Dopaol 2-keto- and 2,3-diketoglycosides from Chelone obliqua

Article abstract of DOI:10.1021/np0499416

Two unique 2-(3,4-dihydroxyphenyl)ethyl glycosides, namely, dopaol β-D-2-ketoglucopyranoside and dopaol β-D-2,3-diketoglucopyranoside, were isolated from Chelone obliqua together with the iridoid glucoside catalpol, dopaol β-D-glucopyranoside, descaffeoylverbascoside, and verbascoside. Glycosides with a diketosugar have not so far been isolated from natural sources.

Full text of DOI:10.1021/np0499416

1
052
J . Nat. Prod. 2004, 67, 1052-1054
Dop a ol 2-Keto- a n d 2,3-Dik etoglycosid es fr om Ch elon e obliqu a
Henrik Franzyk,†,§ Carl Erik Olsen, and Søren Rosendal J ensen*
‡
,†
Department of Chemistry, The Technical University of Denmark, DK-2800, Lyngby Denmark, and Department of Chemistry,
The Royal Danish Agricultural and Veterinary University, DK-1871 Frederiksberg C, Denmark
Received J anuary 29, 2004
Two unique 2-(3,4-dihydroxyphenyl)ethyl glycosides, namely, dopaol â-D-2-ketoglucopyranoside and dopaol
â-D-2,3-diketoglucopyranoside, were isolated from Chelone obliqua together with the iridoid glucoside
catalpol, dopaol â-D-glucopyranoside, descaffeoylverbascoside, and verbascoside. Glycosides with a
diketosugar have not so far been isolated from natural sources.
The genus Chelone (Scrophulariaceae) comprises four
Sch em e 1
species with a geographical distribution limited to North-
1
east America. The two closely related genera Chionophila
2
and Nothochelone with two and one species, respectively,
are both limited to the Western states. There have been
only two previous reports of phytochemical work on Che-
lone, namely, the chromatographic detection of the iridoid
glucosides catalpol and aucubin in C. lyonii Purch., C.
3
4
obliqua L., and C. glabra L. In the course of an investiga-
tion of the chemotaxomy of Scrophulariaceae we now report
on the water solubles of C. obliqua.
The water-soluble part of an ethanolic extract of C.
obliqua was fractionated by reversed-phase chromatogra-
phy with H O-MeOH mixtures to give the iridoid glucoside
2
catalpol. In addition, two novel glycosides (1 and 2) were
5
isolated together with dopaol â-D-glucopyranoside (3),
decaffeoylverbascoside,6 and the more common caffeoyl
7
phenylethyl glucoside (CPG) verbascoside.
Compound 1 was obtained as an amorphous glass, prone
to decompose partially upon high-vacuum drying, and it
was thus only characterized by NMR spectroscopy and by
1
13
2
HRFABMS. From the H and C NMR spectra of 1 in D O,
8
tion between the â-D-glucoside (3), â-D-alloside (4), â-D-
mannoside (5), and â-D-altroside (6) of dopaol was ca. 2:4:
8
it was obvious that it was a glycoside closely related to 3.
The observed [M - H] ion of m/z 311.0767 showed the
molecular formula to be C14
-
:1; the last two compounds proved to be inseparable even
16 8
H O and demonstrated the
by repeated HPLC. This showed that hydride addition to
the R-face of the 2-keto group was only slightly prevalent,
while hydride addition to the R-face of the 3-keto group
was more dominant. The structures of 5 and 6 were based
upon 2D NMR assignment of the 13C NMR spectra and
comparison of these with reported data for methyl â-D-
presence of two additional unsaturations when compared
to 3. Besides the signals from the dopaol aglycon, the 13
C
NMR spectrum revealed two low-intensity resonances (δ
4.3 and 95.8), signals from an anomeric carbon at δ 101.0,
two oxygenated methines at δ 70.2 and 76.0, and finally a
hydroxymethyl signal at δ 62.4. The H NMR signals were
consistent with this and displayed the coupling system
9
1
9
mannopyranoside and methyl â-D-altropyranoside. The
present isolation of a naturally occurring 2,3-diketoglyco-
side is to our knowledge unprecedented. However, the
dihydrate of the corresponding free sugar, â-D-erythro-
HOCH -CHOR-CHOH- together with a signal from an
2
anomeric proton (δ 4.66, linked to the carbon at δ 101.0
according to HSQC) appearing as a sharp singlet. Thus we
concluded that the carbohydrate part of 1 in solution was
the dihydrate of a 2,3-diketo-â-D-glucopyranoside (i.e., a
â-D-erythrohexos-2,3-diulopyranoside), which would ex-
plain the two low-intensity signals. Full assignments were
performed by using the HSQC and HMBC spectra; in the
latter correlations were seen between C-2 (δ 94.3) and H-1
hexos-2,3-diulopyranose (7), has been prepared enzymati-
cally and its X-ray structure determined.1
0,11
The free
diketoaldose was shown to exist as a single dihydrate form
even in DMSO solution; it was stable for several hours,
and the 2-monoketo form could only be detected by NMR
spectroscopy after equilibration for 1 day.11 Similar treat-
ment of 1, however, led to partial decomposition of the
compound. Comparison of the NMR data of the free 2,3-
diketosugar (δ 94.3, 94.7, 93.5, 69.4, 75.7, 61.8) in DMSO
with those of 1 showed a high degree of similarity, even
allowing for the difference in solvent, except for the
expected downfield C chemical shift for C-1 in the
glycoside.
(
δ 4.66) as well as between C-3 (δ 95.8) and H-4 (δ 3.63).
Further proof was obtained by subjecting 1 to sodium
borohydride reduction, which gave rise to all four possible
fully reduced dopaol glycosides. The approximate distribu-
1
1
1
3
*
To whom correspondence should be addressed. Tel: +45 45252103.
Fax: +45 45933968. E-mail: srj@kemi.dtu.dk.
†
The Technical University of Denmark.
Compound 2 could not be obtained in an entirely pure
state, partly because 1 and 2 eluted in overlapping frac-
tions, and even by repeated HPLC only a 10:1 mixture of
‡
The Royal Danish Agricultural and Veterinary University.
Present address: Dept. of Medicinal Chemistry, Danish University of
§
Pharmaceutical Sciences, Copenhagen, Denmark.
1
0.1021/np0499416 CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/25/2004

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 6 1053
Sch em e 2
Neither Chelone lyonii nor Nothochelone nemorosa (Doug-
las ex Lindl.) Straw appeared to contain any dopaol glyco-
sides as seen by an initial screening by NMR and HPLC.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. 1H and 13C NMR
spectra were recorded on a Bruker Avance DRX600 instrument
at 600 and 150 MHz, respectively, in D
1.4 and 2.23) as the standard. HRFABMS (J EOL J MS-
AX505W) were recorded in negative mode, using a bis-
2
O using acetone (δ
3
(hydroxyethyl)disulfide matrix.
P la n t Ma ter ia l. Flowering stems of Chelone obliqua were
collected from plants cultivated in The Botanical Garden of
The University of Copenhagen, Denmark. A voucher (IOK-4/
2
and 1 was obtained. Hence 2 was also only characterized
2
000) has been deposited at the Botanical Museum, The
by NMR spectroscopy and by HRFABMS. Again, the
overall appearances of the 1H and C NMR spectra of
compound 2 were similar to those of 1 and 3. The observed
University of Copenhagen, Denmark (C).
Extr a ction a n d Isola tion . Frozen flowering stems (50 g)
of C. obliqua were homogenized with EtOH (0.3 L), and the
13
-
[
C
M - H] ion of m/z 313.0924 gave the molecular formula
extract was partitioned in H
aqueous layer was concentrated to yield a water-soluble extract
(2.44 g), which was separated on a Merck Lobar (RP-18)
2
2
O-Et O (1:10, 150 mL). The
14
H
18
O
8
, corresponding to one less unsaturation than in
1
3
1
2
. As above, the C NMR spectrum of 2 in D O showed an
unusual carbohydrate moiety to be present in addition to
the signals arising from a dopaol aglycon. The glycon
comprised six carbon atoms, of which one appeared as a
low-intensity signal at unusually low field (δ 93.9), while
the other signals seemed to constitute three oxygenated
methines, a hydroxymethyl group, and an anomeric carbon
reversed-phase column (size C). Elution with H
mixtures (25:1 to 1.5:1) gave, after the polar front, first catalpol
105 mg), then a fraction of slightly impure dopaol â-D-2,3-
2
O-MeOH
(
diketoglucoside (1; 60 mg), a ca. 1:1 mixture (20 mg) of 1 and
dopaol â-D-2-ketoglucoside (2), followed by dopaol â-D-glucoside
(3; 120 mg), descaffeoylverbascoside (30 mg), a fraction (200
mg) containing di- or oligomeric hemiketals of 1, and finally
verbascoside (330 mg) was obtained. The above fraction (200
mg) containing hemiacetalic ketoglucosides was dissolved in
(δ 102.3). As was the case with 1, the anomeric proton (δ
4
.47) appeared as a sharp singlet, and consequently the
quaternary carbon at δ 93.9 could be assigned as a
hydrated 2-keto group. Using the COSY correlations and
starting from the typical hydroxymethyl signals (δ 3.90 and
2
H O and allowed to stand for 24 h, upon which analytical
HPLC showed the sample to be an approximately 1:1 mixture
of 1 and the original hemiketalic mixture. From this fraction
a further amount of 1 could be obtained by chromatography.
3
.71) with the usual large geminal coupling constant typical
1
Dop a ol â-D-2,3-d ik etoglu cosid e (1): H NMR δ 4.66 (1H,
of aldohexoses, it was possible to assign the remaining
s, H-1), 3.63 (1H, d, J ) 10.1 Hz, H-4), 3.55 (1H, ddd, J )
three one-proton signals in the region δ 3.9-3.4 to a single
1
0.1, 6.1 and 2.1 Hz, H-5), 3.89 (1H, dd, J ) 12.2 and 2.1 Hz,
coupling system, namely, HOCH -CHOR-CHOH-CH-
2
H-6a), 3.71 (1H, dd, J ) 12.2 and 6.1 Hz, H-6b), 2.86 (2H, t, J
7.0 Hz, H-R), 4.10 (1H, dt, J ) 10.1 and 7.0 Hz, H-â), 3.90
1H, dt, J ) 10.1 and 7.0 Hz, H-â), 6.89 (1H, d, J ) 2.1 Hz,
OH-. Thus, 2 was the hydrated form of a 2-ketoaldopy-
ranoside. This also allowed assignment of the 13C NMR
signals using the HSQC and HMBC spectra. Additional
proof for the structure of 2 was obtained by sodium
borohydride reduction, which gave a 1:3 mixture of dopaol-
â-D-glucoside (3) and dopaol-â-D-mannoside (5). In this case,
hydride addition on the 2-keto group had proceeded with
the expected preference for the less hindered R-face.
Keto forms of monosaccharides in aqueous solution exist
mainly as the hydrates, as seen from data for both anomers
)
(
H-2′), 6.88 (1H, d, 7.9 Hz, H-5′), 6.78 (1H, dd, 7.9 and 2.1 Hz,
H-6′); 13C NMR δ 101.0 (C-1), 94.3 (C-2), 95.8 (C-3), 70.2 (C-
4), 76.0 (C-5), 62.4 (C-6), 35.7 (C-R), 72.2 (C-â), 132.7 (C-1′),
1
6
3
18.0 (C-2′), 145.0 (C-3′), 143.5 (C-4′), 117.5 (C-5′), 122.4 (C-
-
′); HRFABMS m/z 311.0767 [M - H] (calcd for C14
15 8
H O ,
11.0757).
Dop a ol â-D-2-k etoglu cosid e (2). Fractions from several
workups containing mixtures of 1 and 2 were rechromato-
graphed three times. Finally, a fraction containing ca. 90% of
1
2
of 2-ketoglucose (glucosone) and also for methyl 3-keto-
was obtained: 1H NMR δ 4.47 (1H, s, H-1), 3.48 (1H, d, J )
.5 Hz, H-3), 3.42 (1H, t, J ) 9.5 Hz, H-4), 3.42 (1H, obsc, H-5),
2
9
1
3
glucosides obtained from the free sugars by enzymatic
processes.1
tected methyl glycosides has produced several 2-keto-,
-keto-, and 4-ketoglycosides, and NMR data (in D O) for
2-14
Likewise, chemical oxidation
15,16
of unpro-
3.90 (1H, dd, J ) 12.1, 1.7 Hz, H-6a), 3.71 (1H, dd, J ) 12.1,
5.7 Hz, H-6b), 2.85 (2H, t, J ) 7.0 Hz, H-R), 4.07 (1H, dt, J )
10.1, 7.0 Hz, H-â), 3.86 (1H, dt, J ) 10.1, 7.0 Hz, H-â), 6.87
3
2
(
8
3
1H, d, J ) 1.8 Hz, H-2′), 6.86 (1H, d, 8.1, H-5′), 6.77 (1H, dd,
the dihydrate form of methyl â-D-2-ketoglucopyranoside (8)
were also reported. We have found only two reports of
naturally occurring 2-ketoglycosides in the literature,
namely, on two saponins (gymnemic acids) from Gymnema
sylvestre18 (Asclepediaceae) as well as on antibiotics from
.1, 1.8 Hz, H-6′); 13C NMR δ 102.3 (C-1), 93.9 (C-2), 77.5 (C-
), 69.9 (C-4), 77.1 (C-5), 62.1 (C-6), 35.6 (C-R), 72.2 (C-â), 132.7
(C-1′), 118.0 (C-2′), 145.1 (C-3′), 143.5 (C-4′), 117.5 (C-5′), 122.4
-
(
3
C-6′); HRFABMS m/z 313.0924 [M - H] (calcd for C14
13.0923).
Dop a ol â-D-glu cosid e (3): 13C NMR δ 103.4 (C-1), 74.3 (C-
15 8
H O ,
1
9
a Streptomyces sp. However, several examples of iridoid
-ketoglucosides are known. In the genus Penstemon
3
(
2), 77.0 (C-3), 70.9 (C-4), 77.1 (C-5), 62.0 (C-6), 35.7 (C-R), 72.1
(C-â), 132.7 (C-1′), 118.0 (C-2′), 145.1 (C-3′), 143.5 (C-4′), 117.5
(C-5′), 122.4 (C-6′), essentially as reported,5 except for the
closely related to Chelone), serruloside1 has been isolated
9
2
0
from P. serrulatus, and dihydroserruloside has been
reported from P. confertus. Also, suspensolide21 from
different solvent used (MeOH-d ).
4
Viburnum suspensum (Sambucaceae) and clandonoside22
Sod iu m Bor oh yd r id e Red u ction of Ketoglu cosid es. A
sample of the above mixture of 1 and 2 (27 mg) was treated
together with 8-O-acetylclandonoside from Caryopteris ×
with NaBH
4 2
(5 mg) in H O (2 mL) at 0 °C for 0.5 h followed
Clandonensis (Lamiaceae) are naturally occurring iridoid
by an additional 4 h at room temperature. Upon addition of
3
-ketoglucosides. All of these iridoid compounds were
1
1
0% HOAc (2 mL), the reaction mixture was loaded on an RP-
8 Lobar column (size B), which was eluted with H O and then
shown to exist as the keto forms when their NMR spectra
were recorded in methanol; however, the last two com-
pounds were found to be hydrated when dissolved in
water.22
2
H O-MeOH (15:1 to 6:1). This gave an approximately 1:2
2
mixture (9 mg) of dopaol â-D-glucoside (3) and dopaol â-D-
alloside (4), an intermediary fraction containing an approxi-

1
054 J ournal of Natural Products, 2004, Vol. 67, No. 6
Notes
mately 1:1 mixture (3 mg) of 3 and dopaol â-D-mannoside (5),
and finally a fraction of 5 (13 mg) containing a small amount
of dopaol â-D-altroside (6), as seen by NMR analysis of the
fractions.
Ack n ow led gm en t. We thank the staff of The Botanic
Garden of The University of Copenhagen for providing the
plant material and Dr. O. Ryding, The Botanical Museum, for
the authentification.
Exp er im en t A. 1 (11 mg) was reduced with NaBH
at 0 °C for 0.5 h, then more NaBH (2 mg) was added and the
4
(2 mg)
4
Refer en ces a n d Notes
temperature was allowed to rise to 20 °C. Analysis of the
1
reaction mixture by H NMR and analytical HPLC showed an
(
1) Nelson, A. D.; Elisens, W. J . Am. J . Bot. 1999, 86, 1487-1501.
approximately 4:2:8:1 mixture of 4, 3, 5, and 6 (the last two
coeluting).
(2) Wolfe, A. D.; Elisens, W. J .; Watson, L. E.; Depamphilis, C. W. Am.
J . Bot. 1997, 84, 555-564.
(
3) Kooiman, P. Acta Bot. Neerl. 1970, 19, 329-340.
4) Bowers, M. D.; Boockvar, K.; Collinge, S. K. J . Chem. Ecol. 1993, 19,
Exp er im en t B. An approximately 1:1 mixture of 1 and 2
(
1
(
18 mg) was reduced as above with NaBH
4
(2 × 4 mg). H NMR
8
15-823.
and analytical HPLC showed the reaction mixture to be an
approximately 1:1:3 mixture of 3, 4, and 5.
(5) Shimomura, H.; Sashida, Y.; Adachi, T. Phytochemistry 1987, 26,
2
49-251.
(
(
6) Kikuchi, M.; Yamauchi, Y. Yakugaku Zasshi 1985, 105, 542-546.
7) (a) Scarpati, M. L.; Delle Monache, F. Ann. Chim. (Rome) 1963, 53,
Exp er im en t C. An approximately 1:1 mixture of 2 and 3
(
9 mg) was reduced as above with NaBH
4
(2 × 2 mg). Analysis
3
56-367.
(8) Birkofer, L.; Kaiser, C.; Thomas, U. Z. Naturforsch. 1968, 23b, 1051-
058.
9) Toyota, M.; Saito, T.; Asakawa, Y. Phytochemistry 1996, 43, 1087-
088.
demonstrated the reaction mixture to be an approximately 5:3
mixture of 3 and 4.
1
(
P u r ifica tion of Dop a ol Glycosid es. The above reaction
mixtures from experiments A-C were combined and purified
first by chromatography on an RP-18 Lobar column (size B),
1
(10) Bock, C,; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41,
27-66.
(
11) Volc, J .; Sedmera, P.; Havlicek, V.; Prikrylov a´ , V.; Daniel, G.
Carbohydr. Res. 1995, 278, 59-70.
2 2
which was eluted with H O and then with H O-MeOH (4:1)
to give a dopaol glycoside mixture, which together with the
previously obtained impure fractions (25 mg) from the initial
reduction experiment were subjected to further HPLC puri-
(
12) Sedmera, P.; Volc, J .; Havlicek, V.; Pakhomova, S.; J egorov, A.
Carbohydr. Res. 1997, 297, 375-378.
(13) Freimund, S.; Baldes, L.; Huwig, A.; Giffhorn, F. Carbohydr. Res.
2
002, 337, 1585-1587.
fication using a Merck Hibar RP-18 column (eluent: H O-
2
(
14) Freimund, S.; Huwig, A.; Giffhorn, F.; K o¨ pper, S. Chem. Eur. J . 1998,
MeOH, 10:1), which afforded successive fractions of 4 (3 mg),
a mixture of 4 and 3 (3 mg), pure 3 (3 mg), and finally a ca.
4
, 2442-2455.
(15) Volc, J .; Sedmera, P.; Halada, P.; Prikrylov a´ , V.; Daniel, G. Carbohydr.
Res. 1998, 310, 151-156.
1
0:1 mixture (13 mg) of 5 and 6.
Dop a ol â-D-a llosid e (4): 13C NMR δ 101.2 (C-1), 72.3 (C-
(16) Liu, H.-M.; Sato, Y.; Tsuda, Y. Chem. Pharm. Bull. 1993, 41, 491-
5
01.
2
), 71.5 (C-3), 68.1 (C-4), 74.8 (C-5), 62.4 (C-6), 35.7 (C-R), 72.1
(
17) Tsuda, Y.; Hanajima, M.; Matsuhira, N.; Okuno, Y.; Kanemitsu, K.
(
(
C-â), 132.8 (C-1′), 118.0 (C-2′), 145.1 (C-3′), 143.6 (C-4′), 117.5
Chem. Pharm. Bull. 1989, 37, 2344-2350.
(18) V e´ rtesy, L.; Aretz, W.; Fehlhaber, H.-W.; Kogler, H. Helv. Chim. Acta
8
C-5′), 122.5 (C-6′), essentially as reported, except for the
1
995, 78, 46-60.
4
different solvent used (MeOH-d ).
(
19) Kiuchi, F.; Liu, H. M.; Tsuda, Y. Chem. Pharm. Bull. 1990, 38, 2326-
Dop a ol â-D-m a n n osid e (5): 3C NMR δ 101.0 (C-1), 71.8
1
2328.
(
7
1
C-2), 74.2 (C-3), 68.1 (C-4), 77.5 (C-5), 62.3 (C-6), 35.7 (C-R),
(20) J unior, P. Planta Med. 1984, 50, 417-420.
(
21) Gering, B.; J unior, P.; Wichtl, M. Phytochemistry 1987, 26, 3011-
1.6 (C-â), 133.1 (C-1′), 118.0 (C-2′), 145.3 (C-3′), 143.7 (C-4′),
17.5 (C-5′), 122.5 (C-6′).
3
013.
(
22) Iwagawa, T.; Hase, T. Phytochemistry 1989, 28, 2393-2396.
Dop a ol â-D-a ltr osid e (6): 13C NMR δ 99.5 (C-1), 71.3 (C-
), 71.2 (C-3), 66.2 (C-4), 76.0 (C-5), 62.8 (C-6), 35.7 (C-R), 71.6
(23) Hannedouche, S.; J acquemond-Collet, I.; Fabre, N.; Stanislas, E.;
2
(
(
Moulis, C. Phytochemistry 1999, 51, 767-769.
C-â), 133.1 (C-1′), 118.0 (C-2′), 145.3 (C-3′), 143.7 (C-4′), 117.5
C-5′), 122.5 (C-6′).
NP0499416