Streamlined access to carbohydrate building blocks: methyl 2,4,6-tri-O-benzyl-α-D-glucopyranoside

Abstract

Presented herein is an improved synthesis of a common 3-OH glycosyl acceptor. This compound is a building block that is routinely synthesized by many research groups to be used in glycosylation refinement studies. The only known direct synthesis by Koto lacks regioselectivity and relies on chromatography separation using hazardous solvents. Our improved synthetic approach relies on Koto’s selective benzylation protocol, but it is followed by acylation-purification-deacylation sequence. Although this approach involves additional manipulations, it provides consistent results and is superior to other indirect strategies. Also obtained, albeit in minor quantities, is 4-OH acceptor, another common building block.

Graphical Abstract

The development of accessible methods for glycan production is essential for further innovations and practical applications in all areas of glycosciences.¹ Poor accessibility to sugar building blocks hampers development of all synthetic methodologies and strategies platforms. “Unlike the synthesis of peptides and oligonucleotides, there are no universal building blocks or methods for the synthesis of all glycans.”² Researchers experience significant setbacks because they have to continue to remake simple building blocks. As Seeberger noted “differentially protected monosaccharide building blocks is currently the bottleneck for chemical synthesis.”³ Some 15 years later, most bench time is still dedicated to making building blocks. In contribution to the global effort of the glycoscience community towards making building blocks more accessible, reported herein is the development of selectively protected building blocks of D-glucose as advanced synthetic intermediates. D-Glucose, the predominant monosaccharide in Nature, is also among major components of bacterial glycans and the mammalian glycome.⁴ D-Glucose building blocks are often the first compounds tested in new reactions and applications. Every synthetic glycoscience lab makes glucose building blocks. Paradoxically, it remains the hardest sugar for selective protection due to its trans-trans-trans, all-equatorial 2,3,4-triol arrangement.

The benzyl protecting group is widely used in carbohydrate chemistry due to its stability. As once brilliantly stated by Fraser-Reid, “protecting groups do more than protect.”⁵ Indeed, protecting groups of both glycosyl donors and glycosyl acceptors are known to control all types of selectivity: regio-, stereo-, and chemoselectivity in glycosylation. Protecting groups may also have a powerful effect on the building block reactivity in glycosylation.^6,7 It is benzylated building blocks that created the basis for discovering the armed-disarmed approach by Fraser-Reid.^8–10 Partially benzylated compounds are known to give rise to differential levels of reactivity ranging from the electronically superdisarmed¹¹ to electronically superarmed building blocks.^12–15 Partially benzylated building blocks are also common glycosyl acceptors that are routinely synthesized by many research groups to be used in glycosylation reaction. Recently, we described an improved direct synthesis of glucose 1,3,4,6-tetra-O-acetate (2-OH glucose),^16,17 and herein we report a streamlined synthesis of 3-OH glucose. Also obtained alongside, albeit in minor quantities, is 4-OH glucose, another common building block.

A partial regioselective benzylation of polyols is relatively rare.¹⁸ One such example was described by Koto et al. wherein methyl glucoside 1 was converted into methyl 2,4,6-tri-O-benzyl-α-d-glucopyranoside 2 in one step in 61% yield.¹⁹ According to Koto et al., this was achieved by controlled heating with neat BnCl in the presence of NaH added portionwise. To adapt this approach to a large-scale preparation of compound 2, we attempted to reproduce Koto’s protocol. Benzylation of methyl α-d-glucopyranoside 1 was carried out with benzyl chloride in presence of sodium hydride for 3 h at 100 °C as depicted in Scheme 1. We indeed found that the main component of the reaction mixture was 3-OH derivative 2, along with its 4-OH regioisomer 3, and per-benzylated derivative 4. The presence of the side products was not disclosed by Koto et al.

Scheme 1.

Selective benzylation of methyl α-d-glucopyranoside 1

In an effort to separate the reaction mixture by column chromatography, we managed to separate off compound 4 fairly easily, and it was isolated in 5 or 20% yield, depending on the reaction conditions employed. However, separation of regioisomers 2 and 3 was problematic. It should be noted that the chromatography solvent system reported by Koto, benzene/2-butanone, was neither available to us nor desirable for safety reasons. Our best attempt involved column chromatography using slow 0.2% gradient elution of ethyl acetate in dichloromethane. This allowed us to isolate pure 2 in 29% yield. A better separation of the mixture of compounds 2 and 3 by TLC was achieved using a ternary system comprising hexane/ DCM/ ethyl acetate (2.0/ 2.5/ 0.25, v/v/v). However, we could not achieve improved separation using column chromatography using this solvent-system.

To achieve a more reliable separation of the mixture of compounds 2 and 3, we decided to derivatize the mixture. In the first route, benzoylation was chosen, which was affected using benzoyl chloride in the presence of N,N-dimethylaminopyridine (DMAP) in dry pyridine for 2.5 h at 50 °C. This afforded a mixture of compounds 5 and 6, which was easily separated by column chromatography to afford 3-O-benzoylated compound 5²⁰ in 71% yield. Also obtained was 4-O-benzoylated compound 6²¹ in 15% yield. Alternatively, benzoylation could be achieved using benzoic acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and DMAP in 1,2-dichloroethane at 50 °C. Under these reaction conditions, compounds 5 and 6 were obtained in 65% and 12% yield, respectively.

On the other hand, acetylation of a mixture of compounds 2 and 3 also allowed a straightforward chromatographic separation of the products leading to 3-O-acetyl derivative 7 in 74% yield and its 4-O-acetyl counterpart 8²² in 14% yield (Scheme 2). Finally, deprotection of compounds 5, 6 and 7 under Zemplen conditions produced the respective derivatives 2 and 3 in 91–93% yield as depicted in Scheme 2.

Scheme 2.

Separation of the mixture of 2 and 3 via acylation-purification-saponification sequence

In conclusion, reported herein is an improved synthesis of partially benzylated compound 2. Routinely synthesized by many research groups, this building block is commonly used as a model 3-OH glycosyl acceptor. The only known direct synthesis by Koto lacks regioselectivity, and could not be reproduced accurately because it relies on chromatography using uncommon and hazardous solvents. This is the main reason others still synthesize 3-OH acceptor via multistep derivatization approaches.^23,24 While our synthetic approach relies on Koto’s selective benzylation, it is followed by acylation-purification-deacylation sequence. These additional steps are straightforward and high-yielding, and the main payoff is in the simplified separation of the mixture of regioisomers. Also obtained, albeit in minor quantities, is 4-OH acceptor, another common building block.

Experimental Part

General.

The reactions were performed using commercial reagents. 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 5% sulfuric acid in methanol. Solvents were removed under reduced pressure at <40 °C. ClCH₂CH₂Cl (1,2-DCE) was distilled from CaH₂ directly prior to application. Optical rotations were measured at ‘Jasco P-2000’ polarimeter. ¹H NMR spectra were recorded in CDCl₃ at 300 or 400 MHz, ¹³C NMR spectra were recorded at 75 or 100 MHz. The ¹H NMR chemical shifts are referenced to tetramethyl silane (TMS, δ_H = 0 ppm) or CDCl₃ (δ_H = 7.26 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. Compound purity or compound ratios were accessed or calculated by comparing of the integration intensities of the relevant signals in their ¹H NMR spectra. Accurate mass spectrometry determinations were performed using Agilent 6230 ESI TOF LCMS mass spectrometer.

Methyl 2,4,6-tri-O-benzyl-α-d-glucopyranoside (2) and methyl 2,3,6-tri-O-benzyl-α-d-glucopyranoside (3).

Conditions A.

A 60% suspension of sodium hydride in mineral oil (1.6 g, 40.01 mmol) was added to a mixture containing methyl α-d-glucopyranoside (1, 5.0 g, 25.75 mmol) and benzyl chloride (23.7 mL, 206 mmol), and the resulting mixture was stirred under argon for 5 min at rt. After that, additional sodium hydride (60% suspension in mineral oil, 1.07 g, 26.76 mmol) was added, and the resulting mixture was heated for 3 h at 100 °C. Additional sodium hydride (60% suspension in mineral oil, 1.50 g, 36.0 mmol) was then added, and the resulting mixture was heated for 2 h at 100 °C. The reaction mixture was allowed to cool to rt, poured into ice-water (100 mL) and stirred for 30 min. The mixture was extracted with CH₂Cl₂ (3 × 100 mL), and the combined organic extract was washed with water (2 × 50 mL). The organic phase was separated, dried with Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate – toluene, 5% gradient elution). Eluted first was methyl 2,3,4,6-tetra-O-benzyl α-d-glucopyranoside 4 isolated in 20% yield (2.84 g, 5.12 mmol). Also eluted was a mixture of compounds 2 and 3 as a clear syrup in a combined yield of 67% yield (7.95 g, 17.11 mmol, ratio 5.0/1). Second separation of the mixture of compounds 2 and 3 (3.0 g, 6.46 mmol) by column chromatography on silica gel (ethyl acetate–dichloromethane, 0.2% gradient elution) allowed pure compound 2 as a clear syrup in 29% yield (0.88 g, 1.9 mmol). Analytical data for 2: R_f = 0.45 (ethyl acetate/ hexane, 3/7, v/v); [α]_D²⁰ +40.1 (c = 1.0, CHCl₃); ¹H n.m.r. (300 MHz): δ 2.36 (s, 1H, 3-OH), 3.33 (s, 3H, CH₃), 3.43 (dd, 1H, J_2,3 = 9.6 Hz, H-2), 3.69 (dd, 1H, J_4,5 = 9.2 Hz, H-4), 3.69–3.74 (m, 3H, H-5, 6a, 6b), 4.10 (dd, 1H, J_3,4 = 9.2 Hz, H-3), 4.10–4.81 (m, 7H, H-1, 3 × CH₂Ph), 7.20–7.37 (m, 15H, aromatic) ppm; ¹³C{¹H} NMR (75 MHz): 55.1, 68.3, 69.5, 73.0, 73.4, 73.5, 74.5, 77.3,79.3, 97.4, 127.5, 127.6, 127.7 (x2), 127.8 (x2), 127.9, 128.0 (x2), 128.3 (x4), 128.5 (x2), 137.7, 137.7, 138.3 ppm; HRMS [M + Na]⁺: calcd for [C₂₈H₃₂O₆Na]⁺ 487.2097; found: 487.2098. Partial characterization data for compound 2 has been reported previously.^19,24,25

Conditions B.

A 60% suspension of sodium hydride in mineral oil (1.03 g, 25,75 mmol) was added portionwise to a vigorously stirred mixture of methyl α-d-glucopyranoside (1, 10.1 g, 52 mmol) in benzyl chloride (100 mL, 869 mmol) under argon at 85 °C. After 15 min, the reaction temperature was increased to 105 °C and additional NaH (60% in mineral oil, 5.24 g, 131 mmol) was added portionwise over a period of 15 min, and the resulting mixture was stirred for 3 h at 105 oC. After that, the reaction mixture was allowed to cool to rt and subjected to a work-up sequence described in Conditions A. The crude residue was purified by column chromatography on silica gel (hexane, hexane-toluene, ethyl acetate – toluene, 5% gradient elution). Eluted first was methyl 2,3,4,6-tetra-O-benzyl α-d-glucopyranoside 4 isolated in 5% yield (2.0 g, 2.44 mmol). Also eluted was a mixture of compounds 2 and 3 as a clear syrup in a combined yield of 60% yield (14.5 g, 31.1 mmol, ratio 5.3/1).

Methyl 3-O-benzoyl-2,4,6-tri-O-benzyl-α-d-glucopyranoside (5) and methyl 4-O-benzoyl-2,3,6-tri-O-benzyl-α-d-glucopyranoside (6).

Method A.

N,N-dimethylaminopyridine (DMAP, 0.11 g, 0.86 mmol) and benzoyl chloride (3.6 g, 25.83 mmol) were added to a solution of compounds 2 and 3 (2.0 g, 4.3 mmol) in dry pyridine (25 mL), and the resulting mixture was stirred under argon for 2.5 h at 50 °C. After that, the reaction mixture was allowed to cool to rt, MeOH (~5 mL) was added, the volatiles were removed under reduced pressure, and the residue was co-evaporated with toluene (2 × 20 mL). The resulting residue was diluted with dichloromethane (~200 mL) and washed with water (2 × 50 mL). The organic phase was separated, dried with anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-dichloromethane, 1% gradient elution). Eluted first was compound 6 obtained as a clear syrup in 15% yield (0.37 g, 0.64 mmol). Also eluted was compound 5 obtained as a clear syrup in 71% yield (1.74 g, 3.06 mmol). Analytical data for 5: R_f = 0.40 (ethyl acetate/ toluene, 15/85, v/v); [α]_D²⁰ +10.4 (c = 1.0, CHCl₃); ¹H n.m.r. (300 MHz): δ 3.40 (s, 3H, CH₃), 3.61–3.75 (m, 2H, H-2, 6b), 3.76–3.83 (m, 3H, H-4, 5, 6a), 4.36 (d, 1H, ²J = 10.7 Hz, CHPh), 4.45–4.56 (m, 4H, 2 × CH₂Ph), 4.69 (d, 1H, ²J = 12 Hz, CHPh), 4.76 (d, 1H, J_1,2 = 3.4 Hz, H-1), 5.80 (dd, 1H, J_3,4 = 9 Hz, H-3), 6.97–8.03 (m, 20H, aromatic) ppm; ¹³C{¹H} NMR (75 MHz): δ 55.2, 69.6, 72.5, 73.5, 74.2, 74.3, 75.8, 76.9, 76.9, 97.7, 127.5, 127.6, 127.7, 127.8 (x2), 127.9 (x2), 128.0 (x2), 128.1 (x2), 128.2 (x4), 128.3 (x2), 129.6 (x2), 130.2, 132.8, 137.4, 137.6 (x2), 165.3 ppm; HRMS [M + Na]⁺: calcd for [C₃₅H₃₆O₇Na]⁺ 591.2359; found:591.2359. Analytical data for 6: R_f = 0.55 (ethyl acetate/ toluene, 15/85, v/v); [α]_D²⁰ −10.3 (c = 1.0, CHCl₃); ¹H n.m.r. (300 MHz): δ 3.44 (s, 3H, CH₃), 3.51–3.55 (m, 2H, H-6a, 6b), 3.69 (dd, 1H, J_1,2 = 3.5 Hz, H-2), 3.99 (m, 1H, H-5), 4.1 (dd, J_3,4 = 9.5 Hz, H-3), 4.47–4.85 (m, 7H, H-1, 3 × CH₂Ph), 5.35 (dd, 1H, J_4,5 = 9.9 Hz, H-4), 7.96–7.10 (m, 20 H, aromatic) ppm; ¹³C{¹H} NMR (75 MHz): δ 55.3, 68.8, 70.8, 73.4, 73.5, 75.3, 77.2, 79.1, 79.4, 98.2, 127.3, 127.4, 127.6 (x2), 127.9 (x4), 128.0 (x3), 128.1 (x4), 128.2, 128.4 (x2), 129.6 (x2), 133.0, 137.6.0, 137.9, 138.0.4, 165.2 ppm; HRMS [M + Na]⁺: calcd for [C₃₅H₃₆O₇Na]⁺ 591.2359; found: 591.2367.

Method B.

Benzoic acid (6.26 g, 51.3 mmol), N,N-dimethylaminopyridine (DMAP, 0.418 g, 3.42 mmol), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 6.5 g, 34.2 mmol) were added to a solution of compounds 2 and 3 (7.95 g, 17.11 mmol) in 1,2-dichloroethane (35 mL), and the resulting mixture was stirred under argon for 96 h at 50 °C. The resulting mixture was allowed to cool to rt, diluted with dichloromethane (~400 mL), and washed with water (50 mL), Na₂CO₃ (2 × 50 mL), and water (2 × 50 mL). The organic phase was separated, dried with anhydrous Na₂SO₄, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-toluene, 5% gradient elution). Eluted first was compound 6 obtained as a clear syrup in 12% yield (1.14 g, 2.0 mmol). Also eluted was compound 5 obtained as a clear syrup in 65% yield (6.31 g, 11.09 mmol).

Methyl 3-O-acetyl-2,4,6-tri-O-benzyl-α-d-glucopyranoside (7) and methyl 4-O-acetyl-2,3,6-tri-O-benzyl-α-d-glucopyranoside (8).

Method C.

Acetic anhydride (4.6 mL, 48.7 mmol) was added to a solution of compounds 2 and 3 (7.09 g, 15.3 mmol) in dry pyridine (30 mL), and the resulting mixture was stirred for 2 h at rt. After that, MeOH (~5 mL) was added, the volatiles were removed under reduced pressure, and the residue was co-evaporated with toluene (2 × 20 mL). The residue was purified by column chromatography on silica gel (ethyl acetate-hexane, 5% gradient elution). Eluted first was compound 8 obtained as a clear syrup in 14% yield (1.10 g, 2.17 mmol). Also eluted was compound 7 obtained as a clear syrup in 14% yield (5.75 g, 11.35 mmol). Analytical data for 8: R_f = 0.40 (ethyl acetate/ hexane, 1/3, v/v); [α]_D²² +1.84 (c = 1.0, CHCl₃); ¹H n.m.r. (400 MHz): ¹H NMR (400 MHz, CDCl₃): δ 1.81 (s, 3H, COCH₃), 3.40 (s, 3H, OCH₃), 3.44 (dd, 1H, J_6a,6b = 10.7 Hz, H-6a), 3.49 (dd, 1H, H-6b), 3.58 (dd, 1H, J_2,3 = 9.6 Hz, H-2), 3.81 (m, 1H, J_5,6a = 5.0, J_5,6b = 2.9 Hz, H-5), 3.91 (dd, 1H, J_3,4 = 9.4 Hz, H-3), 4.48 (dd, 2H, ²J = 11.2 Hz, CHPh), 4.61 (d, 1H, J = 3.6 Hz, H-1), 4.71 (dd, 1H, ²J = 12.1 Hz, CH₂Ph), 4.76 (dd, 1H, ²J = 11.7 Hz, CH₂Ph), 5.02 (dd, 1H, J_4,5 = 10.2 Hz, H-4), 7.24–7.36 (m, 15H, aromatic) ppm; ¹³C{¹H} NMR (100 MHz): δ 20.8, 55.4, 68.2, 69.0, 70.6, 73.5, 73.6, 75.3, 79.3, 79.6, 98.2, 127.6 (x2), 127.9 (x2), 128.0, 128.2, 128.4, 128.5, 137.9, 138.0, 138.7, 169.7 ppm; HRMS [M + Na]⁺: calcd for [C₃₀H₃₄NaO₇]⁺ 529.2202; found: 529.2164. Analytical data for 7: R_f = 0.30 (ethyl acetate/ hexane, 1/3, v/v); [α]_D²² +8.17 (c = 1.0, CHCl₃); ¹H NMR (400 MHz, CDCl₃): δ 1.93 (s, 3H, COCH₃), 3.36 (s, 3H, OCH₃), 3.48 (dd, 1H, J_2,3 = 10.0 Hz, H-2), 3.64 (dd, 1H, J_6a,6b = 10.7 Hz, H-6a), 3.66 (dd, 1H, J_4,5 = 9.6 Hz, H-4), 3.73 (dd, 1H, H-6b), 3.79 (m, 1H, J_5,6a = 2.1, J_5,6b = 3.3 Hz, H-5), 4.46 (dd, 2H, ²J = 11.2 Hz, CH₂Ph), 4.54 (dd, 2H, ²J = 12.1 Hz, CH₂Ph), 4.59 (s, 2H, CH₂Ph), 4.70 (d, J_1,2 = 3.5 Hz, H-1), 5.51 (dd, 1H, J_3,4 = 9.6 Hz, H-3), 7.12–7.14 (m, 2H, aromatic), 7.24–7.34 (m, 13H, aromatic) ppm; ¹³C{¹H} NMR (100 MHz): δ 21.1, 55.3, 68.2, 69.8, 72.8, 73.6, 74.3, 76.2, 77.5, 97.9, 127.8, 127.9, 128.5, 137.8, 138.0, 138.1, 169.9 ppm; HRMS [M + Na]⁺: calcd for [C₃₀H₃₄NaO₇]⁺ 529.2202; found: 529.2153.

Methyl 2,4,6-tri-O-benzyl-α-d-glucopyranoside (2).

A 1 M solution of sodium methoxide in methanol (~25 mL) was added dropwise to a solution of compound 5 (6.31 g, 11.09 mmol) in methanol (120 mL) till pH > 9, and the resulting mixture was stirred for 3 h at rt. After that, the reaction mixture was neutralized with Amberlite (H⁺) ion exchange resin. The resin was filtered off and rinsed successively with methanol. The combined filtrate (~250 mL) was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane, 10% gradient elution) to afford the title compound as a clear syrup in 93% yield (4.79 g, 10.31 mmol). A similar protocol was applied to compound 7 (4.54 g, 8.97 mmol) that was converted in compound 2 in 93% yield (3.86g. 8.32 mmol).

Methyl 2,3,6-tri-O-benzyl-α-d-glucopyranoside (3).

A 1 M solution of sodium methoxide in methanol (5 mL) was added dropwise to a solution of compound 5 (1.14 g, 2.00 mmol) in methanol (45 mL) till pH > 9, and the resulting mixture was stirred for 3 h at rt. After that, the reaction mixture was neutralized with Amberlite (H⁺) ion exchange resin. The resin was filtered off and rinsed successively with methanol (7 × 10 mL). The combined filtrate (~75 mL) was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate-hexane, 10% gradient solution) to afford the title compound as a clear syrup in 91% yield (0.85 g, 1.83 mmol). Analytical data for 3: R_f = 0.45 (ethyl acetate/ hexane, 3/7, v/v); [α]_D²⁰ +7.1 (c = 1.0, CHCl₃); ¹H n.m.r. (300 MHz): δ 2.33 (s, 1H, 4-OH), 3.38 (s, 3H, CH₃), 3.55 (dd, 1H, J_2,3 = 3.5 Hz, H-2), 3.60–3.72 (m, 4H, H-4, 5, 6a, 6b), 3.78 (dd, 1H, J_3,4 = 8.9 Hz, H-3), 4.51–5.02 (m, 7H, H-1, 3 × CH₂Ph), 7.25–7.37 (m, 15H, aromatic) ppm; ¹³C{¹H} NMR (75 MHz): δ 55.2, 69.3, 69.8, 70.6, 73.1, 73.5, 75.4, 79.5, 81.4, 98.7, 127.6, 127.8, 127.9 (x2), 128.0 (x2), 128.1, 128.3 (x2), 128.4 (x4), 128.5 (x2), 137.9, 137.7, 138.7 ppm; HRMS [M + Na]⁺: calcd for [C₂₈H₃₂O₆Na]⁺ 487.2097; found: 487.2102.

• Regioselective benzylation;

• Improved synthesis of a common building block;

• 3-OH glycosyl acceptor synthesis revised