Efficient synthesis of kinsenoside and goodyeroside A by a chemo-enzymatic approach

Article abstract of DOI:10.3390/molecules191016950

Kinsenoside (1) and goodyeroside A (2), two naturally occurring stereoisomers with diverse biological activities, have been synthesized efficiently by a chemo-enzymatic approach with a total yield of 12.7%. The aglycones, (R)- and (S)-3-hydroxy-γ-butyrolactone, were prepared from D- and L-malic acid by a four-step chemical approach with a yield of 75%, respectively. These butyrolactones were then successfully glycosidated using β-D-glucosidase as a catalyst in a homogeneous organic-water system. Under the optimized enzymatic conditions, the yields of kinsenoside and goodyeroside A in the enzymatic steps both reached 16.8%.

Full text of DOI:10.3390/molecules191016950

Molecules 2014, 19, 16950-16958; doi:10.3390/molecules191016950
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Letter
Efficient Synthesis of Kinsenoside and Goodyeroside A by a
Chemo-Enzymatic Approach
Yang Zhang 2,†, Yihong Xia 2,†, Yongji Lai 2, Fang Tang 1, Zengwei Luo 2, Yongbo Xue 2,
Guangmin Yao 2, Yonghui Zhang 2 and Jinwen Zhang 1,*
1
Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, China; E-Mail: nagexiaoshui@163.com
2
Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation,
School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan 430030, China; E-Mails: zyhust123@163.com (Y.Z.); missxia55@163.com (Y.X.);
laiyongji1024@163.com (Y.L.); luozengwei@hust.edu.cn (Z.L.); bobbyschro@126.com (Y.X.);
gyap@mail.hust.edu.cn (G.Y.); zhangyh@mails.tjmu.edu.cn (Y.Z.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: tjzhangjinwen@163.com or
2009597@hust.edu.cn; Tel.: +86-27-8366-3253; Fax: +86-27-8369-2762.
External Editor: Derek J. McPhee
Received: 24 September 2014; in revised form: 12 October 2014 / Accepted: 15 October 2014 /
Published: 22 October 2014
Abstract: Kinsenoside (1) and goodyeroside A (2), two naturally occurring stereoisomers
with diverse biological activities, have been synthesized efficiently by a chemo-enzymatic
approach with a total yield of 12.7%. The aglycones, (R)- and (S)-3-hydroxy-γ-butyrolactone,
were prepared from D- and L-malic acid by a four-step chemical approach with a yield of
75%, respectively. These butyrolactones were then successfully glycosidated using
β-D-glucosidase as a catalyst in a homogeneous organic-water system. Under the optimized
enzymatic conditions, the yields of kinsenoside and goodyeroside A in the enzymatic steps
both reached 16.8%.
Keywords: kinsenoside; goodyeroside A; chemo-enzymatic synthesis; glucosidase;
biocatalysis

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1. Introduction
3-(R)-3-β-D-Glucopyranosyloxybutanolide (kinsenoside, 1, Figure 1) has been isolated from three
Anoectochilus species (Orchidaceae), namely, A. koshunensis [1], A. formosanus [2], and A. roxburghii [3].
3-(S)-3-β-D-Glucopyranosyloxybutanolide (gooderoside A, 2, Figure 1), the epimer of kinsenoside,
was first isolated from Crocus sativus (Iridaceae) [4], and later from three Goodyera species
(Orchidaceae) [5]. Preliminary in vitro pharmacological studies indicated that both compounds 1 and 2
had protective effects on primary cultured rat liver cells suffering from carbon tetrachloride exposure [5,6].
Moreover, pharmacological investigations on rats demonstrated that 1 possessed several other
significant effects, including antihyperliposis [7], anti-inflammatory [8], antihyperglycemic [3],
vascular protection [9], ovariectomy-induced bone loss preventive, and osteoclastogenesis suppressing
properties [10]. In light of their effective physiological activities in vitro and in vivo, preparations of
compounds 1 and 2 to meet the need of these compounds for further pharmacological studies have
been described in the literature. In one of these approaches, the key chiral intermediates (R)- and (S)-3-
hydroxy-γ-butyrolactone were initially synthesized, and then reacted with 2,3,4,6-tetra-O-benzyl-β-D-
glucopyranosyl trichloroacetimidate to afford 1 and 2 in a total yield of 0.8% after seven steps [11]. In
another study, optically inactive 1,2,4-butanetriol and 2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl
trichloroacetimidate as protected glucose were used to prepare 1 and 2 with a total yield of 10.7% over
nine steps [12]. However, both these routes need protection and deprotection steps in the glycosylation
process and have relatively circuitous synthetic routes. On the other hand, it is difficult to separate
stereoisomeric products. In order to overcome these challenges, we have adopted a chemical process to
synthesize 3-hydroxy-γ-butyrolactones from malic acid in three steps according to a reference
procedure with some modifications. The obtained (R)- and (S)-3-hydroxy-γ-butyrolactones were
reacted with β-D-glucose as glycosylation agent and β-D-glycosidase as catalyst to prepare kinsenoside
(1) and goodyeroside A (2) separately. At the same time, reaction conditions for the enzymatic step
were optimized by single factor and orthogonal experimental design.
Figure 1. Structures of compounds 1 and 2.
2. Results and Discussion
Kinsenoside and goodyeroside A have been prepared efficiently by a five-step chemo-enzymatic
route with a total yield of 12.7%, while the yield of a chemical approach to the 3-hydroxy-γ-butyrolactone
intermediates was improved to 75%. Reaction conditions for the subsequent enzymatic step
were optimized.

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2.1. Improvement of the Chemical Approach
The synthesis of 3-hydroxy-γ-butyrolactone was significantly improved compared to previous
methods (Scheme 1). Firstly, for post-processing of anhydride 4, the acetic acid formed in the reaction
was removed via the 1,4-dioxane-acetic acid azeotrope. Moreover, the duration of the reaction for
preparing monomethyl ester 5 was remarkably reduced to 30 minutes, far less than the previously
reported 12 h [13,14]. Additionally, in the last synthetic step, the reagent mixture composed of
HCl-MeOH was replaced by HCl -H2O-1,4-dioxane, which significantly enhanced the purity of the
ultimate products and simplified the separation step [13,14]. Under our optimized conditions, the yield
of 75% was as high as described in prior references [13,14].
Scheme 1. Chemical synthesis of compound 6 from compound 3.
Reagents and conditions: a: AcCl, 40 °C, 4 h; b: MeOH, r.t., 30 min; c: NaBH4, i-BuOH, 85 °C, 2 h; d: HCl,
1.4-dioxane, 70 °C.
2.2. Construction and Optimization of Enzymatic Synthesis
(R)- and (S)-3-Hydroxy-γ-butyrolactones, respectively, were successfully converted to their
glucosides in a homogeneous organic-water medium (Scheme 2). Reaction conditions for the
enzymatic step were optimized by single factor and orthogonal experimental design.
Scheme 2. Enzymatic synthesis of compound 2.
2.2.1. Single Factor Experiments
In the initial study, three organic solvents and their concentrations at three levels (80%, 90% and
95%, v/v) were investigated for the enzymatic synthesis. As shown in Figure 2A, 1,4-dioxane was
consistently the best solvent in comparison with tert-butanol and acetonitrile, regardless of
concentrations. In particular, the solvent system consisting of 90% 1,4-dioxane and 10% buffer (v/v)
displayed the best results in promoting the reaction result. Surprisingly, our results clearly
demonstrated that acetonitrile was not applicable as a co-solvent for enzymatic glycosidation,
regardless of buffer ratios. These results are in contrast with those of related studies [15,16], in which
90% tert-butanol or acetonitrile gave better results than 1,4-dioxane.
In order to further optimize the reaction and then use less amount of β-D-glucosidase, a series of
reaction systems containing a range of enzyme amounts were tentatively established. As shown in

Molecules 2014, 19
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Figure 2B, the process with the enzyme at a dose of 5 mg was favorable to accumulate 2. No increased
amount of glucoside was observed with more enzyme, but the amount of glucoside was reduced with
the decreased enzyme dosage. Thus the dose of 5 mg glucosidase was chosen in this study.
Figure 2. (A) Effects of different organic solvents and contents on the synthesis of
2 catalyzed by almond β-D-glycosidase in homogeneous systems; (B) effects of almond
β-D-glycosidase dosages on the synthesis of 2; (C) effects of (S)-3-hydroxy-γ-butyrolactone
concentrations on the synthesis of 2; (D) Dependence of 2 formation on reaction time. For
single factor experiments, when one factor was investigated, the best condition of the three
other factors was chosen.
12
10
8
12
10
8
Acetonitrile
Tertiary butanol
1,4-dioxane
A
B
6
6
4
4
2
2
0
0
3
4
5
6
7
8
8:2
9:1
9.5:0.5
Enzyme dosage (mg/mL)
Organic solvent : buffer(v/v)
12
10
8
12
C
D
10
8
6
6
4
4
2
2
0
0
1:2
1:4
Glucose
1:8
1:10
1:12
12
24
48
72
96
120
132
:
lactone ( mol/mol)
Reaction time (h)
Because enzymatic glycosidation is a thermodynamically-controlled reversable hydrolysis process,
high concentrations of glucose and (S)-3-hydroxy-γ-butyrolactone were expected to give more glucoside.
The maximum glucose concentration was 0.25 M in our chosen reaction medium consisting of
1,4-dioxane and buffer (9:1, v/v) [15]. The effect of the concentration of (S)-3-hydroxy-γ-butyrolactone
on the yield of compound 2 was investigated, and the results showed that in the presence of 0.25 M
glucose, the yield of 2 increased with the content of lactone and reached its maximum at a ratio of 1:10
(glucose: lactone) (Figure 2C). This ratio was finally chosen as the best condition in our study. In our
organic-water medium, the reverse hydrolysis needed more time to reach thermodynamic equilibrium.
As shown in Figure 2D, after 48 h, the reaction reached equilibrium and the concentration of products
were also found to be 10.5 mg/mL. When the reaction duration was extended excessively, some
glucoside might be converted back to glucose and 3-hydroxy-γ-butyrolactone.

Molecules 2014, 19
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2.2.2. Orthogonal Experiments
As mentioned above, the reverse of the glucoside-producing reaction needed a certain amount of
glucosidase and aglycone, as well as a defined duration to reach equilibrium. In order to expound
on the intrinsic relationships among them, three factors including β-D-glucosidase dosage,
(S)-3-hydroxy-γ-butyrolactone concentration and reaction time at four levels were chosen to carry out
an orthogonal experiment (L16). According to intuitive analysis of the orthogonal experiment results
(Table 1), the amount of lactone was the most remarkable factor within a certain range, followed by
reaction time and enzyme dosage. The best estimated conditions include 5 mg glucosidase, 10 equivalents
of lactone in 1 mL reaction medium and a reaction duration between 44 and 68 h, consistent with the
experimental group 8, in which the concentration of 2 was 11.05 mg/mL, and the yield reached 16.8%.
In the same way, we conducted the orthogonal experiment to synthesize 1. Results were similar to that
of experiments of 2 (Table S1 in Supporting Information). The above results demonstrated that there
was no stereoselectivity for (S)- and (R)-3-hydroxy-γ-butyrolactone in the enzymatic step.
Table 1. Orthogonal experiment to determine the optimal enzymatic reaction conditions of 2.
Enzyme Dosage
Lactone
Reaction Time
Concentration of 2
Yield
(%)
9.1
Effect Factor
(mg)
Equivalents
(h)
32
(mg/mL)
6.0
1
2
4
7
4
8
44
8.3
12.6
14.2
13.9
12.7
11.8
13.3
16.8
11.4
14.1
13.2
13.8
12.3
12.9
14.5
13.8
3
4
9
56
9.4
4
4
10
68
9.2
5
5
7
8
44
8.4
6
5
32
7. 8
8.8
7
5
9
68
8
5
10
56
11.1
7.5
9
6
7
56
10
11
12
13
14
15
16
k1
k2
k3
k4
R
6
6
8
68
9.3
9
32
8.7
6
10
44
9.1
7
7
68
8.1
7
8
56
8.5
7
9
44
9.6
7
10
32
9.1
8.217
8.990
8.641
8.841
0.773
7.507
8.478
9.097
9.608
2.101
7.876
8.822
9.115
8.877
1.239
ki (i = 1, 2, 3, 4): Mean concentration of 2 at case i level of corresponding factor; R: range, namely
kimax–kimin
.

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3. Experimental Section
3.1. Materials
β-D-Glucosidase (EC 3.2.1.21, 2.31 U/mg) from almond was purchased from Sigma Co. (St. Louis,
MO, USA), and used without any further treatment; Malic acids were purchased from Aladdin
Chemistry Co. Ltd. (Shanghai, China); Chemical reagents were obtained from Sinopharm Chemical
reagent Co. Ltd. (Shanghai, China).
3.2. NMR, MS and HPLC-ELSD Analysis
The NMR spectra of 3-hydroxy-γ-butyrolactone, kinsenoside and goodyeroside A, were recorded in
CDCl3 and pyridine-d5, respectively, using a Bruker 400 MHz spectrometer (Bruker, Karlsruhe,
Germany). The corresponding solvent signals δH 7.24/δC 77.23 (CDCl3) and δH 8.74/δC 150.35
(pyridine-d5) were used as references. The MS spectra were recorded on a Thermo LCQ DECA. XPplus
ESI-MS (Thermo Fisher, Palo Alto, CA, USA). HPLC analysis of the glucoside was carried out on a
Hitachi HPLC L-2000 instrument (Hitachi, Tokyo, Japan) equipped with an Alltech ELSD 2000
detector (Alltech, Vienna, VA, USA). Compounds 1 and 2 were analyzed on a 4.6 mm × 250 mm
Ultimate AQ-C18 column with 100% ultrapure water as the eluent at a flow-rate of 0.6 mL/min. The
temperature of the detector’s drift tube was 115 °C and the flow rate of N2 was 3.0 L/min.
Concentrations of 1 and 2 were calculated from peak areas by using a calibration curve.
3.3. Chemical Synthesis of 3-Hydroxy-γ-butyrolactone
(S)- and (R)-3-hydroxy-γ-butyrolactone were synthesized using D- and L-malic acid as starting
materials, respectively (Scheme 1). To a 100 mL flask was added L-malic acid (3, 13.4 g, 0.1 mol) and
redistilled acetyl chloride (60 mL). The mixture was stirred at 40 °C for 4 h. Excess solvent was
evaporated with added 1,4-dioxane and white solid 4 (15.6 g, 0.0987 mol) was obtained from the
residue via recrystallization with a chloroform and petroleum ether system (v/v = 1:1, CHCl3/PE).
Compound 4 was then dissolved in redistilled methanol (120 mL) and stirred 30 min at room
temperature. Excess methanol was evaporated to yield the crude product monomethyl ester 5 (18.6 g,
0.0979 mol). After refluxing of NaBH4 (9.5 g, 0.245 mol) in t-BuOH (125 mL) for 2 h, a mixture of
t-BuOH (150 mL) and MeOH (10 mL) containing compound 5 (18.6 g) was added dropwise to the
mixture and then reflux was continued for 2 h. A white solid 6 was obtained by rotary evaporation. A
mixed solvent of HCl/H2O/1,4-dioxane (v/v/v = 55:165:290) was added dropwise to the white solid at
70 °C to prepare (S)-3-hydroxy-γ-butyrolactone (7). After evaporation of the solvents, the white
residue was extracted with THF (200 mL). The organic layers were combined, dried over anhydrous
Na2SO4 and concentrated under vacuum to yield the crude product, which was further purified by flash
chromatography on silica gel using petroleum ether/ethyl acetate (4:1) as the eluent to afford
(S)-3-hydroxy-γ-butyrolactone (7.67 g, 75%) as a colorless or yellowish oil. [α]1D7 = +86.9 (c = 0.24,
EtOH); [α]1D4 = +72.2 (c = 1.25, EtOH) in the reference [11]; 1H-NMR (CDCl3): δH 4.64–4.68 (m, 1H,
H-3), 4.39 (dd, J = 10.3, 4.5 Hz, 1H, H-4b), 4.27 (d, J = 10.3 Hz, 1H, H-4a), 2.72 (dd, J = 18.0, 6.1 Hz,
1H, H-2b), 2.45–2.56 (m, 1H, H-2a). 13C-NMR (CDCl3): δC 37.8, 67.5, 76.0, 176.1.

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The synthesis steps of (R)-3-hydroxy-γ-butyrolactone from D-malic acid were similar to those of
(S)-3-hydroxy-γ-butyrolactone, with a yield of 75%. Colorless oil. [α]1D7 = −83.7 (c = 0.24, EtOH);
[α]1D1 = −72.2 (c = 1.25, EtOH) in the reference [11]. 1H-NMR (CDCl3): δH 4.64–4.68 (m, 1H, H-3), 4.39
(dd, J = 10.3, 4.5 Hz, 1H), 4.24–4.29 (m, 1H, H-4a), 2.73 (dd, J = 18.0, 6.1 Hz, 1H), 2.50 (ddd, J = 18.0,
1.7, 1.1 Hz, 1H). 13C-NMR (CDCl3): δC 37.8, 67.5, 75.9, 176.1.
3.4. Enzymatic Synthesis of Compounds 1 and 2
β-D-Glucosidase from almond (5 mg) was dissolved in phosphate buffer (Na2HPO4-KH2PO4, 70 mM,
pH 6.0, 100 μL), then organic solvent (900 μL), 3-hydroxy-γ-butyrolactone (0.255 g, 2.5 mmol), and
glucose (0.045 g, 0.25 mmol) were added to the reaction mixture. The reaction vessel was sealed and
kept at 50 °C in thermostatic shaker with a shaking speed of 120 rpm for 48 h (Scheme 2). A water-bath
set at 90 °C was used to quench the reaction. Concentrations of 1 and 2 were calculated from peak
areas by using a calibration curve, and products were purified by silica gel column chromatography
with chloroform/ethanol (4:1) as the eluent.
Kinsenoside (1): Colorless oil. [α]1D7 = +21.0 (c = 0.10, EtOH ); [α]1D1 = +17.9 (c = 1.24, EtOH) in the
reference [11]. 1H-NMR (pyridine-d5): δH 4.91 (d, J = 7.8 Hz, 1H, H-1'), 4.84–4.87 (m, 1H, H-3), 4.70
(d, J = 10.1 Hz, 1H, H-4a), 4.55 (d, J = 11.8 Hz, 1H, H-6'a), 4.34–4.45 (m, 2H, H-4b and H-6'b),
4.20–4.24 (m, 2H, H-4' and H-5'), 3.93–4.01 (m, 2H, H-3' and H-2'), 2.83–2.92 (m, 2H, H-2a and
H-2b). 13C-NMR (pyridine-d5): δC 36.0, 63.0, 71.8, 75.1, 75.2, 75.6, 78.7, 79.1, 104.4, 176.4. ESI-MS:
m/z 309.10 [M+HCOO]− (Calcd for C11H17O10: 309.08).
Goodyeroside A (2): White crystals. mp 165–166 °C. [α]1D7 = −68.7 (c = 0.10, H2O); [α]D11 = −69.9
1
(c = 0.55, H2O) in the reference [11]. H-NMR (pyridine-d5) δH 4.94 (d, J = 7.7 Hz, 1H, H-1'),
4.87–4.91 (m, 1H, H-3), 4.66 (d, J = 10.1 Hz, 1H, H-4a), 4.55 (d, J = 11.8 Hz, 1H, H-6'a), 4.33–4.41
(m, 2H, H-4b and H-6'b), 4.19–4.26 (m, 2H, H-4' and H-5'), 3.92–4.01 (m, 2H, H-3' and H-2'),
2.86–2.95 (m, 2H, H-2a and H-2b). 13C-NMR (pyridine-d5): δC 36.8, 63.0, 71.8, 74.4, 75.0, 75.1, 78.7,
79.1, 104.0, 176.7. ESI-MS: m/z 309.10 [M+HCOO]− (Calcd for C11H17O10: 309.08).
4. Conclusions
Kinsenoside (1) and goodyeroside A (2) were efficiently synthesized by a chemo-enzymatic
approach with an overall yield of 12.7%. Aglycones were prepared by chemical approach with a yield
of 75%. For the enzymatic approach, the yield of kinsenoside and goodyeroside A can reach to 16.8%
under optimal reaction conditions, in which a 1 mL reaction system consisted of 1,4-dioxane (900 μL),
phosphate buffer (Na2HPO4-KH2PO4, 70 mmol/L, pH 6.0, 100 μL), β-D-glucose (0.25 mmol), (R) or
(S)-3-hydroxy-γ-butyrolactone (2.5 mmol) and β-D-glucosidase (5 mg); the reaction duration was
44–68 h, under 50 °C in thermostatic shaker with a shaking speed of 120 rpm.

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Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/10/16950/s1.
Acknowledgments
This work was financially supported by Program for New Century Excellent Talents in University
(No. NCET-2008-0224) and National Science Foundation of China (Nos.31370372, 81202423,
81102334, 30400585) and National science and technology major project (No. 2011ZX09102-004).
Author Contributions
Jinwen Zhang, Yonghui Zhang and Zengwei Luo designed research; Yang Zhang, Yihong Xia,
Yongji Lai and Fang Tang performed research and analyzed the data; Jinwen Zhang, Yongji Lai,
Yongbo Xue and Guangmin Yao wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interests.
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distributed under the terms and conditions of the Creative Commons Attribution license
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