Selective synthesis of cinnamyl alcohol by cyanobacterial photobiocatalysts

Article abstract of DOI:10.1016/j.tetlet.2015.01.092

The chemo-selective reduction of cinnamaldehyde was examined using 7 strains of cyanobacterial cells using light energy for NADPH regeneration possibly through photosynthetic electron transport. Cinnamyl alcohol was synthesized selectively with 6 strains, and without the formation of any byproducts with 3 strains. Especially, reaction by Synechocystis sp. PCC 6803 resulted in an extremely high yield (>98%) under illumination while that under dark scarcely proceeded.

Full text of DOI:10.1016/j.tetlet.2015.01.092

Tetrahedron Letters 56 (2015) 1089–1091
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Selective synthesis of cinnamyl alcohol by cyanobacterial
photobiocatalysts
b
c
d
Rio Yamanaka a, , Kaoru Nakamura , Masahiko Murakami , Akio Murakami
⇑
a Faculty of Pharmaceutical Science, Himeji Dokkyo University, 7-2-1 Kami-Ohno, Himeji, Hyogo 670-8524, Japan
b Graduate School of Human Development and Environment, Kobe University, 3-11 Tsurukabuto, Nada, Kobe, Hyogo 657-8501, Japan
c College of Science and Technology, Nihon University, 7-24-1 Narashinodai, Funabashi, Chiba 274-8501, Japan
d Kobe University Research Center for Inland Seas and JST-CREST, 2746 Iwaya, Awaji, Hyogo 656-2401, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemo-selective reduction of cinnamaldehyde was examined using 7 strains of cyanobacterial cells
using light energy for NADPH regeneration possibly through photosynthetic electron transport. Cinnamyl
alcohol was synthesized selectively with 6 strains, and without the formation of any byproducts with 3
strains. Especially, reaction by Synechocystis sp. PCC 6803 resulted in an extremely high yield (>98%)
under illumination while that under dark scarcely proceeded.
Received 24 October 2014
Revised 10 January 2015
Accepted 14 January 2015
Available online 17 January 2015
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Photobiocatalyst
Reduction
Cyanobacteria
Cinnamyl alcohol
NADPH
Introduction
hol dehydrogenase (ADH) is used.8 Recently, the synthesis of cin-
namyl alcohol by ADH from Bacillus strearothermophilus (BsADH)
a
,b-Unsaturated alcohols are important synthetic intermediates
expressed in recombinant Escherichia coli cells has been reported.9
Furthermore, a serious issue associated with the use of biocata-
lysts is the successive supply of cofactors NADH or NADPH, which
provide reducing power and chemical energy. In non-photosyn-
thetic organisms, chemical energy is produced using an organic-
carbon source such as a saccharide. When using isolated enzymes
as biocatalysts, a cofactor-regenerating system should be intro-
duced to ensure the reducing power. In the synthesis of cinnamyl
alcohol by isolated Baker’s yeast alcohol dehydrogenase (ADH), a
cofactor-recycling system (substrate-coupled recycling system of
NADH) was used.8 In this case, ADH also reduces NAD+ to NADH
in the presence of ethanol. In the synthesis of cinnamyl alcohol
by ADH from Bacillus strearothermophilus (BsADH) expressed in
recombinant E. coli cells, NADH is regenerated by BsADH and 2-
propanol (substrate-coupled NADH-regenerating system).9 The
main issues related to these approaches are that an isolation of
enzyme, transgenesis, and/or an excess of organic carbons and bio-
catalysts are required.
used in the pharmaceutical industry, and fragrance and flavoring
manufacturing. In particular, cinnamyl alcohol is an indispensable
ingredient for the anticancer drug Taxol1 and the antibiotic Chloro-
mycetin.2 It is also an essential intermediate for the synthesis of
aroma-related compounds.3 Typically, cinnamyl alcohol 2 is syn-
thesized from cinnamaldehyde 1, a natural substance present in
the bark of the cinnamon tree. However, a synthetic approach for
the selective hydrogenation of C@O over C@C is difficult. Therefore,
the saturated aldehyde 3 and the saturated alcohol 4 are co-syn-
thesized as byproducts (Scheme 1).4
To address the aforementioned issue, biocatalysts have been
recently proposed as an efficient alternative, because biocatalytic
reactions using microorganisms are expected to be more selective
and environmentally friendly.5,6 However, the microorganisms do
not always provide selective and effective reactions. To control bio-
catalytic reactions, several methods have been used.6 The reduc-
tion of cinnamaldehyde by dry yeast (Whole cell) gave the
saturated alcohol as well as cinnamyl alcohol.7 To accomplish
selective synthesis of cinnamyl alcohol, isolated Baker’s yeast alco-
A different type of cofactor-regenerating system is used in
oxygenic photosynthetic (photoautotrophic) organisms such as
cyanobacteria, algae, and plants. In the system, the solar energy,
captured by photosynthetic pigments such as chlorophyll
(Chl a), is converted into electrochemical energy to regenerate
a
⇑
Corresponding author. Tel./fax: +81 79 223 6871.
E-mail address: rioy@himeji-du.ac.jp (R. Yamanaka).
http://dx.doi.org/10.1016/j.tetlet.2015.01.092
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

1090
R. Yamanaka et al. / Tetrahedron Letters 56 (2015) 1089–1091
Results and discussion
We conducted the reduction of cinnamaldehyde 1 to synthesize
cinnamyl alcohol 2 by cyanobacteria under continuous illumina-
tion provided by a red LED15 (660 nm, 160 mol photon/m2 s) at
l
20 °C, on a rotary shaker (110 rpm). Seven strains of cyanobacteria
were used in this study, as listed in Figure 1. As for the reduction of
cinnamaldehyde by Synechocystis sp. PCC 6803 and Synechocystis
sp. PCC 6714, the results in detail are shown in Supplementary Fig-
ure 1 and Supplementary Figure 2. These cyanobacteria were cul-
tivated in a BG-11 medium16 under continuous illumination with
day-light type fluorescent light (20 l
mol photon/m2 s) at 20 °C.
Scheme 1. Reduction pathway of cinnamaldehyde.
The cell amount of cyanobacteria was estimated by the amount
of the main photosynthetic pigment, that is, chlorophyll a (Chl a).
This was quantified by extraction with 100% MeOH and visible
absorption spectrometry ([Chl a] (mg/L) = A665–750 Â 13.4).17 After
incubation, the reaction mixture was extracted with ether, and
the yields of the reaction products were determined by gas chro-
matography (GC), with naphthalene as the internal standard (GC
conditions: HR-52 50 m, He 1.5 mL/min, 170 °C. Retention times:
cinnamaldehyde 1, 7.9 min; cinnamyl alcohol 2, 8.5 min; com-
pound 3, 6.1 min; compound 4, 7.1 min; naphthalene, 6.9 min).
The reductions of cinnamaldehyde in the dark by cyanobacteria
were also conducted as shown in Figure 1, Supplementary Figure 1,
and Supplementary Figure 2.
Cinnamyl alcohol 2 was preferentially synthesized by six strains
of cyanobacteria under illumination by a red LED (Fig. 1). The
reduction of cinnamaldehyde 1 by Synechocystis sp. PCC 6803
and Synechocystis sp. PCC 6714, and Fischerella muscicola UTEX
1301 gave only cinnamyl alcohol 2. Among these three strains, Syn-
echocystis sp. PCC 6803 gave cinnamyl alcohol 2 most efficiently
after ca. 4 days incubation. On the other hand, in the reduction
of cinnamaldehyde 1 by Anabaena sp. PCC 7120 and Plectonema
boryanum IAM M101, 4 was obtained as a byproduct, which may
mean the enone reductase was involved in the reactions. Surpris-
ingly, the reduction of cinnamaldehyde 1 by Synechococcus sp.
PCC 7942 preferentially gave 3 (Fig. 1). Since Synechococcus sp.
NADPH from NADP+ via photosynthetic electron-transfer reactions.
NADPH, a strong reductant in photosynthetic organism cells, can
be also used for the reduction of exogenous artificial substrates.
In a previous report,10 we demonstrated that NADPH in cyanobac-
terial cells can efficiently reduce the exogenously added ketones
(acetophenone derivatives). We also showed that the reduction
of these ketones is mostly light dependent.11 Therefore, cyanobac-
teria are promising and highly efficient candidates for novel
‘photobiocatalysts’.
To date, photosynthetic microorganisms have been used as pho-
tobiocatalysts leading to highly selective hydrogenation.12 In par-
ticular, the C@C bond in enones was preferentially reduced by
the cyanobacterium Synechococcus sp. PCC 7942, and the corre-
sponding saturated aldehyde and saturated alcohol were
obtained.13,14 The critical point in the reduction of
a,b-unsaturated
aldehydes is the regio- or chemo-selectivity. To date, there are no
reports on the selective hydrogenation of C@O in enones by photo-
biocatalysts such as photosynthetic microorganisms.
Here, we report the efficient reactivity and high selectivity
for different functional groups in the reduction of enones
(cinnamaldehyde 1) by several strains of cyanobacteria to give an
allylic alcohol, cinnamyl alcohol 2.
Strain of cyanobacteria
Time
(h)
Conversion (%)
under red LED illuminaꢀon
Conversion (%)
In the dark
(mg Chla)
0
20
Synechocysꢀs sp. PCC 6803
(0.056)
Synechocysꢀs sp. PCC 6714
(0.067)
Fischerella muscicola UTEX 1301
(0.039)
Anabaena cylindrica IAM M1
2 (Cinnamyl alcohol)
(0.035)
3 (3-Phenylpropanal)
4 (3-Phenylpropan-1-ol)
Plectonema boryanum IAM M101
(0.023)
Anabaena sp. PCC 7120
(0.059)
Synechococcus sp. PCC 7942
(0.058)
Figure 1. Reduction profile of cinnamaldehyde by several strains of cyanobacteria under light and dark conditions. The inset figure illustrates the reaction vessel consists of
the red LED (Sharp GL5UR3K, emission peak at 660 nm with 22 nm full-width at half-maximum, light intensity: 160
mol photon/m2 s) illumination system and the flask
l
with a green-colored cyanobacterial suspension. Cinnamaldehyde: 1 mg, medium: BG-11 (20 mL), reaction temperature: 20 °C, for reduced products, 2, 3, and 4, see
Scheme 1.

R. Yamanaka et al. / Tetrahedron Letters 56 (2015) 1089–1091
1091
PCC 7942 contains the enone reductase as reported by Fu et al.,14
Conclusion
C@C in cinnamaldehyde 1 was preferentially reduced. Synechococ-
cus sp. PCC 7942 also has the ketoreductase18 that can reduce C@O
and the reduction of 3 was subsequently occurred to give 4. Thus
the reduction of cinnamaldehyde 1 by Synechococcus sp. PCC
7942 resulted in the mixed products, 3 and 4.
In this work, we proposed a novel method for the chemoselec-
tive synthesis of cinnamyl alcohol from cinnamaldehyde using
cyanobacterial photobiocatalysts such as Synechocystis sp. PCC
6803 and some other strains. Synechocystis sp. PCC 6803 was the
best prospect with highest yield (>98%) and strict chemoselectivity
for the synthesis of cinnamyl alcohol.
The reduction of cinnamaldehyde 1 by cyanobacterial cells may
be partly affected by respiratory activity, because a certain amount
of NADH is produced through dark respiration.19 Therefore, we also
investigated the reductions of cinnamaldehyde 1 by cyanobacteria
simultaneously in the dark (Fig. 1, Supplementary Fig. 1 and
Supplementary Fig. 2). In six cyanobacterial strains, the reduction
somewhat proceeded in the dark. While, only in Synechocystis sp.
PCC 6803, the reduction in the dark was scarce; the reduction
was nearly light-dependent. These results showed that the reduc-
tion of cinnamaldehyde was remarkably enhanced by photosyn-
thetic process of cyanobacteria. In the previous study10 we
proposed that NADPH generated through photosynthetic electron
transport was an essential cofactor in the cyanobacterial reduction
of exogenous substrates such as aromatic ketones.
Acknowledgments
This work was supported by JSPS KAKENHI Grant Numbers
25410189 (R.Y.) and 23370013 (A.M.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.01.
092.
To establish the efficient synthesis of cinnamyl alcohol 2, the
condition for the photobioreduction of cinnamaldehyde 1 by Syn-
echocystis sp. PCC 6803 was studied. Synechocystis sp. PCC 6803
(0.076 mg Chl a, 3.8 mg dried cell weight) was incubated in
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