REVISTA BOLIVIANA DE QUÍMICA CHEMICAL EDUCATION : STRUCTURAL ELUCIDATION OF WITHANOLIDE GLYCOSIDES

REVISTA BOLIVIANA DE QUÍMICA VOLUMEN 20, No.1 - 2003

CHEMICAL EDUCATION : STRUCTURAL ELUCIDATION OF WITHANOLIDE GLYCOSIDES

José Antonio Bravo;a* Michel Sauvain;b Alberto Giménez;c Georges Massiot d;Catherine Lavaud;d

aLaboratorio de Química de Productos Naturales, Instituto de Investigaciones Químicas-IRD, Universidad Mayor de San Andrés, CP 303, La Paz;

bInstitut de Recherche pour le Développement (ex-ORSTOM), 213 rue Lafayette, 75480 Paris cedex 10, France; dInstituto de Investigaciones Fármaco Bioquímicas, Universidad Mayor de San Andrés, CP 20606, La Paz, Bolivia; dLaboratoire de Pharmacognosie UMR 6013 CNRS

Bâtiment 18, BP 1039, 51097 Reims, Cedex 2, France; *Corresponding author: [email protected]

Key Word Index: Withanolide glycosides, 2DNMR, FAB, LSIMS, Tandem MS, structural elucidation RESUMEN Los withanólidos glicosilados están creciendo como grupo de sustancias naturales, tanto en número como en interés desde el punto de vista de su tratamiento espectroscópico para la resolución de sus estructuras. Ponemos en consideración una metodología para el tratamiento abstracto de información espectral de RMN para lograr la elucidación estructural de withanólidos glicosilados, ésta incluye también las técnicas de espectrometría de masas como una herramienta complementaria para asegurar la propuesta estructural. ABSTRACT Withanolide glycosides is a group of substances that are growing in quantity and in structural interest. We submit to consideration a methodology for the abstract treatment of a set of NMR spectra to effect structural elucidation of glycoside withanolides, this includes as well mass spectrometry data as a complementary tool to ensure structural proposals. INTRODUCTION Withanolide glycosides are a group of natural substances, increasing in number and in structural complexity. For a structural study we adapt ourselves to known structural elucidation methodologies for glycosides, like those to solve structures of saponins.1 Consequently structural elucidation starts distinguishing two primary and clearly different elements: the genine or steroidal aglycone and the sugar chain. Strategies for structural elucidation of these two elements depend on the further utility envisioned for the compound (for instance a biological activity to be evaluated, or if in contrast, it regards only the fact of establishing the global structure). If a chemical degradation is not an inconvenient, a partial hydrolysis and further differential miscibility in appropriate solvents may conduct to the separation of the sugar chain from the aglycone. Sugar chain’s partial hydrolysis and subsequent comparison to patrons by TLC with concomitant NMR analyses are applied to effect sugar units’ identification. Spectroscopic analyses follow in order to establish all these partial structures. However, degradation methods can contribute to the apparition of artifacts, particularly out of the aglycone moiety. On the other hand, the aglycone and the sugar chain (the glycoside as a whole) can be structurally depicted throughout extensive use of bi-dimensional NMR techniques avoiding the hydrolytic treatment and preserving the natural character of the molecule. 2D techniques permit besides establishment of the aglycone structure, the description of the sugar chain throughout characterization of sugar units and the establishment of the sugar units’ internal order. Finding the anchor point for the sugar chain into the aglycone concludes the global structural elucidation. DISCUSSION A. Structure of the aglycone. From literature sources there are five steroidal lactones reported to present that support carbohydrates as side chains.2 Physalolactone B or (20R,22R)-1α-acetoxy-3β-20-trihydroxy-witha-5,24-dienolide in dunawitanines A and B,3, 4 and dunawithanines C, D and E.4 Withaferin A in sitoindosides IX and X.5 Tubocapsigenin A monoacetate in tubocapsides A and B.6 12β-hydroxyphysalolactone B composing dunawithanines F,4 and H7 and 12β-acetoxyphysalolactone B in dunawithanine G.7 Many spectroscopic tools are useful to structural

1


approaches to steroidal lactones, from IR spectrometric measurements, the more profitable information are absorption band values corresponding to the stretching of carbonyl of α,β-unsaturated δ-lactone and the enone function, appearing near 1660 cm-1 to 1710 cm-1. A saturated γ-lactone in perulactone2 containing as well an ester function exhibits carbonyl absorption bands at 1762 and 1732 cm-1. Jaborolactone2 with an enone (1740 cm-1), an α,β-unsaturated γ-lactone (1720 cm-1) and a α-hydroxy ketone (1680 cm-1). A vast majority of withasteroids contain two isolated chromophores (an enone or steroidal 2-en-1-one and an α,β-unsaturated δ-lactone). The summation of the UV absorption characteristics of these two chromophores is manifested by a single maximum near 220 nm (214 ~ 240) with high molar absorptivity.2 Exceptionally high values are recorded for withaphysalin E2 (312 nm) and withametelin B2 (314 nm). 1HNMR spectra show a withasteroid-characteristic-cluster corresponding to multiplets and many fine singlets in the interval of 0.5 to 3.0 ppm. Picking out dunawithanine G7 as an example, the 1HNMR spectrum exhibits for the aglycone part signals at low frequencies for six methyl groupings. Two angular methyl groups at δ 0.91 (H-18) and 1.03 (H-19), a tertiary methyl at δ 1.18 (H-21) and two methyl groups as substituents in the α,β-unsaturated δ-lactone function at δ 1.81 and 1.91 (H-27 and H-28). Lastly a methyl ester group resonates at δ 2.02. Other assignable signals are: a double doublet at δ 4.36 (1H, dd, J22, 23a = 13.3 Hz, J22, 23e= 3.5 Hz, H-22) for a geminal-to-lactone-function proton. A double doublet and a triplet for geminal-to-methyl-ester-function protons, δ 4.57 (1H, dd, J12, 11a = 10.3 Hz, J12, 11e = 4.0 Hz, H-12) and δ 4.85 (1H, t, J1, 2e,a = 2.3 Hz, H-1) respectively. A broadened double doublet for an ethylene proton appearing at δ 5.45 (1H, bdd, J6, 7a = 3.4 Hz, J6, 7e = 1.4 Hz, H-6).

AcOC1

28,AcOC12

6

A.1. Planar Structustarting point and thestablished. CH3-19C-5, C-6, C-3 and Cto the methyl ester The analysis of thethese correlate to Hconnects to CH2-4. Tfunction is put in ev

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1H NMR spectrum of dunawithanine G (300 MHz, CDCl3-CD3OD)

re of the aglycone. Targeting CH3-19 proton signals in the 1H NMR spectrum at δ 1.03 as a anks to 2JCH y 3JCH correlations in the HMBC spectrum, the decalyn partial structure AB can be correlates to C-1, C-9, C-5 and C-10. This correlates to H-4 and H-6. Protons H-4 correlate to -2, and H-6 correlates to C-4 and C-8. H-1 δ 4.85 (t, J1, 2e,a = 2.3 Hz) correlates to C-5, C-10 and carbonyl (δ 171,1 ppm), fact witnessing the presence of this ester function substituting on C-1. COSY H-H spectrum permits establishing a connectivity between H-1 and the two H-2, then -3 deshielded at 3.87 ppm for its geminal-to-oxygen-function character. H-3 through 3JH-H he ethylenic H-6 is neighbor of CH2-7 situated in 2.0 – 2.3 ppm. The existence of the δ-lactone

idence by the following correlations in the HMBC stack plot chart: H-22 (4.36 ppm) to lactone

2


carbonyl (167.3 ppm), C-26 to methyl H-27 (1.81 ppm). CH3-27 to C-25 (121.7 ppm), C-25 to CH3-28 (1.91 ppm), and from CH3-28 to C-24 (150.5 ppm) and to C-23 at 31.5 ppm (B).

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Main correlations revealed from the HMBC chart for the aglycone of dunawithanine G (cycles A and B)

The COSY spectrum guides us to the cloture of the lactone cycle through the 3JH-H correlation between H-23 and H-22 (A). Placing the steroidal side chain comes out from the establishment of a 3JH-C correlation between CH3-21 (1.18 ppm) and C-17 (55.2 ppm). In the HMBC chart it can be noticed that CH3-21 are correlated to C-22 (81.2 ppm) and to C-20 (74.9 ppm) as well. The partial structure corresponding to the fusioned cycles C and D an the steroidal side chain can be established (B). The HMBC analysis conducts to the establishment of correlations from the angular CH3-18 (0.91 ppm) to C-12, C-13, C-14 and C-17 (B). The singlet corresponding to protons of a second methyl ester function is overlapped in the proton spectrum by the CH3-28 singlet at 1.91 ppm. This fact is revealed by the a 3JH-C correlation that connects this signal (singlet at 1.91 ppm) with the carbonyl at δ 170.6. This acetate is connected to C-12 according to the cross peak for H-12 δ 4.57 ant the carbonyl at 170.6 ppm (B).

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Main correlations in the COSY chart (A) and in the HMBC chart (B) for the aglycone of dunawithanine G

A.2 Sterochemical structure of the aglycone. In the aglycone of a glycoside of withanolide we must generally define the stereochemistry at the following points: C-3, C-1, C-12, C-20 and C-22. The establishment of stereochemical definitions comes out basically from NMR data and from CD curves. A profound bibliographic research becomes shortcutting an some times indispensable as well. For our example dunawithanine G, we present the following sterochemical definitions: Proton H-12 is defined as α-axial due to its diaxial coupling constant (J = 10.3 Hz) with H-11 that rests so defined in an axial position. Proton H-1 possesses a coupling constant of J = 2.3 Hz with each H-2. This let suggest its β equatorial position. In the ROESY experiment an Overhauser effect of H-1 and CH3-19 as well as a second with H-11 (1.55 ppm) corroborates this position. No through-space effects are observed between H-1 and H-3. The chemical shift of H-3 (δ 3.87) indicates its α axial orientation according to a similitude existing between this value and those reported in the literature for geminal-to-oxygen-functions protons in α-axial positions:

3


Proton δ (ppm) Withanolide

H-3α axial 3.80 1α-acetoxydunawithagenine (physalolactone B)3,8

H-3α axial 3.87 (20S,22R)-1α-acetoxy-3β-hydroxy-witha-5,24-dienolide8

H-3α axial 3.87 3β,20αF -dihydroxy-1-oxo-20R,22R-witha-5,24-dienolide9

H-7α axial 3.85 (20R:22S)- 4β,7β,20-trihydroxy-1-oxo-witha-2,5,24-trienolide10

According to the literature11,12 all withanolides with non modified or even modified α-oriented side chains invariably possess a 17β-OH group. This means that no α-oriented side chain withanolides without a 17β-OH group are known to present. The simple fact of observing the chemical shift of C-17 let establish its non oxygenated nature. Comparing this value in dunawithanine G with those reported in the literature mentioned above permit to define the orientation of the side chain as β. The proximity in values of chemical shifts of methyl groups H-18, H-21 and proton H-22 with those reported in the literature is very useful and corroborating at the moment of defining the side chain orientation.2 Stereochemistry at C-20 was stated as 20R. Justification follows a comparative analysis of the δ value of H-21 with those reported for various 20-OH cholesterol.13 This calculation applied to dunawithanine G conducts to the establishment of a differential of ∆δ =+0,28 ppm for H-21, fact leading to the conclusion of an α orientation for H-21 (20β-OH). To define the 22R configuration in the lactone ring, it is enough to observe a Cotton (+) effect at 250 nm in the DC curves.

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Complementarily, according to published data, the identification of the aglycone of dunawithanine G as a derivative of dunawithagenine,9 aids in adopting all defined stereochemistry for this one, previously investigated by crystallographic methods.14 B .Structure of the sugar chain. Determination of the structure of the sugar chain and the establishment of its anchor point on the aglycone is accomplished using the COSY and the HOHAHA experiments (for the identification of the sugar units) and the HMBC and ROESY experiments (to depict the internal sequence and the anchor point of the sugar chain). This task can be assisted by applying the same NMR techniques to the acetylated derivative of dunawithanine G. The proton spectrum of dunawitanine G shows an ensemble of signals of sugar protons in the interval 3.10 to 3.80 ppm. The COSY spectrum reveals that this ensemble is correlated with two anomeric proton signals namely δ 4.34 (1H, d, Jg1,g2 = 7.6 Hz, g1) and δ 4.81(1H, d, Jr1,r2 = 1.6, r1). The proton spectrum presents a doublet at δ 1.26 (3H, d, Jr6,r5 = 6.1 Hz, r6) correlated through a COSY cross peak to a sugar proton at δ 3.83 (qd, Jr5,r4 = 9.4 Hz, Jr5,r6 = 6.6 Hz, r5) supporting the hypothesis of the presence of rhamnose. The anomeric protons correlate to anomeric carbons at δ 101.1 (d, g1) and δ 101.9 (d, r1) in the XHCORR 1JCH spectrum. In the J-modulated 13CNMR spectrum, ten sugar carbon signals appear between 61 and 79 ppm, corresponding to nine oxymethynes and an oxymethylene. A rhamonse methyl signal is situated at δ 17.4 (q, r6). These values indicate the presence of two hexoses in the sugar chain, one of them a rhamnose. In the COSY chart we evidenced a system of coupled spins for four CHOH and one CH2OH, established from the anomeric proton at δ 4.34 (1H, d, Jg1,g2 = 7.6 Hz, g1). The HOHAHA spectrum corroborates the assignment of sugar signals from the anomeric proton. The following signals for the unknown hexose are defined: δ 4.34 (1H, d, J = 7.6 Hz, g1); δ 3.21 (1H, t, J = 8,3 Hz, g2); δ 3.44 (1H, m, g3); δ 3.51 (1H, m, g4); δ 3.28 (1H, m, g5); δ 3.62 (1H, dd, Jg6,g6’ = 11.9 Hz, Jg6,g5 = 4.9 Hz, g6); δ 3.73 (1H, dd, Jg6’,g6 = 11.9 Hz , Jg6’,g5 = 1.7 Hz, g6’).

4


r3 g6

r4g3

g4 g1 r2,r5

r1 g5 g2 g6’

g1-g2 g6’-g5 g6-g5 g5-g4

g6’-g6

r3-r4

r2-r3

r5-r4

g4-g3

g3-g2

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r1-r2 r2

g1 g2-g1

r1

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1 g1 12

22

r1 g1 r1

g1

12 22 22 12 g4

1 1

Zoom for the COSY spectrum(sugar zone), and for the XHCORR spectrum of direct heteronuclea

5

r correlations, dunawithanine G


The only proton signals defined regarding their coupling constants are g1, g2, g6 and g6’ for which g1 and g2 are axial. To establish the identity of the sugar unit, a proton and a COSY spectra must be run over the acetylated derivative of dunawithanine G. From anomeric proton g1 through all protons until g6, 3JH-H correlations are established. The measurement of the now more readily coupling constants on the split pattern for each proton signal let arrive to transdiaxial values for protons g1 to g5. So we are dealing with triacetyl-β-D-glucose (for an information regarding constant values see final table).

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Homonuclear correlations revealed by the COSY spectrum, dunawithanine Gacetylated

The diequatorial conformation between r1→r2 at δ 4.81 (1H, d, Jr1,r2 = 1.6 Hz, r1) and δ 3.82 (1H, dd, Jr2,r3 = 3.2 Hz, Jr2,r1 = 1.9 Hz, r2) can be seen in the HOHAHA and COSY spectra of the acetylated derivative. The diaxial character orientation for r3→r4 and r4 →r5 is obtained thanks to the reading of the vicinal coupling constants that are superior to 9 Hz. The equatorial-axial conformation of the couple r2eq→r3ax is deduced form de constant J =3,2 Hz. The existence of α-L-rhamnose has been so, proved. The assignment of 13C NMR sugars’ resonances was achieved through the XHCORR C-H direct correlation spectrum.

r2,r5

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r3 g4 g6’ g6 g2

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Zoom for the HOHAHA spectrum (sugar zone) of dunawithanine G

6


g3,r3 r2,r4 g2,r1 g4,r5

g1 g6’

22 g6 1

12 g5

g4-g5 g6-g5 g5

g6’-g5 g4,r5

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22 g6’

g2-g1 g6-g6’ g5-g6’

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g2,r1 1

r3-r2 r3,r4 r2,r4 r1-r2 r5-r4

r2-r3 g2-g3 g4-g3

Zoom for the COSY spectrum (sugar zone) of dunawithanine G acetylated

The quest of the anchor point of the sugar chain over the aglycone moiety is driven by the observation of a cross peak in the ROESY chart putting in evidence an Overhauser ROE effect of the anomeric proton of glucose g1 at 4.34 ppm with H-3 of the genine at 3.87 ppm. In this manner we have established that the sugar chain is branched to the genine moiety by the C-3. However due to overlapping of sugar signals in the proton spectrum the highly performing roesy experiment did not give a solution to the question about the inter – sugar branching point. Then the spectrum shows ambiguously two possible branching points: rhamnose(1→4)glucose o rhamnose(1→3)glucose. The definitive answer is found in the HMBC experiment where proton r1 is coupled to carbon g4 through a 3JH-C correlation.

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This chaining [rhamnose(1→4)glucose] can also be discovered by the observation of a great deshielding of ∆δ +1.76 ppm of proton g3 at δ 5.18 ppm of glucose in the acetylated derivative of dunawithanine G. This fact proves that rhamnose is not a substituent in position 3 of glucose. Proton g4 at δ 3.51 (m) in native dunawithanine G remains

7


practically unchanged at δ 3,83 (t, J = 9.3 Hz, g4) in the acetylated derivative, fact proving that this position was not affected by the acetylation reaction. Hence the nature of the sequence of sugars in dunawithanine is: α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl(1→3)-genine. After the global structural analysis undertaken, Dunawithanine G is (20R,22R)-O-(3)-[α-L-rhamnopyranosyl(1→4)-β-D-glucopyranosyl]-1α,12β-diacetoxy-20-hydroxy-witha-5,24-dienolide. C. Contribution of mass spectroscopy techniques to the establishment of structure Mass spectra were run under different techniques: bombardment by heavy ions (Cs+) in a matrix containing the sample (LSIMS), or bombardment with accelerated atoms (Ar), (FAB). Mass spectrometry in tandem (MS/MS) is also used to analyze fragmentation of fragments of withanolide glycosides.

C.1 FAB and LSIMS. The FAB positive spectrum of dunawithanine G describes a pseudo molecular ion [M+Li]+ at m/z 873 analyzed for de C44H66O17Li. Loss of the sugar chain on one hand and loss of the acetalic bond at C-3 on the other, produce an ion at m/z 547 and at m/z 563. This let conclude a mass of 556 mau for the aglycone analyzed as C32H44O8. The loss of the terminal rhamnose is evident from ion at m/z 720 [M+H-rhamnose]+ and at m/z 726 [M+Li-rhamnose]+. Peaks at m/z 593 for un ion of type 1,5Xglu and at m/z 755 for un ion of type 1,5Xrha, confirm the chaining glucose-rhamnose. The peak at m/z 369 (C21H30O5Li) corresponds to a retro-Diels-Alder fragmentation in cycle B of the aglycone.

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C39 H56 O14 Li

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563

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R=Ac 116710cR=H 112510a

m/z Dunawithanine G acetylated R=H 1125 Dunawithanine G peracetylated R=Ac 1167

According to the LSIMS spectrum run on acetylated dunawithanine G, this one possesses its lithiated molecular ion [M+Li]+ at m/z 1125 (C56H78O23Li). This derivative is present in a mixture with another minor peracetylated derivative (AcO-20) at m/z 1167 ([M+42+Li]+, C58H80O24Li).

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C24H34 O16 LiC32H44O8Li

C44H60 O15 Li

C45 H62O17Li (0,2 Xrha)

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835

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585

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593

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[M+Li]+

Extract from the LSIMS spectrum of dunawithanine G acetylated MS/MS spectrum C.2 MS/MS. Spectra MS/MS of pseudo molecular ion [M+Li]+ at m/z 1125 (C56H78O23Li) of dunawithanine G acetylated fragments into fragments at peaks at m/z 593 (1,5Xglu) and at 881 (1,5Xrha) that confirm the chaining glucose-rhamnose at position 3. Peaks at m/z 835 [M +Li-C12H18O8]+ and 777 [M+Li-C12H17O8-OAc]+ characterize

8


the loss of rhamnose and at the same time its terminal position. The excision of the acetalic bond at C-3 conducts to a series of ions: m/z 563 [aglycone+Li]+, m/z 547 [aglycone+Li-O]+ making possible deduction of the aglycone composition as C32H44O7,and m/z 585 [disacharide +Li]+ (C24H34O16). Two units AcOH are lost from m/z 547 to form the ion at m/z 427.

NMR data for dunawithanine G and its acetylated derivative

H Dunawithanine G¶

Dunawithanine G acetilada*

H sugars

Dun. ¶ Dun. acet* C Dun. ¶ C sugars

Dun. ¶

1e 4.85 t (2.3)

4.95 t (2.3)

Glu 1 75.4 Glu

2e 2.14 dd (16.2)

1 4.34 d (7.6)

4.57 d (7)

2 33.8 1 101.1

2a 1.75 dd (15. 3)

2 3.21 t (8.3)

4.80 dd (8,7, 7)

3 73.8 2 73.6

3a 3.87 m 3.80 m 3 3.44 m 5.18 t (9)

4 37.9 3 75.6

4e 2.48 dd (14, 5.5)

4 3.51 m 3.83 t (9.3)

5 137.0 4 79.1

4a 2.27 t (13.3)

5 3.28 m 3.58 m 6 124.6 5 75.2

6 5.45 brdd (3.4, 1.4)

5.52 brd (3.4, 1.4)

6 3.62 dd (11.9, 4.9)

4.27 dd (11.7, 3.3)

7 31.4 6 61.1

7e 2.00 dd (13.3, 3.3)

6’ 3.73 dd (11.9, 1.7)

4.41 dd (11.7, 2.7)

8 30.3

7a 2.27 t (13.3)

Rha 9 41.0 Rha

8a 1.48 m 1 4.81 d (1.6)

4.80 d (1.6)

10 40.5 1 101.9

9a 1.52 m 2 3.82 dd (3.2, 1.9)

5.03 m 11 27.0 2 71.1

11e 1.55 dd (13.3, 3.3)

3 3.60 dd (9.3, 3.6)

5.18 dd (9.3, 3.3)

12 81.0 3 71.2

11a 1.30 t (13.3)

4 3.39 t (9.5)

5.06 t (9.3)

13 47.2 4 72.6

12a 4.57 dd (10.3, 4)

4.63 dd (10.3, 4)

5 3.83 qd (9.4, 6.6)

3.83 m 14 56.1 5 69.7

14a 1.05 m 6 1.26 d (6.1)

1.16 d (6)

15 23.4 6 17.4

17a 1.75 m 16 23.3 18 0.91 s 0.98 s 17 55.2 19 1.03 s 1.06 s 18 10.1 21 1.18 s 1.26 s 19 19.3 22a 4.36 dd

(13.3, 3.5) 4.31 dd

(13.3, 3.5) 20 74.9

23e 2.09 dd (16.6, 4)

21 21.0

23a 2.45 t (16.6)

22 81.2

27 1.81 s 1.88 s 23 31.5 28 1.91 s 1.93 s 24 150.5

C1OAc 2.02 s 25 121.7 C12OAc 1.91 s 26 167.3

27 12.3 28 20.6 C1OAc 171.1 C1OAc 21.0 C12OAc 170.6 C12OAc 21.7

* in CDCl3; ¶ in CDCl3-CD3OD 9:1; Coupling constants (J) in Hz. REFERENCES 1 MASSIOT, G., LAVAUD, C. Structural Elucidation of Saponins

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Studies in Natural Products Chemistry Structure and Chemistry, 1995, 15, (Part C), Atta-ur-Rahman editor ,

Elsevier Science B.V., Oxford,. 1995. 2 RAY, A. B., GUPTA, M.

In Progress in the Chemistry of Organic Natural Products; Herz, W., Kirby, G. W., Moore, R. E., Steglich, W., Tamm, Ch., Eds.; Springer-Verlag: Wien, 1994; Vol. 63, 16-58.

3 ADAM, G., CHIEN, N.Q., KHOI, N.HW Dunawithanines A and B, the First withanolide Glycosides from Dunalia australis. Phytochemistry, 1984, 23, 2293-2297 4 LISCHEWSKI, L., HANG, N. T. B., PORZEL, A., ADAM, G., MASSIOT, G., LAVAUD, C. Withanolide Glycosides from Dunalia australis. Phytochemistry, 1992, 31, 939-942. 5 GHOSAL, S., KAUR, R., SRIVASTAVA, R., S. Sitoindosides IX and X, New Glycowithanolides from Withania somnifera, Indian J. of Nat. Prod., 1988, 4, 12-13 6 YOSHIDA, K., SHINGU, K., YAHAR, S., NOHARA T. A New Class of Ergostane Glycosides from Tubocapsicum anomalum, Tetrahedron Letters, 1988, 29, 675-676 7 BRAVO, B. J. A., SAUVAIN, M., GIMÉNEZ, A., BALANZA E., SERANI, L., LAPREVOTE, O., MASSIOT, G., LAVAUD, C. Trypanocidal Withanolides and Withanolide Glycosides from Dunalia brachyacantha. J. Nat. Prod, 2001, 64, 720-725 8 LISCHEWSKI, L., HANG, N. T. B., PORZEL, A., ADAM, G., MASSIOT, G., LAVAUD, C. Withanolides from Dunalia australis. Phytochemistry, 1991, 30, 4184-4186. 9 VANDE VELDE, V., LAVIE, D. New withanolides of biogenetic interest from Withania somnifera. Phytochemistry, 1981, 20, 1359-1363 10 ADAM G, HESSE M Strukturaufklärung eines C28-steroidlactons von withaferin-typ aus Dunalia australis (Griseb.)sleum. Tetrahedron, 1972, 28, 3527-3534. 11 GOTTLIEB, H. E., KIRSON, I.

13CNMR spectroscopy of the withanolides and other highly oxygenated C28 steroids. Org. Mag. Res. 1981, 16, 20-25 12 GLOTTER E.

Withanolides and related ergostane-type steroids. Nat. Prod. Rep. 1991, 8, 414-440. 13 MIJARES, A.; CARGILL, D. I.; GLASEL, J. A.; LIEBERMAN, S.

Studies on C-20 epimers of 20-hydroxycholesterol. J. of Org. Chem., 1967, 32, 810-812. 14 RECK, G., KHOI, N. H., ADAM, G. (20R:22R)-1α,3β,20-trihydroxy-witha-5,24-dienolide monohydrate (Dunawithagenine monohydrate) C28H42O5·H2O. Cryst Struct Comm, 1982, 11, 355-364.

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