Synthesis and characterization of novel 2, 2'-bipyrimidine fluorescent derivative for protein binding

Padalkar et al. Chemistry Central Journal 2011, 5:72 http://journal.chemistrycentral.com/content/5/1/72 RESEARCH ARTICLE Open Access Synthesis and characterization of novel 2, 2’bipyrimidine fluorescent derivative for protein binding Vikas S Padalkar, Vikas S Patil and N Sekar* Abstract Background: Fluorescent dyes with biocompatible functional group and good fluorescence behavior are used as biosensor for monitoring different biological processes as well as detection of protein assay. All reported fluorophore used as sensors are having high selectivity and sensitivity but till there is more demand to synthesized new fluorophore which have improved fluorescence properties and good biocompatibility. Results: Novel 4, 4’-(1, 1’-(5-(2-methoxyphenoxy)-[2, 2’-bipyrimidine]-4, 6-diyl)bis(1H-pyrazol-3, 1-diyl)) dianiline fluorescent dye was synthesized by multistep synthesis from 2-phenylacetonitrile, 2-chloropyrimidine and 2methoxyphenol. This dye has absorption at 379 nm with intense single emission at 497 nm having fairly good quantum yield (0.375) and Stokes shift. The intermediates and dye were characterized by FT-IR, 1H NMR, 13C NMR and Mass spectral analysis. The pyrazole bipyrimidine based fluorescent dye possessing two amino groups suitable for binding with protein is reported. Its utility as a biocompatible conjugate was explained by conjugation with bovine serum albumin. The method is based on direct fluorescence detection of fluorophore-labelled protein before and after conjugation. Purified fluorescent conjugate was subsequently analyzed by fluorimetry. The analysis showed that the tested conjugation reaction yielded fluorescent conjugates of the dye through carbodiimide chemistry. Conclusion: In summery synthesized fluorophore pyrazole-bipyrimidine has very good interaction towards protein bovine serum albumin and it acts as good candidate for protein assay. Background Protein recognition by synthetic molecules is a challenging endeavour, since these materials must bind to a large relatively flat surface domain and recognize a unique distribution of amino acid residues of varying charge, size and shape [1]. Identification and quantification of specific proteins is an important issue in medical and clinical research as many diseases have a specific change in protein expression [2-5]. The most commonly used technique in clinics is enzyme-linked immunosorbent assay (ELISA), which requires specific storage of active enzymes and tedious protein modification [6]. Different strategies have been developed to simplify the detection procedure, which involve specific metal coordination, epitope-docking on miniature proteins, aptamer * Correspondence: Department of Intermediates and Dyestuff Technology, Institute of Chemical Technology, N. P. Marg, Matunga, Mumbai - 400 019, India © 2011 Padalkar et al selection, non-natural peptide isosteres, functionalized platforms, secondary structure mimetics, molecular imprinting and receptors embedded in lipid layers. Recognition of protein binding or change in the structure is detected with the help of fluorescence [7], electrochemistry [8], Raman spectroscopy [9], chemiluminescence [10], flow cytometry [11] and micro fluidic methods [12]. However, most of these methods require sophisticated instrumentation and proficient manipulation, which highly motivated the development of simple and reliable protein detection systems. In general, the preferred fluorescent labels should have high fluorescence quantum yields and retain the biological activities of the parent unlabeled biomolecules. A fluorescent dye can be attached to a peptide at a specific point through a covalent bond depending on the sequence of peptide. The linkage between dye and peptide is a covalent bond, which is stable and not Padalkar et al. Chemistry Central Journal 2011, 5:72 http://journal.chemistrycentral.com/content/5/1/72 destructive under most biological conditions. In some cases, a functional linker is introduced between dye and peptide to minimize the alteration of peptide biological activity. For all the peptide labeling, the dye needs to be attached at a defined position: N-terminus, C-terminus, or in the middle of a sequence. Several fluorescence probes have been reported in the literature to investigate biological process through fluorescence measurements [13-15]. For use as reporter molecules in biological systems many organic dyes have been studied, like coumarin derivatives [16], fluorescein isothiocyanates [17,18], anthracene derivatives [19] and b-naphthol [20]. Amine-containing dyes are used to modify peptides using water-soluble carbodiimides (such as EDC) to convert the carboxy groups of the peptides into amide groups. Either NHS or NHSS may be used to improve the coupling efficiency of EDC-mediated protein-carboxylic acid conjugations. A large excess of the amine-containing dyes is usually used for EDC-mediated bioconjugations in concentrated large peptide solutions at low pH to reduce intra- and inter-protein coupling residues, a common side reaction. The spectral changes observed on the binding of fluorophores with proteins are an important tool for the investigations of the topology of binding sites, conformational changes and characterization of substrate to ligand binding. Besides, determination of protein quantity in biological liquids is of great importance in biology and medicine and fluorescent probes are successfully applied for this approach [21]. As a part of our ongoing research to develop novel materials for high tech applications [22-24], here we report the synthesis, characterization and photophysical properties of a novel fluorescent biocompatible fluorescent probe for protein assay having a pyrazole bipyrimidine framework. The novel fluorophore was prepared by multistep synthesis from 2-chloropyrimidine, phenylacetonitrile and 2-methoxyphenol. Results and Discussion Z-2-(4’-Nitrophenyl)-3-hydroxypropenal 5 was prepared form phenylacetonitrile by nitration, hydrolysis followed by Vilsmeier Hacck formylation. The amidine 8 was prepared in two steps from 2-chloropyrimidine 6. Reaction of dimethyl chloromalonate 10 with guaiacol 9 afforded malonate 11. Pyrimidinedione 12 was then constructed via an amidine 8 and malanoate 11 condensation. The pyrimidinedione 12 was converted to the dichloropyrimidine 13 with phosphorous oxychloride, reaction of hydrazine hydrate with dichloropyrimidine 13 yielded the desired 4, 6-dihydrazinyl-5-(2-methoxyphenoxy)-2, 2’-bipyrimidine 14. Intermediate 4, 6-dihydrazinyl-5-(2methoxyphenoxy)-2, 2’-bipyrimidine 14 reacted with an intermediate Z-2-(4’-nitrophenyl)-3-hydroxypropenal 5 which was prepared from phenyl acetonitrile through Page 2 of 7 multistep process yielded 5-(2-methoxyphenoxy)-4, 6-bis (3-)4-nitrophenyl)-1H-pyrazole-1-yl)-2, 2’-bipyrimidine fluorophore 15 contains nitro group which is not biocompatible. The basic requirements for fluorophore to be good candidate are that it sho (...truncated)