Transformation of naturally-occurring 1,9-trans-9,5-cis sweroside to all trans sweroside during acetylation of sweroside aglycone

Article abstract of DOI:10.1016/S0031-9422(00)00460-X

From the rootstock of Scabiosa columbaria L. loganin and sweroside were isolated. Sweroside showed moderate antibacterial activity. Pure sweroside was converted to the sweroside aglycone l-acetoxy derivative (DABCO/Ac₂O) after hydrolysis of the glucose unit. X-ray crystallography of the monoacetate showed unambiguously that it had been transformed to a new seco-iridoid having the novel trans diaxial configuration for the protons on C-1, C-9 and C-5. The mechanism for the rearrangement is discussed.

Full text of DOI:10.1016/S0031-9422(00)00460-X

Phytochemistry 57 (2001) 51±56
www.elsevier.com/locate/phytochem
Transformation of naturally-occurring 1,9-trans-9,5-cis sweroside to
all trans sweroside during acetylation of sweroside aglycone
Marion M. Horn a, Siegfried E. Drewes a,*, Nicola J. Brown a, Orde Q. Munro a,
J.J. Marion Meyer b, Abbey D.M. Mathekga b
aSchool of Chemical and Physical Sciences, University of Natal, Pietermaritzburg, P. Bag X01, Scottsville, 3209, South Africa
bDepartment of Botany, University of Pretoria, Pretoria 0001, South Africa
Received 26 April 2000; received in revised form 3 November 2000
Abstract
From the rootstock of Scabiosa columbaria L. loganin and sweroside were isolated. Sweroside showed moderate antibacterial
activity. Pure sweroside was converted to the sweroside aglycone 1-acetoxy derivative (DABCO/Ac2O) after hydrolysis of the glu-
cose unit. X-ray crystallography of the monoacetate showed unambiguously that it had been transformed to a new seco-iridoid
having the novel trans diaxial con®guration for the protons on C-1, C-9 and C-5. The mechanism for the rearrangement is dis-
cussed. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Scabiosa columbaria; Dipsaceae; Loganin; Sweroside; Rearrangement; Sweroside aglycone-1-acetate
1. Introduction
of their glycosides. Fractionation of the extract yielded
pure loganin 1a. This compound was ®rst isolated from
the fruits of Strychnos nux vomica (Dunstan and Short,
1883). The full structure, stereochemistry and biosynthesis
was only elucidated some 86 years later (Battersby and
Hall, 1969).
Also present in the extract we found the seco-iridoid
sweroside 2a (Inouye et al., 1970) and this is the ®rst
isolation from the family Dipsaceae. It was accompanied
by a complex mixture of other iridoids, all possessing an
aldehyde functionality. This latter mixture and sweroside,
2a, showed only moderate antibacterial activity when
tested against a selection of gram-positive and gram-
negative bacteria.
Scabiosa columbaria L. is a perennial herb, which grows
to a meter in height, ¯owers in midsummer, and has a
large woody rootstock in the form of a tuber. Traditionally
root decoctions are used for sterility. Ointments made
from the charred tubers are also applied to venereal sores
(Hutchings, 1996). Chemically almost nothing is known
about the plant although the glycoside scabiosin has
been isolated from it (Watt and Breyer-Brandwijk, 1962).
We started investigating the plant because it grows
profusely in many parts of South Africa, and, despite its
frequent use by the Zulu people, the chemistry of its
constituents is almost unknown (Van Wyk, 1997).
2.2. Rearrangement of sweroside on acetylation and
X-ray analysis of the resultant product
2. Results and discussion
2.1. Isolation of loganin and sweroside
Iridoids and seco-iridoids all possess the labile acetal
moiety and are frequently subjected to varying degrees
of acid or base treatment during work-up or subsequent
manipulation. In this connection Bianco (1994) has
published a review article which deals speci®cally with
acid hydrolysis and the subsequent rearrangements
which can occur.
It was immediately clear from the preliminary exam-
ination of the extracts (CHCl3)/EtOH) that Scabiosa
columbaria contained several iridoids, mostly in the form
* Corresponding author. Tel.: +27-33-260-5243; fax: +27-33-260-
5009.
E-mail address: drewes@science.unp.ac.za (S.E. Drewes).
We report here on the rearrangement which sweroside
aglycone 2b undergoes during acetylation in the presence
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(00)00460-X

52
M.M. Horn et al. / Phytochemistry 57 (2001) 51±56
of the base diazabicyclooctane (DABCO). (DABCO is
easier to remove at the end of the reaction than pyri-
dine). The reaction a€orded the highly crystalline 1-
acetoxy sweroside aglycone, a new compound with
novel stereochemistry. This seco-iridoid 3, has the pro-
tons at C-1, C-9 and C-5 in the axial orientation, giving
rise to an all trans-con®guration at these three carbon
atoms. This stands in contrast to the ``normal'' 1,9-
trans-9,5-cis arrangement found in iridoids and seco-iri-
doids in which the protons on C-1, C-9 and C-5 assume
eq.eq. and ax orientations respectively. Since 3 was
obtained as colourless orthorhombic crystals, amenable
to X-ray analysis, the relative stereochemistry could be
established with certainty (Fig. 1). Comparison with the
extensive published NMR data (Ma et al., 1994) con-
®rmed the view that ``our'' parent sweroside indeed
had the accepted 1,9-trans-9,5-cis stereochemistry.
Subsequent formation of 3, with its 1,9-trans-9,5-trans-
con®guration was, therefore, attainable only if rearran-
gement had occurred during acetylation.
The seco-iridoid decentapicrin A, a compound for
which X-ray data was available is shown in (4) (Van der
Sluis and Labadie, 1981; Kojic-Prodic et al. 1985). This
compound is identical in all respects to sweroside and
di€ers only in the nature of the glycoside at C-1. A
comparison of the magnitude of the coupling constants
J1,9 and J5,9 respectively for 4 and 3 shows clearly a
H-1/H-9 (trans diequatorial), H-5/H-9 (cis axial, equa-
torial) stereochemistry for 4, compared with a trans diaxial,
trans diaxial, arrangement for the same proton on 3.
We were reassured that the proposed rearrangement
occurs during the acetylation procedure, and not earlier,
by showing that the sweroside aglycone (2b) had a
stereochemistry unchanged from that of the parent
sweroside (2a). A comparison of 1H NMR spectral data
for sweroside (2a), sweroside aglycone (2b) (see Section
3), and the rearranged acetate (3) (Table 1) con®rms the
Fig. 1. Two crystallographically-distinct conformations of sweroside aglycone-1-acetate 3.

M.M. Horn et al. / Phytochemistry 57 (2001) 51±56
53
Table 1
NMR spectral data of rearranged acetate 3 (CDCl3, ꢀ ppm, J=Hz)
stereochemistry shown for (2a), (2b) and 3. There is no
doubt that the aglycone still retains the 1,9 trans dieq.
arrangement prior to acetylation. It is suggested that the
rearrangement during acetylation proceeds as shown in
Scheme 1.
No.
1H
13C
1
3
4
5
6
6.11 (1H, d, 9.3)
7.64 (1H, d, 2.2)
93.1
154.1
104.4
34.0
±
1. The aglycone of sweroside, 2b, under in¯uence of
the base (DABCO) ring opens to generate 2c.
2. In a ®nal step, prior to attachment of the acetyl
moiety, 2c loses an acidic proton at C-9, undergoes
keto-enol tautomerism and ring-closes to 2d (and also
presumably the isomer of opposite con®guration).
2.56 (1H, ddd, 4.5,2.3)
6a 1.56 (1H, m)
6b 2.06 (1H, m)
27.2
7
7a 4.27 (1H, ddd, 24.1, 12.5, 2.4)
7b 4.48 (1H, ddd, 11.5, 4.5, 2.0)
5.52 (1H, ddd, 16.9, 10.3, 9.3)
2.21 (1H, ddd, 9.2, 7.4, 1.6)
10a 5.29 (1H, ddd, 16.9, 1.5, 0.7)
10b 5.33 (1H, ddd, 10.4, 1.4)
68.1
132.6
46.2
8
9
10
We suggest that a series of products, all in equilibrium
with one another, are formed. On acetylation, and quite
fortuitously, one of these products, 3, crystallised out
from the reaction mixture in reasonable yield. The product
was amenable to X-ray and NMR analysis.
121.4
169.1
164.7
20.7
CO2 lactone
Acetate CO
OCH3
2.13 (s)
Scheme 1. Mechanism of inversion at C-1 and C-9.

54
M.M. Horn et al. / Phytochemistry 57 (2001) 51±56
Since acetylation usually occurs in the presence of
rearrangement than the seco-iridoid system as in
sweroside aglucone.
pyridine (rather than DABCO/Ac2O) the reaction on
sweroside aglucone was also carried with this reagent.
To our pleasant surprise the rearranged all trans swero-
side 3 was obtained in 73% yield.
2.6. Biological activity tests
Since aq. root decoctions of Scabiosa columbaria ®nd
a traditional use in the treatment of venereal sores
(Hutchings, 1996), some of the isolated components
(sweroside, loganin and a semi-puri®ed mixture) were
tested for antibacterial activity. At concentrations of
0.001±0.50 mg/ml none of the compounds showed any
activity. At a conc. of 1.0 mg/ml (minimum inhibitory
concentration, MIC), sweroside and the ``mixture'' were
active against Bacillus cereus, B. pumulus, B. subtilis,
Micrococcus kristinae, Staphylococcus aureus (gram
positive) and Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aerugenosa, Enterobacter cloacae (gram
negative). Neither inhibited the growth of Serratia mar-
cescence. These activities can be regarded as ``moder-
ate'' only.
2.3. Rearrangement in the NMR tube
In order to study the e€ect of DABCO on sweroside
aglycone 2b (with intact stereochemistry at C-1, C-9 and
C-5) the compound was dissolved in CDCl3 in an NMR
tube and DABCO only added. Progress of the reaction
was monitored over 10 h. Small changes in the NMR
spectrum were apparent within minutes. The H-1 signal
disappeared rapidly, and after 40 min several new peaks
appeared in the region ꢀ 6.5±9.55. The most signi®cant
of these was a doublet at ꢀ 9.4. (J=3.82 Hz). This peak
gradually increased in amplitude and after 4.5 h a fur-
ther peak was visible at ꢀ 9.87. Both peaks are presumed
to be aldehyde peaks and their presence is regarded as
evidence that ring opening occurs (Scheme 1). The e€ect
is in line with earlier proposals (Bianco, 1994).
3. Experimental
2.4. Iridoids with an all-trans ring fusion in nature
3.1. General
The ®rst naturally-occurring iridoid of the above nat-
ure was reported by Foderaro et al. (1992). These iri-
doids are rare and the authors make the observation
that ``there are no known instances of epimerisation at
C-5 among the hundreds of iridoid isolations''. In a
subsequent report Foderaro and Stermitz (1992) present
detailed spectral data to support their ®nding. They
record a J1,9 coupling constant of 9.9 Hz for the com-
pound (5dH)-6-epidihydrocornin. The X-ray structure
of a suitable derivative revealed clearly the all trans
diaxial nature of the protons on C-1, C-9 and C-5.
The seco-iridoid 3, produced synthetically, thus joins
a small group of naturally-occurring iridoid compounds
with distinctive stereochemical features and hopefully
will alert other researchers to stereochemical changes
which can occur during manipulation of iridoids.
1H and 13C NMR spectra were recorded on Varian-
200 and Varian-500 spectrometers. High resolution
mass spectra were recorded on a Kratos MS 80 RF
double-focussing magnetic sector instrument at 70 eV.
For optical rotations a Perkin Elmer 241 digital polari-
meter was used.
3.2. Plant material
Scabiosa columbaria (rootstock) was collected in an
open ®eld in the Hay®elds area of Pietermaritzburg
during January 1999. It was identi®ed by Dr Trevor
Edwards, Curator of the Bews Herbarium at the Uni-
versity of Natal. A voucher specimen (SED No. 6) of
the whole plant has been deposited in the Herbarium.
2.5. Attempts to ring-open loganin with DABCO
3.3. Extraction and isolation
Loganin aglycone 1b (from ꢁ-glucosidase hydrolysis
of loganin) was subjected to the same acetylation con-
ditions of DABCO/acetic anhydride as described for
sweroside aglycone. No rearrangement occurred and the
1-acetoxy derivative was isolated in good yield and high
Freshly-collected tubers (850 g) were milled and
extracted with 95% EtOH±CHCl3 (1:1) for 6 days at
RT. The solution was ®ltered and the lower phase
separated o€ and concentrated (16 g). A portion of this
material (9 g) was fractionated on a dry silica gel
(Merck Silica Gel 9385) column using CH2Cl2±MeOH
(100: 0!85: 15) as eluent. Three fractions were col-
lected: fraction (i) a€orded loganin aglycone (1b) (35
mg) after puri®cation on the chromatron [CH2Cl2±
Et2O(1: 1)]; fraction (ii) gave loganin (1a) (70 gm) fol-
lowing separation on the chromatotron [CH2Cl2±
MeOH (93: 7)]; fraction (iii) gave a crude mixture
1
purity. The H NMR spectrum di€ers very little from
that of the parent aglucone apart from H-1 which
showed the anticipated down®eld shift from ꢀ 4.98 to
ꢀ 6.07. The J1,9 coupling constant is 3.5 Hz and in loga-
nin itself it is 4.5 Hz (Boros and Stermitz, 1990). In this
instance it is possible that the iridoid skeleton (as in
loganin) is less susceptible to ring opening and subsequent

M.M. Horn et al. / Phytochemistry 57 (2001) 51±56
55
(726 mg) which on separation as above [CH2Cl2±MeOH
(90:10!85:15)] a€orded impure sweroside (2a) (140 mg)
and a mixture of other compounds unidenti®ed as yet
(280 mg). Further separation on the chromatotron
[CH2Cl2±MeOH (93:7!90:10)] gave pure sweroside
(2a) (77 mg).
m, H-5), 4.32 (1H, m, H-7a), 4.48 (1H, m, H-7b), 5.21±
5.28 (2H, m, H-10), 5.44 (1H, broadened s, H-1), 5.73
(1H, ddd, J=17.3, 10.3, 9.2 Hz, H-8), 7.63 (1H, d,
J=2.1 Hz, H-3). 13C NMR (125 Hz, CDCl3): ꢀ ˆ 26:7
(C-6), 28.5 (C-5), 46.9 (C-9), 68.5 (C-7), 94.4 (C-1),
104.8 (C-4), 119.8 (C-10), 134.7 (C-8), 153.6 (C-3), 166.3
(COO-). EI±MS m/z (rel int.): 120 (.5), 85 (62), 83 (100).
3.4. Studies on loganin
3.6. Sweroside aglucone-1-acetate (3)
The properties of loganin (above, mp 220ꢀC) were
identical in all aspects (NMR, mass spectra) to values
published in the literature (Boros and Stermitz, 1990).
The optical rotation, [a]2D5=À62.4ꢀ (c=0.0004, MeOH),
is somewhat lower than the literature value of À82.8ꢀC
(El-Naggar and Beal, 1980).
Loganin (1a) (230 mg) was suspended in water pH 6.8
(5 ml) and b-glucosidase (30 mg) added. The solution
was stirred at 37ꢀC for 5 h, then exhaustively extracted
with ether to give impure loganin aglucone. The latter
was puri®ed on the chromatotron [(CH2Cl2±Et2O (5:4)]
to give pure loganin aglucone (1b) (98 mg), oil. 1H
NMR (200 MHz, CDCl3): ꢀ=1.15 (3H, d, J=6.6 Hz,
Me), 1.53 (1H, m, H-6a), 2.30 (1H, m, H-6b), 1.95 (1H,
m, H-8), 1.95 (1H, m, H-9), 3.17 (1H, m, H-5), 3.72 (3H,
s, CO2Me), 4.13 (1H, m, H-7), 4.98 (1H, d, J=4.9 Hz,
H-1), 7.41 (1H, d, J=1.3 Hz, H-3). 13C NMR (50 MHz,
CDCl3): ꢀ=13.3 (C-10), 31.5 (C-5), 41.4 (C-6), 42.0 (C-
8), 46.2 (C-9), 51.4 (OMe), 74.5 (C-7), 95.6 (C-1), 115.5
(C-4), 151.7 (C-3), 168.2 (C-11).
Loganin aglycone-1-monoacetate 1(c). Loganin agly-
cone (43 mg) was treated with DABCO (6 mg) and
shaken to dissolve in THF (2.5 ml). Ac2O (0.2 ml) was
added and the solution stirred at RT overnight.
Removal of solvent, followed by separation on a small
column (silica gel) eluted with CHCl3, followed by
CH2Cl2±Et2O (7:3) gave a pure oil, (35.4 mg). EI±MS
m/z (rel. int.): 270 (M+, 2.1), 210 (M-OAc, 19), 192 (M±
OAc±H2O, 22), 150 (21), 139 (100), 125 (24), 97 (19). ꢀ H
NMR (200 MHz, CDCl3): ꢀ ˆ 1:15 (3H, d, J=6.8 Hz,
Me), 1.80 (1H, m, H-6a), 2.11 (3H, s, OCOMe), 2.20
(2H, m, H-8, H-9), 2.32 (1H, m, H-6b), 3.18 (1H, m, H-
5), 3.73 (3H, s, CO2Me), 4.14 (1H, m, H-7), 6.07 (1H, d,
J=3.5 Hz, H-1), 7.32 (1H, d, J=0.8 Hz, H-3).
Sweroside aglucone (2b) (above, 45 mg) was treated
with DABCO (6.2 mg), THF (2.5 ml) and acetic anhy-
dride as detailed for loganin aglycone to give the 1-
acetory derivative (3) (31 mg), mp 112±113ꢀC, (obtained
only after several recrystallisations from 95% EtOH),
[a]2D0 ˆ ‡36:4ꢀ (c=0.112, CHCl3); IR (KBr): ꢂ : 3160,
ꢁmu†
1765, 1701, 1618, 1404, 1284, 1201, 1189, 1060. EI±MS
m/z (rel int.): 238 (M+, 2.4), 196 (1.4). 178 (2.6), 160
1
(8.5), 127 (100), 112 (2.5), 97 (8.8). H and 13C NMR
(500 and 125 MHz, CDCl3) are collected in Table 1.
For the X-ray study on (3) di€raction data were col-
lected on a Nonius CAD 4 di€ractometer with graphite
monochromated MoK radiation. The unit cell para-
meters were determined by a least squares ®t. The
structure was resolved by direct methods. Further
details for 1-acetoxy sweroside aglucone (3) are:
C12H14O5. Final R value 0.0470, colourless orthorhom-
bic crystals, cell parameters (Aꢀ) a=7.9637 (7),
b=10.2678 (8), c=29.326 (2), crystal dimensions (mm)
0.58Â0.46Â0.35, space group P212121, V(Aꢀ ) 2398.0(3),
3
Z=8. (Fig. 1).
The dihedral angles H-1/C-1±C-9/H-9 and H-5/C-5±
C-9/H-9 were 177.9 and 174.5ꢀ respectively, in good
accord with the stereochemistry predicted by the 1H
NMR data.
Acknowledgements
We thank the National Research Foundation for
®nancial support.
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