Synthesis and detailed examination of spectral properties of (S)- and (R)-higenamine 4-O-β-D-glucoside and HPLC analytical conditions to distinguish the diastereomers

Article abstract of DOI:10.3390/molecules22091450

Higenamine is a tetrahydroisoquinoline present in several plants that has β-adrenergic receptor agonist activity. Study of the biosynthesis of higenamine has shown the participation of norcoclaurine synthase, which controls the stereochemistry to construct the (S)-isomer. However, when isolated from nature, higenamine is found as the racemate, or even the (R)-isomer. We recently reported the isolation of higenamine 4-O-β-D-glucoside. Herein, its (R)- and (S)-isomers were synthesized and compared to precisely determine the stereochemistry of the isolate. Owing to their similar spectral properties, determination of the stereochemistry based on NMR data was considered inappropriate. Therefore, a high-performance liquid chromatography method was established to separate the isomers, and natural higenamine 4-O-β-D-glucoside was determined to be a mixture of isomers.

Full text of DOI:10.3390/molecules22091450

molecules
Article
Synthesis and Detailed Examination of Spectral
Properties of (S)- and (R)-Higenamine
40-O-β-D-Glucoside and HPLC Analytical
Conditions to Distinguish the Diastereomers
Eisuke Kato *, Ryohei Iwata and Jun Kawabata
Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8589, Japan; ryochanboys@eis.hokudai.ac.jp (R.I.);
junk@chem.agr.hokudai.ac.jp (J.K.)
* Correspondence: eikato@chem.agr.hokudai.ac.jp; Tel./Fax: +81-11-706-2496
Received: 17 August 2017; Accepted: 31 August 2017; Published: 31 August 2017
Abstract: Higenamine is a tetrahydroisoquinoline present in several plants that has β-adrenergic
receptor agonist activity. Study of the biosynthesis of higenamine has shown the participation
of norcoclaurine synthase, which controls the stereochemistry to construct the (S)-isomer.
However, when isolated from nature, higenamine is found as the racemate, or even the (R)-isomer.
0
We recently reported the isolation of higenamine 4 -O-
β-D-glucoside. Herein, its (R)- and (S)-isomers
were synthesized and compared to precisely determine the stereochemistry of the isolate. Owing to
their similar spectral properties, determination of the stereochemistry based on NMR data was
considered inappropriate. Therefore, a high-performance liquid chromatography method was
established to separate the isomers, and natural higenamine 40-O-
β-D-glucoside was determined to
be a mixture of isomers.
Keywords:
higenamine;
tetrahydroisoquinoline;
β-adrenergic receptor agonist;
diastereomer separation
1. Introduction
Higenamine (synonym norcoclaurine) is a tetrahydroisoquinoline found in several plants,
including aconite root [ ], lotus (Nelumbo nucifera) [ ], Gnetum parvifolium [ ], Tinospora crispa [ ],
and Tinospora cordifolia [ ]. It possesses -adrenergic receptor agonist activity owing to its
structural similarity to catecholamine, has been shown to have a tracheal relaxation effect
on guinea pig trachea [ ]; it can protect against myocyte apoptosis in ischemia/reperfusion
injury [ ], collagen-induced arthritis, and spinal cord injury [ ]; and it is clinically studied for
therapeutic effects [10,11].
Biologically, higenamine is synthesized from dopamine and 4-hydroxyphenylacetic acid by the
catalytic action of norcoclaurine synthase (NCS) [12 13]. The stereochemistry of higenamine is regulated
by NCS, with its (S)-isomer known as the enzymatic product [12 13]. However, higenamine is often
isolated from the plant as the racemate [ 14]. Furthermore, in the case of lotus, the (S)-enantiomer
1
2
3
4
5
β
6
7
8,9
,
,
3,
was isolated from the leaves [15], while the (R)-enantiomer was found in the seed embryo located near
the plumule [16].
We previously reported the isolation of higenamine 40-O-
β
-D-glucoside (
as a glucose uptake inducer against muscle cells, and indicated that its stereochemistry is (S) [17].
However, during a following research related to the structure-activity relationship study of , a question
1) from lotus plumule
1
arose about the stereochemistry of natural 1 isolated from lotus plumule, and the above reports have
inspired us to precisely examine the stereochemistry of natural 1.
Molecules 2017, 22, 1450; doi:10.3390/molecules22091450
www.mdpi.com/journal/molecules

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As the correct assignment of stereochemistry in natural products is important, in this article,
we chemically synthesized diastereomers 1R and 1S and compared their spectral properties.
Careful examination of the spectral properties of the synthetic diastereomers indicated that
distinguishing them using spectral methods was difficult. Therefore, we established high-performance
liquid chromatography (HPLC) analysis conditions to separate the diastereomers, and used it to
analyze the stereochemistry of natural 1 isolated from lotus plumule.
2. Results and Discussion
To distinguish the diastereomers of
1 and determine their stereochemistry unequivocally,
samples of 1R and 1S were required. Therefore, 1R was selectively synthesized using a previously
reported procedure and purified using reverse-phase HPLC [18], while 1S was prepared using the
same procedure by employing RuCl[R,R-TsDPEN(p-cymene)] as the catalyst for enantioselective
reduction (Scheme 1).
Scheme 1.
HCOOH, TEA, DMF; (
) 2,3,4,6-tetra-O-acetyl-
(4 Å), CH2Cl2; (f) K2CO3, MeOH, THF; (g) Pd(OH)2, H2, THF, MeOH.
Synthesis of 1S. (
) CbzCl, TEA, DMAP, CH2Cl2; (
-D-glucopyranosyl 2,2,2-trichloroacetimidate, BF3
a
) POCl3, CHCl3, reflux;
(
b
) RuCl[R,R-TsDPEN(p-cymene)],
) Pd(Ph3P)4, K2CO3, MeOH, THF;
Et2O, molecular sieves
c
β
d
(
e
·
Nuclear magnetic resonance (NMR) spectra of synthetic diastereomers 1R and 1S were obtained.
However, in deuterium oxide, no apparent difference was found between the diastereomers (Table 1).
This result might be due to (i) the stereochemistry of the synthetic products not being constructed
as intended, resulting in the same diastereomer being isolated from both procedures; or (ii) the two
diastereomers being indistinguishable under these NMR conditions.
To avoid the former possibility, the stereochemistry of the higenamine moiety was confirmed
by deprotection of 3S and 3R to obtain 5R and 5S, respectively, followed by HPLC purification
and specific rotation measurements. The specific rotation values (5S
5R: +21.4 (c = 0.58, methanol)) were in good correlation with previous reports, which confirmed
their stereochemistry [19 20]. Furthermore, the isomers were analyzed using a Chiral CD-Ph column
(Shiseido Co., Tokyo, Japan) to determine their retention times (Figure 1). The specific rotations of
synthetic 1S 32.4 (c = 1.5, methanol)) and 1R 16.3 (c = 1.5, methanol)) showed distinct values,
which excluded the possibility of isomerization during the synthesis of 5R and 5S.
:
−
20.0 (c = 0.68, methanol);
,
(
−
(
−

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Table 1. NMR data for synthetic 1R and 1S in deuterium oxide.
Synthetic 1R Synthetic 1S
No.
δ
δC
δ
δC
H
H
1
3
4.64 (dd, J = 5.7, 8.8 Hz)
3.46–3.52 (m)
3.28 (td, J = 6.3, 13.2 Hz)
56.78
39.93
4.61 (dd, J = 5.7, 9.1 Hz)
3.47 (td, J = 6.3, 12.6 Hz)
3.27 (td, J = 6.3, 12.6 Hz)
2.95–3.00 (m)
56.75
39.90
4
2.96–3.00 (m)
24.69
24.67
2.91 (td, J = 6.3, 17.3 Hz)
6.74 (s)
2.90 (td, J = 6.3, 17.3 Hz)
5
116.49
143.53
144.77
114.53
123.84
124.47
130.30
131.61
117.72
156.74
39.13
6.73 (s)
116.48
143.51
144.76
114.50
123.79
124.45
130.27
131.58
117.74
156.75
39.14
6 or 7
6 or 7
8
-
-
-
-
6.66 (s)
6.64 (s)
9 or 10
9 or 10
-
-
-
-
-
-
0
1
0
0
0
0
2 , 6
7.26 (2H, d, J = 8.5 Hz)
7.13 (2H, d, J = 8.5 Hz)
-
7.25 (2H, d, J = 8.6 Hz)
7.12 (2H, d, J = 8.6 Hz)
-
2.97 (dd, J = 9.1, 14.5 Hz)
3.36 (dd, J = 5.7, 14.5 Hz)
5.09 (d, J = 7.3 Hz)
3.58 (dd, J = 7.3, 9.5 Hz)
3.50 (dd, J = 9.1, 9.5 Hz)
3.61 (dd, J = 8.2, 9.1 Hz)
3.60–3.63 (m)
3 , 5
0
0
4
7
3.00 (dd, J = 8.8, 14.5 Hz)
3.40 (dd, J = 5.7, 14.5 Hz)
5.12 (d, J = 7.6 Hz)
3.58 (dd, J = 7.6, 9.5 Hz)
3.50 (dd, J = 8.6, 9.5 Hz)
3.61 (dd, J = 8.6, 9.5 Hz)
3.63 (ddd, J = 1.9, 5.7, 9.5 Hz)
3.93 (dd, J = 1.9, 12.5 Hz)
3.75 (dd, J = 5.7, 12.5 Hz)
Glc-1
Glc-2
Glc-3
Glc-4
Glc-5
Glc-6
100.78
73.59
76.25
70.11
76.82
61.23
100.84
73.60
76.26
70.12
76.81
61.26
3.93 (dd, J = 2.2, 12.6 Hz)
3.76 (dd, J = 5.7, 12.6 Hz)
Figure 1. HPLC analysis of 5R, 5S, and their mixture. Conditions: column, Chiral CD-Ph
(4.6
×
250 mm)◦; mobile phase, 20 mM aq. KH2PO4/methanol = 50/50; flow rate, 0.5 mL/min;
temperature, 40 C; detection, 280 nm.
Deuterated solvents other than deuterium oxide were then tested for their suitability to separate
the NMR signals of diastereomers of . Methanol-d4 gave a result similar to that of deuterium oxide,
1
and was therefore considered unsuitable (Table 2). In contrast, pyridine-d5 gave relatively separated
1H-NMR signals for the two diastereomers (Table 3). To determine whether these 1H-NMR signal
differences in pyridine-d5 provided adequate evidence to determine the stereochemistry of 1, a mixture
of 1R and 1S was analyzed by 1H-NMR. The mixture showed distinguishable signals (Figure 2), but a
problem was observed that prevented the use of NMR for the determination of stereochemistry.

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Table 2. NMR data for synthetic 1R and 1S in methanol-d4.
Synthetic 1R Synthetic 1S
No.
δ
δC
δ
δC
H
H
1
3
4.61 (dd, J = 6.1, 8.4 Hz)
3.42–3.48 (m)
57.78
40.87
4.59 (dd, J = 5.8, 8.2 Hz)
3.42–3.48 (m)
57.75
40.82
3.25 (td, J = 6.2, 13.0 Hz)
2.98 (td, J = 6.2, 17.3 Hz)
2.90 (td, J = 6.2, 17.3 Hz)
6.62 (s)
3.25 (td, J = 6.4, 13.0 Hz)
2.98 (td, J = 6.4, 17.3 Hz)
2.90 (td, J = 6.4, 17.3 Hz)
6.62 (s)
4
25.69
25.64
5
116.21
158.69
146.91
114.25
123.65
123.60
130.29
131.64
118.43
158.69
40.43
116.21
158.61
146.82
114.27
123.70
123.60
130.32
131.66
118.37
158.61
40.40
6 or 7
6 or 7
8
6.58 (s)
6.56 (s)
9 or 10
9 or 10
0
1
0
0
0
0
2 , 6
7.23 (2H, d, J = 8.6 Hz)
7.11 (2H, d, J = 8.6 Hz)
7.23 (2H, d, J = 8.6 Hz)
7.11 (2H, d, J = 8.6 Hz)
3 , 5
0
0
4
7
3.35–3.42 (m)
3.03 (dd, J = 8.4, 14.5 Hz)
4.90 (d, J = 7.6 Hz)
3.42–3.48 (3H, m)
3.35–3.42 (m)
3.04 (dd, J = 8.2, 14.4 Hz)
4.90 (d, J = 7.7 Hz)
3.42–3.48 (3H, m)
Glc-1
Glc-2,3,5
102.30
78.20
78.01
74.87
71.43
62.59
102.27
78.12
77.94
74.85
71.38
62.53
Glc-4
Glc-6
3.35–3.42 (m)
3.89 (dd, J = 2.2, 12.0 Hz)
3.35–3.42 (m)
3.89 (dd, J = 2.3, 12.0 Hz)
3.68 (dd, J = 5.8, 12.0 Hz)
3.68 (dd, J = 5.8, 12.0 Hz)
Table 3. NMR data for synthetic 1R and 1S in pyridine-d5.
Synthetic 1R Synthetic 1S
No.
δ
δC
δ
δC
H
H
1
3
5.00 (t, J = 5.7 Hz)
3.73 (ddd, J = 6.0, 6.5, 12.3 Hz)
57.11
40.19
5.00 (t, J = 6.5 Hz)
3.73 (ddd, J = 6.0, 6.3, 12.5 Hz)
57.05
40.00
3.46–3.62 (m)
3.51 (ddd, J = 6.0, 6.3, 12.5 Hz)
4
3.17 (ddd, J = 6.5, 6.5, 14.0 Hz)
25.91
3.17 (ddd, J = 6.3, 6.3, 17.0 Hz)
25.81
2.96 (ddd, J = 6.0, 6.0, 14.0 Hz)
2.97 (ddd, J = 6.0, 6.0, 17.0 Hz)
5
6
7
8
9
7.12 (s)
116.98
146.69
147.79
115.04
124.36
124.20
130.61
131.74
117.56
158.21
40.40
7.09 (s)
116.98
146.62
147.82
115.00
124.36
123.64
130.62
131.75
117.53
158.23
40.48
-
-
-
-
7.09 (s)
7.05 (s)
-
-
-
-
-
-
10
0
1
0
0
0
0
2 , 6
7.36 (2H, d, J = 8.4 Hz)
7.15 (2H, d, J = 8.4 Hz)
-
3.46–3.62 (2H, m)
-
5.45 (d, J = 7.9 Hz)
4.27 (dd, J = 7.9, 8.8 Hz)
4.33–4.37 (2H, m)
-
7.34 (2H, d, J = 8.5 Hz)
7.16 (2H, d, J = 8.5 Hz)
-
3.63 (dd, J = 6.5, 14.2 Hz)
3.48 (dd, J = 6.5, 14.2 Hz)
5.43 (d, J = 7.7 Hz)
4.27 (dd, J = 7.7, 9.1 Hz)
4.33–4.38 (2H, m)
-
3 , 5
0
0
4
7
Glc-1
Glc-2
Glc-3, 4
102.44
75.27
78.83
71.62
79.18
62.71
102.53
75.27
78.86
71.64
79.25
62.74
Glc-5
Glc-6
4.06–4.09 (m)
4.51 (dd, J = 2.2, 12.0 Hz)
4.07–4.10 (m)
4.54 (dd, J = 2.2, 12.0 Hz)
4.40 (dd, J = 5.0, 12.0 Hz)
4.41 (dd, J = 5.0, 12.0 Hz)

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1
Figure 2. H-NMR spectra of 1R, 1S, and their mixture (500 MHz, pyridine-d5, r.t.).
As shown in Figure 2, when the 1H-NMR signals of the mixture were compared with those of 1R
or 1S, differences in the chemical shifts (ppm) were apparent. For example, the signal of the anomeric
proton in glucose moiety (Glc-1) is 5.45 ppm for 1R and 5.43 ppm for 1S; however, for the mixture
of the two, the same proton appeared at 5.45 and 5.48 ppm, for which correspondence is unclear.
These shifts in ppm value were presumably due to differences in the solution conditions, pH, or sample
concentration during NMR analysis, which would influence the status of the cyclic amine in the
tetrahydroisoquinoline moiety. They thus indicated that determining the stereochemistry of
1 through
NMR experiments could potentially cause assignment errors, rendering it an inappropriate method.
1
Because of the fluctuation of H-NMR data with analytical conditions, distinguishing the
diastereomers of
1 required an alternative method. HPLC is a method capable of separating and
analyzing diastereomers. In HPLC analysis, analytical conditions are adjusted by changing the eluent
used, which is present in large excess, and should therefore be a reliable method for analyzing the
stereochemistry of 1.
Although a frequently employed C18 column was not capable of separating the isomers, a
COSMOSIL Cholester column (Nakalai Tesque, Inc., Kyoto, Japan) was found to separate 1R and 1S to
a good degree. Under the HPLC condition employed using this column, 1R and 1S showed apparent
separation with retention times of 29 min and 32 min, respectively, indicating its ability to efficiently
distinguish these isomers (Figure 3).
Natural
1
, isolated from lotus plumule, underwent stereochemical analysis using the established
was a mixture of both isomers (1R 1S = 59/41,
HPLC method. The results showed that natural
1
/
Figure 3). This was further confirmed by analyzing the lotus plumule extract to avoid the possibility of
isomerization during isolation, which showed a similar ratio (1R/1S = 60/40, data not shown).
Other than lotus plumule, Phoebe chekiangensis has been reported to contain higenamine
40-O-
β-D-glucoside (1) [21]. The stereochemistry of 1 in P. chekiangensis was determined as (S) by
Wu et al. by comparing with the NMR spectrum and specific rotation of synthetic 1R [18]. Our results
indicate that this result may need to be reassessed.
In conclusion, we synthesized 1R and 1S and directly compared their spectral data. We showed
that the NMR spectra of these diastereomers were similar and their chemical shifts were influenced
by solution conditions. This indicated that NMR was an unreliable method for determining the
stereochemistry of 1, and presumably those of other similar compounds. Therefore, an HPLC method
for separating the diastereomers was established and natural
analyzed and shown to be a mixture of diastereomers.
1, isolated from lotus plumule, was

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Figure 3. HPLC separation of diastereomers of
1. Conditions: column, COSMOSIL Cholester
(4.6 150 mm); mobile phase, 5% aq. methanol containing 0.1% trifluoroacetic acid; flow rate,
×
0.5 mL/min; detection, 280 nm.
3. Materials and Methods
3.1. General
Chemicals were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless
otherwise noted. NMR spectra were obtained using a Bruker AMX 500 (Bruker BioSpin K.K.,
Yokohama, Japan) spectrometer, and residual solvents or tert-butanol were used as the internal
1
1
standard (tert-butanol (deuterium oxide): H 1.24 ppm, 13C 30.29 ppm; methanol-d4: H 3.30 ppm,
13C 49.0 ppm; pyridine-d5: H 8.74 ppm, 13C 150.35 ppm). Mass spectra were obtained using a Waters
1
LCT Premier Spectrometer (Waters Co., Milford, MA, USA). Specific rotations were measured using a
Jasco P-2200 polarimeter (Jasco Co., Tokyo, Japan).
3.2. Synthesis
As the higenamine moiety with a protective group on the cyclic amine contains isomers, which
were either rotamers resulting from the 4-hydroxybenzyl group or diastereomers resulting from the
stereochemistry of the protective group on the cyclic amine, detailed data were not obtained for
compounds 3 and 4.
3.2.1. (S)-N-Cbz-6,7-di-O-benzylhigenamine (3S)
Compound 2 (1.03 g, 2.03 mmol), prepared using a reported procedure [18,22], was dissolved in
chloroform (20 mL) and phosphoryl chloride (1.0 mL, 11.0 mmol) was added. The mixture was stirred
for 17 h under reflux and then cooled to room temperature (r.t.), diluted with saturated NaHCO3
solution, and extracted with chloroform. The organic layer was washed with brine, dried over sodium
sulfate, and concentrated. The residue was dissolved in dry N,N-dimethylformamide (DMF, 40 mL),
RuCl[R,R-TsDPEN(p-cymene)] (35.5 mg, 0.0558 mmol, Kanto Co., Tokyo, Japan) was added, and
the mixture was stirred under nitrogen. The solution was cooled to 0 ◦C, an azeotropic mixture of
formic acid/triethylamine (TEA) (5:2 mole ratio, 1.6 mL) was added, and the mixture was stirred for
20 h at r.t. The reaction mixture was diluted with saturated NaHCO3 solution and extracted with

Molecules 2017, 22, 1450
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ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, and concentrated.
The resultant residue was dissolved in dichloromethane (20 mL) and triethylamine (1.0 mL, 7.2 mmol),
and a catalytic amount of N,N-dimethyl-4-aminopyridine (DMAP) was added. Carbobenzoxy chloride
(CbzCl, 0.3 mL, 2.1 mmol) was then added and the mixture was stirred for 1 h. The reaction mixture
was dried, dissolved in ethyl acetate, and washed with 1 M aq. HCl and brine. The organic layer
was dried over sodium sulfate and concentrated. The residue was dissolved in methanol (20 mL)
and tetrahydrofuran (THF, 5 mL), following which Pd(PPh3)4 (59.1 mg, 0.051 mmol) and potassium
carbonate (422.0 mg, 3.05 mmol) were added. The mixture was stirred for 2 h at 65 ◦C, cooled, and
acidified with 1 M aq. HCl. The solution was extracted with ethyl acetate, washed with 1 M aq. HCl
and brine, dried over sodium sulfate, and concentrated. The residue was purified with silica gel column
chromatography (hexane/ethyl acetate = 2:1) to obtain 3S (639.1 mg, 1.09 mmol, 54%). Electrospray
ionization-mass spectrometry (ESI-MS) (positive): m/z 608 [M + Na]+, [α]D27
−
42.3 (c = 1.5, chloroform).
3.2.2. (S)-N-Cbz-6,7-di-O-benzylhigenamine 40-O-β-D-glucoside (4S)
Compound 3S (614.5 mg, 1.05 mmol) and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl
2,2,2-trichloroacetimidate (1.00 g, 2.34 mmol) were dissolved in dry dichloromethane (10 mL),
powdered activated 4 Å molecular sieves (4 Å MS; 1.00 g) were added, and the mixture was stirred at
−
10 ◦C under nitrogen. BF3
and stirred for 30 min at
·
Et2O (0.1 mL, 0.79 mmol) in dry dichloromethane (0.9 mL) was added
−
10 ◦C under nitrogen. The reaction mixture was diluted with saturated
NaHCO3 solution and passed through a Celite pad. The organic layer was separated, washed with
saturated NaHCO3 solution and brine, dried over sodium sulfate, and concentrated. The resultant
residue was dissolved in methanol (10 mL) and THF (10 mL), then K2CO3 (301.8 mg, 2.18 mmol) was
added, followed by stirring for 2 h. The reaction mixture was filtered, dried, and purified by silica
gel column chromatography (chloroform/methanol = 9:1) to obtain 4S (751.1 mg, 1.01 mmol, 96%).
ESI-MS (positive): m/z 770 [M + Na]+, [α]D26 −57.5 (c = 1.0, acetone).
3.2.3. (S)-higenamine 40-O-β-D-glucoside (1S)
Compound 4S (156.9 mg, 0.210 mmol) was dissolved in THF (10 mL) and methanol (2 mL), after
which 20% palladium hydroxide on carbon (37.4 mg, Sigma-Aldrich Japan K.K., Tokyo, Japan) was
added and stirred for 4 h under hydrogen. The reaction mixture was passed through a Celite pad,
dried, and dissolved in 15% aq. methanol containing 0.1% trifluoroacetic acid (TFA). The solution
was purified by HPLC (column, Inertsustain C18 (20
×
250 mm, GL Science Co., Tokyo, Japan);
mobile phase, 15% aq. methanol containing 0.1% TFA; flow rate, 9.0 mL/min; detection, 280 nm) to
obtain 1S (107.2 mg, 0.196 mmol, 93%, 95% diastereomeric excess (de) from Figure 3) as a TFA salt.
ESI-MS (positive): m/z 434 [M + H]+; [α]2D5 −32.4 (c = 1.5, methanol). See Tables 1–3 for NMR data.
3.2.4. (S)-Higenamine (5S)
Compound 3S (24.6 mg, 0.042 mmol) was dissolved in THF (1 mL) and methanol (1 mL), 20%
palladium hydroxide on carbon (5.4 mg, Aldrich Co.) was added, and the mixture was stirred for 16 h
under hydrogen. The reaction mixture was passed through a Celite pad, dried, and dissolved in 30%
aq. methanol containing 0.1% TFA (0.5 mL). The solution was purified by HPLC (column, Inertsustain
C18 (20
5.0 mL/min; detection, 280 nm) to obtain 5S (13.2 mg, 0.034 mmol, 82%) as a TFA salt.
ESI-MS (positive): m/z 272 [M + H]+; [α]2D2 20.0 (c = 0.68, methanol). 1H-NMR (500 MHz,
×
250 mm, GL Science Co.); mobile phase, 30% aq. methanol containing 0.1% TFA; flow rate,
−
methanol-d4, r.t.): 2.87–3.01 (3H, m), 3.21–3.27 (1H, m), 3.33–3.37 (1H, m), 3.42–3.47 (1H, m),
4.57 (1H, t, J = 5.0 Hz), 6.61 (1H, s), 6.62 (1H, s), 6.80 (2H, d, J = 10.0 Hz), 7.12 (2H, d, J = 10.0 Hz) ppm;
13C-NMR (125 MHz, methanol-d4, r.t.): 25.84, 40.62, 41.01, 58.06, 114.37, 116.33, 117.11, 123.79, 123.89,
127.14, 131.78, 145.92, 147.00, 158.35 ppm.

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3.2.5. (R)-Higenamine 40-O-β-D-glucoside (1R)
Compound 1R was synthesized following the procedure detailed above for 3S
1S. Compound 3S (yield 40% from
(c = 1.5, chloroform); Compound 4S (yield 56%): ESI-MS (positive): m/z 770 [M + Na]+,
[α]2D6 +24.7 (c = 1.0, acetone); Compound 1S (yield 87%, 95% de from Figure 3): ESI-MS (positive):
m/z 434 [M + H]+; [α]2D5 −16.3 (c = 1.5, methanol). See Tables 1–3 for NMR data.
,
4S, and
2
): ESI-MS (positive): m/z 608 [M + Na]+, [α]D26 +44.9
3.2.6. (R)-Higenamine (5R)
Compound 5R was synthesized following the procedure detailed above for 5S. ESI-MS (positive):
m/z 272 [M + H]+; [α]D22 +21.4 (c = 0.58, methanol). The NMR spectrum was identical to that of 5S.
Acknowledgments: This work was supported by JSPS KAKENHI Grant number 25750391. We thank Simon
Partridge, from Edanz Group (www.edanzediting.com) for editing a draft of this manuscript.
Author Contributions: E.K. and J.K. conceived and designed the experiments, E.K. and R.I. performed the
experiments, E.K. prepared the manuscript, and all authors provided comments and revisions.
Conflicts of Interest: The authors declare no conflict of interests.
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Sample Availability: Samples of the compounds 4S, 4R, 1S and 1R are available from the authors.
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2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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