Enzymes-Assisted Generation of Thiols from Thioacetates

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1 Flavur Bisynthesis and Bitechnlgy 2 2., c..- al Enzymes-Assisted Generatin f Thils frm Thiacetates Abstract Rachid Bel Rhlid, Walter Matthey-Dret, Yvette Fleury Rey, Laurent B. Fay, Marcel A. Juillerat, Imre Blank Nestec Ltd., Nestlé Research Centre, Vers-Chez-Les-Blanc, P.. Bx 44,l 00 Lausanne 26, Switzerland. Thils are imprtant cnstituents f the flavur f many fds and beverages. They were prduced efficiently by enzymatic hydrlysis f thiacetates, which are cmmercially available r easily accessible, by reactin f thiacetic acid with alkenes. Enzymatic reactins were perfrmed in water r phsphate buffer resulting in gd verall yields f the target thils. Intrductin Natural flavurs are defined as bilgically derived arma chemicals generated by micrbial fermentatin and/r by the actin f endgenus r technical enzymes (Bakker et al., 1994). Lipases and esterases are the main bicatalysts used in the prductin f flavuring cmpunds, particularly esters that are widely present in fmit flavurs. Amng these esters, thiacetates have received little attentin and very few studies deal with the generatin and enzymatic hydrlysis f thiacetates t prduce thils (Sprull et al., 1997). These thils play, as ptent drants, a key rle in many fd flavurs. They ccur in lw cncentratins and cntribute significantly t characteristic arma ntes due t their lw dur threshlds (Blank, 2002). Hwever, thils are very unstable and easily xidise and plymerise upn strage, even at lw temperature (Hfmann et al., 1996). T vercme this drawback, we investigated the feasibility f enzymatic hydrlysis f thiacetates, which are much mre stable, int the crrespnding thils. This culd be an interesting apprach if the reactin rate and yield are satisfactry. The thils culd be stred in their stable thiacetate befre release by enzymatic hydrlysis. In this study, we reprt n the generatin f 2-methyl-3 -furanthil and 2-furfurylthil by enzymatic hydrlysis f their thiacetates. In additin, we describe a chemenzymatic apprach t prduce 4-mercapt- 4-methyl-2-pentanne and 3-mercapthexanal. Experimental Chemicals and enzymes The chemicals were f analytical grade. S-2-Furfuryl thiacetate, trans-2-hexenal and 4-methyl-3-penten-2-ne were purchased frm Aldrich. Thiacetic acid was frm Fluka and S-3-(2-methylfuryl) thiacetate frm xfrd chemicals. Lipase frm Candida rugsa and esterase frm prcine liver were purchased frm Sigma. Synthesis f thiacetates 10 mml f trans-2-hexenal r 4-methy-3-penten-2-ne were added t a slutin f thiacetic acid in n-hexane (11 mml, 30 ml). Reactins were perfrmed at rm temperature under stirring. Samples were withdrawn at varius time intervals and analysed by gas chrmatgraphy (FID, FPD, MS). After 48 h reactin time, the slvent was evaprated and samples were used fr enzymatic hydrlysis. Enzymatic hydrlysis f thiacetates in water.- v) Varius amunts f enzyme were added t 10 ml f a slutin f thiacetate ( mml) in distilled water r phsphate buffer (0.2 M). Enzymatic reactins were perfrmed

2 366 Flavur Research at the Dawn f the Twenty-first Century under gentle magnetic stirring. Samples were withdrawn at varius time intervals (frm 1 min t 72 h). A slutin f benzyl mercaptan in diethyl ether (00 pl, 2000 ppm) was then added as internal standard fr quantificatin. The mixture was extracted with diethyl ether and the extracts were dried ver sdium sulphate and cncentrated t a vlume f 2 ml using a Vigreux clumn (30 x 1 cm). The cncentrated slutin was then analysed by varius chrmatgraphic techniques., The influence f the fllwing parameters was studied: ph (6.0, 7.0, and 8.0), temperature (4, 23, and 37"C), and the rati enzyme (units) t substrate (mml) (1 3/0.064, 6í0.064, 77/0.064, and 6Y0.64). Gas Chrmatgraphy and Mass Spectrmetry Gas chrmatgraphy analyses were perfrmed using a Carl Erba gas chrmatgraph (Mega 2) equipped with an autmatic cld n-clumn injectr, flame inisatin detectr (FID), flame phtmetry detectr (FPD), and a sniffing prt. Fused silica capillary clumns (DB-1701 and DB-FFAP) were used, bth 30 m x 0.32 mm with a film thickness f 0.2 pm. GC-MS analyses were carried ut using a Finnigan 8430 mass spectrmeter. The MS- EI spectra were generated at 70 ev and MS-CI at 10 ev with ammnia as the reagent gas. Results and discussins Enzymatic hydrlysis f S-2-furfuryl thiacetate As shwn in figure 1, hydrlysis f S-2-furfuryl thiacetate (1) using lipase frm Candida rugsa allwed prducing 2-furfurylthil (2). Enzymatic reactins were perfrmed in water r in phsphate buffer. The influence f ph n the reactin rate and yield and n the prduct stability was studied. Figure 2 shws the generatin f 2- furfurylthil at ph.8, 6.0, 7.0, and 8.0. After 1 h f reactin, the substrate was cmpletely transfrmed. While the reactin rates were similar at all ph values, the yields were different. A maximum yield f 74% was btained at ph.8 while this yield was nly 0% at ph 8.0. Figure I 1 2 Enzymatic hydrlysis f S-2-furfuryl thiacetate W z 60. al y min h. 2 h. 4 h. 24 h. min. min min. Figure 2. Influence f ph n the enzymatic hydrlysis f S-2-fufluryl thiacetate at ph.8 (i), ph 6. (), ph 7. (.) andph 8. (.)..- - L -r L.- al u) J

3 Flavur Bisynthesis and Bitechnlgy 367 The influence f the amunt f enzyme n the reactin rate and n the verall yield was perfrmed in water, at ph.8 and at rm temperature. The range f enzyme quantity varied frm 13 t 262 units fr the same substrate cncentratin (0.064 mml). The quantity f enzyme influenced the reactin rate but had n significant effect n the yield f 2-furfurylthil generated. The influence f temperature n the enzymatic hydrlysis was als studied. Trials were perfrmed at 4"C, 23 C and 37 C using the same enzyme t substrate rati (6 units / mml). As expected, n significant differences were bserved when the reactin tk place at 23 C r 37"C, while at 4 C the reactin rate was much slwer and the prduct mre stable. Rm temperature was fund t be a gd cmprmise between reactin rate, cst, and energy saving. Enzymatic hydrlysis f S-3 -(2-methylfuryl) thiacetate As shwn in figure 3, enzymatic hydrlysis f S-3-(2-methylturyl) thiacetate (3) resulted in 2-methyl-3-turanthil (4). The reactin was perfrmed at rm temperature, in water (PH.8) cntaining mml f substrate. The cncentratin range f the lipase frm Candida rugsa varied frm t 6 units. 3 4 Figure 3. Enzymatic hydrlysis f S-3-(2-methylfuryl) thiacetate. Figure 4 shws the frmatin and degradatin f 2-methyl-3-furanthil frm S-3-(2- methylfuryl) thiacetate as functin f the amunt f enzyme. An ptimal yield f 88% was btained after 1 min f incubatin when 6 units f enzyme were used fr the hydrlysis. This yield was nly 0% and 10% when 26 and 6. units f enzyme were used, respectively. Hwever, the degradatin rate f 2-methyl-3-furanthil int dimer was higher when 6 units f enzyme were used cmparatively t 26 and 6. units ,-- Figure // 'Y"// = h 6h 24h Time (min) Influence f the quantity f enzyme n the hydrlysis f S-3-(2-methylfuryl) thiacetate. Enzyme units: 6 (I), 26 (), 6. (.). c-3 Chemenzymatic synthesis f 4-mercapt-4-methyl-2-pentanne cu Recently, the blackcurrant-like smelling drant 4-mercapt-4-methyl-2-pentanne (4- c L1 g MMP) (8) was reprted as key cnstituent f grapefruit juice (Buettner et al., 1999) and - identified in Sauvignn Blanc (Darriet et al., 199). As shwn in figure, we prduced this \.- 8 arma cmpund in tw steps. Reactin f mesityl xide () with thiacetic acid (6) in n-.$ hexane resulted in S-( 1, 1 -dimethyl-3-xbutyl) thiacetate (7) with a yield f 7%. 2 Thiacetate 7 was characterised n the basis f GC-MS data analysis and by cmparisn f

4 368 Flavur Research at the Dawn f the Twenty-first Century its NMR data with thse reprted in the literature (Trst et al., 197 1). Enzymatic hydrlysis f cmpund 7 using prcine liver esterase (PLE) allwed prducing 4-MMP in 83% yield. This arma cmpund was characterised by cmparisn f its chrmatgraphic prperties with thse f the reference cmpund. AA 6 y u 7 - PLE SH Figure. Chemenzymatic synthesis f3-mercapt-3-methyl-2-pentanne. Chemenzymatic synthesis f 3-mercapthexanal As shwn in figure 6, reactin between 2-hexenal (9) and thiacetic acid (6) resulted in thiacetate (10) with a yield f 78%. This thiacetate was characterised n the basis f GC- MS and NMR data. Enzymatic hydrlysis f cmpund 10 using PLE as bicatalyst allwed prducing 3-mercapthexanal (1 1) in 47% yield. Cmpund 11 was characterised by cmparisn f its chrmatgraphic prperties and dur characteristic with thse reprted in the literature (Werkhff et al., 1996) References I1 Figure 6. Chemenzymatic synthesis f 3-mercapthexanal. Bakker J, Law BA (1994). Cheese flavur. In: Piggt J, Patersn A. Understanding Natural Flavurs. Eds. Blackie, Lndn, Blank I (2002). Sensry relevance f vlatile rganic sulfur cmpunds in fd. In: ACS Sympsium series 826, American Chemical Sciety, Washingtn DC, 2-3. Buettner A, Schieberle P (1999). Characterizatin f the mst dr-active vlatiles in fresh, hand-squeezed juice f grapefruit (Citrus paradisi Macfayden). J. Agric. Fd Chem., 47: Darriet W, Tminaga T, Lavigne V, Bidrn JN, Duburdieu D (199). Identificatin f a pwerful armatic cmpund f Vitis vinifera L. var. Sauvignn wines: 4- mercapt-4-methylpentan-2-ne. Flav. Fragr. J., 10: Hfmann T, Schieberle P, Grsch W (1 996). Mdel studies n the xidative stability f dr-active thils ccuring in fd flavrs. J. Agric. Fd Chem., 44: Sprull KC, Bwman GT, Carta G, Gainer JL (1997). Enzymatic transfrmatins f thiacids and thiesters. Bitechnl. Prg., 13: Trst BM, Schinski WL, Chen F, Mantz IB (1971). Sterechemistry f desulfurizatin (v e f thietane derivatives. J. Amer. Chem. SC., 93: Werkhff P, Brüning J, Güntert J, Kaulen J, Krammer G, Smmer H (1996). Ptent mercapt/methylthi-substituted aldehydes and ketnes in cked beef liver. Adv. Fd Sci., 18: m L! c :i J

5 Reprinted frm : Flavur Research at the Dawn f the Twenty-first Century Prceedings f the 1 th Weurman Flavur Research Sympsium Edited by Jean-Luc LE QUERE Patrick X. ÉTIÉVANT Institut Natinal de la Recherche Agrnmique Unité Mixte ENESAD-INRA de Recherche sur les Arômes Dijn, France , rue Lavisier 7008 Paris LNDRES - PARIS - NEW YRK

METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY

METHOD 8032 ACRYLAMIDE BY GAS CHROMATOGRAPHY 1.0 SCOPE AND APPLICATION 1.1 Methd 8032 is used t determine trace amunts f acrylamide mnmer in aqueus matrices. This methd may be applicable t ther matrices.