Ptesculentoside, a novel norsesquiterpene glucoside from the Australian bracken fern Pteridium esculentum

Article abstract of DOI:10.1016/j.tetlet.2010.02.032

A novel norsesquiterpene glucoside ptesculentoside has been isolated from the Australian bracken Pteridium esculentum, together with the known bracken carcinogen ptaquiloside and lesser amounts of caudatoside. The structure of ptesculentoside is determined by analysis of 1D and 2D NMR spectra, and via its conversion into previously known pterosin G. Crown Copyright

Full text of DOI:10.1016/j.tetlet.2010.02.032

Tetrahedron Letters 51 (2010) 1997–1999
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Ptesculentoside, a novel norsesquiterpene glucoside from the Australian
bracken fern Pteridium esculentum
b
a
b
Mary T. Fletcher a, , Patrica Y. Hayes , Michael J. Somerville , James J. De Voss
*
a Queensland Primary Industries and Fisheries, Locked Mail Bag 4 Moorooka 4105, Australia
b School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel norsesquiterpene glucoside ptesculentoside has been isolated from the Australian bracken Pter-
idium esculentum, together with the known bracken carcinogen ptaquiloside and lesser amounts of cau-
datoside. The structure of ptesculentoside is determined by analysis of 1D and 2D NMR spectra, and via
its conversion into previously known pterosin G.
Received 30 September 2009
Revised 2 February 2010
Accepted 5 February 2010
Available online 11 February 2010
Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
Keywords:
Pteridium esculentum
Ptaquiloside
Caudatoside
Ptesculentoside
Bracken
Pterosin
Sesquiterpene glucoside
Bracken ferns (Pteridium species) are some of the most ubiqui-
tous plants on earth, with an extensive history of poisoning grazing
livestock.1 The norsesquiterpenoid glucoside ptaquiloside (1b) has
previously been isolated from bracken and shown to be responsi-
ble for a number of the associated syndromes, particularly acute
haemorrhagic disease of cattle (bracken poisoning), bright blind-
ness of sheep, bovine enzootic haematuria and upper alimentary
carcinoma.2 The biological activity of this reactive glycoside has
been attributed to the facile elimination of glucose to form an
unstable conjugated dieneone intermediate which acts as a power-
ful alkylating agent of amino acids and DNA.2
We report here the first phytochemical study of P. esculentum,
and the isolation of ptaquiloside (1b), caudatoside (1c) and the pre-
viously unknown hydroxy ptaquiloside analogue, ptesculentoside
(1a) (Fig. 1). Treatment of the individual glucosides (1a–c) with di-
lute base produces the corresponding elimination products, ptero-
sin G (2a), pterosin B (2b) and pterosin A (2c).
P. esculentum fern was collected from a site near Conondale,
Queensland and the species identification confirmed by Queensland
Herbarium (voucher AQ744801). An extract of milled freeze-dried
immature croziers was partially purified by the method of Rasmus-
Taxonomic classification within the genus Pteridium remains
controversial.3,4 Pteridium aquilinum is the most widespread spe-
cies with 11 subspecies occurring predominantly in the northern
hemisphere,4 and initial isolations of ptaquiloside (1b) relate to
P. aquilinum subspecies.5–8 Ptaquiloside and a series of similarly
reactive analogues including caudatoside (1c) have also been iso-
lated from what is now recognised as a separate species, Pteridium
caudatum.9–11 Previous chemical examination of the other three
Pteridium species (Pteridium arachnoideum, Pteridium esculentum
and Pteridium semihastatum) have been limited to surveys of their
ptaquiloside content by HPLC–UV analysis of the more stable elim-
ination product pterosin B (2b).12–17
14
HO
O
13
10b
H
9
R1
8
1
12
7
6
2
R2
14
4
5
3
10a
11
O
12
8
13
O
HO
7
9
4
HO
4'
O
10b 1
1'
R
1
6'
5'
2
R2
6
OH
2'
3
10a
HO
11
3'
5
HO
2a: R1 = H; R2 = CH2OH
2b: R1 = H; R2 = CH3
1a: R1 = H; R2 = CH2OH
1b: R1 = H; R2 = CH3
2c: R1 = CH3; R2 = CH2OH
1c: R1 = CH3; R2 = CH2OH
* Corresponding author. Tel.: +61 7 3362 9426; fax: +61 7 3362 9429.
E-mail address: mary.fletcher@deedi.qld.gov.au (M.T. Fletcher).
Figure 1. Glucosides isolated from Pteridium esculentum and their elimination
products.
0040-4039/$ - see front matter Crown Copyright Ó 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.02.032

1998
M. T. Fletcher et al. / Tetrahedron Letters 51 (2010) 1997–1999
sen et al.18 to provide a glassy solid. A portion of this material
(25.8 mg) was then subjected to repeated rounds of reverse phase
HPLC to afford pure ptaquiloside (1b) (5.7 mg), caudatoside (1c)
14
8
13
(3.2 mg) and ptesculentoside (1a) (6.7 mg). Ptaquiloside (1b) ([a]
D
ꢀ170.8 (c, 0.42, MeOH)) and caudatoside (1c) ([
a
]
ꢀ94.8 (c, 0.18,
D
MeOH)) were identified by comparison of their 13C and 1H NMR
spectral data with literature data for these compounds.8,9 The mea-
sured opticalrotationfor ptaquiloside(1b) wasconsistentwith liter-
ature,7,10 but the optical rotation for caudatoside (1c) has not
previously been reported.
12
7
11
6
9
4
1
3
2
Ptesculentoside (1a) ([
a
]
ꢀ172.6 (c, 0.6, MeOH)) provided an
D
5
ion at 437.1792 ([M+Na]+) corresponding to a molecular mass for-
mula of C20H30O9 (calculated M+Na: 437.1782). Both proton and
carbon spectra of ptesculentoside (1a) (Table 1) presented many
signals characteristic of ptaquiloside (1b),8 with a noted absence
of the H-10 methyl singlet in the 1H NMR and C-10 resonance in
the 13C NMR spectra. The presence of a CH2OH group at this posi-
tion was suggested by the existence of a characteristic dC 60.4 car-
bon resonance and two non-equivalent protons at dH 3.62 and dH
3.85 both with couplings to H-2 (dH 2.34), and is in agreement with
the molecular formula. The only other significant differences in the
NMR shifts of ptesculentoside (1a) relative to ptaquiloside (1b) are
a downfield shift of 7.7 ppm for C-2 and an upfield shift of 5.7 ppm
10
6'
3β
3α
5'
1'
4'
2'
3'
for C-3, which are consistent with b- and
c-hydroxy substituent
effects.19
2D NMR techniques confirmed the proposed structure for pte-
sculentoside (1a) and enabled assignment of all resonances
(Table 1), in agreement with previous assignments for ptaquiloside
(1b).8 Assignment of the protonated carbons of 1a was performed
by analysis of its HSQC spectrum, with the gross structure deter-
mined from HMBCs. The one-proton doublet at dH 4.61 (dC 99.4
via HSQC) is characteristic of H-10 of a glycoside and the H-10/H-
20 coupling of 7.9 Hz is indicative of the b-anomer. Measured cou-
pling constants for the other protons of the sugar ring established
the stereochemistry at each of the ring carbons and confirmed the
depicted b-glucosyl moiety.
Figure 2. Selected NOE correlations for ptesculentoside (1a).
NOESY correlation with one of the H-3 resonances (dH 2.33) which
is thus H-3
a.
Therefore, the other H-3 resonance (dH 2.45) must be H-3b.
NOESY correlations between H-5/H-3b and H-5 and the single
H-2 resonance (dH 2.34) indicated that these hydrogens were on
the same b face of the molecule, and hence the CH2OH group at
C-2 lies in the
a position. Ptesculentoside (1a) thus has the 2S
configuration.20
NOESY correlations ( Fig. 2) between H-9/H-10 and H-9/H-14
The structure and C-2 stereochemistry of ptesculentoside (1a)
was further confirmed by chemical conversion. Treatment of pta-
quiloside (1b) in aqueous base followed by weak acid readily
established that the A/B rings were cis fused with an
a orientation
of the b-glucosyl group, H-9 and C-14 methyl. H-10 also showed a
Table 1
13C and 1H NMR chemical shifts (d, ppm) and coupling constants (Hz) determined for ptesculentoside (1a) compared with literature data for ptaquiloside (1b)
8,a,c
No.
Ptesculentoside (1a)a,b
Ptaquiloside (1b)
13C
1H
13C
1H
1
2
3
4
5
6
7
8
9
10
11
12
13
14
10
20
30
40
50
60
222.0
52.9
39.7
82.3
123.1
144.7
30.1
72.1
63.4
60.4
19.5
6.1
10.7
26.9
99.4
75.2
78.1
71.8
77.8
62.9
224.0
45.2
45.2
82.0
123.1
144.5
30.1
71.89
62.5
13.6
19.5
5.9
10.6
27.0
99.3
75.2
77.7
71.92
78.2
62.9
2.34, m, overlapping
H-3 : 2.33, overlapping; H-3b: 2.45, dd (5.3, 9.2)
2.20, ddq (6.5, 7.5, 12)
H-3 : 1.92, dd (12, 12); H-3b: 2.47, dd (8, 12)
a
a
5.78, br s
5.75, dq (1, 1.5)
2.64, br s
2.63, d (1.5)
1.05, d (6.5)
1.52, d (1)
3.62, dd (3.4, 12.0); 3.85, dd (4.4, 12.0)
1.54, s
H-12
H-13
a
a
: 0.89, m; H-12b: 0.69, m
: 0.53, m; H-13b: 0.88, m
H-12
H-13
a
a
: 0.85, m (overlap H-13b); H-12b: 0.68, m
: 0.48, m; H-13b: 0.85, m (overlap H-12a)
1.28, s
4.61, d (7.9)
3.20, dd (7.8, 8.9)
3.35, dd (8.9, 8.9)
3.26, dd (8.9, 8.9)
3.30, m
H-60a: 3.91, dd (3.2, 12.4); H-60b: 3.61, dd (1.8, 12.4)
1.27, s
4.59, d (8)
3.19, dd (8, 8.5)
3.32
3.30
3.32
H-60a: 3.88, dd (2, 12); H-60b: 3.66, dd (5.5, 12)
a
Measured in CD3OD. Coupling constants are in parentheses.
1H NMR spectra were recorded at 278 K at 500 MHz and 13C NMR at 125 MHz with the residual CD3OD protonated signal (dH 3.31) and the central peak of the CD3OD
b
septet (dC 49.00) as internal standards.
c
NMR data obtained for ptaquiloside (1b) from P. esculentum is in excellent agreement with the reported data (see Supplementary data)

M. T. Fletcher et al. / Tetrahedron Letters 51 (2010) 1997–1999
1999
HO
O
Supplementary data
O
H
O
HO
O
H
2S
H
Supplementary data (Detailed description of experimental pro-
cedures and 1D and 2D NMR spectra of ptesculentoside (1a), pta-
quiloside (1b), caudatoside (1c) and pterosin G (2a)) associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2010.02.032.
CH2OH
NaOH
CH2OH
HO
HO
OH
(1a)
H2SO4/H2O
O
HO
HO
H
References and notes
2S
CH2OH
1. Vetter, J. Acta Vet. Hung. 2009, 57, 183–196.
2. Yamada, K.; Ojika, M.; Kigoshi, H. Nat. Prod. Rep. 2007, 24, 798–813.
3. Thomson, J. A.; Mickel, J. T.; Mehltreter, K. Bot. J. Linn. Soc. 2008, 157, 1–17.
4. Der, J. P.; Thomson, J. A.; Stratford, J. K.; Wolf, P. G. Am. J. Bot. 2009, 96, 1041–
1049.
(2a)
Figure 3. Elimination of ptesculentoside (1a) to form pterosin G (2a).
5. Niwa, H.; Ojika, M.; Wakamatsu, K.; Yamada, K.; Hirono, I.; Matsushita, K.
Tetrahedron Lett. 1983, 24, 4117–4120.
6. Van der Hoeven, J. C. M.; Lagerweij, W. J.; Posthumus, M. A.; Van Veldhuizen,
A.; Holterman, H. A. J. Carcinogenesis 1983, 4, 1587–1590.
7. Ojika, M.; Wakamatsu, K.; Niwa, H.; Yamada, K. Tetrahedron 1987, 43, 5261–
5274.
8. Oelrichs, P. B.; Ng, J. C.; Bartley, J. Phytochemistry 1995, 40, 53–56.
9. Castillo, U. F.; Wilkins, A. L.; Lauren, D. R.; Smith, B. L.; Towers, N. R.; Alonso-
Amelot, E.; Jaimes-Espinoza, R. Phytochemistry 1997, 44, 901–906.
10. Castillo, U. F.; Ojika, M.; Alonso-Amelot, M.; Sakagami, Y. Bioorg. Med. Chem.
1998, 6, 2229–2233.
11. Castillo, U. F.; Wilkins, A. L.; Lauren, D. R.; Smith, B. L.; Alonso-Amelot, M. J.
Agric. Food Chem. 2003, 51, 2559–2564.
12. Alonso-Amelot, M. E.; Rodulfo-Baechler, S.; Jaimes-Espinoza, R. Biochem. Syst.
Ecol. 1995, 23, 709–716.
13. Agnew, M. P.; Lauren, D. R. J. Chromatogr. 1991, 538, 462–468.
14. Smith, B. L.; Seawright, A. A.; Ng, J.; Hertle, A. T.; Thomson, J. A.; Bostock, P. D..
In Colegate, S. M., Dorling, P. R., Eds.; Plant-associated Toxins: Agricultural,
Phytochemical and Ecological Aspects; CAB International: Wallingford UK,
1994; pp 45–50.
generates pterosin B (2b),12 and caudatoside (1c) likewise provides
pterosin A (2c).9 Similar treatment of ptesculentoside (1a) (2 mg)
afforded, after solvent partitioning, pure pterosin
(0.78 mg) ([
ꢀ13.6 (c, 0.5, MeOH)) (Fig. 3).
G (2a)
a]
D
Pterosin G has previously been characterised as a component
from P. aquilinum var. latiusculum ([
a
]
D ꢀ14.6)21,22 and from Pteris
podophylla ([
a
]
D ꢀ14),23 and 13C and 1H NMR data obtained here is
consistent with that previously reported for this compound.23 Fu-
kuoka et al.21 demonstrated that (ꢀ)-pterosin G has the 2S configu-
ration by reduction with lithium aluminium hydride and oxidation
with chromicanhydrideto affordthe sameindanone as derivedfrom
(ꢀ)-2R-pterosin B.20 The optical rotation obtained here for 2a is in
agreementwith literature21–23 indicatingthesame2Sstereochemis-
try in this compound, and hence also in ptesculentoside (1a).
Determination of the absolute configuration of the glucose unit
was achieved by hydrolysis of ptesculentoside (1a) with 10% HCl in
methanol followed by treatment with trifluoroacetic anhydride.24
In enantioselective GC-coinjection studies, identical retention
times were observed between the hydrolysate of ptesculentoside
15. Smith, B. L.; Seawright, A. A.; Ng, J. C.; Hertle, A. T.; Thomson, J. A.; Bostock, P. D.
Nat. Toxins 1994, 2, 347–353.
16. Smith, B. L.; Embling, P. P.; Agnew, M. P.; Lauren, D. R.; Holland, P. T. N. Z. Vet. J.
1988, 36, 56–58.
17. Rasmussen, L. H.; Lauren, D. R.; Smith, B. L.; Hansen, H. C. B. N. Z. Vet. J. 2008, 56,
304–309.
18. Rasmussen, L. H.; Bruun Hansen, H. C.; Lauren, D. Chemosphere 2005, 58, 823–
835.
(1a) and authentic D
-glucose.25
19. Williams, D. H.; Fleming, I. Spectroscopic Methods in Organic Chemistry, 5th ed.;
McGraw-Hill: Glasgow, 1995.
This study has revealed the presence of the previously unknown
norsesquiterpene glucoside ptesculentoside (1a) in P. esculentum,
together with comparable proportions of ptaquiloside (1b) and les-
ser amounts of caudatoside (1c). These three compounds demon-
strate similar chemical reactivity and presumably have similar
biological activity.
20. The methyl substituent at C-2 in ptaquiloside (2a) [and pterosin (2b)] also lies
in the
a
position,7,8 but the C-2 stereochemistry in these compounds is 2R as
the methyl group is of lower priority order.
21. Fukuoka, M.; Kuroyanagi, M.; Yoshihira, K.; Natori, S. Chem. Pharm. Bull. 1978,
26, 2365–2385.
22. Yoshihira, K.; Fukuoka, M.; Kuroyanagi, M.; Natori, S.; Umeda, M.; Morohoshi,
T.; Enomoto, M.; Saito, M. Chem. Pharm. Bull. 1978, 26, 2346–2354.
23. Tanaka, N.; Murakami, T.; Saiki, Y.; Chen, C.-M.; Gomez, P. L. D. Chem. Pharm.
Bull. 1981, 29, 3455–3463.
Acknowledgements
24. König, W. A.; Benecke, I.; Bretting, H. Angew. Chem., Int. Ed. Engl. 1981, 20, 693–
694.
The authors thank: Barry Blaney (Queensland Primary Indus-
tries and Fisheries) for plant collection, Mark Edginton (Queens-
land Herbarium) for plant identification and Lynette Lambert
(Centre for Magnetic Resonance, University of Queensland). Lars
Rasmussen (Frederiksberg, Denmark) provided authentic ptaquilo-
side and pterosin B for comparison. This study was partly funded
by the Meat and Livestock Australia (Project AHW.017).
25. Enantioselective GC analyses were performed on a Chirasil-
column (25 m ꢁ 0.32 mm ꢁ 0.20 m). The retention times for the standards
were: -Glc (26.10 and 29.79 min), -Glc (26.17 and 29.96 min). For the
L-Val capillary
l
L
D
hydrolysate of ptesculentoside (1a), peaks were observed at 26.10 and
29.90 min. During coinjection studies, identical retention times were
observed between the hydrolysate of (1a) and authentic
peaks observed are a result of the formation of and/or b anomers of pyranose
forms and coincide with the number previously reported for this sugar.24
D-Glc. The multiple
a