Efforts to total synthesis of philinopside E: convergent synthesis of the sulfated lanostane-type tetraglycoside

Abstract

As an important step to total synthesis of philinopside E with important antitumor activities (Ed₅₀ 0.75–3.50 μg mL⁻¹), we described herein convergent synthesis of a triterpene glycoside composed of the sulfated tetrasaccharide residue identical to that of philinopside E and the aglycone of lanost-7-en-3β-ol. The stereocontrolled synthesis of the aglycone from 24,25-dihydrolanosterol was accomplished relying on the stereoselective reductions of the 2,3-unsaturated-1,4-diketone system assisted by a C3-tert-butyldimethylsilyloxy group and convenient installation of the required 7(8)-double bond via syn elimination of triflate. Sequencial convergent coupling of monoglycoside, prepared by reacting the aglycone with orthogonally protected xylosyl thioglycoside, with trisaccharide thioglycoside originated from glucose, xylose and quinovose derivatives, incorporation of sulfation and deprotection afforded the target molecule. The features of our work are that the four 1,2-trans glycosidic bonds were stereoselectively constructed and the precious aglycone was introduced in the late-stage synthesis, which would facilitate the total synthesis of philinopside E and related natural products.

---

Introduction

Triterpene glycosides are a large family of secondary metabolites in plants and marine organisms that have been used in traditional medicines for thousands of years.¹ To date, more than 100 sea cucumber triterpene glycosides have been isolated and characterized.² These saponins share a common aglycone skeleton, 3β,20S-dihydroxy-5α-lanostano-18,20-lactone. Their oligosaccharide moieties, being frequently sulfated, are composed of several different monosaccharide residues, including D-xylose, D-quinovose, D-glucose, 3-O-methyl-D-glucose, and in rare cases, 3-O-methyl-D-xylose.² Most sea cucumber saponins exhibit potential therapeutic effects in the treatment of cancer, inflammation and infections.^2b,c Recently, Yi and co-workers³ isolated four new sulfated saponins, philinopside A, B, E and F, from the sea cucumber Pentacta quadrangularis, which is widely distributed throughout the South China Sea (Fig. 1). As a representative of sea cucumber triterpene glycosides, philinopside E exhibits anti-angiogenesis and antitumor activities with Ed₅₀ values ranging from 0.75–3.50 μg mL⁻¹.^3b Studies on its mode of action revealed that philinopside E interacts with the extracellular portion of the kinase insert domain-containing receptor (KDR) to block its interaction with vascular endothelial growth factor (VEGF) and the resultant downstream signaling. This mechanism is distinct from that of conventional small-molecule inhibitors which exclusively target the cytoplasmic kinase domain of KDR.⁴ In addition, philinopside E markedly suppresses α_νβ₃ integrin-driven downstream signaling.⁴ However, further studies were hampered by the limited availability of this natural product. Chemical synthesis has proved to be a powerful tool to make structurally complex saponins of importance in sufficient amounts,⁵ however, there is no report on the total synthesis of sea cucumber triterpene glycosides such as philinopside E.⁶

Fig. 1 Structure of philinopside A, B, E, F and target triterpene glycoside 1.

Inspection of philinopside E suggested that a few key problems including construction of sulfated oligosaccharide residue and synthesis of highly multi-functionalized aglycone that have to be solved before a successful total synthesis could be performed. Thus, as the first attempt to gain knowledge about the synthesis of philinopside E, we undertook the synthesis of an analogue of philinopside E, the lanostane-type saponin 1 consisting of the sulfated tetrasaccharide found in philinopside E and the aglycone of lanost-7-en-3β-ol (Fig. 1). We hoped that the developed protecting-group and assembly strategies for stereocontrolled formation of glycosidic bonds and the extension of sugar chain, coupled with the strategies for synthesis of lanost-7-en-3β-ol, would pave a practical way to the total synthesis of philinopside E.

Results and discussion

As shown in Fig. 1, target molecule 1 has four 1,2-trans glycosidic bonds and is sulfated at the 4-OH position of the xylose unit. Normally, the formation of 1,2-trans glycosidic linkages is facilitated by the participation of neighboring ester protecting groups such as acetate, benzoate, and pivalate,⁷ and sulfate groups are introduced in the penultimate step of the synthesis.⁸ According to these guidelines, we proposed our plan for the synthesis of saponin 1. As outlined in Scheme 1, we envisioned that 1 could be obtained by removal of 2-naphthyl methyl (Nap) in 2, subsequent sulfation of the resulting hydroxyl and global hydrolysis of all the benzoates. The intermediate 2 could be convergently constructed by coupling lanost-7-en-3-yl xyloside 3 with the trisaccharide donor 4. Monoglycoside 3, in turn, could be synthesized from lanost-7-en-3β-ol 5 as the aglycone and the orthogonally protected xylosyl donor 6. Trisaccharide 4 could be prepared by joining three monosaccharide building blocks 7, 8 and 9. Benzoate and 2-azidomethyl benzoate are chosen as the protecting groups for the C-2 hydroxyl groups of 6–9 to ensure the highly stereoselective formation of 1,2-trans glycosidic linkages. Since the extraction of the aglycone 5 from natural sources such as Dracaena cinnabari^9a and Azorella trifurcata^9b is difficult and there are few methods¹⁰ available to its synthesis, we therefore would prepare the aglycone 5 from 24,25-dihydrolanosterol by designing a new stereoselective route.

Scheme 1 Retrosynthetic analysis of target triterpene glycoside 1.

The synthesis of saponin 1 commenced with the synthesis of 5 en route to monoglycoside 3 (Scheme 2). Thus, tert-butyldimethylsilyl (TBS) protection of the 3-OH of 24,25-dihydrolanosterol 10 (ref. 11) under the forcing conditions, followed by allylic oxidation with KMnO₄ in the presence of 18-crown-6 (ref. 12) afforded the unsaturated ketone 11 in 67% yield over two steps. After that, conjugate reduction of 11 with zinc dust in refluxing acetic acid¹¹ gave diketone 12 in an excellent 96% yield. Simultaneous reduction of two carbonyl groups in 12 by LiAlH₄ led to diol 13 as the single product in 85% yield. Notably, bulky TBS is crucial for the highly stereoselective reduction of diketone 12 because the congeners of 12 with tetrahydropyranyl and acetyl groups as the protecting group of 3-OH led to a diastereomeric mixture by reducing the carbonyl groups.¹³ By treating 13 with acetic anhydride in pyridine with N,N-dimethyl pyridine (DMAP) as a catalyst acetate 14 was obtained with a 91% yield. The absolute stereochemistry of 14 was unambiguously confirmed by X-ray crystallographic analysis (Scheme 2).¹⁴

Scheme 2 Preparation of the aglycone 5. Reagents and conditions: (a) (i) TBSCl, imidazole, N,N-dimethylformamide, 45 °C, 5 h; (ii) KMnO₄, 18-crown-6, CH₂Cl₂/H₂O, 18 °C, 24 h, 67% for two steps; (b) zinc dust, AcOH, 120 °C, 2 h, 96%; (c) LiAlH₄, THF, 13 °C, 3 h, 85%; (d) Ac₂O, pyridine, DMAP, 17 °C, overnight, 91%; (e) MsCl, Et₃N, CH₂Cl₂, 15 °C, 89%; (f) HF·pyridine, THF, 40 °C, 88%; (g) H₂ (130 atm), Pd/C, AcOH, MeOH, 120 °C, 72 h, 95%; (h) (i) BzCl, pyridine, DMAP, 18 °C, 6 h; (ii) AcCl, CHCl₃/MeOH, 40 °C, 24 h, 78% for two steps; (i) MsCl, Et₃N, CH₂Cl₂, 13 °C, 5 h, 87%; (j) NaOAc, AcOH, 120 °C, 0.5 h, 87% (20/21 = 1/12.5); (k) Tf₂O, pyridine, CH₂Cl₂, −10 °C, 5 h, 96% (20/21 = 1/20); (l) LiAlH₄, THF, 21 °C, 4 h, 89%.

On the basis of the crystal structure, the synthesis of 14 deserves some comments. The ORTEP view of the crystal illustrates that two angular methyl groups at C-10 and C-13 are both β-oriented, which can cause steric hindrance to 11β-OH on the same face of the rings. This explains why the acetylation of diol 13 preferentially occurred to the 7β-OH. It should be also pointed out that in ¹H NMR spectrum of 14 the axial 7α-H resonates at 4.91 ppm as a doublet of triplets, while the equatorial 11α-H appears at 4.22 ppm as a broad singlet. These splitting patterns can be predicted by Karplus equation¹⁵ on the basis of stereochemical assignment of 14. The same trends were observed for 7α-H and 11α-H in 13.

With the access to 14 secured, we initially sought to remove the equatorial 11β-OH through a Barton–McCombie radical deoxygenation reaction¹⁶ which involves conversion of the alcohol to a xanthate, followed by reduction with tri-n-butyltin hydride in refluxing toluene. However, subjecting the alcohol 14 to the standard conditions did not afford the xanthate required for the deoxygenation step. Inspired by the work of Deslongchamps et al.,¹⁷ we then tended to use the LiAlH₄ reduction of mesylate to remove the 11β-OH. Unexpectedly, treatment of alcohol 14 with mesyl chloride and triethyl amine resulted in the formation of alkene 15 (Scheme 2), which is probably caused by the elimination of the mesylate generated in situ. The ready availability of alkene 15 led to an alternative strategy for removing the 11β-OH, namely, hydrogenation of the 9(11)-double bond in 15. After many attempts, we found that the presence of the bulky TBS group at the C-3 position significantly hampered the hydrogenation reaction. This issue can be simply solved by removing the TBS group. After deprotection, alcohol 16 could undergo hydrogenation under 130 atmospheric pressures at 120 °C to provide 17 in an excellent 95% yield.

With alcohol 17 in hand, we turned our attention to the synthesis of 5 by a syn elimination (Scheme 2). Thus, benzoylation of the 3-OH followed by a facile chemoselective deacetylation using methanolic hydrogen chloride¹⁸ afforded alcohol 18. Inspired by the synthetic work of Purdy and co-workers,¹⁹ who utilized syn elimination of mesylate to form the desired double bond, mesylate 19, prepared by mesylation of 18, was treated with sodium acetate in refluxing acetic acid to furnish a mixture of olefins 20 and 21 in 87% yield with a ratio of 1/12.5. The ratio was determined by ¹H NMR spectroscopy based on comparing integration values for the H-7 of 20 (5.53 ppm) and 21 (5.22 ppm). It is well established that triflate is a better leaving group than mesylate,²⁰ and triflates have been widely used in formation of double bonds in steroids.²¹ Consequencely, in order to improve the regioselectivity of the elimination reaction, 18 was converted into a triflate using triflic anhydride and pyridine in CH₂Cl₂, which underwent rapid elimination to form 21 with an exclusive selectivity (20/21 = 1/20). This result implies that an E1 mechanism might be responsible for the elimination of triflate. Finally, reductive deprotection of benzoate with LiAlH₄ supplied 5 in 89% yield, ¹H data of which are identical to the reported.²² Thus the transformation of 24,25-dihydrolanosterol into 5 was achieved in 12 steps with an overall 24% yield.

With the aglycone 5 in hand, it is necessary to synthesize pyranoxylosyl donor 6 to make monoglycoside 3. Briefly, Nap protection of the 4-OH in thioglycoside 22 (ref. 23) followed by removal of isopropylidene with methanol in the presence of camphorsulfonic acid (CSA) furnished diol 23 with a 96% yield over two steps. Dibutylstannylene-mediated regioselective benzoylation²⁴ of 23 afforded benzoate 24 in a satisfactory 60% yield. Condensation of alcohol 24 with 2-azidomethyl benzoic acid (AzmbOH) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDCI) gave the xylosyl thioglycoside 6. The glycosylation of 5 with 6 promoted by N-iodosuccinimide (NIS) and trimethylsilyl triflate (TMSOTf) proceeded smoothly to furnish 25 in 91% yield. Notably, this procedure left the 7(8)-double bond on 5 intact. The newly formed 1,2-trans glycosidic linkage on 25 is confirmed by the coupling constant of ³J_{H1′–H2′} = 7.0 Hz. In addition to allowing for the selective formation of 1,2-trans glycosidic bonds through neighboring group participation, Azmb is often used as a temporary protecting group in carbohydrate synthesis because it can be selectively removed in the presence of other acyl groups including benzoyl group (Bz).²⁵ As expected, the Azmb group on 25 was selectively cleaved in the presence of a Bz group by first reducing the azide with Bu₃P and then intramolecular aminolysis of benzoate²⁵ to give alcohol 3 in 92% yield (Scheme 3).

Scheme 3 Synthesis of monoglycoside 3. Reagents and conditions: (a) (i) NapBr, NaH, N,N-dimethylformamide, 27 °C, 6 h; (ii) CSA, CH₂Cl₂/MeOH, 28 °C, 5 h, 96% for two steps; (b) (i) Bu₂SnO, toluene, 120 °C, 2 h; (ii) BzCl, 0 °C, 5 h, 60%; (c) AzmbOH, EDCI, DMAP, CH₂Cl₂, 13 °C, 11 h, 91%; (d) 5, NIS, TMSOTf, CH₂Cl₂, 4 Å molecular sieves, 0 °C, 2 h, 91%; (e) Bu₃P, H₂O, THF, 50 °C, 1 h, 92%.

With the synthesis of 3 fully realized, the next significant step as outlined in Scheme 1 for convergent synthesis of 1 is the extension of the sugar chain on 3 by trisaccharide donor 4. It was initially expected that thioglycoside 4 could be assembled by a reaction sequence involving coupling of xylosyl acceptor 7a with the trichloroacetimidate donor 8, transformation of the resultant disaccharide into the corresponding imidate and subsequent glycosylation with thioglycoside 9. For this purpose, 7a was synthesized by first selective acetylation²⁶ of the 3-OH of thioglycoside 26,²³ benzoylation of both 2-OH and 4-OH, and final removal of the acetyl group with methanolic hydrogen chloride.¹⁸ Glucosyl trichloroacetimidate 8 was then prepared from hemiacetal 28.²⁷ After that, TMSOTf-promoted glycosylation of 7a with 8 was performed. Disappointingly, the reaction did not produce any of the desired disaccharide and only gave glucosyl thioglycoside 29 in 56% yield (Scheme 4 eqn (1)). From this observation, we assumed that the vicinal electron-withdrawing benzoates render the 3-OH of 7a significantly less nucleophilic than the anomeric thioether of the same molecule. Thus aglycone transfer²⁸ of thioglycoside 7a preferentially occurred in the reaction.

Scheme 4 Preparation of disaccharide 34. Reagents and conditions: (a) (i) Ac₂O, TBAOAc, CH₃CN, 40 °C, 6 h; (ii) BzCl, pyridine, DMAP, 25 °C, 4 h, 59% for 27, 60% for 31; (b) AcCl, CH₂Cl₂/MeOH, 26 °C, 24 h, 95% for 7a, 93% for 7b; (c) Cl₃CCN, DBU, CH₂Cl₂, 25 °C, 8 h, 95%; (d) TMSOTf (0.1 equiv.), 4 Å molecular sieves, CH₂Cl₂, 0 °C, 1 h, 56%; (e) TMSOTf (0.1 equiv.), 4 Å molecular sieves, CH₂Cl₂, 0 °C, 1 h 35% for 32; (f) TBSOTf (0.3 equiv.), 4 Å molecular sieves, CH₂Cl₂, 0 °C, 1 h, 39% for 33β, 23% for 33α; (g) TMSOTf, 4 Å molecular sieves, CH₂Cl₂, 0 °C, 1 h, 80%.

To circumvent this unexpected aglycone migration, p-methoxyphenyl (PMP) group, being less nucleophilic than phenylthio group, was introduced as an anomeric protecting group of xylosyl acceptor. In an analogous route to that of 7a, xylosyl acceptor 7b was obtained from 30.²⁹ Unfortunately, upon treatment of 7b with 8 in the presence of TMSOTf (0.1 equiv.), orthoester 32 was obtained in 35% yield (Scheme 4 eqn (2)). By switching the activation agent from TMSOTf to tert-butyldimethylsilyl triflate (TBSOTf) (0.3 equiv.), the reaction did produce the desired β-linked disaccharide 33β in 39% yield, but along with α-isomer 33α in 23% yield. Although it is often disfavored by the presence of a neighboring participatory substituent at the C-2 position of the glycosyl donor, the formation of 1,2-cis glycosidic linkages is not unknown in similar reactions.^7,30 The formation of undesired 33α was ascribed to the decreased reactivity of 3-OH due to intramolecular hydrogen-bond between the anomeric PMP with 3-OH on 7b. ¹H NMR data of 7b demonstrates that 7b partially adopts a ¹C₄ chair conformation, thus offering opportunities for intramolecular hydrogen-bond formation. ¹H NMR studies³¹ and theoretical calculations³² on molecules similar to 7b lend support to those inferences.

Not satisfied with the relatively poor stereochemical outcome of the reaction of 7b with 8, we sought another approach to the desired disaccharide. Previous work by other groups³³ illustrates that benzyl α-xylosides with 3-OH free are competent glycosyl acceptors in a wide variety of glycosylation events. On the basis of these results, TMSOTf-promoted coupling of benzyl α-xyloside 7c (ref. 24b) with trichloroacetimidate 8 was conducted. Happily, this reaction afforded the desirable disaccharide 34 in 80% yield (Scheme 4 eqn (3)). Conversion of benzyl glycoside 34 into disaccharide trichloroacetimidate 35 was achieved by removing the benzyl group of 34 with hydrogenolysis and subsequently treating the resultant alcohol with trichloroacetonitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (Scheme 5). Gratifyingly, coupling of 35 and quinovosyl thioglycoside acceptor 9 proceeded smoothly providing the expected trisaccharide thioglycoside 4 in 70% yield. Thioglycoside 9, in turn, could be readily prepared by selective iodination of primary hydroxyl of 36 (ref. 34) using Ph₃P/I₂/imidazole and the following deiodination of the resulting 37 by hydrogenolysis.

Scheme 5 Synthesis of trisaccharide 4. Reagents and conditions: (a) PPh₃, imidazole, I₂, THF, 29 °C, 1 h; (b) H₂, 10% Pd–C, NaHCO₃, N,N-dimethylformamide, 40 °C, 24 h, 83%; (c) (i) H₂, 10% Pd/C, EtOH, 50 °C, 24 h; (ii) Cl₃CCN, DBU, CH₂Cl₂, 5 h, 74% for two steps; (d) TMSOTf, 4 Å molecular sieves, CH₂Cl₂, 0 °C, 1 h, 70%.

With key two building blocks 3 and 4 available, the synthesis of our target molecule 1 was in sight. As shown in Scheme 6, NIS-promoted reaction of 3 and 4 in the presence of TMSOTf gave rise to the tetrasaccharide saponin 2 in a satisfactory 67% yield. Oxidative removal of the Nap group was then effected by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) in MeOH/CH₂Cl₂ to give alcohol 38 in 88% yield. Using pyridine·SO₃ as the sulfating agent, sulfated product 39 was then obtained in an excellent 94% yield by microwave-assisted sulfation³⁵ of 38 in pyridine at 100 °C. After treatment of 39 with MeONa in methanol to globally deprotect benzoyl groups and subsequent dialysis against water, target molecule 1 was finally obtained. ¹H and ¹³C spectroscopic data due to the tetrasaccharide moiety of 1 match those of philinopside E.

Scheme 6 Synthesis of saponin 1. Reagents and conditions: (a) NIS, TMSOTf, CH₂Cl₂, 4 Å molecular sieves, 2 h, 0 °C, 67%; (b) DDQ, CH₂Cl₂/MeOH, 20 °C, 5 h, 88%; (c) pyridine·SO₃, pyridine, microwave, 100 W, 100 °C, 2 h, 94%; (d) NaOMe, MeOH, 25 °C, 24 h, 86%.

Conclusions

In conclusion, a tritepene saponin, comprised of the same tetrasaccharide residue as that of philinopside E and the aglycone of lanost-7-en-3β-ol, has been accomplished by a convergent coupling of trisaccharide thioglycoside donor with monoglycoside acceptor. Lanost-7-en-3β-ol was efficiently prepared from commercially available 24,25-dihydrolanosterol in a stereocontrolled manner involving stereoselective reduction of unsaturated 1,4-diketone system and facile installation of 7(8)-double bond by syn elimination of triflate. The successful access to trisaccharide thioglycoside demonstrated that a benzyl α-xyloside was a critical glycosyl acceptor for the extension of sugar chain at its 3-OH. Since the sugar fragment of philinopside E is found in more than 40 sea cucumber triterpene glycosides,^2,3 the protecting-group and assembly tactics in this work, used to stereoselectively construct glycosidic linkages and to extend sugar chain, sets the foundation for the synthesis, structural modification and biological evaluation of philinopside E and its congeners.

Experimental

General information

All nonaqueous reactions were carried out under an atmosphere of argon in flame- or oven-dried glassware with magnetic stirring unless otherwise indicated. Dichloromethane for glycosylation reactions was distilled from calcium hydride. All other commercially obtained reagents were used as received, except where specified otherwise. Flash column chromatography was performed on Silica Gel H (300–400 mesh, Qingdao, China). Analytical thin layer chromatography was performed on Silicycle SiliaPlate glass-backed plates coated with silica gel (60 Å pore size, F-254 indicator) and visualized by exposure to ultraviolet light and/or staining with aqueous 8% sulfuric acid in methanol. Optical rotations were determined with a digital polarimeter. High-resolution mass spectral (HRMS) data were determined with a LTQ Orbitrap. ¹H and ¹³C NMR spectra were recorded on a 500 or 600 MHz NMR spectrometer with Me₄Si as the internal standard. Chemical shifts are recorded in δ values and J values were given in Hz.

3β-(tert-Butyldimethylsilyloxy)-lanost-8-en-7,11-dione (11)

To a solution of 10 (3.70 g, 8.63 mmol) in N,N-dimethylform-amide (12 mL) were added TBSCl (2.60 mL, 17.26 mmol) and imidazole (2.07 g, 34.52 mmol). After the reaction mixture was warmed at 45 °C, and stirred for 5 h, the volatile was evaporated in vacuo. The residue was taken up in CH₂Cl₂ and washed with 1 M HCl, saturated aqueous NaHCO₃ and brine. The collected organic phase was dried over Na₂SO₄, filtered, and concentrated under the reduced pressure. The resultant crude product was used next step. A 100 mL round bottom flask open to atmosphere was charged with KMnO₄ (1.16 g, 7.37 mmol) and 18-crown-6 (1.95 g, 7.37 mmol), followed by the addition of 30 mL of water. A solution of the obtained above product in 30 mL of CH₂Cl₂ was then added to the resulting suspension by a quick syringe transfer. The reaction mixture was vigorously stirred under argon at rt for 24 h, then the solid was filtered off with a Buche funnel and washed with CH₂Cl₂. The filtrate was separated, and the aqueous layer was extracted with CH₂Cl₂. The combined organic layers were dried over Na₂SO₄, filtered and concentrated. The residues were purified by silica gel column chromatography (petroleum ether/CH₂Cl₂ = 3/1) to give 11 (478 mg, 0.84 mmol, 67% for two steps). [α]²⁴_D = +56.4 (c 1.10, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 3.23 (dd, J = 11.4, 4.3 Hz, 1H), 2.88–2.78 (m, 1H), 2.73 (d, J = 16.0 Hz, 1H), 2.59 (d, J = 16.0 Hz, 1H), 2.56–2.40 (m, 2H), 2.21–2.07 (m, 1H), 2.03–1.90 (m, 1H), 1.29 (s, 3H), 1.16 (s, 3H), 0.93 (s, 3H), 0.91–0.84 (m, 18H), 0.84 (s, 3H), 0.80 (s, 3H), 0.03 (s, 3H), 0.02 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 202.70, 202.65, 152.2, 150.7, 78.4, 51.9, 50.4, 49.3, 49.1, 47.5, 39.9, 39.6, 39.5, 36.9, 36.4, 36.3, 34.2, 32.4, 28.4, 28.2, 28.1, 27.5, 26.02, 26.00, 24.1, 23.0, 22.7, 18.7, 18.2, 17.8, 17.0, 16.1, −3.6, −4.8; HRMS (ESI): m/z calcd for C₃₆H₆₃O₃Si [M + H]⁺ 571.4541, found 571.4538.

3β-(tert-Butyldimethylsilyloxy)-lanost-7,11-dione (12)

To the reflux solution of 11 (478 mg, 0.84 mmol) in a glacial acetic acid (30 mL), zinc dust (2.10 g) was added portionwise during 1 h and the stirring was continued for another 1 h. At this stage the solid was filtered off and washed with CH₂Cl₂. The filtrate was concentrated in vacuo. The residues were purified by silica gel column chromatography (petroleum ether/CH₂Cl₂ = 3/1) to give 12 (463 mg, 0.81 mmol, 96%). [α]²⁴_D = +33.4 (c 1.05, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 3.19 (dd, J = 11.5, 4.3 Hz, 1H), 2.85–2.74 (m, 1H), 2.63 (d, J = 13.1 Hz, 1H), 2.55 (d, J = 13.6 Hz, 1H), 2.42–2.28 (m, 3H), 2.24–2.10 (m, 2H), 2.09–1.97 (m, 1H), 1.26 (s, 3H), 1.19 (s, 3H), 0.92–0.81 (m, 21H), 0.79 (s, 3H), 0.70 (s, 3H), 0.03 (s, 3H), 0.02 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 209.8, 209.7, 79.0, 60.8, 53.2, 52.69, 52.65, 49.1, 48.8, 46.6, 40.1, 39.7, 39.6, 36.9, 36.4, 36.1, 36.0, 33.2, 28.8, 28.3, 28.1, 27.8, 26.0, 24.1, 23.0, 22.7, 18.6, 18.2, 17.7, 16.3, 15.5, 13.9, −3.7, −4.8; HRMS (ESI): m/z calcd for C₃₆H₆₅O₃Si [M + H]⁺ 573.4697, found 573.4691.

3β-(tert-Butyldimethylsilyloxy)-lanost-7β,11β-diol (13)

To a solution of 12 (700 mg, 1.22 mmol) in anhydrous THF (25 mL) at rt was added LiAlH₄ (465 mg, 12.20 mmol). After stirring for 3 h, the reaction mixture was cooled in an ice bath and quenched by the addition of 5% HCl. The resulting solution was extracted with EtOAc. The combined organic phase was washed with brine, dried with Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 25/1) to afford compound 13 (599 mg, 1.04 mmol, 85%). [α]²⁴_D = +47.4 (c 0.70, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.20 (s, 1H), 3.72–3.55 (m, 1H), 3.19 (dd, J = 11.4, 3.6 Hz, 1H), 1.19 (s, 3H), 1.01 (s, 3H), 0.92–0.83 (m, 24H), 0.81 (s, 3H), 0.04 (s, 3H), 0.03 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 79.6, 72.8, 68.7, 52.4, 51.6, 50.4, 48.2, 45.3, 44.6, 42.7, 39.6, 39.5, 37.44, 37.40, 36.5, 36.2, 32.1, 29.0, 28.5, 28.2, 27.9, 26.1, 24.2, 23.0, 22.7, 19.0, 18.3, 18.1, 17.3, 17.1, 16.3, −3.6, −4.8; HRMS (ESI): m/z calcd for C₃₆H₆₈O₃SiNa [M + Na]⁺ 599.4830, found 599.4817.

7β-Acetoxy-3β-(tert-butyldimethylsilyloxy)-lanost-11-ol (14)

Diol 13 (264 mg, 0.458 mmol) was dissolved in pyridine (20 mL), then DMAP (6 mg, 0.05 mmol) and Ac₂O (130 μL, 1.37 mmol) was sequentially added at 0 °C. The reaction was allowed to stir overnight under argon atmosphere. The reaction was quenched with water and extracted with EtOAc (2 × 40 mL). The combined organic phases were washed thrice with hydrochloric acid (1 M) and brine. The collected organic phase was dried over Na₂SO₄, filtered and concentrated under reduced pressure, which was purified by silica gel column chromatography (petroleum ether/EtOAc = 50/1) to give 14 (258 mg, 0.42 mmol, 91%). [α]²⁴_D = +46.4 (c 0.85, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.91 (td, J = 10.5, 5.8 Hz, 1H), 4.22 (s, 1H), 3.19 (dd, J = 11.4, 4.1 Hz, 1H), 2.25 (dd, J = 12.4, 10.2 Hz, 1H), 2.01 (s, 3H), 1.21 (s, 3H), 1.01 (s, 3H), 0.92–0.82 (m, 21H), 0.81 (s, 3H), 0.78 (s, 3H), 0.04 (s, 3H), 0.03 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 170.7, 79.6, 74.3, 68.6, 52.0, 51.5, 50.6, 48.1, 45.1, 42.7, 40.9, 39.61, 39.57, 37.3, 37.2, 36.7, 36.2, 35.2, 29.0, 28.4, 28.3, 28.1, 27.7, 26.1, 24.2, 23.0, 22.7, 22.1, 19.0, 18.3, 17.8, 17.3, 17.0, 16.3, −3.7, −4.8; HRMS (ESI): m/z calcd for C₃₈H₇₀O₄SiNa [M + Na]⁺ 641.4936, found 641.4932.

7β-Acetoxy-3β-(tert-butyldimethylsilyloxy)-lanost-9-ene (15)

To a mixture of 14 (80 mg, 0.13 mmol) and Et₃N (36 μL, 0.26 mmol) in CH₂Cl₂ (2 mL) was added methanesulfonyl chloride (MsCl, 20 μL, 0.26 mmol) at 0 °C. After the reaction had been completed, the mixture was diluted with CH₂Cl₂ and washed with saturated aqueous NH₄Cl and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residues were purified by silica gel column chromatography (petroleum ether/EtOAc = 20/1) to give 15 (80 mg, 0.11 mmol, 89%). [α]²⁴_D = +34.6 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.33 (d, J = 6.2 Hz, 1H), 4.92 (td, J = 11.0, 5.2 Hz, 1H), 3.18 (dd, J = 10.8, 4.9 Hz, 1H), 2.43 (d, J = 11.0 Hz, 1H), 2.04 (s, 3H), 1.08 (s, 3H), 0.90–0.84 (m, 21H), 0.77 (s, 3H), 0.76 (s, 3H), 0.67 (s, 3H), 0.04 (s, 3H), 0.03 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 170.5, 145.8, 118.0, 79.3, 77.2, 74.5, 50.4, 48.2, 46.7, 46.4, 45.0, 39.6, 39.0, 36.9, 36.6, 36.3, 36.2, 35.8, 28.61, 28.57, 28.3, 28.2, 27.9, 26.1, 24.2, 23.0, 22.7, 22.1, 22.0, 18.6, 18.5, 18.3, 16.1, 14.5, −3.7, −4.8.

7β-Acetoxylanost-9-ene (16)

To a solution of compound 15 (157 mg, 0.26 mmol) in THF (3.0 mL) in a TEFLON tube was added a solution of 70% HF·pyridine (0.88 mL). The reaction mixture was stirred for 6 h at 40 °C, and quenched with saturated NaHCO₃ solution. The resultant mixture was extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaHCO₃, dried over Na₂SO₄, filtered and concentrated. The crude product was purified by silica gel column chromatography (petroleum ether/EtOAc = 5/1) to give 16 (112 mg, 0.23 mmol, 88%). [α]²⁴_D = +48.8 (c 0.55, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.34 (d, J = 6.1 Hz, 1H), 4.92 (td, J = 11.0, 5.2 Hz, 1H), 3.31–3.14 (m, 1H), 2.43 (d, J = 10.6 Hz, 1H), 2.04 (s, 3H), 1.08 (s, 3H), 0.99 (s, 3H), 0.90–0.83 (m, 9H), 0.81 (s, 3H), 0.77 (s, 3H), 0.67 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 170.5, 145.5, 118.3, 78.8, 77.2, 74.4, 50.4, 48.3, 46.7, 46.4, 45.0, 39.6, 39.1, 36.9, 36.6, 36.3, 36.2, 35.8, 28.6, 28.20, 28.15, 27.8, 27.7, 24.2, 23.0, 22.7, 22.1, 22.0, 18.6, 18.5, 15.7, 14.5; HRMS (ESI): m/z calcd for C₃₂H₅₄O₃Na [M + Na]⁺ 509.3965, found 509.3965.

7β-Acetoxy-24,25-dihydrolanosterol (17)

To a solution of olefin 16 (65 mg, 0.13 mmol) in MeOH (12 mL) was added 10% Pd/C (100 mg) and AcOH (600 μL). The reaction vessel was evacuated and backfilled with hydrogen (130 atm). The mixture was kept at 120 °C for 72 h, then filtered through a thin plug of Celite. The filtrate was washed with EtOAc (2 × 50 mL) and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 5/1) to afford 17 (62 mg, 0.13 mmol, 95%). [α]²⁴_D = +37.7 (c 0.90, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 4.85 (td, J = 10.6, 5.3 Hz, 1H), 3.21 (dd, J = 11.4, 3.2 Hz, 1H), 2.00 (s, 3H), 1.96–1.77 (m, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.88–0.84 (m, 9H), 0.83 (s, 3H), 0.79 (s, 3H), 0.78 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 170.6, 78.9, 77.2, 74.6, 51.6, 50.1, 47.3, 46.9, 45.9, 43.0, 39.6, 38.9, 37.4, 36.7, 36.2, 35.5, 31.9, 28.5, 28.24, 28.16, 28.1, 27.7, 24.2, 23.0, 22.7, 22.1, 20.3, 18.9, 16.7, 15.6, 14.5, 13.9; HRMS (ESI): m/z calcd for C₃₂H₅₆O₃Na [M + Na]⁺ 511.4122, found 511.4123.

3β-Benzoyloxylanost-7β-ol (18)

To a solution of 17 (1.30 g, 3.03 mmol) in pyridine (20 mL) was added DMAP (37 mg, 0.30 mmol) and BzCl (528 μL, 4.55 mmol) at 0 °C. The reaction was stirred for 6 h at room temperature and quenched with water. After removal of solvent, the residue was dissolved in CH₂Cl₂ and washed with 0.1 M HCl, saturated aqueous NaHCO₃. The organic phase was dried over anhydrous Na₂SO₄, filtered and concentrated. The resulting residue was dissolved in 65 mL of CH₃OH/CHCl₃ (v/v = 1.6/1) followed by dropwise addition of acetyl chloride (4.80 mL) at 0 °C. The reaction mixture was stirred for 24 h at 40 °C, and then quenched with Et₃N. The volatile was evaporated in vacuo. The resulting residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 15/1) to afford 18 (1.04 g, 1.89 mmol, 62%) with the recovery of acetate (363 mg, 0.61 mmol). [α]²⁴_D = +35.7 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 7.7 Hz, 2H), 7.55 (t, J = 7.3 Hz, 1H), 7.44 (t, J = 7.6 Hz, 2H), 4.72 (dd, J = 11.4, 4.3 Hz, 1H), 3.64 (td, J = 10.0, 4.9 Hz, 1H), 1.05 (s, 3H), 1.00 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.79 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.4, 132.9, 131.0, 129.6, 128.4, 81.5, 77.2, 73.1, 52.1, 49.8, 47.4, 46.7, 46.4, 46.1, 39.6, 38.2, 37.2, 36.8, 36.7, 36.6, 36.2, 31.9, 31.6, 28.7, 28.3, 28.1, 24.2, 22.9, 22.7, 20.3, 18.9, 17.0, 16.8, 14.5, 14.1; HRMS (ESI): m/z calcd for C₃₇H₅₈O₃Na [M + Na]⁺ 573.4278, found 573.4283.

Lanost-7β-O-methylsulfony-3β-yl benzoate (19)

To a solution of 18 (100 mg, 0.18 mmol) in CH₂Cl₂ (2.0 mL) was added MsCl (42 μL, 0.55 mmol) and Et₃N (126 μL, 0.91 mmol) dropwise at 0 °C. The reaction mixture was stirred for 5 h at room temperature, and then quenched with MeOH. The volatile was evaporated in vacuo. The resulting residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 15/1) to afford 19 (99 mg, 0.16 mmol, 87%). [α]²⁴_D = +33.4 (c 0.55, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.03 (d, J = 7.3 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.7 Hz, 2H), 4.79 (td, J = 10.6, 5.3 Hz, 1H), 4.71 (dd, J = 11.6, 4.3 Hz, 1H), 3.01 (s, 3H), 2.41–2.26 (m, 1H), 1.05 (s, 3H), 1.02 (s, 3H), 0.95 (s, 3H), 0.94 (s, 3H), 0.90–0.84 (m, 9H), 0.79 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 133.0, 130.9, 129.7, 128.5, 83.2, 81.2, 51.9, 50.0, 47.1, 47.0, 46.1, 44.0, 40.1, 39.6, 38.2, 37.0, 36.7, 36.5, 36.2, 35.4, 31.6, 29.6, 28.5, 28.3, 28.1, 24.2, 24.1, 23.0, 22.7, 20.4, 19.0, 17.0, 16.5, 14.5, 14.0.

3β-Benzoyloxylanost-7-ene (21)

Method A. 19 (50 mg, 0.08 mmol) was dissolved in acetic acid (2.0 mL) and then added NaOAc (46 mg, 0.56 mmol). The reaction mixture was stirred for 0.5 h at 120 °C. After removal of solvent, the residue was dissolved in CH₂Cl₂ and washed with brine, dried over anhydrous Na₂SO₄, filtered and concentrated for flash column chromatography (petroleum ether/CH₂Cl₂ = 3/1) to afford a mixture of 20 and 21 (41 mg, 0.07 mmol, 87%) at the ratio of 20/21 = 1/12.5.

Method B. To a solution of 18 (1.50 g, 2.73 mmol) in CH₂Cl₂ (50 mL) was added pyridine (2.64 mL, 32.8 mmol) at −10 °C followed by Tf₂O (1.15 mL, 6.81 mmol). The reaction mixture was stirred for 5 h at −10 °C, and then quenched with H₂O. The solvent was evaporated in vacuo. The resulting residue was purified by column chromatography (petroleum ether/CH₂Cl₂ = 3/1) to afford a mixture of 20 and 21 (1.38 g, 2.62 mmol, 96%) at the ratio of 20/21 = 1/20. [α]²⁴_D = +41.3 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.05 (d, J = 7.3 Hz, 2H), 7.55 (t, J = 7.4 Hz, 1H), 7.44 (t, J = 7.7 Hz, 2H), 5.22 (d, J = 5.3 Hz, 1H), 4.77 (dd, J = 11.4, 4.3 Hz, 1H), 1.13 (s, 3H), 0.99 (s, 3H), 0.95 (s, 3H), 0.94 (s, 3H), 0.91–0.85 (m, 9H), 0.66 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 145.1, 132.7, 131.0, 129.5, 128.3, 116.4, 81.8, 52.0, 50.8, 50.4, 47.1, 44.3, 39.5, 37.9, 37.7, 36.5, 36.4, 35.5, 32.2, 32.1, 28.3, 28.0, 27.6, 24.8, 24.10, 24.08, 22.8, 22.8, 22.5, 20.1, 19.0, 16.9, 16.1, 14.2.

Lanost-7-en-3β-ol (5)

To a solution of 21 (700 mg, 1.31 mmol) in THF (15 mL) was added LiAlH₄ (250 mg, 6.57 mmol) at 0 °C. The reaction was stirred for 4 h at room temperature and quenched with 0.1 M HCl. The mixture was extracted with ethyl acetate. The organic phase was washed with 0.1 M HCl, brine, dried over anhydrous Na₂SO₄, filtered and concentrated. The residue was applied to silica gel column chromatography (petroleum ether/EtOAc = 15/1) to afford 5 (501 mg, 1.17 mmol, 89%). [α]²⁴_D = +3.8 (c 0.70, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.21 (d, J = 4.3 Hz, 1H), 3.25 (dd, J = 11.0, 4.5 Hz, 1H), 0.99 (s, 3H), 0.97 (s, 3H), 0.91–0.84 (m, 15H), 0.64 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 145.1, 116.8, 79.5, 52.1, 51.0, 50.4, 47.4, 44.5, 39.7, 38.8, 38.3, 36.7, 36.6, 35.8, 32.3, 32.2, 28.4, 28.2, 27.8, 27.6, 25.0, 24.3, 23.1, 23.0, 22.7, 20.2, 19.1, 16.2, 15.6, 14.3; HRMS (ESI): m/z calcd for C₃₀H₅₂ONa [M + Na]⁺ 451.3910, found 451.3908.

p-Tolyl 4-O-(2-naphthylmethyl)-1-thio-β-D-xylopyranoside (23)

Alcohol 22 (1.69 g, 5.71 mmol) was dissolved in N,N-dimethylformamide (50 mL) followed by addition of NaH (457 mg, 11.42 mmol) at 0 °C. After stirring for 20 min under argon atmosphere, NapBr (1.55 mL, 6.85 mmol) was added to the reaction mixture. With stirring for another 6 h, the reaction was quenched by methanol and poured into water. The mixture was extracted with CH₂Cl₂. The organic phase was collected and washed with HCl (1 M), saturated NaHCO₃, dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was dissolved in 50 mL of CH₂Cl₂/MeOH (v/v = 1/1), and then CSA (1.69 g, 5.71 mmol) was added. The reaction was stirred at room temperature for 5 h, then quenched by Et₃N and concentrated. The residue was purified by silica gel column (petroleum ether/EtOAc = 2/1) to afford 23 (2.17 g, 5.48 mmol, 96% for two steps). [α]²⁴_D = −61.8 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.87–7.72 (m, 4H), 7.52–7.35 (m, 5H), 7.11 (d, J = 7.8 Hz, 2H), 4.85 (d, J = 11.9 Hz, 1H), 4.79 (d, J = 11.9 Hz, 1H), 4.47 (d, J = 9.0 Hz, 1H), 4.09 (dd, J = 11.5, 4.8 Hz, 1H), 3.69 (t, J = 8.4 Hz, 1H), 3.56–3.45 (m, 1H), 3.35 (t, J = 8.7 Hz, 1H), 3.29 (t, J = 10.7 Hz, 1H), 2.33 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 138.6, 135.5, 133.5, 133.3, 133.2, 123.0, 128.6, 128.0, 127.8, 126.9, 126.4, 126.2, 125.8, 89.0, 76.9, 76.7, 73.3, 72.0, 67.3, 21.3; HRMS (ESI): m/z calcd for C₂₃H₂₈O₄NS [M + NH₄]⁺ 414.1734, found 414.1727.

p-Tolyl 3-O-benzoyl-4-O-(2-naphthylmethyl)-1-thio-β-D-xylopyranoside (24)

Diol 23 (1.88 g, 4.74 mmol) and dibutyltin oxide (1.30 g, 5.22 mmol) was dissolved in anhydrous toluene (45 mL). The resulting mixture was refluxed for 2 h with a Dean–Stark trap to remove the formed water during the reaction. At this stage the reaction was cooled to room temperature followed by addition of benzoyl chloride (605 μL, 5.22 mmol) dropwise. After the mixture was stirred for another 5 h, the volatile was removed in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 8/1) to afford 24 (1.42 g, 2.84 mmol, 60%). [α]²⁴_D = −90.9 (c 0.75, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.11 (d, J = 7.5 Hz, 2H), 7.84–7.76 (m, 1H), 7.76–7.67 (m, 3H), 7.58 (t, J = 7.4 Hz, 1H), 7.52–7.33 (m, 7H), 7.13 (d, J = 8.0 Hz, 2H), 5.40 (t, J = 6.2 Hz, 1H), 4.99 (d, J = 5.5 Hz, 1H), 4.84 (d, J = 12.1 Hz, 1H), 4.80 (d, J = 12.1 Hz, 1H), 4.40 (dd, J = 12.2, 3.3 Hz, 1H), 3.77 (t, J = 5.8 Hz, 1H), 3.75–3.70 (m, 1H), 3.65 (dd, J = 12.2, 6.2 Hz, 1H), 2.34 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.0, 138.3, 134.7, 133.5, 133.3, 133.2, 133.0, 130.2, 130.0, 129.6, 128.59, 128.56, 128.0, 127.8, 127.0, 126.4, 126.2, 125.8, 89.5, 73.7, 72.5, 70.4, 63.5, 21.3; HRMS (ESI): m/z calcd for C₃₀H₂₈O₅SNa [M + Na]⁺ 523.1550, found 523.1554.

p-Tolyl 2-O-[2-(azidomethyl)benzoyl]-3-O-benzoyl-4-O-(2-naphthyl-methyl)-1-thio-β-D-xylopyranoside (6)

To a solution of 24 (200 mg, 0.40 mmol) in dry CH₂Cl₂ (5 mL) were added AzmbOH (106 mg, 0.60 mmol), EDCI (153 mg, 0.80 mmol), and DMAP (97 mg, 0.80 mmol) under argon atmosphere. The mixture was stirred for 12 h, and diluted with CH₂Cl₂ followed by washing with 1 M HCl, saturated aqueous NaHCO₃, and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 7/1) to give 6 (239 mg, 0.36 mmol, 91%). [α]²⁴_D = +33.4 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.98 (dd, J = 8.2, 1.0 Hz, 2H), 7.92 (dd, J = 7.8, 1.0 Hz, 1H), 7.79–7.71 (m, 1H), 7.69–7.59 (m, 3H), 7.59–7.53 (m, 1H), 7.51–7.33 (m, 8H), 7.30 (dd, J = 8.4, 1.5 Hz, 1H), 7.28–7.22 (m, 1H), 7.11 (d, J = 8.0 Hz, 2H), 5.62 (t, J = 8.1 Hz, 1H), 5.27 (t, J = 8.3 Hz, 1H), 4.95 (d, J = 8.4 Hz, 1H), 4.78 (d, J = 12.2 Hz, 1H), 4.72 (d, J = 12.2 Hz, 1H), 4.64 (d, J = 14.8 Hz, 1H), 4.54 (d, J = 14.8 Hz, 1H), 4.32 (dd, J = 11.9, 4.7 Hz, 1H), 3.90–3.76 (m, 1H), 3.58 (dd, J = 11.9, 8.9 Hz, 1H), 2.33 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 165.7, 165.3, 138.6, 137.6, 135.0, 133.5, 133.4, 133.21, 133.15, 133.0, 131.3, 130.1, 130.0, 129.41, 129.35, 128.7, 128.6, 128.4, 128.20, 128.15, 128.0, 127.8, 126.9, 126.3, 126.2, 125.8, 86.9, 74.4, 74.1, 73.0, 70.7, 66.6, 52.8, 21.3; HRMS (ESI): m/z calcd for C₃₈H₃₃O₆N₃SNa [M + Na]⁺ 682.1988, found 682.2000.

Lanost-7-en-3β-yl 2-O-[2-(azidomethyl)benzoyl]-3-O-benzoyl-4-O-(2-naphthylmethyl)-β-D-xylopyranoside (25)

A solution of alcohol 5 (24 mg, 0.057 mmol) and thioglycoside 6 (50 mg, 0.074 mmol) in dry CH₂Cl₂ (2 mL) was stirred vigorously in the presence of activated 4 Å molecular sieves (200 mg) for 20 min. The mixture was cooled to 0 °C, then NIS (26 mg, 0.114 mmol) and TMSOTf (1 μL, 0.0057 mmol) were added. After stirring for 2 h, the reaction mixture was quenched with Et₃N, and the solid was filtered off. The filtrate was washed with saturated aqueous Na₂S₂O₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 17/1) to give 25 (51 mg, 0.052 mmol, 91%). [α]²⁴_D = +45.6 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.94 (d, J = 7.4 Hz, 3H), 7.77–7.71 (m, 1H), 7.70–7.59 (m, 3H), 7.53 (t, J = 7.4 Hz, 1H), 7.50–7.39 (m, 4H), 7.36 (t, J = 7.8 Hz, 2H), 7.32–7.27 (m, 2H), 5.62 (t, J = 8.8 Hz, 1H), 5.30 (dd, J = 9.0, 7.2 Hz, 1H), 5.13 (d, J = 5.3 Hz, 1H), 4.77 (d, J = 12.3 Hz, 1H), 4.74–4.68 (m, 2H), 4.62 (s, 2H), 4.14 (dd, J = 11.9, 4.9 Hz, 1H), 3.90–3.80 (m, 1H), 3.50 (dd, J = 11.8, 9.5 Hz, 1H), 3.12 (dd, J = 11.7, 3.5 Hz, 1H), 0.94 (s, 3H), 0.90–0.84 (m, 9H), 0.83 (s, 3H), 0.76 (s, 3H), 0.72 (s, 3H), 0.61 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 165.9, 165.1, 145.0, 137.9, 135.1, 133.4, 133.23, 133.17, 133.0, 131.3, 130.0, 129.6, 129.2, 128.50, 128.45, 128.0, 127.9, 127.8, 127.0, 126.3, 126.2, 125.9, 116.7, 103.1, 90.2, 75.0, 73.7, 73.1, 72.2, 63.4, 52.9, 52.1, 51.0, 50.6, 47.3, 44.5, 39.7, 38.8, 38.2, 36.7, 36.6, 35.4, 32.3, 32.2, 28.15, 28.13, 27.7, 26.2, 24.9, 24.3, 23.0, 22.9, 22.7, 20.2, 19.1, 16.4, 16.2, 14.2; HRMS (ESI): m/z calcd for C₆₁H₇₇O₇N₃Na [M + Na]⁺ 986.5659, found 986.5671.

Lanost-7-en-3β-yl 3-O-benzoyl-4-O-(2-naphthylmethyl)-β-D-xylopyranoside (3)

To a solution of 25 (400 mg, 0.41 mmol, 1 equiv.) in THF (10 mL) were added Bu₃P (815 μL, 3.26 mmol, 8 equiv.) and water (0.40 mL) under argon atmosphere. The reaction mixture was stirred for 1 h at 50 °C, when TLC monitoring indicated that the reactant was completely consumed. The reaction mixture was diluted with EtOAc, washed with saturated aqueous NaHCO₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/CH₂Cl₂/EtOAc = 20/5/1) to give 3 (300 mg, 0.38 mmol, 92%). [α]²⁴_D = −29.1 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 7.4 Hz, 2H), 7.82–7.75 (m, 1H), 7.75–7.66 (m, 3H), 7.57 (t, J = 7.4 Hz, 1H), 7.50–7.34 (m, 5H), 5.37 (t, J = 7.0 Hz, 1H), 5.18 (d, J = 2.6 Hz, 1H), 4.81 (d, J = 12.2 Hz, 1H), 4.78 (d, J = 12.2 Hz, 1H), 4.60 (d, J = 5.2 Hz, 1H), 4.14 (dd, J = 12.1, 3.8 Hz, 1H), 3.78–3.71 (m, 1H), 3.71–3.65 (m, 1H), 3.51 (dd, J = 12.0, 7.2 Hz, 1H), 3.18 (dd, J = 11.7, 3.4 Hz, 1H), 2.87 (d, J = 6.3 Hz, 1H), 0.97 (s, 3H), 0.96 (s, 3H), 0.91–0.84 (m, 15H), 0.64 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.1, 145.1, 135.0, 133.31, 133.26, 133.2, 130.1, 129.9, 128.5, 128.4, 128.0, 127.8, 127.0, 126.3, 126.2, 125.9, 116.7, 104.7, 90.0, 74.1, 73.1, 72.6, 71.3, 61.7, 52.1, 51.0, 50.6, 47.3, 44.5, 39.7, 39.0, 38.3, 36.7, 36.6, 35.4, 32.3, 32.2, 28.5, 28.2, 27.8, 26.3, 25.0, 24.3, 23.0, 22.9, 22.7, 20.2, 19.1, 16.6, 16.2, 14.3; HRMS (ESI): m/z calcd for C₅₃H₇₂O₆Na [M + Na]⁺ 827.5221, found 827.5230.

p-Tolyl 3-O-acetyl-2,4-di-O-benzyl-1-thio-β-D-xylopyranoside (27)

26 (100 mg, 0.39 mmol) was allowed to react with acetic anhydride (41 μL, 0.43 mmol) in dry acetonitrile (1.5 mL) at 40 °C for 12 h in the presence of tetrabutylammonium acetate (35 mg, 0.12 mmol). The solution was concentrated in vacuo and directly purified by flash column chromatography (petroleum ether/EtOAc = 1.5/1). The resulting residue was dissolved in pyridine (2 mL) and then benzoyl chloride (111 μL, 0.93 mmol) and DMAP (3 mg, 0.023 mmol) were added. After the reaction went to completion, the mixture was concentrated in vacuo. The residue was diluted with CH₂Cl₂ and washed with 1 M HCl, saturated aqueous solution of NaHCO₃ and brine. The combined organic phase dried over Na₂SO₄, filtered and concentrated. The obtained residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 8/1) to afford 27 (116 mg, 0.23 mmol, 59% for two steps). [α]²⁴_D = −52.1 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.03 (d, J = 7.4 Hz, 2H), 8.00 (d, J = 7.5 Hz, 2H), 7.62–7.53 (m, 2H), 7.47–7.33 (m, 6H), 7.12 (d, J = 7.9 Hz, 2H), 5.54 (t, J = 7.7 Hz, 1H), 5.26 (t, J = 7.6 Hz, 1H), 5.21–5.13 (m, 1H), 5.01 (d, J = 7.6 Hz, 1H), 4.54 (dd, J = 11.9, 4.6 Hz, 1H), 3.64 (dd, J = 11.9, 8.0 Hz, 1H), 2.34 (s, 3H), 1.97 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 169.9, 165.6, 165.2, 138.7, 133.62, 133.56, 133.54, 130.1, 130.0, 129.9, 129.4, 129.3, 128.7, 128.65, 128.60, 86.9, 71.4, 70.3, 69.2, 65.0, 21.3, 20.8; HRMS (ESI): m/z calcd for C₂₈H₃₀O₇NS [M + NH₄]⁺ 524.1737, found 524.1723.

p-Tolyl 2,4-di-O-benzoyl-1-thio-β-D-xylopyranoside (7a)

To a solution of 27 (2.86 g, 5.65 mmol) in 50 mL of MeOH/CH₂Cl₂ (v/v = 1/2) was added acetyl chloride (2.0 mL) dropwise at 0 °C. The reaction mixture was stirred for 24 h, and then quenched with Et₃N. The volatile was removed in vacuo. The resulting residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 6/1) to afford 7a (2.58 g, 5.37 mmol, 95%). [α]²⁴_D = −59.3 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.06 (d, J = 8.1 Hz, 2H), 8.02 (d, J = 8.1 Hz, 2H), 7.65–7.50 (m, 2H), 7.49–7.31 (m, 6H), 7.11 (d, J = 7.8 Hz, 2H), 5.19–5.06 (m, 2H), 5.00 (d, J = 7.5 Hz, 1H), 4.48 (dd, J = 11.9, 4.4 Hz, 1H), 4.14 (t, J = 7.5 Hz, 1H), 3.61 (dd, J = 11.8, 8.0 Hz, 1H), 2.34 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 138.6, 133.63, 133.58, 133.52, 130.2, 130.0, 129.9, 129.57, 129.55, 128.9, 128.61, 128.59, 86.4, 73.1, 72.4, 71.7, 64.6, 21.4; HRMS (ESI): m/z calcd for C₂₆H₂₅O₆S [M + H]⁺ 465.1366, found 465.1353.

2,4,6-Tri-O-benzoyl-3-O-methyl-α-D-glucopyranosyl trichloroacetimidate (8)

To a solution of hemiacetal 28 (2.90 g, 5.73 mmol) in CH₂Cl₂ (50 mL) were added Cl₃CCN (5.75 mL, 57.3 mmol) and DBU (0.17 mL, 1.15 mmol). The resulting mixture was stirred at room temperature for 6 h followed by concentration in vacuo. The residue was purified by silica gel flash column chromatography (petroleum ether/EtOAc = 7/1) to give 8 (3.53 g, 5.44 mmol, 95%). [α]²⁴_D = +81.5 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.60 (s, 1H), 8.16–7.95 (m, 6H), 7.67–7.50 (m, 3H), 7.51–7.34 (m, 6H), 6.71 (d, J = 3.7 Hz, 1H), 5.58 (t, J = 9.8 Hz, 1H), 5.43 (dd, J = 9.8, 3.8 Hz, 1H), 4.59 (dd, J = 12.2, 2.4 Hz, 1H), 4.44–4.54 (m, 1H), 4.39 (dd, J = 12.2, 5.2 Hz, 1H), 4.18 (t, J = 9.6 Hz, 1H), 3.50 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.1, 165.3, 165.1, 160.4, 133.5, 133.5, 133.1, 129.9, 129.8, 129.7, 129.6, 129.2, 129.2, 128.5, 128.5, 128.3, 93.4, 79.0, 72.10, 70.6, 70.0, 62.7, 60.8; HRMS (ESI): m/z calcd for C₃₀H₂₆O₉NCl₃Na [M + Na]⁺ 672.0565, found 672.0555.

p-Tolyl 2,4,6-tri-O-benzoyl-3-O-methyl-1-thio-β-D-glucopyranoside (29)

A solution of compound 7a (38 mg, 0.082 mmol) and 8 (80 mg, 0.12 mmol) in anhydrous CH₂Cl₂ (2 mL) was stirred vigorously in the presence of activated 4 Å molecular sieves (2 g) for 20 min. The mixture was cooled to 0 °C and TMSOTf (1.5 μL, 0.0082 mmol) was added. After 1 h, the reaction mixture was quenched with Et₃N, and the solid was filtered off. The filtrate was concentrated in vacuo and the resulting residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 7/1) to afford 29 (28 mg, 0.046 mmol, 56%). [α]²⁴_D = +9.4 (c 1.25, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.15–8.08 (m, 2H), 8.03 (m, 4H), 7.65–7.54 (m, 3H), 7.51–7.40 (m, 6H), 7.35 (d, J = 8.1 Hz, 2H), 6.88 (d, J = 8.0 Hz, 2H), 5.41 (t, J = 9.6 Hz, 1H), 5.28 (t, J = 9.4 Hz, 1H), 4.84 (d, J = 10.0 Hz, 1H), 4.64 (dd, J = 12.1, 2.7 Hz, 1H), 4.40 (dd, J = 12.1, 6.3 Hz, 1H), 4.05–3.96 (m, 1H), 3.86 (t, J = 9.1 Hz, 1H), 3.38 (s, 3H), 2.24 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 165.2, 165.1, 138.4, 133.6, 133.5, 133.4, 133.2, 130.0, 129.97, 129.96, 129.8, 129.7, 129.4, 128.7, 128.6, 128.50, 128.48, 86.6, 83.5, 76.3, 71.9, 70.6, 63.5, 60.1, 21.2; HRMS (ESI): m/z calcd for C₃₅H₃₆O₈NS [M + NH₄]⁺ 630.2156, found 630.2149.

4-Methoxyphenyl 3-O-acetyl-2,4-di-O-benzoyl-β-D-xylopyranoside (31)

Following the procedure for the preparation of compound 27, 30 (1.35 mg, 5.27 mmol) was converted into 31 (1.6 mg, 3.16 mmol, 60%) over two steps by purification with silica gel column (petroleum ether/EtOAc = 6/1). [α]²⁴_D = −34.2 (c 0.90, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.03 (d, J = 7.7 Hz, 4H), 7.63–7.51 (m, 2H), 7.48–7.34 (m, 4H), 6.96 (d, J = 9.0 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 5.60 (t, J = 7.6 Hz, 1H), 5.47 (dd, J = 7.6, 5.8 Hz, 1H), 5.31–5.20 (m, 2H), 4.45 (dd, J = 12.2, 4.4 Hz, 1H), 3.75 (s, 3H), 3.70 (dd, J = 12.2, 7.3 Hz, 1H), 2.02 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 170.0, 165.6, 165.3, 155.7, 150.8, 133.7, 133.6, 130.04, 130.02, 129.31, 129.29, 128.7, 128.6, 118.8, 114.7, 99.9, 70.5, 70.3, 69.3, 61.9, 55.8, 20.9; HRMS (ESI): m/z calcd for C₂₈H₂₆O₉Na [M + Na]⁺ 529.1469, found 529.1458.

4-Methoxyphenyl 2,4-di-O-benzoyl-β-D-xylopyranoside (7b)

Following the procedure for the preparation of 7a, 7b (85 mg, 0.18 mmol, 93%), purified by silica gel column chromatography (CH₂Cl₂/EtOAc = 10/1), was prepared from compound 31 (100 mg, 0.20 mmol). [α]²⁴_D = −49.0 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 8.0 Hz, 2H), δ 8.03 (d, J = 8.0 Hz, 2H), 7.62–7.50 (m, 2H), 7.44–7.30 (m, 4H), 7.02 (d, J = 8.9 Hz, 2H), 6.83 (d, J = 8.9 Hz, 2H), 5.41 (d, J = 4.1 Hz, 1H), 5.34 (t, J = 4.9 Hz, 1H), 5.24–5.15 (m, 1H), 4.45 (dd, J = 12.8, 3.4 Hz, 1H), 4.25 (t, J = 5.6 Hz, 1H), 3.81 (dd, J = 12.8, 5.0 Hz, 1H), 3.77 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.2, 166.0, 155.8, 150.4, 133.6, 133.5, 130.1, 130.0, 129.7, 129.4, 128.5, 118.6, 114.8, 99.0, 71.5, 71.1, 69.4, 60.4, 55.8; HRMS (ESI): m/z calcd for C₂₆H₂₄O₈Na [M + Na]⁺ 487.1363, found 487.1355.

4,6-Di-O-benzoyl-3-O-methyl-α-D-glucopyranose-1,2-diyl (4-methoxyphenyl 2,4-di-benzoyl β-D-xylopyranoside)-3-yl orthobenzoate (32)

Following the procedure for the preparation of compound 29, the coupling of 7b (38 mg, 0.083 mmol) and 8 (70 mg, 0.108 mmol) in CH₂Cl₂ in the presence of TMSOTf (1.5 μL, 0.0083 mmol) furnished 32 (28 mg, 0.029 mmol, 35%) after purification by silica gel column chromatography (toluene/EtOAc = 20/1). [α]²⁴_D = −5.3 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.99 (d, J = 7.6 Hz, 2H), 7.94 (d, J = 7.6 Hz, 2H), 7.85 (d, J = 6.9 Hz, 2H), 7.83 (d, J = 6.9 Hz, 2H), 7.69 (d, J = 7.4 Hz, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.42 (t, J = 7.5 Hz, 3H), 7.38–7.28 (m, 7H), 7.17 (t, J = 7.6 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 6.78 (d, J = 9.0 Hz, 2H), 6.04 (d, J = 5.2 Hz, 1H), 5.37 (t, J = 4.6 Hz, 1H), 5.28 (m, 1H), 5.21–5.15 (m, 1H), 5.12 (d, J = 8.9 Hz, 1H), 4.91–4.84 (m, 1H), 4.39 (dd, J = 12.4, 3.3 Hz, 1H), 4.28–4.17 (m, 2H), 4.11 (t, J = 5.3 Hz, 1H), 3.86–3.78 (m, 1H), 3.75 (s, 3H), 3.68 (dd, J = 12.5, 5.2 Hz, 1H), 3.59 (s, 3H), 3.55 (s, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 166.1, 165.5, 165.4, 165.2, 155.4, 150.8, 135.5, 133.5, 133.4, 133.3, 133.0, 130.0, 129.90, 129.87, 129.81, 129.78, 129.76, 129.51, 129.49, 128.5, 128.4, 128.34, 128.29, 126.7, 122.0, 118.4, 114.7, 98.7, 97.9, 77.1, 73.3, 70.8, 70.5, 69.4, 68.5, 67.4, 64.5, 60.6, 58.6, 55.8; HRMS (ESI): m/z calcd for C₅₄H₅₂O₁₆N [M + NH₄]⁺ 970.3281, found 970.3283.

4-Methoxyphenyl 2,4,6-tri-O-benzoyl-3-O-methyl-α-D-glucopyranosyl-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranoside (33α) and 4-methoxyphenyl 2,4,6-tri-O-benzoyl-3-O-methyl-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranoside (33β)

According to the protocol for the synthesis of 29, TBSOTf (6.0 μL, 0.025 mmol)-catalyzed glycosylation of 7b (38 mg, 0.083 mmol) and 8 (70 mg, 0.108 mmol) in anhydrous CH₂Cl₂ (2 mL) to afford 33α (18 mg, 0.019 mmol, 23%) and 33β (31 mg, 0.032 mmol, 39%) with the purification of silica gel column chromatography (toluene/EtOAc = 30/1) 33α: [α]²⁴_D = +66.8 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 7.3 Hz, 2H), 7.99 (d, J = 7.2 Hz, 2H), 7.95 (d, J = 7.3 Hz, 2H), 7.89 (d, J = 7.2 Hz, 2H), 7.79 (d, J = 7.3 Hz, 2H), 7.63–7.42 (m, 7H), 7.39 (s, 2H), 7.35–7.27 (m, 6H), 7.00 (d, J = 9.1 Hz, 2H), 6.81 (d, J = 9.1 Hz, 2H), 5.64 (d, J = 3.7 Hz, 1H), 5.60–5.54 (m, 1H), 5.46 (t, J = 9.7 Hz, 1H), 5.27 (d, J = 4.9 Hz, 1H), 5.19–5.04 (m, 2H), 4.47–4.28 (m, 4H), 4.07 (t, J = 9.7 Hz, 1H), 3.91 (dd, J = 12.2, 4.0 Hz, 1H), 3.77 (s, 3H), 3.56 (dd, J = 12.3, 6.2 Hz, 1H), 3.44 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.2, 165.4, 165.2, 165.0, 155.6, 150.8, 133.8, 133.52, 133.49, 133.1, 129.98, 129.93, 129.86, 129.85, 129.5, 129.22, 129.21, 129.0, 128.7, 128.6, 128.53, 128.46, 128.4, 118.5, 114.8, 99.3, 96.9, 78.8, 73.9, 72.8, 70.7, 70.6, 70.5, 68.7, 62.5, 60.9, 60.8, 55.8; HRMS (ESI): m/z calcd for C₅₄H₅₂O₁₆N [M + NH₄]⁺ 970.3281, found 970.3261. 33β: [α]²⁴_D = −35.6 (c 0.75, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 7.6 Hz, 2H), 8.00 (d, J = 7.5 Hz, 2H), 7.95–7.89 (m, 6H), 7.60–7.40 (m, 6H), 7.36–7.22 (m, 7H), 7.12 (t, J = 7.7 Hz, 2H), 6.73 (d, J = 9.1 Hz, 2H), 6.67 (d, J = 9.1 Hz, 2H), 5.52–5.42 (m, 2H), 5.32 (s, 1H), 5.24 (s, 1H), 5.19–5.12 (m, 2H), 4.57 (dd, J = 12.0, 2.9 Hz, 1H), 4.50–4.37 (m, 3H), 4.15–4.08 (m, 1H), 3.89 (t, J = 9.2 Hz, 1H), 3.78–3.71 (m, 4H), 3.39 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 165.5, 165.3, 165.2, 165.1, 155.1, 150.3, 133.6, 133.5, 133.2, 133.1, 132.9, 130.00, 129.97, 129.81, 129.78, 129.72, 129.4, 129.3, 128.7, 128.5, 128.4, 128.33, 128.27, 118.5, 114.4, 110.1, 101.2, 97.2, 82.0, 73.0, 72.69, 72.68, 70.7, 69.1, 68.7, 63.6, 59.6, 58.9, 55.8; HRMS (ESI): m/z calcd for C₅₄H₅₂O₁₆N [M + NH₄]⁺ 970.3281, found 970.3288.

Benzyl 2,4,6-tri-O-benzoyl-3-O-methyl-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzoyl-α-D-xylopyranoside (34)

Following the procedure for the preparation of compound 29, the coupling of 7c (1.38 g, 3.07 mmol) and 8 (3.0 g, 4.61 mmol) in anhydrous CH₂Cl₂ (20 mL) in the presence of TMSOTf (56 μL, 0.022 mmol) afford 34 (2.30 g, 2.46 mmol, 80%) by silica gel column chromatography (toluene/EtOAc = 30/1). [α]²⁴_D = +1.0 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.06 (d, J = 7.2 Hz, 2H), 8.02–7.95 (m, 4H), 7.93 (d, J = 7.2 Hz, 2H), 7.69 (t, J = 7.4 Hz, 1H), 7.63–7.50 (m, 5H), 7.49–7.35 (m, 6H), 7.33–7.22 (m, 3H), 7.20–7.12 (m, 5H), 7.12–7.04 (m, 2H), 5.30 (t, J = 9.5 Hz, 1H), 5.27–5.19 (m, 2H), 5.11 (d, J = 3.5 Hz, 1H), 5.06 (d, J = 8.0 Hz, 1H), 4.96 (dd, J = 9.4, 3.6 Hz, 1H), 4.69 (d, J = 12.3 Hz, 1H), 4.60 (t, J = 9.1 Hz, 1H), 4.48–4.39 (m, 2H), 4.31 (dd, J = 11.9, 5.9 Hz, 1H), 4.08–3.98 (m, 2H), 3.78 (t, J = 10.6 Hz, 1H), 3.70 (t, J = 9.3 Hz, 1H), 3.24 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.4, 165.6, 165.2, 165.1, 164.9, 137.0, 133.6, 133.3, 133.2, 133.0, 130.0, 129.93, 129.88, 129.79, 129.77, 129.68, 129.62, 129.59, 129.40, 129.35, 128.6, 128.5, 128.38, 128.35, 128.34, 128.0, 127.8, 101.5, 95.1, 81.9, 75.5, 73.9, 72.7, 72.2, 70.8, 69.9, 69.7, 63.9, 59.5, 59.2; HRMS (ESI): m/z calcd for C₅₄H₅₂O₁₅N [M + NH₄]⁺ 954.3331, found 954.3329.

2,4,6-Tri-O-benzoyl-3-O-methyl-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzoyl-α-D-xylopyranosyl trichloroacetimidate (35)

To a solution of 34 (2.58 mg, 2.75 mmol) in EtOH (70 mL) was added 10% Pd/C (1 g). The reaction vessel was evacuated and backfilled with hydrogen (1 atm). The mixture was stirred at 50 °C for 24 h, at this stage TLC indicated the reaction was completed. The mixture was filtered through a pad of Celite. The filtrate was concentrated and the residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 2.5/1) to afford the desired hemiacetal as a white foam which was directly used in the next step. To a soln of the above hemiacetal (1.91 g, 2.26 mmol) and Cl₃CCN (2.3 mL, 22.6 mmol) in CH₂Cl₂ (2.5 mL) was added DBU (171 μL, 1.15 mmol) at 0 °C. The mixture was stirred overnight at ambient temperature, and then concentrated in vacuo. The resulting residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 3.5/1) to afford 35 (2.02 g, 2.04 mmol, 74% over two steps). [α]²⁴_D = −15.7 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.47 (s, 1H), 8.11 (d, J = 7.5 Hz, 2H), 8.01 (d, J = 8.5 Hz, 2H), 7.99 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 7.5 Hz, 2H), 7.71–7.54 (m, 4H), 7.54–7.45 (m, 5H), 7.42 (t, J = 7.7 Hz, 2H), 7.39–7.28 (m, 4H), 7.19 (t, J = 7.7 Hz, 2H), 6.49 (d, J = 3.4 Hz, 1H), 5.38–5.30 (m, 1H), 5.30–5.17 (m, 3H), 5.04 (d, J = 8.0 Hz, 1H), 4.60 (t, J = 9.2 Hz, 1H), 4.54 (dd, J = 12.0, 2.9 Hz, 1H), 4.29–4.17 (m, 2H), 4.12–4.04 (m, 1H), 3.87 (t, J = 10.8 Hz, 1H), 3.70 (t, J = 9.3 Hz, 1H), 3.23 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.4, 165.5, 165.3, 164.9, 164.8, 160.5, 133.73, 133.65, 133.5, 133.4, 133.1, 129.98, 129.95, 129.7, 129.6, 129.5, 129.30, 129.29, 129.0, 128.7, 128.6, 128.42, 128.38, 101.6, 93.7, 81.8, 77.2, 75.4, 72.6, 72.55, 72.48, 70.7, 68.8, 64.0, 61.7, 59.3; HRMS (ESI): m/z calcd for C₄₉H₄₆O₁₅N₂Cl₃ [M + NH₄]⁺ 1007.1958, found 1007.1957.

p-Tolyl 2,3-di-O-benzoyl-6-deoxy-6-iodo-1-thio-β-D-glucopyranoside (37)

To a solution of 36 (1.13 g, 2.29 mmol), Ph₃P (1.20 g, 4.58 mmol) and imidazole (0.62 g, 9.16 mmol) in anhydrous THF (20 mL) was added a solution of I₂ (0.76 g, 2.98 mmol) in anhydrous THF (3 mL). After stirring for 1 h, monitoring by TLC indicated that the reaction went to completion. The mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography to give 37 (petroleum ether/EtOAc = 5/1, 1.26 g, 2.08 mmol, 91%). [α]²⁴_D = +52.4 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.98 (d, J = 8.0 Hz, 2H), 7.94 (d, J = 8.0 Hz, 2H), 7.57–7.45 (m, 4H), 7.44–7.32 (m, 4H), 7.13 (d, J = 7.9 Hz, 2H), 5.45–5.31 (m, 2H), 4.89 (d, J = 9.1 Hz, 1H), 3.79–3.65 (m, 2H), 3.47 (dd, J = 10.7, 6.4 Hz, 1H), 3.42–3.34 (m, 1H), 3.21 (s, 1H), 2.35 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 168.0, 165.3, 138.9, 134.4, 133.9, 133.5, 130.2, 130.0, 129.8, 129.4, 128.7, 128.6, 128.5, 127.5, 86.0, 79.0, 78.4, 73.3, 70.0, 21.4, 5.6; HRMS (ESI): m/z calcd for C₂₇H₂₉O₆NIS [M + NH₄]⁺ 622.0755, found 622.0743.

p-Tolyl 2,3-di-O-benzoyl-6-deoxy-1-thio-β-D-glucopyranoside (9)

To a solution of 19 (1.38 g, 2.28 mmol) in anydrous N,N-dimethylformamide (45 mL) was added 10% Pd/C (700 mg) and NaHCO₃ (633 mg, 7.53 mmol). The reaction vessel was evacuated and backfilled with hydrogen (1 atm). The mixture was stirred at 40 °C for 24 h. The solid was filtered on Celite and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether/EtOAc = 5/1) to give 9 (904 mg, 1.89 mmol, 83%). [α]²⁴_D = +89.7 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.02–7.90 (m, 4H), 7.56–7.48 (m, 2H), 7.42–7.33 (m, 6H), 7.11 (d, J = 8.1 Hz, 2H), 5.40 (t, J = 9.6 Hz, 1H), 5.32 (t, J = 9.0 Hz, 1H), 4.83 (d, J = 9.9 Hz, 1H), 3.65–3.49 (m, 2H), 2.93 (d, J = 4.4 Hz, 1H), 2.34 (s, 3H), 1.46 (d, J = 5.8 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 167.9, 165.4, 138.6, 133.7, 133.6, 133.4, 130.1, 129.9, 129.8, 129.5, 129.0, 128.6, 128.50, 128.49, 86.3, 78.8, 74.7, 70.6, 21.3, 18.0; HRMS (ESI): m/z calcd for C₂₇H₃₀O₆NS [M + NH₄]⁺ 496.1788, found 496.1786.

p-Tolyl 2,4,6-tri-O-benzoyl-3-O-methyl-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3-di-O-benzoyl-6-deoxy-1-thio-β-D-glucopyranoside (4)

A solution of 35 (1.24 g, 1.25 mmol) and 9 (400 mg, 0.84 mmol) in anhydrous CH₂Cl₂ (12 mL) was stirred vigorously in the presence of activated 4 Å molecular sieves (1 g) for 20 min. The mixture was cooled to 0 °C and TMSOTf (15 μL, 0.084 mmol) were added. After 1 h, the reaction mixture was quenched with Et₃N and filtered. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 3/1) to give 4 (756 mg, 70%). [α]²⁴_D = +2.6 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.01 (s, 2H), 7.98 (s, 2H), 7.96 (d, J = 7.7 Hz, 2H), 7.93 (d, J = 7.7 Hz, 2H), 7.89 (d, J = 7.8 Hz, 4H), 7.80 (d, J = 7.7 Hz, 2H), 7.60–7.07 (m, 24H), 5.48 (t, J = 9.2 Hz, 1H), 5.37 (t, J = 9.4 Hz, 1H), 5.32 (t, J = 8.3 Hz, 1H), 5.23 (t, J = 9.7 Hz, 1H), 5.03–4.97 (m, 1H), 4.97–4.88 (m, 2H), 4.74 (d, J = 2.8 Hz, 1H), 4.63 (d, J = 10.1 Hz, 1H), 4.47 (dd, J = 11.9, 3.0 Hz, 1H), 4.33 (dd, J = 12.0, 6.3 Hz, 1H), 4.21 (t, J = 4.5 Hz, 1H), 4.06–3.96 (m, 1H), 3.93 (dd, J = 12.7, 2.4 Hz, 1H), 3.73 (t, J = 9.2 Hz, 1H), 3.42 (t, J = 9.2 Hz, 1H), 3.32 (s, 3H), 3.22–3.15 (m, 1H), 3.11 (dd, J = 12.7, 4.0 Hz, 1H), 2.35 (s, 3H), 1.10 (t, J = 6.0 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.2, 165.6, 165.5, 165.3, 165.2, 164.9, 164.7, 138.4, 133.6, 133.44, 133.37, 133.27, 133.26, 133.19, 133.00, 132.95, 130.02, 129.94, 129.91, 129.8, 129.7, 129.6, 129.4, 129.2, 129.0, 128.63, 128.59, 128.5, 128.43, 128.40, 128.32, 128.27, 101.2, 100.0, 86.2, 82.7, 81.7, 75.4, 74.8, 74.0, 72.7, 72.5, 71.5, 70.6, 70.3, 68.8, 63.7, 59.6, 18.0; HRMS (ESI): m/z calcd for C₇₄H₇₀O₂₀NS [M + NH₄]⁺ 1324.4206, found 1324.4198.

Lanost-7-en-3β-yl 2,4,6-tri-O-benzoyl-3-O-methyl-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3-di-O-benzoyl-6-deoxy-β-D-glucopyranosyl-(1→2)-3-O-benzoyl-4-O-(2-naphthylmethyl)-β-D-xylopyranoside (2)

A solution of 3 (62 mg, 0.076 mmol) and 4 (150 mg, 0.115 mmol) in anhydrous CH₂Cl₂ (2 mL) was stirred vigorously in the presence of activated 4 Å molecular sieves (200 mg) for 20 min. The mixture was cooled to 0 °C and NIS (34 mg, 0.152 mmol) and TMSOTf (2 μL, 0.008 mmol) were added. After stirring for 2 h, the reaction mixture was quenched with Et₃N, and the solid was filtered off. The filtrate was washed with saturated aqueous Na₂S₂O₃ and brine. The organic phase was dried over Na₂SO₄, filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 3.5/1) to give 2 (134 mg, 67%). [α]²⁴_D = +10.6 (c 0.75, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.00 (d, J = 7.3 Hz, 2H), 7.94–7.81 (m, 10H), 7.78 (d, J = 7.3 Hz, 2H), 7.74–7.65 (m, 3H), 7.62 (t, J = 7.4 Hz, 1H), 7.60–7.49 (m, 5H), 7.49–7.43 (m, 5H), 7.43–7.35 (m, 8H), 7.32–7.24 (m, 8H), 7.18 (t, J = 7.8 Hz, 2H), 7.14 (dd, J = 8.4, 1.2 Hz, 1H), 5.36–5.23 (m, 4H), 5.21 (s, 1H), 5.16 (t, 8.1 Hz, 1H), 5.03–4.97 (m, 1H), 4.97–4.93 (m, 1H), 4.87 (d, J = 7.8 Hz, 1H), 4.71 (d, J = 7.9 Hz, 1H), 4.64 (d, J = 4.0 Hz, 1H), 4.59–4.46 (m, 3H), 4.42 (dd, J = 12.0, 3.2 Hz, 1H), 4.28 (dd, J = 11.9, 6.3 Hz, 1H), 4.17 (t, J = 5.6 Hz, 1H), 4.04–3.90 (m, 2H), 3.86–3.75 (m, 2H), 3.67 (t, J = 9.2 Hz, 1H), 3.52–3.61 (m, 1H), 3.39–3.31 (m, 2H), 3.29 (s, 3H), 3.25–3.16 (m, 1H), 3.09 (dd, J = 11.7, 3.6 Hz, 1H), 3.05 (dd, J = 12.6, 5.0 Hz, 1H), 2.04–1.88 (m, 4H), 1.82–1.71 (m, 2H), 1.04 (s, 3H), 1.02–0.97 (m, 6H), 0.91–0.83 (m, 15H), 0.64 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.2, 165.6, 165.5, 165.3, 165.2, 164.8, 164.7, 145.1, 135.2, 133.6, 133.42, 133.40, 133.2, 133.1, 133.04, 133.00, 132.8, 132.7, 130.1, 130.0, 129.97, 129.93, 129.90, 129.87, 129.8, 129.7, 129.6, 129.4, 129.2, 128.7, 128.6, 128.5, 128.4, 128.3, 128.23, 128.19, 128.0, 127.7, 126.8, 126.1, 126.0, 125.9, 116.8, 103.7, 101.5, 100.5, 100.4, 90.2, 83.1, 81.7, 76.4, 75.2, 74.6, 74.4, 73.7, 72.70, 72.68, 72.4, 72.1, 71.0, 70.9, 70.6, 69.1, 63.7, 62.5, 60.2, 59.3, 52.1, 51.0, 50.7, 47.3, 44.5, 39.7, 39.2, 38.3, 36.7, 36.6, 35.4, 32.32, 32.26, 28.2, 27.8, 26.3, 25.0, 24.3, 23.0, 22.7, 20.2, 19.1, 17.6, 16.4, 16.2, 14.2; HRMS (ESI): m/z calcd for C₁₂₀H₁₃₀O₂₆Na [M + Na]⁺ 2009.8743, found 2009.8736.

Lanost-7-en-3β-yl 2,4,6-tri-O-benzoyl-3-O-methyl-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3-di-O-benzoyl-6-deoxy-β-D-glucopyranosyl-(1→2)-3-O-benzoyl-β-D-xylopyranoside (38)

To a solution of 2 (100 mg, 0.05 mmol) in CH₂Cl₂ (2.80 mL) and MeOH (0.80 mL) was added DDQ (34 mg, 0.15 mmol) in three portions at half an hour interval. After 5 h, the reaction was completed and the volatile was removed by evaporation. The residue was taken up in dichloromethane and washed with saturated aqueous NaHCO₃ and brine. The collected organic phase was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 2.5/1) to give 38 (81 mg, 88%). [α]²⁴_D = −2.5 (c 1.05, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.11–7.97 (m, 6H), 7.96–7.83 (m, 8H), 7.79 (d, J = 7.3 Hz, 2H), 7.66–7.53 (m, 3H), 7.52–7.38 (m, 10H), 7.38–7.19 (m, 9H), 7.15 (t, J = 7.8 Hz, 2H), 5.48 (t, J = 9.4 Hz, 1H), 5.42–5.31 (m, 2H), 5.26–5.20 (m, 1H), 5.18 (s, 1H), 5.03–4.98 (m, 1H), 4.98–4.95 (m, 1H), 4.94 (d, J = 7.8 Hz, 1H), 4.88 (t, J = 4.0 Hz, 1H), 4.82 (s, 1H), 4.76–4.67 (m, 2H), 4.47 (dd, J = 12.0, 3.2 Hz, 1H), 4.34 (dd, J = 12.0, 6.3 Hz, 1H), 4.25 (dd, J = 10.1, 1.8 Hz, 1H), 4.22 (t, J = 4.7 Hz, 1H), 4.04–3.97 (m, 1H), 3.94 (dd, J = 12.6, 2.6 Hz, 1H), 3.90 (s, 1H), 3.75 (t, J = 9.2 Hz, 1H), 3.64–3.56 (m, 1H), 3.53 (dd, J = 12.3, 2.9 Hz, 1H), 3.43 (t, J = 9.2 Hz, 1H), 3.33 (s, 3H), 3.19–3.03 (m, 4H), 1.01 (d, J = 6.0 Hz, 3H), 0.97 (s, 3H), 0.91–0.85 (m, 12H), 0.84 (s, 3H), 0.67 (s, 3H), 0.64 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.2, 166.04, 166.00, 165.5, 165.3, 165.2, 164.9, 164.7, 145.2, 133.6, 133.6, 133.5, 133.4, 133.2, 133.2, 133.1, 133.0, 130.1, 130.00, 129.96, 129.9, 129.78, 129.75, 129.74, 129.6, 129.42, 129.38, 129.2, 128.7, 128.6, 128.5, 128.44, 128.43, 128.33, 128.29, 116.6, 102.1, 101.3, 101.0, 100.1, 89.6, 82.9, 81.7, 77.4, 73.9, 73.7, 73.2, 72.72, 72.69, 72.5, 71.2, 70.6, 70.3, 68.8, 67.1, 63.7, 61.7, 59.6, 59.3, 52.1, 51.0, 50.5, 47.3, 44.5, 39.7, 39.1, 38.3, 36.7, 36.6, 35.4, 32.3, 32.2, 28.3, 28.2, 27.8, 26.2, 25.0, 24.3, 23.00, 22.98, 22.7, 20.2, 19.1, 17.5, 16.3, 16.2, 14.3; HRMS (ESI): m/z calcd for C₁₀₉H₁₂₆O₂₆N [M + NH₄]⁺ 1864.8563, found 1864.8521.

Lanost-7-en-3β-yl 2,4,6-tri-O-benzoyl-3-O-methyl-β-D-glucopyranosyl-(1→3)-2,4-di-O-benzoyl-β-D-xylopyranosyl-(1→4)-2,3-di-O-benzoyl-6-deoxy-β-D-glucopyranosyl-(1→2)-3-O-benzoyl-4-O-sulfo-β-D-xylopyranoside (39)

To a mixture of alcohol 38 (60 mg, 0.032 mmol) in anhydrous pyridine (2.0 mL) was added sulfur trioxide/pyridine complex (103 mg, 0.65 mmol). The sealed Pyrex tube was irradiated in the microwave (100 W, CEM corporation, Matthews, North Carolina, USA) at 100 °C for 2 h. After cooling, water (1 mL) was added to the mixture, which was then concentrated in vacuo. The residue was purified by silica gel column chromatography (CH₂Cl₂/MeOH = 13/1) to give 39 (60 mg, 0.03 mmol, 94%). [α]²⁴_D = +2.8 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 5.4 Hz, 2H), 7.99 (d, J = 7.5 Hz, 2H), 7.97 (d, J = 7.5 Hz, 2H), 7.93 (d, J = 7.6 Hz, 2H), 7.87 (d, J = 6.5 Hz, 4H), 7.79 (d, J = 7.5 Hz, 2H), 7.63 (d, J = 7.6 Hz, 2H), 7.60–7.52 (m, 3H), 7.50 (t, J = 7.3 Hz, 1H), 7.47–7.30 (m, 11H), 7.29–7.19 (m, 4H), 7.15 (t, J = 7.6 Hz, 2H), 7.10–7.00 (m, 1H), 6.73–6.54 (m, 2H), 5.46 (t, J = 9.0 Hz, 1H), 5.39–5.24 (m, 3H), 5.19 (s, 1H), 5.04–4.97 (m, 2H), 4.97–4.81 (m, 3H), 4.72 (s, 1H), 4.61 (d, J = 7.8 Hz, 1H), 4.52–4.39 (m, 2H), 4.34 (d, J = 11.9 Hz, 1H), 4.29 (dd, J = 11.8, 6.1 Hz, 1H), 4.20 (t, J = 4.8, 1H), 3.99–3.88 (m, 2H), 3.88–3.78 (m, 2H), 3.76–3.60 (m, 1H), 3.49 (t, J = 9.0 Hz, 1H), 3.29 (s, 3H), 3.16–3.08 (m, 1H), 3.08–2.98 (m, 2H), 1.00 (d, J = 5.5 Hz, 3H), 0.98 (s, 3H), 0.83 (s, 3H), 0.64 (s, 3H), 0.62 (s, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 166.2, 165.7, 165.4, 165.3, 165.2, 164.9, 164.7, 145.1, 133.5, 133.4, 133.3, 133.2, 133.0, 130.3, 130.03, 129.93, 129.88, 129.71, 129.70, 129.6, 129.5, 129.4, 129.2, 128.9, 128.64, 128.60, 128.5, 128.4, 128.3, 128.0, 127.9, 116.6, 102.16, 102.14, 101.1, 100.5, 90.0, 82.6, 81.7, 75.0, 74.4, 73.7, 72.8, 72.7, 72.4, 71.4, 71.2, 70.7, 70.6, 68.8, 63.7, 62.9, 60.0, 59.3, 52.1, 51.0, 50.5, 47.3, 44.5, 39.7, 39.1, 38.3, 36.7, 36.6, 35.4, 32.3, 32.2, 32.1, 30.3, 29.8, 29.7, 29.5, 28.5, 28.1, 27.8, 26.1, 25.0, 24.3, 23.0, 22.8, 22.7, 20.2, 19.1, 17.5, 16.2, 16.2, 14.2; HRMS (ESI): m/z calcd for C₁₀₉H₁₂₂O₂₉S [M − Na]⁻ 1926.7787, found 1926.7743.

Sodium salt of lanost-7-en-3β-yl 3-O-methyl-β-D-glucopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-6-deoxy-β-D-glucopyranosyl-(1→2)-4-O-sulfo-β-D-xylopyranoside (1)

To a solution 39 (50 mg, 0.026 mmol) in 9 mL of anhydrous methanol/CH₂Cl₂ (v/v = 2/1) was added a solution of sodium methoxide (1.04 mL, 1.04 mmol, 1 M) in methanol. After the resulting mixture was stirred at rt for 30 h, it was diluted with water, and then concentrated under reduced pressure. The residue was dialyzed against water followed by lyophilization to afford 1 (25 mg, 86%). [α]²⁴_D = −7.0 (c 0.25, H₂O); ¹H NMR (500 MHz, pyridine-d₅) δ 5.40 (d, J = 7.6 Hz, 1H), 5.35 (s, 1H), 5.26–4.90 (m, 4H), 4.87 (d, J = 7.3 Hz, 1H), 4.81–4.63 (m, 2H), 4.49 (d, J = 10.6 Hz, 1H), 4.40–4.19 (m, 4H), 4.18–3.93 (m, 7H), 3.88 (s, 3H), 3.82–3.53 (m, 5H), 3.26 (d, 1H), 1.74 (d, J = 5.4 Hz, 3H), 1.26 (s, 3H), 1.19 (s, 3H), 1.11 (s, 3H), 0.99 (d, J = 8.6 Hz, 3H), 0.98 (s, 3H), 0.92 (d, J = 6.5 Hz, 6H), 0.76 (s, 3H); ¹³C NMR (126 MHz, pyridine-d₅) δ 145.8, 117.7, 106.0, 105.9, 105.7, 89.6, 88.5, 87.6, 86.5, 84.0, 78.7, 76.8, 76.2, 76.1, 75.8, 75.6, 74.3, 72.3, 71.1, 69.4, 67.1, 64.9, 62.5, 61.4, 52.8, 51.7, 51.4, 48.0, 45.2, 40.3, 40.0, 38.8, 37.33, 37.27, 36.0, 33.1, 32.9, 28.8, 28.7, 28.4, 27.5, 25.5, 25.0, 23.7, 23.5, 23.2, 20.8, 19.7, 18.5, 17.1, 16.8, 14.9; HRMS (ESI): m/z calcd for C₅₃H₈₉O₂₁S [M − Na]⁻ 1093.5621, found 1093.5625.

Acknowledgements

We are grateful for the financial support from NSFC-Shandong Joint Fund (U1406402); National Science Funding of China (21272220); Project on Scientific development in Shandong Province (2012GHY11526) and Fundamental Research Funds for the Central Universities. We also thank Dr Patrick Chaffey and Dr Zhongping Tan at the University of Colorado Boulder for their revisions of this manuscript.

Notes and references

1. K. Hostettmann and A. Marston, Saponins, Cambridge University Press, New York, 1995.
2. (a) V. I. Kalinin, A. S. Silchenko, S. A. Avilov, V. A. Stonik and A. V. Smirnov, Phys. Rev., 2005, 4, 221–236; (b) S.-K. Kim, S. W. A. Himaya and K.-H. Kang, Marine Pharmacognosy Trends and Applications, ed. S.-K. Kim, CRC Press, Boca Raton, 2013, pp. 119–127; (c) D. L. Aminin, E. A. Pislyagin, E. S. Menchinskaya, A. S. Silchenko, S. A. Avilov and V. I. Kalinin, Studies in Natural Product Chemistry, ed. A. Rahman, Elsevier, Kidlington, 2014, pp. 75–94.
3. (a) Y.-H. Yi, Q.-Z. Xu, L. Li, S.-L. Zhang, H.-M. Wu, J. Ding, Y.-G. Tong, W.-F. Tan, M.-H. Li, F. Tian, J.-H. Wu, C.-C. Liaw, K. F. Bastow and K.-H. Lee, Helv. Chim. Acta, 2006, 89, 54–63; (b) S.-L. Zhang, L. Li, Y.-H. Yi and P. Sun, Nat. Prod. Res., 2006, 20, 399–407.
4. F. Tian, C.-H. Zhu, X.-W. Zhang, X. Xie, X.-L. Xin, Y.-H. Yi, L.-P. Lin, M.-Y. Geng and J. Ding, Mol. Pharmacol., 2007, 72, 545–552.
5. (a) H. Pellissier, Tetrahedron, 2004, 60, 5123–5162; (b) B. Yu, Y. Zhang and P. Tang, Eur. J. Org. Chem., 2007, 5145–5161; (c) B. Yu and J. Sun, Chem.–Asian J., 2009, 4, 642–654; (d) C. Gauthier, J. Legault and A. Pichette, Org. Chem., 2009, 6, 321–344; (e) C. Gauthier, J. Legault, M. Piochon-Gauthier and A. Pichette, Phytochem. Rev., 2011, 10, 521–544; (f) B. Yu, J. Song and X. Yang, Acc. Chem. Res., 2012, 45, 1227–1236; (g) Y. Yang, S. Laval and B. Yu, Adv. Carbohydr. Chem. Biochem., 2014, 71, 137–226; (h) L. Zu, Y. Zhao and G. Gu, J. Carbohydr. Chem., 2014, 33, 269–297; (i) Y. Yang, X. Zhang and B. Yu, Nat. Prod. Rep., 2015, 32, 1331–1355.
6. For the synthesis of lanostano-18,20-lactone aglycones, see: (a) G. Habermehl, K.-H. Seib and K.-P. Swiderskjj, Liebigs Ann. Chem., 1978, 419–426; (b) G. G. Habermehl and J. H. Kirsch, Toxicon, 1983, 179–181. For the recent efforts to synthesis of sea cucumber triterpene glycosides, see: (c) J. Yu and B. Yu, Chin. Chem. Lett., 2015, 26, 1331–1335.
7. A. V. Demchenko, Handbook of Chemical Glycosylation: Advances in Stereoselectivity and Therapeutic Relevance, ed. A. V. Demchenko, WILEY-VCH, Weinheim, 2008, pp. 1–28.
8. (a) Y.-P. Hu, Y.-Q. Zhong, Z.-G. Chen, C.-Y. Chen, Z. Shi, M. M. L. Zulueta, C.-C. Ku, P.-Y. Lee, C.-C. Wang and S.-C. Hung, J. Am. Chem. Soc., 2012, 134, 20722–20727; (b) G. Xiao and B. Yu, Chem.–Eur. J., 2013, 19, 7708–7712; (c) S. U. Hansen, G. J. Miller, G. C. Jayson and J. M. Gardiner, Org. Lett., 2013, 15, 88–91; (d) Y. Dai and B. Yu, Chem. Commun., 2015, 51, 13826–13829.
9. (a) M. Masaoud, J. Schmidt and G. Adam, Phytochemistry, 1995, 38, 795–796; (b) C. Areche, P. Cejas, P. Thomas, A. San-Martín, L. Astudillo, M. Gutiérrez and L. A. Loyola, Quim. Nova, 2009, 32, 2023–2025.
10. (a) R. E. Marker, E. L. Wittle and L. W. Mixon, J. Am. Chem. Soc., 1937, 59, 1368–1373; (b) R. B. Woodward, A. A. Patchett, D. H. R. Barton, D. A. J. Ives and R. B. Kelly, J. Chem. Soc., 1957, 1131–1144.
11. B. B. Shingate, B. G. Hazra, D. B. Salunke, V. S. Pore, F. Shirazi and M. V. Deshpande, Tetrahedron, 2013, 69, 11155–11163.
12. V. A. Ignatenko and G. P. Tochtrop, J. Org. Chem., 2013, 78, 3821–3831.
13. (a) K. Takahashi, K. Usami, T. Takahashi, T. Okada and M. Morisaki, Chem. Pharm. Bull., 1987, 35, 3467–3469; (b) G. G. Habermehl, J. H. Kirsch and K. J. Reibstein, Heterocycles, 1982, 17, 183–189.
14. CCDC 1419939 (14·MeOH)†.
15. M. Balci, Basic ¹H- and ¹³C-NMR Spectroscopy, Elsevier, Kidlington, 2005, pp. 87–133.
16. (a) D. H. R. Barton and S. W. McCombie, J. Chem. Soc., Perkin Trans. 1, 1975, 1574–1585; (b) N. Martin and E. J. Thomas, Org. Biomol. Chem., 2012, 10, 7952–7964; (c) M. M. Heravi, A. Bakhtiari and Z. Faghihi, Curr. Org. Synth., 2014, 11, 787–823.
17. M. S. Reddy, H. Zhang, S. Phoenix and P. Deslongchamps, Chem.–Asian J., 2009, 4, 725–741.
18. Q. Liu, T. Guo, L. Zhang, Y. Yu, P. Wang, J. Yang and Y. Li, Eur. J. Med. Chem., 2013, 63, 511–522.
19. R. H. Purdy, A. L. Morrow, J. R. Blinn and S. M. Paul, J. Med. Chem., 1990, 33, 1572–1581.
20. (a) P. J. Stang, M. Hanack and L. R. Subramanian, Synthesis, 1982, 85–126; (b) E. R. Binkley and R. W. Binkley, Preparative Carbohydrate Chemistry, ed. S. Hanessian, Marcel Dekker Inc., New York, 1997, pp. 87–104.
21. R. R. Kumar, S. D. Haveli and H. B. Kagan, Synlett, 2011, 1709–1712.
22. T. Itoh, T. Tamura and T. Matsumoto, Steroids, 1976, 27, 275–285.
23. B. Yang, K. Yoshida, Z. Yin, H. Dai, H. Kavunja, M. H. Eldakdouki, S. Sungsuwan, S. B. Dulaney and X. Huang, Angew. Chem., Int. Ed., 2012, 51, 10185–10189.
24. (a) S. David and S. Hanessian, Tetrahedron, 1985, 41, 643–663; (b) R. F. Helm, J. Ralph and L. Anderson, J. Org. Chem., 1991, 56, 7015–7021.
25. (a) K. R. Love, R. B. Andrade and P. H. Seeberger, J. Org. Chem., 2001, 66, 8165–8176; (b) W. Peng, J. Sun, X. Han and B. Yu, Synlett, 2004, 259–262; (c) K. R. Love and P. H. Seeberger, J. Org. Chem., 2005, 70, 3168–3177.
26. B. Ren, M. Rahm, X. Zhang, Y. Zhou and H. Dong, J. Org. Chem., 2014, 79, 8134–8142.
27. I. Lundt, C. Pedersen and B. Tronier, Acta Chem. Scand., 1964, 18, 1917–1922.
28. (a) H. Yu, B. Yu, X. Wu, Y. Hui and X. Han, J. Chem. Soc., Perkin Trans. 1, 2000, 1445–1453; (b) T. Zhu and G.-J. Boons, Carbohydr. Res., 2000, 329, 709–715; (c) Z. Li and J. C. Gildersleeve, J. Am. Chem. Soc., 2006, 128, 11612–11619; (d) S.-S. Chang, C.-H. Shih, K.-C. Lai and K.-K. T. Mong, Chem.–Asian J., 2010, 5, 1152–1162.
29. J. Tamura, A. Yamaguchi, J. Tanaka and Y. Nishimura, J. Carbohydr. Chem., 2007, 26, 61–82.
30. F. Kong, Carbohydr. Res., 2007, 342, 345–373.
31. (a) E. Petráková and J. Schraml, Chem. Commun., 1983, 48, 877–888; (b) A. D. Bruyn, M. Anteunis, R. V. Rijsbergen, M. Claeyssens and P. Kováč, J. Carbohydr. Chem., 1983, 1, 301–309; (c) R. F. Helm and J. Ralph, Carbohydr. Res., 1993, 240, 23–38; (d) H. Yuasa, N. Miyagawa, M. Nakatani, M. Izumi and H. Hashimoto, Org. Biomol. Chem., 2004, 2, 3548–3556.
32. (a) S. Karamat and W. M. F. Fabian, J. Phys. Chem. A, 2006, 110, 7477–7484; (b) Ž. J. Vitnik and W. M. F. Fabian, Comput. Theor. Chem., 2015, 1051, 104–109.
33. (a) B. Yu and Z. Yang, Org. Lett., 2001, 3, 377–379; (b) Q. Yang, M. Lei, Q.-J. Yin and J.-S. Yang, Carbohydr. Res., 2007, 342, 1175–1181.
34. M.-C. Yan, Y.-N. Chen, H.-T. Wu, C.-C. Lin, C.-T. Chen and C.-C. Lin, J. Org. Chem., 2007, 72, 299–302.
35. (a) A. Raghuraman, M. Riaz, M. Hindle and U. R. Desai, Tetrahedron Lett., 2007, 48, 6754–6758; (b) R. A. Al-Horani, P. Ponnusamy, A. Y. Mehta, D. Gailani and U. R. Desai, J. Med. Chem., 2013, 56, 867–878; (c) P. Xu, W. Xu, Y. Dai, Y. Yang and B. Yu, Org. Chem. Front., 2014, 1, 405–414.

---

Footnote

† Electronic supplementary information (ESI) available. CCDC 1419939. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5ra25845f

---