W.H. Perera et al.
PhytochemistryLetters25(2018)163–170
acquired by direct infusion using an Agilent Model G6530A quadrupole
time of flight mass detector equipped with an electrospray ionization
interface and was controlled by Agilent software (Agilent MassHunter
Work Station, A.05.00). All acquisitions were performed under negative
ionization mode with a capillary voltage of 3500 V. N2 was used as
nebulizer gas (30 psig), as well as drying gas at 11 L/min at drying gas
temperature of 325 °C. The voltage of PMT, fragmentor and skimmer
was set at 750 V, 100 V and 65 V respectively. Full scan mass spectra
were acquired from m/z 100–1700. Data acquisition and processing
was done using the MassHunter Workstation software (Qualitative
Analysis Version B.07.00).
1D and 2D spectra (COSY, gHSQC and gHMBC) were recorded on a
Bruker Avance 500 NMR spectrometer at 298 K, operating at
500.13 MHz for 1H and 125.75 MHz for 13C using Pyridine-d5. The
chemical shifts are given in δ (ppm) and were calibrated using the re-
sidual solvent signals.
unique data and 239 refined parameters. The Flack parameter is
A very small, lower-quality twinned crystal of a mixed water-me-
thanol solvate of endo-steviolbioside 6 was used to confirm its con-
nectivity and relative configurations. Data were collected at T = 100 K
on a Bruker Kappa APEX-II DUO CCD diffractometer, equipped with a
Cu microfocus X-ray source. Structure solution and refinement was
carried out as for 3, except that disordered solvent was removed using
the SQUEEZE procedure, water H atoms could not be located, and OH
hydrogen atoms were not refined. Crystal data: C32H50O13. H2O,
Mr = 705.27, monoclinic space group P21, a = 12.025(2),
b = 7.7676(13), c = 18.882(13) Å, β = 90.597(12)°, V = 1763.6(6) Å3,
Z = 2, Dx = 1.328 Mg m−3, θmax = 59.2°, R = 0.110 for 4153 observed
(of 6542 unique) data and 423 refined parameters.
Supplementary crystallographic data for
3 are contained in
Cambridge Structural Database deposition CCDC-1539128, and for 6
CCDC-1539676. This data can be obtained free of charge via www.ccdc.
or
from
the
Cambridge
2.6. Physicochemical parameters of the new compounds
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK;
2.6.1. 13-ent-kaur-15-en-19-oic acid (Endocyclic steviol isomer) (3)
Crystalline off-white solid; [α]2D5 −60.0 (c 0.1, MeOH), melting
point 198–202 °C HRESIMS/MS m/z 317.2124 [M−H]− (calculated for
3. Results and discussion
C
20H30O3, 317.2122). 1H and 13C NMR spectroscopic data are sum-
Vigorous acid hydrolysis of rebaudioside A yielded three main
aglycones. Compound 1 showed a HRESIMS spectrum with a deproto-
nated molecular ion at m/z 317.2128 [M−H]− (calculated for
2.6.2. 13-[(β-D-glucopyranosyl)oxy]ent-kaur-15-en-19-oic acid (Endo-
steviolmonoside) (4)
C
20H30O3, 317.2122). This compound was isolated in gram quantities
Crystalline white solid; [α]D25 −58.0 (c 0.1, MeOH), melting point
and identified as isosteviol (4.2 g, 21% of rebaudioside A starting
weight). Compound 2 was purified as the minor compound of the
aglycone mixture, showing a HRESIMS spectrum with a deprotonated
molecular ion at m/z 317.2122 [M−H]− (calculated for C20H30O3,
317.2122) and was identified as steviol (46 mg, 0.23% of rebaudioside
A starting weight), the aglycone core of the tetracyclic diterpene gly-
cosides of S. rebaudiana. Chemical shifts of both compounds were as-
signed based on NMR experiments and by comparison with carbon
chemical shifts reported in literature for methyl ester derivatives.
Compound 3 was isolated from the mixture of reaction products
(438 mg, 2.19% of rebaudioside A starting weight) displaying a de-
protonated molecular ion at m/z 317.2124 [M−H]− (calculated for
101–102 °C. HRESIMS/MS m/z 479.2639 [M−H]− (calculated for
C
26H40O8, 479.2650), m/z 317.2027 (collision energy, 40 eV; intensity,
41%) (−162 Da), loss of one hexose. 1H and 13C NMR spectroscopic
2.6.3. 13-[(3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-15-
en-19-oic acid (Endo-rebaudioside G1) (5)
Crystalline white solid; [α]D25 −52.0 (c 0.1, MeOH), melting point
196–197 °C. HRESIMS/MS 641.3196 [M−H]− (calculated for
C
32H50O13, 641.3179), m/z 479.2653 (94.8%) and m/z 317.2120
(14.9%) at collision energy, 40 eV; (−2 × 162 Da), loss of two hexoses.
1H and 13C NMR spectroscopic data are summarized in Table 1.
C
20H30O3, 317.2122) and identified by 1D and 2D NMR experiments as
the endo isomer of steviol (13-ent-kaur-15-en-19-oic acid), Table 1.
Chemical structures of compounds 1-3 are shown in Fig. 1. We were
fortunate to grow high quality crystals from compound 3 allowing X-
Ray structural confirmation (Fig. 2).
2.6.4. 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-15-
en-19-oic acid (Endo-steviolbioside) (6)
Crystalline white solid; [α]D25 −54.0 (c 0.1, MeOH), melting point
192–193 °C. HRESIMS/MS 641.3166 [M−H]− (calculated for
Compound 3 was previously obtained after vigorous acid hydrolysis
mixture using chromatography (silica gel modified with 10% AgNO3)
after preparation of their methyl esters. Herein, we were able to sepa-
rate the three isomers underivatized using a two steps gradient in a high
2014). Isosteviol is the main byproduct from vigorous acid hydrolysis of
rebaudioside A followed by the endocyclic steviol isomer and in a very
low yield steviol. Most probably, the same three aglycones in similar
ratios should be found after hydrolysis of other steviol glycosides. Acid
hydrolysis is not a convenient approach to produce steviol in quantities
since it is the minor product of the reaction.
Additionally, acid hydrolysis of rebaudioside A (28 g) and stevioside
(21 g) were both set at room temperature and at 40 °C (1 N HCl). No
hint of degradation products under mild acidic conditions was observed
at room temperature after one week. However, several degradation
products were observed at 40 °C after 24 h and 66 h for rebaudioside A
and 20 h for stevioside. RP-C18 HPLC chromatograms of rebaudioside A
sampling after acid hydrolysis are shown in Fig. 3.
C
32H50O13, 641.3179), m/z 479.2633 (58.7%) and m/z 317.2121
(10.5%) at collision energy, 40 eV; (−2 × 162 Da), loss of two hexoses.
1H and 13C NMR spectroscopic data are summarized in Table 1.
2.7. X-ray crystallography of compound (3) and (6)
The crystal structure of the endocyclic steviol isomer 3 was de-
termined from a colorless crystal of the methanol solvate having di-
mensions 0.06 × 0.11 × 0.33 mm, using data collected at T = 100 K
with MoKα radiation on a Bruker Kappa APEX-II DUO CCD dif-
fractometer, equipped with an Oxford Cryostream cooler. The structure
was solved using the program SHELXS-97 and refined anisotropically
by full-matrix least squares on F2 using SHELXL2014/7 (Sheldrick,
2008). H atoms were visible in difference maps, but were placed in
idealized positions for the refinement, except for those of OH groups,
which were refined. The absolute configuration was assigned from
starting materials and is consistent with the Flack (1983) parameter
based on resonant scattering of the light atoms. Crystal data: C20H30O3
CH3OH, Mr = 350.48, orthorhombic space group P212121,
a = 7.4846(2), b = 14.5254(4), c = 17.4806(4) Å, V = 1900.44(8) Å3,
Mild acid hydrolysis of rebaudioside A at 40 °C at 24 h showed a
mixture of rebaudioside A/Endo-rebaudioside A and rebaudioside B/
Z = 4, Dx = 1.225 Mg m−3
, θmax = 30.5°, R = 0.037 for all 5813
166