An approach to the synthesis and attachment of scillabiose to steroids

Article abstract of DOI:10.1016/j.steroids.2011.02.010

Hellebrin and transvaalin are two naturally occurring saponins with biological activity. In the present paper, we describe a high yielding route to the synthesis and coupling of their shared glycone, scillabiose, to a model steroid. A convergent coupling

Full text of DOI:10.1016/j.steroids.2011.02.010

Steroids 76 (2011) 588–595
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
An approach to the synthesis and attachment of scillabiose to steroids
Filip S. Ekholma, Gyula Schneiderb, János Wölflingb,∗, Reko Leinoa,∗∗
a Laboratory of Organic Chemistry, Åbo Akademi University, Biskopsgatan 8, FI-20500 Åbo, Finland
b Department of Organic Chemistry, University of Szeged, H-6720-Szeged, Dóm tér 8, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Hellebrin and transvaalin are two naturally occurring saponins with biological activity. In the present
paper, we describe a high yielding route to the synthesis and coupling of their shared glycone, scill-
abiose, to a model steroid. A convergent coupling strategy utilizing a scillabiose-based glycosyl donor
was devised for the glycosylation. This convergent approach is appealing due to its high efficiency and
simple deprotection procedure and may find further use in total synthesis of naturally occurring saponins
and related compounds sharing the same glycone. Due to the widespread occurrence of this glycone in
nature, the complete NMR spectroscopic characterization of all compounds prepared herein is provided
as reference material. In addition, glycosylations were performed with the monosaccharide constituents
of scillabiose, thereby providing a limited series of glycosylated steroids for potential future evaluation
of the effects of the glycone on the overall biological activity.
Received 16 December 2010
Received in revised form 3 February 2011
Accepted 16 February 2011
Available online 23 February 2011
Keywords:
Carbohydrates
NMR spectroscopy
Glycosylation
Scillabiose
© 2011 Elsevier Inc. All rights reserved.
Androst-5-en-3␤-ol-17-one
Saponins
1. Introduction
erythromycine and daunomycin antitumor agents when the car-
bohydrate part is modified or removed [7]. Due to the occurrence
Hellebrin and transvaalin are bufenolides,
a
subclass of
of scillabiose and closely related compounds in several naturally
occurring biomolecules and pharmaceutically active species, we
became interested in the synthesis of this glycone. In the present
paper, we describe a high yielding, convergent strategy for the
synthesis of the scillabiose glycone and its monosaccharide con-
stituents, as well as their coupling reaction to a model steroid.
Being widely distributed in nature, the scillabiose moiety is relevant
from both the synthetic and analytical points of view. The synthetic
methodology presented herein should thus be of interest for sev-
eral areas of research. Importantly, for reference purposes, we also
describe here the fully characterized 1H and 13C NMR spectra of all
of the building blocks as well as the glycosylated steroids.
saponins, and are well known for their pharmacological activities.
Hellebrin has been isolated from Helleborus niger and is notori-
ous for its potent cytotoxic activity [1]. In addition, hellebrin has
been found to display T-cell suppressive effects and has found fur-
ther use as an immunoregulatory molecule [2]. Transvaalin, also
called scillaren, has been isolated from Urginea sanguinea and has
been extensively used as a constituent of herbal medicines to purify
blood, remove abdominal pain and backache and as an abortifacient
[3]. Transvaalin is also known for its poisonous effect on livestock
[4].
As shown in Fig. 1, the carbohydrate parts, scillabiose (4-O-(␤-
d-glucopyranosyl)-␣-l-rhamnopyranoside), of these two saponins
are identical. The scillabiose moiety has also been found in Scilla
maritime as the glycone part of glucoscilliphaeoside [5]. In addition
to the presence in these saponins, a closely related glycon contain-
ing glucuronic acid instead of glucose has been found in molecules
isolated from Acrosiphonia centralis, ulva lactuca and klebsiella [6].
It is well known that the biological interactions displayed by
biomolecules depend on both their aglycone and glycone struc-
tures, as exemplified by the complete lack of activity of the
2. Experimental
Reaction solvents were dried and distilled prior to use when nec-
essary. All reactions containing moisture- or air-sensitive reagents
were carried out under argon atmosphere. The NMR spectra
were recorded with a Bruker Avance spectrometer operating at
600.13 MHz (1H: 600.13 MHz, 13C: 150.90 MHz). The probe tem-
perature during the experiments was kept at 25 ◦C unless indicated
otherwise. All products were fully characterized by utilization of
1H, 1D-TOCSY and 13C 1D-NMR techniques in combination with
DQF-COSY, NOESY, HSQC and HMBC 2D-NMR techniques by using
pulse sequences provided by the manufacturer. Chemical shifts
are expressed on the ı scale (in ppm) using TMS (tetramethylsi-
lane), residual chloroform, acetone, H2O or methanol as internal
∗
Corresponding author. Fax: +36 62 454200.
Corresponding author. Tel.: +358 400 707195.
E-mail addresses: wolfling@chem.u-szeged.hu (J. Wölfling), reko.leino@abo.fi
∗∗
(R. Leino).
0039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.02.010

F.S. Ekholm et al. / Steroids 76 (2011) 588–595
589
Fig. 1. Structures containing the scillabiose glycon: hellebrin (left) and transvaalin (right).
ꢀ
ꢀ
standards. Coupling constants are given in Hz and provided only
once when first encountered. Coupling patterns are given as s (sin-
glet), d (doublet), t (triplet) etc. The following indexes are used to
distinguish between protons on the same carbon: eq (equatorial)
and ax (axial). The computational analysis of the 1H NMR of all
compounds was performed by use of PERCH NMR software with
starting values and spectral parameters obtained from the various
NMR techniques applied [8]. HRMS were recorded using a Bruker
Micro Q-TOF with ESI (electrospray ionization) operated in posi-
tive mode. Optical rotations were measured at 23 ◦C with a Perkin
Elmer 241 polarimeter equipped with Na-lamp (589 nm) and are
reported in units of 10−1 degree cm2 g−1. TLC was performed on
aluminium sheets precoated with silica gel 60 F254 (Merck). Flash
chromatography was carried out on silica gel 60 (0.040–0.060 mm,
Merck). Spots were visualized by UV followed by charring with 1:10
H2SO4/MeOH and heating.
5.66 (dd, 1 H, J4 ,5 = 10.0 Hz, H-4ꢀ), 5.66 (d, 1 H, J1,2 = 0.9 Hz, H-1),
5.52 (dd, 1 H, J2 ,1 = 8.0 Hz, H-2ꢀ), 5.34 (d, 1 H, H-1ꢀ), 4.66 (dd, 1
ꢀ
ꢀ
ꢀ
ꢀ
H, J6 a,5 = 3.1 Hz, J6 a,6 b
ꢀ
ꢀ
= −12.1 Hz, H-6ꢀa), 4.48 (dd, 1 H, J6 b,5
ꢀ
ꢀ =
5.7 Hz, H-6ꢀb), 4.20 (dd, 1 H, J2,3 = 5.6 Hz, H-2), 4.15 (ddd, 1 H, H-5ꢀ),
4.04 (dq, J5,6 = 6.2, J5,4 = 10.0 Hz, H-5), 4.03 (dd, 1 H, J3,4 = 7.5 Hz, H-
3), 3.67 (dd, 1 H, H-4), 1.48 and 1.26 (each s, each 3 H, O2C(CH3)2),
1.22 (d, 3 H, H-6) ppm.
13C NMR (150.9 MHz, CDCl3): ı 166.1 (6ꢀ-OCOPh), 165.8 (3ꢀ-
OCOPh), 165.3 (2ꢀ-OCOPh, 4ꢀ-OCOPh), 133.5–127.7 (arom. C), 109.5
(O2C(CH3)2), 100.5 (C-1ꢀ), 83.6 (C-1), 81.0 (C-4), 77.7 (C-3), 76.6 (C-
2), 73.1 (C-3ꢀ), 72.3 (C-5ꢀ), 72.1 (C-2ꢀ), 69.9 (C-4ꢀ), 65.5 (C-5), 63.2
(C-6ꢀ), 27.9 and 26.3 (O2C(CH3)2), 17.3 (C-6) ppm.
HRMS: calcd. for C49H46O13SNa [M+Na]+ 897.2557; found
897.2554.
2.4. Phenyl 4-O-(2ꢀ,3ꢀ,4ꢀ,6ꢀ-tetra-O-benzoyl-ˇ-d-
glycopyranosyl)-1-thio-˛-l-rhamnopyranoside
(6)
2.1. General procedure for glycosylation
To a solution containing the corresponding acceptor (1 equiv.)
and pre-activated 4 A MS in dry CH2Cl2 (1.6 ml/0.1 mmol substrate)
A solution containing 5 (300 mg, 0.34 mmol) in 80% AcOH
(5 ml) was stirred at 80 ◦C for 5 h, brought to rt and concentrated.
The crude product was purified by column chromatography with
hexane–EtOAc 2:3 as eluent to yield 6 as a colorless oil (210 mg,
95%). Rf = 0.28 (hexane–EtOAc 1:1); [␣]D −125.0 (c 0.1, CHCl3).
1H NMR (600.13 MHz, CDCl3): ı 8.04–7.20 (m, 25 H, arom. H),
˚
was added TMSOTf (0.2 equiv.) at −20 ◦C. The reaction mixture
was stirred for 10 min and the corresponding donor (1.4 equiv.)
dissolved in dry CH2Cl2 (1.7 ml/0.1 mmol substrate) was added
dropwise to the solution. The resulting mixture was stirred for
1.5–2 h, brought to rt, diluted with CH2Cl2 (20 ml) and washed with
sat. NaHCO3-solution (20 ml). The organic phase was dried over
Na2SO4, filtered and concentrated. The crude product was puri-
fied by column chromatography (hexane–EtOAc, 4:1) to give the
corresponding glycosteroid.
5.95 (dd, 1 H, J3 ,4
ꢀ = 9.5 Hz, J3 ,2
ꢀ = 9.8 Hz, H-3ꢀ), 5.69 (dd, 1 H,
ꢀ
ꢀ
ꢀ = 9.9 Hz, H-4ꢀ), 5.56 (dd, 1 H, J2 ,1
ꢀ = 7.9 Hz, H-2ꢀ), 5.42 (d, 1
ꢀ
ꢀ
J4 ,5
H, J1,2 = 1.4 Hz, H-1), 5.29 (d, 1 H, H-1ꢀ), 4.70 (dd, 1 H, J6 a,5
ꢀ = 3.1 Hz,
ꢀ
J6 a,6 b
ꢀ
ꢀ
= −12.1 Hz, H-6ꢀa), 4.46 (dd, 1 H, J6 b,5
ꢀ
ꢀ = 5.3 Hz, H-6ꢀb), 4.18
(ddd, 1 H, H-5ꢀ), 4.18 (dq, J5,6 = 6.2, J5,4 = 9.6 Hz, H-5), 4.06 (dd, 1 H,
J2,3 = 3.4 Hz, H-2), 3.81 (dd, 1 H, J3,4 = 9.3 Hz, H-3), 3.74 (dd, 1 H, H-4),
1.26 (d, 3 H, H-6) ppm.
2.2. General procedure for deprotection
13C NMR (150.9 MHz, CDCl3): ı 166.2 (6ꢀ-OCOPh), 165.9 (3ꢀ-
OCOPh), 165.4 (4ꢀ-OCOPh), 165.3 (2ꢀ-OCOPh), 133.9–127.5 (arom.
C), 101.0 (C-1ꢀ), 87.3 (C-1), 81.4 (C-4), 73.1 (C-3ꢀ), 72.6 (C-2), 72.4
(C-2ꢀ), 72.3 (C-5ꢀ), 71.4 (C-3), 69.7 (C-4ꢀ), 67.5 (C-5), 62.9 (C-6ꢀ), 17.5
(C-6) ppm.
To a solution containing the protected glycosteroid (1 equiv.) in
dry MeOH or dry MeOH:THF mixture (2:1) (6.3 ml/0.1 mmol sub-
strate) was added NaOMe (2 equiv.) and the resulting mixture was
stirred for 3–20 h at rt, neutralized with DOWEX 50 H+-form, fil-
tered and concentrated. The crude product was purified by column
chromatography (CH2Cl2 → CH2Cl2–MeOH 3:1) to yield the depro-
tected glycosteroid.
HRMS: calcd. for C46H42O13SNa [M+Na]+ 857.2544; found
857.2215.
2.5. Phenyl 2,3-di-O-benzoyl-4-O-(2ꢀ,3ꢀ,4ꢀ,6ꢀ-tetra-O-benzoyl-ˇ-
d-glycopyranosyl)-1-thio-˛-l-rhamnopyranoside
(7)
2.3. Phenyl 2,3-O-isopropylidene-4-O-(2ꢀ,3ꢀ,4ꢀ,6ꢀ-tetra-O-
benzoyl-ˇ-d-glycopyranosyl)-1-thio-˛-l-rhamnopyranoside
(5)
6 (207 mg, 0.25 mmol) was dissolved in a mixture of pyridine
(3 ml) and BzCl (1.5 ml) and allowed to stir until TLC indicated
complete disappearance of starting material. The reaction was
quenched after 4 h with MeOH, concentrated, dissolved in CH2Cl2
(40 ml) and washed with water (2× 20 ml) and brine (20 ml). The
organic phase was dried over anhydrous Na2SO4, filtered and con-
Synthesized from 4 (91 mg, 0.3 mmol) and 2 (295 mg, 0.4 mmol)
according to the general procedure for glycosylation providing 5
as a white foam (290 mg, 85%). Rf = 0.41 (hexane–EtOAc 2:1); [␣]D
−73.3 (c 0.2, CHCl3). 1H NMR (600.13 MHz, CDCl3): ı 8.04–7.23 (m,
25 H, arom. H), 5.94 (dd, 1 H, J3 ,4 = 9.5 Hz, J3 ,2 = 9.8 Hz, H-3ꢀ),
ꢀ
ꢀ
ꢀ
ꢀ

590
F.S. Ekholm et al. / Steroids 76 (2011) 588–595
J5 ,6 = 6.2 Hz, J5 ,4 = 9.5 Hz, H-5ꢀ), 3.50 (dddd, 1 H, J3,2eq = 4.4,
J3,4eq = 4.8, J3,2ax = 11.4, J3,4ax = 11.4 Hz, H-3), 3.39 (dd, 1 H, H-4ꢀ),
2.48 (ddd, 1 H, J16eq,15eq = 0.4, J16eq,15ax = 8.8, J16eq,16ax = −19.5 Hz,
H-16eq), 2.38 (ddd, 1 H, J4eq,2ax = −2.1, J4eq,4ax = −13.1 Hz, H-
4eq), 2.20 (ddddd, 1 H, J4ax,7ax = 3.2, J4ax,7eq = 3.2 Hz, H-4ax), 2.13
ꢀ
ꢀ
ꢀ
ꢀ
centrated. The crude product was purified by flash chromatography
with hexane–EtOAc (1:0 → 4:1) as eluent to give 7 as a slightly
yellowish foam (230 mg, 89%). Rf = 0.60 (hexane–EtOAc 1:1); [␣]D
−35.0 (c 0.2, CHCl3). 1H NMR (600.13 MHz, CDCl3): ı 8.07–7.05 (m,
35 H, arom. H), 5.84 (dd, 1 H, J2,1 = 1.7, J2,3 = 3.3 Hz, H-2), 5.80 (dd, 1
H, J3 ,4 = 9.6 Hz, J3 ,2 = 9.9 Hz, H-3ꢀ), 5.67 (dd, 1 H, J4 ,5 = 9.9 Hz,
(dddd,
H, J16ax,15eq = 8.9, J16ax,15ax = 9.3 Hz, H-16ax), 1.98 (dddd,
1
H, J7eq,8 = 5.4, J7eq,7ax = −17.4 Hz, H-7eq), 2.11 (ddd,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
H-4ꢀ), 5.55 (dd, 1 H, J2 ,1 = 7.9 Hz, H-2ꢀ), 5.55 (d, 1 H, H-1), 5.55 (dd, 1
1
1
ꢀ
ꢀ
H, J3,4 = 9.7 Hz, H-3), 5.15 (d, 1 H, H-1ꢀ), 4.80 (dd, 1 H, J6 a,5 = 3.2 Hz,
H, J15eq,14 = 5.8, J15eq,15ax = −12.5 Hz, H-15eq), 1.88 (ddd, 1 H,
ꢀ
ꢀ
J6 a,6 b
ꢀ
ꢀ
= −12.1 Hz, H-6ꢀa), 4.48 (dd, 1 H, J6 b,5
ꢀ
ꢀ = 5.3 Hz, H-6ꢀb), 4.48
J1eq,2eq = 3.3, J1eq,2ax = 3.7, J1eq,1ax = −13.5 Hz, H-1eq), 1.87 (ddd,
(dq, J5,6 = 6.2 Hz, J5,4 = 9.4 Hz, H-5), 4.25 (ddd, 1 H, H-5ꢀ), 4.14 (dd, 1
H, H-4), 1.48 (d, 3 H, H-6) ppm.
1
H, J2eq,1ax = 3.8, J2eq,2ax = −12.6 Hz, H-2eq), 1.84 (ddd,
1 H,
J12eq,11eq = 2.8, J12eq,11ax = 4.3, J12eq,12ax = −13.5 Hz, H-12eq), 1.70
(dddd, J11eq,12ax = 4.2, J11eq,9 = 5.3, J11eq,11ax = −13.7 Hz, H-11eq),
1.69 (dddd, 1 H, J8,7ax = 10.3, J8,9 = 10.9, J8,14 = 11.2 Hz, H-8), 1.68
(ddd, 1 H, H-7ax), 1.59 (ddddd, 1 H, J2ax,1ax = 14.0 Hz, H-2ax), 1.58
(dddd, 1 H, J15ax,14 = 12.7 Hz, H-15ax), 1.51 (dddd, 1 H, J11ax,9 = 12.3,
J11ax,12ax = 13.8 Hz, H-11ax), 1.32 (ddd, 1 H, H-14), 1.28 (d, 1 H, H-6ꢀ),
1.29 (ddd, 1 H, H-12ax), 1.11 (ddd, 1 H, H-1ax), 1.05 (s, 3 H, H-19),
1.03 (ddd, 1 H, H-9), 0.91 (s, 3 H, H-18) ppm.
13C NMR (150.9 MHz, CDCl3): ı 166.1 (6ꢀ-OCOPh), 165.7 (3ꢀ-
OCOPh), 165.2 (4ꢀ-OCOPh), 165.1 (2ꢀ-OCOPh, 2-OCOPh), 164.8
(3-OCOPh), 137.8–125.3 (arom. C), 101.3 (C-1ꢀ), 85.7 (C-1), 77.6 (C-
4), 72.8 (C-3ꢀ), 72.5 (C-3), 72.0 (C-5ꢀ, C-2), 71.8 (C-2ꢀ), 69-7 (C-4ꢀ),
68.5 (C-5), 62.9 (C-6ꢀ), 17.9 (C-6) ppm.
HRMS: calcd. for C60H50O15SNa [M+Na]+ 1065.2768; found
1065.2763.
13C NMR (150.9 MHz, CD3OD and CDCl3): ı 222.8 (C-17), 140.4
(C-5), 121.0 (C-6), 97.9 (C-1ꢀ), 76.2 (C-3), 72.8 (C-4ꢀ), 71.2 (C-3ꢀ),
72.1 (C-2ꢀ), 68.1 (C-5ꢀ), 51.6 (C-14), 50.1 (C-9), 47.6 (C-13), 38.2 (C-
4), 37.1 (C-1), 36.7 (C-10), 35.7 (C-16), 31.3 (C-8), 31.2 (C-12), 30.6
(C-7), 29.1 (C-2), 21.7 (C-15), 20.1 (C-11), 19.1 (C-19), 17.1 (C-6ꢀ),
12.3 (C-18) ppm.
2.6. 2,3-Di-O-benzoyl-4-O-(2ꢀ,3ꢀ,4ꢀ,6ꢀ-tetra-O-benzoyl-ˇ-d-
glycopyranosyl)-˛-l-rhamnopyranosose trichloroacetimidate
(3)
To a solution containing 7 (157 mg, 0.15 mmol) in acetone:H2O
(10:1, 4.4 ml) was added NBS (40 mg, 1.5 equiv.) at 0 ◦C. The
reaction was brought to rt and stirring was continued for 1 h.
This process was repeated twice after which TLC indicated the
reaction to be complete. The reaction was quenched with sat.
NaHCO3-solution (15 ml), diluted with CH2Cl2 (30 ml) and washed
with brine (20 ml). The organic phase was dried over anhy-
drous Na2SO4, filtered and concentrated. The crude product was
purified by column chromatography with Hexane–EtOAc 4:1 as
eluent to yield 2,3-di-O-benzoyl-4-O-(2ꢀ,3ꢀ,4ꢀ,6ꢀ-tetra-O-benzoyl-
HRMS: calcd. for
457.2549.
C
25H38O6Na [M+Na]+ 457.2566; found
2.8. 3ˇ-(ˇ-d-Glucopyranosyloxy)-androst-5-en-17-one (12)
Synthesized from (37 mg, 0.13 mmol) and (133 mg,
8
2
0.18 mmol) according to the general procedure for glycosylation.
Pure product was, however, not obtained and the deprotection step
was carried out according to the general procedure for deprotec-
tion providing 12 as a white powder (41 mg, 78% over two steps).
Rf = 0.60 (MeOH–CH2Cl2 1:5). 1H NMR (600.13 MHz, CD3OD and
CDCl3): ı 5.42 (ddd, 1 H, J6,4ax = −1.6, J6,7ax = 2.5, J6,7eq = 5.3 Hz, H-
␤-d-glycopyranosyl)-␣-l-rhamnopyranosose as
a white foam
(131 mg, 92%). 2,3-Di-O-benzoyl-4-O-(2ꢀ,3ꢀ,4ꢀ,6ꢀ-tetra-O-benzoyl-
␤-d-glycopyranosyl)-␣-l-rhamnopyranosose (97 mg, 0.10 mmol)
was dissolved in CH2Cl2 (2 ml) and DBU (3 ␮l, 0.1 equiv.) and
Cl3CCN (25 ␮l, 2.4 equiv.) was added at 0 ◦C. The reaction mixture
was stirred for 1.5 h, brought to rt, diluted with CH2Cl2 (30 ml)
and washed with brine (20 ml). The organic phase was dried over
anhydrous Na2SO4, filtered and concentrated. The crude product
was purified by column chromatography with Hex:EtOAc:Et3N
3:1:0.01 as eluent to give 3 as a white foam (86 mg, 77%).
Rf = 0.64 (hexane–EtOAc 1:1); [␣]D +25.2 (c 0.2, CHCl3). 1H NMR
(600.13 MHz, CDCl3): ı 8.71 (s, 1 H, OCNHCCl3), 8.06–7.05 (m, 30 H,
arom. H), 6.33 (d, 1 H, J1,2 = 2.0 Hz, H-1), 5.76 (dd, 1 H, J2,3 = 3.4 Hz,
6), 4.40 (d, 1 H, J1 ,2 = 7.8 Hz, H-1ꢀ), 3.86 (dd, 1 H, J6 a,5 = 2.6 Hz,
ꢀ
ꢀ
ꢀ
ꢀ
J6 a,6 b
ꢀ
ꢀ
= −11.9 Hz, H-6ꢀa), 3.71 (dd, 1 H, J6 b,5
ꢀ
ꢀ = 5.5 Hz, H-6ꢀb), 3.62
(dddd, 1 H, J3,2eq = 4.4, J3,4eq = 4.7, J3,4ax = 11.3, J3,2ax = 11.6 Hz, H-
3), 3.40 (dd, 1 H, J3 ,4 = 8.9 Hz, J3 ,2 = 9.3 Hz, H-3ꢀ), 3.35 (dd, 1
ꢀ
ꢀ
ꢀ
ꢀ
H, J4 ,5 = 9.8 Hz, H-4ꢀ), 3.29 (ddd, 1 H, H-5ꢀ), 3.21 (dd, 1 H, H-2ꢀ),
2.48 (ddd, 1 H, J16eq,15eq = 1.1, J16eq,15ax = 8.9, J16eq,16ax = −19.6 Hz,
H-16eq), 2.46 (ddd, 1 H, J4eq,2ax = −2.4, J4eq,4ax = −13.2 Hz, H-4eq),
2.30 (dddddd, 1 H, J4ax,2eq = −2.4, J4ax,7ax = 2.6, J4ax,7eq = 2.7 Hz,
H-4ax), 2.14 (dddd, 1 H, J7eq,8 = 5.0, J7eq,7ax = −17.3 Hz, H-7eq),
2.11 (ddd, 1 H, J16ax,15eq = 8.9, J16ax,15ax = 9.4 Hz, H-16ax), 1.99
(dddd, 1 H, J15eq,14 = 5.9, J15eq,15ax = −12.4 Hz, H-15eq), 1.96 (ddddd,
J2eq,1eq = 2.4, J2eq,1ax = 3.8, J2eq,2ax = −12.9 Hz, H-2eq), 1.90 (ddd,
ꢀ
ꢀ
H-2), 5.75 (dd, 1 H, J3 ,4 = 9.5 Hz, J3 ,2 = 9.9 Hz, H-3ꢀ), 5.62 (dd, 1 H,
ꢀ
ꢀ
ꢀ
ꢀ
J4 ,5 = 9.9 Hz, H-4ꢀ), 5.52 (dd, 1 H, J3,4 = 9.6 Hz, H-3), 5.49 (dd, 1 H,
ꢀ
ꢀ
J2 ,1 = 7.9 Hz, H-2ꢀ), 5.12 (d, 1 H, H-1ꢀ), 4.78 (dd, 1 H, J6 a,5 = 3.2 Hz,
ꢀ
ꢀ
ꢀ
ꢀ
J6 a6 b
ꢀ
ꢀ
= −12.0 Hz, H-6ꢀa), 4.45 (dd, 1 H, J6 b,5
ꢀ
ꢀ = 5.4 Hz, H-6ꢀb), 4.21
1
H, J1eq,2ax = 3.6, J1eq,1ax = −13.4 Hz, H-1eq), 1.82 (ddd, 1H,
J12eq,11eq = 2.6, J12eq,11ax = 4.2, J12eq,12ax = −12.8 Hz, H-12eq), 1.71
(dddd, H, J8,7ax = 10.9, J8,14 = 10.9, J8,9 = 11.1 Hz, H-8), 1.70
(ddd, 1 H, H-5ꢀ), 4.18 (dq, J5,6 = 6.2, J5,4 = 9.6 Hz, H-5), 4.12 (dd, 1 H,
H-4), 1.49 (d, 3 H, H-6) ppm.
1
HRMS: calcd. for C56H46O16Cl3Na [M+Na]+ 1116.1780; found
1116.1755.
(dddd, J11eq,12ax = 4.1, J11eq,9 = 4.8, J11eq,11ax = −13.9 Hz, H-11eq),
1.68 (dddd, 1 H, H-7ax), 1.64 (ddddd, 1 H, J2ax,1ax = 13.9 Hz, H-
2ax), 1.60 (dddd, 1 H, J15ax,14 = 12.8 Hz, H-15ax), 1.53 (dddd, 1 H,
J11ax,12ax = 12.5, J11ax,9 = 13.0 Hz, H-11ax), 1.34 (ddd, 1 H, H-14), 1.29
(ddd, 1 H, H-12ax), 1.11 (ddd, 1 H, H-1ax), 1.07 (s, 3 H, H-19), 1.03
(ddd, 1 H, H-9), 0.91 (s, 3 H, H-18) ppm.
2.7. 3ˇ-(˛-l-Rhamnopyranosyloxy)-androst-5-en-17-one (10)
Compound 10 was synthesized from 8 (32 mg, 0.12 mmol) and
1 (100 mg, 0.16 mmol) according to the general procedure for
glycosylation. Pure product was, however, not obtained and the
deprotection step was carried out according to the general proce-
dure for deprotection providing 10 as a white solid (40 mg, 89%
over two steps). Rf = 0.23 (EtOAc). 1H NMR (600.13 MHz, CD3OD
and CDCl3): ı 5.39 (ddd, 1 H, J6,4ax = −1.9, J6,7ax = 2.0, J6,7eq = 5.2 Hz,
13C NMR (150.9 MHz, CD3OD and CDCl3): ı 222.4 (C-17), 140.2
(C-5), 120.5 (C-6), 100.7 (C-1ꢀ), 78.1 (C-3), 76.1 (C-3ꢀ), 75.8 (C-5ꢀ),
73.1 (C-2ꢀ), 69.8 (C-4ꢀ), 61.1 (C-6ꢀ), 51.2 (C-14), 49.8 (C-9), 47.1 (C-
13), 38.0 (C-4), 36.7 (C-1), 36.2 (C-10), 35.2 (C-16), 30.9 (C-8), 30.8
(C-12), 30.2 (C-7), 28.9 (C-2), 21.2 (C-15), 19.7 (C-11), 18.4 (C-19),
12.6 (C-18) ppm.
C
25H38O7Na [M+Na]+ 473.2515; found
H-6), 4.88 (d, 1 H, J1 ,2 = 1.7 Hz, H-1ꢀ), 3.82 (dd, 1 H, J2 ,3
=
ꢀ
ꢀ
ꢀ
ꢀ
HRMS: calcd. for
473.2513.
3.4 Hz, H-2ꢀ), 3.71 (dd, 1 H, J3 ,4 = 9.4 Hz, H-3ꢀ), 3.69 (dq, 1 H,
ꢀ
ꢀ

F.S. Ekholm et al. / Steroids 76 (2011) 588–595
591
2.9. 3ˇ-(2ꢀ,3ꢀ-Di-O-benzoyl-4ꢀ-(2ꢀꢀ,3ꢀꢀ,4ꢀꢀ,6ꢀꢀ-tetra-O-benzoyl-ˇ-d-
glucopyranosyl)-˛-l-rhamnopyranosyloxy)-androst-5-en-17-one
(13)
2.08 (ddd, 1 H, J16ax,15eq = 9.0, J16ax,15ax = 9.4 Hz, H-16ax), 1.97
(dddd, 1 H, J15eq,14 = 5.8, J15eq,15ax = −12.5 Hz, H-15eq), 1.91 (ddd,
1 H, J1eq,2eq = 3.4, J1eq,2ax = 3.8, J1eq,1ax = −13.4 Hz, H-1eq), 1.85
(dddd, 1 H, J2eq,1ax = 3.8, J2eq,2ax = −12.7 Hz, H-2eq), 1.79 (ddd,
1 H, J12eq,11eq = 2.6, J12eq,11ax = 4.2, J12eq,12ax = −12.7 Hz, H-12eq),
1.70 (dddd, 1 H, J8,7ax = 10.5, J8,14 = 11.1, J8,9 = 11.2 Hz, H-8), 1.70
(dddd, J11eq,12ax = 4.3, J11eq,9 = 5.0, J11eq,11ax = −13.4 Hz, H-11eq),
1.68 (dddd, 1 H, H-7ax), 1.59 (dddd, 1 H, J15ax,14 = 12.7 Hz, H-
15ax), 1.58 (ddddd, 1 H, J2ax,1ax = 13.9 Hz, H-2ax), 1.54 (dddd, 1 H,
J11ax,9 = 12.6, J11ax,12ax = 13.5 Hz, H-11ax), 1.34 (ddd, 1 H, H-14), 1.31
(d, 1 H, H-6ꢀ), 1.27 (ddd, 1 H, H-12ax), 1.11 (ddd, 1 H, H-1ax), 1.06
(s, 3 H, H-19), 1.04 (ddd, 1 H, H-9), 0.89 (s, 3 H, H-18) ppm.
13C NMR (150.9 MHz, CD3OD): ı 223.8 (C-17), 141.9 (C-5), 122.4
(C-6), 105.7 (C-1ꢀꢀ), 99.6 (C-1ꢀ), 83.6 (C-4ꢀ), 78.2 (C-3ꢀꢀ), 78.0 (C-5ꢀꢀ),
77.9 (C-3), 76.1 (C-2ꢀꢀ), 72.6 (C-2ꢀ), 72.4 (C-3ꢀ), 71.5 (C-4ꢀꢀ), 68.5 (C-
5ꢀ), 62.7 (C-6ꢀꢀ), 53.0 (C-14), 51.8 (C-9), 48.8 (C-13), 39.5 (C-4), 38.5
(C-1), 38.0 (C-10), 36.7 (C-16), 32.8 (C-8), 32.7 (C-12), 31.9 (C-7),
30.6 (C-2), 22.8 (C-15), 21.5 (C-11), 19.8 (C-19), 18.1 (C-6ꢀ), 13.9
(C-18) ppm.
Synthesized from
3 (17 mg, 0.06 mmol) and 8 (82 mg,
0.07 mmol) according to the general procedure for glycosylation
providing 13 as a white foam (82 mg, 88%). Rf = 0.48 (hexane–EtOAc
1:1); [␣]D +15.0 (c 0.2, CHCl3). 1H NMR (600.13 MHz, CDCl3):
ꢀꢀ ꢀꢀ
ı 8.06–7.07 (m, 30 H, arom. H), 5.74 (dd, 1 H, J3
= 9.7 Hz,
,4
= 9.8 Hz, H-3ꢀꢀ), 5.63 (dd, 1 H, J4
= 9.9 Hz, H-4ꢀꢀ), 5.49
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
,5
J3
,2
(dd, 1 H, J3 ,2 = 3.4 Hz, J3 ,4 = 9.7 Hz, H-3ꢀ), 5.49 (dd, 1 H, J2 ,1
=
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1.5 Hz, H-2ꢀ), 5.49 (dd, 1 H, J2
= 7.9 Hz, H-2ꢀꢀ), 5.34 (ddd, 1 H,
ꢀꢀ ꢀꢀ
,1
J6,4ax = −1.8, J6,7ax = 2.1, J6,7eq = 5.2 Hz, H-6), 5.08 (d, 1 H, H-1ꢀꢀ), 5.01
(d, 1 H, H-1ꢀ), 4.78 (dd, 1 H, J6
= 3.3 Hz, J6ꢀꢀ ꢀꢀ = −12.0 Hz,
ꢀꢀ
ꢀꢀ
a,5
a,6
b
H-6ꢀꢀa), 4.46 (dd, 1 H, J6ꢀꢀ ꢀꢀ = 5.3 Hz, H-6ꢀꢀb), 4.21 (ddd, 1 H,
b,5
H-5ꢀꢀ), 4.06 (dq, J5 ,6 = 6.2 Hz, J5 ,4 = 9.5 Hz, H-5ꢀ), 3.49 (dddd,
1 H, J3,2eq = 4.5, J3,4eq = 4.8, J3,4ax = 11.3, J3,2ax = 11.5 Hz, H-3), 2.46
(ddd, 1 H, J16eq,15eq = 1.0, J16eq,15ax = 8.9, J16eq,16ax = −19.3 Hz, H-
16eq), 2.35 (ddd, 1 H, J4eq,2ax = −2.3, J4eq,4ax = −13.3 Hz, H-4eq),
2.27 (dddddd, 1 H, J4ax,2eq = −1.1, J4ax,7eq = 2.7, J4ax,7ax = 3.3 Hz,
H-4ax), 2.10 (dddd, 1 H, J7eq,8 = 5.0, J7eq,7ax = −16.8 Hz, H-7eq),
2.08 (ddd, 1 H, J16ax,15eq = 8.9, J16ax,15ax = 9.3 Hz, H-16ax), 1.94
(dddd, 1 H, J15eq,14 = 5.7, J15eq,15ax = −12.4 Hz, H-15eq), 1.93 (dddd,
ꢀ
ꢀ
ꢀ
ꢀ
HRMS: calcd. for
619.3080.
C
31H48O11Na [M+Na]+ 619.3094; found
1
H, J2eq,1ax = 3.9, J2eq,2ax = −12.2 Hz, H-2eq), 1.88 (ddd,
1
H,
3. Results and discussion
J1eq,2eq = 3.3, J1eq,2ax = 3.7, J1eq,1ax = −13.8 Hz, H-1eq), 1.86 (ddd, 1
H, J12eq,11eq = 2.7, J12eq,11ax = 4.3, J12eq,12ax = −13.1 Hz, H-12eq), 1.69
(dddd, J11eq,12ax = 4.1, J11eq,9 = 4.5, J11eq,11ax = −13.8 Hz, H-11eq),
1.67 (ddddd, 1 H, J2ax,1ax = 13.4 Hz, H-2ax), 1.66 (dddd, 1 H,
J8,7ax = 10.2, J8,14 = 10.8, J8,9 = 11.0 Hz, H-8), 1.62 (dddd, 1 H, H-
7ax), 1.54 (dddd, 1 H, J15ax,14 = 12.9 Hz, H-15ax), 1.50 (dddd, 1 H,
J11ax,9 = 11.0, J11ax,12ax = 13.4 Hz, H-11ax), 1.42 (d, 1 H, H-6ꢀ), 1.29
(ddd, 1 H, H-12ax), 1.28 (ddd, 1 H, H-14), 1.08 (ddd, 1 H, H-1ax),
1.05 (s, 3 H, H-19), 1.00 (ddd, 1 H, H-9), 0.89 (s, 3 H, H-18) ppm.
13C NMR (150.9 MHz, CDCl3): ı 221.2 (C-17), 166.1 (6ꢀꢀ-OCOPh),
165.7 (3ꢀꢀ-OCOPh), 165.4 (2ꢀ-OCOPh), 165.2 (4ꢀꢀ-OCOPh, 2ꢀꢀ-OCOPh),
164.8 (3ꢀ-OCOPh), 140.7 (C-5), 133.5–128.1 (arom. C), 121.1 (C-6),
101.4 (C-1ꢀꢀ), 95.9 (C-1ꢀ), 77.9 (C-3), 77.8 (C-4ꢀ), 72.8 (C-3ꢀꢀ), 72.3
(C-3ꢀ), 71.9 (C-5ꢀꢀ), 71.8 (C-2ꢀꢀ), 71.2 (C-2ꢀ), 69.8 (C-4ꢀꢀ), 67.0 (C-5ꢀ),
63.0 (C-6ꢀꢀ), 51.8 (C-14), 50.2 (C-9), 47.6 (C-13), 38.5 (C-4), 37.2 (C-
1), 36.9 (C-10), 35.9 (C-16), 31.5 (C-8), 31.4 (C-12), 30.8 (C-7), 29.3
(C-2), 21.9 (C-15), 20.3 (C-11), 19.4 (C-19), 18.0 (C-6ꢀ), 13.5 (C-18)
ppm.
3.1. Synthesis
Previously, at our laboratories, we have completed a thorough
investigation on finding an optimal glycosyl donor for glycosyla-
tion of steroids. After screening of several donors, containing both
electron withdrawing and electron donating protective groups,
promoters and reaction conditions, benzoylated imidate donors
were identified as superior in terms of efficiency and minimal
waste generation [9]. Additional advantages of the imidate donors
are the simple and rapid methods applied for their preparation
[10]. Glycosylation of steroids has received significant attention
in recent years as exemplified by several papers dealing with this
subject [11]. While several donors have been successfully applied
for glycosylation of sapogenins earlier, the high efficiencies dis-
played by benzoylated imidate donors led us to implement them
in the present study [12]. In previous work, we have reported the
improved synthesis of donors 1 and 2 according to a modified liter-
ature procedure [9,13]. In the present study, these procedures were
applied to provide donors 1 and 2 in 60% overall yield over three
steps, starting from the corresponding unprotected sugars [13a]
(Fig. 2).
HRMS: calcd. for C73H72O17Na [M+Na]+ 1243.4667; found
1243.4642.
2.10. 3ˇ-(4ꢀ-(ˇ-d-Glucopyranosyl)-˛-l-rhamnopyranosyloxy)-
androst-5-en-17-one
(14)
While the synthesis of the methyl glycoside of scillabiose has
been published earlier [14], glycosylations utilizing scillabiose
based donors have appeared in one report only [15]. In the pre-
vious work, however, the thioglycoside utilized resulted in an ␣:␤
mixture of the product. Overall, in the synthesis of glycosylated
biomolecules, minimization of the number of synthetic steps incor-
porating the aglycone is often preferred. This is mainly to ensure
that the reactive centers in the aglycone moiety remain untouched.
Therefore, wherever possible, glycosylations by convergent routes
are preferred over linear ones [16]. Furthermore, protective groups
should essentially be removable in one step and under mild condi-
tions in order to avoid multiple deprotection sequences which may
affect the sensitive functional groups. With these prerequisites in
mind, we devised an approach to the preparation of a scillabiose
glycosyl donor that could be coupled to a biomolecule by a con-
vergent route and contained only one type of protective groups
removable in a single reaction step. The synthesis of the donors 7
and 3 is illusrated in Scheme 1.
Synthesized from 13 (55 mg, 0.05 mmol) according to the
general procedure for deprotection providing 14 as a white
solid (24 mg, 90%). Rf = 0.35 (MeOH–CH2Cl2 1:5); [␣]D −32.1
(c 1.0, MeOH). 1H NMR (600.13 MHz, CD3OD): ı 5.42 (ddd,
1 H, J6,4ax = −1.8, J6,7ax = 2.2, J6,7eq = 5.2 Hz, H-6), 4.82 (d, 1 H,
J1 ,2 = 1.8 Hz, H-1ꢀ), 4.58 (d, 1 H, J1
= 7.8 Hz, H-1ꢀꢀ), 3.87
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
,2
H, J3 ,2 = 3.3 Hz, J3 ,4 = 9.7 Hz, H-3ꢀ), 4.84 (dd,
1 H,
ꢀ
ꢀ
ꢀ
ꢀ
(dd,
1
= 2.4 Hz, J6ꢀꢀ ꢀꢀ = −11.9 Hz, H-6ꢀꢀa), 3.77 (dd, 1 H, H-
ꢀꢀ
J6
ꢀꢀ
a,5
a,6
b
2ꢀ), 3.70 (dq, J5 ,6 = 6.4 Hz, J5 ,4 = 9.5 Hz, H-5ꢀ), 3.69 (dd, 1 H,
ꢀ
ꢀ
ꢀ
ꢀ
J6ꢀꢀ ꢀꢀ = 5.3 Hz, H-6ꢀꢀb), 3.60 (dd, 1 H, H-4ꢀ), 3.44 (dddd, 1 H,
b,5
J3,2eq = 4.4, J3,4eq = 4.7, J3,2ax = 11.2, J3,4ax = 11.6 Hz, H-3), 3.36 (dd,
1 H, J3
= 9.2 Hz, J3
= 9.6 Hz, H-3ꢀꢀ), 3.30 (dd, 1 H, J4
=
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
,2
,4
,5
9.0 Hz, H-4ꢀꢀ), 3.26 (ddd, 1 H, H-5ꢀꢀ), 3.21 (dd, 1 H, H-2ꢀꢀ), 2.45
(ddd, 1 H, J16eq,15eq = 0.4, J16eq,15ax = 8.9, J16eq,16ax = −19.5 Hz, H-
16eq), 2.38 (ddd, 1 H, J4eq,2ax = −2.3, J4eq,4ax = −13.1 Hz, H-4eq),
2.19 (dddddd, 1 H, J4ax,2eq = −2.2, J4ax,7eq = 2.9, J4ax,7ax = 3.2 Hz,
H-4ax), 2.13 (dddd, 1 H, J7eq,8 = 4.7, J7eq,7ax = −16.6 Hz, H-7eq),
In more detail, the present strategy was based on the prepa-
ration of
a thiophenyl rhamnoside acceptor. The thiophenyl

592
F.S. Ekholm et al. / Steroids 76 (2011) 588–595
Fig. 2. Structures of the benzoylated imidate donors 1–3.
Scheme 1. Synthesis of donor 3: (i) 2, TMSOTf, DCM, 85%; (ii) 80% AcOH, 80 ◦C, 96%; (iii) BzCl, pyridine, 89%; (iv) (1) NBS, Acetone:H2O 10:1, 92%; (2) DBU, Cl3CCN, CH2Cl2,
77%.
Table 1
A summary of the performed glycosylations and deprotections: (i) TMSOTf, corresponding donor, −20 ◦C, CH2Cl2; (ii) NaOMe, MeOH:THF 1:1.
.
Entry Glycosyl acceptor Glycosyl donor Product
Glycosylation yielda Deprotected product
Deprotection yielda
O
O
H
H
H
H
H
H
O
O
O
O
BzO
BzO
HO
9
10
HO
1
8
1
Not determinedb
89 (over two steps)
OBz
OH
O
O
H
H
BzO
BzO
HO
HO
H
H
H
H
O
O
O
O
BzO
HO
BzO
HO
Not determinedb
78 (over two steps)
11
12
2
8
2
O
O
H
H
H
H
H
H
O
O
BzO
BzO
HO
HO
O
O
O
O
O
O
BzO
HO
13
14
BzO
HO
BzO
HO
OBz
OH
3
8
3
88
90
a
Isolated yields.
b
Due to purification difficulties the deprotection was carried out on a mixture of product and starting material.

F.S. Ekholm et al. / Steroids 76 (2011) 588–595
593
Fig. 3. Spectral simulation of the 2.52–0.85 ppm region of the 1H NMR spectrum of 14 using PERCH NMR software; top: simulated spectrum, bottom: observed spectrum.
functionality has been shown to be stable under various reac-
tion conditions and, furthermore, it can be selectively activated in
the presence of other functionalities and under mild conditions,
thereby making it an excellent anomeric protective group [17].
In order to minimize the protective group manipulations needed,
the cis C-2 and C-3 hydroxyl groups were protected in one step
as an isopropylidene acetal [18]. Conveniently, the hydroxyl group
at C-4 remains untouched thus directly providing acceptor 4. In
the previous synthesis by Bebault and Dutton, the isopropylida-
tion was followed by an acetylation/deacetylation sequence at the
C-4 hydroxyl group [14]. These two steps are, however, not neces-
sary for preparation of acceptor 4 as also shown in a more recent
report [19]. In short, the synthesis of acceptor 4 commenced by
slightly modified literature procedures involving BF3·OEt2 pro-
moted glycosylation of peracetylated l-rhamnose with thiophenol
[20], followed by deacetylation under Zemplén conditions [21] and,
as the final step, formation of the 2,3-O-isopropylidene acetal in
61% overall yield. Glycosylation of acceptor 4 with donor 2 using
TMSOTf as promoter provided the disaccharide 5 in 85% isolated
yield [9,12,16a]. While compound 5 could in principle be used
as a donor molecule as such, in previous work a similar donor
was shown to yield a mixture of products with poor selectiv-
ity [15]. In addition, removal of the 2,3-O-isopropylidene acetal
requires harsh conditions which may affect sensitive functionalities
in biomolecules [22]. Furthermore, in contrast to ester protec-
tive groups at C-2, the isopropylidene acetal does not enhance
␣-selectivity in the glycosylation. For improving the desired prop-
erties, in particular neighboring group participation, displayed by
ester protective groups, the isopropylidene acetal was cleaved
under acidic conditions (80% AcOH) at 80 ◦C providing 6 in 96%
yield. The newly formed hydroxyl groups were benzoylated thus
providing the thiodonor 7 in 89% yield. In our previous work, the
best results in glycosidation of steroid alcohols were obtained by
TMSOTf promoted activation of an imidate donor [9]. Consequently,
in the present study, the scillabiose thiodonor was converted
to an imidate donor. The thiophenyl group was activated with
NBS, hydrolyzed [23] and the newly formed hemiacetal converted
to the corresponding imidate with DBU and trichloroacetonitrile
[10], thus providing donor 3 in 71% isolated yield over two steps
(Table 1).
donors glycosylations proceeded smoothly and, after deprotection
under Zemplén conditions [21], the unprotected glycosteroids 10
and 12 were isolated in high yields (77–89% over two steps). Next,
we explored the possibility of utilizing donor 7 in the glycosida-
tion. The use of this donor would be desirable as it shortens the
reaction sequence by two steps. Due to the presence of alkene
functionality in steroid 8, the commonly employed activation pro-
cedure using NIS/TfOH was not feasible [17]. Instead, activation of
the thioglycoside using the methodology of Crich was employed
[24]. The key features of this method are based on low tempera-
ture activation of a thioglycoside with BSP, TTBP and Tf2O, thereby
generating a reacting triflate in situ. By use of this activation proto-
col, moderate conversion (∼50%) was obtained. In order to evaluate
and compare the reactivity of the scillabiose donors 7 and 3, the
imidate donor was tested next. When the glycosylation reaction
was performed using donor 3 and following the standard activa-
tion protocol, the glycosylated steroid could be isolated in 88%
yield. One step debenzoylation under Zemplén conditions provided
the deprotected glycosteroid 14 in 90% yield. THF was required as
co-solvent in the deprotection due to the amphiphilicity of this
molecule in the deprotected state. With the final products being
amphiphilic (Rf values of ∼0.3–0.7 in MeOH:CH2Cl2 1:5), they were
also amenable to purification by column chromatography thus
enabling a simple method for removal of the methyl benzoate
formed during the final deprotection step.
3.2. Characterization by NMR spectroscopy
Steroids and glycosteroids commonly exhibit complicated NMR
spectra. Thus, special attention was focused on the NMR spec-
troscopic characterization of the compounds prepared in present
work. While some reports on complete NMR spectroscopic char-
acterization of this class of compounds exist, the spectral data
is of importance as reference material, providing also valuable
structural information on the solution phase properties of the com-
pounds. The NMR spectroscopic assignation is exemplified by the
characterization of compound 14 with all other compounds being
characterized in an analogous fashion.
The glycone part of 14 was characterized first starting with the
well separated anomeric protons of rhamnose (d at 4.82 ppm) and
glucose (d at 4.58 ppm). Since some of the signals from these two
sugars were overlapping in the 1H NMR-spectrum (especially the
region between 3.90 and 3.65 ppm), the anomeric signals where
targeted by selective excitation (400 ms spinlock time) utilizing the
1D-TOCSY experiment [25]. By use of this measurement technique,
these two spin-systems could be easily separated thus providing
a convenient way of identifying the signals corresponding to each
sugar unit. By use of COSY and HSQC, all signals from the glycone
part were identified next in both 1H and 13C NMR-spectra. From the
HMBC spectrum the correlation between H-1ꢀ and C-3 (77.9 ppm)
With donors 1, 2, 3 and 7 at hand, the focus was shifted to
the glycosylation study. 3␤-Hydroxy-androst-5-en-17-one (8) was
used as a model substrate in the glycosylation reaction. This steroid
carries identical stereochemistry (at C-3, C-8, C-9, C-13 and C-14)
to the aglycones of transvaalin and hellebrin and was therefore
selected as a suitable model [1,3]. As mentioned earlier, TMSOTf
promoted glycosylation of steroids with imidate donors was, in
our previous study, the most efficient methodology [9]. In order to
explore the suitability of this strategy to the present study, we first
evaluated the monosaccharide donors 1 and 2. With both of these

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F.S. Ekholm et al. / Steroids 76 (2011) 588–595
and H-3 (3.44 ppm) and C-1ꢀ (99.6 ppm) was clearly visible, thus
providing a starting point for the characterization of the aglycone.
By use of COSY, both of the H-2 and H-4 protons were identified. The
corresponding signals in the 13C NMR spectra were easily identified
by use of HSQC. Next, the spectral assignment was continued from
the well separated signals of H-6 (ddd at 5.42 ppm), C-6 (122.4 ppm)
and C-5 (141.9 ppm). From the HMBC correlation of C-5, H-19 (s
at 1.06 ppm) and H-1eq (ddd at 1.91 ppm) were identified. By use
of HSQC, the corresponding carbon signals were solved. From the
COSY spectra the correlations between H-6 and both of the H-7 pro-
tons were visible. Furthermore, the HMBC correlation between H-6
and C-10 was used to completely solve the A-ring of the steroid. The
HMBC correlation of H-19–C-9 and H-6–C-8 enabled the complete
characterization of the B-ring. For solving the C- and D-rings, the
carbonyl carbon of C-17 (223.8 ppm) and the methyl group con-
nected to C-13 were selected as starting points. From the HMBC
correlations of C-17, both of the H-15 protons could be found. From
the HMBC correlations of H-18 it was possible to identify C-12, C-
13 and C-14 carbon atoms. By use of COSY and HSQC, the C- and
D-rings could now be completely solved. Although assignations of
this type can be considered straightforward or routine, obtaining
accurate coupling constants from such systems is extremely diffi-
cult. This is mainly due to the severe overlap of signals in the steroid
region. We have in previous work described the complete assigna-
tion of a ␤-1,2-linked mannotetraose by use of the NMR simulation
software PERCH [8c]. Also in the present work this program was
utilized for complete assignment of the proton spectra of the com-
pounds prepared, exemplified by the high field region of the 1H
NMR spectrum of 14 shown in Fig. 3. The standard NMR spectro-
scopic techniques (1H, 13C, 1D-TOCSY, COSY, HSQC and HMBC), in
combination with the simulation software, thus enabled the com-
plete assignation of all glycosteroids synthesized in the present
work, being also applicable to the characterization of other types
of molecules in the future.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2011.02.010.
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