Enzyme-mediated syntheses of the enantiomers of γ-irones

Article abstract of DOI:10.1002/1522-2675(20011219)84:12<3650::AID-HLCA3650>3.0.CO;2-5

An enzymatic approach to the synthesis of all the possible stereoisomers of (E) and (Z), cis and trans-γ-irones in enantiomerically pure form from commercial Irone Alpha is described. A very efficient resolution of racemic trans-γ-irone, affording both the enantiomers in high ee and chemical purity, is also presented. Olfactory evaluation of (+)- and (-)-3b and full configuration assignment of the irone isomers contained in samples of Italian iris oil are reported.

Full text of DOI:10.1002/1522-2675(20011219)84:12<3650::AID-HLCA3650>3.0.CO;2-5

3650
Helvetica Chimica Acta Vol. 84 (2001)
Enzyme-Mediated Syntheses of the Enantiomers of g-Irones
by Elisabetta Brenna*, Claudio Fuganti, Sabrina Ronzani, and Stefano Serra*
Dipartimento di Chimica del Politecnico, Centro CNR per lo Studio delle Sostanze Organiche Naturali,
Via Mancinelli 7, I-20131 Milano
An enzymatic approach to the synthesis of all the possible stereoisomers of (E) and (Z), cis and trans-g-
¾
irones in enantiomerically pure form from commercial Irone Alpha is described. A very efficient resolution of
racemic trans-g-irone, affording both the enantiomers in high ee and chemical purity, is also presented.
Olfactory evaluation of (‡)-and ( À)-3b and full configuration assignment of the irone isomers contained in
samples of Italian iris oil are reported.
1. Introduction. The essential oil known as −orris root oil× or −essence d−iris× is a
rather precious ingredient of scents, perfumes, and other cosmetics [1]. Its odoriferous
principle was first isolated by Tiemann in 1893 and named irone [2]. Forty years later
Ruzicka and co-workers [3a] determined the correct elemental analysis of irone
(C14H22O); then Ruzicka and Naves and co-workers [3b,c] independently found that at
least three isomers of irone were present in natural iris oil, their structures being
determined as 1, 2, and 3. In 1971, Rautenstrauch and Ohloff [4] completed the structure
determination by establishing the configuration of the irone isomers they had isolated
from the Italian iris oil (probably from Iris pallida) first used by Ruzicka and co-
workers. They found that the oil contained the following four isomers: (‡)-cis-a-irone
((‡)-1a), (‡)-trans-a-irone ((‡)-1b), (‡)-b-irone ((‡)-2), and (‡)-cis-g-irone ((‡)-
3a). Later, in the same oil, Ohloff and co-workers [5] were able to detect traces of the
trans-g-irone, together with some other isomers, probably showing (Z) configuration at
the CˆC bond in the side chain. The absolute configuration of the trans-g-irone found
in Italian iris oil is apparently still unknown.
In some previous reports [6], we described the enzyme-mediated syntheses of (‡)-
and (À)-cis-a- , ‡( )-and ( À)-trans-a- , ‡( )-and ( À)-cis-g, and (‡)-and ( À)-b irone
¾
isomers, starting from Irone Alpha , a 55 :45 mixture of racemic trans-a-and cis-a-
irone, with a 5% content of b-irone. We wish now to report on the enzymatic approach
we have optimized to obtain the last isomers of the series, (‡)-and ( À)-trans-g-irone
¾
((‡)-and ( À)-3b), using once again commercial Irone Alpha as a starting material.
The key step is the photochemical isomerization of the C(4)ˆC(5) bond of
enantiomerically pure trans-a-irol acetates (‡)-and ( À)-4, obtained by enzymic
resolution, to afford trans-g-irol acetates (‡)-and ( À)-5. We describe also an
alternative biocatalysed synthetic path, which allowed us to prepare both the
enantiomers from racemic trans-g-irone in high chemical purity, in order to clarify
the diverse values for the optical rotation reported in the literature for (‡)-and ( À)-3b.
For this purpose, we optimized a large-scale synthesis of racemic trans-g-irone. The
external olfactory evaluation and the threshold values of the two enantiomers of trans-

Helvetica Chimica Acta Vol. 84 (2001)
3651
g-irone are reported. Full configuration assignment of the irone isomers found in four
samples of Italian iris oil is also given.
2. Results and Discussion. 2.1. Syntheses. Four different enantioselective syntheses
[7a d] of trans-g-irone, from components of the so-called −pool of chirality× as starting
materials, were reported in the literature; none of them was applied to afford both the
enantiomers1). A rough comparison of these publications put into evidence a certain
disagreement in the determination of the optical rotatory power. We now devised two
new synthetic approaches for the preparation of both enantiomerically pure (‡)-and
(À)-3b: in both cases optical activation was obtained by means of lipase-PS-mediated
kinetic resolution of suitable intermediates.
Approach A. We had already reported [6a] on the possibility to prepare (‡)-trans-
and (À)-trans- and (‡)-cis- and (À)-cis-a-irone stereoisomers from commercial Irone
Alpha¾, using as key intermediates the enantiomerically pure epoxy derivatives (À)-6
and (‡)-7, (‡)-8 and (‡)-9, obtained by lipase-PS-mediated acetylation of (Æ)-6 and
(Æ)-8, respectively, in tert-butyl methyl ether (tBuOMe) solution in the presence of
vinyl acetate as an acyl donor. In this work we now report alcohol derivatives (‡)-and
(À)-6 and (‡)-and ( À)-8 were deoxygenated by reaction with Zn and NaI in AcOH at
room temperature, to give (‡)-and ( À)-4a and (À)-and ( ‡)-10a, respectively
(Schemes 1 and 2, resp.). Acetylation of these derivatives in pyridine and Ac2O
afforded a-irol acetates (‡)-and ( À)-4b and (À)-and ( ‡)-10b2), in the trans and cis
1
)
)
For a recent large-scale synthesis of both enantiomers of cis-g-irone by chemical resolution, see [7m].
In [6c], we reported [a]2D0 ˆ À73 (c ˆ 1.50, CH2Cl2) for a sample of (2R,6S,9R)-a-irol acetate with an
impurity of 13% 4-chloro-4,5-dihydro-cis-a-irol acetate.
2

3652
Helvetica Chimica Acta Vol. 84 (2001)
series, respectively. During the preparation of (‡)-and ( À)-cis-g-irone [6c], we found
that 4.5 :2.5 :2 mixtures of cis-g-, cis-a-, and b-irol acetates can readily be converted
i
into 78 82% of cis-g-irol isomer by photoisomerization in PrOH solution in the
presence of xylene as a photosensitizer. Thus, we exploited this shift of the endocyclic
double bond to convert the enantiomerically pure trans-a-irol acetates (‡)-and ( À)-4b
into the trans-g isomers (‡)-and ( À)-5. Simultaneously, in the same experimental
apparatus (see Exper. Part), cis-a derivatives (‡)-and ( À)-10b were irradiated as well,
in order to make a rough comparison between the photochemical isomerizations of the
two sets of diastereoisomers (cis-and trans-a-irol acetates). In both cases, irradiation
OH
OAc
OH
OAc
O
O
O
O
(-)-6
(+)-7
(+)-8
(+)-9
Scheme 1
OH
OH
O
O
(+)-6
i), ii)
(-)-6
i), ii)
OR
OR
(-)-4a R = H
(-)-4b R = Ac
(+)-4a R = H
(+)-4b R = Ac
iii)
iii)
OAc
+
OAc
+
OAc
OAc
(-)-5
11a
(+)-5
11b
iv), v)
iv), v)
O
+
O
+
O
O
(+)-14
(-)-3b
(+)-3b
(-)-14
i
i) Zn, NaI, AcOH. ii) Ac2O, pyridine. iii) hn, PrOH, xylene. iv) KOH, MeOH. v) Manganese(IV) oxide,
CH2Cl2.

Helvetica Chimica Acta Vol. 84 (2001)
3653
Scheme 2
OH
OH
O
O
(-)-8
(+)-8
i), ii)
i), ii)
OR
OR
(-)-10a R = H
(-)-10b R = Ac
(+)-10a R = H
(+)-10b R = Ac
iii)
iii)
OAc
OAc
+
+
OAc
OAc
13b
iv), v), vi)
12a
13a
iv), v), vi)
12b
O
O
O
O
(-)-3a
(+)-15
(+)-3a
(-)-15
i
i) Zn, NaI, AcOH. ii) Ac2O, pyridine. iii) hn, PrOH, xylene. iv) KOH, MeOH. v) Manganese(IV) oxide,
CH2Cl2. vi) Column chromatography.
was prolonged until the ratio between a-and g-isomers, evaluated by GC/MS, did not
change further.
i
Isomerization of trans-a-acetates (À)-and ( ‡)-4b (0.04m solution in PrOH with
10% of xylene, Scheme 1) took ca. twelve days: a photostationary equilibrium was
reached, characterized by 2 7% of still unreacted a-isomer. The trans-g-isomers
obtained were found to be 1:1 mixtures of derivatives (À)-and ( ‡)-5 and of the
corresponding (7Z) diastereoisomers 11a and 11b3) only when their 1H-NMR spectra
were recorded. As a matter of fact, no distinction was possible by GC/MS.
As for the cis-a derivatives (À)-and ( ‡)-10b (0.04m solution in iPrOH with 10% of
xylene, Scheme 2), after 5 h, ca. 40% of cis-g-isomer 12a or 12b respectively3) was
detected by GC, and no traces of (7Z) isomers, neither in the a nor in the g series, were
found. Within 27 h from the beginning, the cis-a-irol acetates had completely
3
)
We use the labels −a× and −b× to distinguish the two enantiomers of derivatives 11 13. They could not be
obtained as single pure compounds, so it was not possible to determine the right sign of the optical rotation
for each of them.

3654
Helvetica Chimica Acta Vol. 84 (2001)
disappeared, and ca. 30% of (7Z) isomers 13a and 13b3) had formed. The appearance
of (7Z)-isomers (5%) was first detected after an irradiation time of 7 h.
The rate constants for the photoisomerization processes were estimated from the
disappearance of the corresponding a-irol acetate, as measured by GC/MS in the
presence of ca. 5% of dodecane as an internal standard: ktrans ˆ 2 ¥ 10À6 sÀ1 and kcis ˆ 6 ¥
10À5 sÀ1. Owing to inherent design limitations of the reactor, a constant temperature
could not be maintained. A difference of ca. one order of magnitude was observed
between the rate constants of cis- and trans-a-irol acetate isomerization. This could be
tentatively attributed to the so-called −steric factor×. This process is probably very
sensitive to the molecular geometry of the substrate itself, since triplet-triplet transfer
generally needs a collision between sensitizer and substrate.
Photosensitized irradiation of cis- and trans-a-irol acetates is similar to that
described for dihydro-a-ionol acetate [8], which readily undergoes isomerization to the
g-isomer. The presence of the C(7)ˆC(8) bond does not inhibit a ! g isomerization;
this CˆC bond is only involved in an (E) ! (Z) process, which seems to happen after
the C(4)ˆC(5) bond shift. This photochemical behavior somewhat resembles that of
cycloalkenes. Several studies have established that transfer of triplet energy to acyclic
or macrocyclic olefins from suitable photosensitizers results in cis ! trans isomerization
of the CˆC bond [9]. By contrast, small-ring cyclic olefins, characterized by highly
strained trans isomers, are known to undergo a different fate. For example, irradiation
of 1-alkylcycloalkenes in the presence of aromatic-hydrocarbon photosensitizers
induces isomerization to the analogous exocyclic olefins [9]. Our substrates show both
a hindered endocyclic CˆC bond responsible for g-isomerization, and a CˆC bond in
the side chain suitable for (E)/(Z) isomerization.
A completely different behavior is reported in the literature for ionone derivatives
subjected to direct irradiation (Scheme 3). a-Ionone is known to afford (E/Z)-retro-a-
Scheme 3. Products of Photochemical Irradiation of a-Ionone, b-Ionone, and b-Ionol

Helvetica Chimica Acta Vol. 84 (2001)
3655
ionone via g-abstraction at C(6) [10], while b-ionone is described to give a mixture of
retro-g-ionone and an a-pyran isomer upon direct irradiation, and only this latter upon
triplet-sensitized reaction [11]. Direct irradiation of b-ionol affords retro-g-ionol [12].
The photoisomerization mixtures were hydrolysed in methanolic KOH solution,
and the corresponding irol derivatives were oxidized in CH2Cl2 in the presence of
manganese(IV) oxide. Both in the trans and in the cis series, (7E)-irone could be
separated from the (7Z) diastereoisomer by column chromatography on SiO2. The
following products were thus obtained:
i) From (À)-4b: (‡)-(7E)-trans-g-irone ((‡)-3b): [a]2D0 ˆ ‡10 (c ˆ 2.8, CH2Cl2),
chemical purity 74% (GC/MS), with 8% of (À)-trans-a-irone, ee 99% (HPLC); (À)-
(7Z)-trans-g-irone ((À)-14): [a]2D0 ˆ À43.2 (c ˆ 1.25, CH2Cl2) [7c]: [a]D20 ˆ À42 (c ˆ 0.4,
CH2Cl2; ee 76%), chemical purity 70 % (GC/MS).
ii) From (‡)-4b: (À)-(7E)-trans-g-irone ((À)-3b): [a]2D0 ˆ À16 (cˆ 3.0 CH2Cl2), chem-
ical purity 79% (GC/MS), with 3% of trans-a-irone, ee 99% (HPLC); (‡)-(7Z)-trans-
g-irone ((‡)-14): [a]2D0 ˆ ‡48.7 (c ˆ 1.32, CH2Cl2), chemical purity 76% (GC/MS).
iii) From (À)-10b: (À)-(7E)-cis-g-irone ((À)-3a): [a]2D0 ˆ À2.54 (c ˆ 4.9, CH2Cl2),
chemical purity 96 % (GC/MS), ee 99% (GC); (‡)-(7Z)-cis-g-irone ((‡)-15): [a]D20
‡50.5 (c ˆ 1.08, CH2Cl2), chemical purity 90% (GC/MS).
ˆ
iv) From (‡)-10b: (‡)-(7E)-cis-g-irone ((‡)-3a): [a]2D0 ˆ ‡1.78 (c ˆ 4.3, CH2Cl2),
chemical purity 93% (GC/MS), ee 99% (GC); (À)-(7Z)-cis-g-irone ((À)-15): [a]D20
À48.3 (c ˆ 1.2, CH2Cl2), chemical purity 91% (GC/MS).
ˆ
This approach allowed us to obtain all eight stereoisomers of (E)-and ( Z)-cis- and
¾
(E)-and ( Z)-trans-g-irones from commercial Irone Alpha . However, in the samples
of (‡)-and ( À)-3b, the presence of a-isomers, showing high values of optical rotation of
the opposite sign (see [6a]: (‡)-1b, [a]2D0 ˆ ‡427 (c ˆ 0.95, CH2Cl2); (À)-1b, [a]2D0
ˆ
À400 (c ˆ 1.05, CH2Cl2)) could not be avoided and strongly affected the values of the
measured optical rotation. An alternative synthetic path affording enantiomerically
pure (‡)-and ( À)-3b with high chemical purity was thus devised.
Approach B. This approach was based on the resolution of (Æ)-trans-g-irone by
enzymic methods. We thus optimized a large-scale synthesis of this racemic material4),
which is depicted in Scheme 4. Commercial 2,3-dimethylbut-2-ene was brominated with
N-bromosuccinimide (NBS) in CCl4 solution, in the presence of catalytic amounts of
dibenzoyl peroxide, to give bromo derivative 16 [13]. This latter was employed as an
alkylating agent of ethyl acetoacetate (ˆethyl 3-oxobutanoate) to afford 175),
according to a general procedure for the alkylation of dianions of b-keto esters
reported in [14b]. Cyclization of substrate 17 with SnCl4 in CH2Cl2 (see, e.g., [15])
allowed us to obtain the known cyclic keto ester 18 [16] as a 2 :1 mixture of trans/cis
diastereoisomers, which had been previously used in a synthesis of racemic trans-g-
irone [7k]. This derivative was subjected6) to reaction with PPh3ˆCH2, followed by
reduction of the ester function with LiAlH4, to afford trans-6-methyl-g-cyclogeraniol
(19)7) as a single diastereoisomer (GC/MS, 1H-NMR). Oxidation to the corresponding
4
)
)
)
)
For previous syntheses of racemic trans-g-irone, see [7e k].
For an analogous terminal olefinic acetoacetate derivative, see [14a].
For a similar synthetic procedure, see [17b].
5
6
7
For optically active 19, see [7b].

3656
Helvetica Chimica Acta Vol. 84 (2001)
Scheme 4
COOEt
i)
ii)
iv), v)
iii)
64%
81%
84%
34%
COOEt
O
16
17 O
18
Br
trans/cis 2:1
O
CH2OH
vi), vii)
58%
19
(±)-3b
i) NBS, CCl4, benzoyl peroxide. ii) Ethyl acetoacetate, 1 equiv of NaH, THF; then 1 equiv of BuLi, 08. iii) SnCl4,
CH2Cl2. iv) PPh3 ˆ CH2, THF, reflux. v) LiAlH4, THF, 08. vi) ClCOCOCl, DMSO; Et3N, CH2Cl2. vii) PPh3 ˆ
CHCOMe3, toluene, reflux.
aldehyde and condensation with PPh3ˆCHCOMe3, by analogy with [7b,i], completed
the synthetic path to racemic trans-g-irone (Æ)-3b (only 2% of cis-g isomer, GC/MS).
As for the resolution of the so-obtained (Æ)-trans-g-irone (Æ)-3b (Scheme 5), we
took advantage of a scheme we had already exploited for a-and g-ionone [17].
Reduction with NaBH4 afforded a 1:1 mixture of g-irols (Æ)-20 and (Æ)-21, which were
converted to the corresponding crystalline 4-nitrobenzoates (Æ)-22 and (Æ)-23
(Scheme 5). These latter derivatives could be separated by fractional crystallization
from hexane. The less soluble diastereoisomer (Æ)-23, obtained as a precipitate from
the first crystallization, was crystallized twice and reached a de of 92% (GC/MS). The
mother liquors from the first crystallization, surprisingly, afforded diastereoisomer (Æ)-
22 with a de of 98% (GC/MS) after four recrystallizations. This latter was the major
product, and was then hydrolysed to give (Æ)-20. The relative configuration depicted in
structural formula (Æ)-20 was deduced from the outcome of the synthetic sequence, as
we will explain below.
After four days, lipase-PS-mediated acetylation of racemic g-irol (Æ)-20 in tBuOMe
in the presence of vinyl acetate gave g-irol acetate (‡)-5, and left unreacted alcohol
(‡)-20. Hydrolysis of (‡)-5, followed by manganese(IV) oxide oxidation afforded (À)-
trans-g-irone (À)-3b with an [a]D20 ˆ À81 (c ˆ 1, CH2Cl2), a chemical purity of 99%
(GC/MS), and an ee > 99% (chiral HPLC). Oxidation of the unreacted alcohol (‡)-20
allowed us to obtain (‡)-trans-g-irone (‡)-3b with an [a]2D0 ˆ ‡76.4 (c ˆ 1.2, CH2Cl2), a
chemical purity of 99% (GC/MS), and an ee > 99% (chiral HPLC). The reference
values of [a]2D0 reported in the literature for (‡)-and ( À)-3b are rather low and not fully
comparable: a) (À)-3b, ee 70% (NMR with chiral shift reagents), [a]2D0 ˆ À43.3 (c ˆ
0.8, CH2Cl2) [7a]; b) (À)-3b, ee 76% (ee of the starting material), [a]2D0 ˆ À53.4 (c ˆ
2.34, CHCl3) [7b]; c) (‡)-3b, ee 80% (ee of the starting material), [a]2D0 ˆ ‡57 (c ˆ 0.33,
CH2Cl2) [7c]; d) (‡)-3b, ee 99% (HPLC), [a]2D0 ˆ ‡59.4 (c ˆ 1.2, CHCl3) [7d].
We have previously observed and experimentally verified that lipase PS usually
reacts with (R) alcohols [6][17][18]: we thus assumed that acetate (‡)-5 had (R)
configuration at C(9). As (‡)-5 is the precursor of (À)-3b, (S) configuration was
assigned to both C(2) and C(6). The configurations of the compounds reported in
Scheme 4 are thus justified.

Helvetica Chimica Acta Vol. 84 (2001)
3657
Scheme 5
OH
OH
+
(±)-21
(±)-23
(±)-20
(±)-22
i)
OAr
OAr
+
ii)
precipitate
mother liquors
OAr
OAr
(±)-23
(±)-22
After 3 crystallizations
from hexane:
de 98%, yield 31%
After 2 crystallizations
from hexane:
de 92%, yield 10%
iii)
OH
(±)-20
iv)
OAc
OH
(+)-20
v)
(+)-5
iii), v)
O
O
(+)-3b
(-)-3b
i) 4-Nitrobenzoyl chloride, pyridine. ii) Fractional crystallization from hexane. iii) KOH, MeOH. iv) Lipase PS,
vinyl acetate, tBuOMe, column chromatography. v) MnO2 in CH2Cl2.
2.2. Olfactory Evaluation. Thanks to the synthetic Approach B, we had in our hands
enantiomerically pure samples of (‡)-and ( À)-trans-g-irone of high chemical purity
suitable for odor evaluation and threshold determination. This work was performed at
Givaudan D¸bendorf AG, Fragrance Research (D¸bendorf, Switzerland). Thus, (‡)-
trans-g-irone ((‡)-3b) is very weak, of a woody odor tonality with a threshold value of

3658
Helvetica Chimica Acta Vol. 84 (2001)
113.5 ng/l; (À)-trans-g-irone ((À)-3b) is also not very powerful, but it possesses a soft
orris-butter-type odor, with a threshold value of 26.35 ng/l.
2.3. Determination of Irone-Isomer and -Enantiomer Distributions in Italian Iris-Oil
Samples. Having accomplished the syntheses described in Sect. 2.1, we had in our hands
all ten possible irone isomers in enantiomerically pure form, and had optimized chiral
GC and HPLC methods to distinguish them. We then had the chance to determine the
configuration of the irone isomers generated from fresh Italian iris rhizomes by an
accelerated procedure, based on an enzymatic process [19].
It is well-known that freshly harvested rhizomes do not contain irones, but their
triterpenoid precursors called −iridals× [20]. According to the traditional procedure,
decorticated rhizomes are kept in a dry and aerated environment for 2 3 years, then
powdered, incubated with diluted sulfuric acid, and finally steam-distilled to provide
the precious −orris butter×. The mechanism of the oxidative degradation affording
irones from iridals is still unknown.
Ten years ago, Gil et al. patented an enzymatic procedure for the development of
irones [19]. The ethanolic extract of fresh rhizomes is treated with a lipoxydase under
O2, in the presence of linoleic acid as a hydroperoxide donor. Steam distillation of the
reaction mixture affords, after extraction with CH2Cl2 and evaporation, a residue
containing various irone isomers. The isomer and enantiomer composition of the iris oil
prepared according to this accelerated method has never been fully characterized.
We had a generous gift of four batches (a d) of fresh iris rhizomes, sold by Aboca
(Sansepolcro, Toscana, Italy) as Iris pallida. Rhizomes were cut into slices and
extracted according to the procedure described in the Exper. Part. The residue was
suspended in H2O in the presence of soy bean flour as a source of lipoxidase and of
linoleic acid. The reaction mixture was vigorously stirred at room temperature under
O2 for 24 h. Steam distillation allowed the recovery of an odorous oil, which was
chromatographed (silica gel) to isolate the fraction containing irone isomers. The four
irone samples thus obtained were submitted to GC and HPLC analysis (see Table).
Table. Isomer and Enantiomer Distribution of Irone-Oil Samples
a
trans-a-Irone
trans-g-Irone
cis-a-Irone
cis-g-Irone
b-Irone Others )
b
c
b
b
% GC main % ee ) % GC main % ee ) % GC main % ee ) % GC main % ee ) % GC % GC
enan-
enan-
enan-
enan-
tiomer
tiomer
tiomer
tiomer
Sample a: 3.0
[a]2D0 ˆ ‡6
(‡)
(‡)
(‡)
(‡)
99
99
99
99
0.61
0.52
0.56
0.49
(À)
(À)
(À)
(À)
99
99
99
99
39.6
29.9
29.1
28.0
(À)
(‡)
(‡)
(‡)
26
26
65
99
39.8
48.5
55.6
55.3
(‡)
(‡)
(‡)
(‡)
99
99
99
99
0.38
0.28
16.6
17.7
8.86
12.2
d
(c ˆ 0.55 ))
Sample b: 3.4
[a]2D0 ˆ ‡25
d
(c ˆ 0.88 ))
Sample c: 5.6
[a]2D0 ˆ ‡11
d
(c ˆ 1.2 ))
Sample d: 4.0
[a]2D0 ˆ ‡26
d
(c ˆ 0.54 ))
a) Unidentified irone components. b) Chiral GC. c) Chiral HPLC. d) In CH2Cl2 solution.

Helvetica Chimica Acta Vol. 84 (2001)
3659
GC Analysis (HP5 column) allowed us to recognize irone isomers by comparison
with the corresponding synthesized samples: traces of trans-g-irone could be revealed.
The abundances of the main irone isomers 1 3 in the four samples were determined.
The corresponding percentages calculated from GC-peak integrals are reported in the
Table.
The two enantiomers of trans-a-, cis-a, and cis-g-irone could be distinguished by
chiral GC analysis, while HPLC was employed to separate the two antipodes of trans-g-
irone. No chromatographic method was found for the separation of the b-irone
enantiomers.
The average composition of the irone oil produced by the employed enzymatic
method was the following: 4% of (‡)-trans-a-irone (ee 99%), 0.6% of (À)-trans-g-
irone (ee 99%), 31.6% of prevalently dextrorotatory cis-a-irone, 49.8% of (‡)-cis-g-
irone (ee 99%), traces of b-irone, and 13.8% of other unidentified components. Among
the latter, traces of (7Z)-cis-g-irone were identified by GC. This composition is quite
similar to the one reported for Iris pallida butter obtained via the classical method [21].
In these extracts, we found only the laevorotatory trans-g-irone, that is to say, the
enantiomer showing at the stereogenic atoms C(2) and C(6) the same configuration
(2S,6S) as naturally occurring (‡)-trans-a-irone. We could suppose that the 0.6% of g-
isomer might derive from partial isomerization of the corresponding a-isomer.
3. Conclusions. In this report, we have described the synthesis of enantiomerically
¾
pure (E/Z)-trans-g-irone isomers starting from Irone Alpha . We have thus completed
a series of studies devoted to the preparation of all ten stereoisomers of (E)-irones
from the commercial mixture of racemic trans- and cis-a-irone. The synthetic
procedure took advantage of the lipase-mediated kinetics resolution of some epoxy
intermediates, and of the photoisomerization of the endocyclic a-to exocyclic g-double
bond. The behavior of trans-a-irol acetates under triplet-excited irradiation, which had
never been investigated, was studied and compared from kinetics point of view to that
shown by cis-a-irol derivatives. A difference of one order of magnitude was found
between the two rate constants. The samples of trans-g-irone obtained by this method
could not be purified from the starting a-isomers. A more efficient procedure for the
preparation of trans-g-irones of higher purity was thus devised. This latter consisted of
separating of trans-g-irol diastereoisomers (Æ)-20 and (Æ)-21 via fractional crystal-
lization of the corresponding 4-nitrobenzoate esters. Diastereoisomerically pure (Æ)-20
was then recovered and submitted to lipase-PS-mediated kinetics resolution. At the
end of this sequence, chemically pure (‡)-and ( À)-3b showing an ee higher than 99%
were obtained. These samples allowed us to describe precise optical-rotation values of
both the enantiomers and to give the complete odor description.
The experience we had collected during the last three years in the analytical
identification of irone isomers induced us to try the complete characterization of irone-
oil samples prepared according to the enzymatic procedure of Gil et al. This work
allowed us to identify the so-called −missing g-irone× [5] and to assess its configuration.
In the extracts produced by the action of lipoxydase on irone precursors, we could
detect (À)-trans-g-irone, the one described to possess a soft orris-butter-type odor,
according to Swiss researchers (see Sect. 2.2).

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Helvetica Chimica Acta Vol. 84 (2001)
The authors are indebted to Dr. Philip Kraft, Mrs. Caroline Denis, Mr. Heinz Koch, and Mr. A. F¸ckiger
(Givaudan D¸bendorf, Fragrance Research, Switzerland) for the odour descriptions and threshold evaluation of
trans-g-irone samples. COFIN-Murst is acknowledged for financial support.
Experimental Part
General. Epoxy derivatives (À)-6, (‡)-7, (‡)-8, and (‡)-9 are described in [6a]; 1-bromo-2,3-dimethyl-but-
¾
2-ene (16) was prepared according to [13]. Irone Alpha was purchased from IES (Allauch, France). Lipase PS
Pseudomonas cepacia (Amano Pharmaceuticals Co., Japan) was employed in this work. TLC: Merck silica gel 60
F254 plates. Column chromatography (CC): silica gel. Chiral GC analysis: tR in min. Optical rotations: Dr.
.
Kernchen-Propol digital automatic polarimeter. IR: in cmÀ1 1H-NMR Spectra: CDCl3 solns. at r.t. unless
otherwise stated; Bruker-AC-250 spectrometer (250 MHz 1H); chemical shifts d in ppm rel. to internal SiMe4, J
values in Hz. GC/MS: in m/z (rel. %); tR in min. Microanalyses were determined on a Carlo Erba Analyzer 1106.
1. Deoxygenation of Epoxy Derivatives 6 and 8. 1.1. General Procedure 1(GP 1) . NaI (3.20 g, 0.21 mol) was
added to a stirred mixture of the suitable epoxy-a-irol (2.24 g, 0.010 mol), Zn powder (0.788 g, 0.012 mol), and
NaOAc (2.34 g, 0.029 mol) in AcOH (50 ml) at r.t. The mixture was stirred at r.t. for 2 24 h, diluted with
hexane/AcOEt 1:1, and filtered. The filtrate was treated with H2O, and sat. NaHCO3 soln., the org. phase dried
and evaporated, and the residue submitted to CC (hexane/AcOEt 95 :5).
1.2 (2R,6R,9S)-a-Irol (ˆ(2S,3E)-4-[(1R,5R)-2,5,6,6-Tetramethylcyclohex-2-en-1-yl]but-3-en-2-ol; (À)-4a).
According to GP 1, (À)-6 (5 g, 0.022 mol) gave, after 24 h, (À)-4a (2.83 g, 61%). [a]2D0 ˆ À94 (c ˆ 0.51, CH2Cl2);
ee > 99% by chiral GC of the corresponding acetate (tR ˆ 20.74). Chemical purity 93% by GC/MS (tR 16.06),
with 7% of cis-a-irol (tR 16.77). 1H-NMR: 5.55 (m, HÀC(8), HÀC(7)); 5.36 (m, HÀC(4)); 4.29 (quint., J ˆ 6,
CHOH); 2.10 1.50 (m, 4 H); 1.55 (m, MeÀC(5)); 1.26 (d, J ˆ 6.5, MeCHOH); 0.81 (d, J ˆ 6.5, MeÀC(2)); 0.80
(s, 1 MeÀC(1)); 0.77 (s, 1 MeÀC(1)). 13C-NMR: 136.9; 135.9; 131.2; 121.4; 68.9; 56.1; 35.2; 34.6; 29.7; 26.50;
22.7; 22.4; 15.1; 14.0. GC/MS: 95 (100), 138 (46), 190 (1), 208 (0.6). Anal. calc. for C14H24O: C 80.71, H 11.61;
found: C 80.65, H 11.57.
1.3. (2S,6S,9R)-a-Irol (ˆ(2R,3E)-4-[(1S,5S)-2,5,6,6-Tetramethylcyclohex-2-en-1-yl]but-3-en-2-ol; (‡)-4a).
According to GP 1, (‡)-6 (5 g, 0.022 mol) gave, after 24 h, (‡)-4a (2.97 g, 65%). [a]2D0 ˆ ‡90.7 (c ˆ 0.38,
CH2Cl2); ee > 99% by chiral GC of the corresponding acetate (tR 21.38). Chemical purity 91% by GC/MS (tR
16.06), with 9% cis-a-irol (tR 16.77). MS and 1H-NMR: in accordance with those of (À)-4a. Anal. calc. for
C14H24O: C 80.71, H 11.61; found: C 80.79, H 11.67.
1.4. (2S,6R,9S)-a-Irol (ˆ(2S,3E)-4-[(1R,5S)-2,5,6,6-Tetramethylcyclohex-2-en-1-yl]but-3-en-2-ol; (À)-
10a). According to GP 1, (‡)-8 (5 g, 0.022 mol) gave, after 2 h, (À)-10a (3.29 g, 72%). [a]2D0 ˆ À55.5 (c ˆ 1.0,
CH2Cl2); ee > 99% by chiral GC of the corresponding acetate (tR 22.40). Chemical purity 98% by GC/MS (tR
16.79). 1H-NMR: 5.59 (dd, J ˆ 6.3, 15.2, HÀC(8)); 5.43 (dd ‡ m, J ˆ 10, 15.2, HÀC(7), HÀC(4)); 4.34 (quint.,
J ˆ 6.3, CHOH); 2.35 (m, 1 H); 1.89 (m, 1 H); 1.69 (m, 1 H); 1.51 (m, MeÀC(5)); 1.45 (m, 1 H); 1.29 (d, J ˆ 6.3,
MeCHOH); 0.85 (d ‡ s, J ˆ 6.5, MeÀC(2), 1 MeÀC(1)); 0.65 (s, 1 MeÀC(1)). 13C-NMR: 137.7; 133.8; 130.5;
121.8; 68.8; 55.6; 38.2; 35.1; 31.9; 26.4; 23.5; 22.9; 15.5; 14.6. GC/MS: 95 (100), 138 (42), 190 (1), 208 (0.5). Anal.
calc. for C14H24O: C 80.71, H 11.61; found: C 80.65, H 11.68.
1.5. (2R,6S,9R)-a-Irol (ˆ(2R,3E)-4-[(1S,5R)-2,5,6,6-Tetramethylcyclohex-2-en-1-yl]but-3-en-2-ol; (‡)-
10a). According to GP 1, (À)-8 (5 g, 0.022 mol) gave, after 2 h, (‡)-10a (3.16 g, 69%). [a]2D0 ˆ ‡53.5 (c ˆ 1.1,
CH2Cl2); ee > 99% by chiral GC of the corresponding acetate (tR 22.9). Chemical purity 98% by GC/MS (tR
16.77). MS and 1H-NMR: in accordance with those of (À)-10a. Anal. calc. for C14H24O: C 80.71, H 11.61; found:
C 80.76, H 11.54.
2. Acetylation of Irol Derivatives 4a and 10a. 2.1. General Procedure 2 (GP 2). All irol derivatives (2.08 g,
0.01 mol) were acetylated by treatment with Ac2O (0.05 mol) and pyridine (5 ml) in excess.
2.2 (2R,6R,9S)-a-Irol Acetate (ˆ(2S,3E)-4-[(1R,5R)-2,5,6,6-Tetramethylcyclohex-2-en-1-yl]but-3-en-2-ol
Acetate; (À)-4b). According to GP 2, (À)-4a gave (À)-4b (3.09 g, 95 %). [a]2D0 ˆ À58 (c ˆ 1.2, CH2Cl2); ee >
99% by chiral GC (tR 20.74). Chemical purity 94% by GC/MS (tR 18.44), with 6% of cis-a-irol acetate (tR 19.06).
1H-NMR: 5.60 5.30 (m, HÀC(7), HÀC(8), HÀC(9), HÀC(4)); 2.04 (s, MeCO); 1.90 1.50 (m, 4 H); 1.56 (m,
MeÀC(5)); 1.30 (d, J ˆ 6.1, MeCHOAc); 0.81 (d, J ˆ 6.7, MeÀC(2)); 0.79 (s, MeÀC(1)); 0.76 (s, MeÀC(1)).
13C-NMR: 170.4; 132.5; 131.2; 131.1; 121.6; 70.9; 56.1; 37.8; 33.6; 32.4; 26.4; 22.6; 21.38; 20.9; 15.20; 14.1. GC/MS:
95 (54), 105 (100), 120 (81), 138 (46), 180 (48), 190 (27). Anal. calc. for C16H26O2: C 76.75, H 10.47; found: C
76.69, H 10.42.

Helvetica Chimica Acta Vol. 84 (2001)
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2.3 (2S,6S,9R)-a-Irol Acetate (ˆ(2R,3E)-4-[(1S,5S)-2,5,6,6-Tetramethylcyclohex-2-en-1-yl]but-3-en-2-ol
Acetate; (‡)-4b). According to GP 2, (‡)-4a (2.80 g, 0.013 mol) gave (‡)-4b (3.06 g, 91%). [a]2D0 ˆ ‡56 (c ˆ
1.1, CH2Cl2); ee > 99% by chiral GC (tR 21.38). Chemical purity 91% by GC/MS (tR 18.44), with 9% of cis-a-irol
acetate (tR 19.05). MS and 1H-NMR: in accordance with those of (À)-4b. Anal. calc. for C16H26O2: C 76.75, H
10.47; found: C 76.79, H 10.52.
2.4 (2S,6R,9S)-a-Irol Acetate (ˆ(2S,3E)-4-[(1R,5S)-2,5,6,6-Tetramethylcyclohex-2-en-1-yl]but-3-en-2-ol
Acetate; (À)-10b). According to GP 2, (À)-10a (3.20 g, 0.015 mol) gave (À)-10b (3.38 g, 90%). [a]2D0 ˆ À105
(c ˆ 1.41, CH2Cl2); ee > 99% by chiral GC (tR 22.40). Chemical purity 98% by GC/MS (tR 19.05). 1H-NMR: 5.50
(m, 2 H); 5.47 (m, 1 H); 5.36 (m, HÀC(9)); 2.32 (m, 1 H); 2.04 (s, MeCO); 1.90 (m, 1 H); 1.70 (m, 1 H); 1.50
(m, MeÀC(5)); 1.44 (m, 1 H), 1.32 (d, J ˆ 6.5, MeCHOAc); 0.85 (d ‡ s, J ˆ 6.7, MeÀC(2), MeÀC(1)), 0.65 (s,
1 MeÀC(1)). 13C-NMR: 170.3; 133.6; 133.2; 132.8; 122.0; 71.0; 55.8; 38.1; 35.2; 31.9; 26.3; 22.7; 21.26; 15.53;
14.47. GC/MS: 105 (100), 120 (80), 138 (44), 180 (57), 190 (20). Anal. calc. for C16H26O2: C 76.75, H 10.47;
found: C 76.81, H 10.39.
2.5 (2R,6S,9R)-a-Irol Acetate (ˆ(2R,3E)-4-[(1S,5R)-2,5,6,6-Tetramethylcyclohex-2-en-1-yl]but-3-en-2-ol
Acetate; (‡)-10b). According to GP 2, (‡)-10a (3.10 g, 0.015 mol) gave (‡)-10b (3.32 g, 89%). [a]2D0 ˆ ‡107
(c ˆ 1.55, CH2Cl2) ([6c]: [a]2D0 ˆ À73 (c ˆ 1.50, CH2Cl2; sample containing 13% of 4-chloro-4,5-dihydro-cis-a-
irol acetate); ee > 99% by chiral GC (tR 22.9). Chemical purity 98% by GC/MS (tR 19.05). MS and 1H-NMR: in
accordance with those of (À)-10b. Anal. calc. for C16H26O2: C 76.75, H 10.47; found: C 76.68, H 10.39.
3. Photochemical Isomerization of 4b or 10b. 3.1. General Procedure 3 (GP 3). A soln. of the suitable irol
acetate (1.0 g, 0.004 mol) in iPrOH (90 ml) in the presence of xylene (10 ml) as photosensitizer was irradiated in
quartz vessels, in a Rayonet photochemical reactor equipped with ten 8-W high-pressure Hg lamps. The soln. was
evaporated and the residue purified by CC (hexane/AcOEt 95 :5).
3.2 (2R,6R,7E,9S)-g-Irol Acetate (ˆ(2S,3E)-4-[(1R,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-
en-2-ol Acetate; (À)-5) and (2R,6R,7Z,9S)-g-Irol Acetate (ˆ(2S,3Z)-4-[(1R,3R)-2,2,3-Trimethyl-6-methyli-
denecyclohexyl]but-3-en-2-ol) Acetate; 11a). According to GP 3, (À)-4b (1.0 g, 0.004 mol) gave a 1 :1 mixture
(1H-NMR) (À)-5/11a (0.89 g, 89%). Chemical purity 61% by GC/MS (tR 18.69). FT-IR (neat): 1738, 1645, 1455,
1371, 1250, 1135. 1H-NMR of the mixture: 5.97 (dd, J ˆ 9, 15, HÀC(7) (E)-isomer); 5.82 (t, J ˆ 11, HÀC(7) (Z)
isomer); 5.76 (m, HÀC(9) (Z) isomer); 5.44 (m, HÀC(8) (Z) and (E) isomers); 5.32 (quint., J ˆ 6, HÀC(9)
(E) isomer); 4.67 (m, 1 H of CˆCH2 E isomer, CˆCH2 (Z) isomer); 4.60 (m, 1 H of CˆCH (E) isomer); 2.97
(d, J ˆ 11, HÀC(6) (Z) isomer); 2.46 (d, J ˆ 9, HÀC(6) (E) isomer); 2.10 2.30 (m, 4 H); 2.03 (s, MeCO (E)
isomer); 1.99 (s, MeCO (Z) isomer); 1.40 1.60 (m, 4 H); 1.29 (m, 8 H); 0.80 0.87 (m, 15 H); 0.76 (s, MeÀC(1)
(E) isomer). GC/MS: 83 (100), 105 (97), 123 (66), 175 (62), 190 (60), 250 (5).
3.3 (2S,6S,7E,9R)-g-Irol Acetate (ˆ(2R,3E)-4-[(1S,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-
2-ol Acetate; (‡)-5) and (2S,6S,7Z,9R)-g-Irol Acetate (ˆ(2R,3Z)-4-[(1S,3S)-2,2,3-Trimethyl-6-methylidene
cyclohexyl]but-3-en-2-ol Acetate; 11b). According to GP 3, (‡)-4b (1.0 g, 0.004 mol) gave a 1:1 mixture
(1H-NMR) (‡)-5/11b (0.85 g, 85%). Chemical purity 65% by GC/MS (tR ˆ 18.69). MS and 1H-NMR: in
accordance with those of the corresponding enantiomers.
3.4 (2S,6R,7E,9S)-g-Irol Acetate (ˆ(2S,3E)-4-[(1R,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-
2-ol Acetate; 12a) and (2S,6R,7Z,9S)-g-Irol Acetate (ˆ(2S,3Z)-4-[(1R,3S)-2,2,3-Trimethyl-6-methylidenecyclo-
hexyl]but-3-en-2-ol Acetate; (13a)). According to GP 3, (À)-10b (1.0 g, 0.004 mol) gave a 2 :1 mixture (GC/MS)
12a/13a (0.91 g, 91%). Chemical purity 95% by GC/MS (tR (13a) 18.53, tR (12a) 19.22). FT-IR (neat): 1734, 1648,
1451, 1370, 1254, 1131. 1H-NMR: 12a: 5.81 (dd, J ˆ 10, 15, HÀC(7)); 5.45 (dd, J ˆ 6.5, 10, HÀC(8)); 5.38 (quint.,
J ˆ 6.5, HÀC(9)); 4.73 (m, 1 H, CˆCH); 4.45 (m, 1 H, CˆCH); 2.32 (m, 2 H); 2.03 (s ‡ m, Ac ‡ 1 H); 1.67
1.15 (d ‡ m, J ˆ 6.5, MeÀC(9) ‡ 3 H); 0.86 (d ‡ s, J ˆ 7, Me ÀC(2), 1 MeÀC(1)); 0.63 (s, 1 MeÀC(1)); 13a: 4.74
(m, 1 H, CˆCH); 4.50 (m, 1 H, CˆCH). GC/MS: 13a: 83 (94), 91 (100), 105 (95), 175 (83), 190 (60), 232 (2),
250 (3); 12a: 83 (96), 91 (100), 105 (91), 175 (64), 190 (64), 232 (3), 250 (5).
3.5 (2R,6S,7E,9R)-g-Irol Acetate (ˆ(2R,3E)-4-[(1S,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-
en-2-ol Acetate; 12b) and (2R,6S,7Z,9R)-g-Irol Acetate (ˆ(2R,3Z)-4-[(1S,3R)-2,2,3-Trimethyl-6-methylidene-
cyclohexyl]but-3-en-2-ol Acetate; 13b). According to GP 3, (‡)-10b (1 g, 0.004 mol) gave a 2 :1 mixture (GC/
MS) 12b/13b (0.95 g, 95%). Chemical purity 95% by GC/MS. MS and 1H-NMR: in accordance with those of the
corresponding enantiomers.
4. Saponification of the (E)/(Z) Mixtures of g-Irol Acetates. 4.1. General Procedure 4 (GP 4). A soln. of the
suitable mixture of g-irol acetates (1.64 g, 6.56 mmol) in MeOH (10 ml) in the presence of KOH (0.551 g,
9.84 mmol) was stirred at r.t. for 2 h. The mixture was diluted with H2O and extracted with AcOEt. The org.
phase was dried (Na2SO4) and evaporated and the residue purified by CC (hexane/AcOEt 7:3).

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Helvetica Chimica Acta Vol. 84 (2001)
4.2 (2R,6R,7E,9S)-g-Irol (ˆ(2S,3E)-4-[(1R,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-ol;
(‡)-20) and (2R,6R,7Z,9S)-g-Irol (ˆ(2S,3Z)-4-[(1R,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-
ol). According to GP 4, the 1:1 mixture (À)-5 and 11a (1.64 g, 9.84 mmol) gave a 1 :1 mixture (GC/MS) of the
corresponding irol derivatives (1.23 g, 90%). 1H-NMR: 5.92 (dd, J ˆ 8.6, 15.1, HÀC(7) (E) isomer); 5.79 (t, J ˆ
11, HÀC(7) (Z) isomer); 5.51 (m, HÀC(8) of (Z) and (E) isomers); 4.73 (m, HÀC(9) (Z) isomer); 4.67 (m,
1 H of CˆCH2 (E) isomer, CˆCH2 (Z) isomer); 4.60 (m, 1 H of CˆCH2 (E) isomer); 4.28 (quint., J ˆ 6,
HÀC(9) (E) isomer); 2.94 (d, J ˆ 11, HÀC(6) (Z) isomer); 2.48 (d, J ˆ 8.6, HÀC(6) (E) isomer); 2.10 2.30 (m,
4 H); 1.40 1.60 (m, 4 H); 1.25 (m, 8 H), 0.77 0.88 (m, 18 H)). GC/MS: (2R,6R,7Z,9S)-g-irol (tR 16.20): 107
(100), 135 (56), 175 (48), 190 (13); (2R,6R,7E,9S)-g-irol (tR 16.46): 107 (100), 135 (56), 175 (33), 190 (10).
4.3. (2S,6S,7E,9R)-g-Irol (ˆ(2R,3E)-4-[(1S,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-ol
(À)-20) and (2S,6S,7Z,9R)-g-Irol (ˆ(2R,3Z)-4-[(1S,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-
ol). According to GP 4, the 1:1 mixture (‡)-5/11b (1.61 g, 6.44 mmol) gave a 1:1 mixture (GC/MS) of the
corresponding irol derivatives (1.51 g, 94%). GC/MS and 1H-NMR: in accordance with those of the
corresponding enantiomers.
4.4. (2S,6R,7E,9S)-g-Irol (ˆ(2S,3E)-4-[(1R,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-ol)
and (2S,6R,7Z,9S)-g-Irol (ˆ(2S,3Z)-4-[(1R,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-ol). Ac-
cording to GP 4, the 2 :1 mixture 12a/13a (2.61 g, 0.010 mol) gave a 2 :1 mixture (GC/MS) of the corresponding
irol derivatives (1.94 g, 89%). 1H-NMR (selection of signals of the main (7E) isomer): 5.75 (ddd, J ˆ 0.95, 9.8,
15.3, HÀC(7)); 5.52 (dd, J ˆ 6.7, 15.3, HÀC(8)); 4.74 (m, 1 H, CˆCH); 4.47 (m, 1 H, CˆCH); 4.35 (quint., J ˆ
0.95, 6.7, CHOH); 1.29 (d, J ˆ 6.7, MeCHOH); 0.90 (s, 1 MeÀC(1)); 0.85 (d, J ˆ 6.3, MeÀC(2)); 0.64 (s,
1 MeÀC(1)); selection of signals of the (7Z) isomer: 5.61 (m, 2 H); 4.77 (m, 1 H, CˆCH); 4.60 (m, 1 H,
CˆCH); 4.54 (m, CHOH); 1.24 (d, J ˆ 6.7, MeCHCHOH); 0.87 (s, 1 MeÀC(1)); 0.86 (d, J ˆ 6.3, MeÀC(2));
0.64 (s, MeÀC(1)). GC/MS: (2S,6R,7Z,9S)-g-irol (tR 16.46): 107 (100), 123 (61), 135 (65), 150 (39), 175 (65),
190 (11), 208 (7); (2S,6R,7E,9S)-g-irol (tR 17.00): 107 (100), 123 (69), 135 (65), 150 (72), 175 (31), 190 (7), 208
(2).
4.5. (2R,6S,7E,9R)-g-Irol (ˆ(2R,3E)-4-[(1S,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-ol)
and (2R,6S,7Z,9R)-g-Irol (ˆ(2R,3Z)-4-[(1S,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-ol). Ac-
cording to GP 4, the 2 :1 mixture 12b/13b (2.47 g, 9.88 mmol) gave a 2 :1 mixture (GC/MS) of the corresponding
irol derivatives (1.79 g, 87%). GC/MS and 1H-NMR: in accordance with those of the corresponding
enantiomers.
5. Manganese(IV) Oxide Oxidation. 5.1. General Procedure 5 (GP 5). A mixture of the suitable g-irols
(1.50 g, 7.21 mmol) in CH2Cl2 (25 ml) in the presence of manganese(IV) oxide (1.5 equiv.) was refluxed for 4 h.
The mixture was filtered, the filtrate evaporated, and the residue purified by CC (hexane/AcOEt 97:3).
5.2. (2R,6R,7E)-g-Irone (ˆ(3E)-4-[(1R,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-one;
(‡)-3b) and (2R,6R,7Z)-g-Irone (ˆ(3Z)-4-[(1R,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-
one; (À)-14). According to GP5, the 1 :1 mixture (2R,6R,7E,9S)-g-irol/(2R,6R,7Z,9S)-g-irol (1.13 g,
5.43 mmol) gave (‡)-3b (0.358 g, 32%) and (À)-14 (0.324 g, 29%).
Data of (‡)-3b: ee > 99% by chiral HPLC (tR 24.54). Chemical purity 74% by GC/MS (tR 17.57), with
impurities of 8% (À)-trans-a-irone (tR 17.25) and 5% (‡)-cis-g-irone (tR 17.90). [a]2D0 ˆ ‡10 (c ˆ 2.8, CH2Cl2).
1H-NMR: 7.07 (dd, J ˆ 9.4, 15.6, HÀC(7)); 6.10 (dd, J ˆ 1.1, 15.6, HÀC(8)); 4.76 (m, 1 H, CˆCH2); 4.67 (m,
1 H, CˆCH2); 2.64 (d, J ˆ 9.4, HÀC(6)); 2.25 (s, MeCO); 2.22 (m, 2 H); 1.61 (m, 2 H); 1.31 (m, 1 H); 0.90 (s,
1 MeÀC(1)); 0.86 (d, J ˆ 6.7, MeÀC(2)); 0.81 (s, 1 MeÀC(1)). GC/MS: 121 (100), 149 (40), 163 (57), 178 (14),
191 (11), 206 (8). Anal. calc. for C14H22O: C 81.50, H 10.75; found: C 81.59, H 10.70.
Data of (À)-14: Chemical purity 70% by GC/MS (tR 15.97). [a]2D0 ˆ À43.2 (c ˆ 1.25, CH2Cl2). 1H-NMR:
6.35 (t, J ˆ 12.1, HÀC(7)); 6.17 (d, J ˆ 12.1, HÀC(8)); 4.75 (m, 1 H, CˆCH2); 4.71 (m, 1 H, CˆCH2); 4.06 (d,
J ˆ 12.1, HÀC(6)); 2.21 (s, MeCO); 2.20 1.20 (m, 5 H); 0.88 (d, J ˆ 6.3, MeÀC(2)); 0.87 (s, 1 MeÀC(1)); 0.82
(s, 1 MeÀC(1)). 13C NMR: 199.0; 148.6; 146.9; 127.4; 110.1; 51.9; 37.8; 35.8; 31.9; 31.6; 31.3; 25.8; 21.8; 15.5. GC/
MS: 121 (95), 149 (44), 163 (67), 173 (11), 191 (100), 206 (10). Anal. calc. for C14H22O: C 81.50, H 10.75; found:
C 81.44, H 10.64.
5.3. (2S,6S,7E)-g-Irone (ˆ(3E)-4-[(1S,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-one; (À)-
3b) and (2S,6S,7Z)-g-Irone (ˆ(3Z)-4-[(1S,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en -2-one; (‡)-
14). According to GP 5, the 1 :1 mixture (2S,6S,7E,9R)-g-irol and (2S,6S,7Z,9R)-g-irol (1.40 g, 6.73 mmol) gave
(À)-3b (0.388 g, 28%) and (‡)-14 (0.332 g, 24%). Anal. calc. for C14H22O: C 81.50, H 10.75; found: C 81.57, H
10.69.
Data of (À)-3b: ee > 99% by chiral HPLC (tR 27.46). Chemical purity 79% by GC/MS (tR 17.57), with
impurities of 3% (‡)-trans-a-irone and 5% (À)-cis-g-irone. [a]2D0 ˆ À16 (c ˆ 3.0, CH2Cl2). MS and 1H-NMR: in

Helvetica Chimica Acta Vol. 84 (2001)
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accordance with those of the corresponding enantiomer. Anal. calc. for C14H22O: C 81.50, H 10.75; found: C
81.43, H 10.81.
Data of (‡)-14: Chemical purity 76% by GC/MS (tR 15.97). [a]2D0 ˆ 48.7 (c ˆ 1.32, CH2Cl2). MS and
1H-NMR: in accordance with those of the corresponding enantiomer. Anal. calc. for C14H22O: C 81.50, H 10.75;
found: C 81.41, H 10.68.
5.4. (2S,6R,7E)-g-Irone (ˆ(3E)-4-[(1R,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-one; (À)-
3a) and (2S,6R,7Z)-g-Irone (ˆ(3Z)-4-[(1R,3S)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-one; (‡)-
15). According to GP 5, the 2 :1 mixture (2S,6R,7E,9S)-g-irol/(2S,6R,7Z,9S)-g-irol (1.84 g, 8.84 mmol) gave
(À)-3a (0.765 g, 42%) and (‡)-15 (0.346 g, 19%).
Data of (À)-3a: ee > 99% by chiral GC. (tR 20.86). Chemical purity 96% by GC/MS (tR 17.90). [a]D20
ˆ
À2.54 (c ˆ 4.9, CH2Cl2). 1H-NMR: 6.93 (dd, J ˆ 15.8, 10.3, HÀC(7)); 6.09 (d, J ˆ 15.8, HÀC(8)); 4.80 (m, 1 H,
CˆCH2); 4.43 (m, 1 H, CˆCH2); 2.55 (d, J ˆ 10.3, HÀC(6)); 2.35 (ddd, J ˆ 2.5, 4.4, 13.3, 1 H); 2.28 (s, MeCO);
2.10 (m, 1 H); 1.60 1.20 (m, 3 H); 0.87 (s ‡ d, J ˆ 7, 1 Me ÀC(1), MeÀC(2)); 0.73 (s, MeÀC(1)). GC/MS: 121
(100), 149 (41), 163 (29), 191 (8), 206 (3). Anal. calc. for C14H22O: C 81.50, H 10.75; found: C 81.45, H 10.77.
Data of (‡)-15: Chemical purity 90% by GC/MS (tR 16.33). [a]2D0 ˆ ‡50.5 (c ˆ 1.08, CH2Cl2). 1H-NMR:
6.29 (d , J ˆ 11.8, HÀC(8)); 6.20 (dd, J ˆ 10.3, 11.8, HÀC(7)); 4.72 (m, 1 H, CˆCH2); 4.42 (m, 1 H, CˆCH2);
3.85 (d, J ˆ 10.3, HÀC(6)); 2.30 (m, 1 H); 2.18 (s ‡ m, MeCO ‡ 1 H); 1.51 (m, 2 H); 1.27 (m, 1 H); 0.88 (s,
1 MeÀC(1)); 0.84 (d, J ˆ 6.4, MeÀC(2)); 0.68 (s, 1 MeÀC(1)). 13C-NMR: 199.4; 149.1; 146.7; 128.8; 107.5; 51.5;
41.9; 38.8; 36.3; 31.9; 31.6; 26.9; 15.9; 13.7. GC/MS: 149 (30), 191 (100), 206 (5). Anal. calc. for C14H22O: C 81.50,
H 10.75; found: C 81.43, H 10.78.
5.5. (2R,6S,7E)-g-Irone (ˆ(3E)-4-[(1S,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-one; (‡)-
3a) and (2R,6S,7Z)-g-Irone (ˆ(3Z)-4-[(1S,3R)-2,2,3-Trimethyl-6-methylidenecyclohexyl]but-3-en-2-one; (À)-
15). According to GP 5, the 2 :1 mixture (2R,6S,7E,9R)-g-irol/(2R,6S,7Z,9R)-g-irol (1.65 g, 7.93 mmol) gave
(‡)-3a (0.751 g, 46%) and (À)-15 (0.294 g, 18%).
Data of (‡)-3a: ee > 99% by chiral GC (tR 20.56). Chemical purity 93% by GC/MS (tR 17.90). [a]2D0 ˆ ‡1.78
1
(c ˆ 4.3, CH2Cl2). MS and H-NMR: in accordance with those of the enantiomer. Anal. calc. for C14H22O: C
81.50, H 10.75; found: C 81.42, H 10.74.
Data of (À)-15: Chemical purity 91% by GC/MS (tR 16.33). [a]2D0 ˆ À48.3 (c ˆ 1.2, CH2Cl2). MS and
1H-NMR: in accordance with those of the enantiomer. Anal. calc. for C14H22O: C 81.50, H 10.75; found: C 81.57,
H 10.68.
6. 6,7-Dimethyl-3-oxooct-6-enoic Acid Ethyl Ester (17). Ethyl acetoacetate (50.4 g, 0.388 mol) was added
dropwise to a suspension of NaH (60% mineral oil; 18.6 g, 0.466 mol) in THF (250 ml) at 08. The colorless soln.
was stirred at 08 for 10 min. To this mixture, 10m BuLi in hexane (46.6 ml, 0.466 mol) was added, and the yellow-
orange soln. was stirred at 08 for 10 min. A soln. of bromo derivative 16 (60.0 g, 0.368 mol) in THF (100 ml) was
then added, and the mixture was stirred for 30 min, while temp. was allowed to reach 208. The reaction was
quenched with conc. HCl soln. in H2O and Et2O. The aq. phase was extracted with Et2O, the combined org.
extract washed with H2O until neutral, dried (Na2SO4), and evaporated, and the residue distilled: 17 (63.2 g,
81%). 1H-NMR: 4.20 (q, J ˆ 7, COOCH2Me); 3.45 (s, COCH2COOEt); 2.58, 2.32 (2m, CH2CH2); 1.63 (m,
Me2CˆCMe); 1.28 (t, J ˆ 7, COOCH2Me).13C-NMR: 203; 167.1; 125.4; 61.2; 49.2; 41.4; 28.2; 20.4; 19.9; 17.9;
13.9. Anal. calc. for C12H20O3: C 67.89, H 9.50; found: C 67.78, H 9.45.
7. 2,2,3-Trimethyl-6-oxocyclohexanecarboxylic Acid Ethyl Ester (18). Tin(IV) chloride (114 g, 0.283 mol)
was added to a soln. of 17 (60.0 g, 0.283 mol) in CH2Cl2 (300 ml) at 08. The mixture was stirred at r.t. for 1 h,
poured into H2O, and extracted with CH2Cl2. The org. extracts were washed with sat. NaHCO3 soln., then with
H2O, dried (Na2SO4), and evaporated. The residue was distilled at 1108, 8 Torr to afford derivative 18 (50.4 g,
84%) as a 2 :1 mixture of trans and cis diastereoisomers, which was submitted directly to the subsequent
reaction. 1H-NMR: 4.15 (m, COOCH2Me); 3.11 (m, COCHCOOEt); 2.32 (m, CH2CO); 1.90 (m, MeCH); 1.65
(m, CH2); 1.28 (m, COOCH2Me); 1.2 0.90 (m, 3 Me). GC/MS: trans-18 (tR 15.49): 55 (100), 83 (76), 114 (28),
167 (29), 194 (40), 212 (5); cis-18 (tR 16.28): 55 (100), 83 (78), 114 (29), 167 (30), 194 (24), 212 (5). Anal. calc.
for C12H20O3: C 67.89, H 9.50; found: C 67.53, H 9.85.
8. (1RS,3RS)-2,2,3-Trimethyl-6-methylidenecyclohexane-1-methanol (19). At À 788, 10m BuLi in hexane
‡
(28.6 ml, 0.287 mol ) was added dropwise to a suspension of (Ph3PMe) BrÀ (102.8 g, 0.287 mol) in dry THF
(300 ml) at À 788. The mixture was warmed to r.t. and stirred until almost complete disappearance of the
phosphonium salt. The cyclic keto ester 18 (50.0 g, 0.235 mol) was added, and the mixture was refluxed for 2 h.
After cooling, the mixture was poured into H2O and extracted with Et2O. The org. phase was washed with sat.
NH4Cl soln. and brine, dried (Na2SO4), and evaporated. The residue was dissolved in hexane and the
triphenylphosphine oxide eliminated by crystallization. The mother liquors were diluted with dry THF (300 ml)

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Helvetica Chimica Acta Vol. 84 (2001)
and cooled to 08. LiAlH4 (9.50 g, 0.250 mol) was added under stirring. The mixture was diluted with H2O and
extracted with Et2O. The org. phase was dried (Na2SO4) and evaporated. Purification of the residue by
1
distillation under reduced pressure gave 19 [7b] (13.4 g, 34%). H-NMR: in accordance with that reported in
[7b]; GC/MS (tR 12.18): 83 (100), 95 (90), 107 (92), 137 (70), 150 (40), 168 (5). Anal. calc. for C11H20O: C 78.51,
H 11.98; found: C 78.58, H 11.92.
9. (Æ)-trans-g-Irone ((Æ)-3b). DMSO (14.7 g, 0.189 mol) was added dropwise to a soln. of ClCOCOCl
(12.5 g, 0.098 mol) in CH2Cl2 (100 ml) at À 788. Then 19 [7b] (13.0 g, 0.077 mol) was added and the mixture
stirred for 15 min at the same temp. The suspension was treated with Et3N (30 g, 0.293 mol) and allowed to
warm to r.t. After 2 h, the mixture was diluted with CH2Cl2 and washed with H2O. The org. phase was dried
(Na2SO4) and evaporated. The obtained crude residue was dissolved in toluene (100 ml) and treated with
(acetylmethylene)triphenylphosphorane (30 g, 0.093 mol). The mixture was refluxed for 8 h. Triphenylphos-
phine oxide was eliminated by crystallization from hexane. The mother liquors were evaporated and submitted
to CC (hexane/AcOEt 95 :5): (Æ)-3b (9.19 g, 58%). Chemical purity 98% by GC/MS (tR 17.57), with 2% cis-g-
irone. 1H-NMR: in accordance with that of (‡)-3b (see above). Anal. calc. for C14H22O: C 81.50, H 10.75; found:
C 81.58, H 10.68.
10. ( Æ )-(2RS,6RS,9SR)-g-Irol 4-Nitrobenzoate ((Æ)-22) and (Æ)-(2RS,6RS,9RS)-g-Irol 4-Nitrobenzoate
((Æ)-23). Derivative (Æ)-3b (9.10 g, 0.044 mol) was reduced with NaBH4. The resulting mixture of the two
racemic alcohol diastereoisomers (8.60 g, 0.041 mol) was dissolved in pyridine (30 ml) and treated with 4-
nitrobenzoyl chloride (9.12 g, 0.0492 mol). After the usual workup, the residue was crystallized from hexane.
The crystalline precipitate was crystallized twice from hexane to afford (Æ)-23 (1.47 g, 10%). The mother liquors
were evaporated, and the residue was crystallized thrice from hexane to afford derivative (Æ)-22 (4.54 g, 31%).
Data of (Æ)-23: M.p. 578. GC/MS: (tR 27.82, de 92%. 1H-NMR: 8.24 (m, arom. H); 6.07 (m, HÀC(7)); 5.60
(m, HÀC(8), HÀC(9)); 4.67 (m, 1 H, CˆCH2); 4.61 (m, 1 H, CˆCH2); 2.51 (d, J ˆ 9, HÀC(6)); 2.18 ( m, 2 H);
1.57 (m, 2 H); 1.46 (d, J ˆ 6, MeCHOAr); 1.27 (m, 1 H); 0.86 (s, 1 MeÀC(1)); 0.84 (d, J ˆ 6, MeÀC(2)); 0.77 (s,
1 MeÀC(1)). 13C-NMR: 163.8; 150.4; 149.3; 136.2; 133.2; 130.6; 130.0; 123.3; 109.1; 73.1; 58.6; 37.2; 36.2; 31.4;
31.3; 26.7; 21.9; 20.5; 15.4. GC/MS: 91 (79), 120 (100), 150 (75), 190 (44), 207 (22), 284 (10), 314 (17), 357 (2).
Anal. calc. for C21H27NO4: C 70.56, H 7.61, N 3.92; found: C 70.49, H 7.68, N 3.87.
1
Data of (Æ)-22: M.p. 738. GC/MS: (tR 27.87, de 98%). H-NMR: 8.24 (m, arom. H); 6.11 (dd, J ˆ 9, 14.3,
HÀC(7)); 5.58 (m, HÀC(8), HÀC(9)); 4.69 (m, 1 H, CˆCH2); 4.62 (m, 1 H, CˆCH2); 2.50 (d, J ˆ 9, HÀC(6));
2.18 ( m, 2 H); 1.58 (m, 2 H); 1.45 (d, J ˆ 6, MeCHOAr); 1.38 (m, 1 H); 0.84 (d, J ˆ 6, MeÀC(2)); 0.82 (s,
1 MeÀC(1)); 0.76 (s, 1 MeÀC(1)). 13C-NMR: 163.9; 150.4; 149.5; 136.3; 133.9; 130.6; 129.9; 123.5; 109.1; 73.3;
58.8; 37.2; 36.1; 31.5; 31.4; 26.7; 21.7; 20.4; 15.5. GC/MS: 91 (100), 120 (94), 150 (86), 190 (51), 207 (30), 284 (7),
314 (19). Anal. calc. for C21H27NO4: C 70.56, H 7.61, N 3.92; found: C 70.62, H 7.56, N 4.05.
11. (Æ)-(2RS,6RS,9SR)-g-Irol ((Æ)-20). Saponification of (Æ)-22 (4.54 g, 0.013 mol) according to GP 4
afforded (Æ)-20 (2.51 g, 95%). Chemical purity 98% by GC/MS (tR 16.46). 1H-NMR: 5.92 (ddd, J ˆ 1.1, 8.6, 15.1,
HÀC(7)); 5.54 (ddd, J ˆ 0.7, 6, 15.1, HÀC(8)); 4.66 (m, 1 H, CˆCH2); 4.60 (m, 1 H, CˆCH2); 4.28 (quint., J ˆ 6,
HÀC(9)); 2.48 (d, J ˆ 8.6, HÀC(6)); 2.18 (m, 2 H); 1.59 (m, 2 H); 1.25 (d ‡ m, J ˆ 6.4, MeCHOH ‡ 1 H); 0.86
(s, 1 MeÀC(1)); 0.83 (d, J ˆ 6.7, 1 MeÀC(2)); 0.77 (s, 1 MeÀC(1)). 13C-NMR: 150.0; 135.6; 129.7; 108.7; 68.8;
58.9; 37.1; 36.0; 31.7; 31.4; 26.8; 23.4; 21.7; 15.6. GC/MS: 107 (100), 135 (56), 175 (33), 190 (10). Anal. calc. for
C14H24O: C 80.71, H 11.61; found: C 80.65, H 11.55.
12. (‡)-(2S,6S,9R)-g-Irol Acetate ((‡)-5) and (‡)-(2R,6R,9S)-g-Irol ((‡)-20). A mixture of derivative (Æ)-
t
20 (2.50 g, 0.012 mol) and lipase PS (2.50 g) in BuOMe (30 ml) in the presence of vinyl acetate (5 ml) was
stirred at r.t. for 4 days. The enzyme was filtered off, the filtrate evaporated, and the residue chromatographed
(hexane/AcOEt): (‡)-5 (1.26 g, 42%), followed by (‡)-20 (1.02 g, 41%).
Data of (‡)-5: B.p. 90 1008/0.2 Torr. Chemical purity 98% by GC/MS (tR 18.69). [a]2D0 ˆ ‡46.6 (c ˆ 1.2,
CH2Cl2). 1H-NMR: 5.97 (dd, J ˆ 9, 15, HÀC(7)); 5.44 (ddd, J ˆ 0.7, 7, 15, HÀC(8)); 5.32 (quint., J ˆ 6,
HÀC(9)); 4.67 (m, 1 H, CˆCH2); 4.60 (m, 1 H, CˆCH2); 2.46 (d, J ˆ 9, HÀC(6)); 2.18 (m, 2 H); 2.03 (s,
MeCO); 1.58 (m, 2 H); 1.29 (d ‡ m, J ˆ 7, MeCHOAc ‡ 1 H); 0.83 (s ‡ d, J ˆ 6.7, 1 MeÀC(1), MeÀC(2)); 0.76
(s, MeÀC(1)). 13C-NMR: 170.3; 149.5; 132.5; 130.7; 108.7; 71.2; 58.7; 37.1; 36.0; 31.5; 31.3; 26.7; 21.7; 21.5; 20.3;
15.6. GC/MS: 91 (100), 105 (91), 123 (56), 175 (62), 190 (65), 250 (7). Anal. calc. for C16H26O2: C 76.75, H 10.47;
found: C 76.81, H 10.49.
Data of (‡)-20: B.p. 95 1008/0.2 Torr. Chemical purity 98% by GC/MS (tR 16.46). [a]2D0 ˆ ‡38.2 (c ˆ 1.0,
CH2Cl2). GC/MS and 1H-NMR: in accordance with those of the corresponding racemate. Anal. calc. for
C14H24O: C 80.71, H 11.61; found: C 80.64, H 11.69.
13. (À)-(2S,6S,9R)-g-Irol ((À)-20). Saponification of (‡)-5 (1.20 g, 4.8 mmol) according to GP 4 gave
alcohol (À)-20 (0.938 g, 94%). Chemical purity 97% by GC/MS (tR 16.46). [a]2D0 ˆ À39.8 (c ˆ 1.0, CH2Cl2). MS

Helvetica Chimica Acta Vol. 84 (2001)
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1
and H-NMR: in accordance with those of the racemate. Anal. calc. for C14H24O: C 80.71, H 11.61; found: C
80.81, H 11.58.
14. (‡)-(2R,6R)-Trans-g-Irone ((‡)-3b). According to GP 5, alcohol (‡)-20 (0.95 g, 4.56 mmol) gave (‡)-
3b (0.760 g, 81%): ee > 99% by chiral HPLC (tR 24.54). Chemical purity 99% by GC/MS (tR 17.57). [a]D20
ˆ
‡76.4 (c ˆ 1.0, CH2Cl2). MS and 1H-NMR: in accordance with those described above. Anal. calc. for C14H22O: C
81.50, H 10.75; found: C 81.41, H 10.68.
15. (À)-(2S,6S)-trans-g-Irone ((À)-3b). According to GP 5 alcohol (À)-20 (0.90 g, 4.37 mmol) gave (À)-3b
(0.648 g, 72%): ee > 99% by chiral HPLC (tR 27.46). Chemical purity 99% by GC/MS (tR 17.57). [a]2D0 ˆ À81
(c ˆ 1.0, CH2Cl2). MS and 1H-NMR: in accordance with those already described above. Anal. calc. for C14H22O:
C 81.50, H 10.75; found: C 81.43, H 10.81.
16. Preparation of Irone Oil Samples from Fresh Rhizomes. Fresh rhizomes (Iris pallida) of four different
batches were obtained from Aboca (Toscana, Italy), cut into slices, and extracted with acetone at r.t. (4 l of
acetone/1 kg of fresh rhizomes). The acetone extracts were evaporated, and the residue was suspended in H2O
and extracted with hexane/AcOEt. The combined org. extract was washed with sat. NaHCO3 soln. and H2O,
dried (Na2SO4), and evaporated, and the residue submitted to the enzymatic procedure for the preparation of
irone oil as described in [19]. Soybean flour was employed as a source of lipoxydase.
17. Irone-Isomer and -Enantiomer Distributions in Italian Iris Oil Samples. 17.1. Irone-isomer distribution
was determined by achiral GC analysis of irone oil samples on a HP-5MS column (30 m  0.25 mm  0.25 mm),
installed in a HP-6890 gas chromatograph equipped with a FID detector and a 5973 mass detector. The
following temp. program was employed: 608 (1 min), then 68/min to 1508 (1 min), then 128/min to 2808 (5 min).
Identification of irone isomers was achieved by comparison with pure synthetic samples. The following retention
times were observed: (Z)-cis-g-irone, tR 16.41; trans-a-irone, tR 17.90; trans-g-irone, tR 18.19; cis-a-irone, tR
18.35; cis-g-irone, tR 18.51; b-irone, tR 19.08.
17.2 The enantiomer excesses of trans-a-irone, cis-a-irone, and cis-g-irone, and a-irol acetates were
determined by chiral GC analysis with a Chirasil DEX CB, 25 m  0.25 mm (Chrompack) column, installed in a
DANI-HT-86.10 gas chromatograph, with the following temp. program: 708 (3 min); then 3.58/min to 1408, then
88/min to 1808 (1 min). The following retention times were observed: (À)-trans-a-irone, tR 18.61; (‡)-trans-a-
irone, tR 19.01; trans-g-irone, tR 19.30; (‡)-cis-a-irone, tR 19.89; (À)-cis-a-irone, tR 19.98; (‡)-cis-g-irone, tR
20.56; (À)-cis-g-irone, tR 20.86; b-irone, tR 21.56.
17.3. The enantiomer excess of trans-g-irone was determined by chiral HPLC analysis with a Chiralcel OD
column (Daicel, Japan) installed in a Merck-Hitachi L-6200 apparatus; UV detector (254 nm), hexane/iPrOH
99 :1 with the addition of one drop of Et3N per 100 ml of eluent, 0.6 ml/min. The following retention times were
observed: cis-a-irone, tR 17.98; (À)-trans-a-irone, tR 19.14; (‡)-trans-a-irone, tR 20.53; (‡)-trans-g-irone, tR
24.54; (‡)-cis-g-irone, tR 26.46; (À)-trans-g-irone, tR 27.46; (À)-cis-g-irone, tR 30.48.
REFERENCES
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Received May 29, 2001