A facile method for the preparation of α-methylene-γ-butyrolactones from tulip tissues by enzyme-mediated conversion

Article abstract of DOI:10.1016/j.tetlet.2009.06.018

We developed a facile method of enzyme-mediated conversion of 6-tuliposide to α-methylene-γ-butyrolactone (tulipalin). We used a tuliposide-converting enzyme for the conversion of 6-tuliposides extracted from tulip tissues into the corresponding tulipalins in high yields within 2 h at pH 7.0. The resulting tulipalins were selectively extracted by using several organic solvents.

Full text of DOI:10.1016/j.tetlet.2009.06.018

Tetrahedron Letters 50 (2009) 4751–4753
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Tetrahedron Letters
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A facile method for the preparation of
a-methylene-c-butyrolactones from
tulip tissues by enzyme-mediated conversion
a
b
a
a
a
Yasuo Kato a, , Hiroyuki Yoshida , Kazuaki Shoji , Yukio Sato , Noriyuki Nakajima , Shinjiro Ogita
*
a Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
b Toyama Prefectural Agricultural, Forestry, and Fisheries Research Center, 1124-1 Yoshioka, Toyama, Toyama 939-8153, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We developed a facile method of enzyme-mediated conversion of 6-tuliposide to a-methylene-c-butyro-
Received 7 May 2009
Revised 2 June 2009
Accepted 5 June 2009
Available online 9 June 2009
lactone (tulipalin). We used a tuliposide-converting enzyme for the conversion of 6-tuliposides extracted
from tulip tissues into the corresponding tulipalins in high yields within 2 h at pH 7.0. The resulting
tulipalins were selectively extracted by using several organic solvents.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Tulip
Tuliposide
Tulipalin
Enzymatic transformation
Selective extraction
a-Methylene-c-butyrolactone
All parts of the tulip (Tulipa gesneriana L.) plant contain large
sides found in tulip tissues, especially petals, into tulipalins. In this
study, we focused on the main tuliposides and tulipalins, that is, 6-
tuliposide A (1a) and B (1b), and tulipalin A (2a) and B (2b), respec-
tively. We optimized the conditions of the enzymatic transforma-
tion of 1a and 1b into 2a and 2b, respectively, and selectively
extracted the products 2a and 2b (Scheme 1).
quantities (0.2–2% w/w fresh weight (F.W.)) of antimicrobial glu-
cose esters, namely, tuliposides (1).1–6 These esters are unstable
and are chemically hydrolyzed under basic conditions to give lac-
tones of their aglycones, tulipalin (2), which has antimicrobial
activities. These compounds are also accumulated in the Lilliflorae
plants, such as those belonging to the genera Erythronium, Gabea,
and Alstromeria.7 During the course of our studies on the biosyn-
thesis and the mechanism of action of tuliposides, we discovered
a tuliposide-converting enzyme that catalyzes the stoichiometric
conversion of tuliposides into tulipalins in tulip tissues.8 Tulipalins
At first, we examined the effect of tuliposide-converting en-
zyme on the transformation of tuliposides extracted from tulip
petals. Freeze-dried petals of the tulip cultivar ‘Murasakizuisho’
(1 g dry weight (D.W.)) were homogenized in a mortar with
50 mL of cold MeOH at 4 °C. The extract was filtered, concentrated
in vacuo, and dissolved in 50 mL of cold water affording a tulipo-
side-containing extract of the tulip petals. The concentrations of
1a and 1b in the extract, as determined by HPLC analysis were
have a common structure—a-methylene-c-butyrolactone—and are
used as synthetic intermediates in the production of several bioac-
tive compounds,9 antimutagenic agents,10 insect repellents,11 elec-
trolyte solution of Li-ion battery,12 and as monomers of functional
4.0 mM and 4.2 mM, respectively.15 Subsequently, 5
potassium phosphate buffer (KPB, pH 7.0) was added to 490 lL
lL of 1 M
bioplastics.13 Until now,
a-methylene-
c-butyrolactones have been
prepared according to organic synthesis methods14 based on petro-
leum chemistry; however, these methods are not environment
friendly. In Toyama Prefecture, Japan, millions of tulip bulbs are
produced every year as one of major agricultural products. The pet-
als are cut and used as biomass in order to produce bulbs of excel-
lent qualities. For efficient utilization of this biomass, it is
important to develop an environmentally safe approach involving
the use of the tuliposide-converting enzyme to convert the tulipo-
of the extract and the mixture was incubated at room temperature
with or without the partially purified tuliposide-converting en-
zyme from the tulip bulb.16 Even in the absence of the enzyme,
4% and 33% of 1a and 1b were chemically converted to 2a and
2b, respectively, in 2 h. Increasing the incubation time for more
than 3 h resulted in further hydrolysis of 2b. On the other hand,
in the presence of 5U of the enzyme, both 1a and 1b were almost
quantitatively converted to 2a and 2b, respectively, within 2 h. The
above-mentioned results strongly suggest that the enzyme has a
potential applicability in the production of tulipalins from tulipo-
sides extracted from tulip tissues.
* Corresponding author. Tel.: +81 766 56 7500x514; fax: +81 766 56 2498.
E-mail address: ykato@pu-toyama.ac.jp (Y. Kato).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.06.018

4752
Y. Kato et al. / Tetrahedron Letters 50 (2009) 4751–4753
O
OH
O
O
O
O
O
O
O
HO
HO
1a
2a
OH
OH
2a
Tuliposide-converting
enzyme
Selective
extraction
+
+
O
OH
OH
OH
OH
O
O
O
O
O
HO
HO
2b
1b
OH
OH
2b
Scheme 1. Enzymatic preparation of a-methylene-c-butyrolactone (tulipalin).
Since the petals of the tulip cultivar ‘Murasakizuisho’ contain
both 1a and 1b, the reaction product obtained was a mixture of
2a and 2b. For the separation of 2a and 2b, we screened suitable
solvents that enabled the isolation of these compounds by simple
extraction and concentration, respectively. After the enzymatic
conversion of tuliposides in the tulip petal extracts into tulipalins,
2a was first extracted from the reaction mixture by using a non-
polar organic solvent (solvent-1) under saturating with NaCl.17
The extraction efficiency of tulipalins is shown in Table 1. The yield
of 2a was higher when aromatic hydrocarbons (entries 4–6) were
used than that obtained when an alkane (entry 1) was used as
the solvent, and the amount of 2b co-extracted using these sol-
vents was less than 10%. Extraction with ether- (entry 2) and es-
ter-type (entry 3) solvents also afforded high yields of 2a;
however, significant amount of 2b was also co-extracted, suggest-
ing that such solvents were not suitable for the selective extraction
of 2a. After extraction of 2a with toluene, 2b remaining in the reac-
tion mixture was further extracted with a polar solvent (solvent-2),
namely, propyl acetate, 3-methylbutan-1-ol, 2-propanol, 1-butanol
(BuOH), and 1:1 mixture of BuOH and acetone and 2b was ob-
tained in the yields of 62%, 65%, 65%, 87%, and 98%, respectively.
Pure 2a and 2b can be obtained from the extracts by simple distil-
lation. On the basis of these results, toluene and 1:1 mixture of
BuOH and acetone were selected as the most suitable solvents
for the selective extraction of 2a and 2b, respectively, from the en-
zyme reaction mixture.
pearl column chromatography was 18.7, 11.1, 15.4, 68.0, 42.1, and
11.4 U/mg, respectively. These enzymes extracted from these plant
parts were used for the enzymatic conversion of tuliposides in the
tulip petal extract. The crude enzyme extracted from the roots,
bulbs, stems, leafs, petals, anthers, and pistils converted 86%,
91%, 88%, 93%, 94%, 97%, and 93% of 1a in the petal extract to 2a
and 84%, 82%, 55%, 64%, 83%, 71%, and 74% of 1b of the same origin
to 2b, respectively. These results show that this enzymatic conver-
sion can be performed using enzymes obtained from any of the
abovementioned tulip tissues. We have previously found that the
enzyme in each tulip tissue has different substrate specificities.8
Thus, the differences in the conversion yields were probably due
to the substrate specificity of the enzymes toward 1a and 1b. The
products 2a and 2b were selectively extracted using toluene and
1:1 mixture of BuOH and acetone in 70–83% and 88–98% yields,
respectively.
A MeOH extract of several tulip tissues was prepared and trea-
ted with the tulip bulb enzyme.19 Table 2 shows the concentra-
tions, conversion yields, and extraction efficiencies of 2a and 2b
formed from the extracts of various parts of the plant. Although
the ratio of 1a/1b was different for each tissue, the enzyme enabled
efficient conversion of the reactants into 2a and 2b in 2 h. This sug-
Table 2
The enzymatic preparation of tulipalins (2a, 2b) from tuliposides (1a, 1b) from
different tulip tissues
Next, we investigated the bioconversion ability of the tulipo-
side-converting enzyme extracted from several different tulip tis-
sues. The specific activity of enzyme that was partially purified18
from the crude extract of roots, stems, leaves, petals, anthers,
and pistils of the tulip cultivar ‘Murasakizuisho’ using DEAE-Toyo-
Entry Tissue
2a
2b
Conc.a
(mM)
Yieldb
(%)
Ext. effi.c Conc.a
Yieldb
(%)
Ext. effi.c
(%)
(%)
(mM)
1
2
3
4
5
6
7
Root
Bulb
Stem
Leaf
Petal
Anther
Pistil
1.38
3.64
2.73
3.99
2.26
0.40
10.4
98
93
91
90
96
93
85
82
90
80
85
82
73
84
2.43
<0.01
0.16
2.29
3.47
5.37
4.33
>99
N.D.
67
>99
88
91
N.D.
89
71
97
Table 1
Extraction of tulipalins (2a, 2b) that were obtained by enzymatic transformation from
tuliposides (1a, 1b) from a tulip petal extract using solvent-1
80
>99
>99
78
Entry
Solvent-1
Extraction efficiencya (%) of
Tulip petal extract (490
from the tulip bulb and 1 M KPB (5
NaCl was added to the reaction mixture until saturation, 2a was extracted with
toluene (200
l
L) was added to a solution of the enzyme (5 U) extract
lL). After 2 h of incubation at room temperature,
2a
2b
1
2
3
4
5
6
Hexane
8
70
86
79
84
88
<0.01
26
73
5
3
10
l
L Â 2, 100 lL Â 1), and the 2b remaining in the mixture was
Diethylether
Propyl acetate
Xylene
Toluene
Benzene
extracted with the same volumes of 1:1 mixture of BuOH and acetone. N.D.: not
determined.
a
Concentration in the reaction mixture.
Calculated on the basis of original concentrations of 1a and 1b before enzymatic
b
treatment.
c
a
Calculated on the basis of the concentrations of 2a and 2b in the reaction
Calculated based on the concentrations of 2a and 2b in the reaction mixture
mixture after enzymatic treatment.
after enzymatic conversion.

Y. Kato et al. / Tetrahedron Letters 50 (2009) 4751–4753
4753
13. (a) Grieco, P. A.; Hiroi, K. J. Chem. Soc. Chem. Commun. 1972, 1317; (b) Griffin, G.
W.; Nishiyama, K.; Ishikawa, K. J. Org. Chem. 1977, 42, 180; (c) Murray, T. F.;
Samsel, E. G.; Varma, V.; Norton, J. R. J. Am. Chem. Soc. 1981, 103, 7520.
14. (a) Brzezinski, L. J.; Rafel, S.; Leahy, W. Tetrahedron 1997, 53, 16423; (b)
Schlewer, G.; Stampf, J.-L.; Benezra, C. J. Med. Chem. 1980, 23, 1031; (c)
Hoffmann, H. M. R.; Rabe, J. Angew. Chem., Int. Ed. Engl. 1985, 24, 94; (d) Barbier,
P.; Benezra, C. J. Med. Chem. 1986, 29, 868; (e) Dinprasert, P.; Mahidol, C.;
Thebtaranonth, C.; Thebtaranonth, Y. Tetrahedron Lett. 1989, 30, 1149; (f)
Ohgiya, T.; Nishiyama, S. Hetrocycles 2004, 63, 2349.
15. The reaction product was analyzed by high-performance liquid
chromatography (HPLC; Shimadzu, Japan) at 208 nm, with an ODS-100 V
column (4.6 Â 150 mm) at a flow rate of 0.65 mL/min using an elution solvent
consisting of 10 mM of H3PO4 in 10% MeOH.
gests that this system can be applicable in the case of all the tulip
tissues. Further, the products 2a and 2b were selectively extracted
with toluene and 1:1 mixture of BuOH and acetone, respectively,
and were recovered in sufficiently high amounts. Studies are under
way to develop methods for the production of tulipalins by the
enzymatic conversion of tuliposide-related compounds extracted
from other plant tissues.
In conclusion, we developed a novel approach for the produc-
tion of
a-methylene-c-butyrolactones (tulipalins) from tulipo-
sides, which are compounds found in tulip tissues, by enzyme-
mediated conversion for successive extraction. The reaction pro-
ceeds at room temperature in an aqueous solution. Since tulipo-
side-related compounds are found in several easily available
plants, the application of enzymatic conversion is an attractive op-
tion for the production of tulipalins. In order to use this system for
16. Two grams (D.W.) of freeze-dried bulbs of the tulip cultivar ‘Murasakizuisho’
were pounded in a mortar into which 20 mL of 10 mM potassium phosphate
buffer (KPB, pH 7.0) was added and the solution was centrifuged (10,000g,
15 min, 4 °C) to obtain a cell-free extract. The extract was loaded onto a DEAE-
Toyopearl chromatography column (Tosoh, Japan), which had been
equilibrated with
a 10 mM KPB solution, fractionated by elution with
100 mM KPB, and the active fractions were collected. The obtained crude
enzyme solutions having specific activity of 100 U/mg were used for enzymatic
conversions. One unit (U) of the enzyme activity was defined as described
previously.8
manufacturing a-methylene-c-butyrolactones from plant biomass,
further studies are now under way. Further, plant tissues are also
being screened for the identification of tissues that specifically
accumulate 1a or 1b that enable us to obtain pure 2a and 2b with-
out any selective extraction.
17. After enzymatic conversion, NaCl was added to the mixture until saturation, 2a
was extracted with solvent-1 (200
the mixture was extracted with solvent-2 (200
extracts were combined and analyzed by gas chromatography (DB-5 column;
Agilent technologies).
lL Â 2, 100 lL Â 1), and the 2b remaining in
l
L Â 2, 100 lL Â 1). Both the
References and notes
18. Samples of freeze-dried roots (1.3 g D.W.), stems (2.2), leaves (2.2), petals (1.2),
anthers (2.0), and pistils (1.2) of the tulip cultivar ‘Murasakizuisho’ were
disrupted in a mortar; subsequently, 150 mL of 10 mM KPB was added and the
mixture was stirred vigorously at 4 °C for 1 h. After filtration and centrifugation
(21,500g, 15 min, 4 °C), the obtained supernatant was mixed with 4 g of DEAE-
Toyopearl equilibrated with 10 mM KPB and stirred for 1.5 h at the same
temperature. The gels were placed in a small column and washed with 20 mL
of 10 mM KPB. The fractions exhibiting 1a-converting activity were eluted
using 100 mM KPB or 100 mM KPB + 100 mM NaCl, combined, concentrated by
Centriprep 100 (Millipore, USA), and used for enzymatic conversion. Five
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crude enzyme extracted from the roots (0.86 U), bulbs (5), stems (0.62), leaves
(3.2), petals (3.0), anthers (3.6), and pistils (0.84) and incubated for 2 h. After
adding NaCl to the reaction mixture until saturation, 2a and 2b were extracted
with toluene and 1:1 mixture of BuOH and acetone, as described.16
19. The tissues (50–200 mg F.W.) were disrupted using a multi-beads shocker
(Yasui-Kikai) in 1 mL of MeOH and the solution was centrifuged (10,000g, 4 °C).
The supernatant was evaporated and the residue was dissolved in 1 mL of cold
water. Five microlitre of 1 M KPB and 490 lL of the tissue extract were mixed
with the enzyme extracted from the bulbs (5 U) and incubated for 2 h. The
reaction product was extracted with toluene and 1:1 mixture of BuOH and
acetone and was analyzed as described.16
´ ˇ
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