The spectral and catalytic studies of chlorophylls and pheophytins in mimetic biotransformation of α-pinene

Article abstract of DOI:10.1016/j.jphotochem.2011.07.012

Natural porphyrin pigments isolated from spinach in the form of a crude extract, chlorophyll a, chlorophyll b, pheophytin a and pheophytin b were studied as potential catalysts for the biomimetic transformation of α-pinene to trans-pinocarveol, pinocarvone and myrtenal. The reaction of monoterpene oxidation was performed in an organic solution under visible light irradiation. Significant changes in the distribution of the products were achieved using β-pinene as the substrate. The pheophytins showed the highest catalytic efficiency among the photocatalysts examined. The photographic visualization of α-pinene oxidation course at the beginning of the reaction, after 4 and 24 h exposure to light showed a distinct shift in the colour excited catalysts. This may indicate a change in their structure leading to their degradation. The absorbance and fluorescence spectra revealed that pheophytins are significantly stable photocatalysts in comparison to both chlorophylls. Pheophytin b displayed the highest stability in the biotransformation, whereas the highest rate of photodecomposition was proved for chlorophyll b under the reaction condition. Differences were noticed in the behaviour of the chlorophylls in the photodegradation process. From the changes in the intensity of the singlet oxygen band on infrared fluorescence spectra of pigments upon pinene addition, and from the reactivity experiments with radicals scavengers, it can be deduced that the biotransformation of pinene by chlorophylls and pheophytins proceeds by common oxygenation mechanism involving singlet oxygen and free radicals.

Full text of DOI:10.1016/j.jphotochem.2011.07.012

Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
The spectral and catalytic studies of chlorophylls and pheophytins in mimetic
biotransformation of ␣-pinene
a,∗
b
b
a
c
c
M. Trytek , E. Janik , W. Maksymiec , J. Fiedurek , A. Lipke , M. Majdan
a
Department of Industrial Microbiology, Institute of Microbiology and Biotechnology, Maria Curie-Skłodowska University, Akademicka St. 19, 20-033 Lublin, Poland
Department of Plant Physiology, Institute of Biology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland
Faculty of Chemistry, Maria Curie-Skłodowska University, M. Curie-Skłodowskiej Sq. 2, 20-031 Lublin, Poland
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural porphyrin pigments isolated from spinach in the form of a crude extract, chlorophyll a, chlorophyll
b, pheophytin a and pheophytin b were studied as potential catalysts for the biomimetic transformation
of ␣-pinene to trans-pinocarveol, pinocarvone and myrtenal. The reaction of monoterpene oxidation was
performed in an organic solution under visible light irradiation. Significant changes in the distribution
of the products were achieved using ␤-pinene as the substrate. The pheophytins showed the highest
catalytic efficiency among the photocatalysts examined. The photographic visualization of ␣-pinene oxi-
dation course at the beginning of the reaction, after 4 and 24 h exposure to light showed a distinct shift in
the colour excited catalysts. This may indicate a change in their structure leading to their degradation. The
absorbance and fluorescence spectra revealed that pheophytins are significantly stable photocatalysts in
comparison to both chlorophylls. Pheophytin b displayed the highest stability in the biotransformation,
whereas the highest rate of photodecomposition was proved for chlorophyll b under the reaction con-
dition. Differences were noticed in the behaviour of the chlorophylls in the photodegradation process.
From the changes in the intensity of the singlet oxygen band on infrared fluorescence spectra of pigments
upon pinene addition, and from the reactivity experiments with radicals scavengers, it can be deduced
that the biotransformation of pinene by chlorophylls and pheophytins proceeds by common oxygenation
mechanism involving singlet oxygen and free radicals.
Received 15 February 2011
Received in revised form 11 July 2011
Accepted 19 July 2011
Available online 29 July 2011
Keywords:
Biomimetic catalysis
Biotransformation
Pinene
Chlorophyll
Pheophytin
Photooxidation
UV–vis spectroscopy
©
2011 Elsevier B.V. All rights reserved.
1
. Introduction
ally been in the milligrams per liter range and thus rather low
compared to other terpene biooxidations, possibly due to the more
complex bicyclic structure of the pinenes [4].
Monoterpene hydrocarbons such as ␣-pinene, ␤-pinene and
p-cymene are inexpensively available in large quantities. Oxy-
genated monoterpenes (terpenoids) are valuable compounds used
in flavour and fragrance industries. Biotransformation of monoter-
pene hydrocarbons to oxygenated ones is of interest because it
allows production of stereoselectively pure compounds under mild
conditions and also the products may be considered as natural
products [1]. Pinene exists in two forms, ␣-pinene and ␤-pinene.
Both forms are present in many essential oils but are mostly
obtained from turpentine (obtained by the dry distillation of wood
or other dry plant material).
Microbial transformation of pinenes has been investigated since
the early 1960s [2,3]. However, the traditional biocatalysts have
several limitations in application to oxidative biotransformations,
including stability and activities, especially to those substrates and
products that are toxic and require being performed in reaction
media other than water. Bioconversion yields of pinenes have usu-
For improvement of the activity and selectivity in the bio-
catalytic oxidation of monoterpenes, the active-site of heme
monooxygenases has been engineered by designed mutagenesis
[5,6]. An alternative approach is replacement of expensive natu-
rally occurring biocatalysts or the reproduction of their catalytic
properties by designing and preparation of active site analogues
of heme monooxygenases. It is known that a crucial role in most
transition metal-enzymes is played by the porphyrin prosthetic
groups (e.g., protoporphyrin IX). Therefore, substantial efforts have
been devoted to making use of metalloporphyrins in biomimetic
catalysis to engineer an artificial catalytic system resembling the
function and activity of monooxygenases like cytochromes P-450,
peroxidases, and catalase to catalyze important and difficult trans-
formations such as regioselective hydroxylation of unactivated C–H
bonds [7–11]. The presence of the porphyrins as the active site
of chlorophylls and pheophytins makes them enticing targets for
biocatalysis.
Several interesting examples of the activity of porphyrins
and metalloporphyrins as biomimetic catalysts in oxidation of
monoterpenes have been reported [12–15]. However, to our
∗ Corresponding author. Tel.: +48 81 537 5958; fax: +48 81 537 5959.
E-mail address: mtrytek1@o2.pl (M. Trytek).
1
010-6030/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2011.07.012

M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
15
knowledge, only one paper deals with the utilization for this pur-
pose of natural chlorophyll in the form of a crude leaf extract. The
main disadvantage of this process is the requirements for the use
of hazardous reactants and UV-light, and the final product is in the
form of trans-pinocarvyl hydroperoxide, while pinocarveol rather
than the hydroperoxide is the desired product [16].
Our work is aimed at investigation of plant pigments (chloro-
phyll a, chlorophyll b, pheophytin a and pheophytin b, and in the
form of a crude extract), all isolated from spinach, as novel catalysts
for biotransformation of ␣-pinene to pinocarveol, pinocarvone and
myrtenal under mild conditions.
spectrofluorometer (JASCO) in the 400–750 nm region at room
◦
temperature (21± C). The spectra of solutions with reagent con-
−
5
−3
centration of 10 mol × dm were measured using a 1 cm quartz
cell. The slit of the spectrofluorometer was adjusted automatically.
The absorption, emission and excitation spectra were recorded
digitally and the SigmaPlot (Jandel Corp.) program was used in
manipulation and plotting the data. No filters were used, as the
FP-6300 apparatus had sufficiently high resolutions and the xenon
lamp “lines” did not interfere with our spectra.
The singlet oxygen luminescence spectrum was registered
by using Photon Technology International Inc. spectrofluorome-
ter equipped with an infrared module (NIR PMT Module with
InP/InGaAs photocatode material, Hamamatsu Photonics K.K.)
operating at spectral response range: 950–1700 nm.
2
. Materials and methods
2
.1. Chemicals
The products of biotransformation exogenously added to the
spectral controls, were prepared from the post-reaction solution
by the isolation of one fraction on a short silica gel column eluting
with a methyl chloride/hexane mixture. The products, trans-
pinocarveol, pinocarvone and myrtenal/myrtenol were added as
a stock in chloroform to the chloroform containing the pigments
(2 mM) to a final concentration of 0.5, 1 and 0.3 g/L, respectively.
All pigments for spectroscopic measurements were previously
(
1S)-(−)-␣-Pinene (98%), (1S)-(−)-␤-pinene (99%), (−)-trans-
pinocarveol (96%), (−)-myrtenol (98.5%), and petroleum ether
were purchased from Sigma–Aldrich, USA. 2,6-Di-tert-butylphenol
(
(
BHT), synthetic vitamin E (Trolox), and tert-butyl hydroperoxide
tert-BuOOH) were stored at 5 C. All reagents and solvents were of
◦
analytical grade.
−
4
dissolved to the final concentration of 5 × 10 M.
2
.2. Chlorophyll a and chlorophyll b isolation
2.5. Photooxidation procedure
Fresh spinach leaves were homogenized with acetone (1:4
(
w:v)) and filtered through Miracloth. Next, the acetone extract
was mixed with distilled water (5:1 (v:v)) and petroleum ether
2:1 (v:v)). The ether layer was concentrated by evaporation under
Biotransformation experiments were carried out under con-
◦
trolled temperature (20 C), using an incubator consisting of four
fluorescent visible lamps (Osram Lumilux, Cool White) regularly
placed above the reaction vessel. The catalytic oxidation reaction
was performed at atmospheric pressure, under oxygen dissolved in
the system in closed 12-ml glass vials. 150 ␮mol of ␣-pinene was
dissolved in 3 cm3 of chloroform or 1,4-dioxane containing an iso-
lated crude extract, chlorophyll or pheophytin (in their respective
concentration), and irradiated for 24 h under estimated intensity of
(
a nitrogen stream and chromatographed on HPTLC Kieselgel 60
plates (Merck, Germany) using the mobile phase of petroleum
ether:isopropanol:water (100:10:0.25 (v:v:v)). Chlorophylls dis-
solved in 100% acetone were identified on the basis of spectral
properties.
The content of chlorophyll in the acetone extracts of fresh
spinach leaves was determined using
method [17]. The ratio of chlorophyll a to b and pheophytin a to
b was 1.8 and 3.5, respectively.
a spectrophotometric
2
light ranging from 120 to 150 ␮mol/m s.
After the specified time of photooxidation, 250 ␮l of the reac-
tion medium were taken, diluted rapidly with a 5-fold volume of an
internal standard solution and used for GC-FID and GC–MS analyses
conducted according to the method reported previously [14]. Quan-
tification of volatile compounds was done by comparison with the
added standard. 0.05% (v/v) n-decane (for substrate) and linalool
2
.3. Conversion of chlorophylls to pheophytins
The acetone extract was obtained as described above. Next, the
extract was evaporated to a small volume and supplemented with
hexane to 50 ml and shaken for 1 min with an equal volume of
9
times. After evaporation of hexane from the sample, the extract was
supplemented with petroleum ether to 3 ml and chromatographed
using the HPTLC method. Pheophytin a and pheophytin b dissolved
in 80% acetone were identified on the basis of absorption spectra
and calculated according to Yang et al. [18].
(
for oxidation products) in hexane were used as an internal stan-
dard for gas chromatography. The substances were identified by
comparison of their mass spectra and retention indexes (RI) with
those of authentic sample spectra in a standard library database
system (NIST 2004 and MassFinder ver. 3).
Reference chemical blank experiments were performed in the
same way, but in darkness, to exclude the possibility of any
non-photochemical transformation reactions during the specified
period of time.
Control experiments were carried out in the presence of 1, 2 and
.3 equivalent of tert-BuOOH, BHT and Trolox, respectively.
Biotransformations were performed in two replicate samples
and the analyses were carried out in duplicate. The error associated
with the GC quantification of the samples was ± 5% and is quoted
for a confidence interval of 95%. The data presented are reported as
the average values.
.2% HCl. The hexane layer was washed with 50 ml water three
The purity of chlorophylls and pheophytins was controlled by
means of absorbance spectroscopy according to the literature data
[
19,20], which allowed exclusion of pigment protonation or met-
0
alation. The pigment concentrations used to record the spectra
−3
−3
−3
−3
were 8.3 × 10 g/l, 4.8 × 10 g/l, 7.3 × 10 g/l and 3.8 × 10 g/l
for chlorophyll a, chlorophyll b, pheophytin a and pheophytin b,
respectively.
2
.4. Spectral measurements
UV–vis measurements were carried out using a Shimadzu UV-
60 A (Japan) spectrophotometer with 1 cm Hellma quartz cells.
3. Results and discussion
1
Spectra were recorded between 300 and 700 nm at a temperature
The hydrocarbon ␣-pinene is an attractive precursor substrate
for bioconversion into valuable natural flavour and fragrance com-
pounds (such as verbenone, pinocarvone and myrtenol), because
of its widespread occurrence and low price. Pinenes (␣- and
◦
of 21 ± 1 C.
The emission and excitation spectra of chlorophylls and pheo-
phytins in chloroform solutions were recorded on a FP-6300

1
6
M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
Fig. 1. The example model chromatograms of the products of (A) ␣-pinene and (B) ␤-pinene photocatalytical biotransformation with chlorophylls and pheophytins: upon
exposure to light. (Solid line); without light (dashed line); 1, 2 and 3 – unidentified products.
␤
-) are major components of turpentine, a by-product of the
products: trans-pinocarveol, pinocarvone and myrtenal. Myrtenol
and all three unidentified products noticeable on GC traces (Fig. 1A)
were obtained only in trace amounts. By contrast, the biotransfor-
mation of another isomer ␤-pinene favors myrtenal as the major
product, whereas myrtenol, trans-pinocarveol and pinocarvone
were obtained in substantially lower amounts (Fig. 1B), owing to
the shift of the double bond in the pinene molecule. Addition of
tert-BuOOH to the photocatalytic systems with pheophytins and
chlorophylls had little, if any, effect on the rate or course of the
reaction. Similarly, the reaction performed in the darkness in the
presence of tert-BuOOH as an oxidant, yielded no products.
pulp making industry. One of the approaches in controlled and
selective biotransformation of monoterpenes (under mild condi-
tions) is the use of porphyrins as biomimetic catalysts. It would
help overcome the problem of sensitivity of traditional bio-
catalysts to toxic terpenes [8,14,15]. Therefore, elaboration of
porphyrin macrocycle-containing catalysts, e.g., chlorophylls, imi-
tating enzymes is desirable.
During our initial studies, we examined the ability of natural
tetrapyrrole pigments in the form of a crude extract, chlorophyll a,
chlorophyll b, pheophytin a and pheophytin b – all isolated from
spinach – to biotransform ␣-pinene. The analysis with the use of GC
and GC/MS techniques showed that ␣-pinene formed three main
Among the chlorophyll preparations examined for ␣-pinene
biotransformation, the highest accumulation of oxidative

M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
17
Table 1
The photocatalytic properties of chlorophyll-derived pigments involved in ␣-pinene biotransformation.
Photocatalyst
Trans-pinocarveol
Pinocarvone
Myrtenal
Remaining substrate
C [g/l]
Turnover numberg
C [g/l]
Turnover number
C [g/l]
Turnover number
C [g/l]
a,g
Crude extract
Crude extact
Chlorophyll a
0.94
0.67
0.49
0.30
0.51
0.58
0.40
0.26
0.57
0.71
0.21
(70)
(94)
2.55
2.32
1.96
0.99
1.25
1.39
1.04
0.45
1.51
1.14
0.33
(190)
(330)
1.0
(74)
(24)
0.01
2.07
7.13
6.24
0.02
0.004
2.47
4.52
1.6
b,g
0.17
0.17
0.23
0.59
0.62
0.27
0.02
0.40
0.23
0.008
c
24.2 (1648)
24.8 (1664)
36.7 (2563)
105.4 (7240)
78.1
62.6
113.0
139.8
40.9
96.3 (6550)
82.4 (5520)
89.7 (6260)
253 (17,394)
207.1
107.2
302.0
228.3
66.8
8.5 (578)
18.7 (1254)
42.6 (2970)
113.5 (7798)
53.2
5.4
79.2
47.6
1.7
d
Chlorophyll b
e
Pheophytin a
Pheophytin b
Chlorophyll a
f
h
Chlorophyll a + BHT
h
Pheophytin a
Pheophytin a + Trolox
Pheophytin a + BHT
2.1
7.95
a
Crude extract from spinach, weight of the catalyst used – 13.5 mg, solvent – chloroform.
Crude extract from spinach, weight of the catalyst used – 7.1 mg, solvent – 1,4-dioxane.
Chlorophyll a, weight of the catalyst used – 0.3 mg, solvent – chloroform.
Chlorophyll b, weight of the catalyst used – 0.18 mg, solvent – chloroform.
Pheophytin a, weight of the catalyst used – 0.2 mg, solvent – chloroform.
Pheophytin b, weight of the catalyst used – 0.08 mg, solvent – chloroform.
b
c
d
e
f
g
Turnover number: the moles of products per mole of pigments used in biotransformation; in the parenthesis: effectiveness was calculated as the concentration of the
product per gram of the catalyst used in the reaction.
h
Amount of the catalyst used – 0.1 ␮moles, biotransformation time – 20 h, solvent – chloroform.
derivatives were obtained using extract containing a mixture
of dyes (extract of spinach) as a photocatalyst, due to its larger
number of molecules used in the reaction compared to the purified
dyes (Table 1). A distinctly different ratio of myrtenal/pinocarvone
in two solvents: chloroform and 1,4-dioxane was observed using
spinach extract, but the reason for this phenomenon is hard to
explain. However, the highest catalytic efficiency expressed as
turnover number (here defined as the moles of products per
mole of pigments used in biotransformation) was displayed
by pheophytin b. No oxidative products were detected in the
absence of light. This confirmed the photochemical character of
the biotransformation reactions involving the chlorophyll-derived
catalysts in the organic media. Similar products composition of
pinenes was found under identical conditions with chlorophylls
as well as pheophytins indicating a common oxidation mecha-
4000
Pheophytin a
Pheophytin a + α- pinen
1274
3
3
2
2
500
000
500
000
1272
1500
000
1
5
00
0
1
nism which is largely independent of the used pigments. These
200
1220
1240
1260
1280
1300
1320
1340
1
products are expected to result from the singlet oxygen ( O ) ene
2
Wavelength (nm)
addition to terpenoic olefins, although in the case of both pinenes,
non-ene products were also formed. As has been postulated, the
common mechanism of the photooxidation is rather complex,
and determined by the concurrent involvement of the reactive
oxygen species: singlet oxygen, superoxide radical anions (O · ),
and hydroxyl radicals [21,22]. To gain a better insight into the
nature of photocatalytic biotransformation, the infrared fluores-
cence spectra of the pigments in chloroform with and without
pinene addition were registered. The ability to the 1O2 genera-
tion by pheophytin and chlorophyll in chloroform was directly
demonstrated by spectroscopic monitoring of luminescence at
Fig. 2. Effect of ␣-pinene on the emission spectrum of 1O2 generated in chloroform
containing pheophytin a by light excitation at 410 nm.
2
−
of pinene with singlet oxygen generated by photocatalysts and
(ii) abstraction of an electron from pinene by excited pigments
followed by the generation and subsequent recombination of
pinenyl and oxygen radical intermediates. It remains unclear how
the pinocarvone is formed. So far it has been assumed that the
ketone originates from either rapid decomposition of correspond-
ing hydroperoxide or the abstraction of an allylic H atom from
1
274 nm (Fig. 2). Moreover, we noted that upon pinene presence
alkoxyl radical by O [24,25]. Presumably, in our system, some free
2
in the photocatalytic system, the intensity of fluorescence band
of O2 significantly decreased in relation to the pheophytin a in
chloroform without pinene, showing unambiguous evidence of
singlet oxygen participating in the biotransformation process. On
the other hand, addition of the free-radical inhibitor BHT (with 2
equiv related to pinene) retarded the photooxygenation seriously
radicals participate in pinocarvone formation, since a substantial
decrease in its fraction was noticed after quenching the radicals by
BHT and Trolox (Table 1).
The summary of the biotransformation kinetics of pinene is pro-
vided in Fig. 3. The pinene biotransformation reaction seems to be
1
of the first order type. The dependencies: log (c/c ) (where c and c0
0
(
Table 1), clearly indicating that a free radical oxidative process
are the pinene concentrations at time t and 0) have the character of
a straight line with r determination coefficients 0.98–0.99. One can
2
was also involved in the biotransformation reaction. The typical
radical-initiated autooxidation should be ruled out because a
broad oxygenated terpenes mixture is obtained via this route, e.g.,
pinene epoxides, diols, verbenol, verbenon, myrtenol, myrtenal,
pinocarveol, and hydroperoxides [23,24]. Based on the above
data we concluded that the photocatalytic biotransformation may
involve two different reaction pathways: (i) direct oxygenation
conclude that the pinene biotransformation consists of the paral-
lel reactions: pinene → trans-pinocarveol; pinene → pinocarvone;
pinene → myrtenal.
Additional support for this idea, resulting directly from the
theory of parallel reactions [26], is the presence of straight line
correlations: ctrans-pinocarveol vs cpinocarvone for all the studied

1
8
M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
Fig. 3. The logarithmic change of the pinene concentration with time during the biotransformation of pinene by chlorophyll a; chlorophyll b; pheophytin a; pheophytin b.
systems, i.e., for chlorophylls and pheophytins. The
drawn on the basis of the total turnover number being the higher
for the pheophytins than for chlorophylls after 24 h-photooxidation
(Table 2). These differences in activity can be explained in terms of
the lack of Mg in the core of the pheophytins structure resulting in
the presence of free electrons, which can be excited to higher elec-
tronic states and participate in singlet oxygen formation during
intersystem crossing.
In the case of pinocarveol and pinocarvone, for all pigments, the
highest photooxidation rate was observed during the first 8–10 h
of the reaction (Fig. 4). After that time, the reaction is evidently
retarded, most likely due to accumulation of the products or/and
the photodegradation of pigments. Appearance of myrtenal pro-
duction with time, despite parallel destruction of the pigments,
probably results from extension of the action of free radicals on
pinene in the photocatalytic system.
The photographic visualization of the ␣-pinene oxidation course
by using chlorophyll-derived pigments at the beginning of the
reaction, after 4 and 24 h exposure to light is shown in Figs. 5–7.
A distinct shift in the colour of the catalysts used may indi-
cate a change in their structure leading to their degradation
(last photo). Chlorophyll and a number of related compounds
such as chlorophyllides, chlorophyllins, pheophytins, chlorins
and bacteriochlorophylls have been examined as photosensitiz-
ers for many types of biological systems, including biomolecules,
cells and multicellular organisms. Some of these compounds are
now being examined as sensitizers for photochemotherapy [27].
However, chlorophylls obtained by extraction with an organic
solvent from living leaves have seldom been used as photo-
catalysts because they are quite unstable under illumination. A
photostable PEG-chlorophyllin (polyethylene glycol derivatives)
conjugate, that has the photochemical function of hydrogen gas
evolution and carbon dioxide fixation under visible light, was
reported. PEG-chlorophyllin acquired a photostable nature and
a high photosensitizing ability to produce superoxide anions by
illumination with visible light, as compared to chlorophyllin. The
pinene → myrtenal reaction case is somewhat complex. The lack
of straight line correlations: c
vs c
, c
pinocarvone
myrtenal trans-pinocarveol
vs cmyrtenal together with the different course of pinene biotrans-
formation in the case of elimination of radicals from the reaction
medium, implies an important role of a radical intermediate,
comparable with singlet oxygen, in pinene oxidation, especially,
into the myrtenal. Indeed, as in the presence of BHT or Trolox in the
reaction mixture, the myrtenal/pinocarvone concentration ratio
has dropped from 0.26 as much as to approximately 0.02 ± 0.005,
the distribution of the observed products upon addition of the
radical scavengers provides further evidence for the reaction
mechanism based on two (probably, cooperative) pathways.
We supported the suggestion of parallel reactions in an indepen-
dent reactivity experiment, since neither pinocarvone nor myrtenal
or other derivatives were detected under typical reaction con-
ditions using trans-pinocarveol or myrtenol as the substrates.
Interestingly, only one, unidentified compound arises in myrtenol
photooxidation.
It is easy to notice from the kinetic data (Table 2) that for
the systems with pheophytins almost 100% of pinene was con-
sumed, whereas in the system with chlorophylls approximately
2
0% or 80% of it remained unchanged at the end of the experiment,
which directly results from the higher catalytic activity of pheo-
phytins in the biotransformation process. Compared to ␣-pinene,
the biotransformation of ␤-pinene, for instance, by pheophytin a,
proceeded with higher efficiency and selectivity, yielding 2.56 g/L
myrtenal, 0.176 g/L pinocarvone and 0.098 g/L pinocarveol (with
the total turnover number of 630.6 after 18 h), whereas in the
case of ␣-pinene, 1.51, 0.57 and 0.4 g/L pinocarvone, pinocarveol
and myrtenal, respectively, were obtained (with the total turnover
number of 494 after 20 h). Generally the biotransformation of
pinene is more pronounced for the systems with pheophytins, as
the evidently higher rate constants, i.e., higher slopes of log(c/c0)
values, for this case can be noticed. A similar conclusion can be

M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
19
Table 2
Kinetic data of the ␣-pinene biotransformation using chlorophylls and pheophytins during 24-h irradiation with visible light.
Time of biotransformation (h)
8
Photocatalysta
Chlorophyll a
Total turnover numberb
Pinene consumption (%)
141.6
350.7
118.2
130.2
348.3
495.3
384.2
394.5
24.5
79.8
9.6
20.9
81.8
97.1
89
24
8
24
8
24
8
24
Chlorophyll b
Pheophytin a
Pheophytin b
99.7
a
−5
The concentration of the pigments used in the photocatalytic biotransformation was 3.3 × 10 M; pinene concentration: 35 mM.
Sum of the turnover number for the main ␣-pinene oxidation products.
b
2
2
1
1
50
00
50
00
100
A
B
9
0
80
7
0
60
5
0
40
3
0
5
0
20
1
0
0
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
time (h)
time (h)
3
3
2
2
1
1
50
300
C
D
00
50
00
50
00
2
2
1
1
50
00
50
00
5
0
5
0
0
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
time (h)
time (h)
−
5
Fig. 4. Turnover numbers of porphyrin-containing pigments as a function of irradiation time of 3.3 × 10 M solutions of (A) chlorophyll a; (B) chlorophyll b; (C) pheophytin
a; (D) pheophytin b in the process of pinene biotransformation; (black squares) pinocarvone; (black diamonds) trans-pinocarveol and (white diamonds) myrtenal.
photosensitizing ability may be closely associated with stabiliza-
tion of chlorophyllin conjugated with PEG, although its mechanism
is yet unclear [28,29].
form without ␣-pinene (Figs. 5B and 6B). This effect could be caused
by the negative influence of the light on the chlorophyll molecule,
known as chlorophyll photobleaching, involving irreversible degra-
dation of the pigments. It was reported previously that the excited
chlorophyll molecule can be oxidized by light-induced, very reac-
tive oxygen species, such as free radicals and/or singlet oxygen [31].
As can be seen in Fig. 5A, the chlorophyll a breakdown is associated
with the reduction of light absorption in the Bx band vs to the By
band. The “red”-shifted Qy electronic region was observed concur-
rently. In our work, the photocatalyst degradation was stronger in
the case of chlorophyll b and it was followed by formation of spec-
tral forms absorbing in the 480–600 nm region (Fig. 6). It is worth
noting that, in the chlorophyll b–pinene solution, its characteris-
tic change in colour from green to red took place about 2 h earlier,
compared to the system without pinene. This indicates that the
presence of an electron donor (as most likely pinene is in the reac-
tion with the electrophilic singlet oxygen) may accelerate the initial
step of the chlorophyll b degradation.
In further studies, the stability and physicochemical proper-
ties of the catalysts during the biotransformation of pinene was
investigated by absorption and emission spectroscopy. For a more
detailed study of the low photostability of the natural chlorophyll
a and chlorophyll b under photocatalytic reaction, their absorption
and emission spectra in chloroform in the absence of pinene were
additionally registered during 24-h visible light irradiation.
Chlorophylls and pheophytins are porphyrin pigments having
characteristic absorption bands in “blue” (Soret-band) and “red”
(
Q-band) spectral regions. Two main maxima: Bx and By occur in
the Soret band. Analogously, the Q band is characterized by two
maxima named Qx and Qy. The position of these bands depends on
the pigment solvent. According to Wellburn [30], the main absorp-
tion maxima of chlorophyll a and b in chloroform are located at
4
31, 665 and 460, 647 nm, respectively. Figs. 5A and 6A show the
absorbance spectra of the chlorophyll a- and b-pinene system in
chloroform at the beginning of the experiment and during illumi-
nation with visible light. A gradual decrease in the intensity of Soret
and Q bands of both chlorophylls in chloroform was observed in the
presence of ␣-pinene during illumination. Similar spectral changes
were examined in the case of illumination of chlorophylls in chloro-
The B/Q ratio (where B and Q are defined as the absorbance
intensity of the Soret and Q region, respectively) increased during
illumination for both chlorophylls (data not shown). This phe-
nomenon suggests changes in the molecule structure connected
with a faster decrease in absorbance in the “red” region-band under
light irradiation. The light-induced breakdown of chlorophylls is

2
0
M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
Fig. 5. UV–vis absorption spectra of the chlorophyll a – B, and the chlorophyll a–␣-pinene system in chloroform during light-promoted biotransformation of ␣-pinene – A.
Beside: the photographic visualization of pigment involved in ␣-pinene biotransformation (1) at the beginning of the reaction; (2) after 4 h of the reaction and (3) after 24 h
of the reaction.
in all probability based on the destruction of the porphyrin ring
in position C5 [32] and/or double bond of the phytyl chain of the
pigment molecule, which finally leads to formation of numerous
transient products. Previously, such an effect in a chlorophyll solu-
tion was observed by Jen and MacKiney [33], Hynninen [34] and
Rontani et al. [35]. In our system, the more rapid photodestruc-
tion of chlorophyll b than chlorophyll a probably results from the
presence of the –CHO group in the former pigment. On the basis
of the absorbance spectra of pheophytin a and b, we can con-
clude that, under the biotransformation reaction condition, these
metal free pigments are more stable forms of porphyrin photo-
catalysts in comparison to the chlorophylls (Fig. 7). The complete
decomposition of pheophytins occurred not earlier than after 24 h
of illumination. Also, they showed neither changes in the absorp-
tion spectrum shape during illumination, which suggests lack of a
transient form of this pigment molecule during degradation, nor
any signs of the metallation process of the central nitrogens or pro-
tonation of the macrocycle with formation of dication. In the event
when the metal ion is complexed by a pyrrole ring or dications of
pheophytins are formed, the position and shape of Q bands differs
from that of free based pheophytins [36–41]. This makes them a
new potential photocatalysts not only for the biotransformation of
emission band in the 420–600 nm spectral region. The “short”-
wavelength bands confirm pigment molecule photobleaching and
appearance of chlorophyll degradation products [42]. In the chloro-
phyll a–pinene system with prolonged time of illumination, the
“short”-wavelength band was “blue”-shifted, which suggests that
different products of chlorophyll degradation are formed during
the biotransformation reaction. Interestingly, the location of the
“short”-wavelength band corresponding to degradation products
was not changed in the case of chlorophyll b during illumina-
tion. However, in contrast to chlorophyll a the “red”-shift of the
“long”-wavelength band (to 658 nm) was observed after 8 and 24 h
illumination (Fig. 9). Therefore, we suggest that the photodecompo-
sition process of these chlorophyll forms proceeds in two different
ways.
The fluorescence emission intensity detected also indicates
stronger chlorophyll b photodestruction under the biotransfor-
mation reaction, in comparison to chlorophyll a. The ratio of
fluorescence intensity in the “red”-region to fluorescence intensity
in the “blue”-region after 2 h illumination was 5.4 and 0.2 in the case
of chlorophyll a and b (with pinene), respectively. As can be seen
from the presented spectra, chlorophylls seem to be more stable
in the system, which does not contain ␣-pinene. The above ratio
calculated for chlorophylls in the system without ␣-pinene was
higher than in the system containing ␣-pinene. The considerably
more intensive fluorescence at the short-wavelength bands of the
chlorophyll–pinene systems, as compared to the chlorophylls with-
out pinene, can be explained by stopping the total decomposition
of the chlorophylls probably through stabilization of the tran-
sient compounds by the terpenoid products of the photocatalysis.
This is especially evident for chlorophyll a, which also demon-
strated less decreased band intensity in the long-wavelength at
␣
-pinene but also for other electron rich hydrophobic compounds.
Chlorophylls and pheophytins are able to fluorescence in vis-
ible light. The fluorescence emission spectrum of chlorophyll a
and b in chloroform has a maximum located at 672 and 651 nm,
respectively (Fig. 8). In our experiment, a clear decrease in the
intensity of fluorescence emission of chlorophyll a and b was
observed along with the time of illumination, being more pro-
nounced for chlorophyll b. This effect is connected with the shift
of the chlorophylls emission bands and occurrence of an extra

M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
21
Fig. 6. UV–vis absorption spectra of the chlorophyll b – B, and the chlorophyll b–␣-pinene system in chloroform during light-promoted biotransformation of ␣-pinene – A
inset – enlarged spectra). Beside: the photographic visualization of pigment involved in ␣-pinene biotransformation (1) at the beginning of the reaction; (2) after 4 h of the
(
reaction and (3) after 24 h of the reaction.
Fig. 7. UV–vis absorption spectra of the pheophytin a–␣-pinene – A, and the pheophytin b–␣-pinene – B system in chloroform during light-promoted biotransformation of
␣
-pinene. Beside: The photographic visualization of pigments involved in ␣-pinene biotransformation (1) at the beginning of the reaction; (2) after 4 h of the reaction and
(
3) after 24 h of the reaction.

2
2
M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
6
5
4
3
2
1
0
0
0
0
0
0
0
chlorophyll a +α -pinene
120
100
A
chlorophyll b +α-pinene
A
678
654
0 h
0 h
2 h
8 h
24 h
2
8
2
h
h
4 h
8
6
0
0
40
20
0
1
1
20
651
B
chlorophyll b
1
00
B
chlorophyll a
672
00
8
6
4
2
0
0
0
0
0
8
0
0
6
40
20
0
5
00
550
600
650
700
750
4
50
500
550
600
650
700
750
Wavelength [nm]
Wavelength [nm]
Fig. 9. Emission spectra of the chlorophyll b – B, and the chlorophyll b–␣-pinene
system in chloroform during light-promoted biotransformation of ␣-pinene – A. The
excitation wavelength was at 460 nm.
Fig. 8. Emission spectra of the chlorophyll a – B, and the chlorophyll a–␣-pinene
system in chloroform during light-promoted biotransformation of ␣-pinene – A. The
excitation wavelength was at 460 nm.
1
1
20
00
460
chlorophyll b
0 h illumination
h illumination + exogenous products
2
the presence of monoterpenes. The thesis that products of the
biotransformation reaction may stabilize the transient products
of chlorophyll degradation under the irradiation is particularly
affirmed by the excitation spectrum of chlorophyll b measured
after 2 h of illumination in the presence of exogenously added prod-
ucts (trans-pinocarveol, pinocarvone and myrtenal). This spectrum
has the same shape as that of chlorophyll b one measured with-
out products in the dark (Fig. 10). Similarly, the protective role
of the products was confirmed by the emission spectra of fluo-
rescence of chlorophyll b in their presence (Fig. 11). The ratio of
fluorescence intensity in the “red”-region to fluorescence intensity
in the “blue”-region after 2 h illumination was 17-fold higher in the
case of exogenous addition of the products than directly under the
649
80
60
4
0
0
0
2
4
00
500
600
700
␣
-pinene biotransformation.
In such non-polar solvents as chloroform, chlorophyll at a
concentration range of 2–3 mM appears merely in dimeric forms
43]. Probably due to the decreasing polarity, the ␣-pinene in the
system with chlorophylls promotes enhancement of the pigment
agglomeration. This was confirmed, firstly, by the red-shifted
emission maximum at the beginning of the photoreaction (from
Wavelength [nm]
Fig. 10. Excitation spectra of the chlorophyll b (solid line) in chloroform and the
chlorophyll b in chloroform with biotransformation reaction products (dashed line).
The emission wavelength was at 650 nm.
[
6
72 to 678 nm for chlorophyll a, and from 651 to 654 nm for
two bands located at 435 and 443 nm, respectively, is observed
indicating appearance of structurally aggregated pigment forms.
In contrast to chlorophylls, the fluorescence emission spectra of
pheophytin a and b (Fig. 13) proved high stability of these pigments
during the biotransformation reaction. A clear decrease in fluores-
cence intensity was observed only after 24 h illumination followed
by slight “blue”-shift of the main fluorescence maximum. Interest-
ingly, the destruction of pheophytin a after 24 h was accompanied
by formation of an additional spectral form with band emission at
635 nm. The excitation, emission and absorbance spectra of pheo-
chlorophyll b) and, secondly, by subsequent changes in the posi-
tion of their bands (Figs. 8 and 9) during light excitation associated
with biotransformation progress, in turn leading to a decrease
in pinene concentration. The above results are confirmed also by
the fluorescence excitation spectra measured for the chlorophyll
b in chloroform or chlorophyll b with ␣-pinene in the presence
of exogenously added oxidative derivatives in the dark (Fig. 12).
In the Soret region, chlorophyll b has a band centered at 460 nm.
Upon ␣-pinene addition, excitonic splitting of this Soret band into

M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
23
1
00
676
chlorophyll b
0 h
A
pheophytin a + α-pinene
2
2
2
h
8
6
4
2
0
0
0
0
0
4 h
80
60
40
20
0
0 h
h without exogenous products
2
8
h
h
24 h
658
650
635
5
00
550
600
650
700
750
B
pheophytin b +α-pinene
660
Wavelength [nm]
150
Fig. 11. Emission spectra of the chlorophyll b–␣-pinene in the chloroform system
with exogenously added products after 0, 2 and 24 h of illumination and the chloro-
phyll b–pinene system in chloroform light-promoted biotransformation of ␣-pinene
after 2 h of illumination (thin line). The excitation wavelength was at 475 nm.
1
00
1
1
20
00
460
without α−pinene
with α−pinene
with α−pinene after 2 h illumination
50
chlorophyll b
648
80
60
40
20
0
0
4
50
500
550
600
650
700
750
4
33
Wavelength [nm]
4
35
443
Fig. 13. Emission spectra of the pheophytin a–␣-pinene – A, and the pheophytin
b–␣-pinene – B system in chloroform during light-promoted biotransformation
of ␣-pinene. The excitation wavelengths were at 415 and 440 nm in the case of
pheophytin a and b, respectively.
475
biomimetic catalysts for the photooxidation of monoterpene into
products that attract great interest in the fragrance and flavour
industry. Pheophytins have proved to be the most effective among
the photocatalysts examined. A pheophytin-based photocatalytic
system would be an appropriate, mild and clean method not only
of transformation of ␣-pinene, but also other precursors widely
distributed in nature. The spectral investigations have provided
considerable information on the stability of the porphyrin pigments
during the biotransformation of ␣-pinene. Based on the analysis of
the absorbance and fluorescence emission spectra, we can state
that under 24-h of light-promoted biotransformation, pheophytin
a and b, the metal free pigments, are more stable photocatalysts, in
comparison with chlorophylls.
Kinetic study confirms a weakening in the catalytic activity
together with an increase in the photodecomposition rate of the
pigments, however with the exception of myrtenal formation.
According to our suggestion, this is due to the parallel type of
mechanism of photocatalytic biotransformation, which could be
exploited in the planning of the improvement of the reaction
selectivity, i.e., changing the proportion of the radical to singlet
oxygen pathway. For future work, the optimal pigment concen-
tration should also be taken into consideration, since an increased
concentration of the pigments results in a decrease in their activ-
ity. As follows from the above-presented results, the course of
photodecomposition of chlorophyll a and chlorophyll b in the bio-
transformation process is evidently different. One of the reasonable
solutions aimed at increasing the stability might be the use of
heterogeneous catalysts through the immobilization of the pheo-
phytins or chlorophylls in photostable and light-transparent solid
supports (e.g., sol–gels materials), which would also be advan-
4
00
500
600
700
Wavelength [nm]
Fig. 12. Excitation spectra of the chlorophyll b (solid line) in chloroform and the
chlorophyll b–␣-pinene system in chloroform with exogenously added products
dashed line). The emission wavelength was at 650 nm.
(
phytins did not show a relationship between the destruction of
pigments and presence of exogenously added products of the bio-
transformation reaction (data not shown).
There are many interesting products resulting from oxy-
functionalization of pinenes, due to many diverse degradation
pathways. However, the drawbacks of the classical biotransfor-
mation systems, such as excess volatility of terpenes, insolubility
in aqueous solutions, and their toxicity towards microorganisms
result in low bioconversion yields, which have usually been in mil-
ligrams per liter range [4,44]. Two noteworthy exceptions were the
␣
-pinene biotransformation with Hormonema sp. yielding 0.3 g/L
verbenone and 0.4 g/L verbenol after 96 h [45], and production of
pinene oxide, verbenol and myrtenol with total products of over
1
g/L using recombinant Escherichia coli in an aqueous-organic two-
phase system [46]. In this context, the total product concentrations
of over 2.5 g/L after 24 h of biotransformationpresented in this work
mark a very promising step towards further optimization of the
process.
4
. Conclusions
The significance of the data presented here lies in the fact
that chlorophyll-derived pigments were the first time used as

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M. Trytek et al. / Journal of Photochemistry and Photobiology A: Chemistry 223 (2011) 14–24
tageous with respect to easier recovery and recycling of these
biocatalysts. In the case of chlorophyll b, binding of the –CHO group
should be carried out, for example by employing it in the immobi-
lization process. Further improvement of the catalytic activity of
chlorophylls may be achieved by increasing the photostability by
chemical modification of these catalysts with synthetic polymers
such as polyethylene glycol derivatives or others.
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