Efficient synthesis of a library of heparin tri- and tetrasaccharides relevant to the substrate of heparanase

Abstract

The glycosylation reaction for construction of the challenging α-GlcN–(1→4)-GlcA/IdoA linkages has been investigated carefully. A standard protocol was thus fixed that employed 2-azido-glucopyranosyl N-phenyl trifluoroacetimidates as donors, TMSOTf as a catalyst, toluene as a solvent, and −30 °C as the working temperature. With this protocol, a variety of mono- and disaccharide donors and acceptors were condensed reliably to provide the corresponding coupled tri- and tetrasaccharides in satisfactory yields and α-selectivity, whereas a remote protecting group or a sugar unit in either the donor or the acceptor did affect considerably the outcome. The resulting tri- and tetrasaccharides bearing orthogonal protecting groups were then converted efficiently into the corresponding heparin tri- and tetrasaccharides via a robust approach involving saponification, O-sulfation, azide reduction, N-sulfation/N-acetylation, and global debenzylation. These heparin tri- and tetrasaccharides are structurally relevant to ΔHexA(2S)–GlcN(NS,6S)–GlcA–GlcN(NS,6S), a reported substrate of heparanase, and therefore could be exploited to examine the substrate specificity of this important enzyme.

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Introduction

Heparin (HP) and heparan sulfate (HS) are highly sulfated linear polysaccharides that represent the most complex members of the glycosaminoglycan (GAG) family.¹ HP, found mainly on mast cells, consists of alternating (1→4)-linked uronic acid (either α-L-iduronic acid (IdoA) or β-D-glucuronic acid (GlcA)) and α-D-glucosamine (GlcN) units.^1,2 HS, which is ubiquitously distributed on the surface of animal cells and in the extracellular matrix (ECM), shares the same backbone with HP but carries less sulfate groups and a lower percentage of the IdoA residue.^1,3–5 The interaction of HP/HS and proteins plays a key role in numerous physiological processes such as blood coagulation, angiogenesis, lipid metabolism, tumor metastasis, growth factor binding, cell adhesion, inflammatory response, and bacterial and viral infections.^1,3,6–8 These functions of HP/HS are to a large extent determined by the saccharide sequence, chain length, and sulfation patterns.⁹ In particular, an HP pentasaccharide specifically binding to antithrombin III has been developed as an anticoagulant drug (Fondaparinux).^2,9 Nevertheless, the structure–activity relationships (SAR) of HP/HS in most HP/HS-mediated biological events are still poorly understood.

Heparanase is a mammalian endo-β-D-glucuronidase that degrades HP/HS at specific sites, so as to reduce the integrity of ECM and basement membranes and release the HS-bound growth factors.^10,11 Overexpression of heparanase is closely associated with the disease state of tumor progression and metastasis.¹² Thus, heparanase has become an important target for developing anticancer drugs.¹³ Previous studies on the substrate specificity of the human heparanase revealed the tetrasaccharide ΔHexA(2S)–GlcN(NS,6S)–GlcA–GlcN(NS,6S) as an ideal sequence.¹⁴ An optimal substrate derivative could be exploited to establish an efficient assay of the heparanase activity, thus facilitating the studies on the function of this important enzyme and the development of inhibitors.¹⁵

Chemical (and chemoenzymatic) syntheses of HP/HS have been studied intensively;^5,9,16–19 various protocols/strategies for the preparation of building blocks, glycosylation, and protecting group manipulation have been explored, such as modular assembly,^{17c,j,18h,l,p} one-pot method,^18e,i pre-sulfated assembly,^18jde novo strategy,^18d solid-phase synthesis,^17e,18q and diversity-oriented synthesis.^18b,k,m However, the α-selective formation of the GlcN–(1→4)-GlcA/IdoA glycosidic linkages remains elusive; the difficult-to-separate or inseparable β anomers often result in various amounts.²⁰ 2-Azide (mostly),²¹ 2N,3O-oxazolidinone,²² and N-p-methoxybenzylidene²³ are employed as amino protecting groups in the GlcN donors to ensure a non-participating effect on the glycosylation, nevertheless all other reaction parameters including the protecting patterns of both the donor and the acceptor, the anomeric leaving group, the promoter, the solvent, the temperature, and the additive also affect seriously the coupling yield and α-selectivity.^20,24 Herein, we report the synthesis of a library of tri- and tetrasaccharides (1–20) structurally relevant to ΔHexA(2S)–GlcN(NS,6S)–GlcA–GlcN(NS,6S) (Fig. 1), whereas the α-selective glycosylation with 2-azido-glucopyranosyl donors to construct the GlcN–(1→4)-GlcA/IdoA linkages has been carefully studied.

Fig. 1 An optimal substrate of heparanase and a library of the structurally relevant oligosaccharides (1–20).

Results and discussion

Synthetic strategy

The GlcA/IdoA units in HP/HS are mostly decorated with 2-O-sulfation, whereas the GlcN units are typically substituted with 2-N-acetylation/sulfation and 6-O-sulfation.^25,26 A judicious choice of a set of glycosyl building blocks installed with orthogonal protecting groups is crucial for the efficient construction of a library of HS oligosaccharides.^9,16–18 To synthesize tri- and tetrasaccharides 1–20 in a combinatorial manner, glucose (Glc) building blocks 21 and 22, an idose (Ido) building block 23, and GlcN building blocks 24–28 were designed to meet the requirements of stereoselective glycosylation, chain elongation, and regioselective sulfation (Fig. 2). As such, the 4,6-hydroxyl groups in 21–23 were protected as 4,6-O-benzylidene acetals that would be oxidized to carboxylic acids followed by blocking with methyl groups at the C6 position with the 4-OH being free for chain elongation. The anomeric tert-butyldimethylsilyl (TBS) groups in GlcN 25 and 26 could be selectively removed with tetrabutylammonium fluoride (TBAF) for further conversion into glycosyl leaving groups. Moreover, the benzoyl (Bz), levulinate (Lev),²⁷ and p-methoxybenzyl (PMB) groups were selected to protect the hydroxyl groups destined for sulfation, while the benzyl (Bn) group was employed as the permanent protecting group. The amine group was masked as an azide that could be readily reduced into free amine for N-acetylation or N-sulfation at a late stage.²⁰

Fig. 2 Monosaccharide building blocks 21–28.

Couplings of Glc/Ido 21–23 with GlcN 25–28 would provide a range of Glc/Ido–(1→4)-GlcN disaccharides, where the 2-ester groups (O-Bz or O-Lev) would secure the formation of the required 1,2-trans glycosidic linkages via the aid of neighboring group participation.²⁸ The resulting Glc/Ido–GlcN disaccharides could be readily converted into GlcA/IdoA–GlcN donors and acceptors.²⁹ The α-selective [2 + 2] glycosidic coupling to construct the GlcN–(1→4)-GlcA/IdoA linkages could then be explored. Employing an optimal set of standard conditions, we could assemble all the building blocks into oligosaccharides. Conventional operation of the protecting groups and timely sulfation would lead to the desired tri- and tetrasaccharides (1–20).

Preparation of the disaccharide building blocks

The eight differentially protected monosaccharide building blocks 21–28 were readily prepared in large quantities adopting the literature procedures.^18f,30,31 Glycosylation of Glc trichloroacetimidate³²21 with GlcN 25 and 26 under the catalysis of TMSOTf (0.05 equiv.) provided β-(1→4) linked disaccharides 29 and 30 in >90% yields (Scheme 1). Treatment of 29 and 30 with BH₃·THF and Bu₂BOTf led to regioselective opening of the 4′,6′-O-benzylidene acetal, affording the disaccharides with free 6-hydroxyl groups at the Glc residues.³³ It should be noted that other BH₃·THF-mediated reduction on 29 and 30 using Cu(OTf)₂, Sc(OTf)₃, or TMSOTf as Lewis acid failed to give the desired disaccharides in good yield.³⁴ The resulting 6-hydroxyl groups were then oxidized with 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) in the presence of [bis(acetoxy)iodo]benzene (BAIB),²⁹ and subsequent methylation of the nascent carboxylic acids afforded GlcA–GlcN disaccharides 31 and 32 in 75% and 77% yields, respectively, over three steps. Removal of the anomeric TBS groups in 31 and 32 with TBAF and acetic acid gave the corresponding lactols, which were readily converted into glycosyl o-cyclopropylethynylbenzoate³⁵33, trichloroacetimidate 34, and N-phenyl trifluoroacetimidate³⁶35 and 36, respectively.

Scheme 1 Preparation of the GlcA–GlcN disaccharide donors 33–36 and acceptors 44–46.

On the other hand, TMSOTf-catalyzed glycosylation of Glc imidates 21/22 with GlcN derivatives 27/28 was performed to provide disaccharides 37–39 in high yields (86–97%), whereas the 2-O-Bz or -Lev group in the Glc donors participated in the glycosylation to secure the exclusive formation of β-linkage. The Lev group in 39 was then replaced by the permanent Bn protecting group. Acidic cleavage of the benzylidene acetals in 37, 38, and 40 with aqueous acetic acid at 80 °C followed by TEMPO oxidation and subsequent methylation furnished disaccharide acceptors 44–46 in 77–84% yields over three steps.

Similarly, glycosylation of GlcN derivatives 25 and 27 with Ido trichloroacetimidate 23 proceeded smoothly under the catalysis of TMSOTf (0.1 equiv.), leading to Ido–GlcN disaccharides 47 and 50 in excellent yields (Scheme 2). Following the above procedures for the conversion of 29→35 and 37→44, Ido–GlcN 47 and 50 were easily transformed into IdoA–GlcN 49 and 52 in good yields.

Scheme 2 Preparation of the IdoA–GlcN disaccharide donor 49 and acceptor 52.

Investigation of the α-selective glycosylation to construct the GlcN–(1→4)-GlcA/IdoA linkages

With GlcA–GlcN derivative 44 as an acceptor, we examined the [2 + 2] glycosylation with GlcA–GlcN ortho-alkynylbenzoate 33, trichloroacetimidate 34, and N-phenyltrifluoroacetimidate 35 as donors. The representative results are listed in Table 1. The coupling of 44, which bears a hindered 4′-OH, with ortho-alkynylbenzoate 33 proceeded sluggishly under the catalysis of PPh₃AuOTf (0.1 equiv.) at room temperature. With toluene as the solvent,³⁷ the reaction led to the coupled tetrasaccharide 53 in a moderate 41% yield (α/β = 4.4) (entry 1). CH₂Cl₂ or ClCH₂CH₂Cl was proved to be the worse solvent for this coupling; the reaction hardly took place, with the α-anomer 53α being isolated in only ∼10% yield after ∼8 h (entry 2). In these reactions, the donor and acceptor could be largely recovered.

Table 1 Examination of the [2 + 2] glycosylation of GlcA–GlcN 44 with GlcA–GlcN donors 33–35

Entry	Donor	Promoter^a	Solvent	Temp.	Yield (α/β ratio)
a 0.1 equiv. was used. b 2-Bromothiophene (12 equiv.) was added as an additive.
1	33	PPh3AuOTf	Toluene	rt	41% (α/β = 4.4)
2		PPh3AuOTf	CH2Cl2 or ClCH2CH2Cl	rt	∼10% (α only)
3	34	TMSOTf	CH2Cl2 or THF	−30 °C	Trace
4		TBSOTf	Toluene	−30 °C	62% (α/β = 3)
5	35	TMSOTf	THF	−30 °C	trace
6		TMSOTf	Et2O	−30 °C	54% (α/β = 10)
7		TMSOTf	CH2Cl2	−30 °C	39% (α/β = 4.2)
8		TBSOTf	CH2Cl2	−30 °C	34% (α/β = 6)
9		TBSOTf	Toluene	−30 °C	58% (α/β = 2.5)
10		TMSOTf	Toluene	−30 °C	81% (α/β = 3.8)
11^b		TMSOTf	Toluene	−30 °C	72% (α/β = 6)

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The coupling of GlcA–GlcN trichloroacetimidates with GlcA–GlcN acceptors was reported to provide the coupled tetrasaccharides in moderate to good yields with varied α-selectivity.^18h,38 The condensation of 34 and 44 in the presence of TMSOTf (0.1 equiv.) in CH₂Cl₂ or THF³⁸ at −30 °C proceeded sluggishly, thereby leading to the formation of 53 in only trace amounts (entry 3). This unexpected result implies the mismatch of these two coupling partners.^18l,o,38 Interestingly, when TBSOTf was employed as the catalyst and toluene as the solvent, the reaction of 34 and 44 delivered tetrasaccharide 53 in a good 62% yield with moderate stereoselectivity (α/β = 3) (entry 4). The anomeric configuration of tetrasaccharide 53 was confirmed by comparison of the chemical shifts of the anomeric carbons of 53α (δ 97.2, 98.6, 101.0, 101.4 ppm) with those of 53β (δ 98.6, 101.1, 101.4, 101.9 ppm).³¹ Due to the observation of the corresponding glycosyl trichloroacetamide which was derived from trichloroacetimidate 34, we then focused our attention on the N-phenyl trifluoroacetimidate donor 35 for further optimization of the present [2 + 2] glycosylation reaction.^18f

The coupling of trifluoroacetimidate 35 with 44 in THF (0.1 equiv. TMSOTf, −30 °C) led to a trace amount of the coupled tetrasaccharide 53 (entry 5). Nevertheless, with Et₂O as the solvent the coupling provided 53 in 54% yield and an excellent α selectivity (α/β = 10) (entry 6). Unfortunately, this reaction took two days due to the poor solubility of 35 and 44 in ether. Replacing the solvent with CH₂Cl₂, the reaction under the catalysis of either TMSOTf or TBSOTf led to lower yield of the tetrasaccharide (<40%) and α selectivity (α/β = 4.2 and 6, respectively; entries 7 and 8). Toluene was again proved to be a superior solvent in the present glycosylation, and the coupling of 35 and 44 catalyzed by TBSOTf provided 53 in 58% yield (α/β = 2.5) in ∼2 h (entry 9). The yield of 53 was improved significantly to 81% when TMSOTf was used as the catalyst, although the α selectivity was still moderate (α/β = 3.8; entry 10). The addition of 2-bromothiophene (12 equiv.) indeed improved the α-selectivity (α/β = 6), whereas the yield decreased slightly (72%; entry 11).³⁹

Taking all together the yield, α selectivity, reaction time, and ease of purification, we decided to utilize glycosyl N-phenyl trifluoroacetimidate as a donor, TMSOTf as a catalyst, toluene as a solvent, and −30 °C as the working temperature to further explore the [2 + 2] coupling of the disaccharide building blocks (Scheme 3). Under these fixed conditions, the condensation of GlcA/IdoA–GlcN trifluoroacetimidates 35, 36, and 49 with GlcA/IdoA–GlcN acceptors 44–46 and 52 provided the corresponding tetrasaccharides 54–59 in good yields (58–77%) and satisfactory α selectively (from α/β = 3.2 to α only). In comparison, the glycosylation with IdoA–GlcN donor (49) showed a higher α-selectivity (α/β = 7.5 to α only vs. α/β = 3.2–6.4) than those with GlcA–GlcN donors (35 and 36), irrespective of the acceptors (44–46 and 52). When GlcA–GlcN 35 was used as a donor, the glycosylation with IdoA–GlcN acceptor 52 and GlcA–GlcN acceptor 44, which bear a similar protecting group pattern displayed a similar α-selectivity (α/β = 3.2 vs. α/β = 3.8). The acceptor bearing the 2′-O-Bn group (i.e., 46) gave a slightly higher α-selectivity (α/β = 4.9 vs. 3.2–3.8) than acceptors bearing the 2′-O-Bz group (i.e., 44 and 52) upon coupling with donor 35. In addition, IdoA/GlcN acceptor 52 exhibited a slightly better α-selectivity (α only vs. α/β = 7.5–10.5) than GlcA–GlcN acceptors 44 and 46 when IdoA–GlcN 49 was employed as a donor.

Scheme 3 The [2 + 2] couplings of GlcA/IdoA–GlcN donors 35, 36, and 49 with GlcA/IdoA–GlcN acceptors 44–46 and 52 under the fixed conditions (0.1 equiv. TMSOTf, 5 Å MS, toluene, −30 °C).

To further examine the influence of acceptors on the α-selectivity in the present glycosylation, we carried out the relevant couplings with GlcN trifluoroacetimidate 24 as a simplified donor (Scheme 4). Interestingly, IdoA–GlcN acceptor 52 displayed a lower α-selectivity (2.2 vs. 5.7 to α only) than GlcA–GlcN acceptors 44 and 46 in the [1 + 2] coupling. However, when GlcA 63 and IdoA 65 were employed as acceptors, a reverse α-preference was found in the coupling with 24 compared to the [1 + 2] coupling. These results are consistent with the previous findings.^17c,j,18o Specifically, the coupling of GlcN 24 and IdoA acceptor 65 furnished exclusively the α-disaccharide 66 in 90% yield, while glycosylation of GlcN 24 with GlcA acceptor 63 led to disaccharide 66 in only 48% yield with a moderate α/β ratio (α/β = 5.5) (Scheme 4).⁴⁰ In general, the α-selectivity in the glycosylation with GlcN donors is to a large extent influenced by the additional sugar units in both the donors and acceptors.^17g,38,41

Scheme 4 [1 + 2] and [1 + 1] glycosylation with GlcN donor 24 under the fixed conditions (0.1 equiv. TMSOTf, 5 Å MS, toluene, −30 °C).

Syntheses of a library of the HS tri- and tetrasaccharides

The orthogonally protected tri- and tetrasaccharides 53–62 were converted readily into the corresponding HS tri- and tetrasaccharides (1–20) bearing the desired sequence and sulfation patterns. With the conversion of tetrasaccharide 53α to HS tetrasaccharides 1 and 2 as an example, this final elaboration involves six steps (Scheme 5). (1) The methyl esters were cleaved with LiOH and H₂O₂ in THF;⁴² (2) the remaining benzoyl groups were removed completely with KOH in methanol; (3) the resulting hydroxyl groups were sulfated with SO₃·pyridine; (4) the azides were reduced with Me₃P in the presence of NaOH;⁴³ (5) the resulting amines were N-acetylated with acetic anhydride or N-sulfated with SO₃·pyridine; and (6) the benzyl groups were finally removed via hydrogenolysis over Pd(OH)₂/C in a mixture of methanol and water. For the conversion of 56α to 5/6, an additional step was carried out at the beginning to remove the PMB groups with dichlorodicyanobenzoquinone (DDQ).⁴⁴ All these steps are high-yielding and robust, and were thus applied reliably to the synthesis of all the desired HS tri- and tetrasaccharides (1–20) with the overall yields of 35–78% (Scheme 6).³¹ The NMR spectra, especially the easily diagnostic chemical shifts of the anomeric carbons, of the synthetic 1–20 were found to be virtually identical to those reported for the analogous oligosaccharides in the literature.^16–18 The sulfation numbers were well confirmed by ESI-MS analyses.³¹

Scheme 5 Elaboration of the heparin tetrasaccharides 1 and 2.

Scheme 6 The heparin oligosaccharides (3–20) synthesized from the fully protected precursors.

Conclusion

A library of tri- and tetrasaccharides (1–20) structurally relevant to ΔHexA(2S)–GlcN(NS,6S)–GlcA–GlcN(NS,6S), an optimal substrate of heparanase, has been synthesized efficiently. These HS oligosaccharides are elaborated from the corresponding orthogonally protected tri- and tetrasaccharides (53α–62α) employing robust transformations including saponification, O-sulfation, azide reduction, N-sulfation/N-acetylation, and debenzylation. The advanced oligosaccharide precursors (53α–62α) are synthesized with the [2 + 2] and [1 + 2] glycosylation as the key step in building the difficult α-GlcN–(1→4)-GlcA/IdoA linkages. This particular glycosylation with a variety of the 2-azido-glucopyranosyl donors has been investigated carefully. A standard protocol has been fixed that employs a glycosyl N-phenyltrifluoroacetimidate as a donor, TMSOTf as a catalyst, toluene as a solvent, and −30 °C as the working temperature. With this protocol, a number of coupling partners have been assembled reliably in satisfactory yields and α-selectivity. It is observed that a remote protecting group or a sugar unit in either the donor or the acceptor could affect considerably both the coupling yield and α-selectivity. Nevertheless, the mechanism behind such effects is yet to be elucidated. The availability/accessibility of the HS tri- and tetrasaccharides would enable us to identify an appropriate substrate of heparanase so as to establish an optimal assay for measuring the heparanase activities.

Experimental section

General glycosylation procedure for the synthesis of disaccharides (29, 30, 37–39, 47, 50)

The glycosyl imidate donor (1.1 equiv.) and 2-azido-2-deoxy-D-glucopyranoside acceptor (1.0 equiv.) were combined in a flask and dissolved in CH₂Cl₂ to maintain a concentration of 0.02 M. Freshly activated powdered 4 Å MS (weight equal to the combined weight of the donor and the acceptor) was added. The mixture was stirred for 1 hour at ambient temperature and then cooled to −30 °C. TMSOTf (0.05 equiv.) was added to the mixture, and the stirring was continued until TLC indicated disappearance of the donor (∼30 min). The reaction was quenched by the addition of Et₃N. The mixture was filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography using a gradient of petroleum ether–EtOAc to afford the pure product.

General procedure for the cleavage of the benzylidene acetal (41–43 and 51)

A solution of the 4′,6′-O-benzylidene disaccharide (0.05 M) in HOAc–H₂O (v/v = 4/1) was stirred at 80 °C for 1 h. The mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography using a gradient of petroleum ether–EtOAc to give the pure product.

General procedure for the regioselective opening of the benzylidene acetal

BH₃·THF (10 equiv.) was added to a solution of the 4′,6′-O-benzylidene disaccharide (29, 30, or 47) in THF at 0 °C. After 15 min, Bu₂B·OTf (2 equiv.) was added dropwise and the stirring was continued at 0 °C for 2 h. The reaction was quenched by addition of Et₃N and the excess BH₃·THF was consumed by slowly adding methanol. The solution was concentrated in vacuo to give the crude product.

General procedure for the TEMPO/BAIB-mediated oxidation and methyl ester formation (31, 32, 44–46, 48, 52)

To a vigorously stirring solution of the disaccharide 4′,6′-diol (0.3 M solution) in CH₂Cl₂–H₂O (v/v = 2/1) were added TEMPO (0.1 equiv.) and BAIB (2.5 equiv.). Stirring was continued until TLC indicated complete conversion of the starting material to a spot of lower R_f (∼3 h). The reaction was quenched by the addition of aqueous Na₂S₂O₃ (10 mL). The mixture was acidized with 5% HCl, then extracted with CH₂Cl₂ three times. The combined organic layers were washed with brine (50 mL), and were then dried over Na₂SO₄ and filtered. The filtrate was concentrated in vacuo, and the residue was co-evaporated with toluene twice. The resulting oily residue was dissolved in DMF (0.1 M), and then treated with KHCO₃ (4 equiv.) and methyl iodide (8 equiv.). The excess methyl iodide was quenched by the addition of AcOH. The mixture was concentrated in vacuo, and then diluted with CH₂Cl₂. The solution was washed with brine twice and then evaporated. The residue was purified by silica gel column chromatography using a gradient of petroleum ether–EtOAc to yield the methyl ester.

General procedure for the cleavage of the silyl ethers

The silyl glycoside (31, 32, or 48) was dissolved in THF (0.1 M) followed by the addition of AcOH (3 equiv.) and TBAF in THF (1 M, 2 equiv.). After stirring for 30 min, the mixture was diluted with CH₂Cl₂ (50 mL) and washed with saturated aqueous NaHCO₃ (50 mL) and brine (50 mL), respectively. The organic phase was dried (Na₂SO₄) and filtered. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography using a gradient of petroleum ether–EtOAc to give the pure lactol.

General procedure for the preparation of the glycosyl N-phenyl trifluoroacetimidates (24, 35, 36, 49)

To a solution of the lactol in acetone (2 mL, 0.1 mmol) were added finely powdered anhydrous K₂CO₃ (2.5 equiv.) and N-phenyl trifluoroacetimidoyl chloride (1.3 equiv.). After stirring at room temperature for 1.5 h, the mixture was filtered. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether–EtOAc containing 1% Et₃N) to give the pure product.

General glycosylation procedure for the preparation of the tri- and tetrasaccharides (53–62)

The glycosyl N-phenyl trifluoroacetimidate donor (1.2 equiv.) and disaccharide acceptor (1.0 equiv.) were combined in a flask, co-evaporated with toluene (3 × 3 mL), and then dissolved in toluene to maintain a concentration of 0.04–0.05 M. Freshly activated powdered 5 Å MS (weight equal to the combined weight of the donor and the acceptor) was added, and the mixture was stirred for 1 h at ambient temperature and then cooled to −30 °C. TMSOTf (0.1 equiv.) was added, and stirring was continued until TLC indicated the disappearance of the donor (∼2 hours). The reaction was quenched by addition of Et₃N (0.5 mL). The mixture was filtered. The filtrate was concentrated in vacuo. The residue was purified by silica gel column chromatography using a gradient of toluene–EtOAc to give the pure product. The α and β anomers were fully separated.

General procedure for the saponification of the methyl esters and benzoyl groups

A premixed solution of 30% solution of H₂O₂ in water (100 equiv. per CO₂Me) and 1 M LiOH (50 equiv. per CO₂Me) were added to a solution of the starting material in THF (0.02 M). The mixture was stirred at rt for 24 h. A solution of KOH (3 M) was added until pH ≃ 14. The mixture was left stirring for 24 h at room temperature. The mixture was then brought to pH = 8–8.5 by addition of acidic resin, and then filtered. The filtrate was concentrated in vacuo (bath temperature 20–30 °C). The residue was dissolved in CH₂Cl₂–MeOH (v/v = 1/1). The resulting solution was layered on the top of a Sephadex LH-20 chromatography column and then eluted with CH₂Cl₂–MeOH (v/v = 1/1). The appropriate fraction was concentrated in vacuo to provide the pure product.

General procedure for the O-sulfation

SO₃·pyridine complex (8 equiv. per OH) was added to a solution of the starting material in DMF (1.0 mL, 0.02 mmol). The mixture was stirred at ambient temperature for 4 h until TLC (RP18 silica gel, H₂O–CH₃OH, v/v = 1/2) indicated completion of the reaction. After addition of CH₃OH (0.5 mL), the stirring was continued for 30 min. The mixture was concentrated in vacuo. The residue was vortexed with water and then applied to a small RP-18 silica gel column, which was eluted with a gradient of H₂O–CH₃OH (v/v from 1/0 to 2/1). The fractions containing the product were concentrated in vacuo. The residue was immediately passed through a column of Dowex 50WX4 Na⁺ resin using CH₃OH as an eluent. The appropriate fraction was concentrated in vacuo to provide the product as sodium salt.

General procedure for the reduction of the azide

A solution of Me₃P in THF (1 M, 4 equiv. per azide) was added to a solution of the starting material in THF (1.0 mL, 0.02 mmol). An NaOH solution (0.1 M, 5 equiv. per azido) was added, and the mixture was stirred at room temperature for 5 h. The progress of the reaction was monitored by TLC (RP-18 silica gel, H₂O–CH₃OH, v/v = 1/2). The pH was then adjusted to 7–8 by careful addition of HCl (1.0 M). The mixture was then concentrated in vacuo to give the crude product.

General procedure for the N-sulfation

SO₃·pyridine (5 equiv. per NH₂) was added to the starting material in CH₃OH (1 mL, 0.01 mmol) containing triethylamine (0.3 mL) and NaOH (0.1 M, 2 equiv. per NH₂) at 0 °C. The progress of the reaction was monitored by TLC (RP-18 silica gel, H₂O–CH₃OH, v/v = 1/1). Two additional portions of SO₃·pyridine were added at 0 °C after 30 min and 1 h, respectively. After stirring for an additional 8 h at 0 °C, the mixture was concentrated in vacuo. The residue was vortexed with water and then applied to a small RP-18 silica gel column, which was eluted with a gradient of H₂O–CH₃OH (v/v from 1/0 to 1/1). The fractions containing the product were concentrated in vacuo. The residue was immediately passed through a column of Dowex 50WX4 Na⁺ resin using a mixture of CH₃OH and H₂O (9/1, v/v) as an eluent. The appropriate fraction was concentrated in vacuo to provide the product.

General procedure for the N-acetylation

Acetic anhydride (10 equiv. per NH₂) was added to a solution of the starting material in a mixture of anhydrous CH₃OH (0.02 mmol) and Et₃N (20 equiv. per NH₂) at 0 °C. The progress of the reaction was monitored by TLC (RP18 silica gel, H₂O–CH₃OH, v/v = 1/1). After stirring for 4 h at room temperature, the mixture was concentrated in vacuo. The residue was vortexed with water and applied to a small RP-18 silica gel column, which was eluted with a gradient of H₂O–CH₃OH (v/v from 1/0 to 1/1). The fractions containing the product were concentrated in vacuo. The residue was immediately passed through a column of Dowex 50WX4 Na⁺ resin using CH₃OH–H₂O (v/v = 9/1) as an eluent. The appropriate fraction was concentrated in vacuo to provide the product.

General procedure for the global removal of benzyl groups (1–20)

Palladium hydroxide on carbon (Degussa type, 20%, 1.5–2.0 times the weight of the starting material) was added to a solution of the starting material in CH₃OH and pH = 7 buffer H₂O (1 mL for 10–20 mg, v/v = 1/1). The mixture was placed under an atmosphere of hydrogen for 24 h. The mixture was filtered and concentrated. The residue was diluted with H₂O. The solution was layered on the top of a Sephadex G-10 column that was eluted with H₂O. The fractions containing the product were concentrated in vacuo. The residue was immediately passed through a column of Dowex 50WX4 Na⁺ resin using H₂O as an eluent. The appropriate fraction was freeze dried to provide the final product as a white solid.

Acknowledgements

Financial support from the Ministry of Science and Technology of China (2012ZX09502-002) and the National Natural Science Foundation of China (20932009) is gratefully acknowledged.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4qo00039k

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