Synthesis of 28a-homoselenolupanes and 28a-homoselenolupane saponins

Abstract

A practical synthesis of 28a-homo-28a-selenolupane triterpenes and the corresponding selenosaponins containing D-mannose, L-arabinose, L-rhamnose, and D-idose moieties is described. Selenium containing triterpenes were obtained from the readily available 3-O-allyl-homobetulin mesylate by nucleophilic substitution with the selenocyanate ion which upon reduction of the –SeCN group afforded the free selenol. Glycosylation using classical Schmidt donors gave 1,2-trans selenosaponins as the main product as well as minute amounts of 1,2-cis isomers. This is one of the very few examples of the synthesis of selenoglycosides by direct glycosylation of free selenols. The studied selenol showed high resistance to air oxidation resulting in good stability during the synthesis of selenolupane derivatives. Cytotoxic activities of new homoselenolupane derivatives were also evaluated in vitro and revealed that some triterpenes exhibited an interesting profile against human cancer cell lines.

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Introduction

In the last few decades, the application of organoselenium compounds,¹ including steroid derivatives,² in cancer prevention and treatment became an objective of investigations of many scientific groups. These studies show that organoselenium compounds display a high cytotoxic activity against different cancer cell lines. Moreover, in some cases a positive effect on the cytotoxic properties was achieved by replacing the sulphur atom with selenium without modifying the basic structure of the compound.³ Additionally, some heterocycles containing selenium possess an interesting anti-inflammatory activity profile with a significant analgesic effect.⁴ Furthermore, many organoselenium derivatives demonstrate antimicrobial, antiviral and antifungal activities.⁵

Previously, we reported the structure–activity relationships of the cytotoxic activity of ichopanol (28a-homobetulin) and ichopanothiol (28a-homothiobetulin) derivatives and saponins based on a homobetulin scaffold. We found, that the heteroatom (oxygen or sulphur) located on the C-17 side-chain is necessary for the anticancer activity of the studied compounds.^6–8 Based on the high biological activity of selenium derivatives, we reasoned that replacing these heteroatoms on the C-17 side-chain of the lupane core with selenium could enhance their anticancer effect. These results will broaden our knowledge regarding the synthesis, reactivity and stability of selenium-containing triterpenes and steroids as they are virtually unknown in the literature.^2,9 Herein, we report the first synthesis of homobetulin and homobetulin based saponins bearing a selenium atom. Furthermore, cytotoxic activities of all new derivatives were investigated.

Results and discussion

Mesylate 1, readily prepared via the reaction of 3-O-allyl-ichopanol and mesyl chloride,⁸ was used for the synthesis of selenolupane triterpenes. Standard synthesis of selenocyanate 3 involves the nucleophilic replacement of sulfonyl ester with potassium selenocyanate. Our first attempt using acetone as a solvent failed giving only selenide 2. However, changing the solvent from acetone to DMF resulted in the formation of the desired product 3 in 87% yield; a small amount of by-product selenol 4 (7%) was also isolated. Reduction of 3 with LiAlH₄ gave the corresponding selenol 4 in 93% yield. Compound 5, needed for SAR studies, was prepared in 69% yield from allyl ether 3 by treatment with palladium(II) chloride (Scheme 1).

Scheme 1 Reagents and conditions: (i) KSeCN (5 equiv.), acetone, 60 °C; (ii) KSeCN (5 equiv.), DMF, 60 °C; (iii) LiAlH₄ (2 equiv.), THF; (iv) PdCl₂, CH₃OH, CH₂Cl₂.

Synthesis of selenoglycosides by direct glycosylation of selenols is rarely found in the literature. Due to high susceptibility of selenols to oxidation the introduction of selenium at the anomeric position of a monosaccharide is achieved by the treatment of the sugar component with alkyl or aryl selenolates usually generated in situ from the corresponding diselenides in the presence of a reducing agent.¹⁰ Free selenols were used occasionally to react with anomeric halogenides,¹¹ acetates,^11b,12 orthoesters,¹³ or Schmidt donors.^11a,14

For the synthesis of lupane-type selenoglycosides we chose the Schmidt methodology. TMSOTf promoted glycosylation was carried out by the treatment of selenol 4 with perbenzoylated glycosyl trichloroacetimidates 6,¹⁵7,¹⁶8,¹⁶ and 9,¹⁷ which gave the desired selenoglycosides in 29–51% yield. A significant amount of unreacted starting selenol 4 was also recovered (13–40%). Although yields were rather moderate, further manipulation of the reaction conditions did not improve the yields of the selenoglycosides. As expected, the presence of the benzoyl protecting groups in the donor molecules directed the anomeric selectivity of glycosylation.¹⁸ Regioselectivity was, however, slightly lower than that in the case of the corresponding sulfur analogues.⁸ With the exception of α-D-mannopyranoside 10, mixtures of α and β anomers 11–16 of L-arabino, L-rhamno, and D-ido derivatives were obtained as specified in Scheme 2.

Scheme 2 Reagents and conditions: (i) 6–9, CH₂Cl₂, TMSOTf, −40 °C; (ii) (a) [Ir(COD)(PMePh₂)]PF₆, H₂, THF; (b) p-TsOH, CHCl₃, CH₃OH; (iii) K₂CO₃, CH₃OH.

Selenols are considered highly susceptible to air oxidation leading to diselenides, however, selenol 4 was relatively resistant to oxidation. Recovered selenol 4 was reused for glycosylation without any significant decrease in the reaction yields. It must be noted, however, that prolonged storage slowly decreased its reactivity, although the physicochemical properties of 4 did not change. Considering the above observations which are crucial criteria in the synthesis of selenolupane derivatives, a study examining the stability of selenol 4 was undertaken. First, the independent synthesis of diselenide 27, the expected product of the air oxidation of 4, was conducted. The homogeneous product was prepared by the reaction of mesylate 1 with dilithium diselenide (Li₂Se₂)¹⁹ affording compound 27 in 88% yield (Scheme 3). The same compound containing inseparable unknown byproducts, may also be obtained by the oxidation of selenol 4 with hydrogen peroxide in the presence of NaOH (79%) and from the equimolar mixture of selenocyanide 3 and selenol 4 in the presence of a base (94%) according to Krief's method.²⁰ Surprisingly, in all cases, the physichochemical properties of 27, including ⁷⁷Se NMR, were identical as found for selenol 4 but differences in reactivity were observed. In contrast to compound 4, compound 27 did not react with Schmidt donors. On the other hand, both derivatives were alkylated by treatment with methyl iodide to afford selenide 28. However, in the case of the reaction of compound 27 with methyl iodide a longer reaction time was required (5 days, 70%), whereas the alkylation of freshly prepared selenol 4 in the presence of sodium hydride was complete within a few hours giving compound 28 in 74% yield (Scheme 3).

Scheme 3 Reagents and conditions: (i) Se, Super Hydride, THF, 88%; (ii) NaH, CH₃I, THF, rt, 5 days, 70%; (iii) NaH, CH₃I, THF, rt, 12 h, 74%.

There are two possible explanations for such unusual behaviour. The first is that both compounds (4 and 27) have identical spectroscopic properties but they are different compounds. This speculation may be supported by the result of alkylation described above. In the first case (considering diselenide 27) sodium hydride acting first as a reducing agent (breaking Se–Se bond) and then as a base was required for alkylation.²¹ It significantly prolonged the reaction time. On the other hand, when the formation of the Se–Se bond was not possible due to high steric hindrance, selenol was formed in both cases. However, reaction conditions and/or long storage time promoted the aggregation of selenol and the formation of relatively unreactive polymeric forms which caused a decrease in reactivity. Similar aggregation of betulin derivatives was described in the literature.²²

Attempts to remove the allyl block from 3-O-allylselenosaponins with palladium(II) chloride were unsuccessful and caused decomposition of the starting materials. Therefore, deallylation was performed under neutral conditions by isomerisation of the allyl double bond in the presence of the hydrogen activated iridium complex [Ir(COD)(PMePh₂)]PF₆ followed by hydrolysis of 1-propenyl ether in the presence of p-TsOH.¹⁷ Deallylated saponins 17–21 were obtained in high yields (78–93%) with the exception of 19 which was isolated in slightly lower yield (63%). Debenzoylation with potassium carbonate in methanol gave free saponins 22–26 in good yields (61–88%, Scheme 2).

Structures of all new compounds were confirmed by extended 1D and 2D NMR experiments, as well as elemental analysis and mass spectroscopy. Stereochemistry at the anomeric center was proven by measuring ¹J_C1,H1 coupling constants. Observed ¹J_C1,H1 coupling constants for 11 (170.4 Hz), 13 (171.0 Hz), and 15 (168.0 Hz) proved the equatorial position of the anomeric proton, whereas ¹J_C1,H1 measured for 12 (158.0 Hz), 14 (153.0 Hz), and 16 (154.0 Hz) indicated the presence of axially oriented anomeric protons.²³ In the case of rhamnosides 13 and 14 (6-deoxy-hexopyranosides) the configuration at the anomeric carbon atom was additionally confirmed by the almost forgotten Sinclair–Sleeter rule.²⁴ The signal for the H-6 protons of 13 in which aglycon is in the axial position was observed upfield (1.39 ppm) in comparison with its epimer in which the signal for the H-6 protons appeared downfield (1.46 ppm). In addition, the structure of 3β-O-allyl-28a-selenocyanato-28a-homolup-20(29)-ene (3) was unequivocally confirmed by X-ray analysis (Fig. 1).

Fig. 1 X-ray structure of 3β-O-allyl-28a-selenocyanato-28a-homolup-20(29)-ene (3).

Antiproliferative activities of the studied derivatives of homobetulin were tested in vitro. Activity against normal human BJ fibroblasts was compared with cytotoxicity on cancer cell lines of various histopathological origins, including T-lymphoblastic leukaemia (CEM), breast adenocarcinoma (MCF7), malignant melanoma (G-361) and cervical carcinoma (HeLa) lines. The cells of all these lines were exposed to six serial 3-fold dilutions of each drug for 72 h, the proportions of surviving cells were then estimated and IC₅₀ values (50% inhibitory concentrations) were calculated. A detailed procedure for the cytotoxicity assay was described previously.⁷ The results obtained from Calcein AM assays are presented in Table 1.

Table 1 IC₅₀ (μM) values obtained from Calcein AM assays with the tested cancer and normal cell lines; means ± SD obtained from three independent experiments performed in triplicate. Betulinic acid was used as a positive control

Comp. no.			IC50 [μM]
	R	R′	CEM	MCF7	HeLa	G-361	BJ
2	All	SeCH2COCH3	>50	>50	>50	>50	>50
5	H	SeCN	2.0 ± 0.6	5.2 ± 1.4	3.4 ± 0.6	3.8 ± 0.8	3.7 ± 0.8
29 ^8	H	SCN	4.4 ± 1.8	13.6 ± 4.8	6.7 ± 0.0	3.0 ± 0.1	4.6 ± 0.5
30 ^7	H	OH	15.3 ± 1.4	>50	>50	10.2 ± 0.9	>50
22	H	Se-α-D-Manp	>50	46.4 ± 5.2	>50	>50	>50
23	H	Se-β-L-Arap	>50	>50	>50	>50	>50
24	H	Se-α-L-Arap	>50	>50	>50	>50	>50
25	H	Se-α-L-Rhap	32.5 ± 4.4	45.4 ± 3.0	42.3 ± 0.0	41.0 ± 2.5	46.9 ± 0.1
26	H	Se-α-D-Idop	>50	>50	>50	>50	>50
Betulinic acid	—	—	40.0 ± 2.8	>50	47.6 ± 1.9	>50	>50

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Selenosaponins were practically inactive, except L-rhamnoside 25 which showed low activity (IC₅₀ 32.5–45.4 μM). Similar results were obtained for oxygen⁷ and sulphur analogs⁸ which suggest that homobetulin saponins do not affect the growth of the cancer lines. Compound 30 was selective against malignant melanoma and leukemia cell lines (IC₅₀ 10.2 or 15.3 μM) without affecting the growth of normal human fibroblasts and other cancer cells which suggested a high therapeutic index. By comparison, selenocyanate 5 showed the highest cytotoxic activity of all compounds tested (IC₅₀ 2.0–5.2 μM). Its cytotoxicity was also higher than that measured for the appropriate sulphur analogue 29, and much higher than those observed for ichopanol (30). Sulphur analogue 29 showed an interesting profile as much more higher activity was determined for G-361 melanoma cells than for other cancer cell lines and fibroblasts. Both compounds, however, exhibit a relatively low therapeutic index.

Conclusions

In conclusion, a series of 28a-homo-28a-selenolupane triterpenes and the corresponding homoselenosaponins bearing D-mannose, L-arabinose, L-rhamnose, and D-idose moieties were synthesised. Required selenol was obtained by a nucleophilic substitution of the corresponding mesylate with potassium selenocyanate followed by the reduction of the –SeCN group. Its unusual stability to air oxidation was discussed. Further glycosylation of selenol with Schmidt donors under the standard conditions followed by deprotection afforded homoselenosaponins. All new compounds were evaluated for their cytotoxic activities towards normal and cancer cell lines. Homobetulin selenocyanate was the most cytotoxic compound, whereas the corresponding saponins were mostly inactive. To our best knowledge this is the first synthesis of lupane-type triterpenes containing selenium as the core component.

Experimental section

General procedures

Silica gel HF₂₅₄ and silica gel 230–400 mesh (E. Merck) were used for TLC and column chromatography, respectively. ¹H, ¹³C and ⁷⁷Se NMR spectra were recorded at 298 K with a Varian NMR-vnmrs600 or vnmrs500 spectrometer, using standard experimental conditions and Varian software (ChemPack 4.1). Configurational assignments were based on the NMR measurements, generated using two-dimensional techniques like COSY and ¹H–¹³C gradient selected HSQC (g-HSQC), as well as ¹H–¹³C gradient selected HMBC (g-HMBC). TMS was used as the internal standard for determining ¹H and ¹³C NMR chemical shifts. J values are given in hertz. High-resolution mass spectra (HRMS-ESI) were acquired with Mariner and MaldiSYNAPT G2-S HDMS (Waters) mass spectrometers. Optical rotations were measured with a Jasco P-2000 automatic polarimeter. IR spectra were recorded on a Jasco 6200 FT-IR spectrophotometer.

Single crystal X-ray diffraction measurements were carried out on an Agilent Supernova diffractometer, at 100 K with monochromated Cu Kα radiation (1.54184 Å). The data reduction was made by using CrysAlisPRO software.²⁵ The structures were solved by direct methods and refined on F² by full-matrix least-squares by using SHELXS97 and SHELXL97.²⁶ All non-hydrogen atoms were refined anisotropically while hydrogen atoms were placed in calculated positions, and refined in riding mode. Crystals of 3 suitable for X-ray analysis were obtained by cooling of a hot methanol solution: monoclinic, P2₁, a = 13.729(2), b = 8.3619(9), c = 14.594(3) Å, β = 114.96(2), V = 1518.9(5) ų, Z = 2, D_calc = 1.279 g cm⁻¹, μ = 1.864 mm⁻¹, R₁ = 0.0894 for 2177 [F_o > 4σ(F_o)] and 0.1157 for all data, wR₂ = 0.2644, S = 1.046. Crystallographic data for the structure have been deposited with the Cambridge Crystallographic Data Centre as supplementary material (deposition numbers: CCDC 1422676).

1-[3β-O-Allyl-28a-homolup-20(29)-en-28a-selenoyl]-2-propanone (2). A solution of 1 (50 mg, 0.087 mmol) and KSeCN (63 mg, 0.43 mmol) in acetone (1 mL) was stirred at 60 °C for 48 h. Then the solvents were evaporated, and the residue was dissolved in DCM and washed twice with water. An organic layer was evaporated to dryness; column chromatography of the residue (hexane–ethyl acetate, 40 : 1 → 10 : 1) gave 2 as a white solid (45 mg, 88%). M.p. 166–167 °C; [α]²⁰_D 14.2 (c 0.3, chloroform); ν_max (film): 2942, 2868, 1700, 1641, 1455, 1230, 1072, 757 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.90–5.96 (m, CH=), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.68 (H-29), 4.57 (H-29), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 3.23 (m, SeCH₂COCH₃), 2.80 (dd, J 4.1, 11.7 Hz, H-3), 2.52 (H-28a), 2.42 (H-28a), 2.40 (H-19), 2.35 (s, SeCH₂COCH₃), 1.92 (H-21), 1.85 (H-28), 1.76 (H-13), 1.71 (H-2), 1.71 (H-16), 1.68 (H-1), 1.68 (s, H-30), 1.64 (H-12), 1.64 (H-22), 1.59 (H-15), 1.50 (H-6), 1.48 (H-18), 1.47 (H-2), 1.42 (H-11), 1.38 (H-6), 1.38 (H-7), 1.35 (H-21), 1.30 (H-28), 1.26 (H-9), 1.24 (H-11), 1.17 (H-16), 1.06 (H-12), 1.05 (s, H-26), 1.01 (H-15), 1.00 (H-22), 0.95 (s, H-23), 0.95 (s, H-27), 0.83 (s, H-25), 0.82 (H-1), 0.78 (s, H-24), 0.67 (H-5). ¹³C NMR (CDCl₃) δ: 203.5 (SeCH₂COCH₃), 150.6 (C-20), 135.9 (CH=), 115.9 (=CH₂), 109.5 (C-29), 86.3 (C-3), 70.6 (OCH₂), 55.8 (C-5), 50.4 (C-9), 49.9 (C-18), 47.2 (C-19), 46.8 (C-17), 42.5 (C-14), 40.4 (C-8), 38.8 (C-4), 38.6 (C-1), 37.1 (C-10), 37.1 (C-13), 35.4 (C-22), 34.2 (C-7), 31.6 (SeCH₂COCH₃), 30.7 (C-16), 29.9 (C-21), 28.4 (C-28), 28.1 (C-23), 27.4 (SeCH₂COCH₃), 27.1 (C-15), 24.9 (C-12), 23.1 (C-2), 20.8 (C-11), 20.1 (C-28a), 19.3 (C-30), 18.2 (C-6), 16.3 (C-24), 16.1 (C-25), 16.0 (C-26), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 209.8. HR-MS (ESI) calc. for C₃₇H₆₁O₂Se [M + H]⁺: 616.3759. Found: 616.3758. Anal. Calcd for C₃₇H₆₀O₂Se (615.85): C, 72.16; H, 9.82. Found: C 71.90; H 10.25.

3β-O-Allyl-28a-selenocyanato-28a-homolup-20(29)-ene (3). A solution of 1 (984 mg, 1.71 mmol) and KSeCN (1.245 mg, 8.55 mmol) in DMF (15 mL) was stirred under an argon atmosphere at 60 °C for 24 h. Then the solvents were evaporated, and the residue was dissolved in DCM (50 mL) and washed with water (20 mL). An organic layer was evaporated to dryness; column chromatography of the residue (hexane–ethyl acetate, 40 : 1 → 20 : 1) gave selenol 4 (63 mg, 7%) as a side product and selenocyanate 3 as a cream-white solid (872 mg, 87%). M.p. 178–179 °C; [α]²⁰_D 7.9 (c 0.2, chloroform); ν_max (film): 2943, 2869, 2150 (SeCN), 1641, 1456, 1376, 1070, 883, 756, 548 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.90–5.96 (m, CH=), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.70 (H-29), 4.60 (H-29), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 3.03 (H-28a), 2.88 (H-28a), 2.79 (dd, J 4.2, 11.7 Hz, H-3), 2.43 (H-19), 2.12 (H-28), 1.92 (H-21), 1.78 (H-13), 1.72 (H-2), 1.68 (H-1), 1.68 (H-16), 1.68 (s, H-30), 1.65 (H-12), 1.60 (H-22), 1.59 (H-15), 1.59 (H-28), 1.52 (H-18), 1.50 (H-6), 1.48 (H-2), 1.42 (H-11), 1.40 (H-6), 1.40 (H-21), 1.38 (H-7), 1.26 (H-9), 1.26 (H-16), 1.24 (H-11), 1.08 (H-22), 1.07 (s, H-26), 1.06 (H-12), 1.05 (H-15), 0.96 (s, H-27), 0.95 (s, H-23), 0.84 (s, H-25), 0.82 (H-1), 0.78 (s, H-24), 0.67 (H-5). ¹³C NMR (CDCl₃) δ: 150.0 (C-20), 135.9 (CH=), 115.9 (=CH₂), 109.9 (C-29), 101.5 (SeCN), 86.3 (C-3), 70.6 (OCH₂), 55.8 (C-5), 50.4 (C-9), 50.0 (C-18), 47.1 (C-17), 47.0 (C-19), 42.5 (C-14), 40.9 (C-8), 38.8 (C-4), 38.6 (C-1), 37.1 (C-10), 37.1 (C-13), 35.2 (C-22), 34.2 (C-7), 30.6 (C-16), 29.7 (C-21), 30.1 (C-28), 28.1 (C-23), 27.1 (C-15), 25.5 (C-28a), 25.1 (C-12), 23.1 (C-2), 20.8 (C-11), 19.3 (C-30), 18.2 (C-6), 16.3 (C-24), 16.1 (C-25), 16.0 (C-26), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 232.1. HR-MS (ESI) calc. for C₃₅H₅₆NOSe [M + H]⁺: 585.3449. Found: 585.3450. Anal. Calcd for C₃₅H₅₅NOSe (584.79): C, 71.89; H, 9.48; N, 2.40. Found: C, 71.80; H, 9.45; N, 2.39.

3β-O-Allyl-28a-homolup-20(29)-en-28a-selenol (4). To a suspension of LiAlH₄ (115 mg, 3.00 mmol) in THF (2 mL) cooled in an ice bath, a solution of selenocyanate 3 (341 mg, 0.58 mmol) in THF (5 mL) was added dropwise and left to warm to room temp. Then the reaction was quenched by the addition of ethyl acetate (2 mL) and saturated NH₄Cl (0.5 mL). After 15 min an additional portion of ethyl acetate (10 mL) was added and the solvents were removed by evaporation. Column chromatography of the residue (hexane–ethyl acetate, 40 : 1) afforded 302 mg (93%) of the title compound as a light yellow powder. M.p. 210–213 °C; [α]²⁰_D 41.6 (c 0.2, chloroform); ν_max (film): 3072, 2943, 2868, 1642, 1456, 1376, 1216, 1135, 1106, 1086, 1071, 920, 883, 758 (s) cm⁻¹. ¹H NMR (CDCl₃) δ: 5.90–5.96 (m, CH=), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.68 (H-29), 4.58 (H-29), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 2.88 (H-28a), 2.79 (dd, J 4.1, 11.7 Hz, H-3), 2.72 (H-28a), 2.42 (H-19), 2.00 (H-28), 1.92 (H-21), 1.81 (H-13), 1.71 (H-2), 1.70 (H-16), 1.69 (s, H-30), 1.68 (H-1), 1.64 (H-15), 1.64 (H-22), 1.60 (H-28), 1.51 (H-6), 1.49 (H-18), 1.47 (H-2), 1.39 (H-6), 1.36 (H-21), 1.35 (H-7), 1.26 (H-9), 1.21 (H-16), 1.08 (s, H-26), 1.03 (H-22), 1.01 (H-15), 0.97 (s, H-27), 0.96 (s, H-23), 0.85 (s, H-25), 0.83 (H-1), 0.79 (s, H-24), 0.68 (H-5). ¹³C NMR (CDCl₃) δ: 150.6 (C-20), 109.6 (C-29), 86.3 (C-3), 55.8 (C-5), 50.5 (C-9), 49.9 (C-18), 47.3 (C-19), 46.8 (C-17), 42.5 (C-14), 41.0 (C-8), 38.8 (C-4), 38.6 (C-1), 37.2 (C-13), 37.1 (C-10), 35.6 (C-22), 34.3 (C-7), 30.7 (C-16), 30.2 (C-28), 30.0 (C-21), 28.1 (C-23), 27.2 (C-15), 25.2 (C-11 or 12), 24.9 (C-28a), 23.1 (C-2), 21.0 (C-11 or 12), 19.3 (C-30), 18.3 (C-6), 16.3 (C-24), 16.2 (C-25), 16.2 (C-26), 14.9 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 325.6. HR-MS (ESI) calc. for C₃₄H₅₆OSe [M]⁺: 560.3496. Found: 560.3511. Anal. Calcd for C₃₄H₅₆OSe (559.78): C, 72.95; H, 10.08. Found: C, 73.50; H, 10.48.

28a-Selenocyanato-28a-homolup-20(29)-en-3β-ol (5). To a solution of selenocyanate 3 (77 mg, 0.13 mmol) in a mixture of methanol (1 mL) and DCM (0.5 mL) PdCl₂ (10 mg) was added and stirred at room temperature for 24 h until no more starting material was detected by TLC. The solvents were evaporated under diminished pressure. Column chromatography of the residue (hexane–ethyl acetate, 20 : 1 → 7 : 3) gave the title compound as a white solid (49 mg, 69%). M.p. 232–236 °C; [α]²⁰_D –3.2 (c 0.1, chloroform). ν_max (film): 2943, 2870, 2148 (SeCN), 1455, 1377, 1043, 1033, 882, 757, 547, 521 cm⁻¹. ¹H NMR (CDCl₃) δ: 4.71 (H-29), 4.60 (H-29), 3.18 (dd, J 4.8, 11.5 Hz, H-3), 3.04 (H-28a), 2.88 (H-28a), 2.43 (H-19), 2.14 (H-28), 1.92 (H-21), 1.79 (H-13), 1.68 (H-16), 1.68 (s, H-30), 1.67 (H-1), 1.66 (H-12), 1.60 (H-2), 1.60 (H-15), 1.60 (H-22), 1.60 (H-28), 1.56 (H-2), 1.52 (H-6), 1.52 (H-18), 1.43 (H-11), 1.41 (H-6), 1.40 (H-21), 1.39 (H-7), 1.27 (H-9), 1.26 (H-16), 1.24 (H-11), 1.08 (H-22), 1.07 (H-12), 1.07 (s, H-26), 1.06 (H-15), 0.97 (s, H-27), 0.96 (s, H-23), 0.90 (H-1), 0.83 (s, H-25), 0.76 (s, H-24), 0.68 (H-5). ¹³C NMR (CDCl₃) δ: 150.0 (C-20), 109.9 (C-29), 101.5 (SeCN), 79.0 (C-3), 55.3 (C-5), 50.4 (C-9), 50.0 (C-18), 47.2 (C-17), 47.1 (C-19), 42.5 (C-14), 40.9 (C-8), 38.8 (C-4), 38.7 (C-1), 37.2 (C-10), 37.1 (C-13), 35.3 (C-22), 34.2 (C-7), 30.6 (C-16), 30.1 (C-28), 29.7 (C-21), 27.9 (C-23), 27.2 (C-15), 25.5 (C-28a), 25.1 (C-12), 27.4 (C-2), 20.8 (C-11), 19.3 (C-30), 18.3 (C-6), 16.1 (C-25), 16.0 (C-26), 15.3 (C-24), 14.9 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 232.4. HR-MS (ESI) calc. for C₃₂H₅₁NNaOSe [M + Na]⁺: 568.3034. Found: 568.3029.

General procedure for glycosylation

A solution of glycosyl donor (6–9, 1.00 mmol) and selenol 4 (280 mg, 0.5 mmol) in DCM (10 mL) was stirred for 20 min at room temperature over molecular sieves (4 Å, 400 mg, finely ground), then cooled to −40 °C and TMSOTf (76 μL, 0.42 mmol) was added. After 60 min the reaction was quenched with Et₃N (1 mL), and the solvents were removed under diminished pressure. Column chromatography (hexane–ethyl acetate, 40 : 1 → 5 : 1) of the residue gave the protected saponins as white foams.

3β-O-Allyl-28a-Se-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl)-28a-seleno-28a-homolup-20(29)-ene (10). Recovered starting selenol 4 (112 mg, 40%) and the title compound (171 mg, 30%) were obtained. Data for 10: [α]²⁰_D 59.7 (c 0.25, chloroform); ν_max (film): 2944, 2869, 1730, 1602, 1452, 1282, 1264, 1105, 1094, 757, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 6.24 (t, J 9.8 Hz, H-4′), 5.90–5.96 (m, CH=), 5.95 (br s, H-1′), 5.89 (H-3′), 5.87 (d, J 3.3 Hz, H-2′), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.76 (H-5′), 4.68 (H-29), 4.64 (dd, J 2.5, 12.3 Hz, H-6′), 4.56 (H-29), 4.48 (dd, J 3.8, 12.3 Hz, H-6′), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 2.73 (H-3), 2.72 (H-28a), 2.53 (H-28a), 2.41 (H-19), 1.99 (H-28), 1.90 (H-21), 1.74 (H-13), 1.66 (H-2), 1.66 (H-16), 1.65 (s, H-30), 1.61 (H-22), 1.59 (H-1), 1.59 (H-12), 1.54 (H-15), 1.43 (H-18), 1.41 (H-28), 1.40 (H-2), 1.34 (H-21), 1.33 (H-11), 1.20 (H-7), 1.18 (H-6), 1.14 (H-7), 1.14 (H-9), 1.11 (H-11), 1.11 (H-16), 1.00 (H-12), 0.99 (H-22), 0.90 (H-6), 0.90 (H-15), 0.88 (s, H-27), 0.87 (s, H-23), 0.82 (s, H-26), 0.73 (H-1), 0.67 (s, H-24), 0.55 (s, H-25), 0.53 (H-5). ¹³C NMR (CDCl₃) δ: 150.4 (C-20), 135.9 (CH=), 115.9 (=CH₂), 109.6 (C-29), 86.2 (C-3), 76.3 (C-1′), 72.6 (C-3′), 71.1 (C-2′), 70.6 (C-5′), 70.6 (OCH₂), 66.8 (C-4′), 62.3 (C-6′), 55.7 (C-5), 50.3 (C-9), 50.0 (C-18), 47.2 (C-19), 46.7 (C-17), 42.3 (C-14), 40.9 (C-8), 38.8 (C-4), 38.5 (C-1), 37.1 (C-13), 37.0 (C-10), 35.3 (C-22), 34.0 (C-7), 30.7 (C-16), 29.9 (C-21), 28.3 (C-28), 28.0 (C-23), 27.0 (C-15), 25.0 (C-12), 23.1 (C-2), 20.8 (C-11), 19.5 (C-28a), 19.3 (C-30), 18.0 (C-6), 16.2 (C-24), 15.9 (C-25), 15.6 (C-26), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 227.8. HR-MS (ESI) calc. for C₆₈H₈₂NaO₁₀Se [M + Na]⁺: 1161.4971. Found: 1161.4980. Anal. Calcd for C₆₈H₈₂O₁₀Se × H₂O (1156.38): C, 70.63; H, 7.32. Found: C, 70.58; H, 7.17.

3β-O-Allyl-28a-Se-(2,3,4-tri-O-benzoyl-β-L-arabinopyranosyl)-28a-seleno-28a-homolup-20(29)-ene (11) and 3β-O-allyl-28a-Se-(2,3,4-tri-O-benzoyl-α-L-arabinopyranosyl)-28a-seleno-28a-homolup-20(29)-ene (12). Recovered starting selenol 4 (36 mg, 13%), 11 (44 mg, 9%) and 12 (212 mg, 42%) were obtained.
Data for 11. [α]²⁰_D 125.8 (c 0.3, chloroform); ν_max (film): 2943, 2868, 1729, 1603, 1451, 1281, 1262, 1108, 1086, 1069, 757, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 6.23 (d, J 5.3 Hz, H-1′), 5.90–5.96 (m, CH=), 5.86 (dd, J 5.3, 10.0 Hz, H-2′), 5.77 (dd, J 3.5, 10.0 Hz, H-3′), 5.75 (H-4′), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.66 (H-29), 4.56 (H-29), 4.52 (dd, J 1.0, 13.1 Hz, H-5′), 4.04 (dd, J 2.3, 13.1 Hz, H-5′), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 2.78 (dd, J 4.1, 11.7 Hz, H-3), 2.58 (H-28a), 2.45, (H-28a), 2.35 (H-19), 1.92 (H-13), 1.88 (H-28), 1.71 (H-2), 1.68 (H-1), 1.68 (H-16), 1.66 (s, H-30), 1.63 (H-22), 1.60 (H-11), 1.60 (H-12), 1.52 (H-15), 1.48 (H-2), 1.48 (H-6), 1.44 (H-18), 1.36 (H-6), 1.35 (H-7), 1.34 (H-28), 1.32 (H-21), 1.26 (H-21), 1.29 (H-11), 1.24 (H-9), 1.15 (H-16), 1.02 (H-12), 0.99 (s, H-26), 0.98 (H-22), 0.96 (H-15), 0.95 (s, H-23), 0.93 (s, H-27), 0.83 (s, H-25), 0.81 (H-1), 0.78 (s, H-24), 0.66 (H-5). ¹³C NMR (CDCl₃) δ: 150.5 (C-20), 109.5 (C-29), 86.3 (C-3), 79.8 (C-1′, ¹J_C1,H1 170.4 Hz), 69.6 (C-4′), 69.5 (C-2′), 69.3 (C-3′), 62.5 (C-5′), 55.8 (C-5), 50.4 (C-9), 49.9 (C-18), 47.2 (C-19), 46.8 (C-17), 42.4 (C-14), 40.8 (C-8), 38.8 (C-4), 38.6 (C-1), 37.0 (C-10), 37.0 (C-13), 35.3 (C-22), 34.2 (C-7), 30.6 (C-16), 29.9 (C-21), 28.9 (C-28), 28.0 (C-23), 27.1 (C-15), 25.1 (C-12), 23.1 (C-2), 20.8 (C-11), 19.3 (C-30), 18.5 (C-28a), 18.2 (C-6), 16.2 (C-24), 16.1 (C-25), 15.9 (C-26), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 191.5. HR-MS (ESI) calc. for C₆₀H₇₆NaO₈Se [M + Na]⁺: 1027.4603. Found: 1027.4601.
Data for 12. [α]²⁰_D 45.6 (c 0.2, chloroform); ν_max (film): 2943, 2868, 1729, 1602, 1452, 1261, 1108, 1092, 1069, 757, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.90–5.96 (m, CH=), 5.81 (t, J 6.4 Hz, H-2′), 5.75 (H-4′), 5.71 (dd, J 6.9, 3.3 Hz, H-3′), 5.29 (H-1′), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.69 (H-29), 4.57 (H-29), 4.46 (dd, J 6.1, 12.2 Hz, H-5′), 4.10–4.13 (m, OCH₂), 3.95 (dd, J 2.9, 12.2 Hz, H-5′), 3.87–3.90 (m, OCH₂), 2.79 (dd, J 4.2, 11.7 Hz, H-3), 2.71 (H-28a), 2.42 (H-19), 1.92 (H-28), 1.86 (H-21), 1.76 (H-16), 1.74 (H-13), 1.70 (H-2), 1.67 (H-1), 1.67 (s, H-30), 1.64 (H-22), 1.62 (H-12), 1.57 (H-15), 1.48 (H-6), 1.46 (H-2), 1.46 (H-18), 1.40 (H-11), 1.39 (H-28), 1.35 (H-7), 1.33 (H-6), 1.30 (H-21), 1.24 (H-9), 1.19 (H-11), 1.18 (H-16), 1.04 (H-12), 1.00 (H-15), 0.98 (H-22), 0.96 (s, H-26), 0.95 (s, H-23), 0.94 (s, H-27), 0.81 (H-1), 0.80 (s, H-25), 0.77 (s, H-24), 0.66 (H-5). ¹³C NMR (CDCl₃) δ: 150.6 (C-20), 109.5 (C-29), 86.3 (C-3), 78.9 (C-1′, ¹J_C1,H1 158.0 Hz), 70.9 (C-2′), 69.9 (C-3′), 68.1 (C-4′), 64.3 (C-5′), 55.8 (C-5), 50.4 (C-9), 49.9 (C-18), 47.2 (C-19), 46.9 (C-17), 42.5 (C-14), 40.9 (C-8), 38.8 (C-4), 38.6 (C-1), 37.1 (C-10), 37.0 (C-13), 35.3 (C-22), 34.2 (C-7), 30.7 (C-16), 29.9 (C-21), 29.2 (C-28), 28.1 (C-23), 27.2 (C-15), 25.1 (C-12), 23.1 (C-2), 20.9 (C-11), 20.5 (C-28a), 19.3 (C-30), 18.2 (C-6), 16.3 (C-24), 16.1 (C-25), 16.0 (C-26), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: approx. 292. HR-MS (ESI) calc. for C₆₀H₇₆NaO₈Se [M + Na]⁺: 1027.4603. Found: 1027.4592.

3β-O-Allyl-28a-Se-(2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl)-28a-seleno-28a-homolup-20(29)-ene (13) and 3β-O-allyl-28a-Se-(2,3,4-tri-O-benzoyl-β-L-rhamnopyranosyl)-28a-seleno-28a-homolup-20(29)-ene (14). Recovered starting selenol 4 (84 mg, 30%), 13 (132 mg, 26%) and 14 (31 mg, 6%) were obtained.
Data for 13. [α]²⁰_D 12.4 (c 0.2, chloroform); ν_max (film): 2943, 2869, 1731, 1602, 1452, 1280, 1262, 1104, 1094, 1069, 757, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.90–5.96 (m, CH=), 5.85 (H-2′), 5.83 (H-3′), 5.76 (br s, H-1′), 5.73 (t, J 9.7 Hz, H-4′), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.71 (H-29), 4.58 (H-29), 4.42 (dq, J 6.2, 9.7 Hz, H-5′), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 2.79 (dd, J 4.0, 11.7 Hz, H-3), 2.69 (H-28a), 2.46 (H-19), 2.00 (H-28), 1.92 (H-21), 1.79 (H-13), 1.76 (H-16), 1.72 (H-2), 1.68 (H-1), 1.68 (s, H-30), 1.66 (H-22), 1.65 (H-12), 1.59 (H-15), 1.48 (H-18), 1.47 (H-2), 1.47 (H-28), 1.46 (H-6), 1.43 (H-11), 1.39 (d, J 6.2 Hz, H-6′), 1.36 (H-21), 1.35 (H-7), 1.32 (H-6), 1.26 (H-9), 1.24 (H-11), 1.20 (H-16), 1.05 (H-12), 1.02 (H-15), 1.02 (H-22), 1.02 (s, H-26), 0.96 (s, H-27), 0.95 (s, H-23), 0.82 (H-1), 0.81 (s, H-25), 0.77 (s, H-24), 0.66 (H-5). ¹³C NMR (CDCl₃) δ: 150.6 (C-20), 109.6 (C-29), 86.3 (C-3), 77.8 (C-1′, ¹J_C1,H1 171.0 Hz), 73.4 (C-2′), 70.6 (C-3′), 71.8 (C-4′), 69.5 (C-5′), 55.8 (C-5), 50.4 (C-9), 49.9 (C-18), 47.1 (C-19), 46.9 (C-17), 42.5 (C-14), 40.9 (C-8), 38.8 (C-4), 38.6 (C-1), 37.1 (C-10), 37.1 (C-13), 35.3 (C-22), 34.2 (C-7), 30.7 (C-16), 29.9 (C-21), 29.2 (C-28), 28.0 (C-23), 27.2 (C-15), 25.1 (C-12), 23.1 (C-2), 20.9 (C-11), 20.6 (C-28a), 19.3 (C-30), 18.2 (C-6), 17.6 (C-6′), 16.3 (C-24), 16.1 (C-25), 16.2 (C-26), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 243.6. HR-MS (ESI) calc. for C₆₁H₇₈NaO₈Se [M + Na]⁺: 1041.4760. Found: 1041.4764.
Data for 14. [α]²⁰_D 95.8 (c 0.3, chloroform); ν_max (film): 2941, 2868, 1730, 1602, 1452, 1280, 1261, 1120, 1092, 1069, 757, 711 cm⁻¹. ¹H NMR (CDCl₃) δ: 6.03 (d, J 3.0 Hz, H-2′), 5.90–5.96 (m, CH=), 5.63 (dd, J 9.4, 10.1 Hz, H-4′), 5.60 (dd, J 3.4, 10.1 Hz, H-3′), 5.30 (s, H-1′), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.68 (H-29), 4.57 (H-29), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 3.87 (H-5′), 2.79 (H-3), 2.77 (H-28a), 2.66 (H-28a), 2.43 (H-19), 1.86 (H-21), 1.78 (H-13), 1.72 (H-2), 1.72 (H-16), 1.68 (H-1), 1.68 (s, H-30), 1.64 (H-22), 1.55 (H-15), 1.55 (H-28), 1.51 (H-6), 1.48 (H-2), 1.48 (H-18), 1.46 (d, J 6.1 Hz, H-6′), 1.45 (H-28), 1.43 (H-11), 1.39 (H-6), 1.38 (H-7), 1.36 (H-21), 1.27 (H-9), 1.24 (H-11), 1.20 (H-16), 1.05 (H-12), 1.05 (s, H-26), 1.03 (H-22), 1.02 (H-15), 0.96 (s, H-27), 0.95 (s, H-23), 0.85 (s, H-25), 0.82 (H-1), 0.79 (s, H-24), 0.68 (H-5). ¹³C NMR (CDCl₃) δ: 150.4 (C-20), 109.6 (C-29), 86.3 (C-3), 77.0 (C-1′, ¹J_C1,H1 153.0 Hz), 76.6 (C-5′), 72.5 (C-3′), 72.4 (C-2′), 71.3 (C-4′), 55.8 (C-5), 50.5 (C-9), 49.8 (C-18), 47.2 (C-19), 46.8 (C-17), 42.5 (C-14), 40.9 (C-8), 38.8 (C-4), 38.6 (C-1), 37.1 (C-10), 37.1 (C-13), 35.4 (C-22), 34.3 (C-7), 30.5 (C-16), 29.9 (C-21), 29.0 (C-28), 28.1 (C-23), 27.2 (C-15), 25.0 (C-12), 23.1 (C-2), 20.9 (C-11), 20.3 (C-28a), 19.3 (C-30), 18.2 (C-6), 18.1 (C-6′), 16.3 (C-24), 16.3 (C-26), 16.1 (C-25), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 326.6. HR-MS (ESI) calc. for C₆₁H₇₈NaO₈Se [M + Na]⁺: 1041.4760. Found: 1041.4757.

3β-O-Allyl-28a-Se-(2,3,4,6-tetra-O-benzoyl-α-D-idopyranosyl)-28a-seleno-28a-homolup-20(29)-ene (15) and 3β-O-allyl-28a-Se-(2,3,4,6-tetra-O-benzoyl-β-D-idopyranosyl)-28a-seleno-28a-homolup-20(29)-ene (16). Recovered starting selenol 4 (104 mg, 37%), 15 (108 mg, 19%) and 16 (57 mg, 10%) were obtained.
Data for 15. [α]²⁰_D 83.1 (c 0.2, chloroform); ν_max (film): 2943, 2869, 1726, 1602, 1452, 1265, 1105, 1093, 1067, 1027, 757, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.94 (br s, H-1′), 5.90–5.96 (m, CH=), 5.66 (br s, H-3′), 5.41 (br s, H-2′), 5.40 (m, H-4′), 5.24–5.27 (m, =CH₂), 5.25 (m, H-5′), 5.11–5.13 (m, =CH₂), 4.73 (dd, J 7.7, 11.6 Hz, H-6′), 4.68 (H-29), 4.57 (H-29), 4.56 (H-6′), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 2.78 (dd, J 4.3, 11.7 Hz, H-3), 2.73 (H-28a), 2.52 (H-28a), 2.40 (H-19), 1.86 (H-21), 1.76 (H-13), 1.72 (H-16), 1.70 (H-2), 1.66 (s, H-30), 1.65 (H-12), 1.64 (H-1), 1.64 (H-22), 1.54 (H-15), 1.54 (H-28), 1.45 (H-2), 1.43 (H-11), 1.42 (H-18), 1.41 (H-28), 1.41 (H-6), 1.31 (H-21), 1.28 (H-7), 1.24 (H-11), 1.20 (H-9), 1.20 (H-16), 1.16 (H-6), 1.05 (H-12), 1.02 (H-22), 0.94 (s, H-23), 0.91 (H-15), 0.91 (s, H-27), 0.90 (s, H-26), 0.78 (H-1), 0.75 (s, H-24), 0.66 (s, H-25), 0.62 (H-5). ¹³C NMR (CDCl₃) δ: 150.5 (C-20), 109.6 (C-29), 86.3 (C-3), 77.1 (C-1′, ¹J_C1,H1 168.0 Hz), 69.7 (C-2′), 66.7 (C-4′), 66.3 (C-3′), 66.3 (C-5′), 63.3 (C-6′), 55.8 (C-5), 50.3 (C-9), 49.9 (C-18), 47.2 (C-19), 46.6 (C-17), 42.3 (C-14), 40.9 (C-8), 38.8 (C-4), 38.5 (C-1), 37.0 (C-10), 37.0 (C-13), 35.3 (C-22), 34.2 (C-7), 30.5 (C-16), 29.9 (C-21), 28.8 (C-28), 28.1 (C-23), 27.1 (C-15), 25.1 (C-12), 23.1 (C-2), 20.9 (C-11), 20.3 (C-28a), 19.3 (C-30), 18.2 (C-6), 16.3 (C-24), 16.0 (C-25), 15.9 (C-26), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 265.9. HR-MS (ESI) calc. for C₆₈H₈₂NaO₁₀Se [M + Na]⁺: 1161.4971. Found: 1161.4962. Anal. Calcd for C₆₈H₈₂NaO₁₀Se (1138.33): C, 71.75; H, 7.26. Found: C, 71.88; H, 7.35.
Data for 16. [α]²⁰_D −9.2 (c 0.5, chloroform); ν_max (film): 2942, 2868, 1727, 1602, 1452, 1264, 1107, 1093, 1068, 1027, 756, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.90–5.96 (m, CH=), 5.69 (t, J 2.7 Hz, H-3′), 5.54 (d, J 1.4 Hz, H-1′), 5.44 (br s, H-4′), 5.40 (br s, H-2′), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.67 (H-6′), 4.65 (H-29), 4.58 (H-29), 4.57 (H-6′), 4.54 (H-5′), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 2.78 (H-3), 2.78 (H-28a), 2.40 (H-19), 1.95 (H-28), 1.90 (H-21), 1.74 (H-13), 1.72 (H-16), 1.70 (H-2), 1.67 (s, H-30), 1.65 (H-1), 1.65 (H-12), 1.64 (H-22), 1.54 (H-15), 1.49 (H-6), 1.47 (H-18), 1.45, (H-2), 1.43 (H-11), 1.39 (H-28), 1.38 (H-21), 1.35 (H-6), 1.34 (H-7), 1.24 (H-9), 1.24 (H-11), 1.20 (H-16), 1.05 (H-12), 1.02 (H-22), 1.00 (s, H-26), 0.96 (H-15), 0.95 (s, H-23), 0.94 (s, H-27), 0.80 (H-1), 0.77 (s, H-25), 0.75 (s, H-24), 0.66 (H-5). ¹³C NMR (CDCl₃) δ: 150.3 (C-20), 109.8 (C-29), 86.3 (C-3), 76.6 (C-1′, ¹J_C1,H1 154.0 Hz), 75.0 (C-5′), 69.7 (C-2′), 67.1 (C-3′), 65.7 (C-4′), 63.2 (C-6′), 55.8 (C-5), 50.4 (C-9), 49.7 (C-18), 47.3 (C-19), 46.9 (C-17), 42.5 (C-14), 40.9 (C-8), 38.8 (C-4), 38.6 (C-1), 37.1 (C-10), 37.1 (C-13), 35.4 (C-22), 34.3 (C-7), 30.6 (C-16), 30.0 (C-21), 28.7 (C-28), 28.1 (C-23), 27.2 (C-15), 25.1 (C-12), 23.1 (C-2), 20.9 (C-11), 19.7 (C-28a), 19.2 (C-30), 18.2 (C-6), 16.3 (C-24), 16.2 (C-26), 16.1 (C-25), 14.8 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 324.1. HR-MS (ESI) calc. for C₆₈H₈₂NaO₁₀Se [M + Na]⁺: 1161.4971. Found: 1161.5000.

General procedure for the deallylation reaction

A solution of iridium complex was prepared from [Ir(COD)(MePPh₂)₂]PF₆ (3–5 mg) according to the literature procedure¹⁷ and transferred to a solution of the corresponding saponin in THF (3 mL), stirred at room temperature for 12 h, and then concentrated. The crude vinyl ether was dissolved in a chloroform (2.0 mL) and methanol (2.0 mL) mixture, and then p-TsOH (70 mg) was added. The mixture was stirred for 1 h and concentrated. Column chromatography (hexane–ethyl acetate, 20 : 1 → 7 : 3) of the residue gave the title compound as foam.

28a-Se-(2,3,4,6-Tetra-O-benzoyl-α-D-mannopyranosyl)-28a-seleno-28a-homolup-20(29)-en-3β-ol (17). Starting from 10 (95 mg, 0.083 mmol) the title compound was obtained (81 mg, 89%). [α]²⁰_D 64.5 (c 0.3, chloroform); ν_max (film): 2942, 2868, 1729, 1602, 1451, 1281, 1264, 1104, 1094, 1068, 756, 709 cm⁻¹. ¹H NMR (CDCl₃) δ: 6.23 (t, J 9.8 Hz, H-4′), 5.97 (br s, H-1′), 5.90 (H-2′), 5.88 (H-3′), 4.77 (H-5′), 4.69 (H-29), 4.66 (dd, J 2.4, 12.3 Hz, H-6′), 4.57 (H-29), 4.50 (dd, J 3.8, 12.3 Hz, H-6′), 3.13 (dd, J 4.2, 11.5 Hz, H-3), 2.74 (H-28a), 2.53 (H-28a), 2.42 (H-19), 1.99 (H-28), 1.91 (H-21), 1.75 (H-13), 1.67 (H-16), 1.66 (s, H-30), 1.62 (H-22), 1.61 (H-12), 1.58 (H-1), 1.56 (H-2), 1.55 (H-15), 1.49 (H-2), 1.44 (H-18), 1.42 (H-28), 1.35 (H-21), 1.34 (H-11), 1.23 (H-7), 1.21 (H-6), 1.18 (H-9), 1.16 (H-7), 1.13 (H-11), 1.12 (H-16), 1.01 (H-12), 1.00 (H-22), 0.93 (H-6), 0.91 (H-15), 0.90 (s, H-23), 0.89 (s, H-27), 0.83 (s, H-26), 0.82 (H-1), 0.66 (s, H-24), 0.56 (s, H-25), 0.56 (H-5). ¹³C NMR (CDCl₃) δ: 150.4 (C-20), 109.6 (C-29), 78.9 (C-3), 76.3 (C-1′), 72.6 (C-2′), 71.1 (C-3′), 70.7 (C-5′), 66.8 (C-4′), 62.6 (C-6′), 55.2 (C-5), 50.3 (C-9), 50.0 (C-18), 47.2 (C-19), 46.7 (C-17), 42.4 (C-14), 40.8 (C-8), 38.8 (C-4), 38.6 (C-1), 37.1 (C-13), 37.0 (C-10), 35.3 (C-22), 34.0 (C-7), 30.7 (C-16), 29.9 (C-21), 28.3 (C-28), 27.9 (C-23), 27.4 (C-2), 27.1 (C-15), 25.0 (C-12), 20.8 (C-11), 19.6 (C-28a), 19.3 (C-30), 18.1 (C-6), 15.9 (C-25), 15.6 (C-26), 15.3 (C-24), 14.4 (C-27). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 227.9. HR-MS (ESI) calc. for C₆₅H₇₈NaO₁₀Se [M + Na]⁺: 1121.4658. Found 1121.4677. Anal. Calcd for C₆₅H₇₈O₁₀Se × H₂O (1116.32): C, 69.94; H, 7.22. Found: C, 70.05; H, 7.23.

28a-Se-(2,3,4-Tri-O-benzoyl-β-L-arabinopyranosyl)-28a-seleno-28a-homolup-20(29)-en-3β-ol (18). Starting from 11 (85 mg, 0.084 mmol) the title compound was obtained (56 mg, 87%). [α]²⁰_D 160.5 (c 0.2, chloroform); ν_max (film): 2942, 2867, 1729, 1451, 1280, 1261, 1108, 1087, 1069, 1027, 757, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 6.23 (d, 1 H, J 5.2 Hz, H-1′), 5.87 (dd, 1 H, J 5.2, 10.0 Hz, H-2′), 5.78 (dd, 1 H, J 10.0, 3.4 Hz, H-3′), 5.75 (br s, 1H, H-4′), 4.66 (br s, 1 H, H-29), 4.56 (br s, 1 H, H-29), 4.53 (dd, 1 H, J 13.0 Hz, H-5′), 4.04 (dd, 1 H, J 2.2, 13.0 Hz, H-5′), 3.18 (dd, 1 H, J 4.9, 11.3 Hz, H-3), 2.56–2.60 (m, 1 H), 2.43–2.48 (m, 1 H), 2.31–2.38 (m, 1 H), 1.82–1.92 (m, 2 H), 1.66 (s, 3 H), 1.00 (s, 3 H), 0.96 (s, 3 H), 0.94 (s, 3 H), 0.82 (s, 3 H), 0.76 (s, 3 H), 0.79–1.75 (m, 24 H), 0.66–0.68 (m, 1 H, H-5). ¹³C NMR (CDCl₃) δ: 150.5 (C-20), 109.6 (C-29), 79.8, 79.0, 69.6, 69.5, 69.3, 62.5 (C-5′), 55.3 (C-5), 50.5, 49.9, 47.2, 46.8 (C), 42.5 (C), 40.8 (C), 38.9 (C), 38.7 (CH₂), 37.2 (C), 37.0, 35.3 (CH₂), 34.2 (CH₂), 30.6 (CH₂), 29.9 (CH₂), 29.0 (CH₂), 28.0, 27.4 (CH₂), 27.2 (CH₂), 25.1 (CH₂), 20.9 (CH₂), 19.3, 18.5 (CH₂), 18.3 (CH₂), 16.1, 16.0, 15.4, 14.8. HR-MS (ESI) calc. for C₅₇H₇₂NaO₈Se [M + Na]⁺: 987.4290. Found: 987.4283.

28a-Se-(2,3,4-Tri-O-benzoyl-α-L-arabinopyranosyl)-28a-seleno-28a-homolup-20(29)-en-3β-ol (19). Starting from 12 (110 mg, 0.109 mmol) the title compound was obtained (66 mg, 63%). [α]²⁰_D 37.5 (c 0.2, chloroform); ν_max (film): 2942, 2868, 1728, 1451, 1278, 1261, 1108, 1092, 1069, 1026, 756, 709 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.81 (m, 1 H, H-2′), 5.74–5.76 (m, 1 H, H-4′), 5.71 (dd, 1 H, J 6.9, 3.2 Hz, H-3′), 5.29 (d, 1 H, J 4.4 Hz, H-1′), 4.69 (br s, 1 H, H-29), 4.57 (br s, 1 H, H-29), 4.46 (dd, 1 H, J 12.2, 6.1 Hz, H-5′), 3.95 (dd, 1 H, J 3.0, 12.2 Hz, H-5′), 3.17 (dd, 1 H, J 4.8, 11.4 Hz, H-3), 2.69–2.73 (m, 2 H), 2.39–2.45 (m, 1 H), 1.81–1.96 (m, 2 H), 1.72–1.78 (m, 2 H), 1.66 (s, 3 H), 0.97 (s, 3 H), 0.96 (s, 3 H), 0.95 (s, 3 H), 0.79 (s, 3 H), 0.75 (s, 3 H), 0.74–1.70 (m, 22 H), 0.66–0.68 (m, 1 H, C-5). ¹³C NMR (CDCl₃) δ: 150.5 (C-20), 109.6 (C-29), 79.0, 78.9, 70.9, 69.9, 68.1, 60.4 (C-5′), 55.3 (C-5), 50.4, 49.9, 47.2, 46.9 (C), 42.5 (C), 40.8 (C), 38.8 (C), 38.7 (CH₂), 37.1 (C), 37.0, 35.3 (CH₂), 34.2 (CH₂), 30.7 (CH₂), 29.9 (CH₂), 29.3 (CH₂), 28.0, 27.4 (CH₂), 27.2 (CH₂), 25.1 (CH₂), 21.0 (CH₂), 20.9 (CH₂), 18.3 (CH₂), 16.1, 16.0, 15.4, 14.9. HR-MS (ESI) calc. for C₅₇H₇₂NaO₈Se [M + Na]⁺: 987.4290. Found: 987.4295.

28a-Se-(2,3,4-Tri-O-benzoyl-α-L-rhamnopyranosyl)-28a-seleno-28a-homolup-20(29)-en-3β-ol (20). Starting from 13 (77 mg, 0.075 mmol) the title compound was obtained (58 mg, 78%). [α]²⁰_D 1.8 (c 0.2, chloroform); ν_max (film): 2942, 2869, 1730, 1602, 1452, 1281, 1262, 1094, 1069, 757, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.82–5.86 (m, 2 H, H-2′, H-3′), 5.77 (d, 1 H, J 0.7 Hz, H-1′), 5.73 (t, 1 H, J 9.8 Hz, H-4′), 4.71–4.72 (m, 1 H, H-29), 4.58 (br s, 1 H, H-29), 4.43 (dq, 1 H, J 6.2, 9.8, Hz, H-5′), 3.18 (dd, 1 H, J 4.8, 11.3 Hz, H-3), 2.68–2.71 (m, 2 H), 2.43–2.49 (m, 1 H), 1.88–2.00 (m, 2 H), 1.75–1.83 (m, 2 H), 1.68 (s, 3 H), 1.40 (d, 3 H, J 6.2 Hz, H-6′), 1.03 (s, 3 H), 0.96 (s, 3 H), 0.96 (s, 3 H), 0.80 (s, 3 H), 0.75 (s, 3 H), 0.83–1.66 (m, 22 H), 0.66–0.68 (m, 1 H, H-5). ¹³C NMR (CDCl₃) δ: 150.5 (C-20), 109.6 (C-29), 78.9, 77.9, 73.4, 71.8, 70.7, 69.6, 55.3, 50.4, 49.9, 47.2, 46.9 (C), 42.5 (C), 40.9 (C), 38.8 (C), 38.7 (CH₂), 37.1 (C), 37.1, 35.3 (CH₂), 34.2 (CH₂), 30.7 (CH₂), 29.9 (CH₂), 29.2 (CH₂), 28.0, 27.4 (CH₂), 27.3 (CH₂), 25.1 (CH₂), 20.9 (CH₂), 20.7 (CH₂), 19.3, 18.3 (CH₂), 17.7, 16.2, 16.1, 15.3, 14.9. HR-MS (ESI) calc. for C₅₈H₇₄NaO₈Se [M + Na]⁺: 1001.4447. Found: 1001.4434.

28a-Se-(2,3,4,6-Tetra-O-benzoyl-α-D-idopyranosyl)-28a-seleno-28a-homolup-20(29)-en-3β-ol (21). Starting from 15 (102 mg, 0.089 mmol) the title compound was obtained (91 mg, 93%). [α]²⁰_D 74.4 (c 0.2, chloroform); ν_max (film): 2942, 2868, 1725, 1451, 1264, 1093, 1067, 1027, 756, 710 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.94 (br s, 1 H), 5.66 (br s, 1 H), 5.40–5.41 (m, 2 H), 5.24–5.26 (m, 1 H, H-5′), 4.73 (dd, 1 H, J7.6, 11.6 Hz, H-5′), 4.68 (br s, 1 H, H-29), 4.54–4.57 (m, 2 H, H-5′, H-29), 3.16 (dd, 1 H, J 4.6, 11.4 Hz, H-3), 2.70–2.76 (m, 1 H), 2.50–2.55 (m, 1 H), 2.37–2.43 (m, 1 H), 1.93–1.99 (m, 1 H), 1.82–1.90 (m, 1 H), 1.73–1.78 (m, 1 H), 1.66 (s, 3 H), 0.95 (s, 3 H), 0.91 (s, 3 H), 0.90 (s, 3 H), 0.73 (s, 3 H), 0.65 (s, 3 H), 0.75–1.68 (m, 23 H), 0.61–0.63 (m, 1 H, C-5). ¹³C NMR (CDCl₃) δ: 150.5 (C-20), 109.6 (C-29), 78.9 (C-3), 77.1 (C-1′), 69.8, 66.7, 66.3, 66.3, 63.2 (C-6′), 55.2 (C-5), 50.4, 49.9, 47.2, 46.6 (C), 42.4 (C), 40.8 (C), 38.8 (C), 38.6 (CH₂), 37.1 (C), 37.0, 35.3 (CH₂), 34.1 (CH₂), 30.5 (CH₂), 29.9 (CH₂), 28.8 (CH₂), 28.0, 27.4 (CH₂), 27.2 (CH₂), 25.0 (CH₂), 20.9 (CH₂), 20.3 (CH₂), 19.3, 18.2 (CH₂), 16.0, 15.9, 15.3, 14.8. HR-MS (ESI) calc. for C₆₅H₇₈NaO₁₀Se [M + Na]⁺: 1121.4658. Found: 1121.4679. Anal. Calcd for C₆₅H₇₈O₁₀Se (1098.30): C, 71.08; H, 7.16. Found: C, 71.45; H, 7.56.

General procedure for the debenzoylation reaction

A suspension of the protected saponin (0.10 mmol) and K₂CO₃ (20 mg) in MeOH (10 mL) was stirred for 2 h, then neutralized with Amberlyst 15 resin (H⁺ form) and purified by column chromatography (hexane–ethyl acetate, 5 : 1 → 1 : 1, and then hexane–ethyl acetate–methanol, 5 : 3 : 1) to afford the unprotected saponin.

28a-Se-α-D-Mannopyranosyl-28a-seleno-28a-homolup-20(29)-en-3β-ol (22). Yield 78% (53 mg); m.p. 222–224 °C. [α]²⁰_D 100.8 (c 0.2, chloroform–methanol, 1 : 1); ν_max (film): 2941, 2868, 1640, 1454, 1377, 1104, 1066, 1032, 880, 795, 719 cm⁻¹. ¹H NMR (CDCl₃/CD₃OD, 1 : 1) δ: 5.63 (s, H-1′), 4.68 (H-29), 4.57 (H-29), 4.08 (d, J 2.2 Hz, H-2′), 3.91 (H-6′), 3.87 (H-5′), 3.85 (H-4′), 3.79 (H-6′), 3.78 (H-3′), 3.18 (H-3), 2.63 (H-28a), 2.52 (H-28a), 2.41 (H-19), 1.91 (H-28), 1.90 (H-21), 1.78 (H-13), 1.74 (H-16), 1.69 (s, H-30), 1.68 (H-1), 1.67 (H-22), 1.66 (H-12), 1.59 (H-2), 1.57 (H-15), 1.54 (H-6), 1.49 (H-18), 1.44 (H-11), 1.40 (H-6), 1.40 (H-7), 1.39 (H-28), 1.37 (H-21), 1.29 (H-9), 1.25 (H-11), 1.21 (H-16), 1.07 (H-12), 1.06 (s, H-26), 1.03 (H-22), 1.02 (H-15), 0.98 (s, H-27), 0.97 (s, H-23), 0.92 (H-1), 0.85 (s, H-25), 0.77 (s, H-24), 0.70 (H-5). ¹³C NMR (CDCl₃/CD₃OD, 1 : 1) δ: 150.4 (C-20), 109.3 (C-29), 81.2 (C-1′), 78.6 (C-3), 74.1 (C-5′), 72.7 (C-2′), 72.1 (C-3′), 66.8 (C-4′), 61.1 (C-6′), 55.1 (C-5), 50.3 (C-9), 49.7 (C-18), 47.1 (C-19), 46.6 (C-17), 42.3 (C-14), 40.7 (C-8), 38.6 (C-4), 38.5 (C-1), 36.9 (C-10), 36.9 (C-13), 35.1 (C-22), 34.0 (C-7), 30.4 (C-16), 29.6 (C-21), 28.7 (C-28), 27.6 (C-23), 27.0 (C-15), 26.8 (C-2), 24.8 (C-12), 20.7 (C-11), 19.8 (C-28a), 19.0 (C-30), 18.1 (C-6), 15.8 (C-25), 15.7 (C-26), 15.1 (C-24), 14.6 (C-27). ⁷⁷Se NMR (CD₃OD : CDCl₃, 1 : 1, 114.4 MHz) δ: 226.3. HR-MS (ESI) calc. for C₃₇H₆₂NaO₆Se [M + Na]⁺: 705.3609. Found: 705.3611.

28a-Se-β-L-Arabinopyranosyl-28a-seleno-28a-homolup-20(29)-en-3β-ol (23). Yield 88% (57 mg); m.p. 238–240 °C. [α]²⁰_D 108.3 (c 0.2, chloroform–methanol, 1 : 1); ν_max (film): 2940, 2868, 1640, 1459, 1377, 1083, 1031, 971, 880, 850, 792, 619 cm⁻¹. ¹H NMR (CDCl₃/CD₃OD, 1 : 1) δ: 5.70 (d, J 4.7 Hz, H-1′), 4.68 (H-29), 4.57 (H-29), 4.12 (dd, J 1.4, 12.4 Hz, H-5′), 3.98 (dd, J 4.7, 9.0 Hz, H-2′), 3.93 (H-4′), 3.73 (dd, J 3.0, 12.4 Hz, H-5′), 3.65 (dd, J 3.2, 9.0 Hz, H-3′), 3.17 (dd, J 7.7, 8.5 Hz, H-3), 2.57 (H-28a), 2.45 (H-28a), 2.41 (H-19), 1.93 (H-16), 1.91 (H-28), 1.89 (H-21), 1.77 (H-13), 1.68 (s, H-30), 1.67 (H-1), 1.64 (H-12), 1.58 (H-2), 1.56 (H-15), 1.56 (H-22), 1.52 (H-6), 1.48 (H-18), 1.43 (H-11), 1.40 (H-6), 1.38 (H-7), 1.38 (H-28), 1.34 (H-21), 1.28 (H-9), 1.23 (H-11), 1.18 (H-16), 1.08 (H-22), 1.06 (H-12), 1.06 (s, H-26), 0.99 (H-15), 0.96 (s, H-23), 0.96 (s, H-27), 0.90 (H-1), 0.83 (s, H-25), 0.75 (s, H-24), 0.68 (H-5). ¹³C NMR (CDCl₃/CD₃OD, 1 : 1) δ: 150.4 (C-20), 109.2 (C-29), 83.8 (C-1′, ¹J_C1,H1 165.3 Hz), 78.5 (C-3), 71.1 (C-3′), 69.3 (C-2′), 68.0 (C-4′), 64.7 (C-5′), 55.1 (C-5), 50.2 (C-9), 49.6 (C-18), 47.0 (C-19), 46.6 (C-17), 42.2 (C-14), 42.2 (C-8), 38.5 (C-4), 38.5 (C-1), 36.8 (C-10), 36.8 (C-13), 35.1 (C-22), 33.9 (C-7), 30.3 (C-16), 29.6 (C-21), 28.8 (C-28), 27.6 (C-23), 26.9 (C-15), 26.7 (C-2), 24.8 (C-12), 20.6 (C-11), 18.9 (C-30), 18.1 (C-28a), 18.0 (C-6), 15.7 (C-25), 15.3 (C-26), 15.0 (C-24), 14.5 (C-27). ⁷⁷Se NMR (CD₃OD : CDCl₃, 1 : 1, 114.4 MHz) δ: 328.6. HR-MS (ESI) calc. for C₃₆H₆₀NaO₅Se [M + Na]⁺: 675.3504. Found: 675.3500.

28a-Se-α-L-Arabinopyranosyl-28a-seleno-28a-homolup-20(29)-en-3β-ol (24). Yield 61% (40 mg); m.p. 203–204 °C. [α]²⁰_D −14.1 (c 0.2, chloroform–methanol, 1 : 1); ν_max (film): 2942, 2868, 1453, 1376, 1081, 1043, 992, 883, 860, 798, 757 cm⁻¹. ¹H NMR (CDCl₃/CD₃OD, 1 : 1) δ: 4.76 (d, J 7.5 Hz, H-1′), 4.69 (H-29), 4.57 (H-29), 4.04 (dd, J 4.4, 12.1 Hz, H-5′), 3.95 (H-4′), 3.79 (t, J 7.5 Hz, H-2′), 3.61 (dd, J 3.3, 7.7 Hz, H-3′), 3.56 (dd, J 2.3, 12.1 Hz, H-5′), 3.16 (dd, J 5.1, 11.2 Hz, H-3), 2.62 (H-28a), 2.44 (H-19), 1.96 (H-28), 1.90 (H-21), 1.80 (H-13), 1.76 (H-16), 1.69 (s, H-30), 1.68 (H-1), 1.68 (H-22), 1.67 (H-12), 1.63 (H-15), 1.60 (H-2), 1.54 (H-6), 1.50 (H-18), 1.44 (H-11), 1.42 (H-6), 1.41 (H-7), 1.41 (H-28), 1.36 (H-21), 1.32 (H-9), 1.24 (H-11), 1.21 (H-16), 1.08 (H-12), 1.06 (s, H-26), 1.02 (H-15), 1.02 (H-22), 0.98 (s, H-27), 0.96 (s, H-23), 0.93 (H-1), 0.85 (s, H-25), 0.76 (s, H-24), 0.70 (H-5). ¹³C NMR (CDCl₃/CD₃OD, 1 : 1) δ: 149.9 (C-20), 108.7 (C-29), 81.5 (C-1′, ¹J_C1,H1 156.8 Hz), 77.9 (C-3), 72.4 (C-3′), 71.1 (C-2′), 67.9 (C-5′), 67.2 (C-4′), 54.8 (C-5), 49.9 (C-9), 49.3 (C-18), 46.7 (C-19), 46.2 (C-17), 41.8 (C-14), 41.8 (C-8), 38.1 (C-4), 38.1 (C-1), 36.5 (C-10), 36.5 (C-13), 34.5 (C-22), 33.6 (C-7), 29.9 (C-16), 29.2 (C-21), 28.8 (C-28), 27.0 (C-23), 26.5 (C-15), 26.2 (C-2), 24.5 (C-12), 20.2 (C-11), 18.5 (C-28a), 18.2 (C-30), 17.6 (C-6), 15.3 (C-26), 15.2 (C-25), 14.5 (C-24), 13.9 (C-27). ⁷⁷Se NMR (CD₃OD : CDCl₃, 1 : 1, 114.4 MHz) δ: 287.9. HR-MS (ESI) calc. for C₃₆H₆₀NaO₅Se [M + Na]⁺: 675.3504. Found: 675.3490.

28a-Se-α-L-Rhamnopyranosyl-28a-seleno-28a-homolup-20(29)-en-3β-ol (25). Yield 78% (52 mg); m.p. 201–202 °C. [α]²⁰_D −94.0 (c 0.2, chloroform–methanol, 1 : 1); ν_max (film): 2941, 2869, 1640, 1454, 1376, 1102, 1064, 973, 882, 756, 647 cm⁻¹. ¹H NMR (CDCl₃/CD₃OD, 1 : 1) δ: 5.55 (d, J 1.0 Hz, H-1′), 4.69 (H-29), 4.57 (H-29), 4.07 (dd, J 1.0, 3.4 Hz, H-2′), 3.92 (dq, J 6.2, 9.4 Hz, H-5′), 3.71 (dd, J 3.4, 9.4 Hz, H-3′), 3.46 (t, J 9.4 Hz, H-4′), 3.16 (dd, J 3.1, 10.2 Hz, H-3), 2.56 (H-28a), 2.42 (H-19), 1.92 (H-28), 1.91 (H-21), 1.78 (H-13), 1.76 (H-16), 1.69 (s, H-30), 1.68 (H-1), 1.68 (H-22), 1.66 (H-12), 1.60 (H-15), 1.59 (H-2), 1.54 (H-6), 1.49 (H-18), 1.44 (H-11), 1.41 (H-6), 1.40 (H-7), 1.40 (H-28), 1.36 (H-21), 1.34 (d, J 6.2 Hz, H-6′), 1.29 (H-9), 1.25 (H-11), 1.21 (H-16), 1.07 (H-12), 1.05 (s, H-26), 1.02 (H-15), 1.02 (H-22), 0.98 (s, H-27), 0.96 (s, H-23), 0.92 (H-1), 0.84 (s, H-25), 0.76 (s, H-24), 0.69 (H-5). ¹³C NMR (CDCl₃/CD₃OD, 1 : 1) δ: 150.1 (C-20), 109.0 (C-29), 80.7 (C-1′), 72.6 (C-2′), 72.6 (C-4′), 72.2 (C-3), 71.7 (C-3′), 70.1 (C-5′), 55.0 (C-5), 50.1 (C-9), 49.5 (C-18), 46.8 (C-19), 46.4 (C-17), 42.0 (C-14), 40.4 (C-8), 38.8 (C-4), 38.4 (C-1), 36.7 (C-10), 36.7 (C-13), 34.8 (C-22), 33.8 (C-7), 30.2 (C-16), 29.5 (C-21), 28.6 (C-28), 27.3 (C-23), 26.8 (C-15), 26.4 (C-2), 24.7 (C-12), 20.5 (C-11), 19.1 (C-28a), 18.7 (C-30), 17.8 (C-6), 16.8 (C-6′), 15.6 (C-26), 15.5 (C-25), 14.8 (C-24), 14.3 (C-27). ⁷⁷Se NMR (CD₃OD : CDCl₃, 1 : 1, 114.4 MHz) δ: 229.2. HR-MS (ESI) calc. for C₃₇H₆₂NaO₅Se [M + Na]⁺: 689.3660. Found: 689.3656.

28a-Se-α-D-Idopyranosyl-28a-seleno-28a-homolup-20(29)-en-3β-ol (26). Yield 73% (50 mg); m.p. 206–208 °C. [α]²⁰_D 78.3 (c 0.2, chloroform–methanol, 1 : 1); ν_max (film): 2941, 2868, 1639, 1454, 1376, 1105, 1043, 982, 880, 733 cm⁻¹. ¹H NMR (CDCl₃/CD₃OD, 1 : 1) δ: 5.49 (br s, H-1′), 4.69 (H-29), 4.57 (H-29), 4.42 (ddd, J 1.5, 6.1, 6.1 Hz, H-5′), 3.87 (m, H-3′), 3.84 (m, H-2′), 3.81 (dd, J 5.8, 11.3 Hz, H-6′), 3.77 (dd, J 6.3, 11.3 Hz, H-6′), 3.71 (m, H-4′), 3.14 (dd, J 4.8, 11.5 Hz, H-3), 2.61 (H-28a), 2.52 (H-28a), 2.43 (H-19), 1.94 (H-21), 1.92 (H-28), 1.82 (H-13), 1.76 (H-16), 1.69 (H-1), 1.69 (s, H-30), 1.68 (H-22), 1.67 (H-12), 1.64 (H-15), 1.62 (H-2), 1.58 (H-2), 1.54 (H-6), 1.51 (H-18), 1.45 (H-28), 1.44 (H-11), 1.43 (H-6), 1.42 (H-7), 1.35 (H-21), 1.33 (H-9), 1.25 (H-11), 1.21 (H-16), 1.08 (H-12), 1.08 (s, H-26), 1.03 (H-15), 1.03 (H-22), 0.99 (s, H-27), 0.96 (s, H-23), 0.93 (H-1), 0.86 (s, H-25), 0.76 (s, H-24), 0.71 (H-5). ¹³C NMR (CDCl₃/CD₃OD, 1 : 1) δ: 149.7 (C-20), 108.2 (C-29), 81.5 (C-1′), 77.6 (C-3), 71.7 (C-2′), 68.4 (C-4′), 68.3 (C-5′), 67.9 (C-3′), 60.5 (C-6′), 54.6 (C-5), 49.7 (C-9), 49.1 (C-18), 46.6 (C-19), 45.8 (C-17), 41.5 (C-8), 40.0 (C-14), 37.9 (C-1), 37.8 (C-4), 36.3 (C-13), 36.2 (C-10), 34.3 (C-22), 33.4 (C-7), 29.6 (C-16), 28.9 (C-21), 28.5 (C-28), 26.6 (C-23), 26.3 (C-15), 25.9 (C-2), 24.3 (C-12), 20.0 (C-11), 19.5 (C-28a), 17.7 (C-30), 17.4 (C-6), 14.8 (C-25), 14.7 (C-26), 14.1 (C-24), 13.5 (C-27). ⁷⁷Se NMR (CD₃OD : CDCl₃, 1 : 1, 114.4 MHz) δ: 267.7. HR-MS (ESI) calc. for C₃₇H₆₂NaO₆Se [M + Na]⁺: 705.3609. Found: 705.3605.

Attempt to di-(3β-O-allyl-28a-homolup-20(29)-en-28a-yl) diselenide (27). To grey elemental selenium (powder, 95 mg, 1.20 mmol) 1 M solution of Super Hydride in THF (1.2 mL, 1.20 mmol) was slowly added under an argon atmosphere. The mixture was allowed to stir at 50 °C for 1 h, cooled to room temp and ^tBuOH (200 μL) was added followed by THF (up to 5 mL total volume). The above suspension (1.0 mL) was added to a solution of mesylate 1 (58 mg, 0.10 mmol) in THF (2 mL) and stirred for 3 h at 50 °C. Solvents were removed under reduced pressure and the residue was purified by preparative TLC (hexane–ethyl acetate, 40 : 1). The product was suspended in a hot CHCl₃–MeOH (1 : 3) mixture, cooled to r.t. and the solid was decanted. Pure 27 (49 mg, 88%) was obtained as a yellow powder. All physicochemical properties were identical as desribed for selenol 4. HR-MS (ESI) calc. for C₃₄H₅₆OSe [½M]⁺: 560.3496. Found: 560.3489. Anal. Calcd for C₆₈H₁₁₂O₂Se₂ × 2 × MeOH (1183.65): C, 71.03; H, 10.22. Found: C, 71.25; H, 10.05.

Methyl 3β-O-allyl-28a-homolup-20(29)-en-28a-yl-selenide (28). To a solution of 4 (56 mg, 0.10 mmol) in THF (1 mL) 60% NaH (20 mg, 0.50 mmol) was added and stirred for 15 min. Then methyl iodide (31 μL, 0.50 mmol) was added and stirred at room temp overnight. The reaction was quenched by the addition of saturated solution of NH₄Cl (60 μL), solvents were coevaporated with toluene and the whole mixture was purified by preparative TLC (hexane–ethyl acetate, 40 : 1) to afford 42 mg (74%) of the title compound as light yellow foam. [α]²⁰_D 15.3 (c 0.2, chloroform); ν_max (film): 2942, 2868, 1642, 1455, 1376, 1135, 1086, 1071, 919, 882, 758 cm⁻¹. ¹H NMR (CDCl₃) δ: 5.90–5.96 (m, CH=), 5.24–5.27 (m, =CH₂), 5.11–5.13 (m, =CH₂), 4.57 (H-29), 4.68 (H-29), 4.10–4.13 (m, OCH₂), 3.87–3.90 (m, OCH₂), 2.79 (dd, J 4.2, 11.7 Hz, H-3), 2.47 (H-28a), 2.42 (H-19), 2.39 (H-28a), 2.01 (s, ²J_Se,H 10.0 Hz calculated from satellite signals, SeCH₃), 1.89 (H-21), 1.88 (H-28), 1.79 (H-13), 1.72 (H-2), 1.68 (H-1), 1.68 (s, H-30), 1.64 (H-22), 1.56 (H-15), 1.50 (H-6), 1.48 (H-2), 1.47 (H-18), 1.39 (H-6), 1.38 (H-7), 1.35 (H-21), 1.33 (H-28), 1.26 (H-9), 1.04 (s, H-26), 1.00 (H-15), 1.00 (H-22), 0.96 (s, H-27), 0.95 (s, H-23), 0.84 (s, H-25), 0.82 (H-1), 0.78 (s, H-24), 0.68 (H-5). ¹³C NMR (CDCl₃) δ: 150.6 (C-20), 135.9 (CH=), 115.9 (=CH₂), 109.6 (C-29), 86.3 (C-3), 70.6 (OCH₂), 55.9 (C-5), 50.5 (C-9), 49.9 (C-18), 47.3 (C-19), 46.7 (C-17), 42.5 (C-14), 41.0 (C-8), 38.9 (C-4), 38.6 (C-1), 37.1 (C-10), 37.1 (C-13), 35.4 (C-22), 34.3 (C-7), 30.7 (C-16), 30.0 (C-21), 28.8 (C-28), 28.1 (C-23), 27.2 (C-15), 25.1 (C-12), 23.1 (C-2), 21.0 (C-11), 20.4 (C-28a), 19.4 (C-30), 18.3 (C-6), 16.3 (C-24), 16.1 (C-26), 16.1 (C-25), 14.9 (C-27), 4.1 (SeCH₃). ⁷⁷Se NMR (CDCl₃, 114.4 MHz) δ: 93.4. HR-MS (ESI) calc. for C₃₅H₅₈OSe [M]⁺: 574.3653. Found: 574.3662. Anal. Calcd for C₃₅H₅₈OSe (573.81): C, 73.26; H, 10.19. Found: C, 72.97; H, 10.28.

Acknowledgements

This work was financed by grants from the National Science Centre, Poland (no. 2012/07/B/ST5/00823) and by the Czech Ministry of Education grant from the National Program for Sustainability I (LO1204) and grant GAČR 14-19590S. We thank Olga Hustáková for excellent technical assistance.

References

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Footnote

† Electronic supplementary information (ESI) available: Copies of ¹H and ¹³C NMR spectra of the synthesized compounds. CCDC 1422676. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ob01938b

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