Dimeric cholaphanes with disulfide spacers

Article abstract of DOI:10.1080/17415993.2017.1414820

New dimeric cholaphanes with disulfide spacers were prepared as potential supramolecular hosts. The spacers bind two identical steroidal subunits through C3, C3′ and C24, C24′ positions (head-to-head isomer–‘cis-dimer’; 5) or C3, C24′ and C3′, C24 positions (head-to-tail isomer–‘trans-dimer’; 4). Another cyclic dimer containing both disulfide and sulfide spacers in its structure (6) was also obtained.

Full text of DOI:10.1080/17415993.2017.1414820

Journal of Sulfur Chemistry
ISSN: 1741-5993 (Print) 1741-6000 (Online) Journal homepage: http://www.tandfonline.com/loi/gsrp20
Dimeric cholaphanes with disulfide spacers
Aneta M. Tomkiel, Urszula Kiełczewska, Barbara Seroka & Zenon Łotowski
To cite this article: Aneta M. Tomkiel, Urszula Kiełczewska, Barbara Seroka & Zenon
Łotowski (2017): Dimeric cholaphanes with disulfide spacers, Journal of Sulfur Chemistry, DOI:
10.1080/17415993.2017.1414820
To link to this article: https://doi.org/10.1080/17415993.2017.1414820
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JOURNAL OF SULFUR CHEMISTRY, 2017
https://doi.org/10.1080/17415993.2017.1414820
Dimeric cholaphanes with disulfide spacers
Aneta M. Tomkiel, Urszula Kiełczewska, Barbara Seroka and Zenon Łotowski
Institute of Chemistry, University of Białystok, Białystok, Poland
ABSTRACT
ARTICLE HISTORY
Received 2 August 2017
Accepted 3 December 2017
New dimeric cholaphanes with disulfide spacers were prepared
as potential supramolecular hosts. The spacers bind two identical
steroidal subunits through C3, C3ꢀ and C24, C24ꢀ positions (head-
to-head isomer – ‘cis-dimer’; 5) or C3, C24ꢀ and C3ꢀ, C24 positions
(head-to-tail isomer – ‘trans-dimer’; 4). Another cyclic dimer contain-
ing both disulfide and sulfide spacers in its structure (6) was also
obtained.
KEYWORDS
Bile acids; cholaphanes;
disulfides; sulfides;
supramolecular chemistry
1. Introduction
Bile acids are useful starting materials for the synthesis of macrocyclic host molecules.
Cholic acid (1) in particular is widely used as a building block for the synthesis of molecular
receptors [1,2], enzyme models, and transporters across phospholipid membranes [3,4].
This is an easily available steroidal substrate that perfectly meets the requirements of this
type of systems. It is characterized by structural rigidity, enantiomeric purity, and well-
defined geometry of the molecule. In addition, cholic acid has three hydroxyl groups with
varying chemical reactivity and unique amphiphilicity associated with the presence of two
sides of the molecule: the hydrophilic side α containing –COOH and –OH groups, and
the hydrophobic side β constituted by C18 and C19 methyl groups [3,4]. There are many
kinds of dimeric structures based on bile acids. They can be acyclic, e.g. molecular ‘clefts’
[5], ‘tweezers’ [6], or ‘umbrellas’ [7], and cyclic, e.g. cyclocholates [8,9] and cholaphanes
[1,10]. Cholaphanes are bile acid-based macrocycles consisting of two to four bile acid
subunits joined together by different atom groups as spacers. Dimeric cholaphanes bind
‘guests’ more strongly and more selectively compared to acyclic dimeric structures [4].
CONTACT Aneta M. Tomkiel
a.tomkiel@uwb.edu.pl
Institute of Chemistry, University of Białystok,
Ciołkowskiego 1K, 15-245 Białystok, Poland
© 2017 Informa UK Limited, trading as Taylor & Francis Group

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A. M. TOMKIEL ET AL.
Scheme 1. Structures of cholaphanes synthesized on the basis of cholic acid.
In our previous papers [11,12], we have described the syntheses of new acyclic dimers of
cholic acid that may act as potential supramolecular hosts containing disulfide spacers (2
and 3). Now we report the synthesis of new cyclic isomeric cholaphanes (4 and 5), which
can be potentially useful for molecular or ionic recognition. We have also synthesized a
new cholic acid-derived dimer (6), containing both a disulfide (at C3, C3ꢀ positions) and a
sulfide linkers (at C24, C24ꢀ positions; Scheme 1).
Disulfide spacers play an important role in biological systems – they can readily undergo
reduction and decomposition according to the reversible redox reaction: 2 · thiol ꢀ disul-
fide + 2H+ + 2e−, observed in, e.g. cysteine–cystine, or glutathione–glutathione disulfide
(GSSG) [13]. Their use in the synthesis of supramolecular hosts should facilitate the release
of the guest molecule from the supramolecule. In addition, the conversion of hydrophobic
disulfide to hydrophilic mercapto groups would make the removal of host molecules from
the body easier.
2. Results and discussion
The dimeric cholaphanes (4 and 5) were prepared according to Scheme 2. Methyl cholate
(7) was obtained from cholic acid (1) using MeOH/HCl [14]. Its LiAlH4 reduction afforded
tetraol 8 [15], which was treated with an iodine–triphenylphosphine complex in ben-
zene/pyridine solution [16] to give diiodide 9. The dihalide was subjected to the reaction
with KSCN in acetone, leading to dithiocyanate 10 [11]. Both iodination and SCN− sub-
stitution transformations are SN2-type reactions (3α-OH→3β-I and 3β-I→3α-SCN),
therefore, C3 configuration in the resulting dithiocyanate 10 remained the same as in
the starting alcohol 8, that was confirmed by NMR spectroscopy. Reduction of 10 with
LiAlH4 in THF gave dithiol 11 in satisfactory yield (74%) [12]. The last and the most
crucial step of the synthesis – transformation of 11 to cyclic dimers 4 and 5 – was per-
formed by its oxidation with sodium hypoiodite, generated in situ from iodine and sodium
hydroxide. We obtained a mixture of two isomeric cholaphanes, readily separable by
a column chromatography, in total yield 45% and ‘trans-dimer’(4)/‘cis-dimer’ (5) ratio
of 1:2.
Determining the structure of both dimers (4 and 5) using the X-ray analysis
proved impossible due to the difficulty in obtaining well-formed crystals. To define the

JOURNAL OF SULFUR CHEMISTRY
3
Scheme 2. Synthesis of cyclic dimers (4 and 5) with disulfide spacers.
stereochemistry of 4 and 5, we recorded the 2D NMR spectra (COSY, HSQC, and HMBC),
but the coupling between protons (or carbon atoms) at C3 and C24ꢀ positions in the head-
to-tail dimer (4) was poorly visible due to the large distance between them (up to 5 bonds).
In the case of head-to-head isomer (5), the protons and carbon atoms at C3 and C3ꢀ (and
C24 and C24ꢀ) positions are indistinguishable, so it is impossible to confirm the struc-
ture by this method. For this reason, we decided to prepare the head-to-head dimer (5)
(Scheme 3) exclusively and compare the NMR spectra of compounds obtained by both
syntheses. Performed molecular modeling with the HyperChem 6.0 (by Hypercube, Inc.),
using the semi-empirical AM-1 method, led to the determination of the plausible spatial
distribution of the cholyl units in both macrocycles. Figure 1 shows the arrangement of
atoms in cholaphanes 4 and 5. Due to different compositions of monomer units, dimers
differ not only in the size of the internal gap, but also in the location of the hydroxyl groups
responsible for the complexation process. Both of these factors affect the choice of poten-
tial guest that can be bound by the host molecule. Molecular modeling also allowed us to
define the types of compounds (e.g. pyrogallol and pyrocatechol) that can suit the size of
the cleft and the distribution of the active sites of the molecules.
Scheme 3 shows two strategies tested for the selective preparation of ‘cis-dimer’ (5).
In the first attempt, acyclic disulfide 2 was used as a starting material, which had been
prepared according to the procedure described in our previous paper [11]. The synthesis
plan of 5 assumed iodination of disulfide 2 followed by the reaction with NaSH · 2H2O,
but only the first step of this project (2 to 12 conversion) proved to be successful. Diiodide
12 was obtained using iodine–triphenylphosphine complex in benzene/pyridine solution
(analogously to the preparation of diiodide 9; Scheme 2) and was subjected to reaction with
NaSH · 2H2O in absolute ethanol [11]. Unfortunately, the main product of this reaction was
compound 13, achieved by hydrogen iodide elimination and disulfide group reduction.
Although we have not obtained the expected product 5, this reaction was a direct proof
of easy decomposition of disulfide bond in this kind of structures. This feature is highly
desirable in the synthesis of potential molecular hosts.

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A. M. TOMKIEL ET AL.
Scheme 3. Two strategies tested for the selective preparation of head-to-head cholaphane (5).
Figure 1. The arrangement of atoms in cholaphanes 4 and 5.
Due to the failure of the first approach an alternative route was explored. The second
synthetic strategy was analogous to that already described, but this time acyclic disulfide 3
was used as a starting material. Compound 3 was prepared as we have previously reported

JOURNAL OF SULFUR CHEMISTRY
5
Scheme 4. Tentative mechanism of cholaphane 5 and 6 formation.
[12] and treated with iodine–triphenylphosphine complex (according to general iodina-
tion procedure) to give diiodide 14. This dihalide was reacted with NaSH · 2H2O in ethanol
and a mixture of two cyclic dimers (5 and 6) was received. As we expected, the cholaphane
(5) was formed, but in 39% yield only. The predominant product of this reaction proved
to be compound 6 (48%). Such proportion of products was possible to obtain when 10
equivalents of NaSH · 2H2O were used, whereas the amount of the reagent was increased,
formation of sulfide 6 was favored. The usage of a large excess of NaSH · 2H2O (more than
20 equivalents) resulted only in compound 6 (85%). Contrarily, decreasing the amount of
inorganic reagent (to 5 equivalents) led to disulfide 5 predominantly (49%). The struc-
ture of compound 6 was confirmed by MS spectroscopy, where a molecular ion peak 839
(M+ + Na) was present.
The mechanism for the 5 and 6 formation was proposed in Scheme 4. As soon as one of
the iodine atoms of compound 14 was substituted by the thiol group, the intermediate A
was formed. The use of a large excess of NaSH · 2H2O generates more alkaline conditions
and causes ionization of this mercapto group (compound B). The RS− ion is a stronger
nucleophile than the NaSH-derived SH− ion and, as a result, competes with it in the reac-
tion with the second –CH2I group from the same molecule. When the RS− ion ‘wins’
the competition, an intramolecular nucleophilic substitution takes place and compound
6 is finally formed. Otherwise, an acyclic dithiol (C) is formed. The dimer C can be eas-
ily oxidized, therefore, the expected head-to-head cholaphane (5) was obtained. It should
be noted that compound 6 can be interesting as a potential supramolecular host too. The
presence of the sulfide bond at positions C24, C24ꢀ in its structure changes an internal slot
size and may be able to bind different molecules than those complexed by 4 and 5.
Scheme 5(a,b) shows a new procedure for the preparation of acyclic disulfide 2 and
failed synthetic route of cholaphane 6. The starting compounds 15 and 16 were achieved
by methods described in the literature [17]. Methyl cholate (7) was subjected to silylation
with tert-butyldimethylsilyl chloride in the presence of DMAP in DMF, followed by LiAlH4
reduction in THF to give triol 16. The 3α-hydroxyl group was converted to TBDMS ether
to protect the C3 position from iodination reaction. 24-Iodide (17) was obtained analo-
gously to the preparation of diiodides 9, 12, and 14, this time with the half amount of the

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A. M. TOMKIEL ET AL.
Scheme 5. (a) Synthesis of acyclic disulfide 2; (b) Synthesis of dithiol 24 and macrocycle 25.
iodinating agent. In the next step, iodide 17 was substituted with SH− in absolute ethanol
and, as a result, three new less polar products were formed. The analyses of NMR and MS
spectra confirmed that compounds 18, 19, and 20 have been obtained. While the acyclic
disulfide 19 was formed by oxidation of thiol 18, the sulfide 20 formation can be explained
in a similar way as unexpected synthesis route of dimer 6. Thiol 18 was oxidized by sodium
hypoiodite to disulfide 19, which was then subjected to TBDMS group removal to give
disulfide 2.
After deprotection of the 3α-hydroxyl group in dimer 20, sulfide 21 was obtained.
This compound seemed to be a convenient substrate for the synthesis of macrocycle 6.

JOURNAL OF SULFUR CHEMISTRY
7
Scheme 6. Tentative mechanism of compound 25 formation.
Therefore, steroidal diiodide 22, prepared from sulfide 21 by general iodination proce-
dure, was used as starting material for the nucleophilic substitution of iodide by SCN− ion
using KSCN. The LiAlH4 reduction of received dithiocyanate 23 gave the inseparable mix-
ture of two compounds – dithiol 24 and macrocycle 25. This was confirmed by the NMR
and MS spectra analysis of this mixture. In the mass spectrum two peaks were present,
among which 807 (M+ + Na) from 25 was more intensive. Its formation is possible due to
the fact that SCN− ion, also called ‘pseudohalide’, reacts similar to halide ions [18] and can
be substituted by a thiol group during the reaction as shown in Scheme 6 (Finkelstein-type
reactions). The second signal, 841 (M+ + Na) represented the molecular ion of dithiol 24.
3. Conclusions
Two new cholaphanes with disulfide spacers – head-to-tail dimer (4) and head-to-head
dimer (5) – were achieved in a four-step procedure. Another route of the synthesis allowed
to obtain a different dimeric cholic acid derivative, containing both disulfide and sulfide
linkers in its structure (compound 6). It was also attempted to selectively obtain a head-to-
head isomer (5) from disulfide dimer C24, C24ꢀ (2). Unfortunately, this strategy resulted
in the formation of elimination product 13. Finally, the ‘cis-dimer’ 5 was received from C3,
C3ꢀ disulfide 3, accompanied by the cholaphane 6 with a sulfide linker at C24, C24ꢀ. The
latter was also selectively formed by the use of large excess of NaSH · 2H2O in the reaction
with diiodide 14.
All of the received cholaphanes (4, 5 and 6) will be checked for potential metal ion
complexation and the ability to recognize and bind small molecules. The physicochemical
studies of their properties are in progress.
4. Experimental
4.1. Materials and apparatus
The cholic acid derivatives, methyl cholate (7) [14], 5β-cholane-3α,7α,12α,24-tetraol
(8) [15], di(3α,7α,12α-trihydroxy-5β-cholan-24-yl) disulfide (2) [11], di(7α,12α,24-
trihydroxy-5β-cholan-3α-yl) disulfide (3) [12], methyl 3α-t-butyldimethylsilyloxy-
7α,12α-dihydroxy-5β-cholan-24-ate (15) [17] and 3α-t-butyldimethylsilyloxy-5β-
cholane-7α,12α,24-triol (16) [17], were prepared according to known procedures.
Melting points were determined on a Toledo Mettler-MP70 or a Kofler bench (Boetius
type) melting point apparatus. 1H and 13C NMR (400 and 100 MHz, respectively) spectra
were recorded on a Bruker Avance II spectrometer in CDCl3 (or CDCl3/MeOD mixture)

8
A. M. TOMKIEL ET AL.
solutions with TMS as the internal standard (only selected signals in the 1H NMR spec-
tra are reported). Infrared spectra were recorded on a Nicolet series II Magna-IR 550
FTIR spectrometer. Mass spectra were recorded with a time-of-flight (TOF) AMD-604
and Accurate-Mass Q-TOF LC/MS 6530 spectrometers with electrospray ionization (ESI).
Merck Silica Gel 60, F 256 TLC aluminum sheets were applied for thin-layer chromato-
graphic analysis. For a visualization of the products, a 5% solution of phosphomolybdic
acid in ethanol was used. The reaction products were separated by column chromatography
performed on a 70–230 mesh silica gel (J. T. Baker).
4.2. General procedures and spectral data
4.2.1. Iodination
To a solution of I2 (1.3 g; 5 mmol) in 20 mL anhydrous benzene triphenylphosphine (1.3 g;
5 mmol) and anhydrous pyridine (0.8 mL; 10 mmol) were added and the mixture was
stirred at room temperature for 20 min. Then 1 mmol steroidal substrate (2, 3, 8, 16 or 21)
was added. The reaction mixture was stirred for 1–3 days. After this time, it was poured
into water (200 mL) and extracted with benzene (3 × 100 mL). The extract was dried over
anhydrous Na2SO4 and evaporated to dryness in vacuo. The residue was subjected to silica
gel column chromatography.
4.2.1.1. 3β,24-Diiodo-5β-cholane-7α,12α-diol. 9 was eluted with hexane/ethyl acetate
(9:1) mixture in 54% yield. Colorless crystals, mp 135–137°C (hexane/ethyl acetate); IR
ATR, νmax (cm−1): 3723 and 3392 (–OH), 2925 and 2865 (–CH), 1225, 1168, 1082 and 1027
(C–O), 593 (C–I); 1H NMR (CDCl3), δ (ppm): 4.98 (m, 1H, 3α-H), 3.99 (m, 1H, 12β-H),
3.85 (m, 1H, 7β-H), 3.24-3.11 (m, 2H, 24-CH2I), 2.47 (ddd, J1 = 15.9 Hz, J2 = 12.5 Hz,
J3 = 3.4 Hz, 1H, 4α-H), 1.01 (s, 3H, 19-CH3), 0.99 (d, J = 6.6 Hz, 3H, 21-CH3), 0.71 (s,
3H, 18-CH3);13C NMR (CDCl3), δ (ppm): 72.94 (CH), 68.23 (CH), 47.37 (CH), 46.48 (C),
41.87 (CH), 40.35 (CH), 39.84 (CH2), 39.36 (CH), 38.50 (CH), 36.66 (CH2), 35.68 (C),
34.81 (CH), 34.15 (CH2), 32.66 (CH2), 30.95 (CH2), 30.30 (CH2), 28.23 (CH2), 27.59 (CH),
27.53 (CH2), 23.14 (CH2), 22.68 (CH3), 17.77 (CH3), 12.48 (CH3), 7.75 (CH2); ESI MS,
m/z: 615 [(M + H)+, 100%]; HRMS-ESI (m/z): [M + H]+ calcd for C24H41I2O2 615.1196,
found 615.1175.
4.2.1.2. Di(7α,12α-dihydroxy-3β-iodo-5β-cholan-24-yl) disulfide. 12 was eluted with
hexane/ethyl acetate (75:25) mixture in 43% yield. Colorless crystals, mp 143–145°C
(hexane/CH2Cl2); IR ATR, νmax (cm−1): 3435 (–OH), 2922 and 2853 (–CH), 1221 (C–O),
1
1082 and 1037 (C–O and C–S); H NMR (CDCl3), δ (ppm): 4.99 (m, 2H, 3α-H and
3αꢀ-H), 4.01 (m, 2H, 12β-H and 12βꢀ-H), 3.84 (m, 2H, 7β-H and 7βꢀ-H), 2.72-2.61 (m,
4H, 24-CH2S- and 24ꢀ-CH2S-), 2.48 (ddd, J1 = 15.8 Hz, J2 = 12.4 Hz, J3 = 3.2 Hz, 2H,
4α-H and 4αꢀ-H), 1.01 (s, 6H, 19-CH3 and 19ꢀ-CH3), 1.00 (d, J = 6.2 Hz, 6H, 21-CH3
and 21ꢀ-CH3), 0.71 (s, 6H, 18-CH3 and 18ꢀ-CH3);13C NMR (CDCl3), δ (ppm): 72.92
(2xCH), 68.17 (2xCH), 47.50 (2xCH), 46.55 (2xC), 42.11 (2xCH), 40.35 (2xCH), 39.93
(2xCH2), 39.67 (2xCH2), 39.58 (2xCH), 38.59 (2xCH), 35.68 (2xC), 35.24 (2xCH), 34.67
(2xCH2), 34.00 (2xCH2), 32.68 (2xCH2), 31.04 (2xCH2), 28.34 (2xCH2), 27.87 (2xCH),
27.55 (2xCH2), 25.76 (2xCH2), 23.12 (2xCH2), 22.81 (2xCH3), 17.77 (2xCH3), 12.57

JOURNAL OF SULFUR CHEMISTRY
9
(2xCH3); ESI MS, m/z: 1061 [(M + Na)+, 100%]; HRMS-ESI (m/z): [M + H]+ calcd for
C48H81I2O4S2 1039.3666, found 1039.3637.
4.2.1.3. Di(7α,12α-dihydroxy-24-iodo-5β-cholan-3α-yl) disulfide. 14 was eluted with
hexane/ethyl acetate (95:5) mixture in 55% yield. Colorless crystals, mp 131–133°C
(hexane/CH2Cl2); IR (CHCl3), νmax (cm−1): 3609 and 3444 (–OH), 2941 and 2869 (–CH),
1233 and 1173 (C–O), 1084, 1069 and 1037 (C–O and C–S), 597 (C–I); 1H NMR (CDCl3),
δ (ppm): 3.97 (m, 2H, 12β-H and 12βꢀ-H), 3.86 (m, 2H, 7β-H and 7βꢀ-H), 3.24-3.12 (m,
4H, 24-CH2I and 24ꢀ-CH2I), 2.50 (tt, J1 = 12.3 Hz, J2 = 3.4 Hz, 2H, 3β-H and 3βꢀ-H),
2.45 (bs, 4H, 4xOH), 2.34 (q, J = 12.3 Hz, 2H, 4α-H and 4αꢀ-H), 2.16 (dt, J1 = 12.5 Hz,
J2 = 4.6 Hz, 2H, 9-H and 9ꢀ-H), 1.00 (d, J = 6.5 Hz, 6H, 21-CH3 and 21ꢀ-CH3), 0.90
(s, 6H, 19-CH3 and 19ꢀ-CH3), 0.69 (s, 6H, 18-CH3 and 18ꢀ-CH3);13C NMR (CDCl3),
δ (ppm): 73.09 (2xCH), 68.33 (2xCH), 48.72 (2xCH), 47.33 (2xCH), 46.49 (2xC), 43.42
(2xCH), 41.79 (2xCH), 39.49 (2xCH), 37.33 (2xCH2), 37.21 (2xCH2), 36.75 (2xCH2),
35.00 (2xCH), 34.93 (2xC), 34.64 (2xCH2), 30.48 (2xCH2), 28.36 (2xCH2), 27.67 (2xCH2),
27.54 (2xCH2), 26.52 (2xCH), 23.18 (2xCH2), 22.77 (2xCH3), 17.79 (2xCH3), 12.50
(2xCH3), 7.89 (2xCH2); ESI MS, m/z: 1061 [(M + Na)+, 100%], 483 [22%]; HRMS-ESI
(m/z): [M + H]+ calcd for C48H81I2O4S2 1039.3666, found 1039.3641.
4.2.1.4. 3α-t-Butyldimethylsilyloxy-24-iodo-5β-cholane-7α,12α-diol. 17 was eluted with
hexane/ethyl acetate (95:5) mixture in 97% yield. The iodination reaction was carried out
according to the general procedure with half of the amount of iodinating agent. Colorless
crystals, mp 162–164°C (hexane/ethyl acetate); IR ATR, ν
(cm−1): 3344 (–OH), 2927
max
and 2860 (–CH), 1248, 1184, 1079 and 1039 (C–O); 1H NMR (CDCl3), δ (ppm): 3.97 (m,
1H, 12β-H), 3.84 (m, 1H, 7β-H), 3.46-3.39 (m, 1H, 3β-H), 3.23-3.11 (m, 2H, 24-CH2I),
2.26-2.17 (m, 2H, 4α-H and 9-H), 0.99 (d, J = 6.6 Hz, 3H, 21-CH3), 0.88 (s, 12H, 19-
CH3 and -Si–C(CH3)3), 0.69 (s, 3H, 18-CH3), 0.04 (s, 6H, -Si(CH3)2);13C NMR (CDCl3),
δ (ppm): 72.99 (CH), 72.78 (CH), 68.43 (CH), 47.18 (CH), 46.46 (C), 41.95 (CH), 41.57
(CH), 39.97 (CH2), 39.49 (CH), 36.69 (CH2), 35.35 (CH2), 34.86 (CH), 34.73 (CH2), 34.66
(C), 31.06 (CH2), 30.20 (CH2), 28.25 (CH2), 27.55 (CH2), 26.63 (CH), 25.90 (3xCH3),
23.19 (CH2), 22.56 (CH3), 18.17 (C), 17.73 (CH3), 12.44 (CH3), 7.83 (CH2), −4.58 (CH3),
−4.65 (CH3); ESI MS, m/z: 641 [(M + Na)+, 100%], 619 [(M + H)+, 68%]; HRMS-ESI
(m/z): [M + H]+ calcd for C30H56IO3Si 619.3043, found 619.3020.
4.2.1.5. Di(7α,12α-dihydroxy-3β-iodo-5β-cholan-24-yl) sulfide. 22 was eluted with
hexane/ethyl acetate (7:3) mixture in 57% yield. Colorless crystals, mp 157–159°C (hex-
ane/ethyl acetate); IR ATR, ν
(cm−1): 3405 (–OH), 2924 and 2860 (–CH), 1256 and
max
1183 (C–O), 1082 and 1033 (C–O and C–S); 1H NMR (CDCl3), δ (ppm): 4.98 (m, 2H, 3α-
H and 3αꢀ-H), 4.00 (m, 2H, 12β-H and 12βꢀ-H), 3.84 (m, 2H, 7β-H and 7βꢀ-H), 2.51-2.45
(m, 6H, 24-CH2S-, 24ꢀ-CH2S-, 4α-H and 4αꢀ-H), 1.01 (s, 6H, 19-CH3 and 19ꢀ-CH3), 0.99
(d, J = 6.5 Hz, 6H, 21-CH3 and 21ꢀ-CH3), 0.71 (s, 6H, 18-CH3 and 18ꢀ-CH3); 13C NMR
(CDCl3), δ (ppm): 72.92 (2xCH), 68.18 (2xCH), 47.51 (2xCH), 46.58 (2xC), 42.13 (2xCH),
40.30 (2xCH), 39.96 (2xCH2), 39.62 (2xCH), 38.63 (2xCH), 35.69 (2xC), 35.22 (2xCH),
35.15 (2xCH2), 34.03 (2xCH2), 32.87 (2xCH2), 32.71 (2xCH2), 31.06 (2xCH2), 28.35
(2xCH2), 27.90 (2xCH), 27.53 (2xCH2), 26.40 (2xCH2), 23.13 (2xCH2), 22.80 (2xCH3),

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A. M. TOMKIEL ET AL.
17.81 (2xCH3), 12.58 (2xCH3); ESI MS, m/z: 1029 [(M + Na)+, 12%], 901 [(M + Na-
HI)+, 100%]; HRMS-ESI (m/z): [M + H]+ calcd for C48H81I2O4S 1007.3945, found
1007.3932.
4.2.2. SCN− substitution
Steroidal iodine (9 or 22; 0.4 mmol) was dissolved in 30 mL of dry acetone and 0.8 g
(8 mmol) of powdered KSCN was added to the flask. The reaction mixture was refluxed
for 2 days. After cooling, the solvent was evaporated and the crude product was subjected
to column chromatography.
4.2.2.1. 3α,24-Dithiocyanato-5β-cholane-7α,12α-diol. 10 was eluted with hexane/ethyl
acetate (75:25) mixture in 70% yield. Colorless crystals, mp 67–69°C (hexane/ethyl
acetate); IR (CHCl3), ν
(cm−1): 3610 and 3482 (–OH), 2944 and 2868 (–CH), 2155
max
(–SCN), 1298, 1269 and 1239 (C–O), 1071 and 1036 (C–O and C–S);1H NMR (CDCl3),
δ (ppm): 4.02 (m, 1H, 12β-H), 3.87 (m, 1H, 7β-H), 3.20 (tt, J1 = 12.7 Hz, J2 = 3.8 Hz,
1H, 3β-H), 3.01–2.87 (m, 2H, 24-CH2SCN), 2.56 (q, J = 12.7 Hz, 1H, 4α-H), 2.17 (dt,
J1 = 11.9 Hz, J2 = 5.4 Hz, 1H, 9-H), 1.03 (d, J = 6.6 Hz, 3H, 21-CH3), 0.94 (s, 3H, 19-
CH3), 0.71 (s, 3H, 18-CH3);13C NMR (CDCl3), δ (ppm): 112.47 (C), 111.92 (C), 73.03
(CH), 68.10 (CH), 48.66 (CH), 47.28 (CH), 46.48 (C), 43.24 (CH), 41.78 (CH), 39.15
(CH), 36.78 (CH2), 36.41 (CH2), 35.27 (CH), 34.55 (C), 34.49 (CH2), 34.38 (CH2), 34.07
(CH2), 28.77 (CH2), 28.13 (CH2), 27.61 (CH2), 26.55 (CH2), 26.47 (CH), 23.13 (CH2),
22.40 (CH3), 17.67 (CH3), 12.37 (CH3); ESI MS, m/z: 499 [(M + Na)+, 100%]; HRMS-ESI
(m/z): [M + H]+ calcd for C26H41N2O2S2 477.2609, found 477.2619.
4.2.2.2. Di(7α,12α-dihydroxy-3α-thiocyanato-5β-cholan-24-yl) sulfide. 23 was eluted
with hexane/ethyl acetate (75:25) mixture in 78% yield. Colorless crystals, mp 103–106°C
(hexane/ethyl acetate); IR (CHCl3), ν
(cm−1): 3611 and 3473 (–OH), 2943 and 2868
max
(–CH), 2154 (–SCN), 1261 (C–O), 1070, 1036 and 1021 (C–O and C–S);1H NMR (CDCl3),
δ (ppm): 4.01 (m, 2H, 12β-H and 12βꢀ-H), 3.87 (m, 2H, 7β-H and 7βꢀ-H), 3.20 (tt,
J1 = 12.3 Hz, J2 = 3.7 Hz, 2H, 3β-H and 3βꢀ-H), 2.60-2.42 (m, 6H, 24-CH2S-, 24ꢀ-CH2S-,
4α-H and 4αꢀ-H), 2.30 (bs, 2H, 2xOH), 2.17 (dt, J1 = 12.1 Hz, J2 = 5.0 Hz, 2H, 9-H and
9ꢀ-H), 0.99 (d, J = 6.5 Hz, 6H, 21-CH3 and 21ꢀ-CH3), 0.93 (s, 6H, 19-CH3 and 19ꢀ-CH3),
0.70 (s, 6H, 18-CH3 and 18ꢀ-CH3); 13C NMR (CDCl3), δ (ppm): 112.00 (2xC), 72.84
(2xCH), 68.01 (2xCH), 48.78 (2xCH), 47.43 (2xCH), 46.54 (2xC), 43.38 (2xCH), 41.99
(2xCH), 39.55 (2xCH), 36.88 (2xCH2), 36.65 (2xCH2), 35.23 (2xCH), 35.09 (2xCH2),
34.60 (2xC), 34.18 (2xCH2), 32.78 (2xCH2), 28.67 (2xCH2), 28.34 (2xCH2), 27.54
(2xCH2), 26.81 (2xCH), 26.30 (2xCH2), 23.11 (2xCH2), 22.66 (2xCH3), 17.79 (2xCH3),
12.57 (2xCH3); ESI MS, m/z: 891 [(M + Na)+, 100%]; HRMS-ESI (m/z): [M + H]+ calcd
for C50H81N2O4S3 869.5358, found 869.5371.
4.2.3. Dithiocyanate reduction
To a solution of 0.2 mmol dithiocyanate (10 or 23) in 30 mL of anhydrous THF LiAlH4
(60 mg; 20 mmol) was added and the mixture was stirred at room temperature for 18 h.
Then several drops of water were added and the precipitate was filtered off. The solvent
was evaporated and the crude product was subjected to column chromatography.

JOURNAL OF SULFUR CHEMISTRY
11
4.2.3.1. 3α,24-Dimercapto-5β-cholane-7α,12α-diol. 11 was eluted with hexane/ethyl
acetate (9:1) mixture in 74% yield. Colorless crystals, mp 146–148°C (hexane/ethyl
acetate); IR (CHCl3), νmax (cm−1): 3617 and 3469 (–OH), 2933 and 2869 (–CH), 1260 and
1236 (C–O), 1069 and 1036 (C–O and C–S);1H NMR (CDCl3), δ (ppm): 4.00 (m, 1H, 12β-
H), 3.85 (m, 1H, 7β-H), 2.68–2.54 (m, 1H, 3β-H), 2.54–2.42 (m, 2H, 24-CH2S-), 2.34 (q,
J = 12.9 Hz, 1H, 4α-H), 2.19 (dt, J1 = 12.3 Hz, J2 = 4.5 Hz, 1H, 9-H), 0.99 (d, J = 6.5 Hz,
3H, 21-CH3), 0.89 (s, 3H, 19-CH3), 0.69 (s, 3H, 18-CH3);13C NMR (CDCl3), δ (ppm):
73.09 (CH), 68.36 (CH), 47.46 (CH), 46.51 (C), 43.66 (CH), 41.96 (CH), 41.71 (CH2), 39.45
(CH), 39.38 (CH), 37.60 (CH2), 35.25 (CH), 34.66 (CH2), 34.55 (CH2), 34.41 (C), 33.19
(CH2), 30.73 (CH2), 28.26 (CH2), 27.60 (CH2), 26.65 (CH), 25.03 (CH2), 23.16 (CH2),
22.72 (CH3), 17.76 (CH3), 12.46 (CH3); ESI MS, m/z: 427 [(M + H)+, 100%]; HRMS-ESI
(m/z): [M + H]+ calcd for C24H43O2S2 427.2704, found 427.2723.
4.2.4. Oxidation with sodium hypoiodite
Steroidal substrate (11 or 18; 0.2 mmol) was dissolved in 100 mL of THF and 100 mL 15%
aqueous solution of NaOH was added. Next, I2 (160 mg; 0.6 mmol) was added and the mix-
ture was stirred at room temperature for 1 h. After this time the solvent was evaporated and
aqueous solution of HCl was added. The crude product was extracted with ethyl acetate.
The extract was dried over anhydrous Na2SO4 and evaporated to dryness in vacuo. The
residue was subjected to silica gel column chromatography.
4.2.4.1. S,Sꢀ:Sꢀꢀ,Sꢀꢀꢀ-Di(5β-cholane-7α,12α-diol)-3α,24ꢀ:3αꢀ,24-disulfide. 4 was eluted
with hexane/ethyl acetate (95:5) mixture in 15% yield. Colorless crystals, mp 147–149°C
(heptane/MeOH); IR ATR, ν
(cm−1): 3431 (-OH), 2923 and 2858 (–CH), 1270
max
(C–O), 1076 and 1032 (C–O and C–S);1H NMR (CDCl3), δ (ppm): 3.94 (m, 2H, 12β-
H and 12βꢀ-H), 3.78 (m, 2H, 7β-H and 7βꢀ-H), 2.88-2.81 (m, 2H, 24a-CH2S- and 24aꢀ-
CH2S-), 2.62-2.56 (m, 2H, 24b-CH2S- and 24bꢀ-CH2S-), 2.52–2.37 (m, 4H, 3β-H, 3βꢀ-H,
4α-H, 4αꢀ-H), 2.17 (dt, J1 = 12.3 Hz, J2 = 5.0 Hz, 2H, 9-H and 9ꢀ-H), 1.02 (d, J = 5.8 Hz,
6H, 21-CH3 and 21ꢀ-CH3), 0.86 (s, 6H, 19-CH3 and 19ꢀ-CH3), 0.65 (s, 6H, 18-CH3 and 18ꢀ-
CH3); 13C NMR (CDCl3/CD3OD), δ (ppm): 72.68 (2xCH), 68.16 (2xCH), 49.27 (2xCH),
46.51 (2xC), 45.89 (2xCH), 43.52 (2xCH2), 43.46 (2xCH), 41.50 (2xCH), 39.54 (2xCH),
37.33 (2xCH2), 36.79 (2xCH2), 36.19 (2xCH), 34.94 (2xCH2), 34.62 (2xC), 34.31 (2xCH2),
29.59 (2xCH2), 28.13 (2xCH2), 27.94 (2xCH2), 26.21 (2xCH), 24.92 (2xCH2), 23.35
(2xCH2), 22.59 (2xCH3), 17.48 (2xCH3), 12.25 (2xCH3); ESI MS, m/z: 871 [(M + Na)+,
100%]; HRMS-ESI (m/z): [M + H]+ calcd for C48H81O4S4 849.5018, found 849.5035.
4.2.4.2. S,Sꢀ:Sꢀꢀ,Sꢀꢀꢀ-Di(5β-cholane-7α,12α-diol)-3α,3αꢀ:24ꢀ,24-disulfide. 5 was eluted
with hexane/ethyl acetate (96:4) mixture in 30% yield. Colorless crystals, mp 112–114°C
(heptane/MeOH); IR ATR, νmax (cm−1): 3420 (-OH), 2922 and 2857 (–CH), 1264 (C–O),
1032 (C–O and C–S);1H NMR (CDCl3), δ (ppm): 4.06 (m, 2H, 12β-H and 12βꢀ-H), 3.90
(m, 2H, 7β-H and 7βꢀ-H), 2.81–2.74 (m, 2H, 24a-CH2S- and 24aꢀ-CH2S-), 2.63-2.57 (m,
2H, 24b-CH2S- and 24bꢀ-CH2S-), 2.55-2.34 (m, 4H, 3β-H, 3βꢀ-H, 4α-H, 4αꢀ-H), 1.10
(d, J = 6.5 Hz, 6H, 21-CH3 and 21ꢀ-CH3), 0.93 (s, 6H, 19-CH3 and 19ꢀ-CH3), 0.74 (s,
6H, 18-CH3 and 18ꢀ-CH3); 13C NMR (CDCl3), δ (ppm): 73.12 (2xCH), 68.65 (2xCH),
48.02 (2xCH), 47.72 (2xCH), 46.53 (2xC), 43.02 (2xCH), 41.32 (2xCH), 39.55 (2xCH),
38.71 (2xCH2), 37.51 (2xCH2), 36.88 (2xCH2), 36.61 (2xCH), 34.81 (2xCH2), 31.92

12
A. M. TOMKIEL ET AL.
(2xC), 29.70 (2xCH2), 28.00 (2xCH2), 27.81 (2xCH2), 27.72 (2xCH2), 26.05 (2xCH), 25.65
(2xCH2), 23.42 (2xCH2), 22.51 (2xCH3), 17.61 (2xCH3), 12.43 (2xCH3); ESI MS, m/z: 871
[(M + Na)+, 100%]; HRMS-ESI (m/z): [M + H]+ calcd for C48H81O4S4 849.5018, found
849.5041.
4.2.5. Substitution using NaSH·2H2O
Steroidal substrate (12, 14 or 17; 0.2 mmol) was dissolved in 30 mL of absolute ethanol
and 0.2 g (2 mmol) of powdered NaSH·2H2O was added. The mixture was stirred at room
temperature for 18 h. After cooling, the solvent was evaporated and the crude product was
subjected to column chromatography. Then the precipitate was filtered off. The solvent was
evaporated and the crude product was subjected to column chromatography.
4.2.5.1. 24-mercapto-5β-chol-3-ene-7α,12α-diol. 13 was eluted with hexane/ethyl
acetate (8:2) mixture in 31% yield. Colorless crystals, mp 124–126°C (hexane/ethyl
acetate); IR ATR, νmax (cm−1): 3415 (–OH), 2921 and 2858 (–CH), 1653 (CC), 1223 (C–O),
1090 and 1038 (C–O and C–S);1H NMR (CDCl3), δ (ppm): 5.76–5.69 (m, 2H, 3-H and
4-H), 4.00 (m, 1H, 12β-H), 3.73 (m, 1H, 7β-H), 2.56–2.45 (m, 2H, 24-CH2S-), 0.99 (d,
J = 6.6 Hz, 3H, 21-CH3), 0.98 (s, 3H, 19-CH3), 0.72 (s, 3H, 18-CH3); 13C NMR (CDCl3),
δ (ppm): 132.57 (CH), 127.24 (CH), 73.03 (CH), 69.14 (CH), 47.57 (CH), 46.42 (C), 42.48
(CH), 41.69 (CH), 39.68 (CH), 35.16 (CH), 34.58 (CH2), 34.39 (CH2), 33.53 (CH2), 33.30
(C), 30.76 (CH2), 28.91 (CH2), 27.52 (CH2), 26.63 (CH), 25.06 (CH2), 23.01 (CH2), 22.25
(CH3), 21.83 (CH2), 17.75 (CH3), 12.70 (CH3); ESI MS, m/z: 415 [(M + Na)+, 100%], 312
[28%]; HRMS-ESI (m/z): [M + H]+ calcd for C24H41O2S 393.2827, found 393.2815.
4.2.5.2. S,Sꢀ:Sꢀꢀ-Di(5β-cholane-7α,12α-diol)-3α,3αꢀ-disulfide:24ꢀ,24-sulfide. 6
was
eluted with hexane/ethyl acetate (97:3) mixture in 48% yield (when more than 20 eq of
NaSH · 2H2O was used, 6 was formed as a only product in 76% yield). Colorless crys-
tals, mp 121–123°C (heptane/MeOH);); IR (KBr), ν
(cm−1): 3444 (–OH), 2934 and
max
1
2865 (–CH), 1232 and 1197 (C–O), 1083, 1070 and 1038 (C–O and C–S); H NMR
(CDCl3), δ (ppm): 3.97 (m, 2H, 12β-H and 12βꢀ-H), 3.85 (m, 2H, 7β-H and 7βꢀ-H),
2.63 (bs, 2H, 2xOH), 2.50-2.29 (m, 10H, 3β-H, 3βꢀ-H, 4α-H, 4αꢀ-H, 24-CH2S-, 24ꢀ-
CH2S- and 2xOH), 2.13 (dt, J1 = 12.2 Hz, J2 = 5.1 Hz, 2H, 9-H and 9ꢀ-H), 1.04 (d,
J = 6.4 Hz, 6H, 21-CH3 and 21ꢀ-CH3), 0.91 (s, 6H, 19-CH3 and 19ꢀ-CH3), 0.69 (s, 6H,
18-CH3 and 18ꢀ-CH3); 13C NMR (CDCl3), δ (ppm): 73.30 (2xCH), 68.32 (2xCH), 48.46
(2xCH), 46.37 (2xC), 46.20 (2xCH), 43.43 (2xCH), 41.41 (2xCH), 39.83 (2xCH), 37.48
(2xCH2), 35.73 (2xCH2), 35.61 (2xCH2), 34.75 (2xC), 34.50 (2xCH2), 29.67 (2xCH2),
28.34 (2xCH2), 28.01 (2xCH2), 27.18 (2xCH2), 26.79 (2xCH2), 26.20 (2xCH), 23.21
(2xCH2), 22.62 (2xCH3), 18.15 (2xCH3), 12.75 (2xCH3); ESI MS, m/z: 839 [(M + Na)+,
100%]; HRMS-ESI (m/z): [M + H]+ calcd for C48H81O4S3 817.5297, found 817.5291.
4.2.5.3. 3α-t-Butyldimethylsilyloxy-24-mercapto-5β-cholane-7α,12α-diol. 18 was eluted
with hexane/ethyl acetate (95:5) mixture in 29% yield. Colorless crystals, mp 105–107°C
(hexane/CH2Cl2); IR ATR, νmax (cm−1): 3355 (-OH), 2926 and 2857 (-CH), 1251 (C–O),
1076 and 1043 (C–O and C–S); 1H NMR (CDCl3), δ (ppm): 3.97 (m, 1H, 12β-H), 3.83
(m, 1H, 7β-H), 3.46–3.38 (m, 1H, 3β-H), 2.54–2.46 (m, 2H, 24-CH2S-), 2.26-2.17 (m, 2H,
4α-H and 9-H), 0.98 (d, J = 6.5 Hz, 3H, 21-CH3), 0.88 (s, 12H, 19-CH3 and -Si–C(CH3)3),

JOURNAL OF SULFUR CHEMISTRY
13
0.68 (s, 3H, 18-CH3), 0.04 (s, 6H, -Si(CH3)2); 13C NMR (CDCl3), δ (ppm): 73.11 (CH),
71.91 (CH), 68.43 (CH), 47.20 (CH), 46.36 (C), 41.59 (CH), 41.46 (CH), 39.47 (CH), 39.37
(CH2), 35.30 (CH), 35.28 (CH2), 34.73 (C), 34.61 (2xCH2), 30.78 (CH2), 30.25 (CH2),
28.10 (CH2), 27.62 (CH2), 26.32 (CH), 25.65 (3xCH3), 25.08 (CH2), 23.19 (CH2), 22.44
(CH3), 18.07 (C), 17.69 (CH3), 12.43 (CH3), −2.99 (2xCH3); ESI MS, m/z: 525 [(M + H)+,
100%], 1049 [(2M + H)+, 5%], 357 [22%], 375 [17%]; HRMS-ESI (m/z): [M + H]+ calcd
for C30H57O3SSi 525.3798, found 525.3787.
4.2.5.4. Di(3α-t-butyldimethylsilyloxy-7α,12α-dihydroxy-5β-cholan-24-yl)disulfide. 19
was eluted with hexane/ethyl acetate (92:8) mixture in 39% yield. Colorless crystals, mp
126–128°C (hexane/CH2Cl2); IR (CHCl3), νmax (cm−1): 3613 and 3445 (-OH), 2931 and
2858 (–CH), 1254 (C–O), 1091 and 1058 (C–O and C–S); 1H NMR (CDCl3), δ (ppm): 3.98
(m, 2H, 12β-H and 12βꢀ-H), 3.84 (m, 2H, 7β-H and 7βꢀ-H), 3.47–3.39 (m, 2H, 3β-H and
3βꢀ-H), 2.74-2.61 (m, 4H, 24-CH2S- and 24ꢀ-CH2S-), 2.27–2.17 (m, 4H, 4α-H, 4αꢀ-H, 9-H
and 9ꢀ-H), 1.00 (d, J = 6.5 Hz, 6H, 21-CH3 and 21ꢀ-CH3), 0.88 (s, 24H, 19-CH3, 19ꢀ-CH3
and 2x(-Si–C(CH3)3)), 0.69 (s, 6H, 18-CH3 and 18ꢀ-CH3), 0.05 (s, 12H, 2x(-Si(CH3)2));
13C NMR (CDCl3), δ (ppm): 72.99 (2xCH), 72.91 (2xCH), 68.40 (2xCH), 47.36 (2xCH),
46.47 (2xC), 41.97 (2xCH), 41.61 (2xCH), 40.00 (2xCH2), 39.75 (2xCH2), 39.59 (2xCH),
35.39 (2xCH2), 35.32 (2xCH), 34.74 (2xC), 34.69 (2xCH2), 34.64 (2xCH2), 31.04 (2xCH2),
28.30 (2xCH2), 27.58 (2xCH2), 26.70 (2xCH), 25.94 (6xCH2), 25.80 (2xCH2), 23.17
(2xCH2), 22.61 (2xCH2), 18.24 (2xC), 17.69 (2xCH2), 12.51 (2xCH2), −4.58 (2xCH2),
-4.64 (2xCH2); ESI MS, m/z: 1069 [(M + Na)+, 100%]; HRMS-ESI (m/z): [M + H]+ calcd
for C60H111O6S2Si2 1047.7361, found 1047.7348.
4.2.5.5. Di(3α-t-butyldimethylsilyloxy-7α,12α-dihydroxy-5β-cholan-24-yl) sulfide. 20
was eluted with hexane/ethyl acetate (9:1) mixture in 31% yield. 126–128°C (hexane/
CH2Cl2); 127–128°C (hexane/CH2Cl2); IR (CHCl3), νmax (cm−1): 3613 and 3452 (–OH),
2948, 2931 and 2858 (–CH), 1254 (C–O), 1091 and 1058 (C–O and C–S);1H NMR
(CDCl3), δ (ppm): 3.98 (m, 2H, 12β-H and 12βꢀ-H), 3.84 (m, 2H, 7β-H and 7βꢀ-
H), 3.47–3.40 (m, 2H, 3β-H and 3βꢀ-H), 2.55-2.42 (m, 4H, 24-CH2S- and 24ꢀ-CH2S-),
2.27–2.17 (m, 4H, 4α-H, 4αꢀ-H, 9-H and 9ꢀ-H), 0.99 (d, J = 6.5 Hz, 6H, 21-CH3 and
21ꢀ-CH3), 0.89 (s, 24H, 19-CH3, 19ꢀ-CH3 and 2x(-Si–C(CH3)3)), 0.69 (s, 6H, 18-CH3
and 18ꢀ-CH3), 0.05 (s, 12H, 2x(-Si(CH3)2));13C NMR (CDCl3), δ (ppm): 72.99 (2xCH),
72.85 (2xCH), 68.40 (2xCH), 47.35 (2xCH), 46.48 (2xC), 41.97 (2xCH), 41.59 (2xCH),
39.97 (2xCH2), 39.56 (2xCH), 35.38 (2xCH2), 35.32 (2xCH), 35.12 (2xCH2), 34.67 (2xC),
34.64 (2xCH2), 32.68 (2xCH2), 31.02 (2xCH2), 28.24 (2xCH2), 27.56 (2xCH2), 26.68
(2xCH), 26.34 (2xCH2), 25.92 (6xCH3), 23.17 (2xCH2), 22.60 (2xCH3), 18.22 (2xC), 17.70
(2xCH3), 12.50 (2xCH3), -4.59 (2xCH3), -4.65 (2xCH3); ESI MS, m/z: 1037 [(M + Na)+,
100%]; HRMS-ESI (m/z): [M + H]+ calcd for C60H111O6SSi2 1015.7640, found 1015.7621.
4.2.6. Removal of TBDMS group
Steroidal substrate (19 or 20; 0.5 mmol) was dissolved in a 30 mL mixture of acetic
acid/water/THF (3:1:1). The mixture was stirred at room temperature for 2 days. After
this time THF was evaporated and aqueous solution of NaOH was added. The crude
product was extracted with ethyl acetate. The extract was dried over anhydrous Na2SO4

14
A. M. TOMKIEL ET AL.
and evaporated to dryness in vacuo. The residue was subjected to silica gel column
chromatography.
4.2.6.1. Di(3α,7α,12α-trihydroxy-5β-cholan-24-yl) sulfide. 21 was eluted with chlo-
roform/methanol (92:8) mixture in 98% yield. Colorless crystals, mp 152–154°C
(hexane/ethyl acetate); IR ATR, ν
(cm−1): 3369 (–OH), 2932 and 2864 (–CH), 1256
max
(C–O), 1076 and 1041 (C–O and C–S);1H NMR (CDCl3/CD3OD), δ (ppm): 3.91 (m, 2H,
12β-H and 12βꢀ-H), 3.78 (m, 2H, 7β-H and 7βꢀ-H), 3.36-3.30 (m, 2H, 3β-H and 3βꢀ-H),
2.47-2.40 (m, 4H, 24-CH2S- and 24ꢀ-CH2S-), 2.10–2.01 (m, 4H, 4α-H, 4αꢀ-H, 9-H and 9ꢀ-
H), 0.94 (d, J = 6.5 Hz, 6H, 21-CH3 and 21ꢀ-CH3), 0.85 (s, 6H, 19-CH3 and 19ꢀ-CH3), 0.64
(s, 6H, 18-CH3 and 18ꢀ-CH3); 13C NMR (CDCl3/CD3OD), δ (ppm): 73.37 (2xCH), 71.88
(2xCH), 68.64 (2xCH), 47.65 (2xCH), 46.70 (2xC), 41.99 (2xCH), 41.83 (2xCH), 39.77
(2xCH), 39.56 (2xCH2), 35.88 (2xCH), 35.63 (2xCH2), 35.56 (2xCH2), 35.08 (2xC), 34.84
(2xCH2), 32.99 (2xCH2), 30.22 (2xCH2), 28.42 (2xCH2), 27.96 (2xCH2), 26.82 (2xCH2),
26.74 (2xCH), 23.52 (2xCH2), 22.74 (2xCH3), 17.84 (2xCH3), 12.77 (2xCH3); ESI MS,
m/z: 787 [(M + H)+, 100%]; HRMS-ESI (m/z): [M + H]+ calcd for C48H83O6S 787.5910,
found 787.5876.
Acknowledgements
We are grateful to Professor Jacek W. Morzycki, Dr L. Siergiejczyk, and Dr. D. Czajkowska-
Szczykowska for discussions.
Disclosure statement
No potential conflict of interest was reported by the authors.
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