Amino acids based on 2 , 4 , 5-triarylimidazoles : synthesis and evaluation as new chemosensors for ion recognition

N-tert-butyloxycarbonyl-4-formylphenylalanine methyl ester and appropriate heterocyclic diones were used in the synthesis of novel fluorescent unnatural amino acids, namely 2,4,5-triarylimidazolyl-alanines. These new compounds were fully characterised by the usual spectroscopic techniques, such as IR and NMR. The photophysical properties of the amino acids were evaluated by UV-Vis absorption and fluorescence spectroscopy in solvents of different character. Interaction studies with biologically and analytically important ions such as F , OH , Cu 2+ and Fe 3+ , through spectrophotometric and spectrofluorimetric titrations were carried out to assess their potential as chemosensors.


Introduction
2,4,5-Triaryl-imidazoles have found application in medicinal chemistry due to their properties, for example as ligands for Ru(II) and Pt(II) complexes, as probes of DNA structure or new therapeutic agents due to their capacity to bind or interact with DNA. 1 Recently we reported the synthesis of several 2,4,5-tri(heteroaryl)imidazoles with interesting photophysical properties (high fluorescence quantum yields and large Stokes' shifts) which could be used in diverse optical applications (e.g.nonlinear optics, fluorimetric chemosensors, OLEDs and DNA intercalators). 2tallic cations can be complexed through N, O and S donor atoms in amino acids, at the main and side chains, and in aromatic heterocycles, whereas anion coordination, based on hydrogen bonding and electrostatic interactions, can arise from amino acid side and main chain OH and NH groups, or from NH groups in heterocycles. 3,4Therefore, the insertion of suitable heterocyclic systems at the side chain of natural amino acids can add extra functionality to the amino acid.
Fluorescent sensors are preferred because they are well suited to meet the need for in vivo probes, such as mapping the spatial and temporal distribution of the biological analytes, and they have other advantages including multiple modes of detection (such as fluorescence quenching, enhancing, life time), extremely high sensitivity, relatively low cost and easy availability. 5Our current research interests include the synthesis and characterization of unnatural amino acids, 6 imidazole and benz-X-azole derivatives with interesting optical properties 7 and innovative heterocyclic colorimetric/fluorimetric chemosensors for anions and cations containing (oligo)thiophene, benzoxazole and amino acid moieties. 8We now report the synthesis and photophysical characterization of novel 2,4,5-triarylimidazolylalanines and the interaction study with biologically important ions such as F -, OH -, Cu 2+ and Fe 3+ , through spectrophotometric and spectrofluorimetric titrations.

General procedure for the synthesis of 2,4,5-triarylimidazolyl-alanines 3a-b
N-tert-butyloxycarbonyl-4-formylphenylalanine methyl ester 1 (1 equiv.)and the appropriate heterocyclic dione 2 (1 equiv.)were dissolved in acetic acid (5 mL/equiv), in the presence of ammonium acetate (20 equiv.)and heated at reflux for 2 hours.After cooling, the mixture was poured over crushed ice and extracted with ethyl acetate (3 x 5 mL).After drying the organic layer over anhydrous MgSO 4 , the solvent was removed in a rotary evaporator and a solid was obtained.The crude solid was purified by column chromatography, using mixtures of dichloromethane and methanol of increasing polarity as eluent.

Spectrophotometric and spectrofluorimetric titrations of 2,4,5-triarylimidazolylalanines 3a-b
Solutions of 2,4,5-triarylimidazolyl-alanines 3 (ca.1.0 × 10 -5 to 1.0 × 10 -6 M) and of the ions under study (ca.1.0 × 10 -1 to 1.0 × 10 -3 M) were prepared in UV-grade acetonitrile (in the form of hydrated tetrabutylammonium salt for F -and OH -and hydrated perchlorate salt for Cu 2+ and Fe 3+ ).Titration of the compounds with the several ions was performed by the sequential addition of equivalents of ion to the alanine solution, in a 10 mm path length quartz cuvette and emission spectra were measured by excitation at the wavelength of maximum absorption for each compound.

Synthesis
Novel 2,4,5-triarylimidazolyl-alanines 3a-b were synthesised by reaction of N-tertbutyloxycarbonyl-4-formylphenylalanine methyl ester 1 with the appropriate heterocyclic dione 2, in the presence of ammonium acetate in acetic acid at reflux for 2 hours.The pure compounds were isolated in 57% (3a) and 58% (3b) yield, and were characterized by the usual spectroscopic techniques.The acidic reaction media yielded the N-deprotected form of the amino acids (Scheme).
The overall trend revealed that alanine 3a with furyl pendants was the most fluorescent (with Φ F in the range 0.64-0.77)whereas alanine 3b bearing thienyl pendants displayed the largest Stokes' shifts (between 105-109 nm).

Spectrophotometric and spectrofluorimetric titrations of 3a-b with ions
The modification of phenylalanine through the introduction of an extra UV-active and highly fluorescent heterocycle at its side chain was expected to provide additional binding sites for a variety of ions through the heterocycle donor atoms, as well as improved photophysical properties for the chemosensing studies.With heterocyclic alanines 3 it was intended to assess the influence of the structure in the chemosensing ability of anions and cations.Considering the biological, environmental and analytical relevance of ions such as F -, OH -, Cu 2+ and Fe 3+ , the interaction of 2,4,5-triarylimidazolyl-alanines 3 with these ions was evaluated through UV-vis and fluorescence spectroscopies in spectrophotometric and spectrofluorimetric titrations in acetonitrile.In the spectrophotometric titrations, no changes were seen in the absorption spectra bands of 2,4,5-triarylimidazolyl-alanines 3a-b after addition of up to 400 equiv of each ion.
In the spectrofluorimetric titrations with Cu 2+ and Fe 3+ , a decrease of the fluorescence intensity (a chelation-enhanced quenching, CHEQ effect) was observed for both the alanines, with an almost complete fluorescence quenching.In Figure 1A is shown the spectrofluorimetric titration of alanine 3a with Cu 2+ , where the drastic effect of ion complexation is evident in the band centred at the wavelength of maximum emission at 317 nm.

A B C D
With regard to the other ions Fe 3+ , F -and OH -, a less pronounced CHEQ effect was also observed after ion addition, without complete quenching of fluorescence, with F -and OH -.In Figure 1 B, C and D are shown the spectrofluorimetric titrations of asparagine 3a with Fe 3+ , F - or OH -.For F -, there was a maximum decrease of 75% and 45 % in fluorescence of alanines 3a-b with the addition of 10 equiv of anion; as for OH -, a fluorescence quenching of 75% and 45% was visible with the addition of 10 equiv to alanines 3a-b; alanines 3a-b responded highly to titration with Fe 3+ with a 100% quenching after 20 and 180 equiv of metal were added.
Alanine 3a would be the more interesting candidate as chemosensor due to the higher fluorescence quantum yield, which is important for maximization of response to analyte in the analysis of very dilute samples.

Conclusions
The novel 2,4,5-triarylimidazolyl-alanines 3a-b are highly emissive, with modest to excellent fluorescence quantum yields (Φ F = 0.64-0.77for 3a; Φ F = 0.14-0.30for 3b) and display large Stokes' shifts (between 86 and 109 nm) in organic solvents of different character.Alanine 3a bearing furyl pendants displayed higher fluorescence than alanine 3b, bearing thienyl pendants, relating to the heteroatom, and larger Stokes shifts were seen for alanine 3b, in all the solvents tested.Through spectrophotometric and spectrofluorimetric titration with several ions (F -, OH -, Cu 2+ and Fe 3+ ) it was concluded that alanines 3 show a high sensitivity and ability to interact with Cu 2+ and Fe 3+ in ACN.