Identification of m-Mentha-3(8),6-diene (isosylveterpinolene) in black pepper oil

Article abstract of DOI:10.1002/(SICI)1522-2675(19990113)82:1<53::AID-HLCA53>3.0.CO;2-7

m-Mentha-3(8),6-diene (= isosylveterpinolene; 5), which has not been found in nature before, was shown by GC/MS analysis and co-injection with a synthetic sample to be present to the extent of 0.12-0.17% in black and green pepper oil. The synthetic reference 5 was prepared by deconjugation of 3- methylcyclohex-2-en-1-one (1), subsequent alkylation with lithium 2-lithio- 2-methylpropanoate, and decarboxylative dehydration of the resulting hydroxycarboxylic acid 3 employing dimethylformamide dimethyl acetal. On polar GC columns, 5 was eluted between p-cymene and terpinolene, and was characterized by an MS similar to that of terpinolene, but with the fragment m/z 121 (59, [M - Me]⁺) being less intense than that of the molecular ion at m/z 136 (67, M⁺). Since m-mentha-3(8),6-diene was also found to the extent of 0.24% in the headspace of ground black peppercorns, it can be considered to be of genuine natural origin, and thus is the first established example of a naturally occurring m-menthadiene.

Full text of DOI:10.1002/(SICI)1522-2675(19990113)82:1<53::AID-HLCA53>3.0.CO;2-7

Helvetica Chimica Acta ± Vol. 82 (1999)
53
Identification of m-Mentha-3(8),6-diene
(
Isosylveterpinolene) in Black Pepper Oil
by Cornelius Nussbaumer, Riccardo Cadalbert, and Philip Kraft*.
Givaudan Roure Fragrance R&D, Überlandstrasse 138, CH-8600 Dübendorf
m-Mentha-3(8),6-diene (ˆ isosylveterpinolene; 5), which has not been found in nature before, was shown
by GC/MS analysis and co-injection with a synthetic sample to be present to the extent of 0.12 ± 0.17% in black
and green pepper oil. The synthetic reference 5 was prepared by deconjugation of 3-methylcyclohex-2-en-1-one
(
1), subsequent alkylation with lithium 2-lithio-2-methylpropanoate, and decarboxylative dehydration of the
resulting hydroxycarboxylic acid 3 employing dimethylformamide dimethyl acetal. On polar GC columns, 5 was
eluted between p-cymene and terpinolene, and was characterized by an MS similar to that of terpinolene, but
‡
‡
with the fragment m/z 121 (59, [M � Me] ) being less intense than that of the molecular ion at m/z 136 (67, M ).
Since m-mentha-3(8),6-diene was also found to the extent of 0.24% in the headspace of ground black
peppercorns, it can be considered to be of genuine natural origin, and thus is the first established example of a
naturally occurring m-menthadiene.
Introduction. ± Black pepper oil (Piper nigrum L., Piperaceae) has an intense, spicy,
and peppery odor, reminiscent of cubebs [1] but with a milder taste which lacks the
pungency of the spice. This oil is not only one of the most important flavoring raw
materials, it also becomes increasingly fashionable in fine fragrances. In ꢀExtreme Polo
Sportꢁ (Lauren, 1998), the extreme amount of around 6% was harmoniously
incorporated. ꢀMiss Diorꢁ (Dior, 1947), ꢀComme des garçonsꢁ (Comme des garçons,
1
994), and ꢀContradictionꢁ (C. Klein, 1997) demonstrate that its use is not limited to
masculine notes. Of importance with respect to the application in perfumery are the
dry-spicy, terpenic top-note aspects of black pepper oil that are mainly due to the
monoterpene fraction. In 1992, Kollmannsberger et al. [2] reported on two unidentified
menthadienes in the monoterpene fraction that could contribute to the characteristic
top note of black pepper oil. They assumed these menthadienes to be meta-substituted,
though the natural occurrence of m-menthanes [3] is controversial. Sylvestrene (ˆ m-
mentha-6,8-diene) and sylveterpinolene (ˆ m-mentha-1,3(8)-diene), which have been
isolated from different turpentine oils (Pinus sylvestris L.), could be artifacts resulting
from the acid-catalyzed ring opening of carenes [4].
If m-menthadienes are genuine secondary metabolites, they could arise either from
an enzymatic degradation of carenes or from the cyclization of a head-to-head joined
diisoprene pyrophosphate, e.g., 2,7-dimethylocta-1,6-dien-3-yl pyrophosphate. Aro-
matic products and p-menthadienes predominate in the pyrolysis of car-3-ene [5], while
dehydrohalogenation in AcOH/NaOAc of sylvestrene dihydrochloride, an addition
product of HCl to car-3-ene, was reported to yield mainly a 42:31:27 mixture of m-
mentha-6,8-diene (ˆsylvestrene), m-mentha-1,8-diene (ˆ isosylvestrene), and m-
mentha-1(7),8-diene [6]. On GC/MS analysis of black pepper oil, we found an
‡
unknown GC peak with a molecular ion M at m/z 136 between the peaks of p-cymene

54
Helvetica Chimica Acta ± Vol. 82 (1999)
and terpinolene; however, the MS of this unknown compound was significantly
different from those of the three sylvestrenes mentioned above [7]. Besides the
‡
‡
fragment [M � Me] at m/z 121 which was less significant than M at m/z 136, the MS
of the unknown compound was rather quite similar to that of terpinolene. Therefore,
and since m-mentha-3(8),6-diene (5) would be the expected biogenetic cyclization
product of a head-to-head joined diisoprene pyrophosphate, we decided to synthesize
this m-menthadiene to establish its presence in black pepper oil.
Results and discussion. ± Our three-step synthesis of m-mentha-3(8),6-diene (5)
started with the deconjugation of 3-methylcyclohex-2-en-1-one (1) in the presence of
adipic acid as a mild acidic catalyst [8] (Scheme). Though the equilibrium ratio 1/2 of
9
(
1
8.8 : 1.2 was quite unfavorable, we were able to enrich 3-methylcyclohex-3-en-1-one
2) up to 70% by using a 80-cm vacuum-jacketed Sulzer column for distillation at 176 ±
808. The distillate was further purified by flash chromatography (silica gel) to provide
isomerically pure 3-methylcyclohex-3-en-1-one (2) on a 70-g scale. According to a
general procedure of Krapcho et al. [9], enone 2 was then alkylated with the lithium a-
lithiocarboxylate of isobutyric acid, prepared by treatment of isobutyric acid with 2
equiv. of lithium diisopropylamide. The crude a,a-dimethyl-b-hydroxycarboxylic acid 3
was isolated by extraction in 79% yield in a sufficiently pure state for direct further
transformation.
Scheme. Synthesis of m-Mentha-3(8),6-diene (5) from 3-Methylcyclohex-2-en-1-one (1).
2 2 4 2 2 2 2 2 2 2
a) HO C(CH ) CO H, D, distillation. b) Me CLiCO Li, THF. c) Me NCH(OMe) , CH Cl .
Following the smooth method of Eschenmoser and coworkers [10] for the
decarboxylative dehydration of hydroxycarboxylic acids, the crude b-hydroxy acid 3
was next treated with dimethylformamide dimethyl acetal in CH Cl2 [11]. Via
2
intermediate 4 (Scheme), the isopropylidene group was built up, and we isolated our
target molecule 5 in 71% yield, free from double-bond isomers. Fig. 1 details the
spectroscopic assignments of m-mentha-3(8),6-diene (5) as determined by COSY-
DQF, HMQC, and NOESY experiments. The methyl groups Me(9) and Me(10) were
discriminated by a NOEDIFF spectrum (irradiation of H� C(2) at 2.65 ppm).
GC/MS Analyses and co-injection of the synthetic reference compound 5 with two
commercial black pepper oils unambiguously established the presence of m-mentha-

Helvetica Chimica Acta ± Vol. 82 (1999)
55
Fig. 1. Spectroscopic Data of m-Mentha-3(8),6-diene (5). Assignments by COSY-DQF,
HMQC, NOESY, and NOEDIFF.
3
(8),6-diene (ˆ isosylveterpinolene; 5) to the extent of 0.12 and 0.16%, respectively,
matching the previously unidentified peak between p-cymene and terpinolene (Fig. 2).
We also found 5 in amounts of 0.24% in the headspace of black peppercorns, of 0.17%
in green pepper oil, and of 0.27% in the supercritical fluid extract (SFE) of Muntok
pepper [2]. The percentage of m-mentha-3(8),6-diene (5) in the different pepper oils
relative to the hydrocarbons of the monoterpene fraction is almost constant (0.22 ±
0
.23%; see Table). This almost constant quantity of 5 in different pepper oils as well as
the high content of 5 in the headspace of ground black peppercorns clearly indicate
that m-mentha-3(8),6-diene (5) is of genuine natural origin. Thus, 5 is the first
established example of a naturally occurring m-menthadiene.
m-Mentha-3(8),6-diene (5) was first synthesized in 1972 by Muraleedharan and
Verghese [12] by dehalogenation of 1,3,6,8-tetrabromo-m-menthane with Zn dust. This
tetrabromide was obtained from a 6-% fraction of the dehydrohalogenate of
sylvestrene dihydrochloride with aniline. In another synthetic approach [13], dehy-
dration of m-menth-6-en-8-ol provided 5 as the 5-% component of a mixture with
sylvestrene and the original alcohol. However, no well-directed synthetic approach
towards 5 has been reported. The above described simple, straightforward synthesis of
5
from commercially available 3-methylcyclohex-2-en-1-one (1), should allow the
identification of this unusual meta-monoterpene in other natural products.
Olfactory Evaluation. ± With the exception of highly unsaturated systems such as
the undeca-1,3,5-trienes [14] or the megastigma-4,6,8-trienes [15], hydrocarbons
generally show only relatively weak, uncharacteristic odors. A recent structure-odor-
relationship study of hydrocarbons [16] [17] indicated that at least two sterically
accessible CˆC bonds ± favorably in conjugation ± should be present in a cycloaliphatic

56
Helvetica Chimica Acta ± Vol. 82 (1999)
Fig. 2. GC Trace of the monoterpene fraction of black pepper oil (Rocchia Distillerie s.a.s.).
TM
Supelcowax 10 column.
Table. Content of m-Mentha-3(8)-6-diene (5) in Different Natural Products. Area-% values in the GC (FID).
Black
Black
Ground black Green pepper oil Muntok pepper
pepper oil pepper oil peppercorns
SCF extract
[2]
(
Rocchia) (Kalsec)
(headspace)
(Synthite)
Car-3-ene
12.64
18.76
0.53
7.49
14.75
0.43
9.25
13.44
0.58
11.79
22.88
1.82
22.94
15.78
1.37
Limonene
p-Cymene
m-Mentha-3(8),6-diene (5)
0.16
0.12
0.24
0.17
0.27
(
in % of monoterpene fraction) (0.22)
(0.23)
(0.70)
(0.22)
(0.42)
Terpinolene
0.46
0.44
0.65
1.25
0.64

Helvetica Chimica Acta ± Vol. 82 (1999)
57
hydrocarbon odorant. Limonene with a fresh lemon-like odor and a threshold of
0 ng/l can be considered as a benchmark [16] [17]. m-Mentha-3(8),6-diene (5) with a
7
pleasant green-herbal, terpenic odor reminiscent of parsley and carrot leaves and a
slightly metallic nuance, is much weaker than limonene, with a threshold of only
5
00 ng/l. Because of this high threshold, 5 does not contribute considerably to the
characteristic top note of black pepper oil.
Experimental Part
General. Black pepper oil was obtained from Rocchia Distillerie s.a.s., I-12014 Demonte, Italy, and Kalsec,
Kalamazoo, Michigan 49005-0511, USA, and green pepper oil from Synthite Industrial Chemicals Ltd., Cochin
682 016, India. Reagents and solvents were purchased from Fluka and used without further purification. 3-
Methylcyclohex-2-en-1-one is also commercially available: Fluka 66453, Aldrich M3,910-5, Lancaster 5090.
Flash chromatography (FC): Merck silica gel 60, particle size 0.040-0.063. IR: Nicolet-510 FT-IR spectrometer; nÄ
�
1
1
13
in cm . H- and C-NMR: Bruker-AVANCE-DPX-400 spectrometer, d in ppm rel. to SiMe
Fisons-MD-800 instrument, rel. int. in % of the base peak.
4
, J in Hz. GC/MS:
3
-Methylcyclohex-3-en-1-one (2). A distillation apparatus with an 80-cm vacuum-jacketed Sulzer column
2
and a Perkin triangle was flushed with N and charged with 3-methylcyclohex-2-en-1-one (1; 484 g, 4.40 mol) and
adipic acid (64.3 g, 440 mmol). The mixture was heated to 2138 (inner temp.; heating mantle) and the column
equilibrated at ambient pressure for 4 h. At a rate of ca. 1 drop per 10 s and a stillhead temp. of 176 ± 1808, the
product was collected for 4 h. This distillate was taken up in pentane (1 l), separated from the aq. layer, and
2 4
dried (Na SO ) to provide a 7:3 fraction of 2/1 (213 g, 70% based on recovered starting material). The ratio 1/2
in the distillation bulb was 98.8 :1.2. FC (silica gel, pentane/t-BuOMe, 9:1, R
f
0.52) of the distillate provided
): 1.73 (d, J ˆ 1.4, Me� C(3));
1
isomerically pure 2 (69.2 g, 32%). IR (neat): 1718s, (C ˆ O). H-NMR (CDCl
3
13
2
2
2
.39 ± 2.47 (m, 2 H� C(5), 2 H� C(6)); 2.78 (s, 2 H� C(2)); 5.60 (t, J ˆ 1.4, H� C(4)). C-NMR (CDCl
3
):
2.54 (q, Me� C(3)), 24.85 (t, C(5)), 38.14 (t, C(6)); 44.26 (t, C(2)); 120.78 (d, C(4)); 131.95 (s, C(3));
‡
‡
‡
10.44 (s, C(1)). MS (70 eV): 110 (67, M ), 95 (3, [M � Me] ), 82 (10, [M � CO] ), 67 (100, [M � CO �
‡
‡
‡
7
Me] ), 53 (31, [M � CO � C
2
H
5
] ), 39 (30, [M � CO � C
3
H ] ).
2
-(1'-Hydroxy-3'-methylcyclohex-3'-en-1'-yl)-2,2-dimethylacetic Acid (3). Lithium diisopropylamide
(
272 mmol) was prepared by the addition of 1.6m BuLi in hexanes (170 ml, 272 mmol) at � 408 under N
2
to a
stirred soln. of (i-Pr) NH (39 ml, 276 mmol) in THF (650 ml). Stirring was continued at 08 for 20 min, before
2
isobutyric acid (12.0 g, 136 mmol) was added dropwise at � 408. The mixture was heated to 508 for 2 h and then
recooled to � 408 before 2 (15.0 g, 136 mmol) was added dropwise. Stirring at � 408 was continued for an
additional 2 h. Then the mixture was poured on ice-water (1 l), and extracted with t-BuOMe (4 Â 250 ml). The
aq. layer was acidified with 2n aq. HCl and extracted with t-BuOMe (4 Â 250 ml). The combined org. extracts
were dried (MgSO
4
) and evaporated: 3 (21.4 g, 79%) Crude material sufficiently pure for further trans-
1
formation. IR (neat): 1702s (C ˆ O), 1165m (C� O); 3150, 3400 (br. sh, O� H). H-NMR (CDCl
3
): 1.28,
1
.30 (2 s, 2 Me� C(2)); 1.67 (s, Me� C(3')), 1.48 ± 2.33 (m, OH, 2 H� C(2'), 2 H� C(5'), 2 H� C(6')); 5.49
13
(
m, H� C(4')). C-NMR (CDCl
3
): 20.77, 20.80 (2 q, 2 Me� C(2)); 21.76 (t, C(5')); 23.68 (q, Me� C(3'));
27.21 (t, C(6')); 37.28 (t, C(2')); 49.51 (s, C(2)); 74.13 (s, C(1')); 120.46 (d, C(4')); 130.65 (s, C(3')); 183.39
‡
‡
‡
(
C
s, C(1)). MS (70 eV): 198 (2, M ), 180 (4, [M � H
2
O] ), 110 (50, [M � C
4
H
8
O
2
] ), 93 (53, [M � H O �
2
‡
‡
‡
‡
4
H
7
O
2
] ), 88 (100, C
4
H
8
O ), 67 (66, C
2
5
H
7
), 43 (90, C
3
H ).
7
m-Mentha-3(8),6-diene ( ˆ Isosylveterpinolene ˆ 1-Methyl-5-(1-methylethylidene)cyclohex-1-ene; 5). To a
suspension of crude 3 (21.4 g, 108 mmol) in CH Cl (270 ml), dimethylformamide dimethyl acetal was added
and the clear soln. stirred under reflux for 2 h and at r.t. for 14 h. The mixture was evaporated, the residue taken
up in pentane, and the pentane soln. washed with H O. After evaporation, the crude product (19.7 g) was
2
2
2
purified by FC (silica gel, pentane, R
f
0.87): 5 (9.71 g, 71%). Colorless, odoriferous liquid. IR (neat): 2913s,
1
2
966s, 2842s, 2804s (C� H); 1441m 1375m (CH
2
, CH
3
); 1678w (CˆC). H-NMR (CDCl
3
): 1.67 (s, 3 H� C(9));
1
5
2
.68 (s, 3 H� C(7)); 1.69 (s, 3 H� C(10)); 2.02 (m, 2 H� C(5)); 2.25 (t, J ˆ 6.2, 2 H� C(4)); 2.65 (s, 2 H� C(2));
13
.46 (m, H� C(6)). C-NMR (CDCl
3
): 19.67 (q, C(10)); 20.09 (q, C(9)); 23.41 (q, C(7)); 26.29 (t, C(4));
6.46 (t, C(5)), 34.44 (t, C(2)), 121.27 (d, C(6)), 121.51 (s, C(8)); 128.12 (s, C(3)); 133.84 (s, C(1)). MS
‡
‡
‡
‡
(
C
C
70 eV): 136 (66, M ), 121 (58, [M � Me] ), 107 (18, [M � C
2
H
5
] ), 105 (20, [M � C
2
H
5
� H
2
] ), 93 (100,
‡
‡
‡
‡
‡
‡
‡
‡
7
H
H
9
5
), 91 (48, C
), 51 (10, C
7
H
7
), 79 (39, C
6
H
7
), 77 (33, C
), 41 (31, C
6
H
5
), 67 (18, C
5
H
7
), 65 (9, C
5
H
5
), 55 (12, C
4
H
7
), 53 (19,
‡
‡
‡
4
4
H
3
), 43 (22, C
3
H
7
3
H
5
). Odor: green, herbal, terpenic, parsley, carrot leaves,
metallic.

58
Helvetica Chimica Acta ± Vol. 82 (1999)
Headspace Sampling. Black peppercorns (30 g; origin: Madagascar), purchased in a local drugstore, were
ground and placed in an Erlenmeyer flask. Following the method described by Neuner-Jehle and Etzweiler [18],
the headspace was trapped under quasi-static conditions on a Porapak Q (50 ± 80 mesh) microfilter during 6 h by
sucking air with a small pump (model 222-4, SKC Inc., PA 15330, USA). Subsequently, the adsorbed volatiles
were eluted with hexane/acetone 4:1 (50 ml), and the extract was analyzed by GC/MS.
Identification of m-Mentha-3(8),6-diene (5). By comparison of the GC/MS spectra and co-injection with
TM
synthetic 5 on a Supelcowax 10 column (Supelco; 30 m  0.25 mm, film thickness 0.25 mm). Temperature
program: 3 min at 408, then 208/min ! 708, then 88/min ! 2508. Flow: 1 ml/min He. Black pepper oil
(
(
Rocchia) 0.16 area-% (FID), i.e. 0.22% of the monoterpene fraction. Black pepper oil (Kalsec): 0.12 area-%
FID), i.e. 0.23% of the monoterpene fraction. Headspace of ground black peppercorns: 0.24 area-% (FID),
i.e. 0.70% of the monoterpene fraction. Green pepper oil (Synthite) 0.17 area-% (FID), i.e. 0.22% of the
monoterpene fraction. SCF Extract of Muntok pepper [2]: 0.27 area-% (FID), i.e. 0.42% of the monoterpene
fraction. Retention times: Car-3-ene 7.39, limonene 8.29, p-cymene 9.58, m-mentha-3(8),6-diene (ˆ isosyl-
veterpinolene; 5) 9.67 and terpinolene 9.78 min.
We are indebted to G. Fr a ter for fruitful discussion, and to U. Müller for a generous gift of 3-methyl-
cyclohex-2-en-1-one. We also wish to thank G. Brunner, J. Märki, and J. Schmid for spectroscopic data, W. Grab
for olfactory evaluation and E. Senn for the determination of odor thresholds. Furthermore, we are grateful to S.
Nitz and H. Kollmannsberger for a sample of their supercritical fluid extract of Muntok pepper.
REFERENCES
[
[
[
[
1] A. J. Jouhar, ꢀPoucherꢁs Perfumes, Cosmetics and Soapsꢁ, Chapman & Hall, London, 1991, 9th edn., p. 268.
2] H. Kollmannsberger, S. Nitz, F. Drawert, Z. Lebensm.-Unters. Forsch. 1992, 194, 545.
3] J. Verghese, Perfum. Flavor. 1980, 5 (3), 47.
4] W. F. Erman, ꢀChemistry of the Monoterpenesꢁ, Marcel Dekker, New York, 1985, 1st edn., p. 604 ± 605 and
p. 882 ± 884.
[
[
[
[
[
5] W. Cocker, D. P. Hanna, P. V. R. Shannon, J. Chem. Soc. C 1968, 489.
6] K. Gollnick, G. Schade, Tetrahedron Lett. 1966, 5157.
7] G. von Bünau, G. Schade, K. Gollnick, Freseniusꢀ Z. Anal. Chem. 1969, 244, 7.
8] E. Widmer, to F. Hoffmann-La Roche, Ger. Offen. DE 2508779, 1975; U.S. Pat. 4005145, 1977.
9] A. P. Krapcho, E. G. E. Jahngen, J. Org. Chem. 1974, 39, 1322.
[
[
[
[
[
[
[
10] A. Rüttimann, A. Wick, A. Eschenmoser, Helv. Chim. Acta 1975, 58, 1450.
11] G. Fr a ter, U. Müller, W. Günther, Tetrahedron 1984, 40, 1269.
12] N. V. Muraleedharan, J. Verghese, J. Ind. Chem. Soc. 1972, 49, 289.
13] K. G. Kaimal, J. Verghese, Indian Perfumer 1983, 27 (3&4), 143.
14] Y.-R. Naves, Bull. Soc. Chim. Fr. 1967, 3152.
15] F. B. Whitfield, G. Sugowdz, Aust. J. Chem. 1979, 32, 891.
16] C. Anselmi, M. Centini, M. L. Paoli, A. Sega, ꢀXIII. ECRO Congressꢁ, Siena, September 8th ± 12th, 1998,
Abstract 116.
[17] A. Buonocore, ꢀRelazioni tra odore struttura molecolare negli idrocarburiꢁ, Diploma Thesis, University of
Siena, 1998.
[18] N. Neuner-Jehle, F. Etzweiler, in ꢀPerfumes: Art, Science and Technologyꢁ, Eds. P. M. Müller, D.
Lamparsky, Elsevier Applied Science, London, 1991, p. 172.
Received November 2, 1998