A Streamlined Regenerative Glycosylation Reaction: Direct, Acid-Free Activation of Thioglycosides

Abstract

Our group has previously reported that 3,3-difluoroxindole (HOFox) is able to mediate glycosylations via intermediacy of OFox imidates. Thioglycoside precursors were first converted into the corresponding glycosyl bromides that were then converted into the OFox imidates in the presence of Ag₂O followed by the activation with catalytic Lewis acid in a regenerative fashion. Reported herein is a direct conversion of thioglycosides via the regenerative approach that bypasses the intermediacy of bromides and eliminates the need for heavy-metal-based promoters. The direct regenerative activation of thioglycosides is achieved under neutral reaction conditions using only 1 equiv. NIS and catalytic HOFox without the acidic additives.

Introduction

From their ubiquitous presence in nature to vital roles in biology, complex carbohydrates are essential molecules made by every living organism. The structure diversity and complexity of carbohydrates have intrigued glycoscientists for decades.^[1] Given that glycosylation is one of the most fundamental modifications of carbohydrates, enzymatic and chemical synthesis of glycosidic linkages remains a focus of many research programs around the world. Among significant advances made in the area, automated synthesis using polymer supports has come to the fore as a powerful means to synthesize glycans.^[2] Many glycosylations in solution make use of thioglycosides,^[3] but glycosylations on solid supports commonly demand highly reactive imidates^[4] or phosphates^[5] as glycosyl donors to offset challenges related to the mismatch between reactive solution-based vs. their unreactive solid-phase-immobilized counter-parts. The use of thioglycosides as glycosyl donors for automated solid-phase synthesis has also been reported,^[6] and a recent Glyconeer procedure comprises the treatment of a supported acceptor with 2×5.0 equiv. thioglycoside donor in the presence of 2×5.5 equiv. of NIS and 2×0.2 equiv. of TfOH for 2×30 min.^[7] With the critical issue of making common glycosyl donors commercially available, the use of thioglycosides is attractive due to their superior stability, but a fairly low reactivity profile and the requirement for stoichiometric promoters leaves ample room for improvement.

As a part of the program to develop new methods and strategies for chemical glycosylation, we reported a regenerative glycosylation reaction concept.^[8] In accordance with this concept, 3,3-difluoroxindole (HOFox) reacts with a stable precursor to form a highly reactive OFox imidate (Scheme 1).^[9] The latter will then react with the glycosyl acceptor while regenerating HOFox aglycone. The released HOFox will mediate the next catalytic cycle to obtain a new OFox donor, etc. In our original study, we reacted S-ethyl glycoside with Br₂ to form glycosyl bromide. The latter reacts very slowly in the presence of Ag₂O, but in the presence of HOFox gives reasonable reaction rates (2–3 h) with only 10 mol% of HOFox and a catalytic Lewis acid (BF₃-Et₂O or TMSOTf). The concentration of reactive glycosyl donor can be controlled by the amount of HOFox added. This regenerative concept differs from the two-step activation^[10] and Huang’s preactivation strategy^[11] in the way that the reactive donor is generated in only small amounts. This approach was successfully applied in oligosaccharide synthesis.^[12] However, the two-step conversion, a stoichiometric generation of the glycosyl bromide from the thioglycoside precursor, followed by the heterogeneous activation of the bromide intermediate in the presence of Ag₂O, remained a drawback of this approach. Ultimately, this hampers its application in polymer supported or flow-through approaches. Reported herein is a direct conversion of thioglycosides via the regenerative approach that bypasses the intermediacy of glycosyl bromides and eliminates the need for heavy metal-based promoters.

Scheme 1.

Regenerative glycosylation approach.

Results and Discussion

When activated with N-iodosuccinimide (NIS) only, many thioglycosides react slowly or do not react at all, even in the presence of an excess of NIS (2–3 equiv.). This is typically improved by using strong Lewis (TMSOTf) or protic acids (TfOH) as co-promoters. Our preliminary experimentation showed that some reactive thioglycosides can be converted to OFox imidates using 1 equiv. NIS and catalytic HOFox without the acidic additives. The formation of OFox intermediates can easily be monitored by TLC and NMR (see the Supporting Information for details). Once formed, the OFox imidates rapidly react with the acceptor while liberating HOFox aglycone. In our opinion, the observation that 1.0 equiv. NIS and catalytic HOFox can activate thioglycosides in a regenerative fashion offers a promising venue for developing a new glycosylation reaction. This discovery also hints on feasibility of the activation of thioglycosides under neutral conditions offering a broader functional and protecting group compatibility.

Therefore, we endeavored to optimize these reaction conditions. We began these studies by investigating a highly reactive thiogalactoside donor 1^[13] in reactions with partially benzylated primary glycosyl acceptor 2.^[14] These results are summarized in Table 1. These reactive substrates were able to react in the presence of only 1.0 equiv. NIS, even in the absence of the acidic additive, producing disaccharide 3^[15] in 74% yield (entry 1). However, this reaction slowed down after 3.5 h, and was incomplete even after 12 h indicating that these reaction conditions are insufficient for effective glycosylation. When glycosidation of thiogalactoside 1 with glycosyl acceptor 2 was repeated in the presence of NIS (1.0 equiv.) and catalytic HOFox aglycone (0.05 equiv.) the reaction was smoothly driven to completion in 6 h, and disaccharide 3 was obtained in an excellent yield of 91% (entry 2). Upon increasing the amount of HOFox to 0.10 equiv., no significant change in the reaction time and yield was observed (entry 3). Increasing the amount of HOFox to 0.20 equiv. decreased the reaction time to 3.5 h, and disaccharide 3 was obtained in 92% yield (entry 4). Realizing the effect of catalyst loading, we increased the amount of HOFox to 0.30 and 0.50 equiv. These adjustments led to a decreased reaction time to 3 and 1.5 h (entries 5 and 6). Based on these results, we chose to perform all subsequent experimentation using 20 mol% of HOFox. In our opinion, these reaction conditions offer the best balance between the amount of catalyst, reaction times, and product yields. While TMSOTf (or TfOH) is not required in these reactions, adding as little as 0.05 equiv. TMSOTf reduces the reaction time and expedites both the generation of the OFox imidate and its activation. Thus, glycosylation in the presence of HOFox and 0.05 equiv. of TMSOTf produced disaccharide 3 in 15 min in 72% yield (entry 7).

Table 1.

NIS/HOFox mediated glycosidations of donor 1 with acceptor 2.

After optimization of the basic reaction conditions, we investigated glycosidation of glycosyl donor 1 with other glycosyl acceptors that differ in their reactivity. The key results of this study are summarized in Table 2. Glycosidations of donor 1 were first conducted with hindered secondary glycosyl acceptors 4, 6 and 8.^[14] The respective disaccharides 5, 7 and 9^[15] were obtained within 3–4 h in yields of 42–71% (entries 1–3). Glycosidation with partially benzoylated deactivated primary 6-OH acceptor 10^[16] was also performed. This reaction gave the corresponding disaccharide 11 in 58% yield within 4 h (entry 4). The reactive 6-OH galactosyl acceptor 12 was glycosylated, and disaccharide 13^[17] was obtained in 76% yield (entry 5). Glycosylation of reactive aliphatic acceptors such as primary acceptor n-butanol 14 and more sterically hindered tertiary acceptor 1-adamantanol 16 proceeded smoothly and produced glycosides 15 and 17 nearly quantitatively (99%) in 30 min and 45 min (entries 6 and 7).

Table 2.

HOFox-catalyzed glycosidation of thiogalactoside donor 1 with various glycosyl acceptors.

After constructing different types of β-galactosidic linkages, we moved on to expand this acid-free glycosylation method to glycosidations of thioglycosides of other sugar series. For that, we first proceeded with glycosidation of per-benzylated thiogalactoside donor 18^[13] with acceptor 2 under standard reaction conditions. This glycosylation afforded disaccharide 19^[18] in a good yield of 80% in 2 h, albeit with no stereoselectivity (α/β=1.2/1, entry 1, Table 3). Glycosylation of the secondary 3-OH glycosyl acceptor 6 produced the corresponding disaccharide 20^[15] in a lower yield of 58% in 4 h (α/β=1.2/1, entry 2). Glycosidations of less reactive mannosyl donor 21^[19] under identical reaction conditions were slower. Thus, glycosylation of 6-OH acceptor 2 produced α-linked disaccharide 22^[20] in 50% yield after 12 h (entry 3). Glycosylation of n-butanol 14 afforded α-linked glycoside 23^[21] in 51% yield in 10 h (entry 4). Some of the glycosylation reactions were repeated with increased amount of promoter system (NIS/HOFox), however no significant improvement was noticed (see the Supporting Information for further details). We then moved on to investigate glycosyl donors with the d-gluco configuration. Glycosylation of glucosyl donor 24^[22] with glycosyl acceptor 2 proceeded smoothly and afforded disaccharide 25^[18] in 87% yield in 4 h (entry 5). Glycosidation of benzylated glucosyl donor 26^[23] with acceptor 2 produced disaccharide 27^[18] in 67% yield in 6 h (α/β=2.0/1, entry 6).

Table 3.

Expanding the scope of HOFox-catalyzed glycosylation with various donors and acceptors of other sugar series.

^[a]NIS 2.0 equiv.;

^[b]NIS 1.2 equiv., HOFox 0.5 equiv.

After investigating the activation of various ethylthio glycosides, we were curious to see whether other thioglycoside leaving groups would act accordingly. For this study we selected the S-tolyl (STol) leaving group because STol glycosides are better suited for large-scale preparations,^[24] can be readily pre-activated,^[25] and a comprehensive reactivity database compiled by Wong specifically for STol glycosides is available.^[26] Glycosidation of tolylthio glucosyl donor 28^[27] with acceptor 2 proceeded smoothly and afforded disaccharide 27 in an excellent yield of 97% in 6 h (α/β=1.1/1, entry 7). Glycosidation of glucosyl donor 28 with galactosyl acceptor 12 was somewhat slower, which was reflected in a lower yield (72%) of disaccharide 29^[28] obtained in 8 h (α/β=1.0/1, entry 8). Glycosylation with less reactive secondary 4-OH acceptor 8 proceeded even slower, and the corresponding disaccharide 30^[29] was obtained in 40% yield in 10 h (α/β= 1/1.1, entry 9). Glycosylation of donor 28 with 1-adamantanol 16 gave the corresponding glycoside 31^[30] in an excellent yield of 91% in 4 h (α/β=1.0/1, entry 10).

In further expansion of the approach, we investigated conformationally super-armed donor 32^[31] equipped with silyl protecting groups. Glycosidation of glucosyl donor 32 with acceptor 2 smoothly afforded the desired disaccharide 33^[31] in an excellent yield of 93% with predominant α-stereoselectivity (α/β=5.0/1, entry 11). No loss of TBS protecting groups, which was noted as a major side reaction in our previous unrelated study,^[31] was noted in NIS/HOFox-promoted reactions. Glycosidation of relatively unreactive 2-phthalimido-protected aminosugar donor 34^[32] with electronically deactivated glycosyl acceptor 10 was also proven feasible. However, for the best results, 2.0 equiv. NIS was necessary, and even then, the reaction required 24 h and disaccharide 35 was obtained in 40% yield. To demonstrate the efficacy of the new reaction, we attempted selective activation of the SEt leaving group in donor 1 over the S-thiazolinyl (STaz) anomeric group of acceptor 36. To obtain a better outcome, we used increased amounts of the promoters, 1.2 equiv. NIS and 0.5 equiv. HOFox. Under these reaction conditions, disaccharide 37 equipped with the anomeric STaz leaving group was obtained in 65% yield in 16 h (entry 13).

Conclusion

In summary, presented herein is the discovery of the direct activation of thioglycosides in the regenerative fashion. We demonstrated that in most cases, only 1 equiv. NIS and catalytic HOFox (0.2 equiv.) are needed to promote glycosidation of various thioglycosides. One advantage of this approach is that it does not require a strong acid as a co-promoter, which allows to maintain neutral reaction conditions during glycosylation. One potential disadvantage of this approach is that it is much less efficient with poorly reactive substrates, glycosylation of which would require the use of additional quantities of HOFox and/or Lewis acid additives. We believe that this approach would be particularly advantageous with the HPLC pump-based reagent delivery automated set-up that is being developed in our labs.

Experimental Section

General experimental:

The reactions were performed using commercial reagents, and the ACS grade solvents used for reactions were purified and dried in accordance with standard procedures. Column chromatography was performed on silica gel 60 (70–230 mesh); reactions were monitored by TLC on Kieselgel 60 F₂₅₄. The compounds were detected by examination under UV light and by charring with 10% sulfuric acid in methanol. Solvents were removed under reduced pressure at <40°C. CH₂Cl₂ was distilled from CaH₂ directly prior to the application. Molecular sieves (3 Å), used for reactions, were crushed, and activated in vacuo at 390°C for 8 h in the first instance and then for 2–3 h at 390°C directly prior to application. Optical rotations were measured at “JASCO P-2000” polarimeter. ¹H NMR spectra were recorded at 300 MHz, ¹³C NMR spectra were recorded at 75 MHz. The ¹H NMR chemical shifts are referenced to tetramethylsilane (TMS, δ_C=0 ppm) for ¹H NMR spectra for solutions in CDCl₃. The ¹³C NMR chemical shifts are referenced to the central signal of CDCl₃ (δ_C=77.00 ppm) for solutions in CDCl₃. HRMS analysis was performed using Agilent 6230 ESI TOF LC/MS mass spectrometer.

Synthesis of reagents and building blocks

3,3-Difluoroxindole (HOFox)

3,3-Difluoroxindole (HOFox) was obtained from Isatin and DAST as previously described, and its analytical data were in accordance with that previously reported.^[8,33]

2-Thiazolinyl 2-O-benzoyl-3,4-di-O-benzyl-1-thio-β-d-galactopyranoside (36):

A solution of ethyl 2-O-benzoyl-3-O-benzyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (38,^[34] 590 mg, 1.16 mmol) and activated molecular sieves 3 Å (885 mg) in dry CH₂Cl₂ (20 mL) was stirred under argon for 1 h at rt. The mixture was cooled to 0°C, Br₂ (0.077 mL, 1.51 mmol) was added, and the resulting mixture was kept for 15 min at 0°C. After that, the volatiles were removed under reduced pressure at rt. The crude residue was dissolved in dry MeCN (10 mL), NaSTaz^[35] (650 mg, 4.6 mmol) was added, and the resulting mixture was stirred under argon for 2 h at rt. After that, the solid was filtered-off and rinsed successively with toluene. The combined filtrate (≈35 mL) was washed with 1% aq. NaOH (≈15 mL) and water (3×15 mL). The organic phase was separated, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford 2-thiazolinyl 2-O-benzoyl-3-O-benzyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (39) as a clear film in 51% yield over two steps (330 mg). Analytical data for 39: R_f=0.50 (ethyl acetate/hexane, 2/3, v/v); [α]_D²⁰ +6.9 (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ=3.22–3.29 (m, 2H, CH₂N), 3.63 (s, 1H, H-5), 3.93 (dd, 1H, J_3,4=3.3 Hz, H-3), 4.06 (d, 1H, J_6a,6b =12.2 Hz, 6a), 4.28–4.47 (m, 4H, H-4, 6b, CH₂S), 4.68 (dd, 2H, CH₂Ph), 5.54 (s, 1H, CHPh), 5.75 (dd, 1H, J_2,3=9.5 Hz, H-2), 6.18 (d, 1H, J_1,2=9.3 Hz, H-1), 7.24–7.26 (m, 4H, aromatic), 7.40–7.61 (m, 9H, aromatic), 7.99 ppm (d, 2H, J=7.2 Hz, aromatic); ¹³C NMR (75 MHz, CDCl₃): δ=28.6, 51.3, 68.4, 68.5 (×2), 69.0, 71.4, 73.0, 77.2, 82.8, 101.1, 126.2 (×2), 127.7 (×2), 127.9, 128.3 (×2), 128.4 (×5), 129.2, 130.0 (×2), 133.4, 137.4, 137.5, 165.9 ppm; HR-FAB MS [M+Na]⁺ calcd for C₃₀H₂₉NO₆S₂Na 586.1334, found 586.1339.

A 1m solution of BH₃ in THF (3.0 mL) was added dropwise to a solution of 39 (290 mg, 0.51 mmol) in CH₂Cl₂ (9 mL). The resulting solution was cooled to 0°C, TMSOTf (4.6 μL, 0.025 mmol) was added dropwise, and the resulting mixture was stirred for 1 h at 0°C. The reaction mixture was then allowed to warm to rt and stirred for additional 1 h. After that, the reaction was quenched with NaHCO₃ (≈2 mL), diluted with CH₂Cl₂ (100 mL), and washed with sat. aq. NaHCO₃ (50 mL) and water (2×50 mL). The organic phase was separated, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane gradient elution) to afford the title compound as a white amorphous solid in 53% yield (150 mg, 0.27 mmol). Analytical data for 36: R_f=0.30 (ethyl acetate/hexane, 2/3, v/v); [α]_D²⁰ +117.8 (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ=3.22–3.26 (m, 2H, CH₂N), 3.61–3.70 (m, 2H, H-5, 6a), 3.81 (m, 1H, H-6b), 3.89 (dd, 1H, J_3,4=2.5 Hz, H-3), 3.97 (d, 1H, J_4,5=2.0 Hz, H-4), 4.16–4.34 (m, 2H, CH₂S), 4.59–4.72 (m, 3H, CH₂Ph, CHPh), 5.02 (d, 1H, ²J=11.7 Hz, CHPh), 5.30 (s, 1H, OH), 5.76 (dd, 1H, J_2,3= 9.6 Hz, H-2), 6.11 (d, 1H, J_1,2=9.2 Hz, H-1), 7.21–7.26 (m, 4H, aromatic), 7.37–7.59 (m, 9H, aromatic), 7.99 ppm (d, 2H, J=7.7 Hz, aromatic); ¹³C NMR (75 MHz, CDCl₃): δ=28.5, 51.4, 61.8, 69.5, 72.5 (×2), 74.4, 77.1, 77.3, 80.1, 83.1, 127.7 (×3), 127.9, 128.1, 128.4 (×4), 128.5 (×2), 129.1, 129.9 (×3), 133.4, 137.2, 137.9, 166.0 ppm; HR-FAB MS [M+Na]⁺ calcd for C₃₀H₃₁NO₆S₂Na 588.1490, found 588.1501.

Synthesis of Glycosides

Typical glycosylation procedure:

A mixture of thioglycoside precursor (30.0 mg, 0.03–0.05 mmol), glycosyl acceptor (0.02–0.04 mmol), and freshly activated molecular sieves (3 Å, 90 mg) in dry CH₂Cl₂ (1.0 mL) was stirred under argon for 1 h at rt. After that, N-iodosuccinimide (NIS, 0.03–0.05 mmol) and HOFox (0.005–0.02 mmol) were added and the reaction mixture was stirred for the time specified in Tables 1–3. The solids were filtered off through a pad of Celite and rinsed successively with CH₂Cl₂. The combined filtrate (≈20 mL) was washed with sat. aq. Na₂S₂O₃ (5 mL), sat. aq. NaHCO₃ (5 mL) and brine (2×5 mL). The organic phase was separated, dried with MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-hexanes gradient elution) to afford a glycoside product in yields listed in Tables 1–3 and below. Anomeric ratios (if applicable) were determined by comparison of the integral intensities of relevant signals in ¹H NMR spectra.

Methyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2,3,4-tri-O-benzyl-β-d-glucopyranoside (3)

Methyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2,3,4-tri-O-benzyl-β-d-glucopyranoside (3) was obtained from thioglycoside 1^[13] and glycosyl acceptor 2^[14] by the general glycosylation method as a clear film in 92% yield. Analytical data for 3 was in accordance with that reported previously.^[15]

Methyl 2-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-3,4,6-tri-O-benzyl-α-d-glucopyranoside (5)

Methyl 2-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-3,4,6-tri-O-benzyl-α-d-glucopyranoside (5) was obtained from thioglycoside 1 and glycosyl acceptor 4^[14] by the general glycosylation method as a clear film in 53% yield. Analytical data for 5: R_f=0.60 (ethyl acetate/hexane, 2/3, v/v); [α]_D²¹ +62.9 (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ=3.34 (s, 3H, OCH₃), 3.55–3.72 (m, 9H, H-2, 3′, 4, 5, 5′, 6a, 6b, 6a′, 6b′), 3.87 (dd, 1H, J_3,4=9.2 Hz, H-3), 4.01 (d, 1H, J_4′,5′=2.7 Hz, H-4′), 4.32 (d, 1H, ²J=10.9 Hz, CHPh), 4.40–4.47 (m, 5H, 5 × CHPh), 4.57–4.63 (m, 5H, 5 × CHPh), 4.81 (d, 1H, J_1′,2′=7.8 Hz, H-1′), 4.91 (d, 1H, J_1,2=3.0 Hz, H-1), 4.98 (d, 1H, ²J=11.8 Hz, CHPh), 5.75 (dd, 1H, J_2′,3′=9.1 Hz, H-2′), 6.94–6.99 (m, 4H, aromatic), 7.06–7.45 (m, 29H, aromatic), 7.81 ppm (d, 2H, J=7.8 Hz, aromatic); 13C NMR (75 MHz, CDCl₃): δ=55.2, 68.4, 69.6, 71.6, 71.7, 72.3, 73.4, 73.5, 74.4, 74.8, 75.0, 76.5, 80.1, 80.4, 81.0, 99.5, 102.5, 126.9, 127.1 (×2), 127.4, 127.6 (×6), 127.7 (×4), 127.9 (×6), 128.1 (×5), 128.2 (×5), 128.3 (×2), 128.5 (×2), 129.8 (×3), 132.6, 137.3, 137.6, 137.9, 138.1, 138.4, 138.7, 165.1 ppm; HR-FAB MS [M+Na]⁺ calcd for C₆₂H₆₄O₁₂Na 1023.4295, found 1023.4312.

Methyl 3-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2,4,6-tri-O-benzyl-α-d-glucopyranoside (7)

Methyl 3-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2,4,6-tri-O-benzyl-α-d-glucopyranoside (7) was obtained from thioglycoside 1 and glycosyl acceptor 6^[14] by the general glycosylation method as a clear film in 71% yield. Analytical data for 7 was in accordance with that reported previously.^[15]

Methyl 4-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (9)

Methyl 4-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (9) was obtained from thioglycoside 1 and glycosyl acceptor 8^[14] by the general glycosylation method as a clear film in 42% yield. Analytical data for 9 was in accordance with that reported previously.^[15]

Methyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2,3,4-tri-O-benzoyl-α-d-glucopyranoside (11)

Methyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2,3,4-tri-O-benzoyl-α-d-glucopyranoside (11) was obtained from thioglycoside 1 and glycosyl acceptor 10^[16] by the general glycosylation method as a white amorphous solid in 58% yield. Analytical data for 11: R_f=0.50 (ethyl acetate/hexane, 2/3, v/v); [α]_D²¹ +28.5 (c=1, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ=3.00 (s, 3H, OCH₃), 3.53–3.65 (m, 5H, H-3, 5′, 6a, 6a′, 6b′), 3.98–4.04 (m, 2H, J_4′,5′=2.2, J_6a,6b = 11.1 Hz, H-4′, 6b), 4.16 (dd, 1H, J_5,6a=8.7, J_5,6b=9.7 Hz, H-5), 4.32–4.61 (m, 6H, H-1′, 5 × CHPh), 4.80 (d, 1H, J_1,2=3.6 Hz, H-1), 4.95 (d, 1H, ²J=11.6 Hz, CHPh), 5.06 (dd, 1H, J_2,3=10.2 Hz, H-2), 5.27 (dd, 1H, J_4,5=9.7 Hz, H-4), 5.66 (dd, 1H, J_2′,3′ = 8.7 Hz, H-2′), 6.03 (dd, 1H, J_3,4=9.8 Hz, H-3), 7.17–7.59 (m, 27H, aromatic), 7.78 (d, 2H, J=7.8 Hz, aromatic) 7.85 (d, 2H, J=7.9 Hz, aromatic), 7.90 (d, 2H, J=7.7 Hz, aromatic), 8.01 ppm (d, 2H, J=7.8 Hz, aromatic); ¹³C NMR (75 MHz, CDCl₃): δ=30.9, 54.8, 68.2, 68.5, 69.5, 70.4, 71.6, 71.7, 72.0, 72.2, 73.5, 74.4, 76.5, 79.6, 96.1, 101.9, 127.5, 127.6 (×4), 127.8 (×2), 127.9 (×3), 128.1 (×4), 128.2 (×4), 128.3 (×6), 128.4 (×2), 128.7, 129.0, 129.2, 129.6 (×2), 129.8 (×4), 130.2, 132.9, 133.2, 133.3, 137.6, 137.7, 138.3, 165.3, 165.4, 165.6, 165.7 ppm; HR-FAB MS [M+Na]⁺calcd for C₆₂H₅₈O₁₅Na 1065.3673, found 1065.3691.

6-O-(2-O-Benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-1,2:3,4-di-O-isopropylidene-α-d-galactopyranose (13)

6-O-(2-O-Benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-1,2:3,4-di-O-isopropylidene-α-d-galactopyranose (13) was obtained from thioglycoside 1 and glycosyl acceptor 12 by the general glycosylation method as a clear film in 76% yield. Analytical data for 12 was in accordance with that reported previously.^[17]

n-Butyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranoside (15)

n-Butyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranoside (15) was obtained from thioglycoside 1 and n-butanol 14 by the general glycosylation method as a white amorphous solid in 99% yield. Analytical data for 15: R_f=0.70 (ethyl acetate/hexane, 2/3, v/v); [α]_D²¹ +3.6 (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ=0.69 (t, 3H, CH₃), 1.18–1.49 (m, 4H, 2 × CH₂), 3.43 (m, 1H, OCH^a), 3.62–3.73 (m, 4H, H-3, 5, 6a, 6b), 3.85 (m, 1H, OCH^b), 4.01 (d, 1H, J_4,5=2.7 Hz, H-4), 4.41–4.46 (m, 4H, J_1,2=9.1 Hz, H-1, 3 × CHPh), 4.65 (dd, 2H, CH₂Ph), 4.99 (d, 1H, ²J=11.7 Hz, CHPh), 5.63 (dd, 1H, H-2), 7.14–7.60 (m, 18H, aromatic), 8.03 ppm (d, 2H, J=7.5 Hz, aromatic); ¹³C NMR (75 MHz, CDCl₃): δ=13.5, 18.8, 29.6, 31.3, 68.6, 69.3, 71.5, 71.9, 72.3, 73.5, 74.3, 79.8, 101.5, 127.4, 127.5, 127.6 (×2), 127.8, 127.9 (×2), 128.1 (×2), 128.2 (×3), 128.3 (×2), 128.4 (×2), 129.7 (×2), 130.3, 132.8 (×2), 137.6, 137.8, 138.4, 165.3 ppm; HR-FAB MS [M+Na]⁺ calcd for C₃₈H₄₂O₇Na 633.2828, found 633.2870.

1-Adamantyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranoside (17)

1-Adamantyl 2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranoside (17) was obtained from thioglycoside 1 and 1-adamantanol 16 by the general glycosylation method as a white amorphous solid in 99% yield. Analytical data for 17: R_f=0.70 (ethyl acetate/hexane, 2/3, v/v); [α]_D²¹ +24.7 (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ=1.45–1.78 (m, 12H, Ada), 2.02 (s, 3H, Ada), 3.56–3.69 (m, 4H, H-3, 5, 6a, 6b), 3.98 (d, 1H, J_4,5=2.6 Hz, H-4), 4.40–4.52 (m, 3H, 3 × CHPh), 4.62 (dd, 2H, CH₂Ph), 4.75 (d, 1H, J_1,2=7.9 Hz, H-1), 4.98 (d, 1H, ²J=11.7 Hz, CHPh) 5.58 (dd, 1H, J_2,3=9.8 Hz, H-2), 7.11–7.60 (m, 18H, aromatic), 8.01 ppm (d, 2H, J=7.7 Hz, aromatic); ¹³C NMR (75 MHz, CDCl₃): δ=30.7 (×5), 36.3 (×3), 42.4 (×4), 69.3, 71.6, 72.3, 73.6, 73.7, 74.5, 75.0, 80.3, 94.5, 127.7 (×2), 127.8 (×2), 127.9, 128.0 (×2), 128.3 (×2), 128.4 (×3), 128.6 (×2), 129.9 (×3), 130.6, 132.9, 137.9, 138.1, 138.7, 165.3 ppm; HR-FAB MS [M+Na]⁺ calcd for C₄₄H₄₈O₇Na 711.3298, found 711.3307.

Methyl 6-O-(2,3,4,6-tetra-O-benzyl-α/β-d-galactopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (19)

Methyl 6-O-(2,3,4,6-tetra-O-benzyl-α/β-d-galactopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (19) was obtained from thioglycoside 18^[13] and glycosyl acceptor 2 by the general glycosylation method as a clear film in 80% yield (α/β=1.2/1). Analytical data for 19 was in accordance with that reported previously.^[18]

Methyl 3-O-(2,3,4,6-tetra-O-benzyl-α/β-d-galactopyranosyl)-2,4,6-tri-O-benzyl-α-d-glucopyranoside (20)

Methyl 3-O-(2,3,4,6-tetra-O-benzyl-α/β-d-galactopyranosyl)-2,4,6-tri-O-benzyl-α-d-glucopyranoside (20) was obtained from thioglycoside 18 and glycosyl acceptor 6 by the general glycosylation method as a clear film in 58% yield (α/β=1.2/1). Analytical data for 20 was in accordance with that reported previously.^[15]

Methyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-α-d-mannopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (22)

Methyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-α-d-mannopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (22) was obtained from thioglycoside 21^[19] and glycosyl acceptor 2 by the general glycosylation method as a colorless syrup in 50% yield. Analytical data for 22 was in accordance with that reported previously.^[20]

n-Butyl 2-O-benzoyl-3,4,6-tri-O-benzyl-α-d-mannopyranoside (23)

n-Butyl 2-O-benzoyl-3,4,6-tri-O-benzyl-α-d-mannopyranoside (23) was obtained from thioglycoside 21 and n-butanol 14 by the general glycosylation method as a colorless syrup in 51% yield. Analytical data for 23 was in accordance with that reported previously.^[21]

Methyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-glucopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (25)

Methyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-glucopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (25) was obtained from thioglycoside 24^[22] and glycosyl acceptor 2 by the general glycosylation method as a clear film in 87% yield. Analytical data for 25 was in accordance with that reported previously.^[18]

Methyl 6-O-(2,3,4,6-tetra-O-benzyl-α/β-d-glucopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (27)

Methyl 6-O-(2,3,4,6-tetra-O-benzyl-α/β-d-glucopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (27) was obtained from thioglycoside 26^[36] and glycosyl acceptor 2 by the general glycosylation method as a clear film in 97% yield (α/β=1.1/1). Analytical data for 27 was in accordance with that reported previously.^[18]

6-O-(2,3,4,6-Tetra-O-benzyl-α/β-d-glucopyranosyl)-1,2:3,4-di-O-isopropylidene-α-d-galactopyranose (29)

6-O-(2,3,4,6-Tetra-O-benzyl-α/β-d-glucopyranosyl)-1,2:3,4-di-O-isopropylidene-α-d-galactopyranose (29) was obtained from thioglycoside 28^[27] and glycosyl acceptor 12 by the general glycosylation method as a clear film in 72% yield (α/β=1.0/1). Analytical data for 29 was in accordance with that reported previously.^[28]

Methyl 4-O-(2,3,4,6-tetra-O-benzyl-α/β-d-glucopyranosyl)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (30)

Methyl 4-O-(2,3,4,6-tetra-O-benzyl-α/β-d-glucopyranosyl)-2,3,6-tri-O-benzyl-α-d-glucopyranoside (30) was obtained from thioglycoside 28 and glycosyl acceptor 8 by the general glycosylation method as a clear film in 40% yield (α/β=1/1.1). Analytical data for 30 was in accordance with that reported previously.^[29]

1-Adamantyl 2,3,4,6-tetra-O-benzyl-α/β-d-glucopyranoside (31)

1-Adamantyl 2,3,4,6-tetra-O-benzyl-α/β-d-glucopyranoside (31) was obtained from thioglycoside 28 and 1-adamantanol 16 by the general glycosylation method as a white amorphous solid in 91% yield (α/β=1.0/1). Analytical data for 31 was in accordance with that reported previously.^[30]

Methyl 6-O-(6-O-benzoyl-2-O-benzyl-3,4-di-O-tert-butyldimethyl-silyl-α/β-d-glucopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (33)

Methyl 6-O-(6-O-benzoyl-2-O-benzyl-3,4-di-O-tert-butyldimethyl-silyl-α/β-d-glucopyranosyl)-2,3,4-tri-O-benzyl-α-d-glucopyranoside (33) was obtained from thioglycoside 32^[31] and glycosyl acceptor 2 by the general glycosylation method as a colorless syrup in 93% yield (α/β=5.0/1). Analytical data for 33 was in accordance with that reported previously.^[31]

Methyl 6-O-(4,6-di-O-benzyl-2-deoxy-3-O-fluorenylmethoxycarbonyl-2-phthalimido-β-d-glucopyranosyl)-2,3,4-tri-O-benzoyl-α-d-glucopyranoside (35)

Methyl 6-O-(4,6-di-O-benzyl-2-deoxy-3-O-fluorenylmethoxycarbonyl-2-phthalimido-β-d-glucopyranosyl)-2,3,4-tri-O-benzoyl-α-d-glucopyranoside (35) was obtained from thioglycoside 34^[32] and glycosyl acceptor 10 by the general glycosylation method as a clear film in 40% yield. Analytical data for 35: R_f=0.60 (ethyl acetate/hexane, 2/3, v/v); [α]_D²¹ +120.0 (c 1, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ=3.04 (s, 3H, OCH₃), 3.61 (dd, 1H, J_5,6a=7.8, J_6a,6b =10.7 Hz, 6a), 3.58–3.76 (m, 3H, H-5′, 6_{a′, 6b′}), 3.85–4.03 (m, 3H, H-4′, OCOCH₂CH), 4.07–4.18 (m, 3H, H-5, 6b, OCOCH₂CH), 4.40–4.65 (m, 5H, H-2′, 2 × CH₂Ph), 4.70 (d, 1H, J_1,2=3.6 Hz, H-1), 5.07 (dd, 1H, J_2,3=10.2 Hz, H-2), 5.29 (dd, 1H, J_4,5=9.9 Hz, H-4), 5.40 (d, 1H, J_1′,2′ = 8.4 Hz, H-1′), 5.71 (dd, 1H, J_3′,4′ =8.9 Hz, H-3′), 6.03 (dd, 1H, J_3,4=9.8 Hz, H-3), 7.11–7.51 (m, 28H, aromatic), 7.67–7.93 ppm (m, 9H, aromatic); ¹³C NMR (75 MHz, CDCl₃): δ=29.7, 46.3, 54.4 (×2), 68.0, 68.2, 69.3 (×2), 70.2 (×2), 71.9, 73.4, 74.7, 77.2, 96.3, 98.8, 114.2, 119.8 (×4), 125.0 (×2), 125.2 (×2), 127.1 (×4), 127.6 (×2), 127.8 (×3), 128.1 (×2), 128.2 (×2), 128.3 (×7), 128.6, 128.9, 129.1, 129.6 (×3), 129.7 (×4), 129.8 (×3), 133.0, 133.2, 133.3, 137.6, 137.9, 140.9, 141.0, 142.9, 143.2, 154.6, 165.1, 165.6 ppm (×2); HR-FAB MS [M+Na]⁺ calcd for C₇₁H₆₁NO₁₇Na 1222.3837, found 1222.3858.

2-Thiazolinyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,4-di-O-benzyl-1-thio-β-d-galactopyranoside (37)

2-Thiazolinyl 6-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-β-d-galactopyranosyl)-2-O-benzoyl-3,4-di-O-benzyl-1-thio-β-d-galactopyranoside (37) was obtained from thioglycoside 1 and glycosyl acceptor 36 by the general glycosylation method as a clear film in 65% yield. Analytical data for 37: R_f=0.50 (ethyl acetate/hexane, 2/3, v/v); [α]_D²¹ +25.8 (c=1.0, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ=3.10–3.19 (m, 2H, CH₂N), 3.55–3.69 (m, 7H, H-3, 3′, 5, 5′, 6a, 6a′, 6b′), 3.95 (d, 1H, J_4,5=2.4 Hz, H-4), 3.99–4.03 (m, 3H, H-4′, 6b, SCH^a), 4.18 (d, 1H, ²J=12.4 Hz, CHPh), 4.26 (m, 1H, SCH^b), 4.34 (d, 1H, ²J=12.1 Hz, CHPh), 4.42 (s, 2H, CH₂Ph), 4.56 (d, 1H, J_1′,2′ =7.9 Hz, H-1′), 4.61–6.71 (m, 4H, 2 × CH₂Ph), 4.84 (d, 1H, ²J=1.2 Hz, CHPh), 5.01 (d, 1H, ²J=1.9 Hz, CHPh), 5.57–5.68 (m, 2H, J_2,3 =9.6, J2′,3′ =9.6 Hz, H-2, 2′), 5.95 (d, 1H, J_1,2=9.3 Hz, H-1), 7.01–7.57 (m, 31H, aromatic), 7.91 (d, 2H, J=7.5 Hz, aromatic), 8.04 ppm (d, 2H, J = 7.5 Hz, aromatic); ¹³C NMR (75 MHz, CDCl₃): δ=29.7 (×2), 30.9, 71.5 (×2), 71.6, 71.7, 71.8, 72.1, 72.4, 73.5, 73.6, 74.6, 74.8, 75.3, 79.6, 79.8, 83.0, 101.1, 113.8, 127.4 (×4), 127.5 (×2), 127.6, 127.7 (×3), 127.9 (×3), 128.1 (×6), 128.2 (×5), 128.3, 128.4 (×2), 128.5 (×2), 129.2, 129.8 (×2), 129.9 (×4), 133.2, 137.4, 137.5, 137.7, 138.4 (×2), 165.2, 165.9 ppm; HR-FAB MS [M+Na]⁺ calcd for C₆₄H₆₃NO₁₂S₂Na 1124.3684, found 1124.3717.