Iteamine, the first alkaloid isolated from Itea virginica L. inflorescence

Article abstract of DOI:10.1016/j.phytochem.2014.01.012

Iteamine, o-aminobenzyl β-d-glucopyranoside, is the first alkaloid to be isolated from Itea virginica. Itea is the sole plant source of d-psicose, a rare sugar likely to be a major dietary supplement. The structure of iteamine was established by NMR and confirmed by total synthesis. Iteamine and its galacto-analog (which was not found in Itea plants) showed no strong inhibition of any of the 15 glycosidases tested; unnatural galacto-iteamine was a weak inhibitor of chicken liver α-N-acetylgalactosaminidase.

Full text of DOI:10.1016/j.phytochem.2014.01.012

Phytochemistry 100 (2014) 126–131
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Iteamine, the first alkaloid isolated from Itea virginica L. inflorescence
b
a
c
c
Benjamin J. Ayers a, , Jacqueline Hollinshead , Alexander W. Saville , Shinpei Nakagawa , Isao Adachi ,
⇑
Atsushi Kato c, Ken Izumori d, Barbara Bartholomew b, George W.J. Fleet a, Robert J. Nash b,
⇑
a Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, UK
b Phytoquest Limited, IBERS, Plas Gogerddan, Ceredigion, Aberystwyth SY23 3EB, Wales, UK
c Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
d Rare Sugar Research Center, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-07, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Iteamine, o-aminobenzyl b-
the sole plant source of D-psicose, a rare sugar likely to be a major dietary supplement. The structure of
iteamine was established by NMR and confirmed by total synthesis. Iteamine and its galacto-analog
(which was not found in Itea plants) showed no strong inhibition of any of the 15 glycosidases tested;
D
-glucopyranoside, is the first alkaloid to be isolated from Itea virginica. Itea is
Received 20 November 2013
Received in revised form 16 January 2014
Available online 14 February 2014
unnatural galacto-iteamine was a weak inhibitor of chicken liver
a-N-acetylgalactosaminidase.
Keywords:
Ó 2014 Elsevier Ltd. All rights reserved.
Itea virginica
Grossulariaceae
Iteamine
a-N-acetylgalactosaminidase
D-Psicose
Introduction
Hough et al., 1973; Hough and Stacey, 1963, 1966a,b). The mono-
saccharide content of wet Itea leaves is 10% of which 80% is a mix-
This paper describes the isolation of the first alkaloid iteamine
1, as the major low molecular weight nitrogen-containing compo-
nent, from Itea virginica inflorescences. The syntheses and glycosi-
dase inhibition profiles of iteamine 1 and the galacto-epimer 2 are
also reported. Many rare sugars, which occur naturally in very
small amounts, have become readily available by biotechnology
ture of
mixture of 3 and 5 can be oxidized by Bacillus pallidus Y25 to pure
-psicose 3 (Poonperm et al., 2007). Neither -psicose 3 nor allitol 4
have been found in any other plants than Itea (Plouvier, 1959).
-Psicose 3 has been identified in many species of Itea including
Itea chinensis Hook. & Arn., Itea ilicifolia Oliv., Itea japonica Oliv., Itea
oldhamii C.K. Schneid., Itea parviflora Hemsl, I. virginica L. (Virginia
Sweetspire) and Itea yunnanensis Franch. Itea plants are distributed
widely and are widely used as a hedge in gardens and as a green
tea in Japan; I. virginica grows rapidly and is easy to obtain (Sun
et al., 2006). Harvesting of Itea provides a natural plant food source
D-psicose 3 and allitol 5 (Poonperm et al., 2007). The
D
D
D
(Granstrom et al., 2004; Izumori, 2006).
produced on a multikilogram scale by epimerization of C-3 of
-fructose 4 by -tagatose-3-epimerase (DTE) (Mu et al., 2012;
D-Psicose [D-allulose] 3,
D
D
Takeshita et al., 2000), with zero calorific value is likely to be a
widely used dietary supplement (Fig. 1) (Chung et al., 2012; Iida
et al., 2010; Matsuo and Izumori, 2009; Matsuo et al., 2002; Sun
of D-psicose 3 as an alternative to microbial biotechnology. Some
et al., 2007).
D
-Psicose 3 has a number of beneficial health proper-
250 iminosugars, nitrogen analogs of monosaccharides, have been
isolated from a wide range of plants (Asano et al., 2000; Nash et al.,
2011; Watson et al., 2001). In view of the unique sugars that occur
in Itea, a search found no iminosugars in Itea but resulted in the
isolation of iteamine 1. o-Aminobenzyl alcohol is a reduced form
of anthranilic (o-aminobenzoic) acid, a biosynthetic precursor of
tryptophan, the glucosylation of which, tentatively, may account
for the formation of iteamine 1. Alternative pathways may include;
the formation of the glucosyl ester of anthranilic acid (Quiel and
Bender, 2003), followed by reduction to iteamine 1; or N-glucosy-
lation of anthranilic acid (Kuzumi et al., 1999), with reduction and
rearrangement giving iteamine 1.
ties (Hisiike et al., 2013; Iida et al., 2013; Matsuo and Izumori,
2006), is a potential anthelmintic (Sato et al., 2008) and also affects
some enzymes of plant metabolism (Kato-Noguchi and Okada,
2013; Kato-Noguchi et al., 2005; Moghaddam and van den Ende,
2012). I. virginica L. (Grossulariaceae), Virginia Sweetspire, is a
shrub native to North America in which D-psicose 3 and allitol 5
are the major monosaccharide components (Carr et al., 1968;
⇑
Corresponding authors. Tel.: +44 1865 275645; fax: +44 1865 277378
(B.J. Ayers).
E-mail addresses: benjamin.ayers@chem.ox.ac.uk (B.J. Ayers), rob.nash@
phytoquest.co.uk (R.J. Nash).
http://dx.doi.org/10.1016/j.phytochem.2014.01.012
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.

B.J. Ayers et al. / Phytochemistry 100 (2014) 126–131
127
OH
O
OH
HOH2C
HO
HOH2C
HO
HOH2C
HO
HO
O
OH
HO
O
OH
O
OH
O
OH
OH
OH
CH2OH
O
CH2OH
HO
HO
HO
CH2OH
CH2OH
CH2OH
H2N
H2N
1
2
3
4
5
Fig. 1. Structures of iteamines and monosaccharides.
OAc
O
OAc
R1
OH
R1
OH
O
OAc
R1
R1
R2
CH2OAc
R1
R2
CH2OH
AcO
AcO
AcO
O
HO
O
HO
O
AcO
Br
R2
i
iii
iv
ii
R2
R2
O
CH2OAc
O
CH2OH
O
CH2OAc
H2N
O2N
O2N
6 R1= H, R2 = OAc
7 R1= OAc, R2 = H
8 R1= H, R2 = OAc, 94%
9 R1= OAc, R2 = H, 93%
10 R1= H, R2 = OAc, 91%
11 R1= OAc, R2 = H, 91%
12 R1= H, R2 = OH, 98%
13 R1= OH, R2 = H, 99%
1 R1= H, R2 = OH, 83%
2 R1= OH, R2 = H, 85%
Scheme 1. Reagents & conditions: (i) HBr (33% in AcOH), DCM, 0 °C – rt, 2 h (ii) I2, DDQ, o-nitrobenzyl alcohol, MeCN, 4 Å MS, 70 min (iii) MeONa (cat.), MeOH, rt, 16 h
(iv) 10% Pd-C, H2, EtOH, 30 min.
ꢀ56.1° (c 1.2, MeOH), with identical 1H and 13C
25
The galacto-analog 2 was not present in the plant. Both 1 and 2
were assayed as inhibitors of 15 glycosidases. Natural iteamine 1
showed no inhibition of any of the enzymes, however galacto-ite-
iteamine 1, ½a D
ꢁ
25
NMR spectra to those of the isolated natural product 1, ½a D
ꢁ
ꢀ49° (c 0.17, MeOH), in 83% yield [70% overall yield from glucose
pentaacetate 6]. galacto-Iteamine 2 was prepared by an identical
sequence from galactose pentaacetate 7 in 71% overall yield.
amine 2 was a weak inhibitor of chicken liver
minidase ( -GalNAcase).
a-N-acetylgalactosa-
a
Results and discussion
Isolation, structure determination and synthesis
Enzyme inhibition
No good inhibition of any of 15 glycosidases by iteamine 1 and
its galacto-analog 2 was found (Table 1) by assays as previously
described (Hakansson et al., 2007; Watson et al., 1997). However
the galacto-analog 2 showed weak inhibition of chicken liver
Fresh inflorescences of I. virginica L. (Grossulariaceae) cv.
Henry’s Garnet were homogenized with aqueous ethanol, and ex-
tracted. The filtrate was treated with Amberlite IR-120 (H+ form),
and eluted with 2 M NH4OH. Concentration of the eluate gave a
light yellow oil which was applied to Amberlite CG-400 (OHꢀ form)
to remove neutral and acidic amino acids and pigments. The frac-
tions with the amine were combined and freeze dried to give
20 mg of an alkaloid iteamine 1 (yield 0.067% fresh weight). The
fractionation was monitored by GC–MS analysis of the trimethyl-
silyl-derivative of iteamine 1, which gave a characteristic mass
spectrum. The structure of iteamine 1 was first studied by NMR
analysis, which indicated the presence of an ortho-substituted ben-
zyl derivative and had the characteristic spectrum associated with
b-glucosides. The 1H and 13C NMR spectra of natural iteamine 1
were identical to those of the synthetic material of 1 below. The ga-
lacto-analog 2 was also prepared. The pertrimethylsilyl-derivative
of iteamine 1 had a GC retention time of 17.2 min, compared with
the pertrimethylsilyl-derivative of glucose running at 8 min. The
pertrimethylsilyl-derivative of galacto-iteamine 2 had a retention
time of 16.8 min. with a similar mass spectrum; the galacto isomer
was not present in the plant extract.
a-N-acetylgalactosaminidase (a-GalNAcase). Inhibitors of b-N-ace-
tylhexosaminidases are common (Zhu et al., 2013) and almost all
contain an acetamide group; in contrast, very few compounds in-
hibit
a-GalNAcase (Best et al., 2010; Clark et al., 2012; Glawar
et al., 2012). The specific weak
a-GalNAcase inhibition by 2 paral-
lels that shown by the enantiomer 14 (Hu et al., 2010) of the nat-
ural product steviamine (Fig. 2) (Michalik et al., 2010; Thompson
et al., 2009), and that by DGJ 15 (Clark et al., 2012); it may be sig-
nificant that none of these compounds contains an acetamide but
are specific, though weak, inhibitors of the a-GalNAcase and show
no inhibition of b-N-acetylhexosaminidases.
Conclusion
Itea plants are likely to provide a natural source of D-psicose 3
as a dietary supplement, so that identification of other components
in Itea is of importance in regard to safety and other issues. This
paper reports the isolation of the first alkaloid from I. virginica,
iteamine 1.
Iteamine 1 was prepared from D-glucose pentaacetate 6, which
with 33% HBr in acetic acid in dichloromethane (DCM) gave the
bromide 8 (94%) (Scheme 1). The bromide 8 with o-nitrobenzyl
alcohol in the presence of iodine and dichlorodicyanoquinone
(DDQ) in acetonitrile afforded the o-nitrobenzyl b-pyranoside 10
(91%). The acetyl groups in 10 were removed by treatment with
sodium methoxide in methanol to produce the o-nitrobenzyl
glucoside 12 (98%) which on hydrogenation in the presence of
palladium on carbon and purification by ion exchange resin gave
Experimental
General experimental
For GC–MS analyses, samples were freeze dried and prepared as
pertrimethylsilyl ethers at 60 °C for 20 min using Tri-Sil reagent
(Pierce Biotechnology Inc., Rockford, IL). The column was
a

128
B.J. Ayers et al. / Phytochemistry 100 (2014) 126–131
Table 1
was stopped by addition of 400 mM Na2CO3. The released p-nitro-
phenol was measured spectrometrically at 400 nm. All commercial
reagents were used as supplied. Unless otherwise stated, all
reactions were performed under an inert atmosphere (argon or
nitrogen). Thin layer chromatography (TLC) was performed on alu-
minum sheets coated with 60 F254 silica. Plates were visualized
using a spray of 0.2% w/v cerium(IV) sulfate and 5% ammonium
molybdate solution in 2 M aqueous sulfuric acid. Flash chromatog-
raphy was performed on Sorbsil C60 40/60 silica. Melting points
were recorded on a Kofler hot block and are uncorrected. Optical
Concentration of iminosugars giving 50 % inhibition of various glycosidases.
Enzyme IC50 M)
(
l
a-Glucosidase
NIa (0%)b
NI (48%)
NI (20%)
NI (28%)
rotation concentrations are quoted in g 100 mLꢀ1 1H and 13C
.
Rice
Yeast
NMR spectra were assigned by utilizing 2D COSY, HSQC and HMBC
spectra. Residual signals from the solvents were used as an internal
reference. HRMS measurements were made using a micrOTOF
mass analyzer.
b-Glucosidase
Almond
C. saccharolyticum
Bovine liver
NI (0%)
NI (31%)
NI (23%)
NI (30%)
NI (44%)
NI (15%)
a
-Galactosidase
Coffee beans
Human lysosome
Plant material
NI (14%)
NI (1.4%)
NI (3.8%)
NI (17%)
30 g of fresh inflorescences of I. virginica L. (Grossulariaceae) cv.
Henry’s Garnet were collected for extraction. A herbarium speci-
men was produced (deposited at the WPBS Herbarium at IBERS,
Plas Gogerddan, Aberystwyth SY23 3EB as NashR 2009/1).
b-Galactosidase
Bovine liver
NI (31%)
NI (15%)
NI (29%)
NI (15%)
1000
a-Mannosidase
Jack beans
b-Mannosidase
Snail
NI (22%)
NI (0%)
NI (3.9%)
NI (16%)
NI (2.9%)
985
Extraction and isolation
a-
L
-Fucosidase
Bovine epididymis NI (1.2%)
The inflorescences were homogenized in 300 ml of 50% aqueous
EtOH and extracted for 15 h. The filtrate was applied to a column of
Amberlite IR-120 (3 ꢂ 100 cm, H+ form), washed with 50% aqueous
EtOH followed by 6 column volumes of water and eluted with 2 M
NH4OH. The ammonium hydroxide eluate was concentrated to
give a light yellow oil and this was applied to Amberlite CG-400
(OHꢀ form) column (1 ꢂ 20 cm) to remove neutral and acidic ami-
no acids and pigments. Water was used to elute the amine and
2 mL fractions were collected and iteamine was collected pure in
fractions 13–20. A small amount of a sugar alcohol was detected
Trehalase
Porcine kidney
NI (6.3%)
NI (6.6%)
a-
L
-Rhamnosidase
P. decumbens
b-N-acetylhexosaminidase
Human placenta
NI (0%)
a-N-acetylgalactosaminidase
Chicken liver
NI (0%)
a
NI: No inhibition (less than 50% inhibition at 1000
(): Inhibition % at 1000 lM.
lM).
b
before the amine. The fractions with the amine were combined
25
and freeze dried to give 20 mg of iteamine 1, ½a D
ꢁ
ꢀ49° (c 0.17,
MeOH). The fractionation was monitored by GC–MS analysis of
the trimethylsilyl-derivative (tms), which gave a characteristic
mass spectrum with fragment ions at m/z 106 (base peak), 217,
233, 331, 361 and 377.1 Iteamine 1 had a GC retention time of
17.2 min with glucose (tms) running at 8 min. galacto-iteamine 2
had a retention time of 16.8 min. with a similar mass spectrum.
The 1H and 13C NMR spectra of natural iteamine 1 were identical
to those of the synthetic material 1 below.
Fig. 2. Enantiomer of steviamine and DGJ.
Chemical synthesis
25 m ꢂ 0.25 mm VF-5 ms ‘‘Factor Four’’ (film thickness, 0.25
lm)
capillary column (Varian Inc.), and the 25 min temperature pro-
gram ran from 160 to 300 °C with an initial rate of increase of
10 °C/min and then held at 300 °C. The mass spectrometer was a
Perkin-Elmer TurboMass Gold, with a quadrupole ion filter system,
which was run at 250 °C constantly during analysis, and the mass
2,3,4,6-Tetra-O-acetyl-
Hydrogen bromide (33% in acetic acid, 25 mL) was added
dropwise to a solution of b- -glucose pentaacetate 6 (4.90 g,
a-D-glucopyranosyl bromide 8
D
12.6 mmol) in dry dichloromethane (50 mL) at 0 °C under argon.
The reaction was allowed to warm to RT and stirred for 2 h, after
which TLC analysis (1:1, cyclohexane/ethyl acetate) indicated the
complete consumption of the starting material (Rf 0.30) and the
formation of a single product (Rf 0.60). The reaction mixture was
poured onto ice-water (200 mL) and extracted with cold
dichloromethane (2 ꢂ 100 mL). The organic fractions were com-
bined, washed with a saturated sodium bicarbonate solution
(3 ꢂ 150 mL), a saturated brine solution (150 mL), dried (MgSO4),
filtered and concentrated in vacuo. The crude residue was recrys-
tallized (diethyl ether/cyclohexane) to give the bromide 8 as a
range was set to 100 to 650 amu. The enzymes
rice and yeast), b-glucosidase (from almond, Caldocellum
saccharolyticum, bovine liver), -galactosidase (from coffee beans),
a-glucosidase (from
a
b-galactosidase (from bovine liver),
a
-mannosidase (from jack
bean), b-mannosidase (from snail),
a-L-fucosidase (from bovine
epididymis), trehalase (porcine kidney),
Penicillium decumbens), b-N-acetylhexosaminidase (human pla-
centa), -N-acetylgalactosaminidase (chicken liver) p-nitrophenyl
a-L-rhamnosidase (from
a
glycosides, and various disaccharides were purchased from Sig-
ma–Aldrich Co. Glycosidase activities were determined using an
appropriate p-nitrophenyl glycoside as substrate at the optimum
pH of each enzyme. The reaction mixture contained 2 mM of the
substrate and the appropriate amount of enzyme. The reaction
1
The mass spectrum is included in the supplementary information, available
online.

B.J. Ayers et al. / Phytochemistry 100 (2014) 126–131
129
white crystalline solid (4.75 g, 94%); m.p. 86–89 °C (diethyl ether/
2.02, 2.04, 2.06, 2.09 (4 ꢂ 3H, s, COCH3), 3.76 (1H, ddd, H-5,
J5,6a = 2.3, J5,6b = 4.6, J5,4 = 9.6), 4.15 (1H, dd, H-6a, J6a,5 = 2.3,
J6a,6b = 12.4), 4.30 (1H, dd, H-6b, J6b,5 = 4.6, J6b,6a = 12.4), 4.68 (1H,
d, H-1, J1,2 = 7.8), 5.06 (1H, d, OCH2Ar, Jgem = 14.7), 5.12 (1H, a-t,
H-4, J4,3 = J4,5 = 9.6), 5.15 (1H, dd, H-2, J2,1 = 7.8, J2,3 = 9.4), 5.24
(1H, a-t, H-3, J3,2 = J3,4 = 9.4), 5.27 (1H, d, OCH2Ar, Jgem = 14.7),
7.43 (1H, t, Ar–H, J = 7.6), 7.62 (1H, t, Ar–H, J = 7.6), 7.67 (1H, d,
Ar–H, J = 7.6), 8.07 (1H, d, Ar–H, J = 8.1); 13C NMR spectral data
(100 MHz, CDCl3): 20.6, 20.6, 20.6, 20.7 (COCH3), 61.7 (C-6), 68.2
(OCH2Ar), 68.3 (C-4), 71.2 (C-2), 72.0 (C-5), 72.7 (C-3), 100.5 (C-
1), 124.7, 128.3, 128.8, 133.4 (ArCH), 133.8 (ArCC), 147.0 (ArCNO2),
169.4, 169.4, 170.2, 170.6 (COCH3); HRMS (ESI): found 506.1270
cyclohexane) {Lit. (Weygand et al., 1958) 88–89 °C (diethyl ether/
25
pet. ether)}; ½a D
ꢁ
+186.1° (c 2.0, CHCl3) {Lit. (Weygand et al.,
25
1958) ½a D
ꢁ
+194° (c 3.94, CHCl3)}; m
cmꢀ1: 1759 (s, >C@O); 1H
NMR spectral data (400 MHz, CDCl3): d 2.04, 2.05, 2.10, 2.10
(4 ꢂ 3H, s, COCH3), 4.14 (1H, dd, H-6a, J6a,5 = 2.0, J6a,6b = 12.5),
4.29 (1H, ddd, H-5, J5,6a = 2.0, J5,6b = 4.2, J5,4 = 9.4), 4.34 (1H, dd,
H-6b, J6b,5 = 4.2, J6b,6a = 12.5), 4.84 (1H, dd, H-2, J2,1 = 4.0,
J2,3 = 9.9), 5.16 (1H, a-t, H-4, J4,5 = J4,3 = 9.7), 5.56 (1H, a-t, H-3,
J3,4 = J3,2 = 9.7), 6.61 (1H, d, H-1, J1,2 = 4.0); 13C NMR spectral data
(100 MHz, CDCl3): d 20.5, 20.6, 20.6, 20.6 (COCH3), 60.9 (C-6),
67.1 (C-4), 70.1 (C-3), 70.6 (C-2), 72.1 (C-5), 86.5 (C-1), 169.4,
169.8, 169.8, 170.5 (COCH3); HRMS (ESI): found 433.0110
+
[M+Na]+; C21H25NNaO12 requires 506.1269; EIMS (probe) eV,
[M+Na]+; C14H19BrNaO9 requires 433.0110; EIMS (probe) eV,
m/z (rel. int.): 506 (100% [M+Na]+).
+
m/z (rel. int.): 433 (100% [M+Na]+).
o-Nitrobenzyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranoside 11
2,3,4,6-Tetra-O-acetyl-
Hydrogen bromide (33% in acetic acid, 15 mL) was added drop-
wise to solution of b- -galactose pentaacetate (3.10 g,
a
-
D
-galactopyranosyl bromide 9
A solution of bromide 9 (500 mg, 1.22 mmol) and o-nitrobenzyl
alcohol (560 mg, 3.65 mmol) in acetonitrile (10 mL) was added to a
solution of iodine (155 mg, 0.61 mmol) and 2,3-dichloro-5,6-dic-
yanobenzoquinone (DDQ, 70 mg, 0.30 mmol) in acetonitrile
(10 mL), over 4 Å beaded molecular sieves under argon. The reac-
tion was stirred for 70 min, after which TLC analysis (1:1, cyclohex-
ane/ethyl acetate) indicated the complete consumption of the
starting material (Rf 0.68) and the formation of a single product
(Rf 0.55). A saturated sodium thiosulfate solution was added
(10 mL) and the reaction stirred vigorously. Ethyl acetate (50 mL)
was added, the phases separated and the organic layer washed
with a saturated sodium thiosulfate solution (25 mL). The organic
phase was dried (MgSO4), filtered, concentrated in vacuo and puri-
fied by flash chromatography (19:1 to 1:1, cyclohexane/ethyl ace-
tate) to afford the acetylated galactoside 11 (536 mg, 91%) as a pale
a
D
7
7.94 mmol) in dry dichloromethane (30 mL) at 0 °C under argon.
The reaction was allowed to warm to RT and stirred for 2 h, after
which TLC analysis (1:1, cyclohexane/ethyl acetate) indicated the
complete consumption of the starting material (Rf 0.52) and the
formation of a single product (Rf 0.68). The reaction mixture was
poured onto ice-water (300 mL) and extracted with cold
dichloromethane (2 ꢂ 150 mL). The organic fractions were com-
bined, washed with a saturated sodium bicarbonate solution
(3 ꢂ 150 mL), a saturated brine solution (150 mL), dried (MgSO4),
filtered and concentrated in vacuo. The crude residue was recrystal-
lized (diethyl ether/cyclohexane) to give the bromide 9 as a white
crystalline solid (3.10 g, 93%); m.p. 83–85 °C (diethyl ether/cyclo-
hexane) {Lit. (Mohri et al., 2003) 86–88 °C, diethyl ether/hexane};
yellow oil; ½a D
ꢁ
25 +10.9° (c 1.07, CHCl3); cmꢀ1: 1369 (s, NO2), 1529
m
½
a D
ꢁ
+225.0° (c 0.6, CHCl3) {Lit. (Haskins et al., 1942) ½a D
ꢁ
(s, NO2), 1749 (s, >C@O); 1H NMR spectral data (400 MHz, CDCl3): d
2.00, 2.06, 2.07, 2.17 (4 ꢂ 3H, s, COCH3), 3.98 (1H, a-t, H-5,
J5,6a = J5,6b = 6.7), 4.15 (1H, dd, H-6a, J6a,5 = 6.7, J6a,6b = 11.4), 4.21
(1H, dd, H-6b, J6b,5 = 6.7, J6b,6a = 11.4), 4.66 (1H, d, H-1, J1,2 = 8.1),
5.06 (1H, d, OCH2Ar, Jgem = 14.8), 5.07 (1H, dd, H-3, J3,4 = 3.4,
J3,2 = 10.4), 5.28 (1H, d, OCH2Ar, Jgem = 14.8), 5.34 (1H, dd, H-2,
J2,1 = 8.1, J2,3 = 10.4), 5.43 (1H, d, H-4, J4,3 = 3.4), 7.47 (1H, t, Ar–H,
J = 7.9), 7.65 (1H, t, Ar–H, J = 7.9), 7.73 (1H, d, Ar–H, J = 7.9), 8.09
(1H, d, Ar–H, J = 7.9); 13C NMR spectral data (100 MHz, CDCl3): d
20.6, 20.7, 20.8, 20.9 (COCH3), 61.2 (C-6), 67.0 (C-4), 68.2 (OCH2Ar),
68.8 (C-2), 70.8 (C-3), 70.9 (C-5), 101.0 (C-1), 124.8, 128.4, 128.8,
133.9 (ArCH), 133.5 (ArCC), 147.0 (ArCNO2), 169.6, 170.1, 170.2,
170.4 (COCH3); HRMS (ESI): found 506.1268 [M+Na]+; C21H25-
25
25
+217.0° (c 1.0, CHCl3)};
m
cmꢀ1: 1749 (s, >C@O); 1H NMR spectral
data (400 MHz, CDCl3): d 2.02, 2.07, 2.12, 2.16 (4 ꢂ 3H, s, COCH3),
4.11 (1H, dd, H-6a, J6a,5 = 6.8, J6a,6b = 11.4), 4.19 (1H, dd, H-6b,
J6b,5 = 6.3, J6b,6a = 11.4), 4.49 (1H, a-t, H-5, J5,6a = J5,6b = 6.4), 5.05
(1H, dd, H-2, J2,1 = 3.8, J2,3 = 10.6), 5.41 (1H, dd, H-3, J3,4 = 3.2,
J3,2 = 10.7), 5.52 (1H, d, H-4, J4,3 = 2.8), 6.70 (1H, d, H-1, J1,2 = 4.0);
13C NMR spectral data (100 MHz, CDCl3): d 20.5, 20.6, 20.6, 20.7
(COCH3), 60.8 (C-6), 67.0 (C-4), 67.7 (C-2), 68.0 (C-3), 71.0 (C-5),
88.1 (C-1), 169.7, 169.9, 170.1, 170.3 (COCH3); HRMS (ESI): found
433.0112 [M+Na]+; C14H19BrNaO9+ requires 433.0110; EIMS (probe)
eV, m/z (rel. int.): 433 ([M(79Br)+Na]+, 100%), 435 ([M(81Br)+Na]+,
93%), 843 ([2M(79Br)+Na]+, 15%), 845 ([M(79Br)+M(81Br)+Na]+,
30%), 847 ([2 M(81Br)+Na]+, 14%).
+
NNaO12 requires 506.1269; EIMS (probe) eV, m/z (rel. int.): 501
(96%, [M+NH4]+), 506 (100%, [M+Na]+), 989 (97%, [2M+Na]+).
o-Nitrobenzyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyranoside 10
A solution of bromide 8 (500 mg, 1.22 mmol) and o-nitrobenzyl
alcohol (560 mg, 3.65 mmol) in acetonitrile (10 mL) was added to a
solution of iodine (155 mg, 0.61 mmol) and 2,3-dichloro-5,6-dic-
yanobenzoquinone (DDQ, 70 mg, 0.30 mmol) in acetonitrile
(10 mL), over 4 Å beaded molecular sieves under argon. The reac-
tion was stirred for 4 h, after which TLC analysis (1:1, cyclohex-
ane/ethyl acetate) indicated the complete consumption of the
starting material (Rf 0.65) and the formation of a single product
(Rf 0.50). A saturated sodium thiosulfate solution was added
(15 mL) and the reaction stirred vigorously. Ethyl acetate (50 mL)
was added, the phases separated and the organic layer washed
with a saturated sodium thiosulfate solution (25 mL). The organic
phase was dried (MgSO4), filtered, concentrated in vacuo and puri-
fied by flash chromatography (20:1 to 5:1, cyclohexane/ethyl ace-
o-Nitrobenzyl b-
Sodium methoxide (5 mg, cat.) was added to a solution of o-
nitrobenzyl 2,3,4,6-tetra-O-acetyl-b- -glucopyranoside 10 (75 mg,
0.16 mmol) in methanol (5 mL). The reaction was stirred at RT
for 18 h, after which TLC analysis (9:1, ethyl acetate/methanol)
indicated the complete consumption of the starting material (Rf
0.90) and the formation of a product (Rf 0.30). The reaction mixture
was concentrated in vacuo and the residue was purified by flash
chromatography (9:1, ethyl acetate/methanol) to afford the gluco-
D
-glucopyranoside 12
D
side 12 as a white crystalline solid (48 mg, 98%); m.p. 139–140 °C;
25
{Lit. (Zehavi et al., 1972) m.p. 131 °C}; ½a D
ꢁ
ꢀ30.0° (c 1.3, MeOH)
25
{Lit. (Zehavi et al., 1972) ½a D
ꢁ
ꢀ4.39° (c 0.55, pyridine)};
m
cmꢀ1
:
1341 (s, NO2), 1526 (s, NO2), 3367 (br. s, OH); 1H NMR spectral data
(400 MHz, CD3OD): d 3.29-3.42 (4H, m, H-2, H-3, H-4, H-5), 3.69
(1H, dd, H-6a, J6a,5 = 5.1, J6a,6b = 11.9), 3.87–3.91 (1H, m, H-6b),
4.44 (1H, d, H-1, J1,2 = 7.8), 5.08 (1H, d, OCH2Ar, Jgem = 15.2), 5.28
(1H, d, OCH2Ar, Jgem = 15.2), 7.51 (1H, t, Ar–H, J = 7.8), 7.71 (1H, t,
Ar–H, J = 7.6), 8.02 (1H, d, Ar–H, J = 7.8), 8.07 (1H, d, Ar–H,
tate) to afford the acetylated glucoside 10 (535 mg, 91%) as a pale
25
yellow oil; ½a D
ꢁ
ꢀ0.95° (c 1.26, MeOH) {Lit. (Zehavi et al., 1972)
cmꢀ1: 1367 (s, NO2), 1529 (s,
NO2), 1755 (s, >C@O); 1H NMR spectral data (400 MHz, CDCl3): d
25
½
aꢁD
ꢀ7.5° (c 2.00, CHCl3)};
m

130
B.J. Ayers et al. / Phytochemistry 100 (2014) 126–131
J = 8.3); 13C NMR spectral data (100 MHz, CD3OD): 62.7 (C-6), 68.4
(OCH2Ar), 71.6 (C-4), 75.2 (C-2), 78.0 (C-3), 78.0 (C-5), 104.0 (C-1),
125.5, 129.2, 130.3, 134.7 (ArCH), 135.7 (ArCC), 148.7 (ArCNO2);
was stirred for 30 min, after which TLC analysis (4:1, ethyl ace-
tate/methanol) indicated the complete consumption of starting
material (Rf 0.39) and the formation of a single product (Rf 0.25).
The reaction mixture was filtered, concentrated in vacuo and the
crude residue loaded onto a short column of DowexÒ (50W-X8,
H+). The resin was washed with water, the product liberated with
aqueous ammonia (2 M) and the ammoniacal fractions concen-
trated in vacuo. The residue was co-evaporated with methanol
+
HRMS (ESI): found 338.0849 [M+Na]+; C13H17NaNO8 requires
338.0846; EIMS (probe) eV, m/z (rel. int.): 338 (100% [M+Na]+).
o-Nitrobenzyl b-
Sodium methoxide (20 mg, cat.) was added to a solution of
o-nitrobenzyl 2,3,4,6-tetra-O-acetyl-b- -galactopyranoside 11
D
-galactopyranoside 13
D
(4 ꢂ 15 mL) and recrystallized (acetone) to afford galacto-iteamine
25
(514 mg, 1.06 mmol) in methanol (10 mL). The reaction was stirred
at RT for 16 h, after which TLC analysis (9:1, ethyl acetate/metha-
nol) indicated the complete consumption of the starting material
(Rf 0.95) and the formation of a product (Rf 0.39). The reaction
mixture was concentrated in vacuo and the residue recrystallized
(2-propanol/cyclohexane) to afford the galactoside 13 as a white
2 (247 mg, 85%); m.p. 80–82 °C (acetone); ½a D
ꢁ
ꢀ37.0° (c 1.2,
MeOH);
m
cmꢀ1: 3373 (br. s, OH, NH2); 1H NMR spectral data
(400 MHz, D2O): d 3.41 (1H, dd, H-2, J2,1 = 7.8, J2,3 = 9.9), 3.49 (1H,
dd, H-3, J3,4 = 3.3, J3,2 = 9.9), 3.55 (1H, dd, H-5, J5,6a = 4.3,
J5,6b = 7.8), 3.64 (1H, dd, H-6a, J6a,5 = 4.3, J6a,6b = 11.9), 3.70 (1H,
dd, H-6b, J6b,5 = 7.8, J6b,6a = 11.9), 3.79 (1H, d, H-4, J4,3 = 3.3), 4.33
(1H, d, H-1, J1,2 = 7.8), 4.60 (1H, d, OCH2Ar, Jgem = 11.4), 4.83 (1H,
d, OCH2Ar, Jgem = 11.4), 6.75–6.80 (2H, m, Ar–H), 7.15 (2H, m, Ar–
H); 13C NMR spectral data (100 MHz, D2O): d 61.4 (C-6), 69.0 (C-
4), 69.6 (OCH2Ar), 71.0 (C-2), 73.2 (C-3), 75.6 (C-5), 101.7 (C-1),
117.6, 119.8, 130.6, 131.3 (ArCH), 122.5 (ArCC), 145.8 (ArCNH2);
crystalline solid (322 mg, 99%); m.p. 148–150 °C (2-propanol/
25
cyclohexane); ½a D
ꢁ
ꢀ11.2° (c 1.08, MeOH);
m
cmꢀ1: 1342 (s,
NO2), 1524 (s, NO2), 3450 (br.s, OH); 1H NMR spectral data
(400 MHz, D2O): d 3.47 (1H, dd, H-2, J2,1 = 7.6, J2,3 = 9.9), 3.51 (1H,
dd, H-3, J3,4 = 3.0, J3,2 = 9.9), 3.55 (1H, dd, H-5, J5,6a = 4.5,
J5,6b = 7.6), 3.62 (1H, dd, H-6a, J6a,5 = 4.5, J6a,6b = 11.6), 3.67 (1H,
dd, H-6b, J6b,5 = 7.6, J6b,6a = 11.6), 3.81 (1H, d, H-4, J4,3 = 3.0), 4.35
(1H, d, H-1, J1,2 = 7.6), 5.01 (1H, d, OCH2Ar, Jgem = 14.4), 5.14 (1H,
d, OCH2Ar, Jgem = 14.4), 7.45 (1H, t, Ar–H, J = 7.7), 7.63 (1H, t, Ar–
H, J = 7.7), 7.70 (1H, d, Ar–H, J = 7.7), 7.98 (1H, d, Ar–H, J = 7.7);
13C NMR spectral data (100 MHz, D2O): d 58.5 (C-6), 65.6 (OCH2Ar),
66.2 (C-4), 68.4 (C-2), 70.3 (C-3), 72.8 (C-5), 100.0 (C-1), 122.6,
HRMS (ESI): found 308.1105 [M+Na]+; C13H19NNaO6 requires
+
308.1105; EIMS (probe) eV, m/z (rel. int.): 286 (10%, [M+H]+), 308
(100%, [M+Na]+), 324 (12%, [M+K]+), 593 (85%, [2M+Na]+); (ESI
ꢀve): 284 (55%, [MꢀH]ꢀ), 320 (93%, [M+35Cl]ꢀ), 322 (34%,
[M+37Cl]ꢀ), 330 (73%, [M+HCOO]ꢀ), 569 (100%, [2MꢀH]ꢀ). Found:
C, 54.61; H, 6.83; N, 4.80. C13H19NO6 requires: C, 54.73; H, 6.71;
N 4.91%.
126.6, 127.5, 131.8 (ArCH), 130.4 (ArCC), 144.9 (ArCNO2); HRMS
(ESI): found 338.0851 [M+Na]+; C13H17NNaO8
requires
Acknowledgments
+
338.0852; EIMS (probe) eV, m/z (rel. int.): 338 (100%, [M+Na]+),
653 (85%, [2 M+Na]+); (ESI ꢀve): 314 (45%, [MꢀH]ꢀ), 360 (95%,
[M+HCOO]ꢀ), 629 (100%, [2 MꢀH]ꢀ).
This work was supported by the Newton Abraham Research
Fund (B.J.A.), the Leverhulme Trust (G.W.J.F.) and a Grant-in-Aid
for Scientific Research (C) (No: 23590127) (A.K.) from the Japanese
Society for the Promotion of Science (JSPS).
o-Aminobenzyl b-
D-glucopyranoside [Iteamine] 1
o-Nitrobenzyl b-
D-galactopyranoside 12 (40 mg, 0.17 mmol)
was dissolved in ethanol (5 mL) and palladium (10% on C, 4 mg)
added. The vessel was evacuated and flushed three times with ar-
gon and three times with hydrogen. The reaction mixture was stir-
red for 20 min, after which TLC analysis (4:1, ethyl acetate/
methanol) indicated the complete consumption of starting mate-
rial (Rf 0.40) and the formation of a single product (Rf 0.30). The
reaction mixture was filtered, concentrated in vacuo and the crude
residue loaded onto a short column of DowexÒ (50W-X8, H+). The
resin was washed with water, the product liberated with aqueous
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
01.012.
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