Tetra-glucopyranosyl diterpene ent-Kaur-16-en-19-oic Acid and ent-13(S)-hydroxyatisenoic acid derivatives from a commercial extract of stevia rebaudiana (bertoni) bertoni

Article abstract of DOI:10.3390/molecules23123328

Stevia rebaudiana and its diterpene glycosides are one of the main focuses of food companies interested in developing novel zero calorie sugar substitutes since the recognition of steviol glycosides as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Rebaudioside A, one of the major steviol glycosides of the leaves is more than 200 times sweeter than sucrose. However, its lingering aftertaste makes it less attractive as a table-top sweetener, despite its human health benefits. Herein, we report the purification of two novel tetra-glucopyranosyl diterpene glycosides 1 and 3 (rebaudioside A isomers) from a commercial Stevia rebaudiana leaf extract compounds, their saponification products compounds 2 and 4, together with three known compounds isolated in gram quantities. Compound 1 was determined to be 13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid-β-D-glucopyranosy ester (rebaudioside Z), whereas compound 3 was found to be 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid -β-D-glucopyranosy ester. Two new tetracyclic derivatives with no sugar at position C-19 were prepared from rebaudiosides 1 and 3 under mild alkaline hydrolysis to afford compounds 2 13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid (rebaudioside Z₁) and 4 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid. Three known compounds were purified in gram quantities and identified as rebaudiosides A (5), H (6) and J (7). Chemical structures were unambiguously elucidated using different approaches, namely HRESIMS, HRESI-MS/MS, and 1D-and 2D-NMR spectroscopic data. Additionally, a high-quality crystal of iso-stevioside was grown in methanol and its structure confirmed by X-ray diffraction.

Full text of DOI:10.3390/molecules23123328

molecules
Article
Tetra-glucopyranosyl Diterpene ent-Kaur-16-en-19-oic
Acid and ent-13(S)-Hydroxyatisenoic Acid Derivatives
from a Commercial Extract of Stevia rebaudiana
(Bertoni) Bertoni
Wilmer H. Perera 1 , Ion Ghiviriga , Douglas L. Rodenburg , Kamilla Alves ,
,2
3
1
1
4
4,†
5
4,6
Frank T. Wiggers , Charles D. Hufford , Frank R. Fronczek
Ilias Muhammad , Bharathi Avula , Ikhlas A. Khan and James D. McChesney 1,*
, Mohamed A. Ibrahim
,
4
4
4
1
Ironstone Separations, Inc., Etta, Oxford, MS 38627, USA; wilmer.perera@gmail.com (W.H.P.);
douglasrodenburg@yahoo.com (D.L.R.); Kamilla_07@yahoo.com (K.A.)
ORISE Fellow-Agricultural Research Service, Natural Product Utilization Research Unit,
2
U.S. Department of Agriculture, University of Mississippi, Oxford, MS 38677, USA
Department of Chemistry, University of Florida, Gainesville, FL 32611, USA; ion@chem.ufl.edu
3
4
National Center for Natural Products Research, University of Mississippi, Oxford, MS 38677, USA;
fwiggers@olemiss.edu (F.T.W.); mmibrahi@olemiss.edu (M.A.I.); milias@olemiss.edu (I.M.);
bavula@olemiss.edu (B.A.); ikhan@olemiss.edu (I.A.K.)
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA; ffroncz@lsu.edu
Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Division,
National Research Centre, Dokki, Giza 12622, Egypt
5
6
*
Correspondence: jdmcchesney@yahoo.com; Tel.: +(303)808-4104
†
Charles D. Hufford deceased on 15 May 2017.
Academic Editors: Muhammad Ilias and Charles L. Cantrell
Received: 29 November 2018; Accepted: 14 December 2018; Published: 15 December 2018
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Stevia rebaudiana and its diterpene glycosides are one of the main focuses of food
companies interested in developing novel zero calorie sugar substitutes since the recognition of
steviol glycosides as Generally Recognized as Safe (GRAS) by the United States Food and Drug
Administration. Rebaudioside A, one of the major steviol glycosides of the leaves is more than
2
00 times sweeter than sucrose. However, its lingering aftertaste makes it less attractive as a
table-top sweetener, despite its human health benefits. Herein, we report the purification of
two novel tetra-glucopyranosyl diterpene glycosides and (rebaudioside A isomers) from a
commercial Stevia rebaudiana leaf extract compounds, their saponification products compounds
and , together with three known compounds isolated in gram quantities. Compound
was determined to be 13-[(2-O- -D-glucopyranosyl-6-O- -D-glucopyranosyl- -D-glucopyranosyl)
oxy]ent-kaur-16-en-19-oic acid- -D-glucopyranosy ester (rebaudioside Z), whereas compound
was found to be 13-[(2-O- -D-glucopyranosyl-3-O- -D-glucopyranosyl- -D-glucopyranosyl)
oxy]ent-hydroxyatis-16-en-19-oic acid - -D-glucopyranosy ester. Two new tetracyclic derivatives with
no sugar at position C-19 were prepared from rebaudiosides and under mild alkaline hydrolysis to
afford compounds 13-[(2-O- -D-glucopyranosyl-6-O- -D-glucopyranosyl- -D-glucopyranosyl)
oxy]ent-kaur-16-en-19-oic acid (rebaudioside Z ) and 13-[(2-O- -D-glucopyranosyl-3-O- -D-
glucopyranosyl-β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid. Three known compounds
were purified in gram quantities and identified as rebaudiosides A ( ), H ( ) and J ( ).
1
3
2
4
1
β
β
β
β
3
β
β
β
β
1
3
2
β
β
β
4
β
β
1
5
6
7
Chemical structures were unambiguously elucidated using different approaches, namely HRESIMS,
HRESI-MS/MS, and 1D-and 2D-NMR spectroscopic data. Additionally, a high-quality crystal of
iso-stevioside was grown in methanol and its structure confirmed by X-ray diffraction.
Molecules 2018, 23, 3328; doi:10.3390/molecules23123328
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3328
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Keywords: Stevia rebaudiana; diterpene glycosides; rebaudioside A isomers; 13(S)-hydroxyatisenoic
acid derivative; iso-stevioside X-ray structure
1
. Introduction
Diabetes is a chronic disease that affected 422 million of people worldwide in 2014 and is
increasing considerably every year. Diabetes is characterized by insulin deficit or insulin insensitivity
and consequently produces high blood sugar levels which are associated with other disorders [ ].
1,2
Reducing or eliminating sugar consumption and replacing sucrose with healthier sweeteners is one
approach to prevent and control diabetes.
Stevia rebaudiana (Bertoni) Bertoni and its steviol glycosides were conferred Generally Recognized
as Safe status by the United States Food and Drug Administration in 2008 and in the European Union
in 2011. Steviol glycosides from this Asteraceae plant have been gaining interest from companies and
the general public since these natural compounds are calorie free sweeteners and have shown higher
sweetener potency than sucrose [3,4]. Additionally, it was recently suggested that rebaudioside
2+
A potentiates the activity of a Ca cation channel expressed in type II taste receptor cells and
pancreatic -cells (TRPM5) enhancing glucose-induced insulin secretion in a dependent manner.
However, regardless of the potential of rebaudioside A as a sugar substitute to prevent and treat type 2
diabetes [ ], the lingering aftertaste of this tetra-glucopyranosyl sugar substitute makes it unattractive
to consumers.
In our continuing efforts to discover novel sugar substitutes with potential refined organoleptic
β
5
properties, we describe herein the isolation and structure elucidation of two new tetra-glucopyranosyl
diterpene glycosides, the preparation of their saponification products together with the isolation of
three known compounds in gram quantities.
2
. Results and Discussion
Structure Elucidation
Two new compounds were isolated from a commercial extract of Stevia rebaudiana using
reversed-phase and high performance normal-phase chromatography [6]. Recently, two approaches
have been described for a rapid detection of novel oligosaccharide arrangements linked at position
C-13 in steviol glycosides and infer their C-19 linkage. One is based on tandem mass spectrometry
dissociation patterns with ranging collision energies [7]. Thus, steviol glycosides with one
monosaccharide unit or a less hindered disaccharide linked at C-19 (e.g., rebaudiosides I and U
with 1-3 and 1-6 linkages, respectively) cleave the C-19 ester linkage with low collision energies (10 eV)
while more hindered disaccharides (1-2 linkages) cleave with higher collision energies (40 eV).
2
5
Compound
1
was purified as an amorphous off-white solid with [
showed a molecular ion at m/z 965.4208
H] ), suggesting a molecular formula C H O . Both,
α
]
−
28.0 (c 0.1, MeOH).
D
HR-ESIMS and HR-ESIMS/MS data of compound
1
−
−
[
M − H] (calculated m/z 965.4235 [M
−
44
70 23
the deprotonated molecular ion and an intense product ion m/z = 803.3703 Da resulted from the loss
−
of one hexose at C-19 ([M
three hexoses ([M H]
−
H]
H O – 3
2
−
H O
×
−
162 Da) were observed at 10 eV. Further sequential loss of
162 Da) m/z = 641.3185, m/z = 479.2635 and m/z = 317.2146
2
−
−
−
from the C-13 moiety was observed at 70 eV. Acid hydrolysis of
and only D-glucose which was identified by comparison of the HPLC retention times of thiocarbamoyl
thiazolidine derivatives prepared from sugar standards as previously described [ ]. Hence compound
is a rebaudioside A isomer.
The second approach was based on comparing retention times of a pure steviol glycoside and its
saponification product with those reported by RP-C18 HPLC [10]. Saponification condition was helpful
to detect single monosaccharide, di and oligosaccharides linked at position C-19. Thus, compound
1 furnished a mixture of aglycones
8,9
1
1

Molecules 2018, 23, 3328
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showed a retention time of 3.94 min that did not match with any previously reported steviol glycosides
in a RP-C18 HPLC method [10]. The aglycone-C13 moiety was produced by mild alkaline hydrolysis
corroborating the linkage of a single sugar at position C-19 (9.32 min) as deduced by MS. The aglycone
with the C-13 portion showed an HPLC retention time that did not match with any of the steviol
glycosides reported with a free carboxylic acid at C-19, suggesting the structural novelty of this
compound, probably, in the C-13-oligosaccharide.
The assignments of the signals of the steviol aglycone started by identifying H-18 as the methyl
protons coupling with C-19, which is the most deshielded carbon. H-18 also couples with a quaternary
carbon, C-4, with methylene carbon, C-3, and with a methine carbon, C-5. The sequence H-3–H-2–H-1
could be followed in the COSY spectrum, and the protons on ring A assigned as axial or equatorial
based on the number of large couplings. Both C-1 and C-5 couple with methyl protons at position 20,
which also couples with a quaternary carbon, C-10 and with a methine carbon, C-9. The sequences
H-6—H-7—H-9 and H-9—H-11—H-12 could be followed in the COSY spectrum. The C/D rings were
supported by the HMBC correlations of H-9 with C-11, C-12, C-14 and C-15; H-14 with C-13, C-15 and
C-16; and H-17 with C-13, C-15 and C-16.
We also observed four anomeric protons at
information, all of them showing beta linkages.
δ 5.14, 5.19, 5.30, and 6.12 ppm supporting the MS
The positions of attachment of the sugar moieties were established based on the 2D-NMR
3
0
δ
HMBC spectrum. J HMBC correlations between anomeric proton H-1 (
6.12 ppm) and C-19
0
(δ
177.7 ppm) confirmed the attachment of one glucose unit at C-19. The position of H-2 was
0
0
confirmed through the COSY correlation between H-2 (
δ
4.17 ppm) and H-1 (
5.19 ppm) and C-13 ( 86.4 ppm) confirmed
the attachment of the first glucose unit at C-13. The connections of other two glucoses were
δ 6.12 ppm).
3
00
δ
Additionally, the J HMBC correlations between H-1 (
δ
3
000
0000
also established through J HMBC correlations between H-1
84.4 ppm) and C-6
H-2 was confirmed through the gDQCOSY correlation between H-2
5.19 ppm). The positions of H-2 of the sugars linked at position C-13 were confirmed through
their COSY correlations between H-2 (
(
δ
5.30 ppm), H-1 (δ
70.3 ppm) respectively. In the same way, the position of
5.14 ppm)
0
0
00
(δ
with C-2
(
δ
0
0
00
0
00
(
δ
4.23 ppm) and H-1
(δ
0
0
00
000
δ
4.23 ppm) and H-1 (
δ
5.19 ppm), H-2 (
5.14 ppm). Compound
-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic
δ
4.11 ppm) and
was named as
0
00
0000
0000
H-1
3-[(2-O-
acid-
(
δ
5.30 ppm) and H-2
-D-glucopyranosyl-6-O-
(
δ
4.06 ppm) and H-1
-D-glucopyranosyl-
(
δ
1
1
β
β
β
1
β
-D-glucopyranosy ester (rebaudioside Z) and assigned the structure shown in Figure 1. H- and
C-NMR chemical shifts are shown in Tables 1 and 2, respectively, and are typical signals of the
ent-kaurene core [11].
13
1
Table 1. H chemical shifts of diterpene glycosides 1–4.
1 a
2 b
3 b
δ (ppm) c
4 b
δ (ppm) c
Moiety
Aglycone
Position
δ (ppm)
δ (ppm)
1
2
3
5
6
7
9
0.73, 1.73
2.20, 1.43
2.28, 1.79
1.03
1.91, 2.50
1.28, 1.28
0.87
0.95, 2.13
1.96, 1.37
1.97, 1.54
1.01
1.87, 1.87
1.52; 1.40
0.96
0.92, 1.59
1.92, 1.40
2.20, 1.09
1.12
1.78, 2.05
1.57, 1.16
1.09
0.85, 1.56
2.01, 1.30
2.14, 0.88
0.90
1.80, 1.98
1.53, 1.13
1.04
1
1
1
1
1
1
1
2
1
2
3
4
5
7
8
0
1.68, 1.68
1.02, 2.35
-
1.97, 2.74
2.06, 2.06
5.10, 5.73
1.23
1.79,1.63
0.83, 1.86
-
1.51, 2.27
2.05, 2.10
4.85, 5.24
1.14
1.63, 1.45
2.51
1.60, 1.44
2.49
4.04
4.05
1.15, 2.51
1.89, 2.11
4.70, 4.84
1.24
1.14, 2.51
1.86, 2.08
4.68,4.82
1.11
1.31
1.03
0.89
0.95

Molecules 2018, 23, 3328
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Table 1. Cont.
1 a
2 b
3 b
δ (ppm) c
4 b
δ (ppm) c
Moiety
Glcβ-C19
Position
δ (ppm)
δ (ppm)
0
1
2
3
4
6.12
4.17
3.98
4.33
4.22
-
-
-
-
-
-
5.43
3.39
3.43
3.39
3.39
-
-
-
-
-
-
0
0
0
0
5
0
6
4.43, 4.57
3.85, 3.71
0
0
0
0
0
0
0
0
0
0
0
0
Glc-C13
1
2
3
4
5
6
5.19
4.23
4.27
4.45
4.08
4.59
3.60
3.54
3.35
3.17
4.56
3.65
3.71
3.38
3.34
4.55
3.64
3.71
3.42
3.30
4.51, 4.78
3.78, 4.10
3.91, 3.68
3.85, 3.70
0
00
00
00
00
00
00
Glc(1–2)
1
2
3
4
5
6
5.30
4.11
4.23
4.28
3.91
4.62
3.30
3.46
3.35
3.36
4.81
3.19
3.35
3.22
3.33
4.81
3.18
3.34
3.21
3.30
0
0
0
0
0
4.22, 4.57
3.68, 3.82
3.87, 3.66
3.85, 3.65
Glcβ(1-X) d
1
2
3
4
5
6
0000
5.14
4.06
4.27
4.12
4.03
4.61
3.21
3.46
3.35
3.36
4.65
3.28
3.39
3.31
3.37
4.65
3.27
3.38
3.33
3.36
0
0
0
0
0
000
000
000
000
000
4.22, 4.57
3.68, 3.82
3.90, 3.66
3.89, 3.65
(
1
)
rebaudioside Z;
D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid-
-O- -D-glucopyranosyl- -D-glucopyranosyl)oxy]ent-hydroxyatis-16-en-19-oic acid. All the sugars showed
4
linkage evidenced for the coupling constants of the anomeric protons (7.6 and 8 Hz), in Pyr-d in MeOH-d ,
(
2
)
rebaudioside
Z
1
;
(
3
)
13-[(2-O-
-D-glucopyranosy ester; (
β
-D-glucopyranosyl-3-O-
β
-D-glucopyranosyl-
β-
β
4) 13-[(2-O-
β-D-glucopyranosyl-
3
β
β
β
a
b
5
,
c
diastereotopic protons in the aglycone are given in the order pro-R, pro-S. Protons in position 6 of glucose were not
assigned sterically. Protons in position 17 are given in the order pro-E, pro-Z. d X = 6 for compound
compound 3 and 4.
1 and 2; X = 3 for
1
2
: R = Glcβ
3: R = Glcβ
5: R = H; R = Glcβ-
1 2
1
1
: R = H
4: R = H
6: R = H; R = Glcβ(1-3)Rhaα-
1 2
1
1
7
: R = Rhaα-; R = Glcβ-
1 2
Figure 1. Chemical structures of purified compounds rebaudioside Z (1) and 13-[(2-O-
glucopyranosyl-3-O- -D-glucopyranosyl- -D-glucopyranosyl)oxy]ent-hydroxyatis-16-en-19-oic acid-
-D-glucopyranosy ester ( ) and chemically modified rebaudioside Z1 ) and 13-[(2-O- -D-
-D-glucopyranosyl- -D-glucopyranosyl) oxy]ent-hydroxyatis- 16-en-19-oic acid
β-D-
β
β
β
3
(2
β
glucopyranosyl- 3-O-
β
β
(
4), rebaudioside A (5), rebaudioside H (6) and rebaudioside J (7).

Molecules 2018, 23, 3328
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Table 2. 13C chemical shifts of diterpene glycosides 1–4.
1 a
2 b
3 b
4 b
Moiety
Position
δ (ppm)
δ (ppm)
δ (ppm)
δ (ppm)
Aglycone
1
2
3
4
5
6
7
8
9
41.2
19.9
36.8
44.5
57.8
22.7
42.2
43.3
54.3
40.3
21.1
38.9
86.4
45.1
48.0
155.0
105.4
28.8
177.7
16.0
40.1
20.9
39.0
45.6
58.7
23.7
43.1
43.1
55.3
40.9
21.5
42.6
88.3
45.8
48.8
154.2
105.7
30.1
184.1
17.3
39.5
18.4
37.7
43.7
57.1
19.9
38.8
33.9
51.0
38.1
26.3
41.0
77.8
37.7
47.4
146.7
108.0
27.5
176.8
11.9
40.5
19.3
39.4
44.7
57.6
20.6
39.4
33.8
51.2
38.0
26.3
41.0
77.8
37.7
47.7
147.0
107.8
29.1
184.4
12.2
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
0
Glcβ-C19
Glcβ-C13
Glcβ(1-2)
Glcβ(1-X)
1
2
3
4
5
6
96.3
74.4
79.5
71.4
79.9
62.6
-
-
-
-
-
-
94.2
72.6
77.3
69.7
77.3
61.0
-
-
-
-
-
-
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
98.3
84.4
78.6
71.8
77.8
70.3
97.7
81.8
78.5
71.8
77.8
70.0
100.5
79.0
86.0
68.8
76.1
61.4
100.3
78.8
85.9
68.6
76.1
61.2
0
0
0
0
0
0
00
00
00
00
00
00
1
2
3
4
5
6
106.8
75.7
78.8
72.1
78.3
63.3
104.8
76.3
77.8
71.4
77.9
62.8
102.2
74.6
76.5
70.6
76.6
61.9
102.1
74.5
76.5
70.6
76.6
61.8
0
0
0
0
0
0
000
000
000
000
000
000
1
2
3
4
5
6
105.9
77.2
78.9
72.6
78.5
63.3
104.6
75.3
77.9
71.7
78.3
62.8
103.1
73.9
76.8
70.1
76.8
61.2
103.1
73.9
76.8
70.1
76.8
61.1
0
0
0
0
0
(
1) Rebaudioside Z; (2) rebaudioside Z
1
;
(3) 13-[(2-O-
-D-glucopyranosy ester; (4) 13-[(2-O-
-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid. NMR spectra recorded in
β
-D-glucopyranosyl-3-O-
β
-D-glucopyranosyl-
β-D-
glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid-
β
β-D-glucopyranosyl-
3
-O-
β
-D-glucopyranosyl-
β
a
b
Pyr-d5 and MeOH-d4. X = 6 for compound 1 and 2; X = 3 for compound 3 and 4.
Compound
B isomer with a Glc
the RP-C18 HPLC method that did not match with any of the previously reported steviol-C13
oligosaccharides [10]. HRESIMS/MS experiment of compound showed a molecular ion at m/z
H] ), suggesting a molecular formula C H O .
2
was prepared through mild alkaline hydrolysis of
1 to afford a new rebaudioside
β
(1-6)[Glc (1-2)]Glc 1- arrangement at C-13 with retention of 9.32 min in
β
β
2
−
−
8
03.3721 [M
−
H] (calculated m/z 803.3702 [M
−
38
60 18

Molecules 2018, 23, 3328
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A sequential loss of three glucoses units at C-13 portion were observed at 70 eV collision energy:
m/z 641.3196 ( 162 Da), m/z 479.2648 (
162 Da) and m/z 317.2115 Da. 1H-NMR showed three
anomeric protons corresponding to the oligosaccharide portion linked at position C-13 ( 4.59;
−
−
δ
H
4
.61 and 4.62 ppm). The free carboxylic acid at position C-19 was evidenced by the signal at 184.1
13
ppm in the C-NMR, less shielded than the observed for compound
was performed using 1D- and 2D-NMR (DQCOSY, HSQC and HMBC) experiments. Compound
was named as 13-[(2-O- -D-glucopyranosyl-6-O- -D-glucopyranosyl- -D-glucopyranosyl)
oxy]ent-kaur-16-en-19-oic acid (rebaudioside Z ). H and C chemical shifts are presented in Table 1.
1. Full structural assignment
2
β
β
β
1
13
1
2
5
Compound
3
was purified as an amorphous off-white solid with [
α
]
−
20.0 (c 0.1, MeOH).
D
−
HR-ESIMS/MS data of compound
m/z 965.4235 [M
molecular ion and an intense product ion m/z = 803.3735 Da resultant from the loss of one hexose
3
showed a molecular ion at m/z 965.4297 [M
H] ), suggesting a molecular formula C H O . Both, the deprotonated
−
H] (calculated
−
−
44
70 23
−
at C-19 [M – H − H O − 162 Da] were observed at 10 eV. Further sequential loss of three hexoses
2
−
[
M – H − H O – 3 × 162 Da] m/z = 641.3195, m/z = 479.27.0 and m/z = 317.2141 from C-13 moiety
2
was observed at 70 eV. Compound 3 showed a HPLC retention time of 4.35 min that did not match with
any diterpene glycosides in the RP-C18 method previously reported [10]. It was hydrolyzed under
mild alkaline conditions corroborating the presence of a single monosaccharide attached at position
C-19. Retention time of the saponification product showed a peak at 10.5 min that did not match with
any aglycone-C13 previously described [10]. The rapid elution of compound
3, <7 min, suggested
that it is a highly substituted glycoside with a single sugar unit linked at C-19. Acid hydrolysis of
3
furnished a mixture of aglycones and only D-glucose which was identified by comparison of the HPLC
retention times of thiocarbamoyl thiazolidine derivatives prepared from sugar standards, as previously
described [8,9].
The aglycone signals were similar to those reported for steviol aglycone in compounds
except for 2.51 and 4.04 but signals were in good agreement with those reported
for a similar aglycone previously found in Stevia eupatoria (Spreng.) Willd. and reported as
1
and
2
δ
H
δ
H
1
13
1 α-hydroxy-ent-kaur-16-en-19-oic acid based on the H- and C-NMR data together with chemical
2-
modifications [12]. Additionally, a triglucopyranosyl derivative isolated from S. rebaudiana (stevioside
isomer) was also reported and was suggested a similar aglycone 12- -hydroxy-ent-kaur-16-en-19-oic
α
acid by comparison with previously reported data [13]. More recent NMR techniques allowed us to
assign the aglycone as 13(S)-hydroxyastinoic acid. This type of aglycone was previously reported
by converting microbiologically diterpene acids from Helianthus sp. with Gibberella fujikuroi [14].
The structure of the aglycone in 3 was inferred as described in Figure 2. The COSY spectrum reveals
the sequence H-9–H-11ab–H-12. The coupling of H-17 with C-12 and C-15, seen in the HMBC spectrum,
set fragment a. Coupling of H-15 with both C-8 and C-9 indicated that C-15 is bonded to C-8, as in
fragment b. Couplings between the proton on the carbon carrying the oxygen (H-13, 4.04 ppm) with
H-12 and H-14 were seen in the COSY spectrum, so they are vicinal, also corroborated for the HMBC
correlation between H-13 and C-16. Protons in position 14 were identified by their coupling with
H-13. They couple with C-8, C-9 and C-15, and therefore C-14 is bonded to C-8, as in fragment
c. H-9 displayed a large coupling with C-14 and C-15, and the stereochemistry of fragment d was
inferred. The stereochemistry of C-13 was assigned as S because of the large coupling of H-13 with
C-16. The assignment of the diastereotopic protons was based on the large couplings between H-14
pro-R and C9, H-14 pro-S and C-15, H-15 pro-S and C-9, H-15 pro-R and C-14, H-11 pro-S and C-16,
H-11 pro-R and C-13. A ROESY spectrum of compound
are very similar to those in , confirmed this stereochemistry and these assignments, as it displayed
nOes of H-20 with H-13 and H-14 pro-R, and of H-14 pro-R and H-11 pro-R.
4, in which the chemical shifts of the aglycone
3

Molecules 2018, 23, 3328
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Figure 2. Structure elucidation for the 13(S)-hydroxyatisenoic acid. The arrows indicate cross-peaks in
the HMBC spectrum between protons at the start position and carbons at the end position.
Four anomeric protons at
MS information, all of them showing beta linkages. H and C chemical shifts are shown in Tables 1
δ 4.56, 4.65, 4.81, and 5.43 ppm were also observed supporting the
H
1
13
and 2, respectively. The positions of attachment of the sugar moieties were established based on
3
the HMBC spectrum. J HMBC correlations between anomeric proton H-1’ (
δ
5.43 ppm) and C-19
(
δ 176.8 ppm) confirmed the attachment of one glucose unit at C-19. The order of the protons in a
sugar starting from the anomeric position was seen in the TOCSY spectra with increasing mixing times
3
00
δ
(
Supporting Information). Additionally, the J HMBC correlations between H-1 (
C-13 ( 77.8 ppm) confirmed the attachment of the first glucose unit at C-13. The connections of the
other two glucoses were also established through J HMBC correlations between H-1 4.81 ppm),
4.56 ppm) and
δ
3
000
(δ
0
000
00
00
δ
H-1
(
δ
4.65 ppm) with C-2 (
δ
79.0 ppm) and C-3 (
86.0 ppm) respectively. In the same way,
0
0
00
position of H-2 was confirmed through the gDQCOSY correlation between H-2 (
δ
3.64 ppm) and
0
0
H-1 (δ 4.56 ppm). The order of the protons in a sugar starting from the anomeric position was seen in
the TOCSY spectra with increasing mixing times, (Supporting Information). Compound was named
3-[(2-O- -D-glucopyranosyl-3-O- -D-glucopyranosyl- -D-glucopyranosyl)oxy]ent-hydroxyatis-16-
en-19-oic acid-β-D-glucopyranosy ester and assigned structurally as shown in Figure 1.
3
1
β
β
β
Compound
4
was prepared through alkaline hydrolysis of
3 to afford a rebaudioside B isomer
with a with a Glc
β
(1-3)[Glc (1-2)]Glc 1- arrangement at C-13 with retention of 10.5 min that did not
β
β
match with any of the previously reported diterpene glycosides with a free carboxylic acid previously
reported in the RP-C18 HPLC method [10]. HRESIMS/MS experiment of compound showed a
H] ), suggesting a molecular
formula C H O . A sequential loss of three glucoses units at C-13 portion at collision energy of
4
−
−
molecular ion at m/z 803.3682 [M
−
H] (calculated m/z 803.3702 [M
−
38
60 18
1
7
0 eV was observed: m/z 641.3162 (
showed three anomeric protons corresponding to the oligosaccharide portion linked at position
C-13 ( 4.55; 4.65 and 4.81 ppm). The free carboxylic acid at position C-19 was evidenced by the
−
162 Da), m/z 479.2703 (
−
162 Da) and m/z 317.2111 Da. H-NMR
δ
H
13
signal at 184.4 ppm in the C-NMR, less shielded than that observed for compound
assignment was performed using 1D- and 2D-NMR experiments. Compound
3-[(2-O- -D-glucopyranosyl-3-O- -D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-hydroxyatis-16-
3
4
. Full structural
was named as
1
β
β
1
13
en-19-oic acid. H- and C-NMR chemical shifts are presented in Table 1. Additionally, glycosylation
−
sites, sugar arrangements, retention times and [M
isomers were presented in Table 3.
−
H] of compounds 1-5 including other related

Molecules 2018, 23, 3328
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Table 3. Glycosylation sites, sugar arrangements, HPLC retention times, molecular weights and type of aglycones of rebaudioside A and B isomers.
Glycosylation Sites
C-13 (R2)
RT (min) a
[M − H]− (m/z)
Experimental
DG
Agly b
C-12 (R2)
C-19 (R1)
DG
Rebaudioside Z (1)
Compound 3
Rebaudioside A
Iso-rebaudioside A
-CH2
-CH
-CH2
-CH2
-CH2
-CH
Glcβ(1-2)[Glcβ(1-6)]-Glcβ1-
Glcβ1-
Glcβ1-
Glcβ1-
Glcβ1-
H
H
H
3.94
4.35
7.11
7.91
9.32
10.5
14.32
14.44
I
II
I
III
I
II
I
III
965.4222
965.4297
965.4178
965.4204
803.3721
803.3682
803.3692
803.3682
Glcβ(1-2)[Glcβ(1-3)]-Glcβ - and -H
1
Glcβ(1-2)[Glcβ(1-3)]-Glcβ1-
Glcβ(1-2)[Glcβ(1-3)]-Glcβ1-
Glcβ(1-2)[Glcβ(1-6)]-Glcβ1-
Rebaudioside Z (2)
1
Compound 4
Rebaudioside B
Iso-rebaudioside B
Glcβ(1-2)[Glcβ(1-3)]-Glcβ - and -H
1
-CH2
-CH2
Glcβ(1-2)[Glcβ(1-6)]-Glcβ1-
Glcβ(1-2)[Glcβ(1-3)]-Glcβ1-
H
a
b
Reversed-phase C18 high performance liquid chromatography method.
-O- -D-glucopyranosyl-
β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid.
Aglycone structures are presented in Figure 3. Compound
3
:
13-[(2-O-
β
β
-D-glucopyranosyl-
-D-glucopyranosyl-
3
β
β
-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid-
β
-D-glucopyranosy ester; compound : 13-[(2-O- -D-glucopyranosyl-3-O-
4
β

Molecules 2018, 23, 3328
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Figure 3. Chemical structures of steviol (I), hydroxyatisenoic acid (II) and endo-steviol aglycone (III).
Additionally, three known compounds were purified in gram quantities and identified as
rebaudiosides A ( ), H (6) and J ( ). The purification of the degradation products formed under
acidic condition from rebaudioside A and stevioside was recently reported [ ]. Preparation of a
5
7
8
high-quality crystal of iso-stevioside, one of the by-products, allowed the confirmation of its structure
by X-ray diffraction (Figure 4).
Figure 4. X-ray structure of iso-stevioside dihydrate, with ellipsoids at the 50% level (ORTEP).
Rebaudioside A, one of the major compounds from S. rebaudiana, has found use as the
main table-top and additive in beverages due to it being more than 200 times sweeter than
sucrose and its potential human health benefits [5]. However, this compound certainly interacts
with bitter taste receptors through several possible mechanisms by which the bitter aftertaste
of rebaudioside A may suppress the sweet gustatory receptors activity. As far as we know,
there is no complete study showing the relationship between structure-organoleptic properties of
steviol glycosides. Only a couple of rebaudioside A isomers with sugar arrangement at position
C-13 as follows Glc
The novel rebaudioside A isomers herein described, rebaudioside Z Glc
at C-13 and 13-[(2-O- -D-glucopyranosyl-3-O- -D-glucopyranosyl-
oxy]ent-hydroxyatis-16-en-19-oic acid- -D-glucopyranosy ester Glc (1-3)[Glc
β
(1-6)[Glc
β
(1-3)]-Glc 1
β
- and Glc
β
(1-2)[Fru
β
(1-3)]-Glc 1
(1-6)[Glc
-D-glucopyranosyl)
(1-2)]-Glc 1- at
β
- were reported [14].
β
β
(1-2)]-Glc 1-
β
β
β
β
β
β
β
β
C-13 with a different aglycone may serve as models to provide important findings to better understand
the relationship between sugar arrangement and positions with sweet/bitter flavors. To date,
several steviol glycoside isomers have been isolated from S. rebaudiana mainly with different sugar
arrangements, position of the attachment to the aglycone and also with different aglycones as is the
case of stevioside (with Glc
β
(1-2)Glc
β
1- at C-13)/12-
α
-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)
oxy]ent-kaur-16-en-19-oic acid-
β
-D-glucopyranosyl ester (Glc
β
(1-2)Glc 1- at C-12) [13]; rebaudioside
β

Molecules 2018, 23, 3328
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E (with Glc
F (with Xyl
C-13)/rebaudioside F isomer (with Glc
with Glc (1-2)Glc 1- at C-19)/rebaudioside I (Glc
β
(1-2)Glc 1
β
- at C-19) /rebaudioside Y (Glc
β
(1-6)Glc 1
β
- at C-19) [15]; rebaudioside
β
(1-2)[Glc
β
(1-3)]-Glc 1
β
- at C-13)/rebaudioside R (Glc
(1-2)[Xyl (1-3)]-Glc 1- at C-13) [14
(1-3)Glc 1- at C-19) [17,
β
(1-2)[Glc
16 17]; rebaudioside D
18]. Additionally,
β(1-3)]-Xylβ1- at
β
β
β
, ,
(
β
β
β
β
several compounds differing in the type of sugar in a specific position could also be
compared for better understanding of the organoleptic properties e.g., rebaudioside C
(Rhaα
(1-2)[Glc
β
(1-3)]-Glc 1
β
- at C-13)/6-deoxyGlc
β(1-2)[Glcβ(1-3)]Glcβ1-/rebaudioside F [18,19];
rebaudioside A/Glc
β
(1-2)[Fru
β
(1-3)]-Glc 1- at C-13 among others. Additionally, diterpene glycosides
β
with an endocyclic double bond (C 15) could be compared with their exocyclic double bond isomers
as in the case of iso-rebaudioside A/rebaudioside A; iso-stevioside/stevioside among other pairs
of compounds.
3
. Materials and Methods
3
.1. Chemicals
Acetonitrile and water for HPLC and Silica gel 60 F254 HPTLC plates were purchased from EMD
Millipore (Cincinnati, OH, USA). The bulk acetonitrile, methanol, methyl tert-butyl ether (MTBE) acetic
acid, ethyl acetate, and isopropyl alcohol (IPA) were purchased from Reagents (Nashville, TN, USA).
Flash silica was purchased from Sorbent Technologies (Atlanta, GA, USA).
3
.2. General Experimental Procedures
The mass detector was a quadrupole time of flight (Model G6530A, Agilent, Palo Alto, CA,
USA) equipped with an electrospray ionization interface and was controlled by Agilent software
A.05.00, Agilent MassHunter Work Station, Palo Alto, CA, USA). All acquisitions were performed
(
under negative ionization mode with a capillary voltage of 3500 V. Nitrogen was used as nebulizer
◦
gas (30 psig) as well as drying gas at 10 L/min at drying gas temperature of 300 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).
NMR spectra were acquired either at the University of Mississippi (Oxford, MS, USA) on
an Avance NMR spectrometer (Bruker, Billerica, MA, USA) equipped with a Bruker 5 mm
C13/H1-F19 cryoprobe or at the University of Florida (Gainesville, FL, USA) on an Inova spectrometer
(
Varian, Palo Alto, CA, USA) equipped with a Varian 5 mm H1/C13/P31-N15 indirect detection probe
both operating at 500 MHz for proton and 125 MHz for carbon and using z-axis pulsed-field gradients.
◦
The temperature was set at 25 C and chemical shifts (
δ
) were reported in ppm and referenced to
tetramethylsilane or solvent signals using similar experimental conditions as previously reported [20].
3
.3. Plant Material
The starting material was a partially processed commercially available extract of Stevia rebaudiana
with Lot # SRE50-14091 purchased from American Mercantile (Memphis, TN, USA). HPLC comparison
of that extract with several other S. rebaudiana extracts purchased from various sources showed high
similarities, differing only in the relative concentrations of specific glycosides but not their presence
or absence.
3
.4. Isolation Procedure
Commercially available S. rebaudiana leaf extract (1.5 kg) was dissolved in methanol or 10%
aqueous methanol at about 200 mg/mL and allowed to crystallize. The crystalline products were
rebaudioside A and stevioside, which accounted for approximately 50% of the starting mass.
Pools rich in rebaudioside N from several large-scale chromatographies for isolation of
rebaudioside C in quantity [21] were combined to obtain 140 g of extract. All this material

Molecules 2018, 23, 3328
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was fractionated on a high efficiency reverse-phase chromatography column (7.5 i.d.
×
50 cm,
1
0 µm spherical C18 gel) [20]. The column was loaded with the 140 grams dissolved in distilled water,
the column eluted with 3 liters of 0.5% acetic acid in water and then switched to 15:85 acetonitrile:
0
0
.1% acetic acid in water (3 liters); 25:75 acetonitrile: 0.1% acetic acid in water (3 liters); 40:60 acetonitrile:
.1% acetic acid in water (3 liters); and column washed with 90:10 acetonitrile: 0.1% acetic acid in
water and 100% methanol (1 liter each, washes combined and passed to waste.). 500 mL fractions
collected and analyzed by HPLC. Similar fractions combined and fractions rich in rebaudioside N
yielded ~22 grams. This pool was adsorbed onto celite (120 g), divided into three portions, each portion
packed into a load column and chromatographed on a high efficiency normal-phase chromatography
column (7.5 i.d.
×
50 cm, 10 µm spherical silica gel) using Reb N mobile phase (Reb C mobile phase
[100:18:14; EtOAc:MeOH:H O with 0.1% AcOH] with additional 10 parts methanol and 10 parts water
2
and 5 parts acetic acid). Column analysis [21] allowed combination of fractions rich in rebaudioside N.
This pool spontaneously crystallized, the crystals filtered and the MLs dried, redissolved and a second
crop obtained.
The supernatant second crop (7.02 g) was absorbed onto 70 g of Celite and subjected to a high
efficiency normal-phase chromatography (7.5 i.d.
H O: AcOH (88:12:0.01 v/v/%). 2 1 L forerun were initially collected, followed by 48 fractions
of 120 mL. All fractions were analyzed by HPLC methods and five main fractions were pooled
based on results from column analysis [21]. Chromatography 1 fraction 1.1 (0.093 g); fraction 2.1
5.858 g); fraction 3.1 (0.64 g); fraction 4.1 (0.328 g) and fraction 5.1 (0.109 g). Fraction 2.1 was
re-chromatographed in high efficiency normal-phase chromatography (7.5 i.d. 50 cm) using MTBE:
MeOH: H O: AcOH (100:30:12.5:0.01). 2 1 L forerun and 40 120 mL fractions were collected
and analyzed by HPLC. Five main fractions were selected by column analysis [21]. Chromatography
×
50 cm, 10 µm spherical silica gel) with acetonitrile:
×
2
,
(
×
×
×
2
2
5
, fraction 1.2 (1.3 g); fraction 2.2 (2.09 g); fraction 3.2 (1.49 g); fraction 4.2 (0.088 g) and fraction
.2 (0.065 g). Fraction 1.2 (1.3 g) was chromatographed in a high efficiency reversed-phase column
(
2
7.5 i.d.
×
50 cm, 10 µm spherical C18 gel) using ACN:H O:AcOH (25:75:0.01). 2
×
1 L forerun and
5 × 120 mL fractions were collected and analyzed by HPLC. Column analysis [21] allowed us to
fraction 1.3 (0.208 g); fraction 2.3 (0.922 g). Fraction 2.2
2.09 g) and fraction 2.3 (0.922 g) were combined and digested with MeOH to afford 2.2 g of solids
which were chromatographed in a high efficiency reversed-phase column (7.5 i.d. 50 cm) using
ACN:H O:AcOH (23:77:0.01). 6 1 L + 1 0.7 L forerun, and 37 120 mL fractions were collected
and analyzed by HPLC. Column analysis allowed us to select three main fractions. Chromatography 4
2
select two main fractions. Chromatography 3
(
,
×
×
×
×
2
,
fraction 1.4 (0.546 g); fraction 2.4 (0.713 g) and fraction 3.4 (1.165 g). Fraction 3.4 (1.165 g) was absorbed
onto 10 g of celite and chromatographed in high efficiency normal-phase chromatography (7.5 i.d.
×
50 cm) using “Reb C” mobile phase 2% MeOH [Reb C mobile phase = EtOAc:MeOH:H O:AcOH
2
(
100:18:14:0.1; v/v/v/%)]. 1
HPLC. Column analysis allowed us to select five main pools. Chromatography 5, fraction 1.5 (67.1 mg);
fraction 2.5 (135 mg); fraction 3.5 (226 mg); fraction 4.5 (179 mg) and fraction 5.5 (179 mg). Compound
×
1 L forerun and 48
×
120 mL fractions were collected and analyzed by
1
(226 mg; 0.02% yield) was obtained from fraction 3.5.
Fractions rich in rebaudioside H were pooled (3.7 g) and chromatographed over a RP-C18 (7.5 i.d.
×
50 cm) column with H O:AcOH (100:0.1 v/v/%) and ACN:H O:AcOH (5:95:0.1 v/v/%) to afford
2
2
seven main fractions. Chromatography 6
,
fraction 1.6 (175 mg); fraction 2.6 (120 mg); fraction 3.6
(
7
108 mg); fraction 4.6 (169 mg); fraction 5.6 (1.8 g, rebaudioside H); fraction 6.6 (156 mg) and fraction
.6 (13 mg). Chromatography 7, fraction 1.6 (175 mg) was submitted to a RP-C18 (250 10 mm, 5 m)
chromatography using H O: AcOH (100:0.1) and ACN: H O: AcOH (10:90:0.1) to afford two main
×
µ
2
2
fractions., fraction 1.7 (110 mg; 0.007% yield) and fraction 2.7 (48.3 mg). Compound 3 was obtained
from fraction 1.7. Additionally, several hundred grams from the initial crystallization and subsequent
chromatographies of rebaudioside A after processing 1.5 kg of commercial extract. Rebaudiosides J
(1 g) and H (1.8 g) were also isolated.

Molecules 2018, 23, 3328
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3
.5. Alkaline Hydrolysis of Compounds 1 and 3
Compounds (40 mg) and (30 mg) were heated individually with NaOH (1 N) at 80 C over 1 h.
◦
1
3
Each reaction mixture was cooled over 5 min and neutralized with two or three drops of acetic acid
glacial [18] with further cleanup through a Strata RP-C18-E cartridge (500 mg/6 mL) (Phenomenex,
Torrance, CA, USA). Elution with a stepwise gradient of 1.5 mL volume each, water, acetonitrile:
water (2:8) and methanol to produce clean compounds
2 (27 mg; 68% yield) and 4 (15 mg; 50% yield).
3
.6. Physicochemical Parameters of Compounds
Rebaudioside Z (
1
): Amorphous off-white solid; [
α
]25
−
28.0 (c 0.1, MeOH). HR-ESIMS/MS m/z
D
−
9
65.4222 [M
one hexose (
162 Da), loss of three hexoses from C-13 moiety at 70 eV collision energy. H- and C-NMR
spectroscopic data are shown in Tables 1 and 2.
−
H] (calculated for C H O , 966.4309), m/z 803.3703 at 10 eV collision energy, loss of
44
70 23
−
162 Da) from C-19 moiety, m/z 641.3185 (
−
162 Da), 479.2635 ( 162 Da) and 317.2146
−
1
13
(
−
Rebaudioside Z (
2
): Amorphous off-white solid; [
α
]25
−
46.0 (c 0.1, MeOH). HR-ESIMS/MS m/z
1
D
−
8
03.3721 [M
−
H] (calculated for C H O , 804.3781), m/z 641.3196 (
−
162 Da), 479.2648 (
−
162 Da)
38
60 18
1
and 317.2115 (
−
162 Da), loss of three hexoses from C-13 moiety at 70 eV collision energy. H- and
13
C-NMR spectroscopic data are shown in Tables 1 and 2.
1
3-[(2-O-
oic acid-
HR-ESIMS/MS m/z 965.4297 [M
collision energy (
162 Da) and 317.2141 (
β
-D-Glucopyranosyl-3-O-
β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-
25
β
-D-glucopyranosy ester (
3
−
): Amorphous off-white solid; [
α
]
−
22.0 (c 0.1, MeOH),
D
−
H] (calculated for C H O , 966.4309), m/z 803.3735 at 10 eV
44
70 23
−
162 Da), loss of one hexoses from C-19 moiety, m/z 641.3195 (
−
162 Da), 479.2703
(
−
−
162 Da), loss of three hexoses from C-13 moiety at 70 eV collision energy.
1
13
H- and C-NMR spectroscopic data are shown in Tables 1 and 2.
1
3-[(2-O-
β
-D-Glucopyranosyl-3-O-
β
-D-glucopyranosyl-
β
-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-
20.0 (c 0.1, MeOH). HRESIMS/MS m/z 803.3682
H] (calculated for C H O , 803.3702), m/z 641.3162 ( 162 Da), m/z 479.2703 ( 162 Da)
25
oic acid (
4
): Amorphous off-white solid; [
α
]
−
D
−
[M
−
−
−
38
60 18
1
13
and m/z 317.2111 Da at 70 eV collision energy. H- and C-NMR spectroscopic data are shown in
Tables 1 and 2.
3
.7. RP-C18 HPLC Analysis
Analyses were performed with a Hewlett Packard Agilent 1100 Series system equipped with a
G1311A quaternary pump, a G1322 degasser, a G1316A oven, G1313A autosampler and a G1315A
diode array detector. Acetonitrile and water for HPLC were purchased from EMD Millipore (Cincinnati,
OH, USA). The elution was performed with 0.01 M phosphoric acid (A) and acetonitrile (B) with a flow
rate set at 1 mL/min. All the analyses were performed with Phenomenex columns. After each analysis,
the column was washed and equilibrated appropriately. 10
RP C-18 Luna (2), Phenomenex (250 4.6 mm, 5
µ
L of compound
1
–
4
were injected in the
◦
×
µ
m) column at 30 C. The elution was performed
using gradient of elution as follows: 0–5 min, 32% B; 5–13 min, 32–41% B; 13–16 min, 41–43% B;
6–17 min: 43–50% B; 17–23 min, 50% B. The chromatogram was recorded at 205 nm and the flow rate
set at 1 mL/min [10].
1
3
.8. Determination of the Sugar Unit Absolute Configuration
Compounds and (1 mg) were hydrolyzed with HCl (1 N) at 80 C over 2 h followed by
liquid-liquid partition with ethyl acetate (2 1 mL). The aqueous layer was neutralized with silver
carbonate and the supernatant was recovered and heated with L-cysteine methyl ester in pyridine
◦
1
3
×
◦
◦
for 1 h at 60–70 C. The mixture was dried in a vacuum oven at 40 C. After dryness, 400 µL of

Molecules 2018, 23, 3328
13 of 15
pyridine and 100
µ
L of phenylisothiocyanate were added and heated for an additional hour at
◦
6
0–70 C to form the thiocarbamoyl thiazoline derivatives. Reaction mixtures were analyzed by the
HPLC method previously reported [9]. The absolute configuration of the sugars was determined by
comparison of the HPLC retention times of the prepared thiocarbamoyl thiazolidine derivatives with
appropriate standards.
3
.9. X-ray Crystallography of Iso-Stevioside
The crystal structure and absolute configuration of iso-stevioside dihydrate were determined
from a colorless crystal of dimensions 0.45
×
0.14
×
0.02 mm, using data collected at T = 90 K with Cu
K
α
radiation on an APEX-II DUO CCD diffractometer (Bruker, Madison, WI, USA) equipped with a
microfocus source and a Cryostream cooler (Oxford, Cryosystems, Oxford, UK). The structure was
solved using the program SHELXS-97 (University of Göttingen, Germany) and refined anisotropically
2
by full-matrix least-squares on F using SHELXL-2014/7 (University of Göttingen, Germany) [22].
All H atoms were visible in difference maps, but were placed in idealized positions for the refinement,
except for those of OH groups and water molecules, which were refined. The absolute configuration
was determined from the Flack [23] parameter of 0.01(5) based on resonant scattering of the light atoms
and 2804 quotients. The reported configuration has C4(R), C5(S), C8(R), C9(R), C10(S), C13(S) and
is in agreement with the known configurations of the
β-D-glucopyranose moieties. Crystal data:
C H O 2H O, Mr = 840.89, monoclinic space group P2 , a = 13.2330(6) Å, b = 8.1081(4) Å,
·
3
8
60 18
2
1
◦
3
−3 ◦
, θmax = 68.3 , R = 0.031
c = 19.1556(8) Å
,
β
= 105.655(2) , V = 1979.05(16) Å , Z = 2, Dx = 1.411 g cm
for all 6901 unique data and 571 refined parameters. Supplementary crystallographic data for
iso-stevioside dihydrate are contained in Cambridge Structural Database deposition CCDC-1879103;
this data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from
the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44)
1
223-336-033 or e-mail: deposit@ccdc.cam.ac.uk)
4
. Conclusions
Two new rebaudioside A isomers, rebaudiosides Z (
-D-glucopyranosyl- -D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid-
ester ( ) were isolated from a partially processed commercial extract of S. rebaudiana and
two new rebaudioside B isomers, rebaudiosides Z1 and 13-[(2-O- -D-glucopyranosyl-3-O-
-D-glucopyranosyl- -D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid were prepared
and purified for the first time, respectively. An additional three known compounds,
1) and 13-[(2-O-
β
-D-glucopyranosyl-3-O-
β
β
β-D-glucopyranosy
3
β
β
β
rebaudiosides A, H and J, were purified in gram quantities. Scarce rebaudioside A isomers
have been reported from Stevia rebaudiana differing in sugar arrangement and type of sugar at
position C-13 (Glc
describe the occurrence of two rebaudioside A isomers with (Glc
Glc (1-3)[Glc (1-2)]-Glc 1-) at position C-13. However, compound 3
β
(1-6)[Glc
β
(1-3)]-Glc 1
β
-) and Glc
β
(1-2)[Fru
β
(1-3)]-Glc 1
(1-6)[Glc
showed a different aglycone
β
-. However, herein we
β
β
(1-2)]-Glc 1-) and
β
(
β
β
β
and was found to be a 13(S)-hydroxyatisenoic acid type. This finding may contribute to better
understanding of the relationship between sweet/bitter taste of Stevia glycosides with their sugar
arrangements. Several new compounds have been reported in recent years, however, a systematic
study comparing organoleptic properties with structure of diterpene glycosides is still not available
in literature.
Supplementary Materials: The supporting information are available online.
Author Contributions: W.H.P., C.D.H., I.M., I.A.K., and J.D.M. conceived and designed the study. W.H.P., K.A.,
D.L.R. purified compounds; W.H.P., I.G., D.L.R., K.A., F.T.W., F.R.F., M.A.I., and B.A. conducted the experiments.
All authors shared analyzing the data and writing the manuscript.
Funding: This research received no external funding.

Molecules 2018, 23, 3328
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Acknowledgments: The project was supported by National Center for Natural Product Research, School of
Pharmacy, University of Mississippi, University, MS 38677, USA. This work was supported in part by the USDA
Agricultural Research Service Specific Cooperative Agreement No. 58-6060-6-015.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–7 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).