. The excited state of 2AP is also red-shifted with respect to the natural bases, which allows for selective excitation. Upon incorporation of 2AP into an oligonucleotide, its fluorescence is significantly attenuated. This observation made it apparent that 2AP could be used to monitor changes in an oligonucleotide structure and environment. The molecule was then identified as a powerful tool for studies of DNA and has been used since then in various DNA investigations (for review, see refs. [2], [3], [4], [5]).

Considerable progress has been made to explain the high fluorescence quantum yield of 2AP compared to adenine [6], [7], [8], [9]. In adenine, fast nonradiative deactivation of the ππ*state (S1 state) via a conical intersection to the ground state is accessible through a strong puckering of the six-membered purine ring with pyramidalization of the carbon atom in position 2 [6], [7]. In 2AP, the presence of NH2 in position 2 significantly lowers the energy of the ππ* state, therefore the conical intersection occurs at higher energies than in adenine, giving stability to the fluorescent ππ* state [6], [7], [8], [9]. The close proximity of the ππ* state to a nearby * state in adenine contributes also to the depletion of the former state via additional conical intersections to the latter state which in turn has a nonradiative reaction path to the ground state [6], [8], [9].

The fluorescence behavior of 2AP in different environment remains unclear. In particular, the drastic reduction in 2AP's fluorescence intensity and quantum yield upon incorporation into DNA is still ambiguous [10], [11]. The most acceptable mechanisms for this fluorescence quenching are excited-state charge transfer [12] and the presence of dark states in the base stacking configuration [12], [13], [14], [15]. In the present work, we explain the mechanism controlling the 2AP fluorescence in aqueous solution and we show that the internal rotation of the NH2 group plays a major role in defining the fluorescence behavior of the molecule. This is not surprising since changing the position of the NH2 group from the 6-position (adenine) to the 2-position (2AP) causes the considerable shift in fluorescence characteristics. We correlate the origin of the intense fluorescence of 2AP in aqueous solution to restriction of the NH2 internal rotation. Easing of this restriction in aprotic solvents leads to increasing nonradiative decay that tends to deactivate the initially populated excited state. We support this finding by examining the fluorescence of two derivatives of 2AP in which the hydrogen atoms of NH2 have been replaced by heavy groups. The chemical structures of N,N-dimethyl-2-aminopurine (2DMeAP) and N,N-diethyl-2-aminopurine (2DEtAP), along with the structure of 2AP, are shown in Fig. 1.

Section snippets

Experimental and theoretical methods

2AP (99%) was obtained from Sigma. (2DMeAP) and (2DEtAP) were custom-made by GlycoTeam GmbH, Germany. The purity of both derivatives was estimated to be ≥98%. Acetonitrile (spectroscopic grade), anhydrous 1,4-dioxane and methanol were obtained from Sigma-Aldrich Chemical Co. Anhydrous ethanol was received from Acros Organics. Deionized water (Millipore) was used. The concentration of all materials was kept at 0.02 mM.

Absorption spectra were obtained with an HP 845x Diode Array spectrophotometer.

Characterization of 2AP in different solvents

The steady-state fluorescence spectra of 2AP dissolved in different solvents are shown in Fig. 2. Table 1 summarizes the spectroscopic parameters along with the measured lifetime values in the corresponding solvents. The results are in substantial agreement with previously reported results for some of the solvents [1], [11], [19], [20].

The fluorescence behavior cannot be directly correlated to solvent polarity. In order to understand the fluorescence behavior, we need to discuss the solvent

Conclusions

In summary, the intense fluorescence of 2AP in aqueous solution is attributed to restriction of the NH2 rotation in the excited state by forming a closed complex between one water molecule and 2AP. This configuration restricts the rotation of the 2-NH2 group which subsequently lowers the nonradiative decay rate. This was confirmed by attaching heavy masses to the amino group, dimethyl (2-(N(Me)2)) and diethyl (2-(N(Et)2)) in which fluorescence was restored in dioxane (an apolar, aprotic

Acknowledgement

The author would like to thank Sultan Qaboos University (Grant No. IG/SCI/CHEM/12/01) for supporting this work.

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