Synthesis of trilobatin from naringin via prunin as the key intermediate: Acidic hydrolysis of the α-rhamnosidic linkage in naringin under improved conditions

Article abstract of DOI:10.1080/09168451.2018.1482455

Trilobatin [4-(β-D-glucopyranosyloxy)-2,4”,6-trihydroxydihydrochalcone] was synthesized from commercially available naringin in three steps with an overall yield of 30%. The key step was the acid-catalyzed site-selective hydrolysis of terminal α-rhamnopyranosidic linkage in neohesperidose involved in naringin under controlled conditions, by applying a high-pressure steam sterilizer.

Full text of DOI:10.1080/09168451.2018.1482455

Bioscience, Biotechnology, and Biochemistry
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Synthesis of trilobatin from naringin via prunin as
the key intermediate: acidic hydrolysis of the α-
rhamnosidic linkage in naringin under improved
conditions
Kazuki Kurahayashi, Kengo Hanaya, Shuhei Higashibayashi & Takeshi Sugai
To cite this article: Kazuki Kurahayashi, Kengo Hanaya, Shuhei Higashibayashi & Takeshi Sugai
(2018): Synthesis of trilobatin from naringin via prunin as the key intermediate: acidic hydrolysis of
the α-rhamnosidic linkage in naringin under improved conditions, Bioscience, Biotechnology, and
Biochemistry, DOI: 10.1080/09168451.2018.1482455
To link to this article: https://doi.org/10.1080/09168451.2018.1482455
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2018.1482455
Synthesis of trilobatin from naringin via prunin as the key intermediate:
acidic hydrolysis of the α-rhamnosidic linkage in naringin under improved
conditions
Kazuki Kurahayashi, Kengo Hanaya, Shuhei Higashibayashi and Takeshi Sugai
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Keio University, Shibakoen, Minato-ku, Tokyo, Japan
ABSTRACT
ARTICLE HISTORY
Received 24 April 2018
Accepted 22 May 2018
Trilobatin [4ʹ-(β-D-glucopyranosyloxy)-2ʹ,4”,6ʹ-trihydroxydihydrochalcone] was synthesized
from commercially available naringin in three steps with an overall yield of 30%. The key
step was the acid-catalyzed site-selective hydrolysis of terminal α-rhamnopyranosidic linkage
in neohesperidose involved in naringin under controlled conditions, by applying a high-
pressure steam sterilizer.
KEYWORDS
Trilobatin; dihydrochalcone
glycoside; naringin; site-
selective hydrolysis
Trilobatin [4ʹ-(β-D-glucopyranosyloxy)-2ʹ,4”,6ʹ-trihy-
droxydihydrochalcone, 1, Figure 1] was first synthe-
sized as an artificial sweetener [1], and later isolated
from the leaves of Malus sp. [2]. and other plants.
Structurally similar phlorhizin (2, Figure 1) is known
as a non-selective inhibitor against sodium glucose
co-transporter SGLT-1 and 2 [3]. Fortunately, toward
its development as a safe sweetener, such undesirable
activities have not been reported in trilobatin.
Trilobatin only shows anti-inflammatory activity
[4,5] and α-glucosidase inhibitory action [6].
Among so-far developed dihydrochalcone glycoside
sweeteners [7], we became interested in the simple
structure and such the safe property of trilobatin. We
embarked upon its synthesis starting from naringin
(3) via its selectively de-rhamnosylated form, prunin
(4), as the key intermediate. We are currently
engaged in transformation of naturally abundant fla-
vonoid glycosides into valuable compounds based on
site-selective reactions [8–10]. For us, however, the
partial hydrolysis of disaccharide is the first trial.
recovery of L-rhamnose from the hydrolysate was
achieved.
We recently have developed the use of high pres-
sure steam sterilizer for acid-catalyzed hydrolysis of
glucuronide in baicalin [14]. The aglycone was repro-
ducibly obtained in 66% yield. The advantages of
using high-pressure steam sterilizer over a sealed
tube are as follows. Its safety has been established
over a history of several centuries. Reactions can be
carried out in open vessels such as conical flasks or
beakers. The scale-up of the reaction is accomplished
simply by increasing the number of reaction vessels
in the sterilizer. The most important issue in high-
pressure steam sterilization is to avoid the introduc-
tion of volatile substances. For example, the acid
catalyst should be sulfuric acid, not hydrochloric acid.
We therefore adopted the use of dilute sulfuric
acid (0.3%) and the reaction time was examined
with a fixed temperature of 121°C as shown in
Scheme 1. When the reaction was carried out for
20 min, the ratio of 3, 4, and naringenin (5) in the
crude precipitate was revealed to be 1.0:1.0:0.6, based
on the 1H NMR spectrum (see Experimental). Prunin
(4) was isolated in 30% yield after chromatographic
purification. A longer reaction time brought about
enhanced conversion (0.4:1.0:0.4), improving the iso-
lated yield of 4 to 58% (Scheme 1). It is noted that
under harsher reaction conditions (2% sulfuric acid,
2 h, 121°C), hydrolysis of both the rhamnosidic and
glucosidic bonds completed, and the aglycone 5 was
produced in quantitative yield.
Results and discussion
The first task in the total transformation is the effi-
cient preparation of the key intermediate 4.
Hydrolysis of the glycosidic bonds of 3 in dilute
sulfuric acid at an elevated temperature [11] was
examined, but the products were the aglycone [nar-
ingenin (5)] and the constituent carbohydrates. The
partial hydrolysis of α-rhamnoside to leave the β-
glucoside unit intact was also attempted. The use of
formic acid in cyclohexanol [12] only resulted in a
yield of 4 as low as 11%. In another approach, aqu-
eous trifluoroacetic acid was applied [13], but only
Although the physiological activities of prunin
[15–18] are attractive, its abundance in natural
resources is not high [19–21]. By the present achieve-
ment, more than two grams of prunin (4) became
CONTACT Takeshi Sugai
sugai-tk@pha.keio.ac.jp
© 2018 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
K. KURAHAYASHI ET AL.
Figure 1. Structures of trilobatin (1), phlorizin (2), naringin (3), and prunin (4).
Scheme 1. Synthesis of prunin (4) by acidic hydrolysis of naringin (3) and the synthesis of trilobatin (1).
readily available in one reaction batch by using
8.6 mmol (5.0 g) of the commercially available and
inexpensive substrate 3.
Disappointingly, in the second step, the free chalcone
was prone to cyclize into the flavanone form [22], so
high reproducibility was not achieved. Another pos-
sible route would be the direct hydrogenation of the
salt form [23] of the chalcone in the aqueous phase,
by omitting the acidification and isolation steps. Such
an approach was only effective when the solubility of
the final dihydrochalcone glycoside in water is low.
For example, according to the above-mentioned pro-
tocol, naringin dihydrochalcone precipitated upon
the final acidification and could be easily recovered.
With a sufficient amount of prunin in hand, we
embarked upon the transformation of 4 to 1. The first
reported transformation [1] included: 1) ring opening
of the flavanone accompanied by β-elimination in an
alkaline solution, to furnish the phenoxide salt in
chalcone form; 2) acidification of the resulting mix-
ture to provide free chalcone; and 3) hydrogenation
of the double bond in the resulting chalcone.

SYNTHESIS OF TRILOBATIN FROM NARINGIN
3
On the other hand, in the case of a water-soluble
material such as 1, desalting and lyophilization are
necessary for the isolation.
P-1010 polarimeter. High resolution mass spectra
(HRMS) were measured by a on Jeol JMS-T100LP
AccuTOF. TOMY LSX-500 high-pressure steam steri-
lizer was used for acidic hydrolysis over 100°C.
For the above-mentioned reasons, we planned an
alternative route through temporary blocking of the
resulting phenolic hydroxy groups accompanied by
β-elimination under basic conditions [24]. First, 4
was treated by sodium hydride in N,N-dimethylfor-
mamide (DMF) [25] with benzyl bromide to give
heptabenzyl ether 6 in 55% yield (Scheme 1). The
product was hydrogenated with Pearlman’s catalyst
[Pd(OH)2/C] in a mixture of methanol and ethyl
acetate [26]. The hydrogenolysis of the seven benzyl
groups proceeded smoothly, accompanied by satura-
tion of the double bond in the chalcone [27]. Simple
removal of the catalyst by filtration and evaporation
of the solvent under reduced pressure furnished the
crude product. By simple separation with preparative
TLC, trilobatin (1) was obtained in 93% yield. The
spectral properties were in good accordance with
those in previous reports [4,28].
7-(β-D-Glucopyranosyloxy)-4ʹ,5-dihydroxyflava-
none (prunin, 4). To naringin (3, 5.0 g, 8.6 mmol),
which was measured in 200 mL conical flask, was
added diluted sulfuric acid solution (300 mg in
100 mL of water). The flask was loosely plugged by
urethane sponge and heated in high-pressure steam
sterilizer for 30 min at 121°C. The mixture was
cooled on ice bath and then left to stand in a refrig-
erator overnight at 4°C. The precipitates were col-
lected by decantation and the residue was washed
successively by rinsing with a mixture of water and
acetone (1:1, total 8 mL) and acetone (8 mL) and
dried in vacuo. The ratio of 3:4:5 was estimated to be
0.4:1.0:0.4 by the comparison of the integral of signals
in 1H NMR (DMSO-d6) [δ: 1.14 (d) and 2.72 (dd) for
3, and 2.72 (dd) for 4, and δ: 2.65 (dd) for 5]. The
residue was purified by silica gel column chromato-
graphy. Elution with ethyl acetate-acetone (2:1) furn-
ished 4 as a yellow amorphous solid (2.2 g, 58%). 1H-
NMR (400 MHz, DMSO-d6) δ: 2.71 (0.3H, dd, J = 2.9,
17.0 Hz), 2.72 (0.7H, dd, J = 2.9, 17.0 Hz), 3.09–3.45
(6H, m), 3.64 (1H, dd, J = 5.3, 11.1 Hz), 4.52–4.55
(1H, m), 4.94 (0.7H, d, J = 7.4 Hz), 4.97 (0.3H, d,
J = 7.4 Hz), 5.02 (1H, d, J = 5.3 Hz), 5.09 (1H, d,
J = 4.7 Hz), 5.33 (1H, d, J = 4.8 Hz), 5.48 (1H, dd,
J = 2.9, 12.7 Hz), 6.11 (0.7H, d, J = 2.2 Hz), 6.12
(0.3H, d, J = 2.2 Hz), 6.14 (1H, d, J = 2.2 Hz), 6.78
(2H, d, J = 8.6 Hz), 7.31 (2H, d, J = 8.6 Hz), 9.59 (1H,
s), 12.04 (1H, s). 13C-NMR (100 MHz, DMSO-d6) δ
of the major isomer: 42.5, 61.0, 69.9, 73.5, 76.8, 77.5,
79.1, 95.9, 96.9, 100.0, 103.7, 115.6, 128.9, 129.1,
158.3, 163.2, 163.4, 165.8, 197.7. Signals of minor
isomer were separately observed at δ: 99.9, 163.2,
163.4, 165.7. IR νmax cm–1: 3306, 2904, 1633, 1576,
1519, 1443, 1366, 1294, 1270, 1196, 1168, 1059, 1015,
Conclusion
Prunin (4) was synthesized from naringin (3) in 58%
yield by preferential hydrolysis of the terminal α-
rhamnosidic linkage in the disaccharide. The reaction
was performed in dilute sulfuric acid under con-
trolled conditions, by applying a high-pressure
steam sterilizer at 121°C. Owing to this achievement,
the synthesis of trilobatin (1) was realized in three
steps from naringin (3) with an overall yield of 30%.
In this synthesis, the partial skeleton of naturally
abundant naringin retained in the final product with-
out any step of glycosylation [28], and the ease in the
treatment and isolation of the product in each step
for the transformations was superior to those in the
previous methods for preparation [1,13].
887, 833. HR-MS [ESI+, (M+Na)+]: calculated for
Experimental
21
C21H22NaO10, 457.1100; found, 457.1111. [α]D
–
47.7 (c 1.0, acetone) [lit [19]. [α]D – 41.8 (c 1.2,
acetone)].
Prolonged reaction (see text) provided 5 in a quan-
titative yield, as an insoluble solid in cold water. Its
1H-NMR spectrum was identical with that of an
authentic specimen.
4ʹ-(2,3,4,6-tetra-O-Benzyl-β-D-glucopyranosyloxy)-
2ʹ,4”,6ʹ-tribenzyloxychalcone (6). To a suspension of
sodium hydride from 50% dispersion in mineral oil
(143 mg, 3.0 mmol) in anhydrous DMF (1 mL) was
added a solution of 4 (103 mg, 0.24 mmol) in anhy-
drous DMF (2 mL). After the mixture was stirred for
30 min, benzyl bromide (0.36 mL, 3.0 mmol) was
added dropwise for 5 min. The mixture was stirred
for 8 h at room temperature. The mixture was cooled
in a water bath and added a mixture of methanol and
General. Naringin (N0073) was purchased from
Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan).
Column chromatography was performed with silica
gel (Kanto Chemical Co. Silica Gel 60 N 37,560–79,
spherical and neutral, 40–50 µm). Preparative TLC
was performed with Merck Silica Gel 60 F254 plates
1
(0.5 mm thickness, No. 1.05744.0001). H NMR and
13C NMR spectra were measured at 400 and 100 MHz
on a VARIAN 400-MR spectrometer. DMSO-d6 and
CDCl3 and CD3OD were used as a solvent and the
residual peaks were used as an internal standard (1H
NMR: DMSO-d6 2.48 ppm, CHCl3 in CDCl3 7.26
ppm; 13C NMR: DMSO-d6 39.9 ppm, CDCl3 77.0
ppm, CD3OD 47.6 ppm). IR spectra were measured
as ATR on a Jasco FT/IR-4700 FT-IR spectrometer.
Optical rotation values were measured with a Jasco

4
K. KURAHAYASHI ET AL.
aqueous solution of ammonia to that. The mixture
was stirred for 5 min. Then, hydrochloric acid (2 M)
was added to the mixture and the organic materials
were extracted with dichloromethane twice. The
combined extract was washed with brine, dried over
anhydrous sodium sulfate, filtered, and concentrated
in vacuo. The residue was purified by silica gel col-
umn chromatography. Elution with hexane-ethyl
acetate (10:1) furnished 6 as a yellow amorphous
Disclosure statement
No potential conflict of interest was reported by the
authors.
Funding
This work was supported by JSPS KAKENHI (16K21362)
for K. H. and is gratefully acknowledged with thanks.
1
solid (134 mg, 55%). H-NMR (400 MHz, CDCl3) δ:
3.50–3.71 (6H, m), 4.54 (2H, s), 4.55 (1H, d,
J = 9.8 Hz), 4.79–4.86 (4H, m), 4.94 (4H, s), 4.95
(2H, d, J = 10.7 Hz), 5.11 (2H, s), 6.34 (2H, s), 6.88
(1H, d, J = 16.2 Hz), 6.97 (2H, d, J = 8.8 Hz), 7.15–
7.45 (38H, m). 13C-NMR (100 MHz, CDCl3) δ: 68.9,
70.1, 70.1, 73.6, 75.2, 75.2, 75.8, 77.2, 77.7, 81.9, 84.6,
95.3, 101.6, 114.4, 115.2, 126.9, 126.9, 127.2, 127.5,
127.7, 127.8, 127.8, 127.9, 127.9, 127.9, 128.1, 128.2,
128.2, 128.4, 128.4, 128,4, 128.5, 128.5, 128.7, 128.7,
130.2, 136.5, 136.7, 137.8, 137.8, 138.1, 138.4, 145.0,
157.3, 159.5, 160.6, 194.5. IR νmax cm–1: 3061, 3030,
2863, 1643, 1593, 1508, 1453, 1425, 1377, 1245, 1172,
1068, 1025, 824, 733, 694. HR-MS [ESI+, (M+Na)+]:
calculated for C70H64NaO10, 1087.4405; found,
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21
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–
20
66.7 (c 0.75, ethanol) [lit [2]. [α]D – 70 (c 2.0,
ethanol)].
Author Contribution
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