Synthesis of gentianine N-oxide by enzymatic hydrolysis of swertiamarin in the presence of hydroxylamine and reaction pathway to gentianine and gentianol

Article abstract of DOI:10.1016/j.tetlet.2019.01.061

Gentianine is a metabolite of gentiopicroside and swertiamarin. Several biological activities have been reported for gentianine, such as antiinflammatory and antidiabetic activity, and hypotensive effect. Gentiopicroside is found in 0.9–9.8% content in Gentian root or Gentian scabra root, and Swertiamarin is contained in Swertia herb in 2–10%. These natural products can be potential starting materials for the synthesis of gentianine. This study describes the β-glucosidase-catalyzed hydrolysis of gentiopicroside and swertiamarin in the presence of hydroxylamine to afford gentianine N-oxide, which can be a synthetic precursor of gentianine derivatives. Enzymatic hydrolysis of swertiamarin selectively afforded gentianine N-oxide in 81% yield, whereas gentiopicroside afforded gentianine N-oxide and gentianol N-oxide. Plausible reaction pathways leading to gentianine, gentianol, and their N-oxides were also investigated.

Full text of DOI:10.1016/j.tetlet.2019.01.061

Accepted Manuscript
Synthesis of Gentianine N-Oxide by Enzymatic Hydrolysis of Swertiamarin in
the Presence of Hydroxylamine and Reaction Pathway to Gentianine and Gen-
tianol
Mikio Fujii, Taiki Kuramochi, Yuhi Nakakuki, Rina Hatazawa, Kiju Konno,
Tatsuo Munakata, Yasuaki Hirai
PII:
S0040-4039(19)30108-X
https://doi.org/10.1016/j.tetlet.2019.01.061
TETL 50594
DOI:
Reference:
Tetrahedron Letters
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20 December 2018
25 January 2019
30 January 2019
Please cite this article as: Fujii, M., Kuramochi, T., Nakakuki, Y., Hatazawa, R., Konno, K., Munakata, T., Hirai,
Y., Synthesis of Gentianine N-Oxide by Enzymatic Hydrolysis of Swertiamarin in the Presence of Hydroxylamine
and Reaction Pathway to Gentianine and Gentianol, Tetrahedron Letters (2019), doi: https://doi.org/10.1016/j.tetlet.
2019.01.061
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Synthesis of Gentianine N-Oxide by Enzymatic Hydrolysis of Swertiamarin in the
Presence of Hydroxylamine and Reaction Pathway to Gentianine and Gentianol
Mikio Fujii,a * Taiki Kuramochi,a Yuhi Nakakuki,a Rina Hatazawa,a Kiju Konno,a Tatsuo Munakata a and Yasuaki
Hiraib
a School of Pharmacy, International University of Health and Welfare, 2600-1 Kitakanemaru, Ohtawara, Tochigi, 324-8501, Japan
b Faculty of Arts and Sciences at Fujiyoshida, Showa University, 4562 Kamiyoshida, Fujiyoshida, Yamanashi, 403-0005, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Gentianine is a metabolite of gentiopicroside and swertiamarin. Several biological activities
have been reported for gentianine, such as antiinflammatory and antidiabetic activity, and
hypotensive effect. Gentiopicroside is found in 0.9–9.8% content in Gentian root or Gentian
scabra root, and Swertiamarin is contained in Swertia herb in 2–10%. These natural products can
be potential starting materials for the synthesis of gentianine. This study describes the -
glucosidase-catalyzed hydrolysis of gentiopicroside and swertiamarin in the presence of
hydroxylamine to afford gentianine N-oxide, which can be a synthetic precursor of gentianine
derivatives. Enzymatic hydrolysis of swertiamarin selectively afforded gentianine N-oxide in
81% yield, whereas gentiopicroside afforded gentianine N-oxide and gentianol N-oxide.
Plausible reaction pathways leading to gentianine, gentianol, and their N-oxides were also
investigated.
Available online
Keywords:
Gentiopicroside
Swertiamarin
-Glucosidase-catalyzed hydrolysis
Genatianine N-oxide
2009 Elsevier Ltd. All rights reserved.
1. Introduction
mechanism involved in the production of 1 from 2 or 3 is
depicted in Scheme 1. Accordingly, the hydrolysis of 2 by -
glycosidase gives aglycon 8, which undergoes hemiacetal bond
Gentianine (1) is a metabolite of gentiopicroside (2) and
swertiamarin (3), which are secoilidoid glycosides known as
constituents of Gentianaceae plants (Fig. 1).1-5 For 1, several
biological activities have been reported, such as
antiinflammatory6,7 and antidiabetic activity,8 hypotensive effect,9
and protective effect on hippocampal CA1 neurons in rats.10 The
antidiabetic activity of 1 stems from the fact that it increases
adipogenesis, which has been associated with a significant
increase in the mRNA expression of PPAR-, GLUT-4, and
adiponectin. For further medicinal studies, the synthesis of
derivatives of 1 is desired.
The synthesis of 1 from 4-methyl-5-vinylnicotinonitrile has
been previously reported.11,12 However, several steps are
generally required. On the other hand, 1 was synthesized by
ammonolytic cleavage of above-mentioned glycosylated form (2
and 3) in low yield due to the formation of gentioflavine (4) and
gentianal (5) as byproducts (Fig. 1).13,14,15 Gentiopicroside 2 is
found in 0.9–9.8% content in Gentian root or Gentian scabra
root,16,17 and swertiamarin 3 is contained in Swertia herb in 2–
10%.5 These natural products are potential starting materials for
the synthesis of 1 and its derivatives.
cleavage to yield dialdehyde 9, whose condensation with
ammonia forms imines 10 and/or 10’. Then, intermolecular
cycloaddition affords dihydropyridines 11 and/or 11’, which
provide 1 after aromatization. In a similar manner, the hydrolysis
of 3 gives 1 through 12. Although gentianol 13 is also known to
be produced by biodegradation, the pathway leading to its
formation is unclear.5
In the absence of ammonia, the formation of 1 is a result of
sequential reactions initially triggered by the hydrolysis of 2 or 3
that affords many kinds of byproducts such as erythrocentaurin
(6)4,18 and gentiogenal (7)19 (Fig. 1). Therefore, enzymatic
hydrolysis of 2 or 3 in ammonium buffer can be envisaged as a
very interesting and challenging approach (Scheme 1); the use of
a degradative and harmless natural product and the mild reaction
conditions of an
Fig. 1. Structure of gentianine 1, gentiopicroside 2, sweritamarin 3, and the
derivatives.
enzymatic reaction are preferred from the viewpoint of green
chemistry.20 Several biotransformations or biodegradations of 2
and 3 by intestinal bacteria have been reported,2-5 and the

2
Tetrahedron Letters
hydroxylamine (entry 6–10, Table 1) on the yield and conversion
in the hydrolysis of 3 were investigated. We found that one
equivalent of hydroxylamine reacted with glucose and excessive
addition of hydroxylamine (entry 11, Table 1) resulted in the
occurrence of side reactions with the concomitant decrease of the
yield. It can be extracted from the table that the reaction at pH 6
and the use of 2.4 equivalents of hydroxylamine (entry 3, Table
1) provided the best results (71% yield and 82% conversion). By
adding fresh enzyme every 30 min for 3 h, the isolated yield
reached 81%. The high conversion was achieved because both
oxime formation and -glucosidase-catalyzed hydrolysis are the
preferred processes in the range of pH 4–6. Generally, pyridine
N-oxides are reactive precursors of pyridine derivatives through,
for example, acetyl migration and aromatic electrophilic
substitution.
Scheme 1 Proposed mechanism for the enzymatic hydrolysis of 2 and 3.
In our preliminary experiments regarding the enzymatic
hydrolysis of 2 or 3,21 two problems arose: the low yield of 1 and
inhibition of 2 to enzyme. Hydrolytic intermediate 9 may react
with the lysine residue of the enzyme, since it has been known to
produce an adduct with ethylamine. In this paper, we investigated
the reaction pathway affording 1 and 13 by enzymatic hydrolysis
of 2 and 3 in the presence of ammonia and moreover the
formation of pyridine N-oxide derivatives 14 and 15 (Fig. 1) in
the presence of hydroxylamine expecting to facilitate the
subsequent reactions.
2. Results and discussions
2.1. Kinetics and time course of the enzymatic hydrolysis of 2
Firstly, to investigate the reactivity of 2 and inhibition of the
products to glucosidase, the kinetics and time course of the
enzymatic hydrolysis of 2 using almond -glucosidase purchased
from Oriental yeast Co. Ltd. (Product No. 46361003, lot.
3459401, 36.8 U/mg) were studied. Initial rate kinetics were
performed. The ratio of consumed 2 was measured by 1H NMR
spectroscopy using propane-1,2-diol as an internal standard. The
remaining amount of 2 (or 3) was calculated by using the integral
of the signal at 7.6 ppm (singlet, 3-position). The Km and Vmax
values in the conditions of the experiment were determined to be
17 mmol/L and 15 mmol/L·h, respectively (Fig. 2a). The time
course of the reaction, which was monitored starting from 14
mmol/L of 2, shows that the reaction stops within one hour (Fig.
2b), which is most likely due to product inhibition because the
enzyme could be stored in the same buffer for one hour without
significant loss of activity. Although -glucosidase exhibited
enough ability to hydrolyze 2, successive addition of the enzyme
and the use of a more reactive nitrogen reagent seemed necessary
to enhance the enzymatic hydrolysis of 2 and 3. Unfortunately,
measurement of the kinetics of 3 was hindered by a stronger
inhibition by hydrolyzed products and slower reaction rate of 3
compared with that of 2.
Fig. 2. Kinetics and time course of the enzymatic hydrolysis of 2.
a) Linweaver–Burk plot of initial rate kinetics. Conditions: 2; 1.8–14
mmol/L, almond -glucosidase from Oriental yeast Co. Ltd., 39 U/mg; 55
U/L, 0.2 M ammonium phosphate buffer (pH 5.5); 10 mL, for 30 min at
38 °C (consumed 2; 10%–35%). b) Time course. Conditions: 2; 14 mmol/L,
almond -glucosidase; 110 U/L, 0.2 M ammonium phosphate buffer (pH
5.5); 10 mL, 38 °C.
2.2. Enzymatic hydrolysis of 2 and 3 in the presence of
hydroxylamine
To improve the efficiency of pyridine ring formation,
ammonia was replaced with hydroxylamine due to the higher
nucleophilicity of the latter compound. To the best of our
knowledge, a hydrolysis reaction catalyzed by -glycosidase in
the presence of hydroxylamine has not been reported so far. The
results of the enzymatic hydrolysis are listed in Table 1. Entries 1
and 2 in Table 1 shows the yield and conversion of products and
the recovery of the substrate in the hydrolysis of 2 and 3,
respectively, at pH 5.5. The conversion to 14 was calculated on
the basis of the consumed substrate (added 2 or 3 minus
recovered 2 or 3). Although hydrolysis of 2 afforded gentianine
N-oxide (14) and gentianol N-oxide (15)22 in 32% and 38% yields,
respectively, that of 3 gave 14 as the sole product in 52% yield.
Compound 14 was previously reported as a degradation product
of 1, and the spectral data of 14 was identical to those found in
the literature.2 Swertiamarin 3 proved to be a much better
substrate than gentiopicroside 2 for the synthesis of 14. Next, the
effect of pH (entry 2–5, Table 1) and the amount of
Scheme 2. Enzymatic hydrolysis of 2 in ammonium phosphate or acetate
buffer.
2.3. Reaction pathway toward 1, 13, 14, and 15.
Despite being a known metabolite from 2 and 3, the
degradation pathway of gentianol 13 has not been well
investigated. To understand the selectivity of the hydrolysis
reaction in the presence of hydroxylamine to afford 14 and 15,
the elucidation of the mechanism of the enzymatic hydrolysis
giving 13 is important.
To this aim, the enzymatic hydrolysis of 2 in the presence of
ammonium acetate was tested. Compound 2 (100 mg) in
ammonium acetate buffer (pH 5.5, 0.2 M, 20 mL) was incubated
at 78 rpm and 38 °C, and enzyme solution (0.5 mg/100 L buffer,
10 times) was added every 30 min until the substrate was
consumed according to TLC analysis. Although 1 could not be

isolated, gentianol 13 and gentianol acetate 1623, were obtained in
8.5% and 18%, respectively (Scheme 2). Since the reaction in
ammonium phosphate buffer affords 135 (11%), 414 (5%), and 64
(1%), but not 16, it can be concluded that the acetate group of 16
stems from the acetate buffer. Compounds 1, 13, and 16 were
proved to be noninterchangeable when treated individually under
the same conditions of the enzymatic hydrolysis, which suggests
that the pathways affording 1, 13, and 16 exist independently.
which supported the pathway through nonchiral intermediate 17.
Meanwhile, the enzymatic hydrolysis of 3 would afford 20,
which possesses weaker acidity at the 9-position than 9 due to the
lack of conjugation with the ester and the aldehyde in this
structure. Nevertheless, 20 would be dehydrated to afford 9,
which would in turn lead to 13. On the other hand, the reaction of
20 with ammonia would afford 1 through intermediates 21 and
22. Accordingly, the enzymatic hydrolysis of 3 afforded 1 and 13
in 8% and 12% yield, respectively.
Scheme 3 depicts a plausible pathway to give 13 from 2. In
this pathway, dialdehyde 9, which is derived from aglycon 8,
contains an acidic proton at the 9-position that could be activated
by an electron withdrawing group such as an aldehyde and a
conjugated ester or aldehyde. Thus, once the anion from 9 is
formed, conjugated allyl addition to the aldehyde would afford
erythrocentaurin (6)4 or, alternatively, its isomerization would
afford 17, which could be a precursor of gentiogenal (7).18,19
Since the vinyl group is normally inactive at pH 5.5 and room
temperature, 7 would be most likely obtained through the
intramolecular addition reaction of the activated olefin in 17 with
the carbonyl group. During the 1H NMR monitoring of the
reaction (Fig. 2b), an intermediate possessing a doublet methyl
signal at 1.35 ppm was observed at the beginning of the reaction,
but it disappeared gradually and was no longer observed after
three hours. Since this intermediate was stable enough to be
detected in the NMR experiment, it is not likely to be the
unstable species 17. On the other hand, 13 was not observed at 30
min reaction time, but it appeared and its concentration increased
while the intermediate disappeared, reaching a maximum
conversion of ~3% after four hours. This result supports the
isomerization of the vinyl group in 9 to the conjugated olefin in
17. According to the pathway in Scheme 3, 17 would react with
ammonia giving imine 18 or 18’, which would be then converted
into 19 and 19’, respectively.5,15 Aromatization by elimination of
the hydroxyl group, followed by nucleophilic attack of water or
acetate would afford 13 or 16, respectively. Indeed, the specific
rotation of 13 obtained from 2 was []D = -0.7° (c = 0.4,
CHCl3){lit.5 []D = -64° (c = 1, CHCl3), synthetic sample
prepared by asymmetric reduction of the corresponding ketone.},
Similar reaction pathways are proposed for the hydrolysis of 2
and 3 in the presence of hydroxylamine (Scheme 4). After the
formation of dialdehyde 9 from 2, two possible routes can be
considered: isomerization to 17 or reaction with the highly
reactive hydroxylamine to afford 14. Considering the yield of the
reaction (Table 1, entry 1), the isomerization is more favored
than the reaction with hydroxylamine. On the other hand, in the
reaction of 3, intermediate 20 would react exclusively with
hydroxylamine to afford 14 due to its lower acidity in
comparison with 9.
It is known that the direct chemical reaction of 2 and 3 with
ammonia afford 1, which would proceed through intermediates
10’ and 21, respectively. In a similar manner, the production of
15 from 2 in the presence of hydroxylamine may be caused by
the chemical reaction of 2 and hydroxylamine or by fast oxime
formation. To discriminate between these possibilities, the
reaction of 2 and 3 was performed in the absence of -
glucosidase, finding that the reaction with hydroxylamine hardly
reached 40% in 12 h. However, an unknown side product (~8%)
that could react with lactone was observed. This side product was
not obtained in the presence of -glycosidase, which indicates
that the enzymatic hydrolysis is necessary to achieve high
conversion.
Table 1. Enzymatic hydrolysis of 2 or 3 in the presence of hydroxylamine
Entry
Substrate
pH
5.5
5.5
6.0
6.5
7.0
6.0
6.0
6.0
6.0
6.0
NH2OH (equiv.) Yield of 14 (%) Conversion to 14a (%) Yield % of 15(%)
Recovery (%)
1
2
3
3
3
3
3
3
3
3
3
2.4
2.4
2.4
2.4
2.4
1.0
1.5
2.0
2.5
3.0
32
52
71
41
25
32
43
65
52
41
33
62
82
50
29
71
68
84
64
49
38
3.2
16
12
16
14
54
36
22
19
15
b
2
-
b
3
-
b
4
-
b
5
-
b
6
-
b
7
-
b
8
-
b
9
-
b
10
-
The conditions: substrate; 50 mg, 0.1 M Na-Phosphate buffer; 20 mL, incubated at 38 °C for 12 h. -Glucosidase (0.2 mg in 200 L buffer) was added every 1 h
for 4 h. a Conversion was calculated on the basis of the amount of consumed substrate. b Not isolated.

4
Tetrahedron
Acknowledgments
We thank Ms. Yumi Sato, Ms. Ayaka Sekine, Ms. Kie
Takaura, Ms. Honami Saito, Mr. Tae Hyung Roh and Ms.
Kurumi Menoto (School of Pharmacy, International University of
Health and Welfare) for their technical supports. We also thank
Ms. Satoko Shimbara-Matsubayashi (School of Pharmacy,
Showa University) for measuring HR-ESI MS.
References and notes
1. Proskurnina NF. Zhurnal Obshchei Khimii 1944, 14, 1148–1152.
2. Wu X, Tang S, Jin Y, Wang S, Wang X, Hattori M, Zhang H,
Wang Z. J. Chromatogr. B 2015, 989, 98–103
3. Yang XF, Song CQ. Zhongguo Zhong Yao Za Zhi. 2000, 25, 673–
676.
4. El-Sedawy AI, Shu YZ, Hattori M, Kobashi K, Namba T. Planta
Med. 1989, 55, 147–150.
5. Wang Z, Tang S, Ma C, Toyooka N, Kida H, Kawasaki M, Hattori
M. J. Trad. Med. 2008, 25, 29–34.
6. Chen W, Wang J. Inflammation 2017, 40, 1401–1408.
7. Kwak WJ, Kim J.-H., Ryu KH, Cho YB, Jeon SD, Moon CK. Biol.
Pharm. Bull. 2005, 28, 750–753.
8. Vaidya H, Goyal RK, Cheema SK. Phytother. Res. 2013, 27, 624–
627.
9. Mansoor A, Samad A, Zaidi MI, Aftab K. Pharm. Pharmacol.
Commun. 1998, 4, 229–230.
10. Liu X, Liu S, Wang N, Lu F, Cao M. Neural Regen. Res. 2011, 6,
1130–1135.
11. Kametani T, Takeshita M, Ihara M, Fukumoto K. J. Org. Chem.
1976, 41, 2542–2545.
Scheme 3 Plausible reaction pathways from 2 and 3 affording 1 and 13
12. Kim HO. Lee NK., Kim JH, Rhee HI, Cho Y-B, Ryu JH, Kim
NH, Ryu KH, Yi JB, Jung JY. Patent, WO 2005063768, 2005.
13. Kubota T, Kamikawa T. Bull. Chem. Soc. Jpn. 1962, 35, 1046.
14. Popov SS, Marekov NL, Do TN. J. Nat. Prod. 1988, 51, 765–768.
15. Wang D, Xu M, Zhu HT, Chen KK, Zhang YJ, Yang CR. Bioorg.
Med. Chem. Lett. 2007, 17, 3195–3197.
16. Niiho Y, Yamazaki T, Nakajima Y, Yamamoto T, Ando H, Hirai
Y, Toriizuka K, Ida Y. J. Nat. Med., 2006, 60, 82–88.
17. Hayashi T. Yakugaku Zasshi 1976, 96, 356–361
18. van der Sluis WG, van der Nat JM, Spek AL, Ikeshiro Y, Labadie
RP. Planta Med. 1983, 49, 211–215
19. Ishiguro K, Yamaki M, Takagi S. Planta Med. 1983, 49, 208–210
20. Anastas, P.T.; Warner, J. C. Green Chemistry: Theory and
Practice. Oxford University Press. New York. 2000.
21. Starting materials 2 (5.1%) and 3 (2.1%) was isolated from
Gentian root (100 g) and Swertia herb (200 g), respectively. The
structure of the known compounds in this paper was determined
by comparing their spectral data with those of reported value.
22. Gentianol N-oxide (15): Colorless amorphous solid. IR ( neat ) 
3492(br), 3061, 2971, 1732, 1616, 1474, 1446, 1310, 1115 cm-1.
1H NMR (CDCl3, 400 MHz) 1H NMR (CDCl3, 400 MHz); 8.67 (s,
1H), 8.58 (s, 1H), 5.13 (q, 1H, J = 6.8 Hz), 4.57–4.66 (m, 2H),
3.19–3.30 (m, 2H), 1.47 (t, 3H, J = 6.8 Hz). 13C NMR (CD3OD,
100 MHz) ; 161.8, 142.0, 141.0, 140.9, 137.6, 125.3, 66.8, 55.5,
24.0, 23.1, 16.9. ESI-HR-MS m/z: 210.0778 (Calced for
C10H12NO4: 210.0766).
23. Gentianol acetate (16): yellowish amorphous solid. IR ( neat ) 
2980, 2925, 1731, 1576, 1426, 1298, 1241, 1139 cm-1. 1H NMR
(CDCl3, 400 MHz) ; 9.20 (s, 1H), 8.82 (s, 1H), 5.99 (q, 1H, J =
6.8 Hz), 4.51–4.62 (m, 2H), 3.27 (ddd, 2H, J = 4.5, 6.0, 16.9 Hz),
3.05 (ddd, 2H, J = 5.5, 6.3, 16.9 Hz), 2.08 (s, 1H), 1.59 (d, 3H, J =
6.8 Hz). 13C NMR (CDCl3, 100 MHz) ; 170.3, 163.5, 151.8,
151.3, 145.4, 133.8, 121.2, 67.8, 66.3, 24.0, 21.4, 21.1. ESI-HR-
MS m/z: 370.2884 (Calced for C12H13NO4: 235.0845).
Scheme 4 Plausible routes of the enzymatic hydrolysis of 2 and 3 in the
presence of hydroxylamine affording 14 and 15
3. Conclusion
In this study, an efficient synthesis of gentianine N-oxide 14
was achieved in good yield (81%) by the enzymatic hydrolysis of
3. Interestingly, the enzyme -glycosidase was effective under
conditions including hydroxylamine, and 14 was obtained as a
single product. This procedure could be very useful for the
synthesis of pharmaceutically important gentianine derivatives,
since pyridine N-oxides are generally more nucleophlically
reactive than pyridines.
The reaction pathways to afford 1, 13, 14, and 15 from 2 and 3
were investigated in detail. The results suggest that the formation
of 13 most likely involves an isomerized aglycon of 2 (9). This
pathway provides helpful information for the study of the
degradation of 2 and 3.

3
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Synthesis of Gentianine N-Oxide by Enzymatic
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Hydrolysis of Swertiamarin in the Presence of
Hydroxylamine and Reaction Pathway to Gentianine and Gentianol
Mikio Fujii, Taiki Kuramochi, Yuhi Nakakuki, Rina Hatazawa, Kiju Konno, Tatsuo Munakata, Yasuaki Hirai

4
Tetrahedron
*Swertiamarin and gentiopicroside are natural
Highlights
resource for organic synthesis
*The use of natural products is preferred in view
of green chemistry
*The use of enzymatic reaction is preferred in
view of green chemistry.
*We demonstrated a combination of enzymatic
hydrolysis and oxime form
*The hydrolysis of swertiamarin afford
gentianine N-oxide selectively