2
S. Ceunen et al. / Carbohydrate Research 379 (2013) 1–6
acyl moiety (Table 2). Other signals included nine methylene and
three methine protons between d 0.80 and 2.42 ppm, all of which
correspond well with the ent-kaurane skeleton. This assignment
was further supported by COSY and HMBC correlations.7,20 The ini-
tial ESIMS indications for the presence of an SV skeleton could not
be confirmed by NMR as inferred from the lack of olefinic proton
signals and the lack of the characteristic olefinic signal around d
104 ppm in 13C NMR and DEPT. In addition, the presence of a C-
13 hydroxyl moiety would have increased chemical shifts for C-
12, C-13, and C-14 with at least 13, 37, and 9 ppm, respectively
(compare with values for SV (5) in Table 2).21
analogous ketone 8a or 8b since the required ring expansion would
involve the primary hydroxy group as a leaving group. Instead, the
more favorable loss of the tertiary hydroxy group and a concomi-
tant hydride shift from C-17 to C-16 could produce aldehyde 7
(Scheme 1). The formation of 7 is supported by HREIMS signals
not seen in the spectrum of ISV, such as m/z 288 (loss of formalde-
hyde, Scheme 2). On the other hand, the ion signal at m/z 275 ob-
served for ISV (corresponding to the loss of the C-15/C-16 moiety
as CH3CO), is not detected in the HREIMS of 7. Instead, a homolo-
gous ion appears at m/z 261 due to the loss of the C-15/C-17 moi-
ety as CH3CHCHO. The ion series (m/z 288, 261 and 215 for 7 and
m/z 275 for ISV) can be rationalized by an initial cleavage between
C-8 and C-15 with charge retention at the quaternary carbon cen-
ter C-8. Another series (m/z 274, 273, 123 and 109) may be initi-
ated by cleavage between C-9 and C-10 (Scheme 2).
Unfortunately, no NMR analysis could be made of 7 due to a lack
of material.
Two anomeric protons at d 4.98 and 6.21 ppm were found in the
1H NMR spectrum of 1, indicating the presence of two sugar moi-
eties. This was confirmed by the ESIMS analysis of 1, showing frag-
ment ions at m/z 523 and 361 in positive mode, corresponding to
the loss of a hexose and pentose residue in addition to water (Ta-
ble 1). The positioning of the sugar and acyl moieties was clarified
by the key HMBC correlations as shown in Figure 3, whereas the
large coupling constants observed for the two anomeric protons
at d 6.21 (d, J = 8.0 Hz) and 4.98 (d, J = 7.7 Hz) are in agreement
with the b disposition of both sugars.4 Good similarities were
found between the 13C NMR spectrum of 1 and the published spec-
tral data for related compounds, such as 16b-hydroxy-17-acetoxy-
Based on the results from spectral and chemical analyses, the
structure of 1 was assigned as 16b-hydroxy-17-acetoxy-ent-kau-
ran-19-oic acid-(6-O-b-D-xylopyranosyl-b-D-glucopyranosyl) ester.
This is the first report on the detection of 1 in a natural source.
Beside 1, seven more compounds were detected (Table 1).
Although their small quantities only warranted analysis by ESIMS,
their distinct sets of signals allowed a tentative designation of
molecular masses of both their full structure and aglycone moie-
ties. Given the similarities in their spectral patterns, they might
possibly be related to 1. Their aglycones all appear to have molec-
ular masses of 336 or 378 Da, the difference being likely due to the
presence or absence of acetylation. Only one compound appears to
have an aglycone with the same molecular mass as SV (318 Da,
peak 5). Beside the recurring differences of 42 Da (acetyl moiety),
variations of 162 (hexose) and 294 Da (hexose and pentose) hint
at similar glycosylation patterns as observed in 1. However, due
to a lack of plant material, their chemical identification as well as
their exact structural relationships could not be established and re-
main unknown. Nevertheless, the identification of 1 as the com-
pound most prevalent in the fraction otherwise containing SVgly
standards suggests that S. phlebophylla is unlikely to contain signif-
icant amounts of SVglys, if any.
ent-kauran-19-oic acid-b-D
-glucopyranosyl ester (2),20 suavioside
E (3),7 and the primeverosyl moiety (4; Table 2).22
Acid hydrolysis of crude water extracts followed by a derivati-
zation of carboxylic acids to their fluorometrically detectable
methoxycoumarinyl derivatives23 revealed a compound eluting
at similar retention times and having a similar molecular mass as
derivatized ISV (data not shown). A similar profile was seen after
acid hydrolysis of 1, of which the water-soluble hydrolysate affor-
ded only xylose and glucose occurring in molar concentrations
deviating 9.1% from the ratio of 1:1. HREIMS analysis of the
ether-soluble, underivatized aglycone showed a fragmentation
pattern that had some similarity with that of ISV (common ions
due to M+ at m/z 318, loss of water at m/z 300, and loss of COOH
at m/z 273). However, in contrast to the ISV ketone structure 6 pro-
duced from SVglys, acid hydrolysis of 1 is less likely to yield an
1. Experimental
1.1. General experimental procedures
1H and 13C NMR spectra were recorded on a Bruker Avance
II + 600 MHz spectrometer equipped with a TXI probe and operat-
ing at 600 MHz for proton and 150 MHz for carbon measurements.
Spectra were recorded in C5D5N and chemical shifts (d) are re-
ported in parts per million (ppm) referenced to tetramethylsilane
(1H) or the IS (NMR) solvent signals (13C). ESIMS was used for ini-
tial compound characterization. Extracts were first separated by
LC, using an Alltima C18 column (250 ꢁ 4.6 mm ID, 5
lm particle
size; Grace, Lokeren, Belgium). The ESIMS analyses were carried
out on a Thermo Electron LCQ Advantage LC–MS combined with
Alltech 3300 ELSD detection and UV detection at 200 nm. Mass
spectrometric detection was performed in both positive and nega-
tive ionization modes in the m/z range of 200–2000. HREIMS anal-
ysis was carried out in case the compounds of our interest could be
obtained in larger amounts (>100 lg). Compounds were re-dis-
solved in minimal amounts of MeOH and subjected to 70 eV ioni-
zation energies in a Kratos MS50TC instrument. High-resolution
detection was performed in the m/z range of 50–700 using a MAS-
PEC II data system. Optical rotation was measured using a PolAAr
20 polarimeter at 20 °C. Semi-preparative and analytical HPLC
was carried out on a system consisting of two LC-20AT pumps
Figure 1. Comparison of HPLC traces of S. phlebophylla leaf extracts with a reference
sample of known SVglys originally extracted from S. rebaudiana leaves. A combined
extract of both leaves was further purified and concentrated on SPE-C18 columns
(bottom trace). ESIMS analysis revealed 8 peaks with distinctive fragmentation and
adduct patterns (peaks 1–8; see enlarged HPLC trace, upper right). Of these, only
peak
1 could be fully characterized (1). Abbreviations: Dul: dulcoside; Reb:
rebaudioside; Rub: rubusoside; SB: steviolbioside; ST: stevioside.
Ceunen, Stijn
