JEOR: Santalum spicatum (R. Br.) A. DC. (Santalaceae)-nor-helifolenal and acorenol isomers: Isolation and biogenetic considerations

Santalum spicatum (R. Br.) A. DC. (Santalaceae)-nor-helifolenal and acorenol isomers: Isolation and biogenetic considerations

Braun, Norbert A

Abstract

Six stereoisomers of nor-helifolen-12-al and four isomers of acoren-11-ol ([alpha]-acorenol, epi-[alpha]-acorenol, [beta]-acorenol, epi-[beta]-acorenol) were identified for the first time in Australian sandalwood oil.

Key Word Index

Santalum spicatum, Santalaceae, nor-helifolen-12-al, acoren-11-ol, biogenesis.

Introduction

Australian sandalwood oil is considered to have a great future in perfumery due to its interesting odor properties (1). The oil is manufactured from Santalum spicatum wood, butts or roots by different extraction and/or distillation techniques (2). In a former investigation of Western Australian sandalwood oil, seventy constituents were identified in a range between 13.3% and

We now report about the isolation and characterization of five other minor diastereomers of nor-helifolenal [1a/b/c/d/f] and the identification of four acorenol isomers [2a-d] from Western Australian sandalwood oil. In addition, some thoughts on the nor-helifolenal biogenesis will be discussed.

Experimental

Plant name: Santalum spicatum (R. Br.) A. DC [syn.: Fusanus spicatus R. Br.; Eucarya spicata Sprag. et Summ.; Santalum cygnorum Miq. (Santalaceae)].

Plant material and oil isolation: Australian sandalwood oil was purchased from Mt. Romance Australia Pty. Ltd., Albany (Australia). The oil was produced from S. spicatum root and butt wood chips of Southwestern Australian origin by a three-step procedure: I) hexane extraction, II) co-distillation with glycerol and III) rectification (average yield: 1.7%).

Oil analysis: Analytical GC was carried out on a Hewlett Packard 6890 gas Chromatograph with FID and DB-Wax column (60 m x 0.25 mm, film thickness 0.25 µm, carrier gas He), programmed from 50°-240°C at 4°C/min. A list of special equipment [multidimensional GC, HPLC, GC/MS, ^sup 1^H-/^sup 13^C-NMR (CDCL^sub 3^)] and conditions were provided in the experimental part of our previous publication about Western Australian sandalwood oil (3).

Isolation and characterization: The commercial sandalwood oil was acetylated (Ac^sub 2^O, NEt^sub 3^, 4-DMAP, CH^sub 2^Cl^sub 2^, room temperature) and subsequently rectified via Sulzer column to obtain 25 fractions [for details see (3): experimental part/oil fractionation]. During the isolation process, fractions were combined according to their composition as assessed by GC.

Nor-helifolen-12-al (= 2,7-dimethyl-tricyclo[5.2.2.01,5] undec-8-en-6-carbaldehyde): [1a-f] were isolated from fraction 3 [1a-d] and fraction 5 [1e/f] of the Sulzer distillation by column chromatography (silica gel, hexane/Et^sub 2^O with increasing amounts of Et^sub 2^O 5-100%) and subsequent preparative HPLC (hexane/Et^sub 2^O = 95:5): [1a] 4.0%[arrow right]36%[arrow right]91%, [1b] 2.4%[arrow right]12%[arrow right]94%, [1c] 1.2%[arrow right]17%[arrow right]90%, [1d] 0.8%[arrow right]10%[arrow right]55%, [1e] 4.6%[arrow right]80%[arrow right]96% and [1f] 2.3%[arrow right]34%[arrow right]68%. [1d] and [1f] were further purified by two dimensional preparative GC: [1d] 55%[arrow right]96% and [1f] 68%[arrow right]93%.

(1R*,2S*,5S,6R*,7R*)-[1a]: ^sup 1^H-NMR: 0.92 (ddd, J = 1.5, 5.1, 11.2 Hz, 1H, 11-H), 1.04 (d, J = 7.0 Hz, 3H, 13-H), 1.09 (m, 1H, 4-H), 1.25 (s, 3H, 12-H), 1.27-1.33 (m, 2H, 3-H, 10-H), 1.47 (m, 1H, 3-H), 1.68-1.78 (m, 3H, 2-H, 4-H, 11-H), 1.90 (m, 1H, 10-H), 1.90 (ddd, J = 2.0, 3.7, 5.9 Hz, 1H, 6-H), 2.00 (m, 1H, 5-H), 6.02 (d, J = 8.3 Hz, 1H, 8-H), 6.10 (d, J = 8.3 Hz, 1H, 9-H), 9.78 (d, J = 3.7 Hz, 1H, CHO). ^sup 13^C-NMR: 14.38 (q, C-13), 22.84 (q, C-12), 29.15 (t, C-4), 29.82 (t, C-11), 31.76 (t, C-3), 32.46 (t, C-10), 38.60 (s, C-7), 39.43 (d, C-2), 46.28 (d, C-5), 48.68 (s, C-1), 63.67 (d, C-6), 132.08 (d, C-9), 139.55 (d, C-8), 204.68 (d, CHO). GC/MS m/z (%): 41 (7), 55 (6), 65 (5), 70 (11), 77 (14), 79 (12), 81 (10), 91 (24), 93 (17), 105 (43), 119 (100), 120 (17), 121 (27), 132 (31), 134 (21), 145 (22), 17.1 (5), 204(4) [M]^sup +^.

(1R*,2S*,5S*,OR*,.7R*)-[1b]: ^sup 1^H-NMR: 0.90 (m, 1H, 3-H), 0.93 (d, J = 7.0 Hz, 3H, 13-H), 1.05 (m, 1H, 11-H), 1.23 (m, 1H, 10-H), 1.23 (s, 3H, 12-H), 1.26 (m, 1H, 4-H), 1.59 (m, 1H, 10-H), 1.74 (m, 1H, 3-H), 1.93 (ddd, J = 2.1, 3.7, 5.9 Hz, 1H, 6-H), 2.05-2.14 (m, 3H, 2-H, 4-H, 5-H), 5.85 (d, J = 8.0 Hz, 1H, 6-H), 5.94 (d, J = 8.0 Hz, IH, 9-H), 9.78 (d, J = 3.7 Hz, 1H, CHO). ^sup 13^C-NMR: 18.13 (q, C-13), 22.88 (q, C-12), 28.32 (t, C-10), 28.96 (t, C-11), 30.97 (t, C-3), 33.43 (t, C-4), 38.54 (s, C-7), 38.62 (d, C-2), 42.72 (d, C-5), 48.15 (s, C-1), 62.84 (d, C-6), 138.51 (d, C-9), 140.08 (d, C-8), 204.79 (d, CHO). GC/MS m/z (%): 41 (8), 70 (11), 77 (14), 79 (12), 91 (24), 93 (17), 105 (43), 119 (100), 120 (17), 121 (27), 132 (30), 134 (17), 145 (22), 161 (5), 171 (6), 189 (3), 204 (3) [M]^sup +^.

(1R*,2S*,5S*,6R*,7R*)-[1c]: ^sup 1^H-NMR: 0.98 (d, J = 6.6 Hz, 3H, 13-H), 1.01-1.16 (m, 2H, 4-H, 10-H), 1.17 (s, 3H, 12-H), 1.36-1.44 (m, 4H, 3-H, 4-H, 10-H, 11-H), 1.62 (m, 1H, 5-H), 1.83 (m, 1H, 11-H), 1.96-2.04 (m, 2H, 2-H, 3-H), 2.04 (dd, J = 5.1, 5.9 Hz, 1H, 6-H), 5.88 (d, J = 8.3 Hz, 1H, 9-H), 6.34 (d, J = 8.3 Hz, 1H, 8-H), 9.21 (d, J = 5.1 Hz, 1H, CHO). ^sup 13^C-NMR: 15.15 (q, C-13), 19.03 (q, C-4), 22.92 (t, C-12), 27.59 (t, C-11), 31.80 (t, C-3), 36.35 (t, C-10), 37.87 (s, C-7), 38.35 (d, C-2), 47.72 (s, C-1), 47.80 (d, C-5), 61.77 (d, C-6), 135.17 (d, C-9), 138.40 (d, C-8), 204.48 (d, CHO). GC/MS m/z (%): 41 (8), 70 (11), 77 (14), 79 (13), 81 (11), 91 (25), 93 (18), 105 (52), 119 (100), 120 (17), 121 (28), 132 (28), 134 (12), 145 (23), 161 (10), 189 (3), 204 (4) [M]^sup +^.

(1R*,2S*,5S*,6R*,7R*)-[1d]: ^sup 1^H-NMR: 0.88 (m, 1H, 11-H), 1.02 (d, J = 7.0 Hz, 3H, 13-H), 1.13 (m, 1H, 10-H), 1.17 (s, 3H, 12-H), 1.29 (m, 1H, 3-H), 1.33-1.43 (m, 2H, 10-H, 11-H), 1.62 (m, 1H, 4-H), 1.72 (m, 1H, 5-H), 1.86 (m, 1H, 4-H), 1.99 (dd, J = 5.1, 5.9 Hz, 1H, 6-H), 2.18 (m, 1H, 3-H), 5.89 (d, J = 8.8 Hz, 1H, 9-H), 6.50 (d, J = 8.8 Hz, 1H, 8-H), 9.20 (d, J = 5.1 Hz, 1H, CHO). ^sup 13^C-NMR: 19.82 (q, C-13), 22.92 (q, C-12), 27.38 (t, C-4), 27.56 (t, C-11), 33.38 (t, C-3), 36.41 (d, C-2), 36.91 (t, C-10), 37.87 (s, C-7), 44.54 (d, C-5), 48.36 (s, C-1), 60.85 (d, C-6), 134.73 (d, C-9), 136.80 (d, C-8), 204.58 (d, CHO). GC/MS m/z (%}: 41 (10), 70 (12), 77 (17), 79 (14), 81 (16), 91 (30), 93 (19), 105 (68), 119 (100), 120 (18), 121 (29), 132 (28), 134 (12), 145 (25), 147 (11), 161 (15), 189 (2), 204 (4) [M]^sup +^.

(1R,2S,5S,6R,7R)-[1e]: ^sup 1^H-NMR: 1.06 (d, J = 6.9 Hz, 3H, 13-H), 1.08-1.19 (m, 2H, 4-H, 10-H), 1.15 (s, 3H, 12-H), 1.24-1.34 (m, 2H, 3-H, 11-H), 1.42-1.55 (m, 3H, 4-H, 10-H, 11-H), 1.67 (m, 1H, 2-H), 1.91 (m, 1H, 3-H), 2.10 (in, 1H, 5-H), 2.45 (dd, J = 5.3, 10.8 Hz, 1H, 6-H), 6.15 (d, J = 8.4 Hz, 1H, 8-H), 6.26 (d, J = 8.4 Hz, 1H, 9-H), 9.39 (d, J = 5.3 Hz, 1H, CHO). ^sup 13^C-NMR: 14.83 (q, C-13), 23.08 (q, C-12), 24.52 (t, C-4), 31.46 (t, C-11), 32.53 (t, C-3), 34.07 (t, C-10), 37.41 (s, C-7), 38.54 (d, C-2), 49.54 (s, C-1), 50.23 (d, C-5), 61.77 (d, C-6), 132.97 (d, C-9), 136.55 (d, C-8), 206.31 (d, CHO). GC/MS m/ z (%): 41 (8), 55 (8), 70 (11), 79 (19), 91 (32), 93 (30), 105 (84), 119 (100), 134 (43), 147 (31), 149 (19), 161 (12), 171 (6), 175 (6), 189 (9), 204 (23) [M]^sup +^.

(1R*,2S*,5S*,6R*,7R*)-[1f]: ^sup 1^H-NMR: 0.92 (d, J = 7.0 Hz, 3H, 13-H), 1.08 (m, 1H, 4-H), 1.14 (s, 3H, 12-H), 1.21 (m, 1H, 3-H), 1.40-1.69 (m, 4H, 3-H, 4-H, 10-H), 2.10 (m, 1H1 2-H), 2.20 (m, 1H, 5-H), 2.24 (dd, J = 5.3, 10.7 Hz, 1H, 6-H), 6.01 (d, J = 8.2 Hz, 1H, 8-H), 6.06 (d, J = 8.2 Hz, 1H, 9-H), 9.39 (d, J = 5.3 Hz, 1H, CHO). ^sup 13^C-NMR: 18.55 (q, C-13), 23.02 (q, C-12), 26.42 (t, C-4), 26.43 (t, C-11), 28.12 (t, C-3), 28.13 (t, C-10), 37.34 (s, C-7), 37.95 (d, C-2), 47.03 (d, C-5), 48.62 (s, C-1), 61.16 (d, C-6), 135.55 (d, C-9), 141.61 (d, C-8), 206.45 (d, CHO). GC/MS m/z (%): 41 (17), 55 (20), 70 (12), 79 (20), 91 (34), 93 (33), 105 (81), 119 (100), 120 (17), 121 (36), 132 (24), 133 (16), 134 (45), 145 (24), 147 (28), 149 (23), 189 (12), 204 (19) [M]^sup +^

Acoren-11-ol(=2-(4,8-dimethyl-spiro[4.5]dec-7-en-1-yl)-propan-2-ol): [2a-d] were isolated from fraction 6 of the Sulzer distillation by column chromatography (silica gel, hexane/Et^sub 2^O with increasing amounts of Et^sub 2^O 5-100%): [2a] 6.4%[arrow right]19%, [2b] 0.3%[arrow right]3%, [2c] 0.8%[arrow right]7%, [2d] 1.3%[arrow right]8%. [2a] was further purified by preparative HPLC (hexane/Et^sub 2^O = 85:15): [2a] 19%[arrow right]98%; [2b-d] by column chromatography (silica gel containing 10% AgNO^sub 3^ (5), hexane/Et^sub 2^O = 85:15) and subsequent two dimensional preparative GC: [2b/c] 3%[arrow right]6%[arrow right]92%/7%[arrow right]5%[arrow right]92% (as 35:65 mixture) and [2d] 8%[arrow right]18%[arrow right]93%.

(1R*,4R*,5S*)-[2a] (= [alpha]-Acorenol): ^sup 4^H-NMR: 0.86 (d, J = 6.9 Hz, 3H, 14-H), 1.21 and 1.22 (s, each 3H, 12-H, 13-H), 1.26 (m, 1H, 3-H), 1.39 (m, 1H, 10-H), 1.49-1.57 (m, 2H, 2-H, 10-H), 1.66 (s, 3H, 15-H), 1.76 (m, 1H, 3-H), 1.79-2.04 (m, 6H, 1-H, 2-H, 4-H, 6-H, 9-H), 2.35 (d, J = 17.6 Hz, 1H, 6-H), 5.42 (m, 1H, 7-H). ^sup 13^C-NMR: 15.08 (q, C-14), 23.44 (q, C-15), 26.19 (t, C-2), 28.00 (q, C-12), 28.02 (t, C-9), 29.19 (t, C-10), 30.28 (t, C-3), 30.73 (t, C-6), 31.60 (q, C-13), 41.81 (d, C-4), 45.07 (s, C-5), 54.81 (d, C-1), 74.75 (s, C-11), 121.10 (d, C-7), 135.02 (s, C-8). GC/MS m/z (%): 41 (11), 43 (12), 55 (11), 59 (25), 67 (10), 69 (10), 77 (12), 79 (18), 81 (13), 82 (7), 91 (19), 93 (34), 94 (11), 95 (12), 105 (30), 107 (19), 119 (100), 120 (14), 121 (49), 122 (11), 133 (12), 134 (17), 135 (9), 147 (9), 149 (11), 161 (26), 189 (9), 204 (30) [M-H^sub 2^O]^sup +^.

(1R*,4S*,5S*)-[2b] (= epi-[alpha]-Acorenol): ^sup 1^ H-NMR: 0.87 (d, J = 7.0 Hz, 3H, 14-H), 1.24 and 1.25 (s, each 3H, 12-H, 13-H), 1.12 (m, 1H, 3-H), 1.41 (m, 1H, 10-H), 1.63 (s, 3H, 15-H), 1.52-2.04 (m, 9H, 1-H, 2-H, 3-H, 4-H, 6-H, 9-H, 10-H), 2.45 (m, J = 18.0 Hz, 1H, 6-H), 5.41 (m, 1H, 7-H). GC/MS m/z (%): 41 (12), 43 (12), 55 (13), 59 (33), 67 (12), 69 (8), 77 (13), 79 (21), 81 (15), 82 (8), 91 (20), 93 (34), 94 (10), 95 (12), 105 (35), 107 (22), 119 (100), 120 (14), 121 (51), 122 (12), 133 (13), 134 (18), 135 (9), 147 (12), 149 (15), 161 (35), 189 (12), 204 (38) [M-H^sub 2^O]^sup +^.

(1R*,4R*,5R*)-[2c] ( = [beta]-Acorenol): 1^sup 1^H-NMR: 0.83 (d, J = 6.6 Hz, 3H, 14-H), 0.87 (m, 1H, 3-H), 1.25 (m, 1H, 10-H), 1.26 and 1.32 (s, each 3H, 12-H, 13-H), 1.61 (s, 3H, 15-H), 1.58-2.04 (m, 9H, 1-H, 2-H, 3-H, 4-H, 6-H, 9-H, 10-H), 2.36 (m, J = 17.3 Hz, 1H, 6-H), 5.33 (s, 1H, 7-H). – GC/MS m/z (%): 41 (14), 43 (15), 55 (13), 59 (35), 67 (13), 69 (13), 77 (12), 79 (19), 81 (17), 82 (15), 91 (18), 93 (38), 94 (13), 95 (15), 105 (27), 107 (20), 119 (100), 120 (15), 121 (44), 122 (12), 133 (11), 134 (15), 135 (9), 147 (8), 149 (10), 161 (25), 189 (9), 204 (32) [M-H2O]^sup +^, 222 (1) [M]^sup +^.

(IR*,4S*,5R*)-[2d] (= epi-[beta]-Acorenol): ^sup 1^H-NMR: 0.86 (m, 1H, 3-H), 0.94 (d, J = 7.0 Hz, 3H, 14-H), 1.25 and 1.29 (s, each 3H, 12-H, 13-H), 1.29 (m, 1H, 10-H), 1.64 (s, 3H, 15-H), 1.60-2.03 (m, 9H, 1-H, 2-H, 3-H, 4-H, 6-H, 9-H, 10-H), 2.09 (m, J = 17.6 Hz, 1H, 6-H), 5.31 (m, 1H, 7-H). GC/MS m/z (%): 41 (12), 43 (13), 55 (11), 59 (35), 67 (11), 69 (9), 77 (12), 79 (19), 81 (15), 91 (17), 93 (35), 94 (13), 95 (15), 105 (30), 107 (18), 119 (100), 120 (13), 121 (41), 122 (12), 133 (10), 134 (16), 135 (8), 147 (10), 149 (10), 161 (23), 189 (10), 204 (27) [M-H2O]^sup +^, 222 (2) [M]^sup +^.

Results and Discussion

Nor-helifolenal isomers: The tricyclic nor-helifolenal has four independent stereogenic centers (C-1/C-7, C-2, C-5 and C-6) resulting in a maximum of 16 stereoisomers (2^sup 4^): eight diastereomers [1a-h] and the corresponding eight enantiomers (see Figure 1). GC/MS analysis of Western Australian sandalwood oil revealed that beside nor-helifolenal [1e] five other compounds [1a/b/c/d/f] are present having a nearly identical mass spectrum with a [M]^sup +^ of m/z = 204 (C^sub 14^H^sub 20^O). ^sup 1^H-NMR spectra of [1a-f] clearly showed an aldehydic proton (d) and two olefinic protons as well as 2 methyl group signals (each s and d). In all ^sup 13^C-N MR spectra 14 carbon atoms [2x (s), 6x (d), 4x (t), 2x (q)] were detected. Two-dimensional NMR techniques (^sup 1^H, ^sup 1^H-COSY, gHSQC, gHMBC, 2D-^sup 13^C-INADEQUATE) helped to fully elucidate that all six compounds [1a-f] are 2,7-dimethyl-tricyclo[5.2.2.0^sup 1.5^]undec-8-en-6-carbaldehyde isomers.

For the determination of the relative configuration of the major isomers [1a] and [1e] X-ray diffractometry was used. Aldehydes [1a/e] were reduced with LiAlH^sub 4^ in Et^sub 2^O to give the corresponding alcohols, which were transformed under standard conditions (acid chloride, NEt^sub 3^, 4-DMAP, CH^sub 2^Cl^sub 2^) into the 3,5-dinitrobenzoate [1a’] (6) and the p-bromosulfonate [1e’] (7), respectively (Figure 2). The crystal structures showed that [1a’] and [1e’] are C-6 epimers. Moreover it was possible to calculate the absolute configuration of [1e’] with the help of the heavy atom (Br). Bromo-derivatives of [1a] (e.g. p-bromobenzoate, p-bromosulfonate), however, were not crystalline. To establish the relative configuration of the other four nor-helifolenal isomers [1b/c/d/f] we used beside the information from X-ray crystallography, coupling constants and also NOESY experiments. Comparison of coupling constants of proton 6-H (see Table I) helped to clarify the relative stereochemistry of C-5 and C-6. [1e/f] showed a large coupling constant between 6-H[Lef-right arrow]5-H (ca. 10.7 Hz) pointing to a syn configuration, while a small coupling constant (5.9 Hz) was measured for [1a/b/c/d] indicating trans position (8). These results were verified by the crystal structures of [1a] and [1e]. Small coupling constants between 6-H[Lef-right arrow]CHO (J = 3.7 Hz) and 6-H[Lef-right arrow]11-H (^sup 4^J = ca. 2.0 Hz) were only observed for [1a/b]. Proton 6-H of the C-5 epimers [1g/h] should also possess these two small couplings together with a large cis coupling (ca. 10.7 Hz). However, until now [1g/h] were not detected in Western Australian sandalwood oil. It seems possible that [1g/h] are not formed, due to steric interactions between the aldehyde function and C-4 as well as between protons 5-H and 6-H. In the NOESY spectra of [1a/c/e] a cross-peak between 9-H and the methyl group at C-2 was obvious, while this peak is missing in the NOESY of [1b/d/f]. These results indicate, that [1a]/[1b] and [1c]/[1d] as well as [1e]/[1f] differ only in the stereochemistry at C-2. Compounds [1a-f] occur in Western Australian sandalwood oil in a range between 0.40% and 0.05% pointing to a random distribution (see Table I). Nevertheless, it seems noteworthy that all three sets of epimers occur in nearly the same ratio of 70:30. Alcohols [1a/b/c/d] possess a weak flowery-fresh odor, whereas [1e] has a weak flowery-metallic and [1f] a weak earthy, patchouli-like note.

Acorenol isomers: Moreover we turned our interest to four odorless constituents [2] (Figure 3) of Western Australian sandalwood oil (Table II). The GC/MS pattern of all four compounds [2] was nearly identical, having a base-peak of m/z 119 and a strong fragmentation peak of m/z 204 [M-18]^sup +^. Only compounds [2c/d], however, show a small molecular ion peak of m/z 222 (C^sub 15^H^sub 26^O). The [M-H2O]^sup +^ fragmentation peak together with m/z 59 [C^sub 3^H^sub 7^O]^sup +^ peak strongly indicated the presence of a tertiary hydroxyl group, located on an isopropyl function. The major isomer [2a] was fully characterized. In the ^sup 1^H-NMR of [2a], one olefinic proton at 5.42 ppm and four methyl groups were obvious. ^sup 13^C-NMR showed beside signals for a tri-substituted double bond [135.02 (s) and 121.10 (d)], one singlet at 74.75 (hydroxylated quarternary carbon atom) and another at 45.07 characteristic for a spirocyclic ring system. Two-dimensional NMR experiments (^sup 1^H,^sup 1^H-COSY, gHSQC, gHMBC, NOESY) together with comparison of published data unambiguously proofed that compound [2a] is [alpha]-acorenol. Measurement of the optical rotation ([[alpha]]^sup 20^^sub D^ = + 8.7°, c = 0.75 in CHCl^sub 3^) of [2a] was used to control the optical purity: op [%] = |[[alpha]]^sub obs^|/|[[alpha]]^sub max^ | x 100 with [[alpha]]^sub max^ = – 36.1° (9). The ratio of the [alpha]-acorenol enantiomers in Western Australian sandalwood oil was calculated to 76:24 in favor of the (1S,4S,5R)-isomer ent-[2a]. The relative stereochemistry of the other three acorenol isomers [2b-d] was determined by comparison of selected ^sup 1^H-N MR signals [CH^sub 3^ (d), C(CH^sub 3^)^sub 2^, 7-H] (see Table II) with literature data (10). From this resulted the following retention order on a DB-Wax column: [alpha]-acorenol [2a] epi-[beta]-acorenol [2b] [beta]-acorenol [2c] epi-[beta]-acorenol [2d]. [alpha]-Acorenol [2a] (9), [beta]-acorenol [2c] (11) and epi-[beta]-acorenol [2d] (12) were isolated from several plants before, but epi-[alpha]-acorenol [2b] was not detected in nature. So far at most two acorenol isomers were found in a single plant: [2a/d] in Hedychium gardnerianum (12) and [2a/c] in Pothomorphe umbellata (13) as well as in Juniperus rigida (9,11). Our investigations showed that in some plants all four acorenol isomers [2] are formed. In the Juniperus species J. virginiana and J. mexicana, which are used to produce cedarwood oil (14,15), [2a/c] are the main isomers, but [2b/d] were detected in trace amounts, too. Alcohols [2] are also present in S. spicatum and S. album in a ratio of [2a]/[2b]/[2c]/[2d] = 66:7:7:20 and 29:17:15:39, respectively (see Table II). These results are interesting in view of the nor-helifolenal biogenesis, because nor-helifolenal [1] is formally derived from acorenol [2].

Biogenetic considerations: It can be envisioned that the helifolane skeleton [E] is formed biosynthetically either by a step-wise cationic mechanism [A][arrow right][B][arrow right][C][arrow right][E] (16) or via an intramolecular Diels-Alder reaction [D][arrow right][E] (17) (Figure 4). The “Diels-Alder pathway” is supported by the following facts: (Z)-[gamma]-curcumen-12-ol [D] (R = CH^sub 3^, R^sup 1^ = CH^sub 2^OH) or [gamma]-curcumene [D] (R = R^sup 1^ = CH^sub 3^) were characterized in Western Australian sandalwood oil (3). These 1,4-dienes [D] can serve as starting materials for a [4+2]-cycloaddition, which can be catalyzed by a Diels-Alderase (18-20). Moreover nor-helifolen-12-al [1e] is enantiopure ([[alpha]]^sup 20^^sub D^ = + 35.7°; c = 2.95 in CHCl^sub 3^), favoring a concerted over a step-wise cationic mechanism.

The “cationic pathway” is sustained among other things through the identification of stabilized alcohols of [A] and [B]: [beta]-bisabolol (3) and acorenol [2]. Both alcohols are analogously formed as racemic by-products in the acid-catalyzed cyclization of nerolidol (21). An enantioselective and/or diastereoselective variant of this synthesis can be found in nature. Enzymatic cyclization of farnesylpyrophosphate ( FPP) catalyzed by a sesquiterpene cyclase/synthase (22-24) could lead not only to enantiomerically enriched [A] and [B] but also to enantiopure helifolene [E] (R = R^sup 1^ = CH^sub 3^).

There are indications for both pathways; however, it can only be clarified by means of molecular biological methods (25) whether a Diels-Alderase or a sesquiterpene cyclase or a combination of both is involved in the biogenesis of the helifolane skeleton [E]. Finally, the transformation of helifolene [E] (R = R^sup 1^ = CH^sub 3^) to nor-helifolenal [1] is probably achieved through degradation of a methyl group. The geminal methyl groups R and R^sup 1^ could be oxidized to form a [beta]-oxo-carboxylic acid (R = CHO, R^sup 1^ = COOH), which is subsequently decarboxylated.

In summary, aldehydes [1a-f] and alcohol [2b] are, to the best of our knowledge, new natural products and alcohols [2a/c/d] have not been described as constituents of Western Australian and East Indian sandalwood oil. S. spicatum should be considered not only as a source of new natural products, but also as a model plant to study sesquiterpene biosynthesis.

Acknowledgment

Claudia Valder thanks Symrise, Holzminden, Germany, for generous financial and technical support of her diploma thesis. The authors are grateful to Wilhelm Pickenhagen for helpful discussions.

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Norbert A. Braun,* Manfred Meier, and Birgit Kohlenberg

Symrise GmbH & Co. KG, Corporate Research Division, D-37601 Holzminden, Germany

Claudia Valder and Michael Neugebauer

Pharmazeutisches Institut, Universitat Bonn, Kreuzbergweg 26, D-53115 Bonn, Germany

* Address for correspondence

1041-2905/03/0006-0381$6.00/0-© 2003 Allured Publishing Corp.

Received: November 2002

Revised: January 2003

Accepted: February 2003

Copyright Allured Publishing Corporation Nov/Dec 2003

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