Electrochemical synthesis of glycoconjugates from activated sterol derivatives

Article abstract of DOI:10.1016/j.steroids.2014.01.007

Several derivatives of cholesterol and other 3β-hydroxy- Δ⁵-steroids were prepared and tested as sterol donors in electrochemical reactions with sugar alcohols. The reactions afforded glycoconjugates with sugar linked to a steroid moiety by an ether bond. Readily available sterol diphenylphosphates yielding up to 54% of the desired glycoconjugate were found to be the best sterol donors.

Full text of DOI:10.1016/j.steroids.2014.01.007

Steroids 82 (2014) 60–67
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Electrochemical synthesis of glycoconjugates from activated sterol
derivatives
a
b
b
a
a
´
Aneta M. Tomkiel , Jan Kowalski , Jolanta Płoszynska , Leszek Siergiejczyk , Zenon Łotowski ,
Andrzej Sobkowiak b, Jacek W. Morzycki a,
⇑
a Institute of Chemistry, University of Białystok, Hurtowa 1, 15-399 Białystok, Poland
b Faculty of Chemistry, Rzeszów University of Technology, P.O. Box 85, 35-959 Rzeszów, Poland
a r t i c l e i n f o
a b s t r a c t
Several derivatives of cholesterol and other 3b-hydroxy-D
5-steroids were prepared and tested as sterol
Article history:
Received 26 October 2013
Received in revised form 13 January 2014
Accepted 20 January 2014
Available online 30 January 2014
donors in electrochemical reactions with sugar alcohols. The reactions afforded glycoconjugates with
sugar linked to a steroid moiety by an ether bond. Readily available sterol diphenylphosphates yielding
up to 54% of the desired glycoconjugate were found to be the best sterol donors.
Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
Glycosylation
Cholesterol
Electrochemical oxidation
Sterol glycoconjugates
Activating groups
1. Introduction
O-sulfonyl glycosides [12], glycals [13], phosphate derivatives
[14], O-silylated glycosides [15], and others.
Steroidal glycosides constitute a structurally and biologically
diverse class of biomolecules which have been isolated from a wide
variety of both plant and animal species [1–4]. These compounds
have received considerable attention due to their physiological
and pharmacological activities. Recent developments in glycobiol-
ogy have revealed the important roles of various glycoconjugates
in immune responses, viral and bacterial infections, inflammation
and signal transductions. A number of important groups of drugs
are glycosides. In most cases the presence of a sugar moiety,
though per se biologically inactive, has a dramatic effect on the
physical, chemical and biological properties of drugs [5].
During our study on the direct electrochemical oxidation of
cholesterol [16] we observed the formation of a nonclassical carbo-
cation when the reaction was carried out in a nonpolar solvent (e.g.
dichloromethane) [17]. A mesomerically stabilized carbocation
may be trapped by any nucleophile that is present in the reaction
mixture affording a cholesterol derivative (see Scheme 1). Due to
stereoelectronic reasons, the 3b position is preferentially attacked
by the nucleophile [18]. The anions (chloride, acetate) or neutral
molecules (water, acetonitrile, cholesterol) that are present in the
reaction mixture may serve as nucleophiles. The carbocation may
also react with nucleophilic reagents that are deliberately added
to the reaction mixture, e.g. carbohydrates with free hydroxyl
groups either at the anomeric position (affording glycosides) or
other positions to form glycoconjugates via ether bonds. This ap-
proach allows to synthesize steroid-sugar conjugates without acti-
vating the sugar moiety. However, the recently described [19]
direct electrochemical glycosylation of cholesterol suffers from
several drawbacks. A major drawback is the need to use a large ex-
cess (at least 3 times) of a sugar partner to avoid the reaction of a
carbocation with cholesterol, which would lead to the formation of
dicholesteryl ether. The aim of the present study is to select conve-
nient protection for the 3b-OH group which would simultaneously
activate sterol for electrochemical oxidation. However, it is essen-
tial that the electrooxidation of a sterol derivative generates, as in
Despite considerable progress in carbohydrate chemistry in the
last few decades, the regio- and stereoselective formation of O-gly-
cosidic bonds between carbohydrates and steroids is still
a
demanding process [6]. The glycosylation of steroids involves the
selective reaction of a sterol with a sugar donor, which generally
bears a good leaving group at the anomeric position. Yields are
frequently not satisfactory due to low reactivity of the steroidal
alcohols. Almost all known methods of sugar activation were
applied in the synthesis of steroidal glycosides, including glycosyl
halides [7], trihalogenoacetimidates [8–10], thioglycosides [11],
⇑
Corresponding author. Tel.: +48 85 7457585; fax: +48 85 7457581.
E-mail address: morzycki@uwb.edu.pl (J.W. Morzycki).
http://dx.doi.org/10.1016/j.steroids.2014.01.007
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

A.M. Tomkiel et al. / Steroids 82 (2014) 60–67
61
cholesteryl phenyl sulfide (3) [22], cholesteryl benzyl ether (12)
[21], cholesteryl t-butyl ether (9) [23], t-butyldimethylsilyl cho-
lesteryl ether (10) [24], cholesteryl iodide (6) [25], cholesteryl thio-
cyanate (7) [26,27], thiocholesterol (8) [26,27], were prepared
according to known procedures.
HO
R
R = cholesteryl-O-
AcO-, Cl-, AcNH-
1
Melting points were determined on a Toledo Mettler-MP70
apparatus. 1H and 13C NMR (400 and 100 MHz, respectively) spec-
tra were recorded on a Bruker Avance II spectrometer in CDCl3
solutions with TMS as the internal standard (only selected signals
in the 1H NMR spectra are reported; sugar protons are marked with
index ‘prime’). Infrared spectra were recorded on a Nicolet series II
Magna-IR 550 FTIR spectrometer in chloroform solutions. Mass
spectra were recorded at 70 eV with a time-of-flight (TOF) AMD-
604 spectrometer with electrospray ionization (ESI) or AutoSpec
Premier (Waters) (EI).
nucleophile
-e-
(cholesterol, acetic acid,
chlorides, acetonitrile)
.
- HO
.
+
(+)
HO
(+)
Scheme 1. Electrooxidation of cholesterol in dichloromethane in the presence of
nucleophiles.
Merck Silica Gel 60, F 256 TLC aluminum sheets were applied
for thin-layer chromatographic analysis. For a visualization of the
products a 5% solution of phosphomolybdic acid in ethanol was
used. The reaction products were separated by column chromatog-
raphy performed on a 70–230 mesh silica gel (J.T. Baker).
the case of cholesterol, a mesomeric carbocation that is capable to
react with a sugar partner.
2. Experimental
2.1. Electrochemistry
2.2.1. Synthesis of steroidal diphenylphosphates
2.2.1.1. Procedure
1 [28]. To a solution of cholesterol (0.7 g,
Cyclic-voltammograms were recorded with iR compensation at
25 °C using a three-electrode potentiostat (Princeton Applied Re-
search Model Parstat 2273). The experiments were conducted in a
3-mL electrochemical cell with an argon-purge system. The work-
ing electrode was a Bioanalytical Systems platinum inlay (1 mm
in diameter), the auxiliary electrode a platinum mesh (contained
in a glass tube with a medium porosity glass frit), and the reference
electrode Ag/0.1 M AgNO3 in acetonitrile. The latter was contained
in a Pyrex tube with a cracked soft glass tip which was placed inside
a Luggin capillary. Before each experiment the working electrode
was polished using Buehler Micropolish Alumina Gamma 3B and
a Buehler Microcloth polishing cloth, rinsed with dichloromethane
and dried. In all of the measurements 0.2 M solution of tetrabutyl-
ammonium tetrafluoroborate (TBABF4) from Aldrich in dichloro-
methane was used as a supporting electrolyte.
The preparative electrolyses were performed with a potentio-
statꢀgalvanostat (Princeton Applied Research Model Parstat
2273) under galvanostatic conditions using a current that was
equal to 7–7.5 mA and a reaction time of 3000–5000 s. The current
applied was the maximum current available for the electrolysis
set-up being used (power supply and ohmic resistance). The reac-
tions were monitored by TLC and stopped when no further increase
in the concentration of glycosylation products was observed. A di-
vided H-cell was used in which the cathodic and anodic compart-
ments (3.5 mL of electrolyte each) were separated by a glass frit. In
all measurements 0.1 M solution of tetrabutylammonium tetra-
fluoroborate (TBABF4) from Aldrich in dichloromethane was used
as a supporting electrolyte. The steroid (0.30 mmol) and sugar
(0.36 mmol) substrates were introduced into the anodic compart-
ment together with 0.3 g of a 3 A molecular sieve to eliminate
traces of water, whereas anionite (1.5–2 g, Dowex 2 ꢁ 8, 200–
400 mesh, perchlorate form) was placed in the cathodic compart-
ment to eliminate chloride ions that are formed by the reduction
of dichloromethane. The solutions in both compartments were
stirred during electrolysis and, additionally, a continuous flow of
argon was applied in the anodic compartment. A platinum mesh
was used as a cathode and a platinum plate (2 ꢁ 1.5 cm) was used
as an anode. All measurements were performed at 25 °C.
1.8 mmol) in THF (25 mL) cooled to 0 °C n-BuLi (2.5 M in hexane,
0.8 mL, 2 mmol), and then diphenyl phosphoryl chloride (DPPC,
0.8 mL, 2.7 mmol) was added. The reaction mixture was stirred
for 0.5 h and the solvent was evaporated in vacuo. The residue
was dissolved in ethyl acetate (100 mL), washed with water
(3 ꢁ 150 mL), and dried over anhydrous Na2SO4. The crude product
was purified by silica gel column chromatography affording 15
(1.0 g, 91%) eluted with hexane-AcOEt (96:04).
2.2.1.1.1. Cholest-5-en-3b-yl diphenyl phosphate (15). White crys-
tals, mp 116–119 °C (CH2Cl2/acetonitrile); Rf = 0.53 (hexane-AcOEt
8:2); IR,
(ppm), d (ppm): 7.35 (m, 4H, H-Ar), 7.24 (m, 4H, H-Ar), 7.19 (m,
2H, H-Ar), 5.37 (m, 1H, H-6), 4.45 (m, 1H, H-3 ), 1.02 (s, 3H, H-
m
max: 1592, 1490, 1281, 1192, 1163, 1024, 957; 1H NMR
a
19), 0.92 (d, 3H, J = 6.6 Hz, H-21), 0.880 (d, 3H, J = 6.6 Hz, H-26 or
H-27), 0.876 (d, 3H, J = 6.6 Hz, H-26 or H-27), 0.68 (s, 3H, H-18);
13C NMR (ppm), d: 150.7 (d, J = 7.0 Hz, C), 139.1 (C), 129.7 (CH),
125.2 (CH), 123.3 (CH), 120.1 (d, J = 5.0 Hz, CH), 80.3 (d,
J = 6.6 Hz, CH), 56.6 (CH), 56.1 (CH), 49.9 (CH), 42.3 (C), 39.77 (d,
J = 4.8 Hz, CH2), 39.68 (CH2), 39.5 (CH2), 36.9 (CH2), 36.4 (C), 36.2
(CH2), 35.8 (CH), 31.9 (CH2), 31.8 (CH), 29.5 (d, J = 4.6 Hz, CH2),
28.2 (CH2), 28.0 (CH), 24.3 (CH2), 23.8 (CH2), 22.8 (CH3), 22.6
(CH3), 21.0 (CH2), 19.2 (CH3), 18.7 (CH3), 11.8 (CH3); 31P NMR
(ppm), d: ꢂ12.02; ESI MS, m/z: 1259 [(2ꢁM+Na)+, 100%], 641
[(M+Na)+, 6%], 273 [(diphenylphosphoric acid+Na)+, 49%]; HRMS
(ESI): m/z calcd for (C39H55O4P+Na)+: 641.3736 (M+Na)+; found
641.3728.
2.2.1.2. Procedure 2. To a solution of diosgenin (0.5 g, 1.2 mmol) in
THF (20 mL), Et3N (2 mL, 14 mmol) was added, and then DPPC
(0.7 mL, 2.3 mmol). The reaction was stirred at 40 °C for 4 days,
and then the solvent was evaporated in vacuo. The residue was dis-
solved in ethyl acetate (100 mL) and washed with water
(3 ꢁ 150 mL), dried over anhydrous Na2SO4 and the crude product
was purified by silica gel column chromatography. Elution with
hexane-AcOEt (92:8) afforded pure 26 (0.6 g, 77%).
2.2.1.2.1. 25R-spirost-5-en-3b-yl diphenyl phosphate (26). Colorless
crystals, mp 133–135 °C (AcOEt/hexane); Rf = 0.35 (hexane-AcOEt
8:2); IR,
(ppm), d (ppm): 7.33 (m, 4H, H-Ar), 7.23 (m, 4H, H-Ar), 7.17 (m,
2H, H-Ar), 5.36 (m, 1H, H-6), 4.43 (m, 1H, H-3 ), 3.48 (m, 1H, H-
m
max: 1592, 1490, 1281, 1192, 1025, 957, 897; 1H NMR
2.2. Chemical synthesis
a
26a), 3.37 (t, 1H, J = 10.8 Hz, H-26b), 1.02 (s, 3H, H-19), 0.98 (d,
3H, J = 6.9 Hz, H-21), 0.787 (d, 3H, J = 6.2 Hz, H-27), 0.787 (s, 3H,
H-18); 13C NMR (ppm), d: 150.5 (d, J = 7.3 Hz, C), 138.9 (C), 129.5
The sugar (1,2:3,4-di-O-isopropylidene-
[20] and steroidal substrates, cholesteryl phenyl ether (2) [21],
a-D-galactopyranose)

62
A.M. Tomkiel et al. / Steroids 82 (2014) 60–67
(CH), 125.0 (CH), 122.9 (CH), 120.0 (d, J = 4.9 Hz, CH), 109.0 (C),
80.6 (CH), 80.0 (d, J = 6.6 Hz, CH), 66.6 (CH2), 62.0 (CH), 56.2
(CH), 49.7 (CH), 41.5 (CH), 40.1 (C), 39.6 (d, J = 4.8 Hz, CH2), 39.5
(CH2), 36.7 (CH2), 36.3 (C), 31.8 (d, J = 17.6 Hz, CH2), 31.3 (CH2),
31.2 (CH), 30.1 (CH), 29.33 (CH2), 29.29 (CH2), 28.7 (CH2), 20.7
(CH2), 19.1 (CH3), 17.0 (CH3), 16.1 (CH3), 14.4 (CH3); 31P NMR
(ppm), d: ꢂ12.03; ESI MS, m/z: 1315 [(2ꢁM+Na)+, 12%], 669
[(M+Na)+, 100%], 273 [(diphenylphosphoric acid+Na)+, 29%]; HRMS
(ESI): m/z calcd for (C39H51O6P+Na)+: 669.3321 (M+Na)+; found
669.3314.
1.07 (s, 3H, H-19), 0.93 (d, 3H, J = 6.6 Hz, H-21), 0.881 (d, 3H,
J = 6.6 Hz, H-26 or H-27), 0.876 (d, 3H, J = 6.6 Hz, H-26 or H-27),
0.70 (s, 3H, H-18); 13C NMR (ppm), d: 162.1 (C), 139.5 (C), 122.9
(CH), 91.9 (C), 78.9 (CH), 56.7 (CH), 56.2 (CH), 50.1 (CH), 42.3 (C),
39.7 (CH2), 39.5 (CH2), 37.3 (CH2), 36.9 (CH2), 36.7 (C), 36.2
(CH2), 35.8 (CH), 31.93 (CH2), 31.87 (CH), 28.2 (CH2), 28.0 (CH),
27.1 (CH2), 24.3 (CH2), 23.8 (CH2), 22.8 (CH3), 22.6 (CH3), 21.1
(CH2), 19.4 (CH3), 18.7 (CH3), 11.9 (CH3); 31P NMR (ppm), d:
140.15; EI MS, m/z: 529 (M+, 1%), 494 [(MꢂCl)+, 4%], 369 [(cho-
lest-5-en-3-yl)+, 100%]; elemental analysis calcd (%) for C29H46Cl3-
NO: C 65.59, H 8.73, Cl 20.03, N 2.64; found: C 65.49, H 8.71, Cl
20.07, N 2.65.
2.2.1.3. Analogously compound 27 was obtained from methyl
5-cholenoate (87%).
2.2.1.3.1. Methyl 3b-diphenylphosphorylchol-5-enoate (27). Color-
less crystals, mp 78–80 °C (AcOEt/hexane); Rf = 0.37 (hexane-
2.2.4. Cholest-5-en-3a-yl phenyl selenide (4)
AcoEt 8:2); IR,
957; 1H NMR (ppm), d (ppm): 7.33 (m, 4H, H-Ar), 7.23 (m, 4H, H-
Ar), 7.17 (m, 2H, H-Ar), 5.36 (m, 1H, H-6), 4.44 (m, 1H, H-3 ),
m
max: 1731, 1592, 1490, 1279, 1192, 1163, 1024,
To cholesteryl tosylate (443 mg, 1 mmol) dissolved in dioxane
(25 mL), AcOK (590 mg, 6 mmol), and then phenylselenol were
added (1.6 g, 10 mmol). The reaction mixture was refluxed under
argon for 22 h. After cooling to room temperature it was poured
to 1 M NaOH (200 mL) and extracted with benzene (3 ꢁ 100 mL).
The dried (anhydrous Na2SO4) extract was evaporated in vacuo
and reaction products were separated by silica gel column chroma-
tography with hexane elution. Compound 4 was obtained in 50%
yield accompanied by i-cholesteryl phenyl selenide (34%), which
was eluted first.
a
3.65 (s, 3H, H-methyl ester), 1.00 (s, 3H, H-19), 0.92 (d, 3H,
J = 6.4 Hz, H-21), 0.67 (s, 3H, H-18); 13C NMR (ppm), d: 174.6 (C),
150.6 (d, J = 7.4 Hz, C), 139.0 (C), 129.6 (CH), 125.1 (CH), 123.2
(CH), 120.0 (d, J = 4.9 Hz, CH), 80.2 (d, J = 6.6 Hz, CH), 56.5 (CH),
55.7 (CH), 51.3 (CH3), 49.8 (CH), 42.3 (C), 39.7 (d, J = 4.8 Hz, CH2),
39.5 (CH2), 36.8 (CH2), 36.3 (C), 35.2 (CH), 31.73 (CH2), 31.70
(CH), 30.94 (CH2), 30.91 (CH2), 29.4 (d, J = 4.6 Hz, CH2), 28.0
(CH2), 24.1 (CH2), 20.9 (CH2), 19.2 (CH3), 18.2 (CH3), 11.8 (CH3);
31P NMR (ppm), d: ꢂ12.05; ESI MS, m/z: 1263 [(2ꢁM+Na)+, 17%],
643 [(M+Na)+, 10%], 273 [(diphenylphosphoric acid+Na)+, 100%];
HRMS (ESI): m/z calcd for (C37H49O6P+Na)+: 643.3165 (M+Na)+;
found 643.3159.
Compound 4; colorless crystals, mp 106–108 °C (CH2Cl2/hex-
ane); Rf = 0.25 (hexane); IR,
m
max: 3075, 1578, 535; 1H NMR
(ppm), d: 7.53 (m, 2H, H-Ar), 7.25 (m, 3H, H-Ar), 5.36 (m, 1H, H-
6), 3.80 (m, 1H, H-3b), 1.03 (s, 3H, H-19), 0.94 (d, 3H, J = 6.6 Hz,
H-21), 0.89 (d, 3H, J = 6.6 Hz, H-26 or H-27), 0.88 (d, 3H,
J = 6.6 Hz, H-26 or H-27), 0.70 (s, 3H, H-18); 13C NMR (ppm), d:
139.2 (C), 134.0 (CH), 130.8 (C), 128.9 (CH), 126.9 (CH), 122.4
(CH), 56.7 (CH), 56.2 (CH), 50.1 (CH), 45.5 (CH), 42.3 (C), 39.8
(CH2), 39.5 (CH2), 38.4 (CH2), 37.3 (C), 36.2 (CH2), 35.8 (CH), 35.0
(CH2), 31.80 (CH), 31.76 (CH2), 28.3 (CH2), 28.0 (CH), 27.5 (CH2),
24.3 (CH2), 23.9 (CH2), 22.8 (CH3), 22.6 (CH3), 20.8 (CH2), 19.4
(CH3), 18.7 (CH3), 11.8 (CH3); EI MS, m/z: 526 (M+, 39%), 369 [(cho-
lest-5-en-3-yl)+, 100%]; HRMS (EI): m/z calcd for C33H50Se+:
526.3078 [M+]; found 526.3071.
2.2.2. Cholest-5-en-3b-yl-dimethyl phosphite (16)
2.2.2.1. Procedure analogous to that described in Ref. [29]. To a
solution of cholesterol (0.5 g, 1.3 mmol) in dichloromethane
(20 mL), 1H-tetrazole (0.14 g, 2 mmol) and dimethyl N,N-
diethylamidophosphite (0.3 mL, 1.7 mmol) were added. The reac-
tion mixture was stirred at room temperature for 1 h, and then
poured into water and extracted with ethyl acetate. The extract
was washed with brine (150 mL), water (3 ꢁ 150 mL), and the sol-
vent was evaporated in vacuo affording 16 (0.52 mg, 84%).
Compound 16; White solid; Rf = 0.60 (hexane-AcOEt 9:1); IR,
m
3
max: 1254, 978; 1H NMR, d: 5.39 (m, 1H, H-6), 3.93 (m, 1H, H-
), 3.53 (d, J = 0.8 Hz, 3H, CH3O–), 3.51 (d, J = 0.8 Hz, 3H, CH3O–
2.2.5. p-(Cholest-5-en-3b-yloxy)phenol (13)
a
To cholesteryl tosylate (443 mg, 1 mmol) dissolved in dioxane
(30 mL) p-toluenesulfonic acid (34 mg, 0.2 mmol), and then hydro-
quinone (440 mg, 4 mmol) were added. The reaction mixture was
refluxed under argon for 4 days. After cooling to room temperature
it was poured to 1 M NaOH (150 mL) and extracted with benzene
(3 ꢁ 100 mL). The dried extract (anhydrous Na2SO4) was evapo-
rated in vacuo and the residue was subjected to silica gel column
chromatography. The product 13 (162 mg, 34%) was eluted with
hexane-AcOEt (9.5:0.5).
), 1.02 (s, 3H, H-19), 0.93 (d, 3H, J = 6.5 Hz, H-21), 0.879 (d, 3H,
J = 6.6 Hz, H-26 or H-27), 0.875 (d, 3H, J = 6.6 Hz, H-26 or H-27),
0.69 (s, 3H, H-18); 13C NMR (ppm), d: 140.3 (C), 122.1 (CH), 73.4
(d, J = 15.9 Hz, CH), 56.7 (CH), 56.2 (CH), 50.1 (CH), 48.6 (d,
J = 8.7 Hz, CH3), 42.3 (C), 41.1 (d, J = 3.9 Hz, CH2), 39.8 (CH2), 39.5
(CH2), 37.2 (CH2), 36.5 (C), 36.2 (CH2), 35.8 (CH), 31.90 (CH2),
31.87 (CH), 30.6 (d, J = 4.0 Hz, CH2), 28.2 (CH2), 28.0 (CH), 24.3
(CH2), 23.8 (CH2), 22.8 (CH3), 22.5 (CH3), 21.0 (CH2), 19.3 (CH3),
18.7 (CH3), 11.8 (CH3); ESI MS, m/z: 501 [(M+Na)+, 100%]; HRMS
(ESI): m/z calcd for (C29H51O3P+Na)+: 501.3474 (M+Na)+; found
501.3469.
Compound 13; white crystals; mp 192–194 °C (hexane/AcOEt);
Rf = 0.49 (hexane-AcOEt 8:2); IR,
mmax: 3601, 3336, 1507, 1229,
827; 1H NMR (ppm), d: 6.80 (d, 2H, J = 9.1 Hz, H-Ar), 6.75 (d, 2H,
J = 9.1 Hz, H-Ar), 5.38 (m, 1H, H-6), 4.59 (s, 1H, –OH), 3.98 (m,
2.2.3. Cholest-5-en-3b-yl trichloroacetimidate (14)
1H, H-3a), 1.06 (s, 3H, H-19), 0.93 (d, 3H, J = 6.5 Hz, H-21), 0.882
2.2.3.1. Procedure analogous to that described in Refs. [30,31]. To a
solution of cholesterol (2 g, 5 mmol) and CCl3CN (5 mL, 25 mmol)
in dichloromethane (40 mL) cooled to 0 °C DBU (0.9 mL, 5 mmol)
was added. The reaction was stirred under argon for 1 h and the
solvent was evaporated in vacuo. Dry flash chromatography with
hexane-AcOEt (95:5) + 1% triethylamine elution afforded 14
(2.4 g, 88%).
(d, 3H, J = 6.6 Hz, H-26 or H-27), 0.877 (d, 3H, J = 6.6 Hz, H-26 or
H-27), 0.70 (s, 3H, H-18); 13C NMR (ppm), d: 151.6 (C), 149.7 (C),
140.4 (C), 122.2 (CH), 117.8 (CH), 116.0 (CH), 78.4 (CH), 56.8
(CH), 56.2 (CH), 50.2 (CH), 42.3 (C), 39.8 (CH2), 39.5 (CH2), 38.8
(CH2), 37.2 (CH2), 36.8 (C), 36.2 (CH2), 35.8 (CH), 31.93 (CH2),
31.88 (CH), 28.3 (CH2), 28.2 (CH2), 28.0 (CH), 24.3 (CH2), 23.8
(CH2), 22.8 (CH3), 22.6 (CH3), 21.1 (CH2), 19.4 (CH3), 18.7 (CH3),
11.9 (CH3); EI MS, m/z: 478 (M+, 5%), 369 [(cholest-5-en-3-yl)+,
100%]; HRMS (EI): m/z calcd for C33H50O2+: 478.3811 [M+]; found
478.3806.
Compound 14; white crystals; mp 153–156 °C (hexane/AcOEt);
Rf = 0.51 (benzene-hexane 4:6); IR,
m
max: 1659, 1099, 799; 1H NMR
(ppm), d: 8.24 (s, 1H, @NH), 5.43 (m, 1H, H-6), 4.78 (m, 1H, H-3
a
),

A.M. Tomkiel et al. / Steroids 82 (2014) 60–67
63
2.2.6. Other cholesterol derivatives
2.2.6.1. Cholest-5-en-3b-yl thiocyanate (7). White crystals, mp 126–
128 °C (acetone); Rf = 0.56 (hexane-AcOEt 95:5); IR,
NMR (ppm), d: 5.43 (m, 1H, H-6), 3.10 (m, 1H, H-3 ), 1.04 (s, 3H, H-
(C), 109.8 (C), 84.6 (CH), 81.3 (CH), 79.4 (CH), 78.9 (CH), 62.4
(CH2), 56.79 (CH), 56.77 (CH), 56.2 (CH), 50.32 (CH), 50.27 (CH),
48.4 (CH), 44.6 (CH), 44.1 (CH), 42.3 (C), 40.6 (CH2), 40.2 (CH2),
39.8 (CH2), 39.7 (CH2), 39.5 (CH2), 36.92 (C), 36.89 (C), 36.2
(CH2), 35.8 (CH), 31.91 (CH2), 31.86 (CH2), 29.4 (CH2), 29.3 (CH2),
28.2 (CH2), 28.0 (CH), 27.15 (CH3), 27.14 (CH3), 27.1 (CH3), 26.9
(CH3), 24.3 (CH2), 23.8 (CH2), 22.8 (CH3), 22.5 (CH3), 20.9 (CH2),
19.4 (CH3), 19.3 (CH3), 18.7 (CH3), 11.9 (CH3); ESI MS, m/z: 1069
[(M+Na)+, 100%]; HRMS (ESI): m/z calcd for (C66H110O5S2+Na)+:
1069.7693 (M+Na)+; found 1069.7679.
m
max: 2154; 1H
a
19), 0.92 (d, 3H, J = 6.5 Hz, H-21), 0.878 (d, 3H, J = 6.6 Hz, H-26 or
H-27), 0.873 (d, 3H, J = 6.6 Hz, H-26 or H-27), 0.69 (s, 3H, H-18);
13C NMR, d: 140.0 (C), 123.1 (CH), 111.2 (C), 56.7 (CH), 56.2 (CH),
50.1 (CH), 48.1 (CH), 42.3 (C), 39.7 (CH2), 39.7 (CH2), 39.5 (CH2),
39.4 (CH2), 36.5 (C), 36.2 (CH2), 35.8 (CH), 31.8 (CH2), 31.7 (CH),
30.0 (CH2), 28.2 (CH2), 28.0 (CH), 24.2 (CH2), 23.8 (CH2), 22.8
(CH3), 22.5 (CH3), 20.9 (CH2), 19.2 (CH3), 18.7 (CH3), 11.8 (CH3);
ESI MS, m/z: 877 [(2ꢁM+Na)+, 75%], 482 [(M+MeOH+Na)+, 100%],
450 [(M+Na)+, 95%]; HRMS (ESI): m/z calcd for (C28H45NS+Na)+:
450.3171 (M+Na)+; found 450.3165.
2.2.8.2.
3b-O-(3,4-O-isopropylidene-a-D-galactopyranos-6-yl)-cho-
lest-5-ene (24). Colorless crystals, mp 145–148 °C (CH2Cl2/hex-
ane); Rf = 0.30 (hexane-AcOEt 9:1); IR,
mmax: 3550, 1083, 1064;
1H NMR (ppm), d: 5.37 (s, 1H, H-10), 5.35 (m, 1H, H-6), 4.50 (dd,
1H, J1 = 5.8 Hz, J2 = 5.5 Hz, H-50), 4.46 (dd, 1H, J1 = 7.0 Hz,
J2 = 5.8 Hz, H-40), 4.15 (d, 1H, J = 7.0 Hz, H-30), 4.10 (d, 1H,
J = 7.6 Hz, H-60a), 3.62 (s, 1H, H-20), 3.58 (dd, 1H, J1 = 7.6 Hz,
2.2.6.2. Cholest-5-ene-3b-thiol (8). White crystals, mp 96–97 °C
(hexane/AcOEt); Rf = 0.41 (hexane); IR, mmax
(ppm), d: 5.33 (m, 1H, H-6), 2.70 (m, 1H, H-3
:
a
1467; 1H NMR
), 1.01 (s, 3H, H-
J2 = 5.5 Hz, H-60b), 3.35 (m, 1H, H-3
a), 1.54 (s, 3H, H-isopropyli-
19), 0.93 (d, 3H, J = 6.5 Hz, H-21), 0.881 (d, 3H, J = 6.6 Hz, H-26 or
H-27), 0.876 (d, 3H, J = 6.6 Hz, H-26 or H-27), 0.69 (s, 3H, H-18);
13C NMR, d: 141.9 (C), 121.0 (CH), 56.7 (CH), 56.2 (CH), 50.2 (CH),
44.2 (CH2), 42.3 (C), 39.9 (CH2), 39.8 (CH2), 39.5 (CH2), 39.4 (CH),
36.3 (C), 36.2 (CH2), 35.8 (CH), 34.1 (CH2), 31.788 (CH), 31.787
(CH2), 28.2 (CH2), 28.0 (CH), 24.3 (CH2), 23.8 (CH2), 22.8 (CH3),
22.6 (CH3), 20.9 (CH2), 19.3 (CH3), 18.7 (CH3), 11.8 (CH3); ESI MS
(negative ion mode), m/z: 401 [(MꢂH)ꢂ, 100%].
dene), 1.36 (s, 3H, H-isopropylidene), 1.01 (s, 3H, H-19), 0.93 (d,
3H, J = 6.5 Hz, H-21), 0.880 (d, 3H, J = 6.6 Hz, H-26 or H-27),
0.876 (d, 3H, J = 6.6 Hz, H-26 or H-27), 0.67 (s, 3H, H-18); 13C
NMR (ppm), d: 140.4 (C), 122.1 (CH), 108.4 (C), 100.7 (CH), 79.4
(CH), 75.5 (CH), 75.4 (CH), 72.0 (CH), 69.4 (CH), 63.1 (CH2), 56.8
(CH), 56.2 (CH), 50.2 (CH), 42.3 (C), 39.8 (CH2), 39.5 (CH2), 39.2
(CH2), 37.2 (CH2), 36.8 (C), 36.2 (CH2), 35.8 (CH), 31.94 (CH2),
31.90 (CH), 29.0 (CH2), 28.2 (CH2), 28.0 (CH), 25.8 (CH3), 24.4
(CH3), 24.3 (CH2), 23.8 (CH2), 22.8 (CH3), 22.6 (CH3), 21.1 (CH2),
19.4 (CH3), 18.7 (CH3), 11.9 (CH3); ESI MS, m/z: 593 [(MꢂH2O+Na)+,
100%]; elemental analysis calcd (%) for C36H60O6: C 73.43, H 10.27;
found: C 73.36, H 10.25.
2.2.7. Electrolysis of cholesteryl diphenylphosphates (15) in the
presence of 1,2:3,4-di-O-isopropylidene-
Cholesteryl diphenylphosphate (15; 185 mg; 0.30 mmol)
and 1,2:3,4-di-O-isopropylidene- -galactopyranose (96 mg; 0.37
D-galactopyranose
D
mmol) were dissolved in a 0.1 M solution of tetrabutylammonium
tetrafluoroborate in dichloromethane (3.5 mL) and introduced into
the anodic compartment together with a 0.3 g 3A molecular sieve
to eliminate traces of water. The same supporting electrolyte was
placed in the cathodic compartment with an anionite (1 g, Dowex
2 ꢁ 8, 200–400 mesh, perchlorate form) added to eliminate chlo-
ride ions from forming by the reduction of dichloromethane. A pre-
parative electrolysis was carried out in a divided H-cell in which
the cathodic and anodic compartments (3.5 mL of electrolytes
each) were separated by a glass frit under galvanostatic conditions.
A direct current 7.5 mA was run for 3600 s. A platinum mesh was
used as a cathode and a platinum plate (2 ꢁ 1.5 cm) was used as
an anode. Ag/0.1 M AgNO3 in an acetonitrile electrode was used
as a reference. When the electrolysis was completed the solvent
was removed from the reaction mixture and the products were
separated by silica gel column chromatography. The hexane–ethyl
acetate (94:6) elution afforded unreacted substrate (15; 22 mg;
2.2.8.3. p-Di(cholest-5-en-3b-yloxy)benzene (25). Colorless crystals,
mp 223–226 °C (CH2Cl2/hexane); Rf = 0.41 (hexane-AcOEt 95:5);
IR,
m
max: 1602, 1503, 1033, 1018, 830; 1H NMR (ppm), d: 6.82 (s,
4H, H-Ar), 5.38 (m, 2H, H-6), 3.99 (m, 2H, H-3
a), 1.06 (s, 6H, H-
19), 0.93 (d, 6H, J = 6.5 Hz, H-21), 0.884 (d, 6H, J = 6.6 Hz, H-26 or
H-27), 0.879 (d, 6H, J = 6.6 Hz, H-26 or H-27), 0.70 (s, 3H, H-18);
13C NMR (ppm), d: 151.9 (C), 140.5 (C), 122.1 (CH), 117.4 (CH),
78.2 (CH), 56.8 (CH), 56.2 (CH), 50.2 (CH), 42.3 (C), 39.8 (CH2),
39.5 (CH2), 38.9 (CH2), 37.2 (CH2), 36.9 (C), 36.2 (CH2), 35.8 (CH),
32.0 (CH2), 31.9 (CH), 28.4 (CH2), 28.2 (CH2), 28.0 (CH), 24.3
(CH2), 23.8 (CH2), 22.8 (CH3), 22.6 (CH3), 21.1 (CH2), 19.4 (CH3),
18.7 (CH3), 11.9 (CH3); EI MS, m/z: 846 (M+, 3%), 369 [(cholest-5-
en-3-yl)+, 100%]; elemental analysis calcd (%) for C60H94O2: C
85.04, H 11.18; found: C 84.88, H 11.21.
2.5.8.4. Dicholesteryl disulfide (23). White crystals; mp 104–107 °C
12%) and 3b-O-(10,20:30,40-di-O-isopropylidene-
a-D-galactopyr-
(hexane/AcOEt); Rf = 0.38 (hexane); IR,
m
max: 1110, 648, 517, 508;
anos-60-yl)-cholest-5-ene (17; 101 mg, 54%), followed by choles-
terol (1; 6 mg, 5%) eluted with hexane–ethyl acetate (9:1).
Other electrochemical reactions with different cholesteryl
donors were carried out in a similar manner.
1H NMR, d: 5.38 (m, 2H, H-6), 2.62 (m, 2H, H-3
a
), 1.01 (s, 6H, H-
19), 0.93 (d, 6H, J = 6.5 Hz, H-21), 0.879 (d, 6H, J = 6.6 Hz, H-26 or
H-27), 0.874 (d, 6H, J = 6.6 Hz, H-26 or H-27), 0.69 (s, 6H, H-18);
13C NMR (ppm), d: 141.7 (C), 121.2 (CH), 56.8 (CH), 56.2 (CH),
50.7 (CH), 50.3 (CH), 42.3 (C), 39.8 (CH2), 39.6 (CH2), 39.5 (CH2),
39.1 (CH2), 36.8 (C), 36.2 (CH2), 35.8 (CH), 31.90 (CH2), 31.85
(CH), 29.1 (CH2), 28.2 (CH2), 28.0 (CH), 24.3 (CH2), 23.8 (CH2),
22.8 (CH3), 22.6 (CH3), 21.0 (CH2), 19.3 (CH3), 18.7 (CH3), 11.9
(CH3); EI MS, m/z: 803 [(M+H)+, <1%], 369 [(cholest-5-en-3-yl)+,
100%]; elemental analysis calcd (%) for C54H90S2: C 80.73, H
11.29, S 7.98; found: C 80.69, H 11.32, S 7.96.
2.2.8. Other products of electrochemical reactions
2.2.8.1. 3,4-O-isopropylidene-2-O-[1-methyl-1-(cholest-5-en-3b-yl-
sulfanyl)-ethyl]-1-(cholest-5-en-3b-yl-sulfanyl)-b-
D
-galactopyranose
max: 3482,
(22). White solid; Rf = 0.37 (hexane-AcOEt 8:2); IR,
m
1242, 1161, 1059; 1H NMR (ppm), d: 5.32 (m, 2H, H-6), 3.38 (dd,
J1 = 7.3 Hz, J2 = 1.2 Hz, 1H, H-20), 4.23 (d, 1H, J = 1.3 Hz, H-10), 4.10
(m, 2H, H-30, H-50), 3.81 (m, 3H, H-40, H-60), 2.82 (m, 2H, H-3
a),
1.48 (s, 3H, H-isopropylidene), 1.43 (s, 6H, H-isopropylidene),
1.41 (s, 3H, H-isopropylidene), 1.022 (s, 3H, H-19), 1.017 (s, 3H,
H-19), 0.93 (d, 6H, J = 6.4 Hz, H-21), 0.881 (d, 6H, J = 6.6 Hz, H-26
or H-27), 0.874 (d, 6H, J = 6.6 Hz, H-26 or H-27), 0.69 (s, 6H, H-
18); 13C NMR (ppm), d: 142.0 (C), 141.7 (C), 120.9 (CH), 110.8
3. Results and discussion
As has been shown in our previous paper [19], anodic oxidation
of cholesterol (1) in the presence of a proper sugar affords

64
A.M. Tomkiel et al. / Steroids 82 (2014) 60–67
glycosides or ether glycoconjugates, depending on the sugar struc-
ture. The sterol ethers are of special interest since they are hardly
degradable as opposed to steroidal acetals or esters, which are bio-
logically active compounds but undergo fast metabolic transforma-
tions in vivo. Known methods of etherification are limited in the
steroid field, e.g. Williamson ether synthesis is often unsuccessful
even for unhindered 3-hydroxysteroids [32]. Sugar triflates were
applied for synthesis of sterol glycoconjugates, as an opposite ap-
proach employing sterol triflates and inactivated sugars proved
to be unsuccessful [33].
10
0
I, μΑ
-10
-20
-30
-40
-50
-60
-70
(a)
(b)
(c)
(d)
We have recently described the electrochemical method of syn-
thesis of steroid glycoconjugates using 6b-arylsulfanyl-3a,5a-
cyclosteroid derivatives as donors of a sterol moiety [22]. Since
the preparation of these compounds is troublesome, attempts have
been undertaken to select a suitable sterol donor for electrochemi-
cal glycosylation reactions from readily available 3-cholesteryl
derivatives 2–16 (Scheme 2). All of these compounds are electro-
chemically active; cyclic voltammograms measured in dichloro-
methane on a platinum electrode indicate that the main oxidation
peak for the substances occurs within 1.8–2.2 V (vs Ag/AgNO3 in
MeCN). Cholesteryl phenyl ether (2), phenylsulfide (3), phenylsele-
nide (4), iodide (6), and hydroquinone ether (13) showed additional
anodic peaks which appeared at less positive potentials. They prob-
ably resulted from electrooxidation of a substituent. Fig. 1 presents
a comparison of the electrochemical behavior of substrates [sugar
2.5
2.0
1.5
1.0
0.5
0.0
E, V vs Ag/AgNO3 in MeCN
Fig. 1. Cyclic voltammograms registered in 0.2 M tetrabutylammonium tetrafluo-
roborate (TBABF4) in dichloromethane on a platinum electrode (area 0.008 cm2) of
(a) the supporting electrolyte (black), (b) 1,2:3,4-di-O-isopropylidene-a-D-galacto-
pyranose (red), (c) cholesteryl diphenylphosphate 15 (green), and (d) glycoconju-
gate 17 (blue). Concentrations of all compounds are equal to 5 mM. Scan rate
1 V sꢂ1
, potentials were measured vs Ag/0.1 M AgNO3 in acetonitrile at room
temperature. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
alcohol
– 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose (red
line) and the sterol donor – cholesteryl diphenylphosphate (15 –
green line)] and the glycoconjugate product (17 – blue line). A cyclic
voltammogram of the supporting electrolyte (black line) is also
shown.
An attempt to prepare glycoconjugates by using cholesterol
phenyl ether (2) [21] as a sterol donor failed, as only traces of gly-
coconjugate 17 were detected (Table 1).
C8H17
C8H17
St
St'
Steroidal substrates:
O
HO
O St (13)
NH
Cl3C C O St (14)
C O St (9)
HO St (1)
PhO St (2)
F C
CO St (5)
3
I
St (6)
C Si O St (10)
TsO St (11)
O
PhS St (3)
PhO P O St (15)
OPh
MeO P O St (16)
OMe
NCS St (7)
HS St (8)
CH2O St (12)
PhSe 3α-St (4)
Steroidal products:
Cl St (19)
O
St (17)
O St' (18)
St O St (20)
O St (24)
O
O
O
O
O
O
O
O
O
O
O
St
S S St
O
(23)
O
OH
OH
O
OH
O
C8H17
O
S
St
St
O
O St (25)
O
S
St (22)
21
Scheme 2. Steroidal substrates (1–16) and products of electrochemical reactions (17–25).

A.M. Tomkiel et al. / Steroids 82 (2014) 60–67
65
Table 1
Electrochemical oxidation of cholesterol derivatives (1–16) in the presence of 1,2:3,4-
di-O-isopropylidene-
-D-galactopyranose.a
OH
O
OH
OH
O
O
O
O
a
O
O
O
O
O
- e-
O
O
O
Substrate 3b-O-(10,20:30,40-di-O-isopropylidene-
a
-
D
-
Other steroidal
products (yield)
S
H
St
S
St
OH
St
O
.
.
galactopyranos-60-yl)-cholest-5-ene (17)
+
+
yield
S
H
OH
C8H17
O
1
2
3
4
5
6
7
8
20%
3%
9%
O
O
.
- OH , - H+
O
18 (1.5%)
21 (2%)
St
S
S
St
St (22)
<1%
Not detected
Traces
Traces
6%
Scheme 3. Tentative mechanism of the electrochemical reaction of thiocholesterol
(HS-St; 8).
22 (25%), 23
(11%)
9
10
16%
16%
20 (30%), 1 (29%)
1 (56%), 20 (16%),
19 (2%)
side moiety in 22 was proven by the presence in its 1H NMR spec-
trum of a narrow signal (J = 1.3 Hz) of the anomeric proton at
4.23 ppm. As it could be expected, cholesteryl disulfide (23) was
formed as a minor product of the reaction.
11
12
13
14
2%
2%
4%
24 (6%), 19 (3%)
25 (24%)
32%
21 (5%), 1 (3%),
19 (2%), 20 (2%)
1 (6%)
Further study employed t-butyl and t-butyldimethylsilyl choles-
terol derivatives (9 [23] and 10 [24]). The electrochemical reactions
afforded cholesterol glycoconjugate 17 only in low yields. This was
so because cleavage of the O–C3 bond is not the favored reaction in
these systems. The privileged reaction is scission of the neighboring
bond between the oxygen atom and the adjacent carbon or the sil-
icon tertiary center. These reactions lead to the formation of choles-
terol 1. The reaction with cholesteryl benzyl ether (12) [21] was
also studied. However, conversion of the reaction was very low.
In the next attempt cholesteryl p-tosylate (11) was used as a
cholesterol donor. The conversion was rather low and a mixture
of two glycoconjugates (17 and 24) was formed in addition to
cholesteryl chloride 19. The initially formed glycoconjugate 17
underwent partial deprotection to 30,40-monoacetonide 24 on pro-
longed electrolysis. This result prompted us to study the stability of
glycoconjugate 17 under reaction conditions. The anodic oxidation
of compound 17 was carried out in the same way (galvanostatic
conditions) as for other substrates. To our surprise glycoconjugate
17 did not prove to be resistant to further transformations. The
decomposition products were: 30,40-monoacetonide 24 (18%), cho-
lesteryl chloride 19 (8%), and cholesterol 1 (6%). It is obvious from
this experiment that harsh reaction conditions (high amperage,
long reaction time) should be avoided.
15
16
54%
46%
1 (5%)
a
A divided H-cell was used with the cathodic and anodic compartments (3.5 mL
of electrolyte each) separated by a glass frit. In all of the measurements 0.1 M
solution of TBABF4 in dichloromethane was used as a supporting electrolyte. Gal-
vanostatic conditions were applied (current 5–10 mA) and the reaction time was set
to 3600–6000 s. A platinum mesh was used as a cathode and a platinum plate
(2 ꢁ 1.5 cm) was used as an anode. All measurements were carried out in room
temperature.
It is well known that electrochemical oxidation of alkyl iodides
leads to the loss of a non-bonding electron from the iodine atom,
followed by cleavage of the C–I bond to form a carbonium ion
and an iodine atom. The products described in the literature were
formed by further reactions of the carbonium ion, while two iodine
atoms combined to form I2 [34]. However, to our surprise an
analogous electrochemical reaction of readily available cholesteryl
iodide (6) [25] with 1,2:3,4-di-O-isopropylidene-a-D-galactopyra-
nose failed (mostly unchanged starting material was recovered).
This is probably due to the further reaction of the carbonium
ion with I2 which leads to the starting material and an iodine(I)
species [35,36]. This process competes with the carbocation reac-
tion with the sugar nucleophile and prevents production of the de-
sired product. Most likely the iodine(I) species is further reduced in
the reaction medium to iodide. Attempts to force 6 to react re-
sulted in partial success. The reaction carried out in the presence
of Co(ClO4)2 as a mediator in a dichloromethane/acetonitrile 7:3
mixture (higher acetonitrile content caused solubility problems)
afforded the desired glycoconjugate 17 in a 17% yield.
Hydroquinone glycosides have recently been reported as a new
class of glycosyl donors in electrochemical reactions [39]. We
thought that the analogous sterol hydroquinone ethers can serve
as sterol donors. However, the reaction stopped at the dicholeste-
ryl hydroquinone ether 25 stage (Scheme 4) and further oxidation
of this intermediate did not proceed under the conditions that
were applied.
A similar failed attempt was experienced with cholesteryl tri-
fluoroacetate (5) [37] and thiocyanate (7) [26,27] as sterol donors.
The reason may be their side reactions that occur at the anode.
Also, the conversion was very low in the case of phenylselenide 4
(obtained by substitution of p-toluenesulfonate 11 with phenylsel-
enol) [38]. It should be noticed that this compound shows an
opposite configuration at C3 since the substitution with a highly
nucleophilic phenylselenide followed the SN2 mechanism. The
electrochemical reaction of 4 afforded less than 1% of glycoconju-
gate 17 in addition to the elimination product 21 (2%).
None of the reactions reported so far gave a better yield of gly-
conjugate 17 than the reaction with cholesterol 1 itself.
The best results were achieved with cholesteryl trichloroace-
timidate (14) and diphenylphosphate (15) [27,40]. This can proba-
bly be attributed to the high electrochemical stability of the
leaving groups, especially in the case of diphenylophosphate [41].
C8H17
C8H17
C8H17
Then the electrochemical reaction of thiocholesterol (8) [26,27]
was briefly studied. The major product of a reaction with the gal-
actose derivative proved to be compound 22, which contained
two thiocholesterol molecules attached to the sugar core. This
product was probably formed by two consecutive nucleophilic at-
tacks of thiocholesterol on the electrochemically activated sugar
13
O
- 2e-
- H+
O
- H+
(+)
O
O
HO
13
25
O
C8H17
acetals as depicted in Scheme 3.
a
-Configuration of the thioglyco-
Scheme 4. The electrochemical reaction of a cholesterol hydroquinone ether (13).

66
A.M. Tomkiel et al. / Steroids 82 (2014) 60–67
C8H17
C8H17
.
NH
- e-
+
.
NH
C8H17
-CCl3
- HNCO
Cl3C
PhO
C
O
Cl3C
C
O
14
15
Sugar-OH
- H+
16
C8H17
C8H17
.
+
-
PhO
.
O
PhO P O
+
- PhO-PO2
- e-
O
P
OPh
O
OPh
Scheme 5. Tentative mechanisms of electrochemical reactions of cholesteryl trichloroacetimidate (14) and diphenylphosphate (15).
O
COOMe
O
or
O
P
OPh
O
P
OPh
PhO
O
PhO
OH
O
26
27
O
O
Anodic oxidation
O
O
O
O
O
O
HO
Cl
O
O
30a (2%)
30b (1%)
31a (5%)
31b (4%)
28a (37%)
28b (37%)
29a (7%)
29b (5%)
O
O
a: diosgenin derivative; b: 5-cholenate derivative
Scheme 6. The electrochemical glycosylation of diosgenin and methyl 5-cholenoate diphenylphosphates (26 and 27).
The tentative mechanism of anodic reactions proceeding with
these cholesteryl donors is outlined in Scheme 5. It seems that
these processes involve a splitting of the initially formed steroidal
radical cation into a trichloromethyl radical, isocyanic acid and the
mesomeric cation, which reacts with a sugar partner.
Glycosyl trichloroacetimidates [8] are widely used in sugar
chemistry for glycosylation of various aglycones, including steroids
[9,10]. However, they have never been employed, to the best of our
knowledge, in electrochemical glycosylation. Anodic oxidation of
cholesteryl trichloroacetimidate (14) in the presence of 1,2:3,4-
phite (cholesteryl-O-P(OMe)2; 16) with 1,2:3,4-di-O-isopropyli-
dene- -galactopyranose was also briefly studied. The yield of
glycoconjugate 17 was quite good (46%) but inferior to that
achieved with cholesteryl diphenylphosphate (15) as a cholesteryl
donor. Therefore diphenylphosphates were chosen for further
study of electrochemical glycosylation.
The reactions of other steroidal diphenylphosphates (26 and 27)
derived from diosgenin or methyl 5-cholenoate, respectively, with
the model sugar also provided reasonable yields (37% in both cases)
of the corresponding glycoconjugates 28 (Scheme 6) [22]. The
major products were accompanied by a number of by-products –
parent sterols 29, chlorides 30, and disteroidal dimers 31. It should
be noted that the spiroketal side chain of 26 remained intact during
the electrochemical reaction. We have previously reported [43] that
the isomerization of spiroketals at C20 may occur under similar
conditions. However, such processes take place at higher potential,
a-D
di-O-isopropylidene-a-D-galactopyranose afforded product 17 in
a better yield (32%) than with the cholesteryl donors that were pre-
viously described. However, a number of by-products was also
detected. By using a twofold excess of sugar the reaction yield
was increased to 39%. In another experiment steroid was used in
excess and then the reaction yield amounted to 57%.
Chemical glycosylations based on glycosyl phosphates as glyco-
syl donors were also widely studied [42]. We prepared cholesteryl
diphenylphosphate (15) and its electrochemical reaction with a
model sugar was carried out. The reaction afforded glycoconjugate
17 in a relatively good yield (54%) in addition to a small amount of
cholesterol as the only by-product. A reaction mechanism similar
to that described above for trichloroacetimidate 14 is suggested
(Scheme 5). In this case the initial radical cation underwent
splitting to a phenoxy radical, phenyl metaphosphate and the
mesomeric carbocation. The reaction of cholesteryl dimethylphos-
like electrooxidation of 1,2:3,4-di-O-isopropylidene-a-D-galacto-
pyranose, and do not disturb the glycosylation reaction.
4. Conclusion
A series of cholesterol derivatives was examined as the poten-
tial donors of a steroid moiety in the electrochemical synthesis of
glycoconjugates. Readily available cholesteryl diphenylphosphate
(15) was found to be the best compound for this purpose. With this

A.M. Tomkiel et al. / Steroids 82 (2014) 60–67
67
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Acknowledgment
Financial support from the Polish National Science Centre
(UMO-2011/01/B/ST5/06046) is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2014.01.
007.
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