366 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS University of Pennsylvania and Drexel University communities. Since there is evidence to suggest specific anosmias for both isomers of 3M2H (17,18), all subjects were screened for sensitivity to the odorants used in the experiment. Subjects were paid to participate. Different groups of subjects were used for each 3M2H mixture tested: 12 subjects (six male and six female mean age of 30.0 years) were tested in sessions comparing 10:1 (E)- to (Z)-3M2H with the individual isomers, and 12 subjects (six males and six females mean age of 25.8 years) were tested with the 10:1 (Z) to (E) ratio of 3M2H. A total of 20 subjects (10 males and 10 females mean age of 30.7 years) participated in the threshold procedures for the individual EE3M2H isomers. SYNTHESIS AND PURIFICATION OF ETHYL-3-METHYL-2-HEXENOATES A mixture of the E and Z isomers was synthesized via Wittig methodology in the following manner. Twenty-two ml (0.111 mol) of triethyl phosphonoacetate was added in a dropwise fashion to a stirred slurry of 4.48 g (0.112 mol) Nail (60% in mineral oil) in 100 ml anhydrous toluene under an N 2 blanket. The resulting betaine was allowed to form at room temperature over 30 minutes. To this reactive intermediate, 11.8 ml (0.111 mol) of 2-pentanone was added with addition of heat (40øC). The reaction mixture was stirred for 16 h to yield a viscous biphasic product. Gas chromatographic analysis indicated the formation of the E and Z esters in a 3:1 ratio. The reaction mixture was poured into ice water and extracted with three 100-ml portions of ether, dried over Na2SO4, and filtered and carefully concentrated to yield 20.3 g of clear, fruity-smelling volatile oil. Flash chromatography (1% ether in hexane) was employed to remove re- sidual toluene, leaving 15.5 g (89.6%) of the ethyl ester mixture. GC/MS analysis of the ester mixture confirmed the 1:3 ratio of Z:E isomers and the presence of an exo double- bond rearrangement (ethyl-3-methylidine-hexanoic ester) in a 1.1% yield. No [•/ double-bond migration was observed. Separation of the E and Z ethyl esters was accomplished via preparative HPLC {Zorbax Sil 9.4-mm x 25-cm column (8 pro) mobile phase: 1% ether in hexane 10 ml/min (15 mPa) Varian RI-3 refractive index detector (500 x 10 6 RI/FS sensitivity)}. Injection size was 400 pl of a 20% solution of the esters in mobile phase. Capacity factors for the Z and E esters were: k'x = 3.33 and k' E = 4.33 (o• = 1.3). Combined gas chromatography/mass spectrometry (GC/MS) was employed to determine the purity of the separated ethyl esters. A Finnigan 4510 GC/MS data system equipped with a split/splitless injector and a fused silica capillary column, and with capabilities for operation in both electron impact and chemical ionization modes, was used for analyses. The column employed was a 30-m x 0.32-mm (I.D.) fused silica column with a 1.00-pm coating of Stabilwax (cross-bonded polyethylene glycol, Restek Inc., Bellefonte, PA). The analysis conditions were as follows: 60øC, 10 rain, then 4ø/min to 220øC, held at top temperature for 1 hr. The mass spectrometer was interfaced with a Nova 4X computer, which utilizes the Super Incos software for data acquisition, analysis, and quantitation. The mass range employed during these analyses is typically m/z 40-400, which is scanned once per second. The (E)-ethyl-3-methyl-2-hexenoate was found to be 99.4% in the E, SZ form, and

CROSS-ADAPTATION BY STRUCTURAL ANALOGS 367 0.6% in the E, SE form. GC/MS of the (Z)-ethyl-3-methyl-2-hexenoate showed 93.4% in the Z, SZ form, with the closely eluting exo compound, ethyl-3-methylidine-hexanoic ester (k'•x o = 3.5 ot = 1.05) comprising 5.4% of the sample. The remaining 1.2% exhibited a M + ion at m/z 128, indicating the presence of the free acid. This required additional chromatographic cleanup on the Zorbax Sil column to remove the acid and yield a pure ester sample 94% Z, SZ form 6% exo isomer. Nuclear magnetic resonance (NMR). A Varian XL 300 MHz instrument was employed for HNMR and CMR experiments with tetramethylsilane (TMS) as an internal reference. Resonances are given in delta units as measured downfield from TMS, and are given as: singlet (s), droplet (d), triplet (t), quartet (q), and multipier (m): (E) Ethyl-3-methyl-2-hexenoate: Mass spectrum: m/z (tel. int.) 41(56), 43(37), 53(17), 55(61), 67(17), 69(51), 82(43), 83(21), 84(3), 95(27), 100(37), 111(100), 113(53), 127(5), 128(38), 129(3), 141(10), M+156(43), 157(4). •HNMR: 5.17 (s, ]H), 4.15 (q,J = 7.2 Hz, 2H), 2.14 (s, 3H), 2.10 (dJ = 7.2 Hz, 2H), 1.51 (mJ = 7.2 Hz, 2H), 1.31 (t J = 7.2 Hz, 3H), 0.93 (t J = 7.2 Hz, 3H) •3C NMR: 162.0, 160.058, 115.551, 59.422, 42.931, 20.513, 18.615, 14.316, 13.629. (Z) Ethyl-3-methyl-2-hexenoate + ethyl-3-methylidene-hexanoic ester: •HNMR: 5.17 (s, 1H), 4.18 (q,J = 3.6 Hz, 2H), 4.17 (q,J = 7.2 Hz, 2H), 2.66-2.58 (m, 2H), 2.24-2.14 (m, 2H), 1.88 (s, 3H), 1.60-1.44 (m, 2H), 1.28 (t J = 7.2 Hz, 3H) •3CNMR: 160.533,116.128, 113.660, 59.407, 59.371, 35.215, 30.711, 25.412, 25.127, 21.418, 14.286, 14.075, 11.972 Mass spectrum: Z-ethyl-3-methyl-2-hexenoate: m/z (rel. int.)41(96), 43(81), 45(14), 53(37), 55(93), 67(33), 69(67), 81(21), 82(43), 83(17), 95(41), 99(4), 100(17), 111(100), 113(56), 127(4), 128(14), 141(13), M + 156(46), 157(6). Mass spectrum: Ethyl-3-methylidene-hexenoic ester: m/z (rel. int.)41(69), 43(64), 45(10), 51(9), 53(43), 55(100), 67(26), 69(33), 71(15), 81(36), 82(18), 83(13), 99(60), 100(4), 109(11), 110(22), 111(84), 113(6), 127(6), 128(22), 141(0.6), M+156(50), 157(5). STIMULI PRESENTATION The stimuli used are presented in Figure 1. The 3-methyl-2-hexenoic acids were syn- thesized and purified by column chromatography in a manner described previously (10). Both the acids and esters used as odorants were diluted in odorless, light, white, mineral oil and presented in 270-ml, polypropylene squeeze-bottles with plastic, flip-top caps. Each bottle contained 10 ml of the odorant/mineral oil solution. For the cross-adaptation procedure, the odorants used were a 10:1 (E)- to (Z)-3M2H mixture, a 10:1 (Z)- to (E)-3M2H mixture, and the individual isolated (E)- and (Z)- isomers of EE3M2H. A 12-step binary dilution series was prepared for each odorant. Initially, 20 mg of each isomeric mixture was diluted in 20 ml of odorless, light, white, mineral oil to yield a 1 mg/ml (0.1% w/v) solution with molar concentrations of 7.81 mM of 3M2H, 6.32 mM of EE3M2H, 5.37 mM of EE3M20, and 6.93 mM of EE3M2P. The dilution scheme for each odorant was the same, ranging from 1.0 x 10 •% w/v (step 12) to 4.88 x 10-5% w/v (step 1). For the threshold procedure, the odorants used were the individual (E)- and (Z)-isomers of EE3M2H and a 3:1 (E) to (Z) ratio of EE3M2H. A 24-step binary dilution series was prepared for each odorant, starting at 1.0 x 10 •% w/v (step 24). Cross-adaptation procedure. For each of the 3M2H mixtures, subjects were tested in two 30-minute sessions, which were separated by at least 24 hr. A forced-choice, staircase