J. Agric. Food Chem. 2004, 52, 7075−7083
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Synthesis of Deuterated γ-Lactones for Use in Stable Isotope Dilution Assays JO-ANNA HISLOP, MARTIN B. HUNT, SIMON FIELDER,
AND
DARYL D. ROWAN*
The Horticulture and Food Research Institute of New Zealand Ltd., Private Bag 11030, Palmerston North, New Zealand
Two syntheses of deuterated γ-lactones for use as internal standards in stable isotope dilution assays (SIDA) were developed. [2,2,3,3-2H4]-γ-Octa-, -γ-deca-, and -γ-dodecalactones with >89% deuterium incorporation were prepared in 27, 17, and 19% overall yields, respectively, by the reduction of a doubly protected hydroxypropiolic acid with deuterium gas. [3,3,4-2H3]-γ-Octa- and -γ-dodecalactones were prepared in 6 and 23% yields with >92% deuterium incorporation by the free radical addition of 2-iodoacetamide to [1,1,2-2H3]-1-hexene and [1,1,2-2H3]-1-decene, respectively. Reaction yields were highly dependent upon the purity of the 1-alkene starting material. The deuterated γ-lactones were evaluated as internal standards for SIDA. KEYWORDS: γ-Lactones; deuterium; synthesis; isotope labeling; SIDA; flavor compounds; γ-dodecalactone; γ-decalactone; γ-octalactone
INTRODUCTION
γ-Lactones (Scheme 1) are important flavor compounds that occur in a wide range of foods including stone fruit (1, 2), wine (3), milk (4), and dairy products (5). γ-Lactones are formed by the cyclization of the corresponding γ-hydroxycarboxylic acids (6), with the even-numbered carbon chain lactones predominating in foodstuffs. The intense and characteristic odors of these lactones are described as being “fruity” and in some cases as “reminiscent of coconut” (γ-octalactone), “peach-like” (γ-decaand γ-dodecalactones), and “fatty” (γ-dodecalactone) (7). The respective odor detection thresholds for γ-octa-, γ-deca-, and γ-dodecalactone in water are 7, 11, and 7 ppb (2). To enhance flavor, γ-lactones are added to many foodstuffs at concentrations often comparable to those occurring naturally and within a range of 0.01-60 ppm (7). Accurate quantitation of flavor compounds is most readily achieved through the addition of a known quantity of a stable isotope labeled analogue to the sample as an internal standard (SIDA) (8). Due to the nearly identical physical and chemical properties of the labeled internal standard and the analyte, the labeled and unlabeled materials are recovered with equal efficiencies, and losses of the analyte during isolation and measurement are compensated for. To minimize interferences during GC-MS analysis, deuterium labeling should increase the molecular weight of a labeled internal standard by at least 2 mass units. For γ-lactones, under standard electron impact GCMS conditions (70 eV), loss of the alkyl substituent at C4 is favored, leading to an intense base peak at m/z 85 in the mass * Author to whom correspondence should be addressed (telephone +00-64-6-356-8080; fax +00-64-6-351-7011; e-mail drowan@ hortresearch.co.nz).
spectrum (Scheme 1). For maximum sensitivity in SIDA, a deuterium label should therefore be placed in the lactone ring. Although many syntheses of γ-lactone have been developed (9), few are suitable for the introduction of multiple deuterium atoms regioselectively into the lactone ring. Heiba et al. (10) have reported the synthesis of a range of γ-lactones via a onestep procedure involving the addition of substituted carboxylate salts to alkenes at elevated temperatures in the presence of manganese(III) salts. Curran and Ko (11) reported the one-step synthesis of multiply substituted γ-lactones by the free radical addition of 2-bromoacetic acid derivatives to butyl vinyl ether. Yorimitsu et al. (12) have used Curran’s atom transfer process and reported the isolation of γ-lactones in good yields from the addition of a 2-iodocarbonyl compound to a 1-alken-ω-ol in water in the presence of a radical initiator. Thus, 2-iodoacetamide and 5-hexen-1-ol were reacted in water in the presence of a radical initiator to give 8-hydroxy-γ-octalactone in 95% yield. The reaction failed, however, to produce any lactone products when a water-insoluble 1-alkene was used. Ring-deuterated γ-dodecalactone and γ-decalactone have been reported by Haffner and Tressl (13) as metabolic products of the yeast Soridiobolus salmonicolo. Ring-deuterated d4-oak lactones have been prepared by Pollnitz et al. (14) by deuterium exchange and borodeuteride reduction of 3-methyl-4-oxooctanoic acid. A similar strategy has been used to prepare d5γ- and δ-lactones with deuterium incorporated into both the ring positions (C3 and C4) and the side chain (C5) (15, 16). This paper reports the synthesis of [3,3,4-2H3]- and [2,2,3,32H ]-ring-labeled γ-lactones using two general strategies: (1) 4 hydrogenation of 4-hydroxypropargylic acids (17) with deuterium gas and (2) addition of carboxymethyl radicals to [1,1,2-
10.1021/jf048885b CCC: $27.50 © 2004 American Chemical Society Published on Web 10/15/2004
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J. Agric. Food Chem., Vol. 52, No. 23, 2004
Scheme 1. Chemical Structures of γ-Dodeca- (1), γ-Deca- (2), and
γ-Octalactones (3) and 6-Dodecyne-γ-lactone (4) Showing Origin of the Base Peak m/z 85 in the Mass Spectrum
2H
3]-1-alkenes (10-12) as well as a preliminary evaluation of use of these compounds for SIDAs.
MATERIALS AND METHODS Chemicals. Reagents were obtained from the Aldrich Chemical Co. (Milwaukee, WI) and were used without further purification unless otherwise stated. Reactions were performed under an atmosphere of dry nitrogen in oven-dried glassware. Benzene, THF, and diethyl ether (BDH) were purified by distillation from sodium benzophenone ketyl. Reactions were monitored by thin-layer chromatography on aluminumbacked Kieselgel 60 F254 silica gel plates (Merck) with the specified solvents. Compounds were visualized under an ultraviolet lamp followed by treatment with alkaline potassium permanganate and strong heating. Flash column chromatography was carried out using silica gel (60-62 µm, Merck). Short path distillation was carried out using a GKR-51 Kugelrohr (Bu¨chi). 1 H and 13C nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 on a JEOL HNM-GX270W NMR spectrometer. Chemical shifts (δ) are in parts per million relative to chloroform at 7.27 ppm for 1H (270 MHz) and at 77.0 ppm for 13C (67.8 MHz). The following abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad. Infrared spectra were measured on a Paragon 1000 FT-IR spectrometer as thin films between NaCl plates. Capillary gas chromatography-mass spectrometry (GC-MS) was carried out on a VG70250S magnetic sector instrument operating at a 70 eV ionization potential (spectral interface at 180 °C) coupled to a Hewlett-Packard 5890 series II gas chromatograph fitted with a 30 m × 0.25 mm i.d. DB1 column, film thickness ) 0.25 µm; temperature was programmed from 40 °C (5 min) to 280° C at 5 °C/min (hold for 20 min), helium head pressure ) 5 psi. Synthesis of [2,2,3,3-2H4]-γ-Lactones. 4-Hydroxydec-2-ynoic Acid Methyl Ester. A solution of lithium diisopropylamide was prepared by stirring n-butyllithium (1.45 M [hexanes], 21.7 mL, 31.4 mmol) and
Hislop et al. diisopropylamine (4.12 mL, 31.4 mmol) in THF (20 mL) under argon at 0 °C. This solution was added to a solution of propiolic acid (1.0 g, 14.3 mmol) in THF (10 mL) at -78 °C. N,N,N′,N′-Tetramethylethylenediamine (5 mL) and heptanal (1.63 g, 14.3 mmol) were added, and the mixture was warmed to room temperature with stirring. After 1 h, water (10 mL), saturated NH4Cl (20 mL), and aqueous HCl (10%, 10 mL) were added, and the aqueous layer was washed with ether (3 × 10 mL). The organic phase was evaporated, and the crude oil was dissolved in ether (40 mL) and extracted with 2% NaOH (2 × 10 mL). The aqueous phase was acidified with concentrated HCl (2 × 10 mL) and re-extracted with ether (3 × 10 mL). The combined organic phases were washed with water (2 × 10 mL) and brine (10 mL), dried over MgSO4, and concentrated in vacuo to give 4-hydroxydec-2-ynoic acid as a white power (1.15 g, 44%). This was dissolved in ether (35 mL) and treated with excess diazomethane (prepared from N-methyl-Nnitrosotoluene-p-sulfonamide) at 0 °C for 5 min. Removal of volatiles in vacuo and purification by flash chromatography on silica gel eluting with hexane through to hexane/ethyl acetate (4:1) gave the title compound (0.80 g, 28%) as a pale yellow oil: Rf 0.59 (hexane/ethyl acetate 1:1); νmax (film) 3422, 2956, 2931, 2860, 1720, 1458, 1437, 1254, 1049 cm-1; 1H NMR δ 0.86 (3H, t, J ) 6.2 Hz, H10), 1.26 (6H, s, H7 to H 9), 1.43 (2H, m, H6), 1.73 (2H, m, H5), 3.75 (3H, s, OCH3), 4.46 (1H, t, J ) 6.6 Hz, H4); 13C NMR δ 14.0, 22.5, 24.9, 28.8, 31.6, 36.8, 52.8, 61.9, 75.9, 88.6, 163.8; MS, m/z (rel int %) 197 (52), 181 (45), 113 (28), 69 (37), 55 (72), 43 (100). Found: M+ - H 197.1172, C11H17O3 requires 197.1178. 4-tert-Butyldimethylsiloxydec-2-ynoic Acid Methyl Ester (5). To a stirred solution of 4-hydroxydec-2-ynoic acid methyl ester (0.80 g, 4.0 mmol) in DMF (2 mL) at 0 °C was added imidazole (0.33 g, 4.9 mmol) and tert-butyldimethylsilyl chloride (0.54 g, 5.0 mmol). The solution was brought to room temperature and stirred for 18 h. Water (2 mL) was added to the reaction, and the solution was partitioned against ether (3 × 25 mL). The combined organic extracts were washed with water (2 × 20 mL) and saturated brine (20 mL), dried over MgSO4, concentrated in vacuo, and purified by flash chromatography on silica gel. Eluting with hexane through to hexane/ethyl acetate (5:1) gave 5 (0.84 g, 66%) as a colorless oil: Rf 0.59 (hexane/ethyl acetate 4:1); νmax (film) 2957, 2859, 1720, 1464, 1436, 1362, 1341, 1249, 1096 cm-1; 1 H NMR δ 0.11 (3H, s, SiCH3), 0.14 (3H, s, SiCH3), 0.89 (12H, s, H10 and C(CH3)3), 1.28 (6H, s, H7 to H9), 1.41 (2H, m, H6), 1.71 (2H, m, H5), 3.76 (3H, s, OCH3), 4.44 (1H, t, J ) 6.4 Hz, H4); 13C NMR δ -5.1, -4.5, 14.1, 18.2, 22.6, 25.0, 25.7, 28.9, 31.7, 37.8, 52.6, 62.6, 75.6, 89.1, 153.8; MS, m/z (rel int %) 311 (2), 281 (7), 255 (52), 229 (24), 203 (34), 171(33), 149 (7), 89 (100), 73 (49), 55 (21), 41 (19). Found: M+ - H 311.2043, C17H31O3Si requires 311.2042. [2,2,3,3-2H4]-4-tert-Butyldimethylsiloxydecanoic Acid Methyl Ester 6. To 5 (0.83 g, 2.7 mmol) dissolved in freshly distilled benzene (25 mL) was added Wilkinson’s catalyst (10 mol %, 0.125 g). The flask was fitted to a hydrogenation apparatus and degassed three times before stirring vigorously while the uptake of D2 gas (99.8% atom D) was monitored. After consumption of 2.0 equiv of D2 (60 h), the flask was isolated from the reservoir of deuterium gas. Benzene was removed under reduced pressure and the crude oil filtered through a small plug of Celite with hexane/ethyl acetate (20:1). The product was purified further by flash chromatography on silica gel eluting with hexane through to hexane/ethyl acetate (20:1) to give 6 (0.83 g, 97%) as a colorless oil: Rf 0.56 (hexane/ethyl acetate 4:1); νmax (film) 2956, 2931, 2858, 1743, 1463, 1435, 1361, 1256, 1198, 1091 cm-1; 1H NMR δ 0.04 (6H, s, Si(CH3)2) 0.88 (12H, s, H10 and C(CH3)3), 1.27 (8H, br s, H6 to H9), 1.43 (2H, m, H5), 3.66 (3H, s, OCH3); 13C NMR δ -4.6, -4.4, 14.1, 18.1, 22.6, 25.2, 25.9, 29.5, 31.9, 37.0, 51.4, 71.0, 174.4; MS, m/z (rel int %) 320 (
