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Ann. N.Y. Acad. Sci. 1048: 466–470 (2005). © 2005 New York Academy of Sciences.doi: 10.1196/annals.1342.068

Characterization of Enzymatically Synthesized Diferulate

SRÐAN TUFEGDŽIC′,a JELENA BOGDANOVIC′,b VUK MAKSIMOVIC′,bAND ŽELJKO VU�INIC′,baInstitute of Chemistry, Technology and Metallurgy, Belgrade, Serbia and MontenegrobCenter for Multidisciplinary Studies, University of Belgrade,11000 Belgrade, Serbia and Montenegro

ABSTRACT: Horseradish peroxidase was used to synthesize diferulates by aprocedure in which ethyl ferulate was used as substrate. Four different formswere obtained, of which two dominant were the 5-5′ and 8-5′ diferulate. Fluo-rescence emission spectra of the diferulates (excited at 284 nm) indicate thatthey contain two chromophores, as opposed to the substrate molecule. Fluores-cence excitation spectra with emission at 417 nm further demonstrate the dif-ference between the synthesized diferulates and starting substrates.

KEYWORDS: ferulic acid; diferulate; peroxidase; radical coupling;fluorescence

Diferulates play an important role in plant cell wall formation. Synthesized in vivofrom ferulic acid by the action of oxidoreductases through a radical-based reaction,they cross-link polysaccharide-bonded ferulates and make lignan/lignin residues.Significant effort has been made in the optimization of synthetic strategies to obtainthese compounds.1 However, the only commercially available form is the 5-5′ difer-ulate, present in plants in small quantities compared to overall diferulates.2 The un-availability of purified forms of diferulates is hampering studies of cell wallformation and physiology.

Previous results3 that initiated this study show data on only one diferulate formas a peroxidase reaction product, thus making it hard to interpret the synthetic path-ways. Studies conducted on similar grounds with laccase gave two products.4 Wechoose to test whether the peroxidase will give different products or their distribu-tion. Reports published to date show synthesis of various types of diferulates (5-5′,8-5′, or 8-O-4′ isomers) with low yields and the presence of various byproducts.5,6

In order to mimic the natural metabolic pathway, where carbohydrate esters of feru-lic acid are substrates for enzymatic reaction, we used ethyl ferulate as the enzymesubstrate. The only modification of the reported procedure was the addition ofDMSO as co-solvent for the substrate dissolution, in order to avoid prolonged heat-ing in acid aqueous buffer and possible hydrolysis problems.

Address for correspondence: Srðan Tufegdžic′, IHTM, Department of Chemistry, StudenskiTrg 12-16, YU-11000 Belgrade, Serbia and Montenegro. Voice/fax: +381-11636061.

srdjant@chem.bg.ac.yu

467TUFEGD�IC′ et al.: ENZYMATICALLY SYNTHESIZED DIFERULATE

The IR spectra were recorded on a Perkin-Elmer 457 grating FT instrument. Themass spectra were obtained on a Finnigan ITDS 700 instrument. The NMR spectrawere recorded on a Varian Gemini 200, 1H-NMR at 200 MHz, 13C-NMR at 50 MHz,for samples in deuterated chloroform. Fluorescence spectra were recorded usingFluorolog-3 (Jobin Yvon, Paris). Reverse-phase HPLC was performed on a HewlettPackard HP1100 chromatograph with a HP 1100 Diode Array Detector (Palo Alto,CA) monitoring 210, 280, and 330 nm, with reference signal at 600 nm. Separations

FIGURE 1. Fluorescence emis-sion with (a) excitation at 310 nm and(b) 284 nm and excitation emission at417 nm (c) spectra of ethyl ferulate,ferulic acid, and products 5-5′ and 8-5′diferulate (P1 and P2) in methanol.

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were performed on a Waters Symmetry C-18 RP column 125×4mm with 5 µm par-ticle size (Waters, Milford, MA), with corresponding Symmetry guard column. Themobile phase was a gradient of 0.1% phosphoric acid in acetonitrile. All preparativechromatographic separations were performed on a Merck column chromatographySilica gel 60 and Merck Kieselgel 60 PF254 in toluene/ethyl-acetate gradient. Theperoxidase from horseradish (type II) was purchased from Sigma (St. Louis, MO).Ethyl ferulate was prepared by a variant of Fieser’s esterification procedure. Uponenzymatic reaction buffered aqueous reaction mixture was extracted with ethyl ace-tate, the extract dried, filtered, and evaporated.

Ethyl ferulate coupling reaction proceeds very fast with separation of productsfrom the buffer solution. Four reaction products have been detected, and two ofthem, being dominant, have been characterized. The purity of the products was con-firmed with HPLC. IR spectral data confirmed the presence of functional groupspresent in the starting ethyl ferulate, and both isolated compounds have similar IRspectra with only minor changes in the fingerprint region. Mass spectrometry of iso-lated products demonstrated the presence of dimers, on account of observed molec-ular ion m/z 445. According to 13C-NMR spectra, the first product (P1) issymmetrical, based on number of spectral lines observed. 1H-NMR spectra showedthat it has preserved all C7 and C8 hydrogens and both carbon-carbon double bonds.Comparison with the previously recorded spectra confirmed that (P1) is 5-5′product6 and the second product (P2) is an open form 8-5′ diferulate. 1H-NMR spec-tra of (P2) showed that ratio of hydrogens attached to double bond has changed to2:1 and that both double bonds are preserved. Possible 8-O-4 bonding is ruled outbased on 13C-NMR spectra, leaving as the only solution the C-C type of bonding.Integral of signals given by aromatic hydrogens confirm that bonding is made on ar-omatic core. Recorded fluorescence spectra (FIG. 1) gave us additional structure in-formation. Comparing emission spectra, after excitation at 310 nm (FIG. 1a) and 284nm (FIG. 1b), show that the first gave only simple symmetrical bands of each com-pound, while the excitation at 284 nm, in case of diferulates, showed a small satelliteband, absent in starting compounds. This suggests that diferulates, in contrast to thestarting compounds, contain two different fluorophores. From ferulic acid to diferu-late, high wavelength maximum in excitation spectra (FIG. 1c) had a tendency to in-crease in intensity. Since this is a symmetry-forbidden transition, enhancement ofintensity could be explained in terms of decreasing of symmetry on the chro-mophore, indicating a higher degree of structural asymmetry around aromatic ring.Displacement of position of long wavelength maximum to higher values in diferu-lates is in agreement with increased size of conjugated system (FIG. 1). Since the rad-ical mechanism has been confirmed to participate in the peroxidase–ferulate model,resonant structures of ferulic acid phenoxy radical are presented in FIGURE 2. En-zyme mechanism states that two substrate molecules per cycle are converted to rad-ical per one hydrogen peroxide used.7 Reaction regioselectivity is substrate inherentfeature explained by radical intermediate resonance structures e and f in FIGURE 2.Chiral product formation previously reported3 is incompatible with our findings thattwo regioisomers are synthesized. Reaction stereoselectivity could be explained onlyby the influence of another chiral molecule, an enzyme in this case, indicating itstight bonding of intermediate. Reaction intermediate leaves the enzyme active centerand then freely combines in solution with other substrate molecules or intermediates.Our findings are different than those reported elsewhere3 and can be explained only

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as transport phenomenon caused by small alteration of experimental set-up thatcaused the modified solubility of reactant and products.

ACKNOWLEDGMENT

This work was supported by grants 1911 and 1934 from the Ministry of Scienceand Environmental Protection of the Republic of Serbia.

FIGURE 2. Ferulic anion phenoxy radical resonant structures of the proposed mecha-nism of synthesis of diferulates.

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REFERENCES

1. KAISA, S. & B. GÖSTA. 2001. Regioselectivity in oxidative cross coupling of phenols:application to the synthesis of dimeric neolignans. Tetrahedron 57: 365–370.

2. MICARD, V. et al. 1997. Dehydroferulic acids from sugar-beet pulp. Phytochem. 44:1365–1368.

3. RALPH, J. et al. 1998. Simple preparation of 8-5-coupled diferulate. J. Agric. FoodChem. 46: 2531–2532.

4. CARUNCHIO, F. et al. 2001. Oxidation of ferulic acid by laccase: identification of theproducts and inhibitory effects of some dipeptides. Talanta 55: 189–200.

5. QUIDEAU, S. & J. RALPH. 1994. A biomimetic route to lignin model compounds via sil-ver (I) oxide oxidation. Holzforschung 48: 124–132.

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7. HENRIKSEN, A. et al. 1999. The structures of the horseradish peroxidase C-ferulic acidcomplex and the ternary complex with cyanide suggest how peroxidases oxidizesmall phenolic substrates. J. Biol. Chem. 274: 35005–35011.