Synthesis of sabina δ-lactones and sabina δ-lactams from (+)-sabinene

Article abstract of DOI:10.1071/CH13306

Two sabina δ-lactones (3 and 4) were obtained in a two-step synthesis from (+)-sabinene (1). The oxidation of (+)-sabinene (1) with potassium permanganate and sodium periodate to (-)-sabina ketone (2) was the first step. In the second step, the ketone obtained was subjected to chemical and microbial Baeyer-Villiger oxidation. Chemical Baeyer-Villiger oxidation of this ketone afforded two δ-lactones 3 and 4 whereas microbial Baeyer-Villiger oxidation afforded only 'abnormal' δ-lactone 4. (-)-Sabina ketone was also the starting material for the synthesis of new δ-lactams (7 and 8). They were obtained by Beckmann rearrangement of sabina ketone oximes 5a and 5b. An attempt to separate (-)-sabina ketone oximes 5a and 5b is also presented. CSIRO 2013.

Full text of DOI:10.1071/CH13306

CSIRO PUBLISHING
Aust. J. Chem. 2013, 66, 1399–1405
Full Paper
http://dx.doi.org/10.1071/CH13306
Synthesis of Sabina d-Lactones and Sabina d-Lactams
from (1)-Sabinene
A
A B
,
Radosław Gniłka and Czesław Wawrzen´czyk
A
Department of Chemistry, Wrocław University of Environmental
and Life Sciences, Norwida 25, 50-375 Wrocław, Poland.
Corresponding author. Email: czeslaw.wawrzenczyk@up.wroc.pl
B
Two sabina d-lactones (3 and 4) were obtained in a two-step synthesis from (þ)-sabinene (1). The oxidation of
(þ)-sabinene (1) with potassium permanganate and sodium periodate to (ꢀ)-sabina ketone (2) was the first step. In the
second step, the ketone obtained was subjected to chemical and microbial Baeyer–Villiger oxidation. Chemical Baeyer–
Villiger oxidation of this ketone afforded two d-lactones 3 and 4 whereas microbial Baeyer–Villiger oxidation afforded
only ‘abnormal’ d-lactone 4. (ꢀ)-Sabina ketone was also the starting material for the synthesis of new d-lactams (7 and 8).
They were obtained by Beckmann rearrangement of sabina ketone oximes 5a and 5b. An attempt to separate (ꢀ)-sabina
ketone oximes 5a and 5b is also presented.
Manuscript received: 13 June 2013.
Manuscript accepted: 15 July 2013.
Published online: 26 August 2013.
Introduction
In the present paper, the syntheses of two sabina d-lactones
(3 and 4) and two new sabina d-lactams (7 and 8) from natural
(þ)-sabinene (1) are described.
Natural and synthetic terpenoid and sesquiterpenoid lactones
exhibit many types of biological activity such as antifungal,[1,2]
antibacterial,[1–4] and anticancer.[4–7] They are also known as
good feeding deterrents towards insects.[8–11]
Results and Discussion
We are interested in the synthesis of terpenoid lactones
because of their odoriferous properties and antifeedant activity.
In recent years, starting from monoterpenes,[12–14] monoterpe-
noid alcohols,[15] and ketones[16,17] we have synthesized many
terpenoid lactones. In most of the syntheses carried out, the
Claisen rearrangement of allyl alcohols, halolactonization of
g- and d-unsaturated acids and esters, Baeyer–Villiger oxidation
of ketones, and addition of Meldrum acid to alkenes were
essential steps leading to the final products. Some of the
obtained lactones showed very high feeding-deterrent activity
against insect pests.[17–20] Recently, we published the synthesis
of four new lactones from (ꢀ)-a- and (þ)-b-thujone.[21] Prelim-
inary biological tests indicated that three of these possess
antifeedant activity against peach aphid (Myzus persicae).
Further, a lactone obtained from (ꢀ)-a-thujone by chemical
Baeyer–Villiger oxidation exhibits strong antifeedant activity
against the granary weevil (Sitophilus granarius).
Monoterpenoid ketones such as pinocamphone, caran-2-one,
and menthone were also good starting materials for the synthesis
of lactams via Beckmann rearrangement of the corresponding
oximes.[22–24] Terpenoid lactams could be hydrolyzed to new
chiral amino acids – potential analogues of g-aminobutyric acid
(GABA). Lochyn´ski and coworkers showed that amino acids
obtained from (ꢀ)-cis-caran-4-one and (ꢀ)-menthone via
hydrolysis of corresponding e-lactams were able to cross the
blood–brain barrier and had neurological activity.[25] Further-
more, some natural lactams possess antifeedant activity.[26] To
the best of our knowledge, synthetic terpenoid lactams have not
been tested for antifeedant activity against insect pests so far.
Synthesis of Sabina d-Lactones
The syntheses of sabina d-lactones 3 and 4 were carried out
according to Scheme 1. The oxidation of natural (þ)-sabinene
(1, 70 % purity by GC) using an aqueous solution of sodium
periodate with a catalytic amount of potassium permanga-
nate[27] afforded, after purification by silica-gel column chro-
matography, pure (ꢀ)-sabina ketone 2 in 65 % isolated yield
(Scheme 1). The optical rotation of this product ([a]2D0 ꢀ25.7,
c 1.0 in CHCl3) in comparison with data reported in literature for
(þ)-sabina ketone[28] ([a]D20 þ18.1, c 0.8 in CHCl3, enantio-
meric excess (ee) 80 %) indicates that (ꢀ)-sabina ketone 2
possesses S and R configuration on the C-1 and C-5 carbon
atoms respectively. The spectral (1H and 13C NMR, mass
spectrometry (MS)) data of ketone 2 are the same as presented in
the literature.[28]
The Baeyer–Villiger oxidation of (ꢀ)-sabina ketone 2 with
meta-chloroperbenzoic acid (m-CPBA) afforded a mixture of
two d-lactones (by GC): 3 (75 %) and 4 (25 %). After separation
and purification of these products bysilica-gel columnchromato-
graphy, sabina lactones 3 and 4 were obtained in 43 and 20 %
isolated yield respectively. The structures of these products were
established on the basis of their spectral data (IR, 1H, 13C,
heteronuclear single quantum correlation (HSQC), correlated
spectroscopy (COSY) NMR, and high-resolution (HR)MS).
The presence of two multiplets at 0.76 and 0.87 ppm of the
CH2-7 protons in the 1H NMR spectrum of lactone 3 indicates
that the cyclopropane ring was not affected during the oxidation
process. In the same spectrum, a doublet of doublets (J 6.8, 2.8 Hz)
Journal compilation Ó CSIRO 2013
www.publish.csiro.au/journals/ajc

1400
R. Gniłka and C. Wawrzen´czyk
O
of (ꢀ)-sabina ketone 2 as well as the mechanism of Baeyer–
Villiger oxidation. It is very well documented in the literature
that chemical[31] and microbial oxidation[32,33] of chiral ketones
proceeds with the complete retention of configuration of migrat-
ing groups. So it can be concluded that lactones 3 and 4 possess a
configuration of chiral centres identical to (ꢀ)-sabina ketone 2.
In the literature, we found that compounds 3 and 4 were
obtained as products of the ozonolysis of (þ)-sabinene.[34]
Griesbaum and Miclaus presented the spectral data of these
lactones without assignment of signals in the 1H and 13C NMR
spectra to corresponding protons and carbon atoms. For these
reasons, we present in the Experimental section the complete
KMnO4, NaIO4,
K2CO3
2
5
1
3
4
6
10 % (v/v) CH2Cl2
20ЊC, 48 h
1
2
Fusarium
oxysporum
m-CPBA, CH2Cl2, rt
AM 21
1
spectroscopic (IR, H, 13C NMR, and HRMS) data for sabina
lactones 3 and 4.
O
O
O
2
O
2
5
Synthesis of Sabina d-Lactams
O
O
1
6
1
6
3
4
3
4
7
7
ϩ
(ꢀ)-Sabina ketone 2 was also the starting material for the
synthesis of new d-lactams (7 and 8). The reaction of this ketone
(2) with hydroxylamine hydrochloride afforded a mixture of
oximes (5a : 5b 60 : 40 by GC) (Scheme 2).
5
4
3
4
Difficulties with the separation of the sabina ketone oximes
(5a þ 5b) by silica-gel column chromatography pushed us to
separation of their esters. So the mixture of oximes (5a : 5b,
60 : 40 by GC) was converted by an enzymatic esterification
process into mixture of propionic esters. The lipase from
Thermomyces lanuginosus (Lipozyme TL, Novozymes) was
used as a catalyst with vinyl propionate as an acyl group donor.
As a result of this reaction, a mixture of oxime propionates
(6a : 6b, 60 : 40 according to GC) was obtained. The products
(6a þ 6b) were easily separated by silica gel-column chroma-
tography, giving pure oxime propionates 6a and 6b in 30 and
27 % isolated yields respectively (Scheme 2).
Scheme 1. Oxidation of (þ)-sabinene (1) to sabina d-lactones 3 and 4.
at 3.81 ppm was observed. The value of the chemical shift of this
multiplet and its integration indicate that only one proton (H-1)
is bound to the carbon atom connected with the alkoxy oxygen
atom. Furthermore, the chemical shift (61.20 ppm) of the carbon
atom (C-1) coupled with this proton (H-1) in the HSQC NMR
spectrum of lactone 3 is characteristic for a carbon atom directly
connected with an oxygen atom. These data clearly prove that an
oxygen atom was introduced between the carbonyl group and
more substituted carbon atom (C-1) in ketone 2.
The structures of these compounds were confirmed by their
spectral data (1H, 13C, COSY, and HSQC NMR). The signals of
the carbonyl carbon atoms of the ester group in the 13C NMR
spectra of oxime propionates 6a and 6b are located at 174.44 and
173.46 ppm respectively. Triplets at 1.10 and 1.11 ppm of
methyl group protons and quartets at 2.35 and 2.38 ppm of
methylene group protons for 6a and 6b respectively observed in
the 1H NMR spectra of the oxime propionates proved the
presence of the propionyl group in both (6a and 6b) products.
The E/Z configuration of the C¼N double bond was assigned
taking into account the chemical shifts of the CH2-3 group and
H-1 protons. In the 1H NMR spectrum of 6a, the multiplets of
the CH2-3 protons are located at 2.79 ppm and in the range of
1.80–2.11 ppm, whereas the multiplet of the H-1 proton appears in
the same region as one of the CH2-3 protons (1.80–2.11 ppm). The
difference in chemical shift of CH2-3 protons (Dd ¼ 0.9 ppm)
indicates that one of these, located closer to the C¼N bond
plane, is strongly deshielded by the cis-oriented alkoxycarbonyl
group. These data indicate that (þ)-sabina ketone oxime propi-
onate 6a is the E-isomer.
Taking into account the possibility of migration of the less-
substituted carbon atom to the electrophilic oxygen atom in a
Criegee intermediate during Baeyer–Villiger rearrange-
ment,[29,30] we expected the second, minor product to be the
so-called ‘abnormal’ lactone 4, in which the alkoxy oxygen
atom is located between the carbonyl group and less-substituted
carbon atom. Our expectation was fully confirmed by the
spectral data of this product. The two doublets of doublets of
1
doublets at 4.05 and 4.25 ppm in the H NMR spectrum of
product 4 are present. The chemical shifts of these multiplets
indicate that an oxygen atom was inserted between the carbonyl
group and less-substituted carbon atom (C-3) in (ꢀ)-sabina
ketone 2. Additionally, in the HSQC NMR spectrum of 4, these
two multiplets are coupled with the C-4 carbon atom, whose
chemical shift (64.54 ppm) is characteristic for a carbon atom
directly bound to an oxygen atom. It is worth noting that this
type of lactone is usually obtained by microbial Baeyer–Villiger
oxidation.[29] So (ꢀ)-sabina ketone 2 was also subjected to
microbial Baeyer–Villiger oxidation using whole cells of
Fusarium oxysporum AM21 as catalyst (Scheme 1). This micro-
organism was selected from three fungi strains tried in our
previous studies.[21]
In the case of oxime propionate 6b, the multiplets of the
1
CH2-3 protons in the H NMR spectrum are located closer to
each other (Dd ¼ 0.2 ppm). The signal of one of these protons is
located at 2.12–2.26 ppm (together with the multiplets of the
H-1 proton) and the second at 2.37 ppm. These data indicate that
it is a Z-isomer of sabina ketone oxime.
The microbial Baeyer–Villiger oxidation of (ꢀ)-sabina
ketone 2 afforded only one product (4) in good (32 %) isolated
1
yield. GC analysis and spectral data (IR, H, 13C, HSQC, and
COSY NMR) confirmed that this product is ‘abnormal’
lactone 4.
The configurations of the chiral centres of both obtained
lactones were established taking into account the configuration
The pure E-oxime propionate (6a) was hydrolyzed with a
2.5 % methanolic solution of potassium hydroxide at room
temperature (Scheme 2). As a result of this reaction, E-oxime
5a was obtained in 94 % yield (96 % purity according to GC;

Sabina d-Lactones and Sabina d-Lactams
1401
O
OH
O
N
N
O
2
KOH, MeOH
rt
NH2OH • HCl,
CH3COONa,
rt
6a
5a
O
(de of E isomer ϭ 96 %)
O
OH
O
N
2
N
O
Silica-gel column
chromatography
Lipozyme TL,
2
1
1
3
3
4
DIPE
4
5
5
6
6
rt
HO
N
O
6a ϩ 6b
5a ϩ 5b
N
O
KOH, MeOH
rt
6b
5b
(de of Z isomer ϭ 86 %)
Scheme 2. Resolution of sabina ketone oximes 5a and 5b.
diastereoisomeric excess (de) of E isomer ¼ 96 %). The struc-
ture of this compound was established on the basis of its spectral
(1H, 13C, HSQC, COSY, distortionless enhancement by polari-
zation transfer (DEPT) 135 NMR, and HRMS) data.
0.72 and 0.87 ppm in the 1H NMR spectrum of 5b indicate that
the cyclopropane moiety is present in the molecule. The one-
proton doublet of doublets (J 8.7 and 3.4 Hz) at 2.25 ppm in the
1H NMR spectrum of 5b was assigned to the H-1 proton. The
value of its chemical shift compared with the chemical shift of
1
The two multiplets at 0.55 and 0.78 ppm in the H NMR
1
spectrum of 5a confirm that the cyclopropane ring was not
affected during hydrolysis. The value of the chemical shift
(2.75 ppm) of one of the CH2-3 protons in the 1H NMR spectrum
of 5a indicates that it is strongly deshielded by the hydroxy
group. The second CH2-3 proton is located between 1.71 and
1.85 ppm. Therefore, the first, similarly to one of the CH2-3
protons in 6a, is located closer to the plane of the C¼N moiety.
These facts prove that the configuration of the C¼N double
bonds in oxime 5a is the same as in the oxime propionate 6a.
The value (9.16 ppm) of the chemical shift of the proton of the
hydroxy group in the 1H NMR spectrum of 5a indicates its acidic
character.
the same proton (1.68 ppm) in the H NMR spectrum of 5a
indicates the deshielding effect of the cis-oriented hydroxy
group. This fact clearly confirms the Z-configuration of the
C¼N double bond in 5b.
The signal of the hydroxy group proton is situated at
1
9.13 ppm in the H NMR spectrum of 5b. As in the case of
oxime 5a, E/Z isomerization of 5b to a mixture of oximes
(5a : 5b 60 : 40 by GC) was observed in tetrahydrofuran. The
corresponding oxime propionate (6b) did not undergo E/Z
isomerization in aprotic solvents but quickly isomerized to a
mixture of both oxime propionates (6a : 6b 60 : 40 by GC) in
chloroform solution.
During the workup of the reaction mixture and purification of
E-sabina ketone oxime 5a, E/Z isomerization in an aprotic
solvent (tetrahydrofuran) to a mixture of sabina ketone oximes
(5a : 5b 60 : 40 by GC) was observed. It is well documented in
the literature[35] that compounds containing a carbon–nitrogen
double bond undergo acid-catalyzed E/Z isomerization. It can be
suggested that the acidic proton of the hydroxy group catalyzes
the E/Z isomerization of 5a. Moreover, we did not observe E/Z
isomerization of E-oxime propionate 6a in aprotic solvents.
However, in chloroform solution, this ester (6a) very quickly
undergoes E/Z isomerization to a mixture of both oxime
propionates (6a : 6b 60 : 40 by GC).
These facts prove that the acidic proton of the hydroxy group
in Z-sabina ketone oxime 5b also catalyzes E/Z isomerization.
It can be concluded that sabina ketone oximes 5a and 5b are
unstable in protic and aprotic solvents. Such isomerization also
caused the Beckmann rearrangement of pure Z-oxime propio-
nate 6b with p-toluenesulfonyl chloride in alkaline solution to
give a mixture of lactams (7 : 8 39 : 61 by GC).
For these reasons, the inseparable mixture of sabina ketone
oximes (5a : 5b 60 : 40 by GC) was subjected to Beckmann
rearrangement with p-toluenesulfonyl chloride in alkaline solu-
tion.[36] As a result of this reaction, a mixture of two lactams: 7
(70 %) and 8 (30 %) was obtained (Scheme 3). These com-
pounds were separated by silica-gel column chromatography
and obtained in 23 % (7) and 8 % (8) yields.
The second oxime (5b) was obtained after hydrolysis of 6b in
84 % yield (95 % purity by GC, de of Z isomer ¼ 86 %). The
structure of this compound was also proved by spectral (1H, 13C,
HSQC, COSY, DEPT 135 NMR) data. The two multiplets at
The structures of lactams 7 and 8 were established on the
basis of their spectral (1H, 13C, HSQC, COSY, DEPT 135 NMR,

1402
R. Gniłka and C. Wawrzen´czyk
OH
HO
2. The E-isomer of (ꢀ)-sabina ketone oxime (5a, de of E
isomer ¼ 96 %) and Z-isomer oxime of the same ketone
(5b, de of Z isomer ¼ 86 %) were obtained. Unfortunately,
these compounds were unstable and underwent acid-
catalyzed E/Z isomerization in both aprotic and protic
solvents. It is noteworthy that this isomerization in aprotic
solvents was catalyzed by the acidic proton of the hydroxy
group of the oxime.
N
N
O
H
N
TsCl,
NaOH
O
2
2
5
1
6
3
4
NH
7
1
6
3
4
7
ϩ
ϩ
55ЊC
5
7
8
5a ϩ 5b
3. The (ꢀ)-sabina ketone oximes 5a and 5b are a good sub-
strates for enzymatic esterification catalyzed by lipase from
Thermomyces lanuginosus.
Scheme 3. Beckmann rearrangement of sabina ketone oximes 5a and 5b.
4. The two new d-lactams 7 and 8 were obtained from
(þ)-sabinene 1. Their physical and spectral data are
presented.
and HRMS) data. In the 1H NMR spectrum of 7, a broad singlet
at 6.95 ppm and multiplet at 2.42 ppm were observed. The first
was assigned to the proton of the NH group and the second to the
H-1 proton. The chemical shift of the H-1 proton indicates that it
is bonded to the carbon atom directly connected to the nitrogen
atom. Therefore, the nitrogen atom was introduced between the
carbonyl group and C-1 carbon atom in (ꢀ)-sabina ketone 2.
Furthermore, the H-1 proton is coupled in the COSY NMR
spectrum of product 7 with two protons giving doublets of
doublets at 0.58 ppm (J 5.4, 3.5 Hz) and 0.66 ppm (J 6.5, 5.4 Hz)
Experimental
General
Analytical TLC was performed on aluminium plates coated with
silica gel (Fluka, Kieselgel 60, F254). Most compounds were
detected by spraying plates with solution of: 1 % Ce(SO4)2, 2 %
H3[P(Mo3O10)4] in 10 % H2SO4, followed by heating at 1208C.
Lactams 7 and 8 were detected by spraying the plates with a
solution of: ninhydrin (0.3 g) in n-butanol (100 mL) and acetic
acid (3 mL), followed by heating at 1008C.
Liquid column chromatography was carried out on silica gel
(Kieselgel 60, 230–400 mesh ASTM, Merck) with suitable
mixtures of hexane, ethyl acetate, dichloromethane, diethyl
ether, acetone, chloroform, and methanol as eluents.
1
in the H NMR of this product. These two multiplets were
assigned to the protons of the CH2-7 methylene group and
indicate that the cyclopropane ring was not affected during the
Beckmann rearrangement.
Two multiplets at 3.03 and 3.17 ppm were observed in the
1H NMR spectrum of 8. They are coupled with carbon atom at
37.43 ppm (C-4) in the HSQC NMR spectrum of 8. These facts
allowed the assignment of these signals to the protons of the
CH2-4 methylene group in this lactam (8). The chemical shifts of
the signals of CH2-4 protons indicate that they are bonded to the
carbon atom directly connected with the nitrogen atom. There-
fore, the nitrogen atom was inserted between the carbonyl group
and C-3 carbon atom in (ꢀ)-sabina ketone 2. Two doublets of
doublets located at 0.81 ppm (J 9.3, 5.4 Hz) and 1.34 ppm (J 5.4
and 4.2 Hz) in the 1H NMR spectrum of 8 are coupled with the
H-1 proton (1.43 ppm, dd, J 9.3, 4.2 Hz) in the COSY NMR
spectrum of this compound. The chemical shifts of these protons
confirm the presence of the cyclopropane ring in the molecule.
The configurations of the chiral centres of lactams 7 and 8
were assigned taking into account the configuration of
(ꢀ)-sabina ketone 2 as well as the mechanism of Beckmann
rearrangement. It is well documented that Beckmann rearrange-
ment of enantiomerically pure oximes proceeds with the reten-
tion of configuration of a migrating group.[22–24,37,38] It can be
concluded that the configurations of the chiral centres in
products 7 and 8 have identical configurations to (ꢀ)-sabina
ketone 2. Taking into consideration the mechanism of Beck-
mann rearrangement, the amounts of lactams 7 and 8 obtained,
and their spectral data, it can be seen that compound 7 is a
product of the rearrangement of the trans oxime (5a) whereas 8
must be a product of the rearrangement of the cis oxime (5b)
(Scheme 3).
Gas chromatography analyses were performed with a flame
ionization detector (FID) on an Agilent Technologies 7890A.
The capillary columns used were: for 1–4, HP-5 (30 m ꢁ 0.32mm
ID ꢁ 1.0 mm film); oven temperature: start 908C; programmed
from 90 to 1508C at 58C minꢀ1, hold 2 min; from 150 to 3008C at
408C minꢀ1, hold 3 min; as carrier gas, H2 was used at a flow rate
of 1 mL minꢀ1; split ratio 30 : 1; for 5a, 5b, 6a, 6b, 7, 8:
CP-ChiralSil-L-Val (25 m ꢁ 0.25 mm ID ꢁ 0.12 mm film); oven
temperature: start 1008C; programmed from 100 to 2008C at
158C minꢀ1, hold 3 min; as carrier gas, H2 was used at a flow rate
of 2 mL minꢀ1; split ratio 15 : 1. The injection temperature was
kept at 2508C and FID temperature at 3008C for all analyses.
HRMS (electrospray ionization (ESI)) was carried out on a
high-resolution mass spectrometer micrOTOF-Q II (Bruker).
Optical rotation was measured on a Jasco P-2000-Na with
iRM automatic polarimeter in chloroform solution for com-
pounds 1–4, 7, and 8, and in tetrahydrofuran solution for
compounds 6a and 6b. Concentration is denoted in g per
100 mL.
1H, 13C, 1H–1H COSY, and 1H–13C HSQC NMR spectra for
products 2, 3, and 4 were recorded in CDCl3 solution on a Bruker
Avance DRX 300 MHz; spectra for products 7 and 8 were
recorded in CDCl3 solution on a Bruker Avance 600 MHz;
spectra for products 5a, 5b, 6a, and 6b were recorded in [D8]
tetrahydrofuran solution on a Bruker Avance DRX 300 MHz.
Chemical shifts were referenced to the residual signal of CHCl3
(d 7.26 ppm for 1H NMR, 77.0 ppm for13C NMR) or THF
(d 3.58 ppm for 1H NMR, 67.57 ppm for 13C NMR).
Conclusions
1. One can observe the analogy of biotransformation of
(ꢀ)-sabina ketone 2 and microbial transformation of
(þ)-b-thujone[21] in Fusarium oxysporum AM21 culture.
Both ketones were oxidized only to the ‘abnormal’ lactones,
in which less-substituted carbon atoms migrated to the
electrophilic oxygen atom in Criegee intermediates.
Chemical Synthesis of Sabina Lactones
A solution of (þ)-sabinene (1, 500 mL, mixture of sabinene and
b-pinene 70 : 30 by GC, Treatt PLC) in dichloromethane
(20 mL) was added to 200 mL of an aqueous solution of sodium

Sabina d-Lactones and Sabina d-Lactams
1403
periodate (6.1 g, 28.5 mmol), potassium permanganate (170 mg,
1.08 mmol), and potassium bicarbonate (1.2 g, 9 mmol). The
reaction mixture was stirred for 3 days at 208C. Progress of the
oxidation of (þ)-sabinene (1) was monitored by GC. After this
time, the product was extracted with dichloromethane
(3 ꢁ 100 mL), dried over anhydrous MgSO4, and concentrated
under vacuum. The crude product (2) was separated and purified
by silica-gel column chromatography (hexane/ethyl acetate
10 : 1) to give pure (ꢀ)-sabina ketone 2 (196 mg, 1.4 mmol,
65 % yield).
University. The microorganism was maintained at 48C on
Sabouraud agar slants containing peptone (10 g Lꢀ1), glucose
(40 g Lꢀ1), and agar (15 g Lꢀ1). A loop of fungal strain was used
to inoculate 100 mL of sterile medium (10 g Lꢀ1 peptone,
30 g Lꢀ1 glucose) in a 300-mL Erlenmeyer flask. After 3 days of
agitation on a rotary shaker at 150 rpm at 258C, the culture was
used to inoculate 400 mL of the same medium in a 2-L flat-
bottomed flask. The fungal strain was incubated on a rotary
shaker at 150 rpm at 258C. After 3 days, (ꢀ)-sabina ketone 2
(83 mg, 0.6 mmol) dissolved in acetone (1 mL) was added to the
grown culture. The reaction mixture was shaken for 11 days and
progress of transformation of 2 was monitored by GC. After that
time, the product was extracted with dichloromethane
(3 ꢁ 150 mL). The organic solution was dried over anhydrous
MgSO4 and concentrated under vacuum. Crude product was
purified by silica-gel column chromatography (hexane/ethyl
acetate/diethyl ether 3 : 1 : 1) to give pure lactone 4 (30 mg,
0.19 mmol, 32 % yield).
A
solution of meta-chloroperbenzoic acid (166 mg,
0.96 mmol, 77 %, Aldrich) in dichloromethane (5 mL) was dried
over anhydrous MgSO4 and then evaporated under nitrogen.
This prepared peracid in a small amount of dichloromethane
(100 mL) was added to (ꢀ)-sabina ketone 2 (53 mg, 0.38 mmol)
and the mixture was stirred for 72 h at room temperature. The
progress of the reaction was monitored by TLC and GC. When
the reaction was complete, dichloromethane (5 mL) was added
to the reaction mixture. The solution was washed with sodium
sulfite and sodium bicarbonate, dried (MgSO4), and the solvent
was evaporated under vacuum. The crude products (3 and 4)
were separated and purified by silica-gel column chromatogra-
phy (hexane/ethyl acetate/dichloromethane 10 : 1 : 1) to give
pure lactone 3 (25 mg, 0.16 mmol, 43 % yield) and pure lactone
4 (12 mg, 0.08 mmol, 20 % yield). It is noteworthy that lactone 4
was invisible on TLC plates after spraying with cerium–
molybdenum solution and heating at 1208C. For this reason,
product 4 was detected by GC. The physical and spectral data of
all synthesized products are given below.
Enzymatic Esterification of Sabina Ketone Oximes
The (ꢀ)-sabina ketone 2 (65 mg, 0.5 mmol) dissolved in meth-
anol (3 mL) was added to 5 mL of an aqueous solution of
hydroxylamine hydrochloride (75 mg, 1.1 mmol) and sodium
acetate (150 mg, 1.9 mmol). The reaction mixture was stirred for
48 h at room temperature. After that time, the products (5a þ 5b)
were extracted with diethyl ether (3 ꢁ 25 mL). The organic
solution was washed with sodium bicarbonate, dried over
anhydrous MgSO4, and concentrated under vacuum. The mix-
ture of the two diastereoisomers of sabina ketone oximes
obtained (5a : 5b 60 : 40 according to GC, 83 mg, 0.5 mmol) was
dissolved in diisopropyl ether (1 mL). Lipase from Thermo-
myces lanuginosus(160 mg, Lipozyme TL) and vinyl propionate
(184 mg, 1.8 mmol) were added to this solution. The reaction
mixture was stirred overnight at room temperature. The progress
of the reaction was monitored by GC. After 14 h, the reaction
mixture was filtered through Celite 560 (Sigma–Aldrich) and
washed with diisopropyl ether (20 mL). Then, the organic
solution was washed with sodium bicarbonate, dried over
anhydrous MgSO4, and concentrated under vacuum. The crude
products (6a and 6b) were separated and purified by silica-gel
column chromatography (hexane/acetone 5 : 1) to give pure
E-oxime propionate 6a (29 mg, 30 % yield) and pure Z-oxime
propionate 6b (26 mg, 27 % yield).
(1S,6R)-6-(Propan-2-yl)-2-oxabicyclo[4.1.0]
heptan-3-one 3
Colourless liquid. [a]2D0 40.8 (c 1.2 in CHCl3). nmax (film)/cmꢀ1
1740, 1468, 1174, 757. dH (300 MHz, CDCl3) 0.76 (t, J 6.9, 1H,
one of the CH2-7), 0.87 (dd, J 7.0, 2.8, 1H, one of the CH2-7),
0.91 and 1.00 (two d, J 6.5, 6H, (CH3)2CH–), 1.11 (m, 1H,
(CH3)2CH–), 1.74 (ddd, J 13.9, 8.5, 5.7, 1H, one of the CH2-5),
2.18 (ddd, J 13.9, 6.6, 6.2, 1H, one of the CH2-5), 2.31 (ddd,
J 16.6, 6.6, 5.7, 1H, one of the CH2-4), 2.40 (ddd, J 16.6, 8.5, 6.2,
1H, one of the CH2-4), 3.81 (dd, J 6.8, 2.8, 1H, H-1).
dC (75.5 MHz, CDCl3) 17.94 and 18.88 ((CH3)2CH–), 18.93
(C-7), 21.66 (C-5), 24.02 (C-6), 28.73 (C-4), 34.22 ((CH3)2CH–),
61.20 (C-1), 171.46 (C-3). m/z (ESI) 177.0882 [M þ Na]þ (calc.
for C9H14NaO2 177.0891).
The physical and spectral data of the oxime propionates
obtained are given below.
(1S,6S)-6-(Propan-2-yl)-3-oxabicyclo[4.1.0]
heptan-2-one 4
(1)-(E)-Sabina Ketone Oxime Propionate 6a
Colourless liquid. [a]2D0 18.4 (c 0.5 in CHCl3). nmax (film)/cmꢀ1
1717, 1216, 1085, 770, 750, 668. dH (300 MHz, CDCl3) 0.93
(dd, J 9.5, 5.6, 1H, one of the CH2-7), 0.98 and 0.99 (two d, J 6.8,
6H, (CH3)2CH–), 1.23 (septet, J 6.8, 1H, (CH3)2CH–), 1.58 (dd,
J 5.6, 4.3, 1H, one of the CH2-7), 1.65 (dd, J 9.5, 4.3, 1H, H-1),
1.82 (dd, J 13.9, 3.6, 1H, one of the CH2-5), 2.04 (ddd, J 13.9,
13.5, 6.0, 1H, one of the CH2-5), 4.05 (ddd, J 13.5, 12.0, 3.6, 1H,
one of the CH2-4), 4.25 (ddd, J 12.0, 6.0, 1.4, 1H, one of the CH2-4).
dC (75.5 MHz, CDCl3) 14.96 (C-7), 18.97 and 19.09
((CH3)2CH–), 20.64 (C-5), 23.07 (C-1), 29.81 ((CH3)2CH–),
31.05 (C-6), 64.54 (C-4), 171.94 (C-2). m/z (ESI) 177.0873
[M þ Na]þ (calc. for C9H14NaO2 177.0891).
Colourless liquid. [a]2D0 73 (c 1.355 in THF). dH (300 MHz, [D8]
THF) 0.76 (dd, J 4.7, 3.3, 1H, one of the CH2-6), 0.92 and 0.98
(two d, J 6.8, 6H, (CH3)2CH–), 1.01 (m, 1H, one of the CH2-6),
1.10 (t, J 7.5, 3H, CH3CH2COO–), 1.57 (septet, J 6.8, 1H,
(CH3)2CH–), 1.80–2.11 (m, 4H, one of the CH2-3, CH2-4, and
H-1), 2.35 (q, J 7.5, 2H, CH3CH2COO–), 2.79 (m, 1H, one of the
CH2-3). dC (75.5 MHz, CDCl3) 9.55 (CH3CH2COO–), 17.75
(C-6), 19.86 and 20.02 ((CH3)2CH–), 25.12 (C-3), 26.71
(CH3CH2COO–), 26.82 (C-4), 27.69 (C-1), 33.30 ((CH3)2CH–),
38.52 (C-5), 171.59 (C-2), 174.44 (CH3CH2COO–). m/z (ESI)
232.1293 [M þ Na]þ (calc. for C12H19NNaO2 232.1313).
Microbial Synthesis of Sabina Lactone 4
(1)-(Z)-Sabina Ketone Oxime Propionate 6b
Fusarium oxysporum AM21 was obtained from the Collection
of the Institute of Biology and Botany, Wroclaw Medical
Colourless liquid. [a]2D0 39 (c 0.610 in THF). dH (300 MHz, [D8]
THF) 0.92 and 0.97 (two d, J 6.8, 6H, (CH3)2CH–), 0.95 (m, 1H,

1404
R. Gniłka and C. Wawrzen´czyk
one of the CH2-6), 1.06 (m, 1H, one of the CH2-6), 1.11 (t, J 7.5,
3H, CH3CH2COO–), 1.53 (septet, J 6.8, 1H, (CH3)2CH–),
1.76–1.95 (m, 2H, CH2-4), 2.12–2.26 (m, 2H, one of the CH2-3
and H-1), 2.37 (m, 1H, one of the CH2-3), 2.38 (q, J 7.5, 2H,
CH3CH2COO–). dC (75.5 MHz, CDCl3) 9.56 (CH3CH2COO–),
18.21 (C-6), 19.85 and 19.97 ((CH3)2CH–), 25.15 (C-1),
25.73 (C-4), 26.72 and 27.14 (CH3CH2COO– or C-3),
33.52 ((CH3)2CH–), 40.08 (C-5), 171.98 (C-2), 173.46
(CH3CH2COO–). m/z (ESI) 232.1304 [M þ Na]þ (calc. for
C12H19NNaO2 232.1313).
vacuum and the products (7 and 8) were extracted with diethyl
ether (3 ꢁ 25 mL). The ether extracts were combined, washed
with sodium bicarbonate and brine, dried over anhydrous
MgSO4, and concentrated under vacuum. The products (7 and 8)
were separated and purified by silica-gel column chromato-
graphy (hexane/acetone 2 : 1). The pure lactam 7 (50 mg, 23 %
yield with respect to the ketone) and impure lactam 8 (50 mg)
were obtained. Therefore, compound 8 was additionally purified
by preparative TLC (chloroform/methanol 20 : 1) to give 17 mg
(8 % yield with respect to the ketone) of pure 8. The physical and
spectral data of the lactams obtained are given below:
Hydrolysis of Sabina Ketone Oxime Propionates
(1S,6R)-6-(Propan-2-yl)-2-azabicyclo[4.1.0]
heptan-3-one 7
A methanolic solution of potassium hydroxide (1 mL of 2.5 %
solution) was added to the oxime propionate (6a, 29 mg,
0.14 mmol; 6b, 26 mg, 0.12 mmol). The reaction mixture was
stirred for 30 min at room temperature and then concentrated
under vacuum. The ether solution of crude oxime (3 mL) was
transferred to a flask containing a saturated aqueous solution of
sodium bicarbonate (5 mL). The product was extracted with
diethyl ether (4 ꢁ 3 mL). The ether extracts were combined,
dried over anhydrous MgSO4, and concentrated under vacuum.
As a result, 5a (20 mg, 94 % yield), (96 % purity according to
GC, de of E isomer ¼ 96 %) and 5b (16 mg, 84 % yield), (95 %
purity according to GC; de of Z isomer ¼ 86 %) were obtained.
The spectral data of the oximes obtained are given below.
Colourless liquid. [a]2D0 76.4 (c 1.1 in CHCl3). nmax (film)/cmꢀ1
3210, 1670, 1468. dH (600 MHz, CDCl3) 0.58 (dd, J 5.4, 3.5, 1H,
one of the CH2-7), 0.66 (dd, J 6.5, 5.4, 1H, one of the CH2-7),
0.90 and 0.97 (two d, J 6.7, 6H, (CH3)2CH–), 1.05 (septet, J 6.7,
1H, (CH3)2CH–), 1.60 (m, 1H, one of the CH2-5), 2.08–2.14 (m,
2H, one of the CH2-5 and one of the CH2-4), 2.24 (m, 1H, one of
the CH2-4), 2.42 (m, 1H, H-1), 6.95 (bs, 1H, NH). dC (75.5 MHz,
CDCl3) 18.29 and 19.09 ((CH3)2CH–), 22.61 (C-7), 23.23 (C-5),
24.44 (C-6), 30.62 (C-4), 34.36 (C-1), 35.68 ((CH3)2CH–),
174.11 (C-3). m/z (ESI) 176.1040 [M þ Na]þ (calc. for
C9H15NNaO 176.1051).
(E)-Sabina Ketone Oxime 5a
(1S,6S)-6-(Propan-2-yl)-3-azabicyclo[4.1.0]
heptan-2-one 8
Colourless liquid (20 mg, yield 94 %, de of E isomer ¼ 96 %).
[a]2D0 61 (c 1.105 in THF). dH (300 MHz, [D8]THF) 0.55 (dd,
J 4.5, 3.4, 1H, one of the CH2-6), 0.78 (dd, J 8.6, 4.5, 1H, one of
the CH2-6), 0.90 and 0.96 (two d, J 6.9, 6H, (CH3)2CH–), 1.50
(septet, J 6.9, 1H, (CH3)2CH–), 1.68 (dd, J 8.6, 3.4, 1H, H-1),
1.71–1.85 (m, 3H, one of the CH2-3 and CH2-4), 2.75 (m, 1H,
one of the CH2-3), 9.16 (s, 1H, –OH). dC (75.5 MHz, CDCl3)
16.99 (C-6), 19.99 and 20.14 ((CH3)2CH–), 22.97 (C-3), 26.82
(C-4), 27.63 (C-1), 33.54 ((CH3)2CH–), 36.70 (C-5), 164.57
(C-2). m/z (ESI) 176.1041 [M þ Na]þ (calc. for C9H15NNaO
176.1051).
Colourless liquid. [a]2D0 34 (c 1.0 in CHCl3). nmax (film)/cmꢀ1
3213, 1658, 1495. dH (600 MHz, CDCl3) 0.81 (dd, J 9.3, 5.4, 1H,
one of the CH2-7), 0.97 and 0.98 (two d, J 6.8, 6H, (CH3)2CH–),
1.17 (septet, J 6.8, 1H, (CH3)2CH–), 1.34 (t, J 4.8, 1H, one of the
CH2-7), 1.43 (dd, J 9.3, 4.2, 1H, H-1), 1.78–1.82 (m, 2H,
CH2-5), 3.11 (m, 1H, one of the CH2-4), 3.17 (m, 1H, one of the
CH2-4), 6.51 (bs, 1H, NH). dC (151 MHz, CDCl3) 14.19 (C-7),
19.07 and 19.31 ((CH3)2CH–), 20.00 (C-5), 24.00 (C-1), 30.75
(C-6), 35.71 ((CH3)2CH–), 37.43 (C-4); 174.48 (C-2). m/z (ESI)
176.1042 [M þ Na]þ (calc. for C9H15NNaO 176.1051).
(Z)-Sabina Ketone Oxime 5b
Supplementary Material
Colourless liquid (16 mg, yield 84 %, de of Z isomer ¼ 86 %).
[a]2D0 19 (c 0.755 in THF). dH (300 MHz, [D8]THF) 0.72 (dd,
J 4.4, 3.4, 1H, one of the CH2-6), 0.87 (ddd, J 8.7, 4.4, 1.3, 1H,
one of the CH2-6), 0.90 and 0.95 (two d, J 6.9, 6H, (CH3)2CH–),
1.50 (septet, J 6.9, 1H, (CH3)2CH–), 1.73–1.83 (m, 2H, CH2-4),
1.97 (dd, J 16.2, 9.3, 1H, one of the CH2-3), 2.16 (ddd, J 16.2,
8.4, 2.4, 1H, one of the CH2-3), 2.25 (dd, J 8.7, 3.4, 1H, H-1),
9.10 (s, 1H, –OH). dC (75.5 MHz, CDCl3) 16.60 (C-6), 19.98 and
20.12 ((CH3)2CH–), 23.29 (C-1), 26.00 (C-4), 26.84 (C-3),
33.70 ((CH3)2CH–), 37.88 (C-5), 163.47 (C-2). m/z (ESI)
176.1045 [M þ Na]þ (calc. for C9H15NNaO 176.1051).
The NMR spectra of all obtained compounds are available on
the Journal’s website.
Acknowledgements
This research was supported financially by the European Union through the
European Regional Development Fund (grant No. POIG.01.03.01–00–158/
09–05). We thank Dr Anna Poliwoda (Opole University, Poland) for per-
forming HRMS measurements.
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