Electrochemical synthesis of glycoconjugates from activated sterol derivatives

Steroids 82 (2014) 60–67

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Electrochemical synthesis of glycoconjugates from activated sterol derivatives Aneta M. Tomkiel a, Jan Kowalski b, Jolanta Płoszyn´ska b, Leszek Siergiejczyk a, Zenon Łotowski a, Andrzej Sobkowiak b, Jacek W. Morzycki a,⇑ a b

Institute of Chemistry, University of Białystok, Hurtowa 1, 15-399 Białystok, Poland 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

Article history: Received 26 October 2013 Received in revised form 13 January 2014 Accepted 20 January 2014 Available online 30 January 2014

a b s t r a c t Several derivatives of cholesterol and other 3b-hydroxy-D5-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. Ó 2014 Elsevier Inc. All rights reserved.

Keywords: Glycosylation Cholesterol Electrochemical oxidation Sterol glycoconjugates Activating groups

1. Introduction 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 glycobiology 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]. Despite considerable progress in carbohydrate chemistry in the last few decades, the regio- and stereoselective formation of O-glycosidic 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: [email protected] (J.W. Morzycki). http://dx.doi.org/10.1016/j.steroids.2014.01.007 0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

O-sulfonyl glycosides [12], glycals [13], phosphate derivatives [14], O-silylated glycosides [15], and others. During our study on the direct electrochemical oxidation of cholesterol [16] we observed the formation of a nonclassical carbocation 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 approach allows to synthesize steroid-sugar conjugates without activating 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 excess (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 convenient protection for the 3b-OH group which would simultaneously activate sterol for electrochemical oxidation. However, it is essential that the electrooxidation of a sterol derivative generates, as in

A.M. Tomkiel et al. / Steroids 82 (2014) 60–67

R

HO 1

nucleophile (cholesterol, acetic acid, chlorides, acetonitrile)

-e-

.+

HO

R = cholesteryl-OAcO-, Cl-, AcNH-

. - HO (+) (+)

Scheme 1. Electrooxidation of cholesterol in dichloromethane in the presence of nucleophiles.

the case of cholesterol, a mesomeric carbocation that is capable to react with a sugar partner. 2. Experimental 2.1. Electrochemistry Cyclic-voltammograms were recorded with iR compensation at 25 °C using a three-electrode potentiostat (Princeton Applied Research Model Parstat 2273). The experiments were conducted in a 3-mL electrochemical cell with an argon-purge system. The working 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 tetrabutylammonium tetrafluoroborate (TBABF4) from Aldrich in dichloromethane was used as a supporting electrolyte. The preparative electrolyses were performed with a potentiostatgalvanostat (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 reactions were monitored by TLC and stopped when no further increase in the concentration of glycosylation products was observed. A divided H-cell was used in which the cathodic and anodic compartments (3.5 mL of electrolyte each) were separated by a glass frit. In all measurements 0.1 M solution of tetrabutylammonium tetrafluoroborate (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 compartment 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 compartment 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. 2.2. Chemical synthesis The sugar (1,2:3,4-di-O-isopropylidene-a-D-galactopyranose) [20] and steroidal substrates, cholesteryl phenyl ether (2) [21],

61

cholesteryl phenyl sulfide (3) [22], cholesteryl benzyl ether (12) [21], cholesteryl t-butyl ether (9) [23], t-butyldimethylsilyl cholesteryl ether (10) [24], cholesteryl iodide (6) [25], cholesteryl thiocyanate (7) [26,27], thiocholesterol (8) [26,27], were prepared according to known procedures. Melting points were determined on a Toledo Mettler-MP70 apparatus. 1H and 13C NMR (400 and 100 MHz, respectively) spectra 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) AMD604 spectrometer with electrospray ionization (ESI) or AutoSpec Premier (Waters) (EI). 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 chromatography performed on a 70–230 mesh silica gel (J.T. Baker). 2.2.1. Synthesis of steroidal diphenylphosphates 2.2.1.1. Procedure 1 [28]. To a solution of cholesterol (0.7 g, 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 crystals, mp 116–119 °C (CH2Cl2/acetonitrile); Rf = 0.53 (hexane-AcOEt 8:2); IR, mmax: 1592, 1490, 1281, 1192, 1163, 1024, 957; 1H NMR (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-3a), 1.02 (s, 3H, H19), 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); 13 C 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: 12.02; ESI MS, m/z: 1259 [(2M+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 dissolved 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, mmax: 1592, 1490, 1281, 1192, 1025, 957, 897; 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), 4.43 (m, 1H, H-3a), 3.48 (m, 1H, H26a), 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

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(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: 12.03; ESI MS, m/z: 1315 [(2M+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. 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). Colorless crystals, mp 78–80 °C (AcOEt/hexane); Rf = 0.37 (hexaneAcoEt 8:2); IR, mmax: 1731, 1592, 1490, 1279, 1192, 1163, 1024, 957; 1H NMR (ppm), d (ppm): 7.33 (m, 4H, H-Ar), 7.23 (m, 4H, HAr), 7.17 (m, 2H, H-Ar), 5.36 (m, 1H, H-6), 4.44 (m, 1H, H-3a), 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); 31 P NMR (ppm), d: 12.05; ESI MS, m/z: 1263 [(2M+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. 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,Ndiethylamidophosphite (0.3 mL, 1.7 mmol) were added. The reaction 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 solvent was evaporated in vacuo affording 16 (0.52 mg, 84%). Compound 16; White solid; Rf = 0.60 (hexane-AcOEt 9:1); IR, mmax: 1254, 978; 1H NMR, d: 5.39 (m, 1H, H-6), 3.93 (m, 1H, H3a), 3.53 (d, J = 0.8 Hz, 3H, CH3O–), 3.51 (d, J = 0.8 Hz, 3H, CH3O– ), 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. 2.2.3. Cholest-5-en-3b-yl trichloroacetimidate (14) 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%). Compound 14; white crystals; mp 153–156 °C (hexane/AcOEt); Rf = 0.51 (benzene-hexane 4:6); IR, mmax: 1659, 1099, 799; 1H NMR (ppm), d: 8.24 (s, 1H, @NH), 5.43 (m, 1H, H-6), 4.78 (m, 1H, H-3a),

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 [(MCl)+, 4%], 369 [(cholest-5-en-3-yl)+, 100%]; elemental analysis calcd (%) for C29H46Cl3NO: 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.

2.2.4. Cholest-5-en-3a-yl phenyl selenide (4) 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 chromatography with hexane elution. Compound 4 was obtained in 50% yield accompanied by i-cholesteryl phenyl selenide (34%), which was eluted first. Compound 4; colorless crystals, mp 106–108 °C (CH2Cl2/hexane); Rf = 0.25 (hexane); IR, mmax: 3075, 1578, 535; 1H NMR (ppm), d: 7.53 (m, 2H, H-Ar), 7.25 (m, 3H, H-Ar), 5.36 (m, 1H, H6), 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 [(cholest-5-en-3-yl)+, 100%]; HRMS (EI): m/z calcd for C33H50Se+: 526.3078 [M+]; found 526.3071.

2.2.5. p-(Cholest-5-en-3b-yloxy)phenol (13) To cholesteryl tosylate (443 mg, 1 mmol) dissolved in dioxane (30 mL) p-toluenesulfonic acid (34 mg, 0.2 mmol), and then hydroquinone (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 evaporated 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). 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, 1H, H-3a), 1.06 (s, 3H, H-19), 0.93 (d, 3H, J = 6.5 Hz, H-21), 0.882 (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.

A.M. Tomkiel et al. / Steroids 82 (2014) 60–67

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, mmax: 2154; 1H NMR (ppm), d: 5.43 (m, 1H, H-6), 3.10 (m, 1H, H-3a), 1.04 (s, 3H, H19), 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); 13 C 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 [(2M+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. 2.2.6.2. Cholest-5-ene-3b-thiol (8). White crystals, mp 96–97 °C (hexane/AcOEt); Rf = 0.41 (hexane); IR, mmax: 1467; 1H NMR (ppm), d: 5.33 (m, 1H, H-6), 2.70 (m, 1H, H-3a), 1.01 (s, 3H, H19), 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); 13 C 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 [(MH), 100%]. 2.2.7. Electrolysis of cholesteryl diphenylphosphates (15) in the presence of 1,2:3,4-di-O-isopropylidene-D-galactopyranose Cholesteryl diphenylphosphate (15; 185 mg; 0.30 mmol) and 1,2:3,4-di-O-isopropylidene-D-galactopyranose (96 mg; 0.37 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 chloride ions from forming by the reduction of dichloromethane. A preparative 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; 12%) and 3b-O-(10 ,20 :30 ,40 -di-O-isopropylidene-a-D-galactopyranos-60 -yl)-cholest-5-ene (17; 101 mg, 54%), followed by cholesterol (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. 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-ylsulfanyl)-ethyl]-1-(cholest-5-en-3b-yl-sulfanyl)-b-D-galactopyranose (22). White solid; Rf = 0.37 (hexane-AcOEt 8:2); IR, mmax: 3482, 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-3a), 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, H18); 13C NMR (ppm), d: 142.0 (C), 141.7 (C), 120.9 (CH), 110.8

63

(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. 2.2.8.2. 3b-O-(3,4-O-isopropylidene-a-D-galactopyranos-6-yl)-cholest-5-ene (24). Colorless crystals, mp 145–148 °C (CH2Cl2/hexane); Rf = 0.30 (hexane-AcOEt 9:1); IR, mmax: 3550, 1083, 1064; 1 H 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-60 a), 3.62 (s, 1H, H-20 ), 3.58 (dd, 1H, J1 = 7.6 Hz, J2 = 5.5 Hz, H-60 b), 3.35 (m, 1H, H-3a), 1.54 (s, 3H, H-isopropylidene), 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 [(MH2O+Na)+, 100%]; elemental analysis calcd (%) for C36H60O6: C 73.43, H 10.27; found: C 73.36, H 10.25. 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, mmax: 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-3a), 1.06 (s, 6H, H19), 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); 13 C 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-5en-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 (hexane/AcOEt); Rf = 0.38 (hexane); IR, mmax: 1110, 648, 517, 508; 1 H NMR, d: 5.38 (m, 2H, H-6), 2.62 (m, 2H, H-3a), 1.01 (s, 6H, H19), 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); 13 C 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. 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 structure. The sterol ethers are of special interest since they are hardly degradable as opposed to steroidal acetals or esters, which are biologically active compounds but undergo fast metabolic transformations 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 approach employing sterol triflates and inactivated sugars proved to be unsuccessful [33]. We have recently described the electrochemical method of synthesis of steroid glycoconjugates using 6b-arylsulfanyl-3a,5acyclosteroid 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 electrochemical glycosylation reactions from readily available 3-cholesteryl derivatives 2–16 (Scheme 2). All of these compounds are electrochemically active; cyclic voltammograms measured in dichloromethane 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), phenylselenide (4), iodide (6), and hydroquinone ether (13) showed additional anodic peaks which appeared at less positive potentials. They probably resulted from electrooxidation of a substituent. Fig. 1 presents a comparison of the electrochemical behavior of substrates [sugar 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.

10 I, μΑ

0 -10 -20

(a) (b) (c) (d)

-30 -40 -50 -60 -70 2.5

2.0

1.5

1.0

Fig. 1. Cyclic voltammograms registered in 0.2 M tetrabutylammonium tetrafluoroborate (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-galactopyranose (red), (c) cholesteryl diphenylphosphate 15 (green), and (d) glycoconjugate 17 (blue). Concentrations of all compounds are equal to 5 mM. Scan rate 1 V s1, 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.)

An attempt to prepare glycoconjugates by using cholesterol phenyl ether (2) [21] as a sterol donor failed, as only traces of glycoconjugate 17 were detected (Table 1).

C8H17

St

St'

Steroidal substrates: HO St (1)

O F3C CO St (5)

C O St (9)

PhO St (2)

I St (6)

C Si O St (10)

PhS St (3)

NCS St (7)

HO

O St (13)

NH Cl3C C O St (14) O PhO P O St (15) TsO St (11) OPh CH2O St (12) MeO P O St (16) OMe

HS St (8)

Steroidal products: O

O St (17)

Cl St (19)

O St' (18)

O

St O St (20)

O

O O

O OO

OO

C8H17

O

0.0

E, V vs Ag/AgNO3 in MeCN

C8H17

PhSe 3α-St (4)

0.5

St S S St (23)

OH

O

O St (24) O

O

OH OH

O O

S St St O

O

O St (25)

S St (22) 21 Scheme 2. Steroidal substrates (1–16) and products of electrochemical reactions (17–25).

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A.M. Tomkiel et al. / Steroids 82 (2014) 60–67 Table 1 Electrochemical oxidation of cholesterol derivatives (1–16) in the presence of 1,2:3,4di-O-isopropylidene-a-D-galactopyranose.a Substrate

0

0

0

0

3b-O-(1 ,2 :3 ,4 -di-O-isopropylidene-a-Dgalactopyranos-60 -yl)-cholest-5-ene (17) yield

1 2 3 4 5 6 7 8

20% 3% 9% <1% Not detected Traces Traces 6%

9 10

16% 16%

11 12 13 14

2% 2% 4% 32%

15 16

54% 46%

O

OH O

O O

OH

C8H17

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-galactopyranose 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 reaction with the sugar nucleophile and prevents production of the desired product. Most likely the iodine(I) species is further reduced in the reaction medium to iodide. Attempts to force 6 to react resulted 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. A similar failed attempt was experienced with cholesteryl trifluoroacetate (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 phenylselenol) [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 glycoconjugate 17 in addition to the elimination product 21 (2%). Then the electrochemical reaction of thiocholesterol (8) [26,27] was briefly studied. The major product of a reaction with the galactose 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 attacks of thiocholesterol on the electrochemically activated sugar acetals as depicted in Scheme 3. a-Configuration of the thioglyco-

O S St + OH S St H

O O

18 (1.5%) 21 (2%)

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. Galvanostatic 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.

.

.

+

25 (24%) 21 (5%), 1 (3%), 19 (2%), 20 (2%) 1 (6%) 1 (5%)

O O

O O O S St +H

O

. - OH , - H

OH

O O

-e

OO

Other steroidal products (yield)

22 (25%), 23 (11%) 20 (30%), 1 (29%) 1 (56%), 20 (16%), 19 (2%) 24 (6%), 19 (3%)

OH

-

St

O S St S St (22)

Scheme 3. Tentative mechanism of the electrochemical reaction of thiocholesterol (HS-St; 8).

side moiety in 22 was proven by the presence in its 1H NMR spectrum 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. Further study employed t-butyl and t-butyldimethylsilyl cholesterol 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 silicon tertiary center. These reactions lead to the formation of cholesterol 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 prolonged 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%), cholesteryl 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. 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 dicholesteryl hydroquinone ether 25 stage (Scheme 4) and further oxidation of this intermediate did not proceed under the conditions that were applied. None of the reactions reported so far gave a better yield of glyconjugate 17 than the reaction with cholesterol 1 itself. The best results were achieved with cholesteryl trichloroacetimidate (14) and diphenylphosphate (15) [27,40]. This can probably be attributed to the high electrochemical stability of the leaving groups, especially in the case of diphenylophosphate [41].

C8H17

C8H17 C8H17

- 2e- H+

O HO

13 - H+

(+)

O O

13

25

O O C8H17

Scheme 4. The electrochemical reaction of a cholesterol hydroquinone ether (13).

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A.M. Tomkiel et al. / Steroids 82 (2014) 60–67 C8H17

- e-

NH Cl3C C O

C8H17

.+

.

NH Cl3C C O

C8H17

-CCl3 - HNCO

14 C8H17

- e-

O PhO P O OPh

15

Sugar-OH - H+

C8H17

. PhO

- PhO-PO2

.+ O PhO P O OPh

16

+

Scheme 5. Tentative mechanisms of electrochemical reactions of cholesteryl trichloroacetimidate (14) and diphenylphosphate (15).

O

COOMe

O or

O PhO P O OPh

O PhO P O OPh

26 O

OH O

Anodic oxidation

O O

O O

HO

O O O

O

27

O

O

Cl

28a (37%)

29a (7%)

30a (2%)

31a (5%)

28b (37%)

29b (5%)

30b (1%)

31b (4%)

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 1,2:3,4di-O-isopropylidene-a-D-galactopyranose afforded product 17 in a better yield (32%) than with the cholesteryl donors that were previously 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 glycosyl 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-

phite (cholesteryl-O-P(OMe)2; 16) with 1,2:3,4-di-O-isopropylidene-a-D-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, like electrooxidation of 1,2:3,4-di-O-isopropylidene-a-D-galactopyranose, and do not disturb the glycosylation reaction.

4. Conclusion A series of cholesterol derivatives was examined as the potential 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

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compound moderate yields (up to 54%) of glycoconjugates were achieved. A limitation of the process were consecutive reactions of the glycoconjugate at a growing voltage and its limited stability under electrolysis conditions. Although the electrochemical overoxidation of glycoconjugates seems to be a minor problem at the initial steps due to a relatively large difference in the oxidation potentials and concentrations between substrates (sterol donors, sugar alcohols) and the resulting glycoconjugates, the possibility that side processes will occur increases as the anode potential is more positive and the glycoconjugate concentration grows. Therefore, electrolysis at a constant potential will be attempted in the future. 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. References [1] Hostettmann K, Marston A. Saponins. New York: Cambridge University Press; 1995. [2] Yang C-R, Tanaka O, editors. Studies in plant science. Advances in plant glycosides chemistry and biology, vol. 6. Amsterdam: Elsevier; 1999. [3] Waller GR, Yamasaki K, editors. Saponins used in traditional and modern medicine. New York: Plenum Press; 1996. [4] Waller GR, Yamasaki K, editors. Saponins used in food and agriculture. New York: Plenum Press; 1996. [5] Davis BG. Recent developments in glycoconjugates. J Chem Soc Perkin Trans 1 1999:3215–37. [6] Pellissier H. The glycosylation of steroids. Tetrahedron 2004;60:5123–62. [7] Li C, Yu B, Liu M, Hui Y. Synthesis of diosgenyl a-l-rhamnopyranosyl-(1 ? 2)[b-D-glucopyranosyl-(1 ? 3)]-b-D-glucopyranoside (gracillin) and related saponins. Carbohydr Res 1998;306:189–95. [8] Schmidt RR, Michel J. Facile synthesis of a- and b-O-glycosyl imidates; Preparation of glycosides and disaccharides. Angew Chem Int Ed Engl 1980;19:731–2. [9] Morzycki JW, Wojtkielewicz A. Synthesis of a cholestane glycoside OSW-1 with potent cytostatic activity. Carbohydr Res 2002;337:1269–74. [10] Yu B, Zhang Y, Tang P. Carbohydrate chemistry in the total synthesis of saponins. Eur J Org Chem 2007;31:5145–61. [11] Ma X, Yu B, Hui Y, Xiao D, Ding J. Synthesis of glycosides bearing the disaccharide of OSW-1 or its 1 ? 4-linked analogue and their antitumor activities. Carbohydr Res 2000;329:495–505. [12] Kahne D, Walker S, Cheng Y, Van Engen D. Glycosylation of unreactive substrates. J Am Chem Soc 1989;111:6881–2. [13] Williams IJ, Garbaccio RM, Danishefsky SJ. Carbohydrates. In: Ernst B, Hart GW, Sinay P, editors. Chemistry and biology, vol. 1. Weinheim (Germany): Wiley-VCH Verlag GmbH; 2000. p. 61–92. [14] Yamanoi T, Nakamura K, Takeyama H, Yanagihara K, Inazu T. New synthetic methods and reagents for complex carbohydrates. VIII. Stereoselective a- and b-mannopyranoside formation from glycosyl dimethylphosphinothioates with the C-2 axial benzyloxyl group. Bull Chem Soc Jpn 1994;67:1359–66. [15] Mukaiyama T, Matsubara K. Stereoselective glycosylation reaction starting from 1-O-trimethylsilyl sugars by using diphenyltin sulfide and a catalytic amount of active acidic species. Chem Lett 1992;21:1041–4.

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