Preparative isolation and structural characterization of sucrose ester isomers from oriental tobacco

Article abstract of DOI:10.1016/j.carres.2013.01.021

To date, the structures of the sucrose tetraester (STE) isomers, a main kind of sucrose esters (SEs) in Solanum, have not been conclusively assigned. In this study, three groups of STE isomers with the molecular weight 650, 664 and 678 (designated as STE I, STE II and STE III, respectively) have been isolated and purified from the oriental tobacco-Komotini Basma using a semi-preparative RP-HPLC method. The full characterization of the isomers in the three groups of STE were investigated for the first time by MS (HRMS, MS²) and NMR (¹H, ¹³C, HSQC) spectroscopy combined with alkaline hydrolysis and STE derivation experiments. The STE III (a single compound) was confirmed as a known sucrose tetraester. Furthermore, the STE II was found to contain three isomers and the structures were first unambiguously established as 6-O-acetyl (2,3 or 2,4 or 3,4)-di-O-3-methylvaleryl-(4 or 3 or 2)-O-2-methylbutyryl-α-d-glucopyranosyl-β-d-fructofuranoside. Finally, the STE I was discovered to contain seven isomers and the structures were elucidated as 6-O-acetyl (2 or 3 or 4)-O-3-methylvaleryl-(3,4 or 2,4 or 2,3)-di-O-2-methylbutyryl-α-d-glucopyranosyl-β-d-fructofuranoside, 6-O-acetyl (2 or 3 or 4)-O-3-methylvaleryl-(3,4 or 2,4 or 2,3)-di-O-isovaleryl- α-d-glucopyranosyl-β-d-fructofuranoside and 6-O-acetyl (2,3 or 2,4 or 3,4)-di-O-3-methylvaleryl-(4 or 3 or 2)-O-isobutyryl-α-d-glucopyranosyl- β-d-fructofuranoside (one of the 3 isomers).

Full text of DOI:10.1016/j.carres.2013.01.021

Carbohydrate Research 372 (2013) 73–77
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Preparative isolation and structural characterization of sucrose ester
isomers from oriental tobacco
Chunxiao Jia a, Yingying Wang a, Yonghua Zhu a, Chunping Xu b, Duobin Mao b,
⇑
a Department of Applied Chemistry, College of Material and Chemical Engineering, Zhengzhou University of Light Industry, Zhengzhou, PR China
b Department of Food Engineering, College of Food and Biology Engineering, Zhengzhou University of Light Industry, Zhengzhou, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
To date, the structures of the sucrose tetraester (STE) isomers, a main kind of sucrose esters (SEs) in Sola-
num, have not been conclusively assigned. In this study, three groups of STE isomers with the molecular
weight 650, 664 and 678 (designated as STE I, STE II and STE III, respectively) have been isolated and puri-
fied from the oriental tobacco-Komotini Basma using a semi-preparative RP-HPLC method. The full char-
acterization of the isomers in the three groups of STE were investigated for the first time by MS (HRMS,
MS2) and NMR (1H, 13C, HSQC) spectroscopy combined with alkaline hydrolysis and STE derivation exper-
iments. The STE III (a single compound) was confirmed as a known sucrose tetraester. Furthermore, the
STE II was found to contain three isomers and the structures were first unambiguously established as 6-
Received 20 December 2012
Received in revised form 22 January 2013
Accepted 29 January 2013
Available online 6 March 2013
Keywords:
Sucrose tetraesters
Oriental tobacco-Komotini Basma
Semi-preparative RP-HPLC
Structural analysis
O-acetyl (2,3 or 2,4 or 3,4)-di-O-3-methylvaleryl-(4 or 3 or 2)-O-2-methylbutyryl-
-fructofuranoside. Finally, the STE I was discovered to contain seven isomers and the structures were
elucidated as 6-O-acetyl (2 or 3 or 4)-O-3-methylvaleryl-(3,4 or 2,4 or 2,3)-di-O-2-methylbutyryl-
glucopyranosyl-b- -fructofuranoside, 6-O-acetyl (2 or 3 or 4)-O-3-methylvaleryl-(3,4 or 2,4 or 2,3)-di-
O-isovaleryl- -glucopyranosyl-b- -fructofuranoside and 6-O-acetyl (2,3 or 2,4 or 3,4)-di-O-3-methyl-
valeryl-(4 or 3 or 2)-O-isobutyryl-
a-D-glucopyranosyl-b-
D
a-D-
MS and NMR spectroscopies
D
a-
D
D
a
-
D
-glucopyranosyl-b-
D
-fructofuranoside (one of the 3 isomers).
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
been made to report the full characterization of the SE isomers
with the same molecular weight.5,28
Sucrose esters (SEs) are the second major class of tobacco sur-
face chemicals, which are biosynthesized exclusively in trichome
glands from sucrose and activated acids derived from branched-
chain amino acid metabolism.1–4 The most abundant tobacco leaf
SEs were identified as sucrose tetraesters (STEs).1,5,6 SEs isolated
from the leaf surface extracts are recognized as precursors of
important oriental tobacco smoke flavour components. It has been
proved that SEs readily release fatty acids such as isobutyric acid,
methylbutyric acid, and methylvaleric acid on thermolysis during
tobacco combustion.7,8 SEs also have been shown to possess insec-
ticidal,9–12 antibacterial and plant growth regulating activities.13–17
Studies have shown that some other related Solanum (e.g., potato,
tomato, petunia) can also produce sucrose esters.18–23
In this paper, three groups of STE isomers with the molecular
weight 650, 664 and 678 were isolated and purified from oriental
tobacco-Komotini Basma by semi-preparative RP-HPLC. By direct
NMR and MS spectra along with alkaline hydrolysis and STE deri-
vation experiments, the structures of the three isomers in the
STE group of MW 664 were established unequivocally and the
structures of the seven isomers in the STE group of MW 650 were
elucidated for the first time.
2. Experimental
2.1. Material and reagents
There are already several reports on the separation and struc-
tural characterisation of the different types of SE homologs from
tobaccos by silica gel column or HPTLC combining with spectro-
scopic methods,13,14,24 LC–MS,25,26 and LC–MSn,27 in previous stud-
ies. However, to the best of our knowledge, few attempts have
N,O-Bis-(trimethylsilyl)trifluoroacetamide (BSTFA) (98% purity),
acetic acid, isobutyric acid, 2-methylbutyric acid (99% purity) were
purchased from Acros Organics (USA). Isovaleric acid and 3-meth-
ylvaleric acid (99% purity) were from Tokyo Kasei Kogyo Co., Ltd
(Tokyo, Japan). Acetonitrile and methanol (HPLC grade) purchased
from Siyou Fine Chemical Co. Ltd (Tianjing, China). HPLC grade
water (18 mX) was prepared using a Millipore Milli-Q purification
system (Millipore Corp., USA). The oriental tobacco-Komotini Bas-
ma leaves sample was kindly provided from the R&D center of
⇑
Corresponding author. Tel.: +86 371 86609623; fax: +86 371 63556306.
E-mail address: duobinmao@163.com (D. Mao).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.01.021

74
C. Jia et al. / Carbohydrate Research 372 (2013) 73–77
Hongta Tobacco (Group) Co., Ltd (Yuxi, China). Tobacco sample was
dried at 45 °C and ground to pass through a 40-mesh sieve for fur-
ther analysis.
2.3. TLC of sucrose tetraesters
TLC Silica Gel 60 F254 plates, 6.5 cm ꢀ 2.5 cm, thickness 0.5 mm
(Qingdao Haiyang Chemical Group Co. Ltd, China) were developed
in a twin trough unsaturated chamber using PhMe/MeCOEt/MeOH/
H2O (10:5:4.5:0.2, v/v/v/v). Plates were developed to a distance run
by the solvent of 4 cm at room temperature and relative humidity
of 40%. The developed plates were dried in a stream of cold air for
3 min and then immersed for 20 min into a color reagent of
CO(NH2)2/H3PO4/n-BuOH (1 g of CO(NH2)2 and 4.5 mL 85% (v/v)
H3PO4 dissolved in 48 mL n-BuOH saturated by water), by means
of the Camag chromatogram immersion device II. Heating at
70 °C for 20 min furnished coloured bands for separated
compounds.29
2.2. Extraction and isolation of sucrose tetraesters
A sample of tobacco leaves (50.0 g) was extracted by ultrasonic
assistant extraction with dichloromethane (1000 mL) at 25 °C for
20 min. After filtration, the residue was washed with dichloro-
methane (3 ꢀ 50 mL) and the resulting solution were combined
and concentrated under reduced pressure to give a residue which
was dissolved in CH3OH/H2O (80:20, v/v) (100 mL). This solution
was extracted with n-hexane (3 ꢀ 100 mL), and the CH3OH/H2O
phase was collected and extracted with CHCl3/H2O/saturated aq
KCl (4:2:1, v/v) (3 ꢀ 70 mL). The CHCl3 phase was collected,
washed with water (10 mL), dried over anhydrous sodium sulfate
(15 g) and then concentrated to a volume of 10 mL. This solution
was then subjected to column chromatography (column size
4 cm ꢀ 60 cm) on silica gel (100–200 mesh), Initial elution was
with n-hexane/acetone (1:1, v/v) at a rate of 4 mL/min to afford
fractions 1–7 (40 mL/fraction), and then with n-hexane/acetone
(2:3, v/v) to give fractions 8–25. For the routine TLC screening test
of STEs, each fraction was concentrated to 5 mL before being ap-
plied to the plate.
2.4. Analytical and semi-preparative HPLC separation procedure
The combined fractions containing STEs was concentrated to
20 mL and filtered through a membrane filter (0.45 lm, Millipore).
Two milliliters of the resulting filtrate (named FSTE) was injected
into the semi-preparative HPLC. The HPLC system equipped with
a Waters 600 controller, a Waters Delta 600 pump, a differential
refractive index detector (RID, Waters 2414), and a sunFire™ prep
C18 column (250 mm ꢀ 10 mm i.d.,10
lm, Waters Corp., USA). The
O
OCCH3
OH
O
O
R3O
a
OH
=
R2O
OR1
O
4
OH
4
OH
O
C
O
C
1
2
3
R
=
R2
STE III :
STE II :
=
R3
=
CH2
CH2 CH3
R4
CH3
CH
1
b
( the acetyl was the same in
CH3
STE I , STE II and STE III)
5
O
O
C
6
7
8
2
1
3
4
CH CH2 CH3
C
c
R3
=
R 1
=
R2
=
CH
CH2
CH2 CH3
CH3
CH3
9
5
O
C
O
C
6
7
8
1
2
3
4
R2
R2
=
=
R3
=
CH2 CH3
CH
R1
=
=
=
STE I :
or
CH2
CH2 CH3
CH
CH3
CH3
9
5
O
C
O
C
10
11 12
2
1
3
4
d
CH2
CH3
CH
CH
R3
R3
=
=
R1
CH2
CH2 CH3
CH3
CH3
5
12
O
C
O
C
13
14
1
2
3
4
CH
CH3
or
R1
R2
=
CH2
CH2 CH3
CH
CH3
CH3
14
5
Figure 1. Structures and numbering systems of the isolated STEs, (a) general structural formula of the STEs homologs; (b) structures of the acyl groups in STE III; (c)
structures of the acyl groups at the positions of O-2, O-3 and O-4 of the glucose residue in STE II; (d) structures and combination modes of the acyl groups at the positions of
O-2, O-3 and O-4 of the glucose residue in STE I.

C. Jia et al. / Carbohydrate Research 372 (2013) 73–77
75
2.5. Structural identification of STE isomers
All MS analyses (HRMS, MS2) were performed on a Waters Q-
TOF Micro mass spectrograph (Waters Corp., USA) by full scan
ESI-MS and collision induced dissociation (CID) ESI-MS/MS. The
analytical parameters were spray voltage: 3.5 kV, cone voltage:
35 V, collision voltage: 26.0 V. Data were acquired in the posi-
tive-ion MS and MS2 modes. The sample was injected using a peri-
staltic pump.
1D and 2D NMR spectra were acquired on a Bruker-DPX-400
(400 MHz) NMR spectrometer, with samples (3–10 mg) dissolved
in 0.4–0.6 mL CDCl3, using tetramethylsilane (TMS) as internal
standard. The 1H, 13C, H, H-COSY, HSQC experiments were standard
Bruker implementations.
2.6. Alkaline hydrolysis experiments
Each group of purified STE isomers (5 mg) was dissolved in 2 mL
KOH/CH3OH/H2O solution (1 mol KOH dissolved in 1 L of CH3OH/
H2O (80:20, v/v)), and the solution was heated at 60 °C for
30 min, after which time concentrated hydrochloric acid (0.3 mL)
was added, and the mixture then centrifuged. The fatty acids in
the supernatant layer were analysed by GC–MS. GC–MS analyses
were performed on a GC 6890-MS 5973 N Gas chromatography–
mass spectrometry (equipped with an automatic sampler,
G1701CA MSD chemical workstation and NIST 05 mass spectral
data base, Agilent Corp., USA), GC conditions were as follows:
HP-INNOWax capillary column (30 m ꢀ 250
l
m ꢀ 0.25
lm), with
an initial column temperature of 80 °C, increasing at 5 °C/min to
160 °C, which was then held for 3 min. Injector temperature was
250 °C. The carrier gas was helium at a flow rate of 1.0 mL/min.
Injection volume was 1 lL, with a split ratio of 5:1. MS were mea-
sured with electron impact (EI) ionization energy of 70 eV. Inter-
face temperature was 250 °C, and the quadrupole temperature
was 150 °C; the electron multiplier was voltage 1.34 kV. The MS
measurements were made in the scan method with a mass scan
range of 35–500 m/z. Standard acid solutions (acetic acid, isobu-
tyric acid, 2-methylbutyric acid, isovaleric acid and 3-methylvaler-
ic acid) of different concentrations (1 lg/mL, 20 lg/mL and 40 lg/
mL) were also analyzed under the above conditions.
3. Results and discussion
3.1. TLC analyses
TLC revealed the existences of sucrose tetraesters from the 16th
to the 22nd of the 25 fractions from the tobacco extracts. The
developing agent CO(NH2)2/H3PO4/n-BuOH proved to be a sensitive
reagent for STEs giving bluish grey spots. Rf values on TLC for the
20nd fraction (F20) was 0.69. No material was observed at this Rf
in the 12th fraction (F12) indicated indicating the absence of STEs.
Figure 2. HSQC spectra of the acyl region of STE I–STE III. The numbering of carbons
and the attached protons in HSQC spectra are the same as that shown in Figure 1b, c
and d.
3.2. Analytical and semi-preparative HPLC separation
The crude sample (FSTE) was analyzed as described in Section 2.4
and three groups of STE isomers designated as STE I, STE II and STE
III were observed with retention times of 6.04 min, 7.43 min, and
9.46 min, respectively.
Semi-preparative HPLC separation of the FSTE gave the three
different purified STE fractions as viscous oils. As a result, from
50.0 g of a tobacco leaves sample, 14.8 mg of STE I, 30.4 mg of
STE II and 25.2 mg of STE III were obtained, and the purity of
STE I, STE II and STE III was 99.6%, 99.2% and 99.8%, respectively,
in a single run.
mixed solvent CH3CN/H2O (70:30, v/v) was used as eluent at a flow
rate of 4.0 mL/min.
HPLC analyses of the FSTE and the purified STE fractions were
performed on a Waters 1525 (Waters Corp., USA), using a sunFire™
C18 column (250 mm ꢀ 4.6 mm i.d. 5
lm, Waters). CH3CN/H2O
(70:30, v/v) was used as eluent at a flow rate of 1.0 mL/min. STEs
were detected by evaporation light scattering detector (ELSD,
Waters 2424). The purity of the STE fractions was expressed on
an area percentage basis.

76
C. Jia et al. / Carbohydrate Research 372 (2013) 73–77
3.3. Structural identification of the isomers in the three groups
of STEs
according to the GC–MS analysis results of the standard acid solu-
tions (Section 2.6). The results showed that the alkaline hydrolysis
experiment of STE III gave acetic acid and 3-methylvaleric acid
with the molar ratio being approximately 1:3. Based on the NMR
data and alkaline hydrolysis experiment, it is clear that the four
acyl groups on O-6, O-2, O-3 and O-4 positions of STE III were
one acetyl and three 3-methylvaleryls (Fig. 1b). Therefore, STE III
was essentially a single compound, and the structure was estab-
The HRMS analysis results further confirmed that STE I, STE II
and STE III were three groups of STE isomers since only one major
peak was observed in every case. The molecular formulas of STE I,
STE II and STE III were established as C30H50O15, C31H52O15 and
C
32H54O15 by HRMS analysis (m/z 673.3051 [MSTE I+Na]+, calcd
for C30H50O15Na, 673.3047), m/z 687.3206 [MSTE II+Na]+ calcd for
lished as 6-O-acetyl 2,3,4-tri-O-3-methylvaleryl-
syl-b- -fructofuranoside. It is the only STE that has to date been
characterised in this homologous series.5
a-D-glucopyrano-
C
C
31H52O15Na, 687.3204, and m/z 701.3363 [MSTE III+Na]+, calcd for
32H54O15Na, 701.3360, respectively. On the basis of the molecular
D
formulae and literature data,4,5,27 these STEs probably possessed a
general structure formula which is shown in Figure 1a. In the MS2
spectra, a major peak at m/z 511.2 [MSTE I+Naꢁ162], m/z 525.3
[MSTE II+Naꢁ162] and m/z 539.3 [MSTE III+Naꢁ162], corresponding
to the loss of a fructose unit, were observed for STE I, STE II and
STE III, respectively. This further confirmed that these STEs belong
to the same structural type showed in Figure 1a. It contains an
acetyl group on O-6 of the glucose ring, three acyl groups of 4 to
6 carbons on O-2, O-3 and O-4 of the glucose ring, and four free hy-
droxyl groups on the fructose ring. Furthermore, on the basis of lit-
erature data,4,5,27 the combination of fatty acids bound to the
glucose ring can be C2C5C5C6 or C2C4C6C6 in STE I, C2C5C6C6 in
STE II and C2C6C6C6 in STE III where the subscript denotes the num-
ber of carbons in the acid. Except for STE III, each group contains
several isomers. In this study, the structures of the four acyl groups
on O-6, O-2, O-3, and O-4 were further investigated by 1D and 2D
NMR spectra along with the alkaline hydrolysis and derivation
experiments of STEs, and the structures of the isomers in STE I
and STE II were confirmed or elucidated. Except for the 13C NMR
data of STE III,5 the NMR and HRMS spectra from the authentic
samples of STE I, STE II and STE III have been obtained for the first
time.
The HSQC spectra of the acyl region (Fig. 2) made possible
unambiguous assignments of the chemical shifts of carbon and
the attached protons for all four acyls in STE III and STE II, and al-
low most of the chemical shifts of acyls region to be assigned in
STE I. The 1H NMR and 13C NMR data are summarised in Table 1.
In Table 1 (STE III), the data of the acyl region indicated the
presence of three 3-methylvaleryls and an acetyl in STE III by com-
parison with the data obtained by measurements on standards of
these fatty acids. In addition, the NMR data were confirmed by
the experiment result of alkaline hydrolysis. The hydrolyzates after
acidification were analyzed by GC–MS. By searching with NIST 05
MS mass spectral data base and comparison with the mass spectra
of standard acids, the fatty acid compositions of the STEs could be
achieved. The molar response value of the acids could be calculated
In Table 1 (STE II), the data of the acyl region indicated the pres-
ence of two 3-methylvaleryls, one 2-methylbutyryl and one acetyl
in STE II by comparison with the data from standards of the fatty
acids. Moreover, the analysis of hydrolyzate after acidification by
GC–MS gave acetic acid, 2-methylbutyric acid and 3-methylvaleric
acid with the molar ratio being approximately 1:1:2. Therefore, the
three acyls at O-2, O-3 and O-4 in STE II were two 3-methylvaleryls
and one 2-methylbutyryl (Fig. 1c) and three arrangements of these
acyl groups located at the O-2, O-3 and O-4 positions of the
glucose molecule are possible. Thus three isomers of STE II may ex-
ist, which was confirmed by silylation with BSTFA/DMF (1:1, v/v)
and examination of the product by GC–MS, when three
peaks were observed (data not shown). Therefore the structures
can be assigned as 6-O-acetyl (2,3 or 2,4 or 3,4)-di-O-3-methyl-
valeryl-(4 or 3 or 2)-O-2-methylbutyryl-a-D-glucopyranosyl-b-D-
fructofuranoside.
In Table 1 (STE I), the data of the acyl region also indicated the
presence of 3-methylvaleryls, 2-methyl butyryl and one acetyl in
STE I by comparison with the data from STE II. The complete assign-
ments of carbons and protons in the acyls of STE I were difficult due
to some overlap of the resonance signals in 1H NMR, 13C NMR and
HSQC spectra. This problem would be resolved with the assistance
of the alkaline hydrolysis experiment. The analysis of hydrolyzate
of STE I after acidification by GC–MS gave acetic acid, isobutyric acid,
2-methylbutyric acid, isovaleric acid and 3-methylvaleric acid with
the approximate molar ratio of acetic acid/isobutyric acid/meth-
ylbutyric acid/3-methylvaleric acid = 7:1:12:8. According to the
fatty acid compositions, the rest of the NMR data of STE I (Table 1)
was assigned to isovaleryl and isobutyryl groups, respectively. Over-
all, theabove results suggestedthat the structuresand combinations
of the three acyls at O-2, O-3 and O-4 positions of glucose in STE I
were as shown in Figure 1d. According to the three different struc-
tural arrangements in Figure 1d and the approximate molar
ratio of the fatty acids mentioned above, it is deduced that STE I
may consist of seven isomers, and the structures are 6-O-acetyl (2
or 3 or 4)-O-3-methylvaleryl-(3,4 or 2,4 or 2,3)-di-O-2-methylbuty-
Table 1
13C NMR and 1H NMR date (d ppm) of the acyl region of STE I-STE III
⁄The serial numbers of carbons and attached protons are those for the 3-methylvaleryl (1–5), 2-methylbutyryl (6–9), isovaleryl (10–12), isobutyryl (13 and 14) and acetyl (40)
groups as shown in Fig. 1.

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In summary, three groups of STE isomers with the molecular
weight 650, 664 and 678 have been isolated and purified from
the oriental tobacco-Komotini Basma with a purity of 99.6%,
99.2% and 99.8%, respectively, using a semi-preparative RP-HPLC
method for the first time. The structures of all isomers in the three
groups of STEs were identified by MS (HRMS, MS2) and NMR (1H,
13C, HSQC) spectra along with alkaline hydrolysis and STE deriva-
tion experiments. As a result, the structures of the three isomers
in STE II were identified conclusively. The structures of the seven
isomers in STE I were elucidated. The provision of detailed NMR
and mass spectrometry data will be useful for the future identifica-
tion of other STE compounds and, additionally, using our isolation
method, the groups of STE isomers can be isolated from tobaccos or
other plants on a large scale and may be applied for the bioactivity
studies.
24. Ohya, I.; Sinozaki, Y.; Tobita, T.; Takahashi, H.; Matsuzaki, T.; Koiwai, A.
Phytochemistry 1994, 37, 143–145.
25. Ashraf-Khorassani, M.; Nazem, N.; Taylor, L. T.; Coleman, W. M. Beitr.
Tabakforsch. Int. 2005, 21, 441–450.
Acknowledgements
26. Ashraf-Khorassani, M.; Nazem, N.; Taylor, L. T.; Coleman, W. M. Beitr.
Tabakforsch. Int. 2005, 21, 380–389.
27. Ding, L.; Xie, F. W.; Zhao, M. Y.; Xie, J. P.; Xu, G. W. Rapid Commun. Mass
Spectrom. 2006, 20, 2816–2822.
28. Simonovska, B.; Srbinoska, M.; Vovk, I. J. Chromatogr., A 2006, 1127, 273–277.
29. Li, Y. K.; Zhang, S. F.; Yang, J. Z. Chin. J. Chromatogr. 2002, 20, 476–478.
The authors wish to thank the National Science Foundation
(21176229/B060804) of China for the financial support of this work,
and the Center of Forecasting and Analysis of Zhengzhou University
(Henan, China) for help with NMR and MS measurements.
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