Notes & Tips Single-step synthesis of 4-nitrophenyl ferulate for spectrophotometric assay of feruloyl esterases Shyamala Hegde a,c , Pullabhatla Srinivas b,c , Gudipati Muralikrishna a,c, * a Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570 020, India b Department of Plantation Products, Spices and Flavor Technology, Central Food Technological Research Institute, Mysore 570 020, India c Council of scientific and Industrial Research, New Delhi, India article info Article history: Received 16 November 2008 Available online 13 January 2009 abstract We report here, for the first time, a single-step method for the synthesis of 4-nitrophenyl ferulate (4NPF), a spectrophotometric substrate for the assay of feruloyl esterases by dehydrative coupling of ferulic acid and 4-nitrophenol. Ó 2009 Elsevier Inc. All rights reserved. Feruloyl esterases (EC 3.1.1.73), which hydrolyze ferulic acid es- ters, are important enzymes under both physiological and patho- logical conditions in plants [1] and also have several industrial applications [2,3]. Few methods have been reported for measuring this enzyme activity, but most of them are based on high-perfor- mance liquid chromatography (HPLC) 1 techniques [4,5]. Mastihuba and coworkers reported a spectrophotometric method for easy, quick, and routine assay using 4-nitrophenyl ferulate (4NPF) [4]. Be- cause 4NPF is not commercially available, it was synthesized by a chemoenzymatic procedure [6]. This method, involving four syn- thetic steps, uses expensive lipase enzyme. We report here, for the first time, a single-step method of the synthesis of 4NPF starting from ferulic acid and 4-nitrophenol (4NP). 4NPF was synthesized by dehydrative coupling of ferulic acid and 4NP using dicyclohexylcarbodiimide (DCC) as reagent in a reaction as shown in Scheme 1. To ferulic acid (1 g, 5.15 mmol, melting point [m.p.] 168–170 °C) dissolved in dry dioxane (10 ml), 4NP (0.716 g, 5.15 mmol, m.p. 112–114 °C) dissolved in dioxane was added dropwise. The reaction mixture was made basic by the addition of 1 ml of pyridine and 1 ml of triethylamine. After stirring for 10 min, solid DCC (1.18 g, 5.7 mmol) was added in por- tions of 200 mg at intervals of 3 min under a nitrogen atmosphere. The reaction mixture was stirred under the nitrogen atmosphere at 25 °C and monitored on thin-layer chromatography (TLC). The pre- cipitate of dicyclohexyl urea (DCU) started appearing in the reac- tion mixture after 15 min. The completion of the reaction was assessed by the complete disappearance of the reactants by TLC on silica gel using 10% methanol in chloroform as the developing solvent. R f values of ferulic acid, 4NP, and 4NPF were 0.39, 0.56, and 0.72, respectively. After completion of the reaction (8 h), the reaction mixture was filtered to separate DCU and filtrate was evaporated under reduced pressure. The residue was purified by column chromatography using silica gel (100–200 mesh size), with chloroform as the solvent. Fractions containing 4NPF (R f of 0.72 on TLC) were pooled and evaporated. The final product (50% yield) showed a melting point of 176–178 °C and a single peak on HPLC (reverse phase, C-18 column, methanol/water/trifluoroacetic acid [TFA] 70:30:0.1, flow rate 1 ml/min) at 340 nm. The mass spec- trometry (MS) analysis of the product [electrospray ionization (ESI)–MS] in the negative ion mode exhibited molecular mass ion at 314.3, corresponding to the molecular mass of 4NPF. 4NPF was further characterized by 1 H and 13 C nuclear magnetic resonance (NMR) spectral data recorded on a Bruker Avance 500-MHz spec- trometer using CDCl 3 (Table 1). The 1 H NMR of 4NPF showed sig- nals at 3.94, 5.93, 6.45, 7.82, 6.95, 7.07, and 7.15 from ferulic acid and showed signals at 7.35 and 8.28 from 4NP. The absence of 9.57 (br, 1 H) in the spectra of 4NPF, which represents the –COOH proton of ferulic acid, confirmed the coupling of ferulic acid and 4NP. Similarly, whereas the carbonyl in ferulic acid appeared at 168.13, the product contained the signal at 164.30, corresponding to the ester carbonyl moiety. The 13 C spectrum of the product also contained methoxyl carbon signal at 55.71 as in ferulic acid. In addition to the two olefinic and six aromatic carbon signals from ferulic acid, the compound contained an additional four signals for four aromatic carbons from the nitrophenol moiety. Dehydrative coupling is a chemical reaction often employed in synthetic chemistry for the synthesis of esters. Such a strategy was adopted previously for the synthesis of p-nitrophenyl ester of piperic acid; however, this does not contain the phenolic func- tional group [7,8]. In the current study, we were able to synthesize 4NPF by reaction of ferulic acid with 4NP, where the reaction con- 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.01.001 * Corresponding author. Address: Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570 020, Karnataka, India. Fax: +91 821 2517233. E-mail address: krishnagm2002@yahoo.com (G. Muralikrishna). 1 Abbreviations used: HPLC, high-performance liquid chromatography; 4NPF, 4- nitrophenyl ferulate; 4NP, 4-nitrophenol; DCC, dicyclohexylcarbodiimide; m.p., melting point; TLC, thin-layer chromatography; DCU, dicyclohexyl urea; TFA, trifluoroacetic acid; MS, mass spectrometry; ESI, electrospray ionization; NMR, nuclear magnetic resonance; DMSO, dimethyl sulfoxide. Analytical Biochemistry 387 (2009) 128–129 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio