The new halolactones and hydroxylactone with trimethylcyclohexene ring obtained through combined chemical and microbial processes

Article abstract of DOI:10.1016/j.molcatb.2014.02.012

The commercially available racemic (±) α-ionone was used as a substrate for the four-step chemical synthesis of three new γ- halolactones. During these processes known α-ionol and new compounds: γ,δ-unsaturated ester ((6E)-5-(2,6,6-trimethylcyclohex-2-en-1-yl) oct-6-en-3-one) (3) and γ,δ-unsaturated acid ((6E)-3-(2,6,6- trimethylcyclohex-2-en-1-yl)hex-4-enoic acid) (4), were obtained as intermediates. γ,δ-Unsaturated acid was used as a substrate for obtaining also new compounds: 5-(1-chloroethyl)-4-(2,6,6-trimethylcyclohex-2-en- 1-yl)dihydrofuran-2(3H)-one (5), 5-(1-bromo)-4-(2,6,6-trimethylcyclohex-2-en-1- yl)dihydrofuran-2(3H)-one (6) and 5-(1-iodo)-4-(2,6,6-trimethylcyclohex-2-en-1- yl)dihydrofuran-2(3H)-one (7). At the last step these halolactones were converted into the hydroxylactone by microorganisms. Several fungal strains (Fusarium species, Syncephalastrum racemosum, Botrytis cinerea) were tested. Most of the selected microorganisms transformed these lactones by hydrolytic dehalogenation into a new 5-(1-hydroxyethyl)-4-(2,6,6-trimethylcyclohex-2-en-1- yl)dihydrofuran-2(3H)-one (8), mainly the (+) stereoisomer. The hydroxylactone obtained during biotransformation has been examined for its antimicrobial activity against bacteria, yeasts and fungi. It was found that this compound exhibits growth inhibition against some tested microorganisms. The structure of all the substrates and products was established on the basis of their spectral data.

Full text of DOI:10.1016/j.molcatb.2014.02.012

Journal of Molecular Catalysis B: Enzymatic 102 (2014) 195–203
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
The new halolactones and hydroxylactone with trimethylcyclohexene
ring obtained through combined chemical and microbial processes
a
a
b
Małgorzata Grabarczyka,∗, Wanda Ma˛czka , Katarzyna Win´ ska , Barbara Zarowska ,
˙
Mirosław Anioła
a Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
b Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Chełmon´skiego 37/41, 51-630 Wrocław,
Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 October 2013
Received in revised form 13 January 2014
Accepted 21 February 2014
Available online 6 March 2014
The commercially available racemic
( ) ␣-ionone was used as a substrate for the four-
step chemical synthesis of three new ␥-halolactones. During these processes known ␣-ionol
and new compounds: ␥,␦-unsaturated ester ((6E)-5-(2,6,6-trimethylcyclohex-2-en-1-yl)oct-6-en-3-
one) (3) and ␥,␦-unsaturated acid ((6E)-3-(2,6,6-trimethylcyclohex-2-en-1-yl)hex-4-enoic acid) (4),
were obtained as intermediates. ␥,␦-Unsaturated acid was used as
a substrate for obtaining
also new compounds: 5-(1-chloroethyl)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)dihydrofuran-2(3H)-one
(5), 5-(1-bromo)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)dihydrofuran-2(3H)-one (6) and 5-(1-iodo)-4-
(2,6,6-trimethylcyclohex-2-en-1-yl)dihydrofuran-2(3H)-one (7). At the last step these halolactones were
converted into the hydroxylactone by microorganisms. Several fungal strains (Fusarium species, Syn-
cephalastrum racemosum, Botrytis cinerea) were tested. Most of the selected microorganisms transformed
these lactones by hydrolytic dehalogenation into a new 5-(1-hydroxyethyl)-4-(2,6,6-trimethylcyclohex-
2-en-1-yl)dihydrofuran-2(3H)-one (8), mainly the (+) stereoisomer. The hydroxylactone obtained during
biotransformation has been examined for its antimicrobial activity against bacteria, yeasts and fungi.
It was found that this compound exhibits growth inhibition against some tested microorganisms. The
structure of all the substrates and products was established on the basis of their spectral data.
© 2014 Elsevier B.V. All rights reserved.
Keywords:
␣-Ionone
Halolactones
Hydroxylactone
Hydrolytic dehalogenation
Fusarium species
Antimicrobial activity
1. Introduction
sponge exhibited activity against human protein tyrosine phos-
phatase 1B [5]. These compounds usually exist in living organisms
Ionones are an important group of natural compounds known as
rose ketones. They occur in different essential oils, including rose
oil, and are often used in the fragrance industry because of their
interesting scents. Structural analogues of ionone often occur in
nature and are responsible for the biological properties of different
plants and animals. Such compounds showing cytotoxic activity
have been isolated from tropical [1] or Mediterranean [2] shrubs.
Ichthyotoxic compounds were isolated from the nudibranch of
Southern California [3]. Ionone type bisnorsesquiterpenoids, which
have been isolated from the Spanish sunflower, showed allelo-
pathic activity [4]. Otheranalogues isolated from the Hainan marine
in small quantities, thus isolating them is difficult and expensive. A
better route is using methods of chemical synthesis to obtain poten-
tially bioactive compounds. Some analogues of ␣-ionone, including
a substituted aromatic ring in their structure obtained by Srivastava
[6], showed anti-ischaemic activity. Other heterocyclic derivatives
of ionone synthesized by Balbi [7,8] exhibited anti-inflammatory
properties. Total synthesis of oxy- and hydroxy-derivatives of ␣-
ionone was reported by Kikuchi [9]. There are also publications
about the biotransformation of ␣-ionone which led to receiving an
alcohol or different oxy-derivatives of this ketone [10–14].
Biotransformation is a very good method that can be used to
introduce the hydroxy group to different molecules. Many exam-
ples are known of filamentous fungi being used for this process. The
substrates that were used for biotransformation were terpenoids
[15–21], sesquiterpenoids [22], natural drimenic lactones [23], dri-
manic terpenes [24] and lovastatin [25].
∗
Corresponding author. Tel.: +48 71 3205 252.
E-mail addresses: magrab@onet.pl (M. Grabarczyk), wanda m19@o2.pl
(W. Ma˛czka), katarzyna.winska@up.wroc.p (K. Win´ ska),
Lactones are a group of compounds that are found all over the
world, mainly among plants and sea organisms. Some lactones
˙
barbara.zarowska@up.wroc.pl (B. Zarowska), miroslaw.aniol@up.wroc.pl
(M. Anioł).
http://dx.doi.org/10.1016/j.molcatb.2014.02.012
1381-1177/© 2014 Elsevier B.V. All rights reserved.

196
M. Grabarczyk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 195–203
include atoms of chlorine or bromine in their structure and exhibit
a variety of favourable activities including: cytotoxic [26–29], anti-
fungal [26,30,31], antiviral [26], antibacterial [26,32] or anticancer
[33]. Thanks to their properties these compounds are often used
in traditional medicine as therapeutic agents. Similar activities are
shown by lactones that include a hydroxy group in their structure:
antipyretic or antirheumatic [34], cytotoxic [35–37], antifungal [38]
or antibacterial [39].
When looking for new derivatives of ␣-ionone we decided to
synthesize several compounds, i.e. halolactones as well as hydroxy-
lactones with a trimethylcyclohexene ring, using both the synthetic
way and microbes. The microorganisms were used at the final stage
because of their ability to enantioselectively introduce a hydroxy
group in the place of a halogen.
2.2.2. (2,6,6-Trimethylcyclohex-2-en-1-yl)but-3-en-2-ol (2)
The reduction of ␣-ionone using lithium aluminium hydride
according to the known procedure [40] gave ␣-ionol (9.3 g, 92%).
The product is characterized by the data given below (comparable
with literature data [41]): ˛2D0∼0◦. 1H NMR (300 MHz, CDCl3) ı 0.80
and 0.82 (two s, 3H, CH3-11), 0.88 and 0.89 (two s, 3H, CH3-12), 1.17
(m, 1H, one of CH2-2), 1.26 (d, J = 6.3 Hz, 3H, CH3-10), 1.40 (m, 1H,
one of CH2-2), 1.49 (m, 1H, OH), 1.57 (s, 3H, CH3-13), 1.99 (m, 2H,
CH2-3), 2.08 (d, J = 8.7 Hz, 1H, H-6), 4.30 (dd, J = 6.3, 6.1 Hz, 1H, H-
9), 5.40 (m, 1H, H-4), 5.44 (dd, J = 15.3, 8.7 Hz, 1H, H-7), 5.52 (dd,
J = 15.3, 6.1 Hz, 1H, H-8). IR (KBr, cm−1): 3351, 2914, 1661, 1364,
1064.
2.2.3. (6E)-5-(2,6,6-trimethylcyclohex-2-en-1-yl)oct-6-en-3-one
(3)
Ester (12.1 g, 84%) obtained as a product of Claisen rearrange-
ment (orthoacetate modification) [42,43] is characterized by the
data given below: nD = 1.4765. 1H NMR (300 MHz, CDCl3) 0.87 (s,
3H, CH3-13 cis), 0.90 (s, 3H, CH3-13 trans), 1.02 (s, 3H, CH3-14 cis),
1.04 (s, 3H, CH3-14 trans), 1.25 (t, J = 7.2 Hz, 3H, OCH2CH3 trans),
1.27 (t, J = 7.2 Hz, 3H, OCH2CH3 cis), 1.57 (m, 2H, 2 × H-6), 1.62–1.66
(m, 12H, 2 × CH3-10 and 2 × CH3-15), 1.75 (dd, J = 15.1, 1.5 Hz, 4H,
2 × CH2-2), 1.96 (m, 4H, 2 × CH2-3), 2.22 (two d, J = 11.0 Hz, 2H, CH2-
11 trans), 2.45 (m, 2H, CH2-11 cis), 2.98 (m, 2H, 2 × H-7), 4.11 (q,
J = 7.2 Hz, 2H, OCH2CH3 trans), 4.12 (q, J = 7.2 Hz, 2H, OCH2CH3 cis),
5.33–5.53 (m, 6H, H-4, H-8, H-9); 13C NMR (300 MHz, CDCl3) ı 14.30
and 14.34 (OCH2CH3), 17.93 and 18.01 (C-2), 23.08 and 23.10 (C-
3), 25.30 and 25.88 (C-10), 28.47 and 28.50 (C-14), 28.52 and 28.53
(C-13), 30.52 and 31.00 (C-1), 33.46 and 33.54 (C-15), 38.23 and
38.50 (C-11), 39.67 and 42.54 (C-7), 53.58 and 53.96 (C-6), 60.06
and 60.10 (OCH2CH3), 122.32 and 123.34 (C-9), 123.84 and 125.26
(C-8), 132.98 and 133.11 (C-4), 134.10 and 134.28 (C-5), 172.91 and
173.70 (C-12). IR (KBr, cm−1): 3000, 1719, 1439, 1029. EI-MS m/z
(%) 264 [M+H], 264 (8), 219 (2), 176 (6), 141 (5), 123 (100), 95 (11),
81 (39), 67 (13), 57 (7), 39 (13).
2. Experimental
2.1. General
The progress of chemical reactions and biotransformations
and the purity of the isolated products were monitored by
the TLC technique on silica gel-coated aluminium plates (DC-
Alufolien Kieselgel 60 F254, Merck) and by GC analysis which
was carried out on a Agilent Technologies 6890N instrument
using an DB-17 column (cross-linked methyl silicone gum,
30 m × 0.32 mm × 0.25 ␮m). The temperatures during the GC anal-
ysis were as follows: injector 150 ◦C, detector (FID) 280 ◦C,
column temperature: 120 ◦C, 120–280 ◦C (rate 25 ◦C/min), 280 ◦C
(hold 1 min). The structures of obtained compounds were also
confirmed by gas chromatography–mass spectrometry (GC-MS)
analysis using Varian SATURN 2000 instrument (EI ioniza-
tion) with an HP-1 column (cross-linked methyl silicone gum,
25 m × 0.32 mm × 0.52 ␮m) under the following conditions: injec-
tor 200◦C, detector 300◦C, column temperature: 120◦C (hold 2 min),
120–300◦C (rate 20◦C/min), 300◦C (hold 3 min). The enantiomeric
compositions of the products obtained during biotransforma-
tion were determined by GC analysis using the chiral column
CP-cyclodextrin-B-325 (30 m × 0.25 mm × 0.25 ␮m) under the fol-
lowing conditions: injector 200 ◦C, detector (FID) 250 ◦C, column
temperature: 140 ◦C (hold 45 min), 140–200 ◦C (rate 20 ◦C/min),
and 200 ◦C (hold 1 min). All of the products were purified by means
of preparative column chromatography on silica gel (Kieselgel 60,
230–400 mesh).
2.2.4. (6E)-3-(2,6,6-trimethylcyclohex-2-en-1-yl)hex-4-enoic
acid (4)
Basic hydrolysis of ester 3 according to the procedure described
previously [43] gave 2.5 g (74%) of acid with the following physi-
cal and spectral data: thick oil. 1H NMR (300 MHz, CDCl3) ı 0.85
(s, 3H, CH3-13 cis), 0.88 (s, 3H, CH3-13 trans), 0.99 (s, 3H, CH3-
14 cis), 1.02 (s, 3H, CH3-14 trans), 1.56 (m, 2H, 2 × H-6), 1.60–1.65
(m, 12H, 2 × CH3-10 and 2 × CH3-15), 1.69 (dd, J = 15.5, 1.5 Hz, 4H,
2 × CH2-2), 1.96 (m, 4H, 2 × CH2-3), 2.24 (two d, J = 10.8 Hz, 2H, CH2-
11 trans), 2.47 (m, 2H, CH2-11 cis), 2.97 (m, 2H, 2 × H-7), 5.27–5.53
(m, 6H, H-4, H-8, H-9), 8.80 (m, 2H, 2 × COOH). 13C NMR (300 MHz,
CDCl3) ı 18.00 and 18.07 (C-2), 23.08 and 23.09 (C-3), 25.31 and
25.83 (C-10), 28.48 and 28.53 (C-14), 28.44 and 28.46 (C-13), 30.60
and 31.00 (C-1), 33.49 and 33.55 (C-15), 37.90 and 38.07 (C-11),
39.31 and 42.17 (C-7), 53.72 and 53.79 (C-6), 122. 50 and 123.51
(C-9), 124.21 and 125.52 (C-8), 132.90 and 134.03 (C-4), 135.80
and 136.12 (C-5), 179.00 and 180.00 (C-12). IR (KBr, cm−1): 2968,
1792, 1456, 1172.
1H NMR and 13C NMR spectra were recorded in a CDCl3 solu-
tion on a Bruker Avance DRX 300 spectrometer. The assignments
of 13C chemical shifts were made by means of a C/H correlation het-
eronuclear multiple quantum coherence (HMQC). IR spectra were
determined using FTIR on a Thermo-Mattson IR 300 spectrometer.
Optical rotations were measured on an Jasco P-2000 polarimeter.
2.2. Organic synthesis
The starting material, racemic ( ) ␣-ionone (1), was purchased
from Fluka. All other products were obtained according to proce-
dures described below.
2.2.5. 5-(1-Chloroethyl)-4-(2,6,6-trimethylcyclohex-2-en-1-
yl)dihydrofuran-2(3H)-one
(5)
2.2.1. (2,6,6-Trimethylcyclohex-2-en-1-yl)but-3-en-2-on (1)
1H NMR (300 MHz, CDCl3) ı 0.85 (s, 3H, CH3-11), 0.92 (s, 3H,
CH3-12), 1.22 (ddd, J = 13.4, 4.9, 4.5 Hz, 1H, one of CH2-2), 1.45 (ddd,
J = 13.4, 8.1, 8.1 Hz, 1H, one of CH2-2), 1.56 (d, J = 1.5 Hz, 3H, CH3-
13), 2.04 (m, 2H, CH2-3), 2.25 (s, 3H, CH3-10), 2.29 (m, 1H, H-6),
5.50 (m, 1H, H-4), 6. 04 (dd, J = 15.9 Hz, 1H, H-8), 6.61 (dd, J = 15.9,
9.8 Hz, 1H, H-7).
Chlorolactonization of acid 4 was performed according to the
known procedure [21] led us to obtain 1.98 g (69%) of chlorolactone
5. The product is characterized by the data given below: nD = 1.5180.
1H NMR (300 MHz, CDCl3) ı 0.93 (s, 3H, CH3-13), 0.96 (s, 3H, CH3-
14), 1.20 (m, 2H, CH2-2), 1.54 (d, J = 6.2 Hz, 3H, CH3-12), 1.69 (m, 3H,
CH3-15), 2.03 (m, 2H, CH2-3), 2.22 (m, 1H, H-6), 2.51 (m, 2H, H-7 and
one of CH2-8), 2.65 (m, 1H, one of CH2-8), 3.73 (dd, J = 10.1, 10.1 Hz,

M. Grabarczyk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 195–203
197
1H, H-10), 4.34 (dq, J = 10.1 and 6.2 Hz, 1H, H-11), 5.60 (m, 1H, H-
4). 13C NMR (300 MHz, CDCl3) ␦ 19.98 (C-12), 22.94 (C-3), 25.66
(C-15), 28.14 (C-13), 28.28 (C-14), 31.85 (C-2), 33.03 (C-8), 38.81
(C-7), 49.14 (C-6), 63.86 (C-10), 79.14 (C-11), 88.46 (C-1), 125.76
(C-4), 130.98 (C-5), 170.40 (C-9). IR (KBr, cm−1): 2959, 1776, 1455,
1223, 1061. EI-MS m/z (%) 270 [M+H], 271 (4), 147 (70), 123 (52),
107 (100), 91 (28), 81 (37), 67 (16), 53 (7), 39 (36).
each flask with the grown culture. Incubation of the shaken cul-
tures with the substrate was continued for 7 days. After 3, 5 and
7 days of incubation, the mixture of unreacted substrate, products
and mycelium was extracted with 15 mL of dichloromethane. After
evaporation of the solvent, the residue was dissolved in 2 mL of
acetone and analyzed by TLC (silica gel, hexane:acetone, 3:1) and
GC (DB-17 column).
2.2.6. 5-(1-Bromoethyl)-4-(2,6,6-trimethylcyclohex-2-en-1-
yl)dihydrofuran-2(3H)-one
(6)
2.3.3. Preparative transformations
The halolactones 5–6 (100 mg dissolved in 10 mL of acetone)
were distributed between 10 Erlenmeyer flasks with the 3-day
cultures of fungal strains prepared as described in the screening
procedure. The cultures were incubated with the substrates for 7
days, then the products were extracted with dichloromethane (3×
40 mL). The combined organic solutions were dried over anhydrous
magnesium sulfate and the solvent was evaporated in vacuo. Mix-
ture containing the product, the unreacted substrate and the fungal
metabolites was separated by column chromatography (silica gel,
hexane:acetone 3:1) to obtain a pure product – hydroxylactone 8.
According to the procedure of Grabarczyk [21] we obtained 2.5 g
(74%) of bromolactone 6 from acid 4 with the following physical and
spectral data: nD = 1.5252. 1H NMR (300 MHz, CDCl3) ı 0.94 (s, 3H,
CH3-13), 0.97 (s, 3H, CH3-14), 1.23 (m, 2H, CH2-2), 1.60 (d, J = 6.2 Hz,
3H, CH3-12), 1.69 (m, 3H, CH3-15), 2.03 (m, 2H, CH2-3), 2.24 (m,
1H, H-6), 2.52 (dm, J = 10.2 Hz, 1H, H-7), 2.65 (m, 2H, CH2-8), 3.86
(dd, J = 10.2, 10.2 Hz, 1H, H-10), 4.46 (dq, J = 10.2, 6.2 Hz, 1H, H-11),
5.60 (m, 1H, H-4). 13C NMR (300 MHz, CDCl3) ı 21.16 (C-12), 22.91
(C-3), 25.66 (C-15), 28.22 (C-13), 28.33 (C-14), 31.85 (C-7), 33.29
(C-2), 39.17 (C-8), 35.41 (C-7), 50.50 (C-6), 57.58 (C-10), 79.18 (C-
11), 88.43 (C-1), 125.85 (C-4), 131.00 (C-5), 170.49 (C-9). IR (KBr,
cm−1): 2956, 1783, 1449, 1176, 1008. EI-MS m/z (%) 315 [M+H],
316 (3), 235 (15), 193 (11), 175 (12), 123 (100), 107 (76), 79 (50),
53 (10), 39 (36).
2.3.4. 5-(1-Hydroxyethyl)-4-(2,6,6-trimethylcyclohex-2-en-1-
yl)dihydrofuran-2(3H)-one
(8)
Thick oil. 1H NMR (300 MHz, CDCl3) ı 0.93 (s, 3H, CH3-13), 0.96
(s, 3H, CH3-14), 1.19 (m, 2H, CH2-2), 1.35 (d, J = 6.6 Hz, 3H, CH3-12),
1.69 (s, 3H, CH3-15), 1.70 (m, 1H, H-6), 1.80 (m, 1H, OH), 2.01 (m,
2H, CH2-3), 2.47 (m, 2H, CH2-8), 2.87 (m, 1H, H-7), 3.80 (dq, J = 6.6,
3.3 Hz, 1H, H-11), 4.23 (dd, J = 3.3, 3.3 Hz, 1H, H-10), 5.59 (m, 1H,
H-4). 13C NMR (300 MHz, CDCl3) ı 20.09 (C-12), 23.03 (C-3), 25. 51
(C-15), 27.87 (C-13), 28.12 (C-14), 31.61 (C-2), 32.33 (C-8), 33.12
(C-1), 35.41 (C-7), 51.41 (C-5), 68.05 (C-11), 89.25 (C-10), 125.58
(C-4), 131.73 (C-6), 177.47 (C-9). IR (KBr, cm−1): 3448, 2928, 1772,
1186, 992. EI-MS m/z (%) 252 [M+H], 252 (10), 235 (10), 207 (8),
176 (10), 150 (25), 123 (100), 107 (32), 93 (26), 67 (12), 45 (20), 39
(22).
2.2.7. 5-(1-Iodoethyl)-4-(2,6,6-trimethylcyclohex-2-en-1-
yl)dihydrofuran-2(3H)-one
(7)
Iodolactonization of acid 4 was performed according to the
reported procedure of Grabarczyk et al. [43] gave us 1.98 g (69%)
of iodolactone 7 with the following physical and spectral data:
nD = 1.5432. 1H NMR (300 MHz, CDCl3) ı 0.98 (s, 3H, CH3-13), 1.01
(s, 3H, CH3-14), 1.28 (m, 2H, CH2-2), 1.59 (m, 3H, CH3-15), 1.80 (d,
J = 6.9 Hz, 3H, CH3-12), 2.07 (m, 2H, CH2-3), 2.28 (s, 1H, H-1), 2.55
(m, 1H, one of CH2-8), 2.68 (m, 1H, one of CH2-8), 3.08 (m, 1H, H-
7), 3.89 (dd, J = 10.2, 10.2 Hz, 1H, H-10), 4.51 (m, 1H, H-11), 5.64 (m,
1H, H-4); 13C NMR (300 MHz, CDCl3) ı 21.15 and 21.63 (C-12), 23.06
(C-3), 25.66 (C-15), 28.22 and 28.32 (C-13), 27.20 and 28.44 (C-14),
32.88 (C-2), 31.88 and 33.47 (C-8), 39.16 and 39.63 (C-7), 50.48 (C-
6), 57.56 (C-10), 79.11 (C-11), 88.42 (C-1), 125.85 and 126.12 (C-4),
130.94 and 131.38 (C-5), 170.50 and 176.71 (C-9), IR (KBr, cm−1):
2957, 1788, 1448, 1167, 978. EI-MS m/z (%) 362 [M+H], 362 (6), 306
(40), 235 (60), 179 (36), 137 (24), 123 (89), 94 (86), 79 (100), 57
(15), 39 (46).
2.4. Bioassay tests
Some bacteria strains: Micrococcus flavus C1, Bacillus cereus C3,
Escherichia coli C1, Bacillus subtilis B5, Pseudomonas fluorescens W1,
yeast strains: Debaryomyces hansenii K12a, Saccharomyces cere-
visiae SV30, Yarrowia lipolytica ATCC 20460, Schizosaccharomyces
pombe C-1, Rhodotorula rubra C-9 and also fungal strains: Aspergillus
niger XP, Fusarium linii 3A, Penicillium sp., Alternaria sp. were tested.
The bacterial cultures were carried out in a liquid broth consist-
ing of 15 g of dry bullion (Biocorp) and 10 g of glucose dissolved
in 1 L of distilled water. Cultures of yeast and fungi were grown in
YPG medium, containing10 gofyeast extract, 10 g of bacteriological
peptone and 10 g of glucose dissolved in 1 L of distilled water. The
effects of hydroxylactone 8 on the microbial growth were assessed
using a microbiological apparatus Bioscreen C. The bacterial cul-
tures were carried out in a liquid broth for 48 h (for bacteria strains)
or 48 and 96 h (for yeast and fungi strains). Hydroxylactone 8 was
dissolved in DMSO and used at a concentration of 0.1%. The result-
ing microbial growth curves were compared to control cultures in
medium supplemented with DMSO. Each culture was performed in
3 replications.
2.3. Biotransformation
2.3.1. Microorganisms
The fungal strains which were used in this study were obtained
from a collection of the Institute of Biology and Botany, Medical
University, Wrocław: Fusarium culmorum AM10, Fusarium ave-
naceum AM11, Fusarium oxysporum AM13, Fusarium tricinctum
AM16, Fusarium semitectum AM20, Fusarium solani AM203, Botrytis
cinerea AM235, and Beauveria bassiana AM278. They all are avail-
able in Department of Chemistry, University of Environmental and
Life Sciences. The strains were cultivated on Sabouraud’s agar con-
taining 0.5% of aminobac, 0.5% of peptone, 4% of glucose and 1.5% of
agar dissolved in distilled water at 28 ◦C and stored in a refrigerator
at 4 ◦C.
The data were analyzed using a spreadsheet software (Excel 97)
and the means for the triplicates of each culture medium type were
calculated. The mean values were used to generate the growth
curves for each investigated strain, constituting a function of the
incubation time and the culture medium absorbancy. The resulting
microbial growth curves were compared to control cultures in the
medium supplemented with dimethyl sulfoxide.
2.3.2. Screening procedure
The fungal strains used for microbial transformations were culti-
vated at 25 ◦C in Erlenmayer flasks filled with 100 mL of the medium
containing 3 g of glucose and 1 g of peptobac. After three days,
10 mg of the substrate dissolved in 1 mL of acetone was added to
Evaluation of the impact of compounds 5–7 to increase the
aforementioned strains was not possible in a Bioscreen C apparatus

198
M. Grabarczyk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 195–203
Scheme 1. Synthesis of halolactones 5–7.
due to the fact that in aqueous solutions (culture media) they
underwent turbidity causing precipitation breeding environment
making it impossible to measure the OD during breeding. The
biological activity of these substances have been studied with the
use of platelet diffusion test using paper-disc assay. YM agar (yeast
and fungi) and nutrient broth (for bacteria) were inoculated with
standardized (using Thoma chamber for yeast or McFarland, in the
case of bacteria) cell suspension of selected microorganisms to a
final concentration 5 × 105 cells/mL. Paper disks with a diameter
of 0.5 cm were placed on the surface of sterile plates which were
previously prepared. Then 10 ␮L of a 20% solution of the compound
in DMSO was applied. The paper disks were left at 4 ◦C for 8 h for
diffusion of the test substances to the medium and incubated at
25 ◦C temp (filamentous fungi) or 30 ◦C (yeasts and bacteria) for
48–96 h. The clear zone devoid of cells observed around the disc
with the appropriate compound was identified as the ability of this
compound to inhibit the growth of the strain inoculated into the
medium. Clear zone with less visible micro colonies was identified
as the ability to restrict the growth of the strain.
Scheme 2. Structures of compounds 2–4.
between H-1 and H-7 protons (J = 8.7 Hz) indicated equatorial posi-
tion of H-1, and the coupling constant for H-7 and H-8 (J = 15.3 Hz)
suggested trans configuration of the double bond. The position of
the hydroxyl group–trans relative to H-8 was determined on the
basis of coupling constants for H-9 and H-8 protons (J = 6.1 Hz)
and H-9 and CH3-10 (J = 6.3 Hz). At the next step allylic alcohol 2
was transformed during the Johnson–Claisen rearrangement into
an unsaturated ester 3 (a mixture of the cis-trans in a ratio 56%: 44%
according to GC analysis). 1H NMR analysis confirmed this conclu-
sion, because the doubling singlets coming from the methyl groups
as well as the signals from the ester ethoxy group were detected.
Also, comparison of the integration of the signals derived from the
two protons of the ester ethoxy group and three olefinic protons
(H-4, H-8, H-9) indicated their relationship 2: 3 which suggested
presence of the two isomers. The wide multiplet of H-7 proton sug-
gested its axial position, indicating that CH2-11 group was located
perpendicular to the plane of the ring. The mixture of ester 3 iso-
mers after base hydrolysis gave unsaturated acid 4 (also a mixture
of the cis-trans isomers in a ratio 56%:44%) (Scheme 2).
2.5. Odour evaluation
The odour evaluation was performed for ethanolic solutions
(10%) of samples with the use of a strip blotter.
3. Results and discussion
The racemic ( ) ␣-ionone was used as a substrate for the
four-step chemical synthesis. During these processes ␣-ionol (2)
[41], ␥,␦-unsaturated ester (3) and ␥,␦-unsaturated acid (4), were
obtained as intermediates. ␥,␦-Unsaturated acid was then used for
the preparation of chlorolactone (5), bromolactone (6) and iodolac-
tone (7) (Scheme 1).
Acid 4, without the separation of isomers, was used to obtain
chlorolactone 5, bromolactone 6 and iodolactone 7. Chloro- and
bromolactonization was carried out according to a procedure
described earlier [20,21]. Iodolactonization was performed accord-
ing to the procedure described earlier [43,44]. Both bromo- and
iodolactone were obtained as a mixture of stereoisomers with a 70%
predominance of one of them, while chlorolactone with a 95% pre-
dominance of one of them. The structure of the obtained lactones
was determined on the basis of their spectral data (1H NMR and IR).
The absorption bands at: 1776 (5), 1783 (6) and 1788 cm−1 (7) in
the IR spectra confirmed the presence of the ␥-lactone ring in the
molecules of these compounds. The 1H NMR analysis showed that
the H-1 proton in all three lactones, and all the earlier products, was
The analysis of 1H NMR spectra allowed us to determine the
structure of particular compounds. The signal of H-1 proton in
␣-ionone (1) was a multiplet with small coupling constant that
indicated its equatorial position and an axial position of the side-
chain. The same situation was observed in the case alcohol ␣-ionol
2 [40] obtained by the reduction of ␣-ionone 1 with lithium
aluminium hydride in dry diethyl ether. The coupling constant

M. Grabarczyk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 195–203
199
Scheme 3. Spatial structures of lactones 5–7.
Table 1
Hydrolytic dehalogenation of lactones 5, 6 and 7 after 7 days of incubation.
Fig. 1. Composition of the product mixtures of preparative biotransformations of
lactone 5.
Microorganism
5
6
7
90
80
70
60
50
40
30
20
10
0
82.4
Fusarium culmorum AM10
Fusarium avenaceum AM11
Fusarium oxysporum AM13
Fusarium tricinctum AM16
Fusarium semitectum AM20
Syncephalastrum racemosum AM105
Fusarium scirpi AM199
(+)
(+)
(+)
(−)
(+)
(−)
(+)
(+)
(−)
(+)
(+)
(+)
(+)
(+)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
(−)
80.3
78.2
74.7
61.6
38.4
bromolactone 6
hydroxylactone 8
25.3
21.8
Botrytis cinerea AM235
Cunninghamella japonica AM472
19.7
17.6
(−) – the conversion below 60%
(+) – the conversion 67–86%
in an equatorial position. The coupling constants between H-7 and
H-10 protons (10.1 Hz for 5 and 10.2 Hz for 6) as well as between
H-10 and H-11 protons (10.1 Hz for 5, 10.2 Hz for 6 and 10.2 for
7) indicated their mutual trans position. Analysis of COSY spectra
showed that in the case of lactones 5 and 6 there were no interaction
between H-10 and H-11 protons, thus indicating their anticlinal
conformation. However, in the case of iodolactone 7 it was found
that H-10 and H-11 protons were in synclinal conformation relative
to each other (visible interactions between these two protons).
These observations led us to a conclusion that in lactones 5 and
6 the halogen atom was situated in the same plane as the lactone
ring, whereas in the case of lactone 7 the iodine atom was located
across this plane (Scheme 3). According to Snider [45] and Jayara-
man [46] the difference in diastereoselectivity can be explained by
different mechanisms of chloro-, bromo- and iodolactonization. In
the process of chloro- and bromolactonization the double bond is
attacked by bromine leading to the formation of bromonium ion.
During iodolactonization the complex of iodine and double bond is
attacked by the carboxylate anion.
Another derivative we planned to obtain was hydroxylactone.
We could not receive it by chemical synthesis so we decided to
apply an alternative method consisting of using a biocatalyst, in
which whole cells of filamentous fungi were used. In the first
step nine fungal strains (Fusarium culmorum AM10, Fusarium ave-
naceum AM11, Fusarium oxysporum AM13, Fusarium tricinctum
AM16, Fusarium semitectum AM20, Syncephalastrum racemosum
AM105, Fusarium scirpi AM199, Botrytis cinerea AM235, Cunning-
hamella japonica AM472) from the collection of the Institute of
Biology and Botany at the Medical University in Wrocław were
used for a screening procedure to verify which microorganisms
were capable of converting halolactones 5, 6 and 7 into other prod-
ucts. The results of the GC analyses of hydrolytic dehalogenation of
lactones 5, 6 and 7 are shown in Table 1.
Fig. 2. Composition of the product mixtures of preparative biotransformations of
lactone 6.
Scheme 4. Transformation of halolactones 5–7.
only for chloro- and bromolactone. The results of all the preparative
transformations are presented in Figs. 1 and 2.
Chlorolactone 5 was subjected to the biotransformation on a
preparative scale using seven strains of filamentous fungi (Table 2),
while for bromolactone 6 a total of 5 strains (Table 3) were selected
during the screening tests. All of the chosen microorganisms trans-
formed both halolactones into hydroxylactone 8 (Scheme 4, Fig. 3).
GC and 1H NMR analysis showed that each time only one and
the same compound was obtained. This demonstrates high stere-
ospecificity of the tested microorganisms. The highest degree of
conversion was observed for chlorolactone 5 and bromolactone
6, transformed by two strains in each case: F. oxysporum AM13
and F. tricinctum AM16 (about 90%) and F. avenaceum AM11 and
F. tricinctum AM16 (over 80%), respectively.
The results presented in Table 1 indicated that the best results
were achieved for six microorganisms from the Fusarium species
and Botrytis cinerea, but only for two substrates, i.e. chlorolactone
5 and bromolactone 6 (the degree of conversion was 67–85%). In the
case of the third compound, iodolactone 7, the degree of conversion
into a product did not exceed 50%. Because the main objective of
this step was to obtain the new product with the greatest possible
efficiency, biotransformation on a preparative scale was carried out
Fig. 3. Spatial structure of hydroxylactone 8.

200
M. Grabarczyk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 195–203
Table 2
Data concerning hydroxylactone 8 obtained during preparative biotransformations of lactone 5.
20
D
Entry
Strain
Yield (according to GC)
Isolated yield (g)/(%)
ee (%)
˛
1
2
3
4
5
6
7
F. culmorum AM10
F. avenaceum AM11
F. oxysporum AM13
F. tricinctum AM16
F. semitectum AM20
F. scirpi AM199
64.9
75.1
91.6
89.5
73.9
74.1
74.8
0.022/23.6
0.024/25.7
0.027/28.9
0.016/17.1
0.024/25.7
0.017/18.2
0.0117/12.5
62.5
18.0
11.4
15.8
9.0
+78.69 (c = 0.62%, CHCl3)
+21.43 (c = 0.81%, CHCl3)
+11.24 (c = 0.74%, CHCl3)
+16.31 (c = 0.68%, CHCl3)
+5.47 (c = 0.67%, CHCl3)
+79.34 (c = 0.60%, CHCl3)
+2.56 (c = 0.55%, CHCl3)
57.2
6.8
B. cinerea AM235
Table 3
Data concerning hydroxylactone 8 obtained during preparative biotransformations of lactone 6.
20
˛
Entry
Strain
Yield (according to GC)
Isolated yield (g)/(%)
ee (%)
D
1
2
3
4
5
F. culmorum AM10
F. avenaceum AM11
F. oxysporum AM13
F. tricinctum AM16
F. semitectum AM20
78.2
82.4
74.7
80.3
61.6
0.0137/17.4
0.0260/33.0
0.0237/30.1
0.0306/38.9
0.0236/30.0
42.0
8.2
12.0
8.9
+49.99 (c = 0.79%, CHCl3)
+4.56 (c = 1.62%, CHCl3)
+8.72 (c = 1.36%, CHCl3)
+5.15 (c = 1.49%, CHCl3)
+8.26 (c = 0.84%. CHCl3)
11.8
As we know from previous reports [20,21,47,48] the exchange
of a halogen atom into the hydroxy group under the influence of
enzymes present in the cells of filamentous fungi is similar to the
SN2 substitution with a simultaneous change of the molecule con-
formation. The same situation was also observed in this case, as
evidenced by the structure of the product. The analysis of 1H NMR
spectra showed that the H-1 proton remained in the equatorial
position. The coupling constants between H-7 and H-10 protons
(3.3 Hz) indicated that H-10 proton changed its position from lay-
ing across the plane of lactone ring in the substrates to being in
the same plane as the lactone ring in hydroxylactone. The same
coupling constant between H-10 and H-11 (3.3 Hz) revealed their
synclinal conformation. These observations indicated that both H-
7 proton and the OH group were located in trans positions to each
other, and that the hydroxy group was perpendicular to the lac-
tone ring. Such positioning of the OH group was possible only if
the halogen atom in the substrate was situated in the same plane
as the lactone ring. Such a situation occurred for chlorolactone 5
and bromolactone 6. However, in the case of iodolactone 7 the
iodine atom occupied the place in which the hydroxy group would
be introduced, which explains the low degree of conversion of the
iodolactone during the biotransformation.
1.5
1
0.5
0
0
20
40
60
ꢀme [h]
M.flavus-kontrola
M.flavus+DMSO
M.flavus+DMSO+lactone 8
Fig. 4. Effect of hydroxylactone 8 on the growth of M. flavus C1.
1.5
1
0.5
0
In order to confirm that microorganisms were responsible for
the process of hydrolytic dehalogenation, an experiment similar
to the screening using halolactone 5, 6 or 7 and a sterile medium
(without the participation of microorganisms) was conducted. In
every case only substrates were observed after 7 days in the reac-
tion mixture.
0
20
40
60
ꢀme [h]
B.cereus-control
B.cereus+DMSO
Another parameter taken into account was the enantioselec-
tivity of the biotransformation process. All of the hydroxylactones
were verified in terms of their optical purity.
Fig. 5. Effect of hydroxylactone 8 on the growth of B. cereus C3.
0.8
0.6
0.4
0.2
0
In all cases, the formation of (+) isomer of hydroxylactone 8 was
preferred. The best results were obtained for chlorolactone 5 when
F. culmorum AM10 (de = 62.5%) and F. scirpi AM199 (de = 57.2%)
were used as a biocatalysts; in the case of bromolactone 6 only one
strain, F. culmorum AM10, transformed the substrate into a product
with ee = 42.0%. In all of the cases the (+)-enantiomer was preferred
for the obtained products.
Considering the fact that naturally occurring hydroxylactones
exhibit a variety of biological activities including bactericidal and
fungicidal [38,39] we decided to test the obtained hydroxylactone 8
in this respect. Bioassays performed on selected strains of bacteria
(entries 1–5), yeasts (entries 6–10), and fungi (entries 11–14) are
given in Table 4 and in Figs. 4–11.
0
10
20
30
40
50
60
ꢀme[h]
P.fluorescens-control
P.fluorescens+DMSO
P.fluorescens+DMSO+lactone 8
The above data led us to the conclusion that hydroxylactone
8 showed growth inhibition for five strains: M. flavus C1 (Table 4
Fig. 6. Effect of hydroxylactone 8 on the growth of P. fluorescens W1.

M. Grabarczyk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 195–203
201
Table 4
Effects of hydroxylactone 8 on the bacterial, the yeast and the fungi growth.
2.5
2
1.5
1
Entry
1
Microorganism
Effects
Micrococcus flavus C1
Max OD = 0.60 (control OD
max. = 1.35)
0.5
0
2
Bacillus cereus C3
(Longer lag phase up to
11 h), limiting growth,
increase in OD = 0.49
(control OD max. = 1.037)
No influence on the growth
No influence on the growth
(Longer lag phase up to
10 h) limiting growth,
increase in OD max = 0.32
(control OD max. = 0.75)
Max OD = 0.65 (control OD
max. = 1.13)
0
20
40
60
80
100
ꢀme [h]
3
4
5
Escherichia coli C1
Bacillus subtilis B5
Pseudomonas fluorescens W1
A.niger-control
A.niger+DMSO
A.niger+DMSO+lactoned 8
6
7
Debaryomyces hansenii K12a
Saccharomyces cerevisiae SV30
Fig. 9. Effect of hydroxylactone 8 on the growth of A. niger XP.
(Longer lag phase up to
20 h)
2
8
9
10
11
Yarrowia lipolytica ATCC 20460
Schizosaccharomyces pombe C-1
Rhodotorula rubra C-9
no influence on the growth
No influence on the growth
No influence on the growth
Max OD = 1.06 (control OD
max. = 2.061)
1.5
1
0.5
0
Aspergillus niger XP
12
13
Fusarium linii 3A
Penicillium sp.
No influence on the growth
Max OD = 0.654 (control
OD max. = 1.88)
0
20
40
60
ꢀme [h]
80
100
14
Alternaria sp.
Max OD = 1.3 (control OD
max. = 1.89)
Penicillium-control
Penicillium+DMSO
1.5
1
Penicillium+DMSO+lactone 8
Fig. 10. Effect of hydroxylactone 8 on the growth of Penicillium sp.
0.5
0
2
1.5
1
0
10
20
30
ꢀme [h]
40
50
60
0.5
0
D.hansenii-control
D.hansenii+DMSO
0
20
40
60
ꢀme [h]
80
100
D.hansenii+DMSO+lactone 8
Fig. 7. Effect of hydroxylactone 8 on the growth of D. hansenii K12a.
Alternaria sp.-control
Alternaria sp.+DMSO
2
1.5
1
Alternaria sp.+DMSO+lactone 8
Fig. 11. Effect of hydroxylactone 8 on the growth of Alternaria sp.
Encouraged by these good results we decided to check halolac-
tones 5–7 for their antimicrobial activity. The results of these tests
are given in Table 5.
0.5
0
The above data indicates that halolactones 5–7 were able to
only inhibit the growth of bacteria strains, except E. coli C1 (entry
3). Growth inhibition zones were within 0.79–4.36 mm. The best
growth inhibitor proved to be bromolactone 6 relative to B. cereus
C3 (entry 2), which also caused restriction of the growth of this
strain in lower concentrations. But it is to be noted that the biolog-
ical activity of the tested compounds was significantly lower than
the bactericidal activity of oxytetracycline, which resulted in inhibi-
tion zone of up to 16 mm. To other strains, i.e. yeast and filamentous
fungi, tested compounds proved to be inactive.
In addition to the above tests we also performed an olfactory
analysis of all the new compounds. The results are shown in Table 6.
We found that the most of the tested compounds were char-
acterized by a pleasant fragrance with the exception of acid (4)
and microbiologically obtained hydroxylactone (8). Considering
the fragrance characteristics of the halolactones 5–7, it is clear
that a small change in the structure of the compound (change of a
0
10
20
30
ꢀme [h]
40
50
60
S.cerevisiae-control
S.cerevisiae+DMSO
S.cerevisiae+DMSO+lactone 8
Fig. 8. Effect of hydroxylactone 8 on the growth of S. cerevisiae SV30.
entry 1, Fig. 3), D. hansenii K12a (Table 4 entry 6, Fig. 6) and A.
niger XP, Penicillium sp., Alternaria sp. (Table 4 entry 11, 13 and 14,
Figs. 8–10). In three other cases – B. cereus C3, P. fluorescens W1
(Table 4 entry 4 and 5, Figs. 4 and 5) and S. cerevisiae SV30 (Table 4
entry 7, Fig. 7) – growth retardation with elongated lag phase were
observed. For the rest of microorganisms (E. coli C1, B. subtilis B5,
Y. lipolytica ATCC 20460, R. rubra C-9, S. pombe C-1, F. linii 3A) no
activity with hydroxylactone 8 was noted.

202
M. Grabarczyk et al. / Journal of Molecular Catalysis B: Enzymatic 102 (2014) 195–203
Table 5
Growth inhibition zone size/growth restriction zone size (mm) of selected strains of bacteria under the influence of lactones 5–7. The above data indicates that halolactones
5–7.
Entry
Microorganism
Compound
5
6
7
Oxytetracycline
1
2
3
4
5
Micrococcus flavus C1
Bacillus cereus C3
Escherichia coli C1
Bacillus subtilis B5
Pseudomonas fluorescens W1
2.16/–
1.43/1.60
–/–
1.19/–
0.79/–
2.69/–
4.36/3.32
–/–
2.39/–
1.39/–
1.72/–
–/2.06
–/–
0.80/–
–/–
16.3/–
16.10/–
4.17/–
12.93/–
10.49/–
Table 6
Acknowledgments
Odour characteristic of the new compounds.
This project was financed by European Union from the European
Regional Development Fund. Grant No. POIG. 01.03.01-00-158/09.
Compound
odour
Ester (3)
Acid (4)
Small intensive, pleasant, cream-almond
Medium intensive, unpleasant, dry
Chlorolactone (5)
Medium intensive, pleasant, green and woody,
note of Viola odorata
Appendix A. Supplementary data
Bromolactone (6)
Iodolactone (7)
High intensive, pleasant, vegetable, note of celery
Medium intensive, pleasant, floral, note of Viola
odorata
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2014.02.012.
Hydroxylactone (8)
Small intensive, unpleasant
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