Synthesis of cardenolide glycosides and putative biosynthetic precursors of cardenolide glycosides

Article abstract of DOI:10.1016/S0039-128X(97)00118-9

A rapid and efficient procedure for glycosylation of steroids was established using a modified Koenigs-Knorr procedure. Peracetylated β- glycosides were synthesized by reaction of cardenolides, various pregnanes and 23-nor-5,20(22)E-choldienic acid at room temperature with the peracetylated 1-bromo derivatives of D-glucose, D-galactose, D-fucose and cellobiose. Subsequent deprotection was performed by alkaline hydrolysis with sodium methoxide. Structures of the respective glycosides were established by NMR techniques. The complete protocol was shown to be non-destructive at all stages to the sugar moiety and the steroidal nucleus. The γ-unsaturated lactone ring of the cardenolides was shown to remain intact and no formation of C-14 unsaturated compounds was observed.

Full text of DOI:10.1016/S0039-128X(97)00118-9

Synthesis of cardenolide glycosides
and putative biosynthetic precursors
of cardenolide glycosides
Melitta Luta, Andreas Hensel, and Wolfgang Kreis
Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Institut fu¨r Botanik und Pharmazeutische
Biologie, Erlangen, Germany
A rapid and efficient procedure for glycosylation of steroids was established using a modified Koenigs–Knorr
procedure. Peracetylated ␤-glycosides were synthesized by reaction of cardenolides, various pregnanes and
23-nor-5,20(22)E-choldienic acid at room temperature with the peracetylated 1-bromo derivatives of D-glucose,
D-galactose, D-fucose and cellobiose. Subsequent deprotection was performed by alkaline hydrolysis with sodium
methoxide. Structures of the respective glycosides were established by NMR techniques. The complete protocol
was shown to be non-destructive at all stages to the sugar moiety and the steroidal nucleus. The ␥-unsaturated
lactone ring of the cardenolides was shown to remain intact and no formation of C-14 unsaturated compounds
was observed. (Steroids 63:44–49, 1998) © 1998 by Elsevier Science Inc.
Keywords: cardenolides; glycosylation; norcholenic acid; pregnanes; NMR
thesis of new pharmacological active compounds such as
new cardenolide glycosides or putative biosynthetic precur-
sors of steroid pathways such as pregnane glycosides or
glycosides of norcholenic acids. The glycosidation method
described here is a versatile variation of the Koenigs–Knorr
methodology which was applied to a variety of steroids,
using as carbohydrate components several hexoses, the sen-
sitive 6-deoxyhexose fucose and cellobiose as a model
substance for an oligosaccharide.
Introduction
Despite considerable progress in carbohydrate chemistry in
the past few years, the stereoselective formation of O-
glycosidic bonds between carbohydrates and steroids is
usually a time consuming process with relatively low yields
of the newly formed glycosides.1,2 This is due to the low
reactivity of the secondary alcohol functions in the steroid
moiety and the necessity to activate the glycosyl donors.
Methods described for glycosidation of steroids are mostly
based on the classical Koenigs–Knorr synthesis with ␣-1,2-
cis halogenated carbohydrates as reactants.1 These methods
are not generally applicable but have to be adapted to the
specific requirements of the substrates.3 The catalysts and
solvents employed, which influence stereoselectivity, and
the non-destructive removal of the blocking groups must be
optimized to obtain acceptable yields. In contrast to these
multi-stage procedures the few cases of direct synthesis of
O-glycosides using unprotected carbohydrates,4 highly re-
active glycosyl donors5 and enzymatic glycosylation6 are
very effective, but are restricted to special applications.
The aim of the present study was to establish a method
for O-glycosylation of steroids, capable of condensing any
mono- or oligosaccharide in acceptable yields stereoselec-
tively to the hydroxyl function at C-3 of a given steroid.
Such a method should be applicable widely for rapid syn-
Experimental
General
Chemicals used were purchased from Merck (Darmstadt, Ger-
many) in analytical grade quality. Acetobromoglucose, acetobro-
mogalactose and 21-acetoxy pregnenolone were purchased from
Sigma (Steinheim, Germany). Disilver maleinate was prepared as
described previously.7 23-Nor-5,20(22)E-chol-dienic acid-3␤-ol
was synthesized according to Maier et al.8 1H and 13C NMR
spectra were measured in deuterochloroform and deuterated meth-
anol on a Bruker AM 360 spectrometer at 360 and 90 MHz,
respectively. Chemical shifts are expressed in ␦ ppm downfield
relative to tetramethylsilane as internal standard. Coupling con-
stants are given in Hertz. Thin layer chromatography was per-
formed on precoated plates Silica G60 F254 (Merck, Darmstadt,
Germany). Mobile phase I for the separation of peracetylated
sugars: Ethyl acetate/CH2Cl2 (70:30 v/v); mobile phase II for the
separation of peracetylated and deprotected steroid glycosides:
CH2Cl2/MeOH/H2O (80:18:2 v/v/v). Spots were visualized by
spraying of the developed plates with a mixture of anisaldehyde
(0.5 mL), acetic acid (10 mL), methanol (85 mL) and sulfuric acid
(5 mL) with subsequent heating to 110°C for 5 min. Cardenolide
Address reprint requests to A. Hensel, Friedrich-Alexander-Universita¨t
Erlangen-Nu¨rnberg, Institut fu¨r Botanik und Pharmazeutische Biologie,
Staudtstr. 5, 91058 Erlangen, Germany.
Received January 20, 1997; accepted September 11, 1997.
Steroids 63:44–49, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(97)00118-9

Cardenolide glycosides: Luta et al.
glycosides were analyzed by HPLC.9 Melting points (m.p.) were
determined with a Bu¨chi 535 system and are uncorrected. Column
chromatography was performed on Sephadex LH 20 (Pharmacia,
Upsala, Sweden) with methanol as the mobile phase (1 mL/min);
column dimensions: 80 ϫ 2 cm. Elemental analysis was performed
with a Carlo Erba Elemental System. For additional purity testing
a minimum purity of not less than 95% was specified which was
determined by TLC or HPLC. All compounds described here
conform to this specification.
After neutralization and evaporation the residue was dissolved in 2
mL of methanol and purified by column chromatography on Seph-
adex LH20.
3␤-O-(␤-D-glucopyranosyl)-digitoxigenin (2) from 375 mg of
digitoxigenin (1). Afforded 247 mg (46%) product; m.p.: 226–
228°C (lit.11 228–230°C); TLC: Rf (II) ϭ 0.31; HPLC purity:
100% (tR 12.2 min); 1H-NMR(CD3OD) ␦ ppm: 0.88 (s, 3H, H-18),
0.96 (s, 3H, H-19), 2.83 (m, 1H, H-17), 3.18 (m, 1H, H-2Ј), 3.24
(m, 1H, H-5Ј), 3.30 (m, 2H, H-3Ј, H-4Ј), 3.65 (m, 1H, H-6ЈA), 3.83
(dd, 1H, H-6ЈB), 4.08 (br.s, 1H, H-3), 4.31 (d, J1Ј,2Ј ϭ 6.5 Hz, 1H,
H-1Ј), 4.85 (dd, J ϭ 18.1 Hz, 1H, H-21), 5.89 (t, J ϭ 3 Hz, 1H,
H-22); 13C-NMR(CD3OD) ␦ ppm: 16.40 (C-18), 24.06 (C-19),
62.84 (C-6Ј), 71.76 (C-4Ј), 75.21 (C-2Ј), 75.35 (C-3), 77.85 (C-5Ј),
78.25 (C-3Ј), 102.69 (C-1Ј), 117.78 (C-22), 177.25 (C-23), 178.46
(C-20); data conform to lit.11
General procedure for the peracetylation
of carbohydrates
The dried carbohydrate (5 mmol) was dissolved in absolute pyri-
dine (10 mL) and treated at 0°C with acetic anhydride (5 mL). The
reaction mixture was stirred for 2 h at 0°C and then kept at 4°C for
24 h. The excess of acetic anhydride was destroyed under ice
cooling by dropwise addition of water (1 mL). The solution was
extracted three times with CH2Cl2; the organic phases were com-
bined and subsequently washed with diluted H2SO4, Na2CO3 and
water. Multiple evaporation of the residue with ethanol afforded
pure products. 1,2,3,4-Tetra-O-acetyl-␤-D-fucopyranose from D-
fucose (0.82) was obtained as a colorless syrup (1.58 g, 94%);
TLC: Rf(l) ϭ 0.42. 1,2,2Ј,3,3Ј,4Ј,6,6Ј-Octa-0-acetyl-cellobiose
from D-cellobiose (1.71 g); the crude product was crystallized from
CH2Cl2 with pentane affording white crystals (2.6 g, 77%); m.p.
226–228°C (lit.10 228°C), TLC: Rf(l) ϭ 0.57.
3␤-O-(␤-D-galactopyranosyl)-digitoxigenin (3) from 375 mg of
digitoxigenin (1). Afforded 262 mg (49%) product; m.p. 228°C
(lit.11 211°C); TLC: Rf(II) ϭ 0.29; HPLC purity: 99% (tR 10.3
1
min); H-NMR(CD3OD) ␦ ppm: 0.88 (s, 3H, H-18), 0.96 (s, 3H,
H-19), 2.83 (m, 1H, H-17), 3.28 (m, 1H, H-2Ј), 3.52 (dd, 1H,
H-6AЈ), 3.80 (dd, 1H, H-6BЈ), 4.07 (s, 1H, H-3), 4.27 (d, J1Ј,2Ј
ϭ
7 Hz, 1H, H-1Ј), 4.87 (dd, J ϭ 18.1 Hz, 1H, H-21), 5.89 (t, 1H,
H-22); 13C-NMR(CD3OD) ␦ ppm: 16.44 (C-18), 24.09 (C-19),
62.43 (C-6Ј), 70.30 (C-4Ј), 72.70 (C-2Ј), 75.14 (C-3Ј), 75.36 (C-3),
76.48 (C-5Ј), 103.41 (C ϭ 1Ј), 117.80 (C-22), 177.25 (C-23),
178.48 (C-20); data conform to lit.11
General procedure for the activation of
3␤-O-(␤-D-fucopyranosyl)-digitoxigenin (4) from 375 mg of
digitoxigenin (1). Afforded 223 mg (43%) product; m.p. 195°C
(lit.6: 195°C); TLC: Rf(II) ϭ 0.49; HPLC purity: 95% (tR 27.4
peracetylated carbohydrates to the ␣-D halogenoses
The peracetylated carbohydrates (5 mmol) were dissolved in dry
CH2Cl2 (15 mL) and treated with HBr 33% in glacial acetic acid
(5 mL). The reaction mixture was stirred for 30 min at room
temperature. The solution was extracted three times with CH2Cl2;
the organic phases were combined and subsequently washed with
diluted H2SO4, Na2CO3 and water. The solvent was evaporated
and the residue dried at reduced pressure.
1
min); H-NMR(CD3OD) ␦ ppm: 0.88 (s, 3H, H-18), 0.97 (s, 3H,
H-19), 1.32 (d, J5,6 ϭ 6.5 Hz, 1H, H-6Ј), 2.87 (m, 1H, H-17),
3.56–3.63 (m, 3H, H-2Ј,3Ј,5Ј), 3.74 (m, 1H, H-4Ј), 4.03 (br.s, 1H,
H-3), 4.23 (d, J1Ј,2Ј ϭ 7.6 Hz, 1H, H-1Ј), 4.88 (dd, J ϭ 18.1 Hz,
2H, H-21), 5.87 (s, 1H, H-22); data conform to lit.6; 13C-
NMR(CD3OD) ␦ ppm: 16.41 (C-18), 17.14 (C-6Ј), 24.08 (C-19),
71.81 (C-5Ј), 72.37 (C-4Ј), 73,04 (C ϭ 2Ј), 83.80 (C-3Ј), 75.38
(C-3), 102.4 (C-1Ј), 117.80 (C-22), 177.28 (C-23), 178.50 (C-20).
2,3,4-tri-O-acetyl-␣-D-fucopyranosyl bromide from 1,2,3,4-Tetra-
O-acetyl-␤-D-fucopyranose (1.65 g) was obtained as a slightly yellow
1
syrup (1.74 g, 99%); TLC: Rf(l) ϭ 0.33; H-NMR (CDCl3): ␦ ppm
1.22 (d, J5,6 ϭ 6.5 Hz, 3H H-6), 2.01, 2.11, 2.17 (3 ϫ s, 3 ϫ 3 H, 3 ϫ
CH3-CO), 4.41 (q, J5,6 ϭ 6.5 Hz, 1H, H-5), 5.03 (q, J1,2 ϭ 3.9 Hz, J2,3
ϭ 10.4 Hz, 1H, H-2), 5.2–5.4 (m, 2H, H-3,4), 6.70 (d, J1,2 ϭ 3.9 Hz,
1H, H-1). Data conform to the lit.6 2,2Ј,3,3Ј,4Ј,6,6Ј-Hepta-O-acetyl-
cellobiosyl bromide from 1,2,2Ј,3,3Ј,4Ј,6,6Ј-Octa-O-acetyl-cellobiose
(2.26 g). The crude product was dissolved in acetone and crystallized
from pentane at 4°C (2.0 g, 86%); m.p 175–184°C (lit.10 180°C);
TLC: Rf(l) ϭ 0.45; 1H-NMR (CDCl3): ␦ ppm: 1.99–2.15 (7 ϫ s, 7 ϫ
3H, 7 ϫ CH3-CO), 3.67–3.87 (m, 2H, H-3Ј, H-4Ј), 3.84 (m, 1H,
H-2Ј), 4.05 (m, 1H, H-6BЈ), 4.17 (m, 1H, H-6B), 4.38 (m, 1H, H-6ЈA),
4.52 (m, 1H, H-5Ј), 4.55 (d, J ϭ 8 Hz, H-1Ј), 4.77 (m, 1H, H-6A), 4.95
(m, 1H, H-2), 5.10 (m, 2H, H-3, H-4), 6.53 (d, J ϭ 8Hz, H-1).
3␤-O-(␤-D-cellobiosyl)-digitoxigenin (5) from 375 mg of digi-
toxigenin (1). Afforded 271 mg (39%) product; m.p. 212°C; TLC:
Rf(II)
NMR(CD3OD) ␦ ppm: 0.88 (s, 3H, H-18), 0.95 (s, 3H, H-19), 2.83
(m, 1H, H-17), 4.06 (br.s, 1H, H-3), 4.31 (d, J1Љ,2Љ ϭ 8 Hz, 1H,
H-1Љ), 4.44 (d, J1Ј,2Ј ϭ 8 Hz, 1H, H-1Ј), 4.88 (dd, J ϭ 18.1 Hz, 1H,
H-21), 5.89 (s, 1H, H-22); 13C-NMR(CD3OD) ␦ ppm: 16.42
(C-18), 24.08 (C-19), 72.02 (C-4Љ), 75.29 (C-3), 83.35 (C-4Ј),
103.20 (C-1Љ), 103.60 (C-1Ј), 117.80 (C-22), 177.23 (C-23),
178.48 (C-20).
ϭ
0.12; HPLC purity: 100%, (tR 9.7 min); 1H-
3␤-O-(␤-D-fucopyranosyl)-pregn-5-en-3␤-ol-20-one (11) from
316 mg of pregn-5-en-3␤-ol-20-one (10). Afforded 152 mg
(33%) product; m.p. 173–176°C; TLC: Rf(II) ϭ 0.65; 1H-
NMR(CD3OD) ␦ ppm: 0.63 (s, 3H, H-18), 1.00 (s, 3H, H-19), 1.35
(d, J5Ј,6Ј ϭ 6.5 Hz, 3H, H-6Ј), 2.12 (s, 3H, H-21), 2.53 (t, J16,17 ϭ
9 Hz, 1H, H-17), 3.53–3.66 (m, 3H, H-2Ј, H-3Ј, H-5Ј), 3.72 (br.s,
1H, H-4Ј), 4.15 (m, 1H, H-3), 4.32 (d, J1Ј,2Ј ϭ 7.6 Hz, 1H, H-1Ј),
5.36 (dd, J6,7 ϭ 6 Hz, 1H, H-6); 13C-NMR (CD3OD) ␦ ppm: 13.55
(C-18), 16.77 (C-6Ј), 19.82 (C-19), 71.79 (C-5Ј), 72.59 (C-4Ј),
73.16 (C-2Ј), 75.33 (C-3Ј), 75.91 (C-3), 103.36 (C-1Ј), 122.14
(C-6), 142.44 (C-5), 215.41 (C-20). Analysis calculated for
C27H42O6 (462.64): C 70.10, H 9.15. Found: C, 70.15; H, 9.27.
General procedure for glycosylation of
steroid alcohols
1 mmol of steroid and 2.75 mmol of disilver maleinate were
dissolved in a mixture of 12 mL of dry Et2O/CH2Cl2 (3:1 v/v). 2.5
mmol of peracetylated halogenose were added. The reaction mix-
ture was stirred for 72 h at room temperature. The mixture was
filtered and the clear solution evaporated to dryness. For deacety-
lation the residue obtained was dissolved in 10 mL of methanol
and treated with 50 ␮mol of sodium methoxide at 60°C for 8 h.
Steroids, 1998, vol. 63, January 45

Papers
Table 1 Chemical Shifts (ppm) in 1H- and 13C-NMR Spectra of ␤-D-fucose, Unglycosylated 6 and Fucosylated 7 5␤-pregnan-3␤-ol-20-
one
1H-NMR
13C-NMR
Atom
␤-D-Fucose
6
7
␤-D-Fucose
6
7
3
17
18
19
20
21
1Ј
—
—
3.50
2.57
0.52
0.89
—
3.93
2.57
0.52
0.88
—
2.03
4.16
3.45
3.45
3.57
3.57
1.17
—
—
71.0
63.7
13.4
12.3
209.2
—
79.25
64.82
13.80
12.71
212.40
—
103.27
73.12
75.26
72.43
71.69
16.83
—
—
—
—
—
—
—
2.03
—
—
5.3
3.36
3.52
3.62
3.69
1.15
97.10
72.71
73.92
72.42
71.70
16.59
—
2Ј
—
—
3Ј
—
—
4Ј
—
—
5Ј
—
—
6Ј
—
—
3␤-O-(␤-D-fucopyranosyl)-pregn-5-en-20-one-3␤,21-diol (14)
from 374 mg of pregn-5-en-20-one-3␤,21-diol-21 acetat (13).
Afforded 154 mg (32%) product. m.p. 163–165°C; TLC: Rf(II) ϭ
3␤-O-(␤-D-fucopyranosyl)-5␣-pregnan-3␤-ol-20-one (9) from
318 mg of 5␣-pregnan-3␤-ol-20-one (8). Afforded 178 mg
(39%) product; m.p. 185–187°C; TLC: Rf(II) ϭ 0.61; H-NMR
1
1
0.55; H-NMR(CD3OD) ␦ ppm: 0.73 (s, 3H, H-18), 1.03 (s, 3H,
␦ ppm: 0.60 (s, 3H, H-18), 0.84 (s, 3H, H-19), 1.26 (d, J5Ј,6Ј ϭ
H-19), 1.27 (d, J5Ј,6Ј ϭ 6.5 Hz, 3H, H-6Ј), 2.53 (t, 1H, H-17),
3.53–3.66 (m, 3H, H-2Ј, H-3Ј, H-5Ј), 3.72 (br.s, 1H, H-4Ј), 4.15
(m, 1H, H-3), 4.30 (t, 1H, H-21), 4.40 (d, J1Ј,2Ј ϭ 8 Hz, 1H, H-1Ј),
5.37 (dd, 1H, H-6); 13C-NMR(CD3OD) ␦ ppm: 13.60 (C-18),
16.84 (C-6Ј), 19.85 (C-19), 57.54 (C-17), 71.79 (C-5Ј), 72.35
(C-4Ј), 73.04 (C-2Ј), 73.78 (C-3Ј), 75.25 (C-3), 102.95 (C-1Ј),
122.50 (C-6), 142.11 (C-5), 212.36(C-20). Analysis calculated for
C27H42O7 (478.63): C,67.76; H, 8.85. Found: C, 67.73; H, 8.91.
6.5 Hz, 3H, H-6Ј), 2.10 (s, 1H, H-21), 2.62 (t, 1H, H-17), 3.49
(m, 3H, H-2Ј,3Ј,5Ј), 3.61 (m, 1H, H-4Ј), 4.00 (br. s, 1H, H-3),
4.31 (d, J1Ј,2Ј ϭ 7.6 Hz, 1H, H-1Ј); 13C-NMR(CD3OD) ␦ ppm:
12.71 (C-19), 13.80 (C-18), 16.85 (C-6Ј), 45.37 (C-5), 64.82
(C-17), 71.77 (C-5Ј), 72.35 (C-4Ј), 73.06 (C-2Ј), 75.19 (C-3Ј),
79.25 (C-3), 102.78 (C-1Ј), 212.40 (C-20). Analysis calculated
for C27H44O6 (464.64): C, 69.79; H, 9.55. Found: C, 69.12;
H, 9.55.
Figure 1 General reaction for ␤-glycosylation of steroids.
46 Steroids, 1998, vol. 63, January

Cardenolide glycosides: Luta et al.
3␤-O-(␤-D-fucopyranosyl)-5␤-pregnan-3␤-ol-20-one (7) from
318 mg of 5␤-pregnan-3␤-ol-20-one (6). Afforded 173 mg (38%)
product; m.p. 193°C; TLC: Rf(II) ϭ 0.63; H-NMR (CD3OD) ␦
3␤-O-(␤-D-fucopyranosyl)-23-Nor-5,20(22)E-choladienic
acid (17) from 387 mg 23-nor-5,20(22)E-choladienic acid
ethyl ester8 (16). Afforded 153 mg (30%) product; m.p. 160°C
start of decomposition; TLC: Rf(II) ϭ 0.63; 1H-NMR (CD3OD)
1
ppm: 0.52 (s, 3H, H-18), 0.88 (s, 3H, H-19), 1.25 (d, J5Ј,6Ј ϭ 6.5
Hz, 3H, H-6Ј), 2.03 (s, 1H, H-21), 2.57 (t, 1H, H-17), 3.45 (m, 3H,
H-2Ј,3Ј,5Ј), 3.59 (dd, 1H, H-4Ј), 3.93 (br. s, 1H, H-3), 4,16 (d,
J1Ј,2Ј ϭ 7.6 Hz, 1H, H-1Ј); 13C-NMR (CD3OD) ␦ ppm: 13.78
(C-18), 16.83 (C-6Ј), 23.81 (C-19), 64.89 (C-17), 71.69 (C-5Ј),
72.43 (C-4Ј), 73.12 (C-2Ј), 75.26 (C-3Ј), 75.73 (C-3), 103.27
(C-1Ј), 212.45 (C-20). Analysis calculated for C27H44O6 (464.64):
C, 69.79; H, 9.55. Found: C, 69.36; H, 9.61.
␦ ppm: 0.63 (s, 3H, H-18), 1.02 (s, 3H, H-19), 1.37 (d, J5Ј,6Ј
ϭ
6.5 Hz, 3H, H-6Ј), 2.16 (s, 1H, H-21), 2.37 (t, 1H, H-17), 3.67
(m, 3H, H-2Ј,3Ј,5Ј), 3.84 (br.s, 1H, H-4Ј), 4.02 (br.s, 1H, H-3),
4.32 (d, J1Ј,2Ј ϭ 7.6 Hz, 1H, H-1Ј), 5.44 (dd, 1H, H-6), 5.71 (s,
1H, H-22), 8.54 (s, 1H, -COOH); 13C-NMR (CD3OD) ␦ ppm:
14.29 (C-18), 16.77 (C-61Ј), 122.48 (C-6), 127.97 (C-22),
142.16 (C-5), 161.14 (C-20), 179.65 (C-23). Analysis calcu-
Figure 2 Cardenolides, pregnanes, and their gly-
cosylated products.
Steroids, 1998, vol. 63, January 47

Papers
lated for C29H44O7 (464.64): C, 69.02; H, 8.79. Found: C,
69.13; H, 8.81.
lysis. Removal of the acetyl protecting groups was per-
formed by strong alkaline hydrolysis with 50% sodium
hydroxide or sodium methoxide in methanol. These condi-
tions did not hydrolyze the ␥-lactone system of the buteno-
lide ring in cardenolides nor degraded the sensitive 20-keto-
21-hydroxyfunction of 14. Prolonged reaction times were
necessary when weak bases, such as sodium hydrogen car-
bonate,15 trialkylamine3,16 or N-methylpyrrolidine16 were
used for deprotection; only sodium methoxide and sodium
hydroxide were suitable for almost complete hydrolysis
within 8 h in overall yields of 30 to 50%.
The optimized general reaction sequence followed to
obtain 3␤-O-glycosyl-steroids is displayed in Figure 1. Ac-
cording to this procedure digitoxigenin 1 was glycosylated
easily to the respective ␤-D-glucoside 2, ␤-D-galactoside 3,
␤-D-fucoside 4 and ␤-D-cellobioside 5 in yields of about 40
to 50% referring to the starting material 1 (Figure 2).
Similar glycosydation procedures with D-fucose as carbo-
hydrate component were applied to 5␤-pregnan-3␤-ol-20-
one 6, 5␣-pregnan-3␤-ol-20-one 8 and pregnenolone 10 to
give the respective 3␤-O-D-fucopyranosides 7, 9 and 11 in
an overall yield of about 30% (Figure 2). Prior to the
glycosylation of pregn-5-en-20-one-3␤,21-diol 12 it was
necessary to protect the 21-hydroxyl group by formation of
the ethyl ester 13. The subsequent reaction sequence with
peracetylated fucosyl bromide yielded 21-hydroxypregn-5-
ene-3␤-D-fucoside 14. Norcholenic acids as putative bio-
synthetic precursors of cardenolides are characterized by a
C-23 carboxylic group which has to be protected by ethyl
ester formation prior to glycosylation procedures. Prepara-
tion of the 23-norchola-5,20(22)E-diene acid-3␤-D-
fucoside 17 from 3␤-hydroxy-23-norchola-5,20(22)E-diene
acid ethyl ester 16 showed the robustness and suitability of
the described glycosylation procedure for the rapid and
efficient preparation of various steroid glycosides.
Results and discussion
For the preparation of steroid glycosides the activated ␣-
bromo-derivatives of D-glucose, D-galactose, D-fucose and
D-cellobiose were reacted with the respective steroids. Sil-
ver maleinate as “catalyst” showed a superior purity profile
for the newly formed glycosides than silver perchlorate,
silver salicylate, Fetison’s reagent or mercury cyanide
which resulted in products with higher impurity profiles,
insufficient yield or poor stereoselectivity. Silver maleinate
on the other hand forced the carbohydrate moiety exclu-
sively into the desired ␤-configuration. This was proven by
NMR spectroscopy as exemplified by the respective NMR
data of unglycosylated 6 and fucosylated 7 5␤-pregnan-3␤-
ol-20-on (Table 1). As a result of glycosylation chemical
shifts of C-3 resp. at H-3 of the steroid changed significantly
compared to the unglycosylated steroids. All other signals
correspond well to those of the unglycosylated steroid and
are in good accordance with the published data.12,17 In the
1H-NMR the 3␣-H shifts from 3.5 to 4.0 ppm. In the
13C-NMR shifts for C-3 towards higher frequencies take
place in good accordance with shifts calculated by the
respective increments (8.3 ppm). Formation of the ␤-
glycosidic bond can also be proven by the signals originat-
ing from the carbohydrate moiety. In the 13C-NMR the
signals for C-2Ј to C-6Ј remain nearly unchanged whereas
for C-1Ј a typical downfield shift of about 6 ppm to 103
ppm occurs. Such
a shift is characteristic for ␤-
configuration whereas ␣-bonds are characterized by shifts
to about 100 ppm only.14 In the 1H-NMR a significant
upfield shift of the anomeric proton from about 5 ppm to 4
ppm is seen. All other carbohydrate-related proton signals
remain nearly unchanged. The coupling constants of the
doublet for H-1Ј of about 8 Hz in D-glucose, D-galactose,
D-fucose and D-cellobiose are typical for
a trans-
Acknowledgments
configuration of H-1Ј and H-2Ј and are correlated to ␤-
glycosidic linkage for these glycosides. Applying the Kar-
plus relation13 ␤-configuration of the glycosidic bond was
inferred.
The authors gratefully acknowledge financial support by the
Deutsche Pharmazeutische Gesellschaft and the Deutsche
Forschungsgemeinschaft (Grant He 1642/2-1).
No evidence was found for isomerisation or degradation
of the steroid part or the sugar moiety. The spectral data for
the relevant positions C-3 and C-1Ј of all synthesized fuco-
sides or other glycosides were consistent with the exem-
plary data evaluated for fucosylated 5␤-pregnan-3␤-ol-20-
one as shown in Table 1.
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