Fused oxopyrans as fluorescent labels for neurotransmitter amino acids

Fluorescent conjugates of N-benzyloxycarbonyl protected γ-aminobutyric acid (GABA) were prepared by coupling to its C-terminus several polyheteroaromatic labels, based on the pyran ring. Photophysical properties were evaluated and the results showed that these GABA conjugates presented strong fluorescence with maximum emission wavelengths between 393 and 503 nm, as well as high fluorescence quantum yields in ethanol.


Introduction
The improvement of the sensitivity of analytical methodologies has shifted the research towards the area of fluorescent labelling, as fluorescence is far more sensitive than common UV techniques.The development of novel fluorescent derivatisation reagents and their subsequent evaluation with model compounds has broad application in biology and biochemistry, to investigate the structure and dynamics of living systems. 1,2e analysis of neuroactive amino acids provides a means of diagnosis of disease and possible treatment in neuropsychiatric diseases. 3,4Most of the neurotransmitter amino acids are small aliphatic molecules with neither fluorescence nor strong absorption in the ultraviolet/visible region.Thus, amino acids are derivatized to improve both the selectivity and sensitivity of their detection in biological samples.Amoung the fluorescent labels that have been reported for determination of amino acids are dansyl chloride (Dns), dabsyl chloride, fluorescamine, fluorenylmethyl chloroformate, o-phthalaldehydes (OPA), etc. [5][6][7] Considering these facts in connection with our current research interests in the development of new fluorescent heterocyclic compounds and their application as labels 8,9 we now report the use of different heteroaromatic fluorophores, such as benzopyran derivatives, in the preparation of fluorescent conjugates of γ-aminobutyric acid (GABA), one of the most important transmitters in the central nervous system, with the aim of undertaking a comparative study of their performance as labelling units.
Our purpose being the investigation of compounds 1a-c as fluorescent labels for neurotransmitter amino acids, namely γ-aminobutyric acid (GABA), we synthesised the corresponding conjugates in order to perform a comparative study of their photophysical properties.
Derivatisation at the C-terminus of N-benzyloxycarbonyl-protected GABA with fluorophores 1a-c was carried out in DMF, at room temperature, with the aid of N,N'dicyclohexylcarbodiimide (DCC) assisted by 1-hydroxybenzotriazole (HOBt) under standard conditions 12 (for 1a) or by using potassium fluoride 13 (for 1b and 1c), yielding fluorescent GABA conjugates 2a-c (Scheme 1, Table 1).All conjugates were characterised by IR, 1 H and 13 C NMR spectroscopy and elemental analyses or high resolution mass spectrometry.The IR spectra of labelled GABA 2a-c showed bands due to stretching vibrations of the carbonyl group of the fluorophore-amino acid ester linkage from 1716 to 1739 cm -1 .The spectra also showed the carbonyl bands of the N-benzyloxycarbonyl protecting group (1688 to 1691 cm -1 ), as well as the oxo group at the pyran ring (1622 to 1691 cm -1 ). 1 H NMR spectra showed signals of the amino acid residue, in the form of multiplets for the GABA methylene groups centered at about δ 1.90, 2.55 and 3.30 ppm, and at δ 5.11-5.24ppm the signal for the ester methylene group, as well as the characteristic protons of the heterocyclic moiety at δ 6.31-6.65 ppm for the H-2.The confirmation of the presence of the newly formed ester linkage was also supported by 13 C NMR spectra signals of the ester carbonyl group, which were found at about δ 172 ppm.Signals of the oxo group of the heterocycle (at about δ 160 ppm) and the urethane carbonyl of the Z protecting group (at about δ 155 ppm) were also visualised.
Table 1.Yields, UV/Vis and fluorescence data for GABA ester conjugates 2a-c in absolute ethanol.
The UV/Vis absorption and emission spectra of degassed 10 -5 -10 -6 M solutions in absolute ethanol of compounds 2a-c were measured, absorption and emission maxima, molar absorptivities and fluorescence quantum yields are also reported (Table 1).Fluorescence quantum yields were calculated using 9,10-diphenylanthracene as standard (Φ F = 0.95 in ethanol). 14The wavelength of maximum absorption and emission was red-shifted for conjugates bearing the labels with more fused rings (compare compounds 2a,2b with 2c) (Figure 1).