Biotransformations of terpenes by fungi from amazonian Citrus plants

Article abstract of DOI:10.1002/cbdv.201300112

The biotransformations of (RS)-linalool (1), (S)-citronellal (2), and sabinene (3) with fungi isolated from the epicarp of fruits of Citrus genus of the Amazonian forest (i.e., C. limon, C. aurantifolia, C. aurantium, and C. paradisiaca) are reported. The

Full text of DOI:10.1002/cbdv.201300112

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1909
Biotransformations of Terpenes by Fungi from Amazonian Citrus Plants
by Maria Gabriela Moreno Ruedaa), Alessandra Guerrinia), Pier Paolo Giovanninib), Alessandro
Medicib), Alessandro Grandinia), Gianni Sacchettia), and Paola Pedrini*a)
a
`
) Dipartimento di Scienze della Vita e Biotecnologie, Universita di Ferrara, Via L. Borsari 46, IT-44121
Ferrara (phone þ390532455700; fax þ390532455715; e-mail: pdp@unife.it)
b
`
) Dipartimento di Scienze Chimiche e Farmaceutiche, Universita di Ferrara, Via Fossato di Mortara 17-
27, IT-44121 Ferrara
The biotransformations of (RS)-linalool (1), (S)-citronellal (2), and sabinene (3) with fungi isolated
from the epicarp of fruits of Citrus genus of the Amazonian forest (i.e., C. limon, C. aurantifolia, C.
aurantium, and C. paradisiaca) are reported. The more active strains have been characterized, and they
belong to the genus Penicillium and Fusarium. Different biotransformation products have been obtained
depending on fungi and substrates. (RS)-Linalool (1) afforded the (E)- and (Z)-furanlinalool oxides (7
and 8, resp.; 39 and 37% yield, resp.) with Fusarium sp. (1D2), 6-methylhept-5-en-2-one (4; 49%) with F.
fujikuroi, and 1-methyl-1-(4-methypentyl)oxiranemethanol (6; 42%) with F. concentricum. (S)-
Citronellal (2) gave (S)-citronellol (12; 36–76%) and (S)-citronellic acid (11; 5–43%) with Fusarium
species, while diastereoisomeric p-menthane-3,8-diols 13 and 14 (20 and 50% yield, resp.) were obtained
as main products with Penicillium paxilli. Finally, both Fusarium species and P. paxilli biotransformed
sabinene (3) to give mainly 4-terpineol (19; 23–56%), and (Z)- and (E)-sabinene hydrates (17 (3–21%)
and 18 (11–17%), resp.).
1. Introduction. – Terpenes are secondary metabolites of plants and are produced
both for the defence against microorganisms and insects, and for their pollinator-
attractive properties [1]. In mammals, terpenes are involved in stabilizing cell
membrane, in metabolic pathways, and they act as regulators in some enzymatic
reactions [2]. In particular, mono- and sesquiterpenes are the major constituents of
essential oils and are widely used in the perfumery industry. Moreover, terpenes are
good starting materials for the synthesis of fine chemicals [3], and, in particular, they
are employed to produce flavorings [4][5]. On the other hand, the application of
terpenes as fragrances and flavors depends on the absolute configuration of the
compounds, since the enantiomers present different organoleptic properties. In this
field, the biotransformations allow the regio- and stereoselective production of
compounds under mild conditions [2][6].
The use of microorganisms in terpene biotransformations is relatively recent.
Various fungal and bacterial strains have been employed in order to obtain new
fragrances, and new strains are continuously selected based on their ability to
biotransform terpenes [6]. While various microbial and plant cells are able to
biotransform linalool (i.e., Aspergillus niger [7–9], Botrytis cinerea [9], Corynespora
cassicola [9], Diplodia gossipina [10], Debaryomyces sp. [11], Kluyveromyces sp. [11],
and Pichia sp. [11]) and citronellal (i.e., Saccharomyces cerevisiae [12][13], Pseudo-
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
monas aeruginosa [14], Rhodotorula minuta [15], Petroselinum crispum [16], and
Solanum aviculare [17]), the biotransformations of sabinene have not been studied.
Since these terpenes are significantly present in the essential oils obtained from the
leaves of the plants of Citrus genus that are used in cosmetology, their possible
biotransformations could be applied successively to the essential oils in order to
improve their properties.
In this article, the isolation of 33 fungi (Table 1) from the epicarp of fruits of Citrus
genus (i.e., C. limon, C. aurantifolia, C. aurantium, and C. paradisiaca) harvested from
three Achuar communities1) (i.e., Wasakentsa, Pumpuentsa, and Sewastian) of the
Amazonian forest (Ecuador, Province of Morona-Santiago), and their activities to
biotransform (RS)-linalool (1), (S)-citronellal (2), and sabinene (3) are reported.
Table 1. Fungi from Fruits of Citrus Genus of the Amazonian Forest (Ecuador)
Plant
Origin
Fruit
Fungi
Citrus aurantifolia
Pumpuentsa
1A
2A
1B
2B
1A5
2A2, 2A17
Wasakentsa
1B5, 1B7, 1B13, 1B14, 1B18, 1B20, 1B23, 1B24
2B4
Citrus limon
Wasakentsa
Wasakentsa
1C
1C3, 1C4, 1C5, 1C19, 1C21, 1C22
Citrus paradisiaca
1D
2D
1D2, 1D4, 1D6
2D1, 2D2, 2D4, 2D7, 2D10, 2D15, 2D17
Citrus aurantium
Sewastian
1F
1F1, 1F 2, 1F 5, 1F 6, 1F 9
2. Results and Discussion. – 2.1. Biotranformation Screening of Linalool (1). The
biotransformation screening with fungi was carried out on analytical scale adding the
substrate (1 g lꢀ1) to the cultures grown at 28–308 for 5 d. The reaction mixtures were
analyzed by GC-FID, and the products were characterized by GC/MS. The results
obtained with linalool (1) are compiled in Table 2 and Scheme 1.
Only eleven fungi were able to biotransform linalool (1). Some strains (i.e., 1B14,
1B24, 1C21, and 2D7) afforded 6-methylhept-5-en-2-one (4; 4–62%), obtained by a
loss of vinyl moiety, but only the strain 1B14 gave its reduction product 6-methylhept-5-
en-2-ol (5; 8%). The heptenone 4 has been reported to be obtained by biotransforma-
tion with B. cinerea [18][19], while the heptenol 5 has not been previously detected.
The other fungi produced a mixture of (E)-furanlinalool oxide (7; 10–49%), (Z)-
furanlinalool oxide (8; 8–46%), (E)-pyranlinalool oxide (9; 1–16%), and (Z)-
pyranlinalool oxide (10; 1–2%) in variable amounts. A similar distribution of products
have been obtained with Aspergillus strains [8]. In GC-FID spectra, however, traces
(<1%) of their precursor (i.e., 6,7-epoxylinalool) [7] were present. Together with the
1
)
The Achuar territory represents one of the last primary forests that connect the Andes with the
Amazon forests of Peru and Brazil.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
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Table 2. Screening of Biotransformation of (RS)-Linalool (1) with Fungi (analytical scale)
Fungi
Biotransformation products, yield [%]a)
1
4
5
6
7
8
9
10
1B14
1B24
1C5
1C21
1D2
1D4
1D15
2D7
1F1
30
71
17
92
2
2
55
–
62
29
8
29
36
16
2
8
49
46
46
45
1
3
2
4
45
2
4
47
10
29
45
8
25
1
1
1
1
1
2
80
41
78
1F 2
1F 9
2
22
a) Yields obtained from GC-FID.
mixture of rearranged compounds 7–10, some fungi (i.e., 2D7, 1F2, 1C5, and 1F9)
furnished the 1-methyl-1-(4-methylpentyl)oxiranemethanol (6; 2–45%), which was,
however, the only product obtained with the fungus 1D15 (45%).
2.1. Biotranformation Screening of (S)-Citronellal (2). Almost all the strains
showed a high aptitude to biotransform (S)-citronellal (2; Table 3 and Scheme 2).
Almost all strains produced the reduction product (S)-citronellol (12) in high yields
(> 50%) and, in some cases, in quantitative yields (i.e., 2A17, 1B20, and 2D15) as
previously observed with various microorganisms [12][13][20][21], germinating wheat
seed [22], and tissue cultures of Petrosellinum crispum [16]. Many strains gave also the
oxidation product (S)-citronellic acid (11), although in lower yields (4–62%). On the
other hand, the strain 1D6 produced the mixture of the diastereoisomers (1S,3S,4S)-p-
menthane-3,8-diol (13; 25%) and (1S,3R,4S)-p-menthane-3,8-diol (14; 62%) obtained
by ring closure and subsequent addition of H2O. The yields of compounds 13 and 14 are
very remarkable when compared with those obtained with Solanum aviculare [17].
Both compounds 13 and 14 were also obtained with other strains (i.e., 1B18, 1B24, 2B4,
1C3, 1C22, 1C4, 1D4, 2D1, and 2D4) in variable yields (5–25% and 5–60%, resp.).
Also (1S,3S,4R)-isopulegol (15; 1%) and (1S,3R,4R)-isopulegol (16; 2–13%) were
obtained as minor products by ring closure and proton exchange. The strains 2D4 and
1D4 gave the best yields of the isopulegol 16 (12 and 13%, resp.). A similar behaviour
was reported with Solanum aviculare [17].
2.3. Biotranformation Screening of Sabinene (3). Only nine strains gave biotrans-
formation products with sabinene (3; Table 4 and Scheme 3).
In all cases, 4-terpineol (19; 3–74% yield) was the main product, with poor yields of
(Z)-sabinene hydrate (17; 1–3%) and (E)-sabinene hydrate (18; 1–5%). The most
interesting strain was 1C22 that, together with high yield of 19 (74%) and low yield of
17 (3%) and 18 (2%), furnished traces of other metabolites such as (Z)-p-menth-2-en-
1-ol (21; 1%), (E)-p-menth-2-en-1-ol (22; 1%), and a-terpineol (20; 1%). All of the
products were obtained by an initial protonation of the C¼C bond to give a Thuile

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 1. Biotransformation Products of (RS)-Linalool (1)
carbocation A that is in equilibrium with the terpinen-4-yl cation B, the a-terpinyl
cation C, and finally with the phellandrenyl cation D. All carbocations, upon addition of
H2O, give the biotransformation products 17–22. This proposed mechanism for the
biotransformations was observed in biosynthetic pathways [23][24].
2.4. Identification of Fungi. The fungi 1B14, 1C5, 1C22, 1D2, and 2D15 have been
chosen as representatives of the phenotypes isolated from different origins and as
producers of different biotransformation products. They have been identified by CBS
as belonging to Fusarium and Penicillium genera (Table 5).

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1913
Table 3. Screening of Biotransformation of (S)-Citronellal (2) with Fungi (analytical scale)
Fungi
Biotransformation products, yield [%]a)
2
11
12
13
14
15
16
2A17
1B5
–
–
1
–
1
–
–
1
3
–
–
–
–
2
–
–
–
11
–
10
17
2
23
1
26
1
100
64
83
45
57
100
38
63
40
51
23
69
84
86
10
84
11
36
16
55
7
1B13
1B14
1B18
1B20
1B23
1B24
2B4
1C3
1C4
1C5
1C19
1C21
1C22
1D2
1D4
1D6
1D15
2D1
2D2
2D4
2D7
2D10
2D17
1F1
8
25
2
62
15
5
15
12
19
31
27
35
50
9
2
4
4
22
16
12
25
60
1
2
4
16
28
11
21
25
55
62
13
2
100
2
11
20
45
7
53
34
5
4
8
12
68
11
43
81
74
75
74
72
2
4
24
19
11
1F 2
1F 9
3
17
a) Yields obtained from GC-FID.
In particular the fungi 1C5 and 1D2, identified as Fusarium sp., are new species
although very close to F. concolor Reinking, showing an identical ribosomal ITS but
with different EF (elongation factor 1a) sequence.
2.5. Biotransformation of Terpenes 1–3 with Penicillium and Fusarium sp. The
biotransformations of (RS)-linalool (1), (S)-citronellal (2), and sabinene (3) were
repeated on preparative scale with the selected and identified fungi, and the products
were isolated by chromatography and characterized (GC/MS, and 1H- and 13C-NMR).
The biotransformation of linalool (1) with F. fujikuroi gave 6-methylhept-5-en-2-
one (4; 49%) and 6-methylhept-5-en-2-ol (5; 10%; Table 6), arising from the
degradation of C(1)ꢀC(2) chain (!4) and the subsequent reduction of CO function
(! 5) (Scheme 1). The other Fusarium species, however, afforded oxidation products.
The 1,2-epoxy derivative 6 was obtained in appreciable yield (42%, with F.
concentricum and 22% with Fusarium sp. (1C5)), while the 6,7-epoxy derivative has
been described as the intermediate through which the biotransformations of linalool

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Scheme 2. Biotransformation Products of (S)-Citronellal (2)
Table 4. Screening of Biotransformation of Sabinene (3) with Fungi (analytical scale)
Fungi
Biotransformation products, yield [%]a)
3
17
18
19
20
21
22
1B14
1B24
1C5
1C22
1D2
1D6
2D1
2D4
2D7
86
81
77
18
82
95
85
89
86
3
3
3
3
3
2
3
3
2
5
1
1
2
1
9
13
17
74
10
3
13
7
12
1
1
1
1
1
2
1
a) Yields obtained from GC-FID.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1915
Scheme 3. Biotransformation Products of Sabinene (3)
can afford the diasteroisomeric furan- and pyranlinalool oxides 7–10 [7–9]. In our
case, Fusarium sp. (1C5) gave (E)-furanlinalool oxide (7; 32%), lower amounts of (E)-
pyranlinalool oxide (9; 12%), and traces of (Z)-pyranlinalool oxide (10; 1%), while
Fusarium sp. (1D2) produced practically equimolar amounts of 7 (39%) and (Z)-
furanlinalool oxide (8; 37%). The different behavior of the 1C5 and 1D2 strains
confirmed their different identity. As observed on analytical scale, no biotransforma-

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
Table 5. CBSa) Characterization of Fungi
Fungus
Plant
Identification
1B14
1C5
1C22
1D2
Citrus latifoliab)
Citrus limonb)
Fusarium fujikuroi Nirenberg
Fusarium sp.c)
Citrus limonb)
Penicillium paxilli Balnier
Citrus paradisiacab)
Citrus paradisiacab)
Fusarium sp.d)
2D15
Fusarium concentricum Nirenberg & O’Donnell
a) Central Bureau voor Schimmelcultures, Utrecht, The Netherlands. b) From Wasakentsa. c) Close to
Fusarium concolor Reinking. d) Close to Fusarium concolor Reinking. This strain is identical to strain
1C5 with regard to the nucleotide sequence of the ribosomal ITS but somewhat different in the EF
(elongation factor 1a) sequence.
Table 6. Biotransformations of Terpenes 1–3 with Penicillium and Fusarium Strains
Fungi
Terpene Products (yields [%])
P. paxilli
1
F. fujikuroi
1
1
1
1
4 (49)
5 (10)
F. concentricum
Fusarium sp. (1C5)
Fusarium sp. (1D2)
6 (42)
6 (22)
7 (32)
7 (39)
9 (12) 10 (2)
16 (5)
8 (37)
P. paxilli
F. fujikuroi
F. concentricum
Fusarium sp. (1C5)
Fusarium sp. (1D2)
2
2
2
2
2
12(10)
13 (20) 14 (50) 15 (2)
11 (43) 12 (36)
11 (5) 12 (76)
11 (22) 12 (38)
11 (7)
12 (71)
P. paxilli
F. fujikuroi
F. concentricum
Fusarium sp. (1C5)
Fusarium sp. (1D2)
3
3
3
3
3
17 (3)
18 (13) 19 (56)
17 (14) 18 (11) 19 (23)
17 (21) 18 (17) 19 (30)
17 (15) 18 (12) 19 (38)
17 (16) 18 (13) 19 (35)
tion of linalool (1) was obtained with Penicillium paxilli that showed, on the contrary, a
good activity towards (S)-citronellal (2) affording the diasteroisomeric (1S,3S,4S)-p-
menthane-3,8-diol (13; 20%) and (1S,3R,4S)-p-menthane-3,8-diol (14; 50%), and
traces of (1S,3S,4R)-isopulegol (15; 2%), and (1S,3R,4R)-isopulegol (16; 5%; Table 6
and Scheme 2). These compounds derived from the same cyclic zwitterionic inter-
mediate originating from the nucleophilic attack of the C¼C bond on the aldehydic
function. The zwitterionic intermediate gave the diols 13 and 14 through H2O addition
and the isopulegols 15 and 16 through prototropic rearrangement [17]. Finally, all
Fusarium strains both reduced and oxidized the aldehydic function to give (S)-
citronellol 12 (36–76%) and (S)-citronellic acid 11 (5–43%), respectively.
On the other hand, while various studies regarding the biotransformation of linalool
[7–11] and citronellal [12–17] have been reported, biotransformations of sabinene
were not studied. In this field, Fusarium and Penicillium strains followed a common
pathway (Table 6 and Scheme 3).

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
1917
In particular both P. paxilli and Fusarium strains gave as main product the 4-
terpineol 19 (23–56%). Other products, obtained in appreciable yields, were the
diasteroisomeric (Z)-and (E)-sabinene hydrates, 17 (14–21%) and 18 (11–17%),
respectively.
3. Conclusions. – Various fungi from the Amazonian forest were tested to
biotransform linalool, citronellal, and sabinene. This screening has identified some
strains belonging to Fusarium and Penicillium genera able to biotransform these
terpenes to various products. Two Fusarium strains are new although very similar to F.
concolor Reinking. While various microrganisms have been employed in biotrasfor-
mation of linalool and citronellal, this is the first example of biotranformation of
sabinene to 4-terpineol and (E)- and (Z)-sabinene hydrate in appreciable yields.
Experimental Part
General. (RS)-Linalool (Fluka), (ꢀ)-(S)-citronellal (Aldrich), and sabinene (Extrasynthese) are
commercially available. Various culture media have been used: PDA (potato-dextrose agar; potato
extract, 4 g lꢀ1; glucose, 20 g lꢀ1; agar, 15 g lꢀ1), PDB (potato-dextrose broth; potato extract, 4 g lꢀ1
glucose, 20 g, lꢀ1), and Saboraud broth (trypton, 5 g lꢀ1; pepton, 5 g lꢀ1; glucose, 20 g lꢀ1).
;
Isolation of Fungi. The fungi were isolated from the epicarp of the fruits of Citrus genus harvested
from three Achuar communities (i.e., Wasakentsa, Pumpuentsa, and Sewastian) of the Amazonian forest
(Ecuador, Province of Morona-Santiago). The fruits were cut in two, and 4.0-cm2 segments were cut to
separate flavedo and albedo in order to sample inside and outside the epicarp. The samples were put on
four Petri dishes containing PDA and chloramphenicol (200 mg lꢀ1), incubated at 308 for 48 h. Thirty-
three strains of fungi were isolated and numbered (Table 1).
GC and GC/MS Analysis. GC Analyses were performed on a ThermoQuest GC-Trace gas
chromatograph equipped with a FID detector and a Varian FactorFour VF-5ms poly-5% phenyl-95%-
dimethyl-siloxane-bonded phase column (30 mꢁ0.25 mm; film thickness, 0.15 mm). Operating con-
ditions were as follows: injector temp., 3008; FID temp., 3008; He, carrier gas (flow rate, 1 ml/min and
split ratio, 1:5); oven temp., 55–908 (18/min), 90–2508 (208/min). The percentage composition of
chemical constituents was computed by the normalization method from the GC peak areas, without using
correction factors.
The GC analysis of the mixture of (RS)-linalool (1) displayed the following retention times (tR
[min]): 6-methylhept-5-en-2-one (4), 12.22; 6-methylhept-5-en-2-ol (5), 12.78; (E)-furanlinalool oxide
(7), 18.86; (Z)-furanlinalool oxide (8), 20.37; 1-methyl-1-(4-methylpentyl)oxiranemethanol (6), 20.48; 1,
21.86; (E)-pyranlinalool oxide (9), 28.9; (Z)-pyranlinalool oxide (10), 29.65. The analysis of the mixture
of (S)-citronellal (2) led to the following tR values [min]: (1S,3S,4R)-isopulegol (15), 26.49; (1S,3R,4R)-
isopulegol (16), 27.45; 2, 27.18; (S)-citronellol (12), 35.60; (S)-citronellic acid (11), 38.48; (1S,3S,4S)-p-
menthane-3,8-diol (13), 38.12; (1S,3R,4S)-p-menthane-3,8-diol (14), 38.74. The analysis of mixture of
sabinene (3) exhibited the following tR values [min]: 3, 11.26; (Z)-sabinene hydrate (17), 18.85; (E)-
sabinene hydrate (18), 21.77; (Z)-p-menth-2-en-1-ol (21), 24.03; (E)-p-menth-2-en-1-ol (22), 25.95; 4-
terpineol (19), 30.00; a-terpineol (20), 31.72.
GC/MS Analyses were performed on a Varian GC-3800 gas chromatograph equipped with a Varian
MS-4000 mass spectrometer using electron impact (EI), and hooked to NIST and Abreg libraries. The
´
compounds were identified by comparing their GC tR values, Kovats indices (KI values), and the MS
fragmentation pattern with those of pure standards, or by matching the MS fragmentation patterns and
KI values with those in the above mentioned mass-spectra libraries and those in the literature [25]. The
GC conditions were the same as reported for GC analysis, and the same column was used. The MS
conditions were as follows: ionization voltage, 70 eV; emission current, 10 mAmp; scan rate, 1 scan/s;
mass range, m/z 40–500; trap temp., 1508, transfer line temp., 3008. A mixture of aliphatic hydrocarbons

1918
CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
(C8 –C24) in hexane (SigmaꢀAldrich) was injected, under the above temp. program, to calculate the
retention indices [26].
Screening of Biotransformation of Terpenes 1–3 with Fungi on Anal. Scale. General Procedure.
Sterilized Saboraud medium (20 ml) was inoculated with a mycelium portion of the isolated fungi on
PDA medium. The mixture was incubated for 5 d at 28–308 and 80 rpm. The selected substrate (200 ml;
the soln. was prepared dissolving 0.1 g of substrate in 1 ml of DMSO) was added to the resulting
suspension of grown cells, and the incubation was continued for 5 d. Aliquots (1 ml) were withdrawn and
centrifuged to remove the cells (5000 rpm; 10 min). The supernatant was extracted with Et2O (1 ml),
dried (Na2SO4), and analyzed by GC and GC/MS. The yields of biotransformation of 1 (Table 2), 2
(Table 3), and 3 (Table 4) were calculated by GC-FID, and the biotransformations products were
characterized by GC/MS.
Identification of Fungi. The identification of fungi was performed by CBS (Centraal Bureau voor
Schimmelcultures) Fungal Biodiversity Centre, Utrecht, The Netherlands. Five strains were selected on
basis of the biotransformations of 1, 2, and 3: 1B14, 1C5, 1C22, 1D6, and 2D15. The results are compiled
in Table 5.
Biotranformations of Terpenes 1–3 with Fungi. General Procedure. The biotransformations were
carried out as previously described. Sterilized Saboraud medium (200 ml) was inoculated with a
mycelium portion obtained from the culture of the fungi identified by CBS. The mixture was incubated
for 5 d at 288 and 80 rpm. To the resulting suspension of grown cells, a soln. of selected terpene (0.2 g) in
DMSO (2 ml) was added. After 5 d incubation, the mixture was centrifuged (5000 rpm; 10 min), the
supernatant was extracted with Et2O (3ꢁ5 ml), dried (Na2SO4), and the solvent was removed under
vacuum (bath at r.t.) to obtain the crude mixture (0.17–0.19 g (85–95%) for 1 and 2, 0.11–0.12 g (55–
60%) for 3).
For 1, the crude mixture was subjected to prep. TLC (hexane/Et2O 1:1), and the products were
characterized by GC/MS analysis and NMR spectra, compared with those of authentic samples. In
particular the furanlinalool oxides 6 and 7, and the pyranlinalool oxides 9 and 10 were prepared according
to the method described by Winterhalter [27]. The structures of 6-methylhept-5-en-2-one (4) and its
alcohol 5 in crude extracts were determined by comparison with commercial standards (SigmaꢀAldrich).
The other compounds were identified in GC/MS with the methods reported in Exper. Part and references
therein.
For 2, the mixture was separated by prep. TLC (petroleum ether (PE)/AcOEt 5 :2) to obtain
(1S,3R,4S)-p-menthane-3,8-diol (14) and (1S,3S,4S)-p-menthane-3,8-diol (13), characterized by NMR
spectra and comparison of their GC/MS data with those of the authentic samples prepared according to
the method reported in [17][28]. The minor products were identified only by GC/MS. The structure of
(S)-citronellic acid (11) and (S)-citronellol (12) was confirmed by GC/MS of the reference standards,
prepared by oxidation and reduction of (S)-citronellal, resp.
For 3, 4-terpineol (19) and a-terpineol (20), their GC/MS data were compared with those of the
authentic samples (SigmaꢀAldrich). The other compounds were identified in GC/MS with the method
reported in the Exper. Part and references therein.
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Received April 7, 2013