Infrared Multiple Photon Dissociation Action Spectroscopy and Computational Studies of Mass-Selected Gas-Phase Fluorescein and 2′,7′-Dichlorofluorescein Ions

Abstract

Fluorescein (FL) and its derivative 2',7'-dichlorofluoroescein (DCF) are well-known fluorescent dyes used in many biological and biochemical applications. Although extensive studies have been carried out to investigate their chemical and photophysical properties in different solvent media, little is known about their intrinsic behaviors in the gas phase. Here, infrared multiple photon dissociation (IRMPD) action spectra are reported for the three charged prototropic forms of FL and DCF and compared with computed IR spectra from electronic structure calculations. In each case, the measured spectra show good agreement with the calculated spectra of the lowest energy computed conformer. Moreover, the major bands of the monoanion IRMPD spectra show striking similarities to those of the dianions and are quite different from those of the cations. These experimental results clearly indicate that the gaseous monoanions are predominantly deprotonated on the xanthene chromophore, rather than the benzoate deprotonation site favored in solution. Investigations such as this, which provide a better understanding of intrinsic properties of ionic dyes, forms a baseline from which to elucidate solvent effects and will aid the rational design of dyes possessing desirable fluorescence properties.

Rotational and vibrational dynamics in the excited electronic state of deprotonated and protonated fluorescein studied by time-resolved photofragmentation in an ion trap

“…Interestingly, very similar fragmentation pattern was reported for both [FL + H] + and [FL − H] − in IRMPD experiments 20 . This points, for both excitation schemes (nanosecond IRMPD and femtosecond UV/Vis), toward dissociation starting from highly vibrationally excited electronic ground states.…”

“…This fragmentation pattern was noted before in Refs. 18 and 19 and also observed in IR multiple-photon dissociation (IRMPD) experiments 20 . Furthermore, [FL + H] + exhibits a much lower fragmentation yield than [FL − H] − , even when applying higher pump photon energy and intensity for photoexcitation (425 nm vs. 520 nm excitation wavelength and an intensity of 0.8  μ J vs. 0.3  μ J, respectively).…”

Ionization and tautomerism of methyl fluorescein and related dyes

“…The recent studies of the structure and spectra in the gas phase are of special interest [10][11][12][13][14]. The evident reason of such activities is the expanding applications of the abovementioned compounds in versatile fields, including biochemistry [15][16][17][18].…”

“…Whereas the neutral form exists in solution as a mixture of three tautomers, zwitterion H 2 Z ± , quinonoid H 2 Q, and colorless lactone H 2 L, only the carboxylate tautomer, HQ À , is typical for the monoanion. The phenolate tautomer HX À appears in small quantities only in pure non-hydrogen bond donor solvents, such as DMSO, acetonitrile, and acetone [29], but it predominates in the gas phase [10][11][12][13][14]. For the derivatives bearing halogen atoms in the xanthene moiety, such as eosin, the zwitterionic tautomer is less typical, whereas the monoanion exists predominantly as HX À [25,28].…”

The Gas-Phase Photophysics of Eosin Y and its Maleimide Conjugate

“…Raman spectroscopy, and in the gas-phase by IRMPD. [41][42][43] Narayaman et al found that the FTIR of EY in solution contains many intense absorption features between 1000 and 2000 cm -1 . Yao et al measured gas-phase fluorescein and 2',7'-dichlorofluorescein by IRMPD action spectroscopy and found a similar series of intense vibrational transitions in the same energy range.…”