Highly Efficient Syntheses of Hyaluronic Acid Oligosaccharides

Abstract

Highly efficient syntheses of hyaluronic acid oligosaccharides have been accomplished through the pre-activation based iterative one-pot strategy. A series of oligosaccharides ranging from di- to hexasaccharides were rapidly assembled using only near stoichiometric amounts of the building blocks without aglycon adjustment or purifications of intermediate oligosaccharides. Deprotection and oxidation protocols were developed for protective group removal and oxidation-state adjustment. The availability of such structurally well defined synthetic hyaluronic acid oligosaccharides will greatly facilitate the establishment of detailed structure–function relationships.

Introduction

Glycosaminoglycans (GAG) are a family of linear and negatively charged oligosaccharides, which are involved in many important biological processes such as lymphocyte trafficking, tumor metastasis, inflammatory response, and neuron growth.^[1–6] As the only unsulfated GAG, hyaluronic acid (HA) is comprised of tandem disaccharide repeats of β-1,4-D-glucuronic acid-β-1,3-D-N-acetylglucosamine. Biological studies of HA are receiving increased interests due to their mediatory roles in cell adhesion, cell migration, innate immunity and wound healing.^[2,7]

Endogenous HA can be transformed into hyaluronan oligosaccharides (sHA) in vivo. sHA have novel biological properties^[8–18] distinct from high molecular weight (~2 × 10⁶ D) and low molecular weight (~8 × 10⁴ D) HA polymers. It has been reported that sHA can not only suppress tumor cell growth^[12] but also sensitize multidrug resistant tumor cells towards a variety of chemotherapeutic drugs,^[9] while HA is ineffective. In addition, sHA have been shown to be angiogenic, whereas their high molecular weight counterparts suppress angiogenesis.^[17,18] It has become evident that the biological activities of sHA are sequence specific,^[10,14] an example of which is the report that sHA tetrasaccharides up-regulate a heat-shock protein expression and suppress cell death under stress conditions while the corresponding di-, hexa- and octasaccharides are inactive.^[10] However, in most cases, detailed understandings of structure–activity relationships remain to be established due to the lack of structurally defined sHA. Access to synthetic sHA with distinct length and sequence can greatly facilitate a systematic investigation of its structure–activity relationship, providing exciting opportunities for the development of novel therapeutics for a variety of diseases.

Chemical syntheses of sHA are typically carried out following two general approaches.^[5,19] In the first method, glucuronic acid building blocks are directly utilized to react with a glucosamine derivative.^[20] The newly formed disaccharide was transformed into either an acceptor by selective deprotection or a glycosyl donor through aglycon adjustment. Repetition of the glycosylation, selective deprotection, aglycon adjustment and glycosylation processes led to the impressive synthesis of a sHA octasaccharide.^[20] Due to the low reactivity of glucuronic acid, as an alternative, more reactive glucose can be used as building blocks.^[21–27] With this strategy, a selectively removable protective group must be installed on the 6-hydroxyl group of glucose to allow for oxidation-state adjustment. The complete deprotection and oxidation of 6-OH on all glucose units can present significant challenges.

All current sHA syntheses are characterized by their stepwise nature, which require multiple protective group adjustment, aglycon modification and purification of intermediate oligosaccharides.^[5,19–27] To facilitate biological studies of sHA, a robust method needs to be developed so that fast and efficient assembly of multiple sHA can be achieved.

Results and Discussion

Recently, we have developed a new reactivity independent pre-activation based iterative one-pot oligosaccharide synthesis method, where multiple sequential glycosylations can be carried out in the same reaction vessel without intermediate purification, allowing construction of oligosaccharides within a few hours.^[28,29] Moreover, unlike the traditional reactivity based one-pot approach where glycosylation reactions must be performed in the order of decreasing donor anomeric reactivity values,^[30,31] it is not necessary to fine-tune anomeric reactivities using the pre-activation method.^[29] This is particularly advantageous for syntheses of oligosaccharides, which consist of repeating units, as the same building blocks can be used repeatedly without resorting to anomeric reactivity adjustments.^[28,29] Herein, we explore the applicability of the iterative one-pot method in construction of the highly challenging sHA.

Building block evaluations

Previous syntheses of sHA have been dominated by the usage of powerful glycosyl trichloroacetimidates.^[5,19] We plan to examine the alternative of using thioglycosides as building blocks, because thioglycosides are stable to most functional group transformation and yet can be easily activated by a wide range of thiophilic promoters.^[32,33] Moreover, they can be conveniently stored on bench top for months without decomposition. Despite the fact that thioglycosides have been one of the most popular glycosyl donors in syntheses of complex oligosaccharides,^[32,33] they have been rarely used for sHA assembly.^[34,35]

The direct usage of glucuronic acid as the glycosyl donor was investigated first. Thioglucuronic acid 1 was activated in the absence of an acceptor by p-TolSOTf, formed in situ by reacting p-TolSCl with AgOTf at −65°C,^[29] which was followed by addition of thioglycosyl acceptor 2 (Table 1, entry 1). However, even after the reaction temperature was raised to room temperature, only donor hydrolysis product was isolated without the desired disaccharide 3. In order to improve the nucleophilicity of the acceptor, we replaced the bulky phthalimido (Phth) moiety in 2 with trichloroethyl carbamate (Troc) (acceptor 4).^[36] However, this did not result in any improvements. Glycosylation of acceptor 2 with glycosyl bromide 5 using promoter AgOTf or reaction of acceptor 6^[37] with donor 1 promoted by N-iodosuccinimide (NIS)/AgOTf did not produce significant amounts of disaccharides. These results demonstrated low reactivities of these glucuronic acid donors and pointing to the necessity of using more reactive glucosyl donors.

Table 1.

Evaluations of building blocks.

Entry	Donor	Acceptor	Disaccharide (yield/%)
1	1	2	3 (< 5)
2	7	2	12a (<5)
3	7	4	12b (<10)
4	7	9	12c (<5)
5	7	10	12d (<20)
6	8	2	13a (<5)
7	8	4	13b (<5)
8	8	9	13c (<5)
9	11	2	14β (75) 14α (5)[a]
10	11	2	14β (75)[b]
11	11	6	15 (88)[c]

^[a]Acceptor 2 was added as a solution in diethyl ether to pre-activated donor in diethyl ether.

^[b]Acceptor 2 was added as a solution in CH₂Cl₂ to pre-activated donor in diethyl ether. Final ratio of CH₂Cl₂/diethyl ether 1:1.

^[c]Reaction was carried out by mixing donor and acceptor together in CH₂Cl₂/diethyl ether 1:1 followed by addition of the promoter.

Peracetylated thioglucosyl donor 7^[31] and p-methoxybenzylidene containing donor 8 were examined next with glucosamine acceptors 2, 4, 9, and 10 under the pre-activation condition, which did not produce much desired disaccharides in dichloromethane, diethyl ether or a mixed solvent dichloromethane/diethyl ether system (Table 1, entries 2–8). Finally, we decided to introduce the tert-butyldimethylsilyl (TBS) moiety into the glucosyl donor (donor 11), due to beneficial effects of TBS on glycosylation in our previous chitotetraose syntheses.^[28]

Thioglucosyl donor 11 was prepared from p-tolyl-1-thio-β-D-glucopyranoside (16),^[31] which was first protected with p-methoxybenzylidene (Scheme 1). Dibutyltin oxide mediated selective benzylation^[38] followed by benzoylation gave thioglycoside 8 in 66% yield over three steps. Regioselective opening of the p-methoxybenzylidene group and TBS protection produced donor 11 (90% yield for two steps). The p-methoxybenzyl (PMB) moiety in 11 can be selectively removed by oxidation in the presence of other protective groups, which is crucial for future oxidation-state adjustments.

Scheme 1.

Building block 11: a) p-methoxybenzylidene dimethyl acetal, camphorsulfonic acid, DMF, 60–70°C; b) nBu₂SnO, toluene, reflux; BnBr, CsF, DMF, 140°C; c) BzCl, N,N-dimethylaminopyridine, 66% for three steps; d) NaCNBH₃, TFA, MS-AW 300, DMF; e) TBSOTf, 2,6-lutidine, CH₂Cl₂, 90% for two steps.

The glycosylation of thioglucoside donor 11 with acceptor 2 was carried out first in diethyl ether (Table 1, entry 9). The desired thioglycosyl disaccharide 14β (¹H NMR: δ_H1′ = 4.73 ppm, ³J_H1′,H2′ = 8.4 Hz) was obtained in 75% yield along with 5% of the corresponding α disaccharide 14α (¹H NMR: δ_H1′ = 5.48 ppm, ³J_H1′,H2′ = 4.2 Hz). The structure of 14α was confirmed by the presence of Bz carbonyl in the ¹³C NMR spectrum with a chemical shift of δ_Bz = 164.9 ppm and a strong HMBC correlation between C1′ and H3. The decrease in the stereospecificity of this reaction despite the presence of participating benzoyl moiety on O2′ is due to the competing α-directing effect of ethereal solvents,^[39–44] presumably through stabilization of the oxocarbenium ion by the oxygen lone pair of diethyl ether from the β-face, leading to α-glycoside. To enhance the stereoselectivity, we discovered that by simply adding the acceptor dissolved in dichloromethane to the pre-activated donor in diethyl ether, the formation of disaccharide 14α can be suppressed to negligible amount without affecting the yield of the desired β-disaccharide (Table 1, entry 10).^[45] Disaccharide 15 bearing a methoxy group at the reducing end was also synthesized in good yield by glycosylating methyl glycoside 6 with donor 11 (Table 1, entry 11).

With the thiotolyl moiety at its reducing end, disaccharide 14β was directly used as a glycosyl donor without any aglycon adjustments. Glycosylation of acceptor 17 by disaccharide 14β formed trisaccharide 18 in 50% yield with 20% of the hydrolyzed donor as the major side product (Scheme 2a). Reaction of trisaccharide 18 with the o-glycoside acceptor 6 led to the desired tetrasaccharide 19 in 47% yield (Scheme 2b). Small amount of trisaccharide 20 could also be isolated from the reaction mixture, which was formed due to the assistance of electron rich 6-OPMB in stabilization of the oxocarbenium ion followed by removal of the p-methoxybenzyl moiety. This long range participation by 6-O protective group has been reported when the glycosyl donor contains a participating group on 6-OH.^[39,46,47] Although we have obtained the tetrasaccharide, the yields for formation of tri- and tetrasaccharides are not sufficiently high enough to enable one-pot synthesis. We investigated next the usage of disaccharide as the acceptor. The disaccharides 14β and 15 were deprotected with HF·pyridine producing thioglycoside 21 and disaccharide 22 in 92 and 87% yield, respectively (Scheme 2c). Gratifyingly, glycosylation of 21 by 14β formed tetrasaccharide 23 in 75% yield (Scheme 2d).

Scheme 2.

From results of these glycosylations, we conclude that it is advantageous to perform donor pre-activation in diethyl ether particularly for building blocks having high anomeric reactivities (e.g. 11). This corroborates with our observation that glycosylation of glucoside 24 by the reactive perbenzylated thioglucoside 25 proceeded cleanly in diethyl ether in 75% yield, while the corresponding reaction in dichloromethane led to a lower yield (~48%) with several side products isolated resulting from donor decomposition. This may be due to stabilization of the reactive oxocarbenium ion intermediate by diethyl ether.

Another important factor to consider in the synthesis is solubilities of the building blocks because the reactions are carried out at low temperatures. The TBS moiety in thioglycoside donor 11 renders it more soluble in diethyl ether compared with donor 8, leading to significantly improved yields in reaction with acceptor 2. Higher glycosylation yields obtained with disaccharide 21 (Scheme 2d) versus monosaccharide acceptor 17 (Scheme 2a) can also be partially attributed to better solubility of disaccharide 21 in the reaction medium.

One-pot syntheses of sHA

With suitable building blocks and reaction conditions identified, one-pot syntheses were performed. Pre-activation of donor 11 (1 equiv) by p-Tol-SOTf was followed by addition of 2 (0.9 equiv) and a sterically hindered non-nucleophilic base 2,4,6-tri-tert-butylpyrimidine (TTBP).^[48] The reaction was allowed to proceed for 90 minutes resulting in the complete disappearance of acceptor 2 based on TLC analysis. The reaction mixture was then warmed up to 0°C for 15 minutes to decompose the slight excess of activated donor, and cooled back down to −65°C. Addition of the second acceptor 22 (0.81 equiv) and TTBP to the reaction mixture followed by activation with p-TolSOTf produced sHA tetrasaccharide 19 in 64–75% overall yield in just three hours (Table 2, entry 1), which was the only compound needed purification in this three component one-pot synthesis.

Table 2.

Iterative one-pot synthesis of sHA. The values in the parenthesis denote the number of equivalents of each reagent.^[a]

	Protocol	Donor	Acceptor 1 (A1)	Acceptor 2 (A2)	Acceptor 3 (A3)	Product	Yield/%
1	A	11	2	22		19	64–75
2	A	26	21	22		28	65
3	B	11	2	21	27	29	55
4	B	11	2	21	22	30	54–60

^[a]Reagents and conditions: a) AgOTf, p-TolSCl, −65°C, 10 min; then acceptor, TTBP, 90 min to 0°C; 15 min, 0°C; b) acceptor, TTBP, AgOTf, p-TolSCl, −65→ 0°C in 90 min.

sHA sequences containing odd number of monosaccharide units can also be synthesized. Sequential one-pot reactions of 26,^[28] 21 and 22 following the reaction protocol A produced pentasaccharide 28 (65% yield) (Table 2, entry 2, protocol A). The other possible pentasaccharide sequence 29 with glucose at the reducing end was rapidly synthesized through consecutive condensations of four components, that is, 11, 2, 21 and 27^[25] (Table 2, entry 3, protocol B), in 55% overall yield for the one-pot reactions.

Because it is unnecessary to achieve the precise anomeric reactivities with our pre-activation based one-pot strategy,^[29] building blocks can be pooled to generate various sHA without anomeric reactivity adjustment. Following the exact reaction sequence for the one-pot synthesis of 19 except for the insertion of 21 as the second acceptor, sHA hexasaccharide 30 was produced in 54–60% yield (Table 2, entry 4, protocol B), which corresponded to an average of 90% yield for the five synthetic steps carried out in a one-pot reaction.

With close to stoichiometric amounts of building blocks used for each glycosylation reaction and no oligosaccharide intermediate purification involved, these syntheses can be easily scaled up. Near gram quantity of sHA hexasaccharide 30 was obtained within several hours in similar yields as the smaller scale. In comparison, the overall yield for the traditional stepwise synthesis of a sHA hexasaccharide was 27% for five synthetic steps starting from an advanced disaccharide building block.^[22] In addition, it required multiple chromatography separations of synthetic intermediates. The scalability coupled with the speed of glycoassembly and higher overall yields highlights the advantages of using the iterative one-pot approach for complex oligosaccharide synthesis.

Deprotection of sHA

Deprotection of large complex oligosaccharides can be a highly challenging task due to the presence of large number of protective groups. This is further compounded by the necessity of oxidation-state adjustment in sHA synthesis and thus the need to differentiate 6-hydroxyl groups of the glucose units from others. Treatment of hexasaccharide 30 with HF·pyridine cleanly removed the TBS moiety and the resulting free hydroxyl group was protected by acetate. Although it has been reported that a PMB group was easily removed by cerium ammonium nitrate (CAN) or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation from hyaluronan di- and trisaccharides,^[21,25] cleavage of all three PMB groups in 32 turned out to be very problematic. Treatment of 32 with CAN or DDQ both failed to produce the desired triol 33 in good yields with multiple decomposition products formed. Simultaneous removal of the phthalimide, acetate and benzoyl groups in 32 followed by per-acetylation led to hexasaccharide 34 in 50% yield. Unfortunately, CAN or DDQ oxidation of 34 again did not generate the desired triol 35 either. Surprisingly, we discovered that the presence of TBS moiety was beneficial for deprotection of PMB, as CAN oxidation of hexasaccharide 30 produced triol 36 in 74% yield (Scheme 3a).

Scheme 3.

a) HF·pyridine; b) Ac₂O, pyridine; c) CAN, CH₃CN, H₂O; d) DDQ, H₂O, CH₂Cl₂; e) NH₂CH₂CH₂NH₂, nBuOH, 80 °C then Ac₂O, pyridine; f) TEMPO, NaOCl, NaBr, Bu₄NBr, NaHCO₃, then NaClO₂, 2-methy-2-butene, NaH₂PO₄; PhCHN₂; g) H₂, Pd(OH)₂; CH₃NH₂ then Ac₂O, MeOH.

Subsequent oxidation-state adjustment of 36 to tricarboxylic acid proves to be non-trivial. In contrast to literature results, 2,2,6,6-tetramethylpiperidinyl-1-oxy (TEMPO)/NaOCl^[21,49–56] or TEMPO/iodobenzene diacetate^[57] oxidation led to a mixture of partially oxidized products, which is probably due to the hydrophobic nature of our fully protected oligosaccharides.^[58] A two-step process of Dess–Martin oxidation and subsequent treatment with NaClO₂^[59] also gave inconsistent results. Through extensive experimentation, we discovered that with a two-step one-pot oxidation protocol of NaOCl/TEMPO followed by addition of NaClO₂,^[58] we were able to carry out this transformation cleanly, which was followed by benzyl ester formation with phenyl diazomethane producing ester 37.^[60] Removal of TBS, catalytic hydrogenation, transamidation with methyl amine and selective acetylation led to fully deprotected sHA hexasaccharide 38 in 44% overall yield from 36 (Scheme 3b). Identical deprotection sequences on sHA 15, 19, 28, and 29 provided free sHA oligosaccharides 39–42 in good overall yields (Scheme 3c). sHA sequences obtained through enzymatic degradation are limited to oligosaccharides containing even number of monosaccharide units.^[11,13] This is the first time that the sHA pentasaccharide sequence 41 has been acquired through either chemical synthesis or degradation.

Conclusion

We have developed highly efficient syntheses of sHA oligosaccharides through the pre-activation based iterative one-pot strategy. Glycoassemblies were accomplished rapidly using only near stoichiometric amounts of building blocks without aglycon adjustment or purification of intermediate oligosaccharides. Reaction solvents and protective groups are important to ensure high yielding glycosylations. The sequence of deprotection is found to be crucial for oxidation-state adjustments and protective group removal. Preliminary studies indicate that this methodology can also be applied to syntheses of other GAGs, such as chondroitin and heparin oligosaccharides. We anticipate that the availability of these structurally well defined sHA will greatly facilitate our understanding of the versatile biological roles of sHA.

Experimental Section

General conditions

Chemicals used were reagent grade as supplied except where noted. All reactions were performed under a nitrogen atmosphere unless specified otherwise. Analytical thin-layer chromatography was performed using silica gel 60 F254 glass plates (EM Science); compound spots were visualized by UV light (254 nm) and/or by staining with a solution containing Ce(NH₄)₂(NO₃)₆ (0.5 g) and (NH₄)₆Mo₇O₂₄·4H₂O (24.0 g) in 6% H₂SO₄ (500 mL) or a solution of KMnO₄ (3 g), K₂CO₃ (20 g) and NaOH (0.25 g) in water (300 mL). Flash column chromatography was performed on silica gel 60 (230–400 mesh, EM Science). ¹H NMR and ¹³C NMR spectra were recorded on a Varian VXRS-400 or Inova-600 instrument and were referenced using Me₄Si (0 ppm), residual CHCl₃ (δ ¹H NMR 7.26 ppm) CDCl₃ (δ ¹³C NMR 77.0 ppm), residual CHDCl₂ (δ ¹H NMR 5.32 ppm), CD₂Cl₂ (δ ¹³C NMR 54.0 ppm). ESI mass spectra were recorded on an ESQUIRE LC-MS operated in both positive and negative ion mode. High-resolution mass spectra were recorded on a Micromass electrospray Tof II (Micromass, Wythenshawe, UK) mass spectrometer equipped with an orthogonal electrospray source (Z spray) operated in positive ion mode, which is located at the Mass Spectrometry and Proteomics Facility, the Ohio State University.

General procedure for pre-activation based single-step glycosylation

The reaction mixture of donor (0.321 mmol) and freshly activated MS 4 Å (500 mg) in anhydrous Et₂O (10 mL) was stirred for 1 h at room temperature, then cooled to −65°C. A solution of AgOTf (250 mg, 0.96 mmol) in Et₂O (10 mL) was added directly to the solution without touching the wall of the reaction flask. After 10 min at −65°C, p-TolSCl (50 μL, 0.321 mmol) was added via a syringe. The mixture was stirred for 5 min until the yellow color dissipated and the donor was completely consumed judged by TLC. A solution of acceptor 1 (0.289 mmol) and TTBP (80 mg, 0.321 mmol) in anhydrous CH₂Cl₂ (10 mL) was added slowly to the pre-activated donor along the wall of the reaction flask. The reaction was continued for 90 min from −65 to 0°C and quenched with triethylamine (500 μL). The obtained reaction mixture was concentrated under reduced pressure, resuspended in CH₂Cl₂ (50 mL) and filtered. The filtrate was concentrated and purified by flash column chromatography (hexanes, EtOAc and CH₂Cl₂) to afford the desired product.

General procedures for one-pot synthesis of oligosaccharide

Protocol A

The mixture of donor (0.321 mmol) and freshly activated MS 4 Å (500 mg) in anhydrous Et₂O (10 mL) was stirred for 1 h at room temperature, then cooled to −65°C. A solution of AgOTf (250 mg, 0.96 mmol) in Et₂O (10 mL) was added directly to the solution without touching the wall of the reaction flask. After 10 min at −65°C, p-TolSCl (50 μL, 0.321 mmol) was added via a syringe. The mixture was stirred for 5 min until the yellow color dissipated and the donor was completely consumed judged by TLC. A solution of acceptor 1 (0.289 mmol) and TTBP (80 mg, 0.321 mmol) in anhydrous CH₂Cl₂ (10 mL) was added slowly to the pre-activated donor along the wall of the reaction flask. The reaction was continued for 90 min from −65 to 0°C. A solution of acceptor 2 (0.261 mmol) and TTBP (71 mg, 0.289 mmol) in anhydrous CH₂Cl₂ (10 mL) was added and the resulted solution was cooled down to −65°C followed by the addition of AgOTf (200 mg, 0.783 mmol) in Et₂O (8 mL) and p-TolSCl (45 μL, 0.289 mmol). The reaction was stirred for 90 min from −65 to 0°C and quenched with triethylamine (300 μL). The obtained reaction mixture was concentrated under reduced pressure, resuspended in CH₂Cl₂ (50 mL) and filtered. The filtrate was concentrated and purified by flash column chromatography (hexanes, EtOAc and CH₂Cl₂) to afford the desired product.

Protocol B

The mixture of donor (230 mg, 0.321 mmol) and freshly activated MS 4 Å (500 mg) in anhydrous Et₂O (10 mL) was stirred for 1 h at room temperature, then cooled to −65°C. A solution of AgOTf (250 mg, 0.96 mmol) in Et₂O (10 mL) was added directly to the solution without touching the wall of the reaction flask. After 10 min at −65°C, p-TolSCl (50 μL, 0.321 mmol) was added via a syringe. The mixture was stirred for 5 min until the yellow color dissipated and donor was consumed judged by TLC. A solution of acceptor 1 (0.289 mmol) and TTBP (71 mg, 0.289 mmol) in anhydrous CH₂Cl₂ (10 mL) was added slowly to the pre-activated donor along the wall of the reaction flask. The reaction was continued for 90 min from −65 to 0°C and cooled back down to −65°C. To the reaction mixture, AgOTf (220 mg, 0.866 mmol) in Et₂O (8 mL) and p-TolSCl (45 μL, 0.289 mmol) were sequentially added. The mixture was stirred for 15 min until no donor was left when a solution of acceptor 2 (0.255 mmol) and TTBP (71 mg, 0.289 mmol) in anhydrous CH₂Cl₂ (10 mL) was added slowly. The reaction was kept for 90 min from −65 to 0°C. Then acceptor 3 (0.261 mmol) and TTBP (63 mg, 0.253 mmol) in anhydrous CH₂Cl₂ (10 mL) was added. The mixture was cooled down to −65°C followed by the sequential addition of AgOTf (200 mg, 0.783 mmol) in Et₂O (8 mL) and p-TolSCl (41 μL, 0.261 mmol). The reaction was stirred for 90 min from −65 to 0°C and quenched with triethylamine (400 μL). The obtained reaction mixture was concentrated under reduced pressure, resuspended in CH₂Cl₂ (100 mL) and filtered. The filtrate was concentrated and purified by flash column chromatography (hexanes, EtOAc and CH₂Cl₂) to afford the desired product.

General procedures for deprotection and oxidation-state adjustment

Deprotection of PMB

An aqueous solution (2.5 mL) of CAN (1.16 g, 2.14 mmol) was added at 0°C to a solution of the PMB-protected compound (0.42 mmol) in acetonitrile (10 mL). The reaction mixture was stirred for 1 h from 0°C to room temperature then diluted with EtOAc (100 mL) and washed with water (30 mL) three times. The organic phases were collected and dried over MgSO₄. The obtained residue was purified by flash column chromatography to give the desired product.

Oxidation of alcohols to carboxylic acids

A 1M aqueous solution of NaBr (25 μL), 1M aqueous solution of tetrabutylammonium bromide (50 μL), TEMPO (2.2 mg, 0.014 mmol, 0.3 equiv per hydroxyl group) and a saturated aqueous solution of NaHCO₃ (125 μL) were added to a solution of the alcohol (0.045 mmol) in CH₂Cl₂ (1 mL) and H₂O (170 μL) cooled with an ice-water bath. The resulted mixture was treated with an aqueous solution of NaOCl (150 μL, chlorine content not less than 4%) and continuously stirred for 1 h from 0°C to RT. The reaction media was neutralized with 1N HCl (about 50 μL) to pH 6–7. It is important to keep the acidity of the reaction close to neutral as lower pH had resulted in formation of large amount of hemiacetal side product. After neutralization, tBuOH (0.7 mL), 2M 2-methyl-but-2-ene (1.4 mL) in THF and a solution of NaClO₂ (50 mg, 0.44 mM) and NaH₂PO₄ (40 mg, 0.34 mM) in water (200 μL) were added. The reaction mixture was kept at room temperature for 1–2 h, diluted with saturated aqueous NaH₂PO₄ solution (5 mL) and extracted with EtOAc (10 mL) three times. The organic layers were combined and dried over MgSO₄. After removal of the solvent, the desired compound was purified by flash column chromatography (hexanes, EtOAc + 1% AcOH).

Benzyl ester formation

The crude product from the oxidation reaction was dissolved in dichloromethane (5 mL) and treated with phenyl diazomethane solution in diethyl ether (~2 equiv per acid) for 2–3 h until the disappearance of all starting material as judged by TLC. The residue after evaporation was purified by flash column chromatography to provide the benzyl ester.

Removal of TBS

The TBS-protected compound (0.5 mmol) was dissolved in pyridine (4 mL) in a plastic flask followed by the addition of 65–70% HF·pyridine solution (4 mL, diluted with 2 mL pyridine) at 0°C. The solution was stirred for 48 h until complete disappearance of the starting material as judged from TLC analysis. The reaction mixture was diluted with EtOAc (30 mL) and washed with 10% aqueous CuSO₄ solution (20 mL). The aqueous phase was extracted with EtOAc (30 mL) twice and the combined organic layers were washed with saturated aqueous NaHCO₃ solution (3×50 mL). After drying over MgSO₄ and concentrated, the obtained residue was purified by flash column chromatography (hexanes/EtOAc) to give the desired product.

Hydrogenation

Pd(OH)₂ (80 mg) was added to a solution of the oligosaccharide (0.045 mmol) in THF (1.5 mL), MeOH (2 mL) and acetic acid (1.5 mL). The reaction flask was evacuated using a water aspirator and filled with hydrogen. The process was repeated two times and the reaction mixture was stirred under hydrogen atmosphere until a single desired peak was observed by ESI mass spectrum (~48 h). The solution was filtered and concentrated to provide the crude product.

Removal of benzoyl and phthalimido groups by MeNH₂

The crude product from hydrogenation was treated with 33% methyl amine solution in ethanol (1 mL per μmol of compound) until only the desired peak was observed by ESI mass spectrum (~72 h).

Acetylation

The crude reaction product from methyl amine treatment (0.05 mmol) was dissolved in methanol (2 mL) followed by addition of acetic anhydride (5 equiv) and triethylamine at 0°C. The reaction was run for 2–3 h, and treated with Amberlite IR-120 for 30 min. After filtration, the solvents were removed and the residue was dissolved in water (5 mL), washed with Et₂O (3 mL) and purified by Sephadex G-15 size-exclusion chromatography and anion-exchange chromatography (DE 52, Whatman) to give the desired product.

p-Tolyl-2,3,4-tri-O-acetyl-1-thio-β-D-glucopyranuronic acid methyl ester (1)

p-TolSH (312 mg, 2.52 mmol) and tetrabutylammonium iodide (465 mg, 1.26 mmol) at room temperature were added to a solution of bromo-2,3,4-tri-O-acetyl-α-D-glucopyranuronic acid methyl ester (5; 500 mg, 1.26 mmol) in EtOAc (5 mL) and 1M aqueous Na₂CO₃ (5 mL). The reaction was stirred for 1 h until no starting material was observed by TLC. Extraction by EtOAc and washing of the organic layer with saturated NaHCO₃ followed by flash column chromatography gave compound 1 (499 mg, 90%). ¹H NMR (400 MHz, CDCl₃): δ = 1.99 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.35 (s, 3H, OCH₃), 3.76 (s, 3H, CO₂CH₃), 4.10 (d, ³J(H,H) = 10.0 Hz, 1H), 4.66 (d, ³J(H,H) = 10.0 Hz, 1H), 4.93 (t, 1H, ³J(H,H) = 9.6 Hz, 1H), 5.14 (t, ³J(H,H) = 9.6 Hz, 1H), 5.25 (t, ³J(H,H) = 9.6 Hz, 1H), 7.13 (d, ³J(H,H) = 12 Hz, 2H), 7.39 (d, ³J(H,H) = 12 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃): δ = 20.7, 20.8, 21.0, 21.4, 53.1, 69.4, 69.9, 73.5, 76.4, 86.4, 127.3, 130.0, 134.2, 139.2, 167.1, 169.3, 169.5, 170.3; ESI-MS: m/z: calcd for C₂₁H₂₄NaO₆S: 463.1, found: 463.2 [M+Na]⁺.

p-Tolyl-2-deoxy-2-N-Troc-4,6-O-benzylidene-β-D-glucopyranoside (4)

The solution of compound 2 (1.25 g, 2.48 mmol) and ethylenediamine (2 mL) in n-butanol (15 mL) was refluxed for 1.5 h until no starting material was observed by TLC. The solution was dried under reduced pressure followed by chromatography to afford free amine (881 mg, 95%). The free amine (750 mg, 2.16 mmol) was dissolved in a biphasic system of aqueous saturated NaHCO₃ (15 mL) and THF (15 mL) followed by addition of 2,2,2-trichloroethoxy chloroformate (349 μL, 2.60 mmol) at 0°C. The reaction was stirred for 1 h and diluted with dichloromethane followed by extraction and flash chromatography to provide the desired product 4 (1.40 g, 99%). ¹H NMR (400 MHz, CDCl₃ + 1 drop of CD₃OD): δ = 2.26 (s, 3H, CH₃), 3.36–3.48 (m, 3H), 3.71 (t, ³J(H,H) = 10.0 Hz, 1H), 3.77 (t, ³J(H,H) = 9.2 Hz, 1H), 4.25 (dd, ³J(H,H) = 4.4, 10.0 Hz, 1H), 4.62 (d, ³J(H,H) = 12.0 Hz, 1H), 4.75 (d, ³J(H,H) = 10.0 Hz, 1H), 4.77 (d, ³J(H,H) = 12.0 Hz, 1H), 5.47 (s, 1H, PhCH), 7.03–7.42 (m, 9H); ¹³C NMR (100 MHz, CDCl₃ + 1 drop of CD₃OD): δ = 21.2, 57.4, 68.7, 70.5, 72.3, 74.7, 81.4, 87.8, 95.7, 101.9, 126.4–138.5 (aromatic carbons), 155.2; ESI-MS: m/z: calcd for C₂₃H₂₅Cl₃NNaO₆S: 571.1, found: 571.0 [M+Na]⁺.

p-Tolyl 2-O-benzoyl-3-O-benzyl-4,6-O-p-methoxybenzylidene-1-thio-β-D-glucopyranoside (8)

The solution of p-tolyl-1-thio-β-D-glucopyranoside 16 (10 g, 35 mmol), p-anisaldehyde dimethyl acetal (7.1 mL, 42 mmol) and camphorsulfonic acid (1.6 g, 7 mmol) in anhydrous DMF (80 mL) was swirled on a rotary evaporator under aspirator pressure at 50°C for 3 h, then was warmed up to 70°C to remove methanol generated until the reaction was completed judging by TLC. The resulting solution was diluted with diethyl ether (150 mL) followed by washing with saturated NaHCO₃ and drying over MgSO₄. The residue was purified by flash column chromatography (hexanes/EtOAc 5:5 → 3:7) to afford the desired diol p-tolyl 4,6-O-p-methoxybenzylidene-1-thio-β-D-glucopyranoside (13 g, 92%) after purification. ¹H NMR (400 MHz, CDCl₃): δ = 2.36 (s, 3H, CH₃), 2.71 (br, 1H, OH), 2.81 (br, 1H, OH), 3.42 (t, ³J(H,H) = 8.8 Hz, 1H), 3.46–3.49 (m, 2H), 3.74 (t, ³J(H,H) =10.0 Hz, 1H), 3.77 (s, 3H, OCH₃), 3.82 (t, ³J(H,H) = 8.8 Hz, 1H), 4.36 (dd, ³J(H,H) = 4.0, 10.0 Hz, 1H), 4.54 (d, ³J(H,H) = 10.0 Hz, 1H), 5.47 (s, PhCH), 6.87–7.44 (m, 8H); ¹³C NMR (100 MHz, CDCl₃): δ = 21.4, 55.2, 68.6, 70.6, 72.8, 74.8, 80.4, 88.8, 102.0, 114.0–138.8 (aromatic carbons); ESI-MS: m/z: calcd for C₂₁H₂₅O₆S: 405.3, found: 405.2 [M+H]⁺.

The diol p-tolyl 4,6-O-p-methoxybenzylidene-1-thio-β-D-glucopyranoside (8.5 g, 21 mmol) was heated under reflux with Bu₂SnO (5.7 g, 23 mmol) in a flask equipped with a Dean–Stark device in anhydrous toluene (400 mL) for 3 h and concentrated to approximate 100 mL. After cooling the reaction mixture down to room temperature, anhydrous DMF (150 mL dried over 5% w/v MS 4 Å twice overnight) was added followed by addition of CsF (3.5 g, 23 mmol) and BnBr (2.76 mL, 23.1 mmol) and the obtained solution was stirred for 4 h at 140°C. After the reaction was complete, DMF was removed under reduced pressure. The residue was dissolved in CH₂Cl₂ and extracted by 1 M aqueous solution of KF. The organic phase was dried over MgSO₄ and purified by flash column chromatography (hexanes/EtOAc/CH₂Cl₂ 5:1:1) to offer the desired product p-tolyl 3-O-benzyl-4,6-O-p-methoxybenzylidene-1-thio-β-D-glucopyranoside (8.4 g, 80%). ¹H NMR (400 MHz, CDCl₃): δ = 2.35 (s, 3H, CH₃), 2.52 (br, 1H, OH), 3.44–3.51 (m, 2H), 3.61 (t, ³J(H,H) = 9.2 Hz, 1H), 3.67 (t, ³J(H,H) = 8.8 Hz„ 1H), 3.76 (t, ³J(H,H) = 8.4 Hz, 1H), 3.81 (s, 3H, OCH₃), 4.36 (dd, ³J(H,H) = 4.2, 10.8 Hz, 1H), 4.55 (d, ³J(H,H) = 9.6 Hz, 1H), 4.77 (d, ³J(H,H) = 11.6 Hz, 1H), 4.94 (d, ³J(H,H) = 11.6 Hz, 1H), 5.52 (s, 1H, PhCH), 6.88–7.43 (m, 13H); ESI-MS: m/z: calcd for C₂₈H₃₀NaO₆S: 517.6, found: 517.7 [M+Na]⁺.

p-Tolyl 3-O-benzyl-4,6-O-p-methoxybenzylidene-1-thio-β-D-glucopyrano-side (7.5 g, 15.2 mmol) was treated with benzoyl chloride (2.12 mL, 18.2 mmol), N,N-dimethylaminopyridine (DMAP, 4.4 g, 36.4 mmol) in anhydrous CH₂Cl₂ (50 mL) overnight at room temperature. The reaction mixture was washed with an aqueous NH₄Cl solution (30 mL) and an aqueous saturated NaHCO₃ solution (30 mL) followed by drying over MgSO₄. The desired product 8 was obtained (8.2 g, 90%) after purification by flash column chromatography (hexanes/EtOAc/CH₂Cl₂ 4:1:1). ¹H NMR (400 MHz, CDCl₃): δ = 2.32 (s, 3H, CH₃), 3.75 (dt, ³J(H,H) = 4.8, 9.2 Hz, 1H), 3.78 (t, ³J(H,H) = 9.2 Hz, 1H), 3.82 (s, 3H, OCH₃), 3.78–3.84 (m, 1H), 3.86 (t, ³J(H,H) = 9.2 Hz, 1H), 4.39 (dd, ³J(H,H) = 4.8, 10.0 Hz, 1H), 4.64 (d, ³J(H,H) = 12.0 Hz, 1H), 4.77 (d, ³J(H,H) = 10.0 Hz, 1H), 4.78 (d, ³J(H,H) = 12.0 Hz, 1H), 5.25 (dd, ³J(H,H) = 9.2, 10.0 Hz, 1H), 5.56 (s, 1H, PhCH), 6.90–8.05 (m, 18H); ¹³C NMR (100 MHz, CDCl₃): δ = 21.4, 55.5, 68.8, 70.8, 72.3, 74.4, 79.6, 81.6, 87.4, 101.5, 113.9–138.8 (aromatic carbons), 160.3, 165.2 (carbonyl groups); ESI-MS: m/z: calcd for C₃₅H₃₅O₇S: 599.2, found: 599.0 [M+H]⁺.

p-Tolyl 4,6-O-benzylidene-2-deoxy-2-N-trichloroacetyl-β-D-glucopyrano-side (9)

A solution of compound 2 (1.25 g, 2.48 mmol) and ethylenediamine (2 mL) in n-butanol (15 mL) was heated under reflux for 1.5 h until no starting material was observed by TLC. The solution was dried under reduced pressure followed by chromatography to afford free amine (881 mg, 95%). The free amine (534 mg, 1.43 mmol) was dissolved in anhydrous THF (3 mL) followed by addition of triethylamine (216 mg, 2.14 mmol) and trichloroacetyl chloride (167 μL, 1.50 mmol) at 0°C. The reaction was stirred for 1 h and diluted with dichloromethane followed by extraction and flash chromatography (hexanes/EtOAc 3:1) to provide desire product 9 (740 mg, 99%). ¹H NMR (400 MHz, CDCl₃ + 1 drop of CD₃OD): δ = 2.25 (s, 3H, CH₃), 3.42–3.45 (m, 2H), 3.61 (t, ³J(H,H) = 10.2 Hz, 1H), 3.69 (t, ³J(H,H) = 10.2 Hz, 1H), 3.93 (t, ³J(H,H) = 9.6 Hz, 1H), 4.24 (dd, ³J(H,H) = 4.2, 10.2 Hz, 1H), 4.93 (d, ³J(H,H) = 10.2 Hz, 1H), 5.45 (s, 1H, PhCH), 7.03–7.39 (m, 9H); ¹³C NMR (100 MHz, CDCl₃ + 1 drop of CD₃OD): δ = 21.3, 57.3, 68.7, 70.6, 71.4, 81.5, 86.8, 101.9, 126.4–138.8 (aromatic carbons), 165.6 (carbonyl group); ESI-MS: m/z: calcd for C₂₂H₂₂Cl₃NNaO₅S: 541.8, found: 542.1 [M+Na]⁺.

p-Tolyl-2-deoxy-2-azido-4,6-O-benzylidene-1-thio-α-D-glucopyranoside (10)

Borontrifluoride etherate (15 mL, 120 mmol) was added slowly at 0°C under an atmosphere of N₂ to a solution of 2-deoxy-2-azido-3,4,6-tri-O-acetyl-α/β-D-glucopyranosyl acetate^[61] (7.6 g, 20 mmol) and p-TolSH (3.72 g, 30 mmol) in dichloromethane (50 mL). The reaction was kept at room temperature for 5 d and carefully quenched with saturated aqueous solution of NaHCO₃. The aqueous layer was then separated and washed twice with dichloromethane. The organic layers were combined and dried over Na₂SO₄. p-Tolyl-2-deoxy-2-azido-3,4,6-tri-O-acetyl-1-thio-α/β-D-glu-copyranoside (6.0 g, 13.7 mmol, 70%) was isolated by flash chromatography (hexanes/EtOAc 2:1).

Sodium methoxide in MeOH (5M, 4 mL) was added at room temperature to a solution of p-tolyl-2-deoxy-2-azido-3,4,6-tri-O-acetyl-1-thio-α/β-D-glucopyranoside (6.0 g, 13.7 mmol) in MeOH (20 mL). After 2 d, the solution was neutralized with Amberlite IR-120, filtered and evaporated. The crude mixture of p-tolyl 2-deoxy-2-azido-1-thio-D-glucopyranosides obtained was azotropically dried with anhydrous toluene and then dissolved in anhydrous CH₃CN (30 mL). Benzaldehyde dimethyl acetal (4.2 mL, 27.4 mmol) and camphorsulfonic acid (0.9 g, 4.1 mmol) were added and the reaction mixture was stirred at room temperature overnight. The reaction was quenched with Et₃N and evaporated. Silica gel column chromatography (hexanes/EtOAc 3:1) afforded compound 10 (3.2 g) and the corresponding β-isomer (1.6 g) in 90% total yield. ¹H NMR (400 MHz, CDCl₃): δ = 2.37 (s, 3H), 2.88 (brs, 1H, OH), 3.60 (t, ³J(H,H) = 9.2 Hz, 1H), 3.78 (t, ³J(H,H) = 10.2 Hz, 1H), 3.92 (dd, ³J(H,H) = 5.4, 10.0 Hz, 1H), 4.10 (t, ³J(H,H) = 9.6 Hz, 1H), 4.25 (dd, ³J(H,H) = 5.0, 10.2 Hz, 1H), 4.44 (dt, ³J(H,H) = 5.0, 10.0 Hz, 1H), 5.58 (d, ³J(H,H) = 5.4 Hz, 1H), 5.58 (s, 1H), 7.05–7.56 (m, 9H, ArH); ¹³C NMR (50 MHz, CDCl₃): δ = 21.2, 63.3, 63.9, 68.5, 70.7, 81.7, 88.1, 102.2, 126.3, 128.4, 129.0, 129.5, 130.0, 133.1, 136.8, 138.5; ESI-MS: m/z: calcd for C₂₀H₂₁N₃NaO₄S: 422.1, found: 422.2 [M+Na]⁺.

p-Tolyl 2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside (17)

Acetal 8 (6.5 g, 10.8 mmol) in anhydrous DMF (80 mL) was cooled down to 0°C followed by sequential addition of solid NaCNBH₃ (5.44 g, 86.4 mmol) and TFA (12.4 g, 108 mmol). The resulting suspension was stirred for 48 h until no starting material was found. After neutralization by solid NaHCO₃, the solution was filtered and diluted with EtOAc followed by washing with a saturated aqueous NaHCO₃ solution. The organic phase was collected and applied to flash column to afford alcohol 17 (6.1 g, 95%) containing trace amount of regioisomer p-tolyl 2-O-benzoyl-3-O-benzyl-4-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside. Pure 17 was obtained after removal of TBS from compound 11 following the general procedure for TBS deprotection. ¹H NMR (400 MHz, CDCl₃): δ = 2.31 (s, 3H, CH₃), 3.52–3.60 (m, 1H), 3.69 (t, ³J(H,H) = 8.8 Hz, 1H), 3.76–3.81 (m, 3H), 3.82 (s, 3H, OCH₃), 4.49 (d, ³J(H,H) = 11.6 Hz, 1H), 4.52 (d, ³J(H,H) = 11.6 Hz, 1H), 4.65 (d, ³J(H,H) = 11.6 Hz, 1H), 4.69 (d, ³J(H,H) = 11.6 Hz, 1H), 4.72 (d, ³J(H,H) = 9.6 Hz, 1H), 5.22 (dd, ³J(H,H) = 8.8, 9.6 Hz, 1H), 6.87–8.06 (m, 18H); ¹³C NMR (100 MHz, CDCl₃): δ = 21.4, 55.5, 70.4, 72.3, 72.4, 73.7, 74.9, 78.5, 83.9, 86.9, 114.1, 128.0–138.3 (aromatic carbons), 159.6, 165.4 (carbonyl groups); ESI-MS: m/z: calcd for C₃₅H₃₆NaO₇S: 623.2, found: 623.3 [M+Na]⁺.

p-Tolyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-me-thoxybenzyl-1-thio-β-D-glucopyranoside (11)

A solution of alcohol 17 (5.56 g, 9.26 mmol) in anhydrous CH₂Cl₂ (50 mL) was cooled down to −20°C followed by sequential addition of 2,6-lutidine (1.98 g, 18.5 mmol) and TBSOTf (3.67 g, 13.9 mmol). The resulting solution was warmed to room temperature slowly until no starting material was left judged by TLC. The reaction mixture was diluted with CH₂Cl₂ (50 mL) and washed with saturated NaHCO₃ solution. The organic phase was collected and dried followed by separation by flash column chromatography (hexanes/EtOAc/CH₂Cl₂ 20:1:1) to give target molecule 11 (6.27 g, 90%) along with a 1:1 mixture (~5%) of compound 11 with its regioisomer p-tolyl 2-O-benzoyl-3-O-benzyl-6-O-tert-butyldimethylsilyl-4-O-p-methoxybenzyl-1-thio-β-D-glucopyranoside. ¹H NMR (400 MHz, CDCl₃): δ = −0.05 (s, 3H, CH₃Si), 0.00 (s, 3H, CH₃Si), 0.85 (s, 9H, (CH₃)₃CSi), 2.30 (s, CH₃, 3H), 3.57–3.69 (m, 4H), 3.80–3.84 (m, 1H), 3.82 (s, 3H, OCH₃), 4.45 (d, ³J(H,H) = 11.6 Hz, 1H), 4.56 (d, ³J(H,H) = 11.6 Hz, 1H), 4.61–4.64 (m, 2H), 4.75 (d, ³J(H,H) = 10.0 Hz, 1H), 5.24 (t, ³J(H,H) = 10.0 Hz, 1H), 6.87–7.99 (m, 18H); ¹³C NMR (100 MHz, CDCl₃): δ = −4.5, −3.5, 18.2, 21.4, 26.2, 55.5, 69.5, 71.3, 73.2, 73.4, 75.4, 81.2, 85.0, 86.6, 113.9–138.0 (aromatic carbons), 159.4, 165.4 (carbonyl groups); HRMS: m/z: calcd for C₄₁H₅₀NaO₇SSi: 737.2944, found: 737.2953 [M+Na]⁺.

p-Tolyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside (14β)

The mixture of donor 11 (714 mg, 1 mmol) and freshly activated MS 4 Å (800 mg) in diethyl ether (20 mL) was stirred for 1 h at room temperature, and cooled down to −65°C followed by the addition of AgOTf (771 mg, 3 mmol) in anhydrous Et₂O (10 mL) directly to the solution without touching the wall of the reaction flask. After 5 min, p-TolSCl (157 μL, 1 mmol) was added via a syringe to activate the donor. The yellow color of the reaction disappeared quickly and TLC analysis showed the donor was completely consumed. A solution of acceptor 2 (451 mg, 0.9 mmol) and TTBP (248 mg, 1 mmol) in CH₂Cl₂ (3 mL) was then slowly added to the reaction mixture along the wall of the reaction flask. It was stirred for 90 min until the temperature reached 0°C, and triethylamine (300 μL) was added. The reaction mixture was concentrated to remove most Et₂O, resuspended in CH₂Cl₂ (100 mL), and filtered. The filtrate was concentrated and purified by flash column chromatography using a three solvent system (CH₂Cl₂, hexanes and EtOAc) to give the desired disaccharide 14β (740 mg, 0.675 mmol, 75%). ¹H NMR (400 MHz, CDCl₃): δ= −0.16 (s, 3H, CH₃Si), −0.09 (s, 3H, CH₃Si), 0.74 (s, 9H, (CH₃)₃CSi), 2.28 (s, 3H, p-CH₃ArS), 3.12 (m, 1H), 3.29 (m, 1H), 3.38 (t, ³J(H,H) = 8.4 Hz, 1H), 3.42 (dd, ³J(H,H) = 2.0, 8.4 Hz, 1H), 3.57–3.67 (m, 3H), 3.83 (s, 3H, p-CH₃OAr), 3.87 (t, ³J(H,H) = 8.8 Hz, 1H), 4.28–4.44 (m, 5H), 4.58 (d, J = 12 Hz, 1H), 4.66 (t, ³J(H,H) = 8.8 Hz, 1H), 4.73 (d, ³J(H,H) = 8.4 Hz, 1H), 5.03 (t, ³J(H,H) = 8.4 Hz, 1H), 5.33 (s, 3H, PhCH), 5.44 (d, ³J(H,H) = 10.8 Hz, 1H), 6.90–7.95 (m, 27H); ¹³C NMR (100 MHz, CDCl₃): δ = −4.7, −3.7, 18.2, 21.4, 26.1, 54.7, 55.6, 68.7, 70.8, 71.0, 73.1, 74.4, 74.6, 76.2, 76.5, 81.2, 83.4, 84.7, 99.6, 101.6, 114.0, 126.7–138.5 (aromatic carbons), 159.5, 164.7 (carbonyl groups); HRMS: m/z: calcd for C₆₂H₆₇NNaO₁₃SSi: 1116.4000, found: 1116.3998 [M+Na]⁺.

p-Tolyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-α-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside (14α)

When acceptor 2 was added as a solution in diethyl ether, 14α (~5%) was separated from the reaction along with 14β (75%). ¹H NMR (600 MHz, CDCl₃): δ = −0.20 (s, 3H, CH₃Si), −0.14 (s, 3H, CH₃Si), 0.70 (s, 9H, (CH₃)₃CSi), 2.30 (s, 3H, p-CH₃ArS), 2.74 (dd, ³J(H,H) = 2.4, 10.8 Hz, 1H), 2.78 (dd, ³J(H,H) = 3.0, 10.8 Hz, 1H), 3.04–3.08 (m, 1H), 3.54–3.62 (m, 2H), 3.66 (t, ³J(H,H) = 9.0 Hz, 1H), 3.74 (dd, ³J(H,H) = 9.0, 10.2 Hz, 1H), 3.81 (s, 3H, p-CH₃OAr), 4.21 (dd, ³J(H,H) = 4.8, 10.2 Hz, 1H), 4.22–4.28 (m, 2H), 4.46 (t, ³J(H,H) = 10.2 Hz, 1H), 4.60 (d, ³J(H,H) = 11.4 Hz, 1H), 4.64 (s, 1H), 4.69 (d, ³J(H,H) = 11.4 Hz, 1H), 4.80 (dd, ³J(H,H) = 9.0, 9.6 Hz, 1H), 5.09 (dd, ³J(H,H) = 4.2, 9.6 Hz, 1H), 5.48 (d, ³J(H,H) = 4.2 Hz, 1H), 5.59 (d, ³J(H,H) = 10.8 Hz, 1H), 6.90–7.92 (m, 27H); ¹³C NMR (100 MHz, CDCl₃): δ = −5.1, −3.5, 18.2, 21.4, 26.3, 54.5, 55.6, 67.2, 68.6, 73.1, 73.1, 73.2, 74.0, 75.0, 79.7, 83.0, 85.1, 96.8, 101.3, 113.9, 123.7, 124.2, 126.2–138.7 (aromatic carbons), 159.3, 164.9, 167.4; ESI-MS: m/z: calcd for C₆₂H₆₇NNaO₁₃SSi: 1116.4, found: 1116.2 [M+Na]⁺.

Methyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside (15)

The mixture of donor 7 (714 mg, 1 mmol), acceptor 10 (370 mg, 0.9 mmol) and freshly activated MS 4 Å (800 mg) in diethyl ether (20 mL) and CH₂Cl₂ (10 mL) was stirred for 1 h at room temperature, and cooled down to −65°C. A solution of AgOTf (771 mg, 3 mmol) in anhydrous Et₂O (10 mL) was added directly to the solution without touching the wall of the reaction flask. After 10 min, p-TolSCl (157 μL, 1 mmol) was added via a syringe. The reaction mixture was stirred for 1.5 h until the temperature reached 0°C, and triethylamine (300 μL) was added. The reaction mixture was concentrated to remove most Et₂O, resuspended in CH₂Cl₂ (100 mL), and filtered. The filtrate was concentrated and purified by flash column chromatography (hexanes/EtOAc 4:1 → 2.5:1) to give the desired disaccharide 15 (800 mg, 0.792 mmol, 88%). ¹H NMR (400 MHz, CDCl₃): δ = −0.16 (s, 3H, CH₃Si), −0.08 (s, 3H, CH₃Si), 0.75 (s, 9H, (CH₃)₃CSi), 3.07 (m, 1H), 3.29 (dd, ³J(H,H) = 6.0, 10.4 Hz, 1H), 3.36 (s, 3H, CH₃O), 3.38–3.44 (m, 2H), 3.56–3.61 (m, 2H), 3.68 (t, ³J(H,H) = 10.4 Hz), 3.83 (s, 3H, p-CH₃OAr), 3.87 (m, 1H), 4.27–4.33 (m, 4H), 4.35 (d, ³J(H,H) = 11.2 Hz, 1H), 4.44 (d, ³J(H,H) = 11.2 Hz, 1H), 5.58 (d, ³J(H,H) = 11.2 Hz, 1H), 4.67 (dd, ³J(H,H) = 8.4, 10.0 Hz, 1H), 4.73 (d, ³J(H,H) = 8.4 Hz, 1H), 5.01 (d, ³J(H,H) = 8.4 Hz, 1H), 5.04 (t, ³J(H,H) = 8.4 Hz, 1H), 5.33 (s, 1H, PhCH), 6.91–7.46 (m, 23H); ¹³C NMR (100 MHz, CDCl₃): δ= −4.7, −3.7, 18.1, 21.4, 26.1, 55.4, 55.5, 57.1, 66.7, 68.6, 68.9, 70.9, 73.0, 74.5, 74.6, 75.6, 76.1, 81.8, 83.4, 99.7, 99.7, 101.6, 114.0, 126.7–138.0 (aromatic carbons), 159.5, 164.7 (carbonyl groups); HRMS: m/z: calcd for C₅₆H₆₃NNaO₁₄Si: 1024.3916, found: 1024.3879 [M+Na]⁺.

p-Tolyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-2-deoxy-2-N-phthalimido-4,6-O-benzylidene-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranoside (18)

Compound 18 (100 mg, 50%) was synthesized using donor 14β and acceptor 17 following the general procedure for single-step pre-activation based glycosylation. ¹H NMR (400 MHz, CDCl₃): δ = −0.16 (s, 3H, CH₃Si), −0.11 (s, 3H, CH₃Si), 0.73 (s, 9H, (CH₃)₃CSi), 2.23 (s, 3H, CH₃), 3.10 (m, 1H), 3.16-.20 (m, 2H), 3.27–3.32 (m, 2H), 3.35–3.45 (m, 4H), 3.56–3.68 (m, 3H), 3.75 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.81 (m, 1H), 3.99–4.21 (m, 2H), 4.18 (d, ³J(H,H) = 10.8 Hz, 1H), 4.26 (d, ³J(H,H) = 10.8 Hz, 1H), 4.29–4.33 (m, 2H), 4.35 (d, ³J(H,H) = 11.2 Hz, 1H), 4.43 (d, ³J(H,H) = 11.2 Hz, 1H), 4.98 (d, ³J(H,H) = 8.4 Hz, 1H), 4.57 (d, ³J(H,H) = 11.6 Hz, 1H), 4.61 (d, ³J(H,H) = 11.6 Hz, 1H), 4.67 (dd, ³J(H,H) = 8.8, 9.2 Hz, 1H), 4.74 (d, J = 8.0 Hz, 1H), 4.83 (d, ³J(H,H) = 12 Hz, 1H), 5.03 (t, ³J(H,H) = 8.8 Hz, 1H), 5.10 (t, ³J(H,H) = 9.2 Hz, 1H), 5.25 (d, ³J(H,H) = 8.4 Hz, 1H), 5.29 (s, 1H, PhCH), 6.78–7.94 (m, 37H); ¹³C NMR (100 MHz, CDCl₃): δ = −4.7, −3.7, 18.1, 21.4, 26.0, 55.5, 56.1, 66.4, 68.0, 68.7, 68.8, 71.0, 72.1, 72.6, 73.1, 74.4, 74.5, 74.8, 75.5, 75.7, 76.1, 79.0, 81.4, 81.9, 83.4, 86.2, 97.9, 99.6, 101.4, 113.9–138.5 (aromatic carbons), 159.3, 159.5, 164.7, 165.3 (carbonyl groups); ESI-MS: m/z: calcd for C₉₀H₉₅NNaO₂₀SSi: 1592.6, found: 1592.7 [M+Na]⁺.

Methyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-2-deoxy-4,6-O-benzylidene-2-N-phthalimido-β-D-glucopyranoside (19)

Tetrasaccharide 19 was synthesized by two methods.

Method 1

A mixture of donor 18 (100 mg, 0.063 mmol), acceptor 6 (28 mg, 0.056 mmol) and freshly activated MS 4 Å (200 mg) in diethyl ether (3 mL) and CH₂Cl₂ (3 mL) was stirred for 1 h at room temperature, and cooled down to −65°C. A solution of AgOTf (45 mg, 0.18 mmol) in anhydrous Et₂O (1 mL) was added directly to the solution without touching the wall of the reaction flask. After 10 min, p-TolSCl (9 μL, 1 equiv) was added via a syringe. The reaction mixture was stirred for 1.5 h until the temperature reached 0°C, and triethylamine (100 μL) was added. It was concentrated to remove most Et₂O, resuspended in CH₂Cl₂ (20 mL), and filtered. The filtrate was concentrated and purified by flash column chromatography (hexanes/EtOAc/CH₂Cl₂ 4:1:1 → 2:1:1) to give 19 (49 mg, 0.026 mmol, 47%), along with small amount of trisaccharide 20. Compound 20: ¹H NMR (400 MHz, CDCl₃): δ = −0.16 (s, 3H, CH₃Si), −0.09 (s, 3H, CH₃Si), 0.74 (s, 9H, (CH₃)₃CSi), 3.02–3.08 (m, 1H), 3.22–3.76 (m, 10H), 3.78 (s, 3H, CH₃O), 4.00–4.04 (m, 2H), 4.21 (d, ³J(H,H) = 5.2 Hz, 1H), 4.27 (d, ³J(H,H) = 12 Hz, 1H), 4.33 (dd, ³J(H,H) = 8.4, 10.0 Hz, 1H), 4.35 (d, ³J(H,H) = 11.2 Hz, 1H), 4.42 (d, ³J(H,H) = 11.2 Hz, 1H), 4.52 (dd, ³J(H,H) = 8.4, 10.0 Hz, 1H), 4.56 (d, ³J(H,H) = 12 Hz, 1H), 4.60 (d, ³J(H,H) = 12 Hz, 1H), 4.69 (d, ³J(H,H) = 7.6 Hz, 1H), 4.78 (d, ³J(H,H) = 12 Hz, 1H), 4.84 (s, 1H), 5.05 (d, ³J(H,H) = 8.4 Hz, 1H), 5.22–5.28 (m, 2H), 6.84–8.06 (m, 33H); ESIMS: m/z: calcd for C₇₅H₇₉NNaO₁₉Si: 1348.5, found: 1348.6 [M+Na]⁺.

Method 2

Tetrasaccharide 19 (948 mg, 0.51 mmol) was synthesized in 64–75% yield using building blocks 11 (540 mg, 0.75 mmol), 2 (342 mg, 0.68 mmol), and 22 (870 mg, 0.68 mmol) following protocol A for one-pot synthesis. ¹H NMR (400 MHz, CDCl₃): δ = −0.18 (s, 3H, CH₃Si), −0.12 (s, 3H, CH₃Si), 0.73 (s, 9H, (CH₃)₃CSi), 2.64 (m, 1H), 2.73 (m, 1H), 2.92 (m, 1H), 3.06 (m, 1H), 3.15 (m, 1H), 3.26–3.33 (m, 2H), 3.31 (s, 3H, CH₃O), 3.38–3.43 (m, 3H), 4.38 (m, 1H), 3.55 (t, ³J(H,H) = 8.4 Hz, 1H), 3.60–3.65 (m, 2H), 3.75 (s, 3H, CH₃O), 3.78 (s, 3H, CH₃O), 3.91–3.95 (m, 2H), 4.02 (d, ³J(H,H) = 12 Hz, 1H), 4.17–4.43 (m, 9H), 4.50–4.59 (m, 3H), 4.64 (m, 2H), 4.88 (m, 3H), 4.98 (s, 1H, PhCH), 5.19 (d, ³J(H,H) = 8.4 Hz, 1H), 5.23 (s, 1H, PhCH), 6.79–7.82 (m, 46H); ¹³C NMR (100 MHz, CDCl₃): δ = −4.7, −3.7, 18.1, 26.0, 55.2, 55.5, 55.50, 55.51, 56.1, 57.0, 66.2, 66.4, 66.8, 68.7, 68.9, 71.0, 72.2, 73.1, 73.9, 73.9, 74.1, 74.4, 74.5, 75.2, 75.4, 76.0, 76.1, 80.9, 81.3, 81.6, 83.4, 97.6, 99.4, 99.7, 100.4, 101.4, 101.6, 113.9–138.7 (aromatic carbons), 159.5, 159.4, 164.5, 164.7 (carbonyl groups); HRMS: m/z: calcd for C₁₀₅H₁₀₈N₂NaO₂₇Si: 1879.6806, found: 1879.6783 [M+Na]⁺.

p-Tolyl 2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-1-thio-β-D-gluco-pyranoside (21)

Compound 21 was synthesized in 92% yield from compound 14β following the general procedure for TBS removal. ¹H NMR (400 MHz, CDCl₃): δ = 2.28 (s, 3H, p-CH₃ArS), 2.70 (d, J = 2.4 Hz, 1H), 3.06 (m, 1H), 3.37–3.46 (m, 3H), 3.64–3.68 (m, 2H), 3.68–3.85 (m, 2H), 3.82 (s, 3H, p-CH₃OAr), 4.31–4.38 (m, 4H), 4.44 (d, ³J(H,H) = 11.6 Hz, 1H), 4.50 (d, ³J(H,H) = 11.6 Hz, 1H), 4.66–4.71 (m, 2H), 4.99 (dd, J = 8.4, 9.6 Hz, 1H), 5.44 (s, 1H, PhCH), 5.44 (d, ³J(H,H) = 9.6 Hz, 1H), 6.88–7.81 (m, 27H); ¹³C NMR (100 MHz, CDCl₃): δ = 21.4, 54.5, 55.6, 68.9, 70.4, 70.7, 72.8, 73.2, 73.5, 73.8, 74.2, 81.4, 82.4, 84.7, 100.3, 101.8, 114.1, 126.5–138.6 (aromatic carbons), 159.6, 164.7 (carbonyl groups); HRMS: m/z: calcd for C₅₆H₅₃NNaO₁₃S: 1002.3135, found: 1002.3140 [M+Na]⁺.

Methyl 2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-1-thio-β-D-glucopyranoside (22)

Compound 22 (500 mg) was synthesized in 87% yield from compound 15 following the general procedure for TBS removal. ¹H NMR (400 MHz, CDCl₃): δ = 2.70 (d, ³J(H,H) = 2.4 Hz, 1H), 3.13 (m, 1H), 3.35 (s, 3H, CH₃O), 3.39–3.51 (m, 3H), 3.56–3.71 (m, 2H), 3.78 −3.88 (m, 2H), 3.82 (s, 3H, p-CH₃OAr), 4.27 (dd, ³J(H,H) = 8.8, 10.4 Hz, 1H), 4.30–4.34 (m, 3H, 1H), 4.45 (d, ³J(H,H) = 11.2 Hz, 1H), 4.50 (d, ³J(H,H) = 11.2 Hz, 1H), 4.68 (d, ³J(H,H) = 8.0 Hz, 1H), 4.69 (t, ³J(H,H) = 9.6 Hz, 1H), 5.00 (t, ³J(H,H) = 8.8 Hz, 1H), 5.01 (d, ³J(H,H) = 8.8 Hz, 1H), 5.45 (s, 1H), 6.88–7.47 (m, 23H); ¹³C NMR (100 MHz, CDCl₃): δ = 55.2, 55.5, 57.1, 66.5, 69.0, 70.4, 72.8, 73.1, 73.5, 73.8, 74.2, 76.1, 81.7, 82.4, 99.7, 100.3, 101.8, 114.1, 126.5–138.2 (aromatic carbons), 159.6, 164.7 (carbonyl groups); HRMS: m/z: calcd for C₅₀H₄₉NNaO₁₄: 910.3051, found: 910.3055 [M+Na]⁺.

p-Tolyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-2-deoxy-2-N-phthalimido-4,6-O-benzylidene-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-2-deoxy-2-N-phthalimido-3,6-O-benzylidene-β-D-glucopyranoside (23)

Compound 23 (50 mg) was synthesized in 75% yield using donor 14β and acceptor 21 following the general procedure for single-step pre-activation based glycosylation. ¹H NMR (600 MHz, CDCl₃): δ = −0.19 (s, 3H, CH₃Si), −0.14 (s, 3H, CH₃Si), 0.71 (s, 9H, (CH₃)₃CSi), 2.23 (s, 3H, CH₃), 2.60 (m, 1H), 2.71 (m, 1H), 2.91 (m, 1H), 3.04 (m, 1H), 3.12 (m, 1H), 3.23 (m, 2H), 3.32–3.41 (m, 3H), 3.49–3.62 (m, 4H), 3.70 (m, 1H), 3.71 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.89–3.92 (m, 2H), 4.01 (m, 1H), 4.15–4.21 (m, 3H), 4.24–4.28 (m, 2H), 4.32 (d, ³J(H,H) = 10.8 Hz, 1H), 4.36–4.41 (m, 3H), 4.82–4.56 (m, 3H), 4.63 (d, ³J(H,H) = 11.4 Hz, 1H), 4.67 (d, ³J(H,H) = 8.4 Hz, 1H), 4.85 (t, ³J(H,H) = 8.4 Hz, 1H), 4.95 (t, ³J(H,H) = 7.8 Hz, 1H), 4.96 (s, 1H, PhCH), 5.16 (d, ³J(H,H) = 9.0 Hz, 1H), 5.21 (s, 1H, PhCH), 5.35 (d, J = 10.2 Hz, 1H), 6.78–7.82 (m, 50H); ¹³C NMR (100 MHz, CDCl₃): δ = −4.7, −3.6, 18.1, 21.4, 26.0, 54.4, 55.5, 55.5, 56.1, 66.2, 66.8, 68.6, 68.7, 68.8, 70.5, 71.0, 72.2, 73.1, 73.9, 74.0, 74.0, 74.4, 75.1, 75.4, 76.0, 76.8, 77.0, 80.8, 81.3, 83.4, 84.6, 97.5, 99.4, 100.3, 101.3, 101.6, 113.9–138.6 (aromatic carbons), 159.4, 159.4, 164.5, 164.7 (carbonyl groups); ESI-MS: m/z: calcd for C₁₁₁H₁₁₂N₂NaO₂₆SSi: 1973.3, found: 1973.0 [M+Na]⁺.

Methyl 4,6-O-benzylidene-3-O-tert-butyldimethylsilyl-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-2-deoxy-4,6-O-benzylidene-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (28)

Pentasaccharide 28 (350 mg, 0.156 mmol) was synthesized in 65% yield using building blocks 26 (154 mg, 0.25 mmol), 21 (220 mg, 0.22 mmol), and 22 (200 mg, 0.22 mmol) following protocol A for one-pot synthesis. ¹H NMR (400 MHz, CDCl₃): δ = −0.31 (s, 3H, CH₃Si), −0.16 (s, 3H, CH₃Si), 0.54 (s, 9H, (CH₃)₃CSi), 2.61–2.70 (m, 3H), 2.88–2.94 (m, 2H), 3.09 (d, ³J(H,H) = 10.4 Hz, 1H), 3.18–3.25 (m, 2H), 3.29 (s, 3H, CH₃O), 3.33–3.51 (m, 5H), 3.64–3.69 (m, 3H), 3.73 (s, 3H, p-CH₃OPh), 3.76 (s, 3H, p-CH₃OPh), 3.92–4.15 (m, 9H), 4.24 (dd, ³J(H,H) = 4.8, 10.4 Hz, 1H), 4.25–4.31 (m, 4H), 4.41–4.56 (m, 6H), 4.65–4.77 (m, 4H), 4.95 (d, ³J(H,H) = 8.4 Hz, 1H), 5.08 (s, 3H, PhCH), 5.09 (s, 3H, PhCH), 5.15 (d, ³J(H,H) = 8.8 Hz, 1H), 5.22 (d, ³J(H,H) = 8.4 Hz, 1H), 5.41 (s, 1H, PhCH), 6.89–7.93 (m, 51H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ = −5.1, −4.2, 17.8, 25.4, 55.2, 55.4 (3C), 55.7, 56.9, 58.4, 66.0, 66.1, 66.3, 67.1, 67.4, 68.7 (2C), 68.8, 69.6, 72.2, 72.4, 73.5, 74.0, 74.2, 74.3, 74.3, 74.7, 75.0, 76.1, 76.3, 80.5, 80.6, 81.5, 81.6, 82.8, 97.4, 97.6, 99.6, 100.2, 100.3, 101.6 (2C), 102.0, 113.9–138.5 (aromatic carbons), 159.5, 159.5, 164.6 (carbonyl groups); HRMS: m/z: calcd for C₁₂₆H₁₂₅N₃NaO₃₃Si: 2258.7862, found: 2258.7900 [M+Na]⁺.

Methyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-me-thoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranoside (29)

Pentasaccharide 29 (224 mg, 0.096 mmol) was synthesized in 55% yield using building blocks 11 (156 mg, 0.218 mmol), 2 (99 mg, 0.196 mmol), 21 (173 mg, 0.176 mmol) and 27 (87 mg, 0.176 mmol) following protocol B for one-pot synthesis. ¹H NMR (600 MHz, CD₂Cl₂): δ = −0.14 (s, 3H, CH₃Si), −0.07 (s, 3H, CH₃Si), 0.76 (s, 9H, (CH₃)₃CSi), 2.63 (m, 1H), 2.74 (m, 1H), 2.98 (m, 1H), 3.03–3.05 (m, 2H), 3.09 (m, 1H), 3.17–3.22 (m, 2H), 3.29–3.45 (m, 7H), 3.39 (s, 3H), 3.58 (t, ³J(H,H) = 8.4 Hz, 1H), 3.63 (t, ³J(H,H) = 9.0 Hz, 1H), 3.72 (s, 3H, p-CH₃OPh), 3.73 (s, 3H, p-CH₃OPh), 3.75 (s, 3H, p-CH₃OPh), 3.80–3.83 (m, 2H), 3.91 (dd, ³J(H,H) = 4.8, 10.8 Hz, 1H), 3.98 (t, ³J(H,H) = 10.8 Hz, 1H), 4.03 (dd, ³J(H,H) = 4.8, 10.8 Hz, 1H), 4.08 (d, ³J(H,H) = 7.8 Hz, 1H), 4.13–4.20 (m, 5H), 4.30–4.34 (m, 4H), 4.43 (d, ³J(H,H) = 7.8 Hz, 1H), 4.45 (d, ³J(H,H) = 10.8 Hz, 1H), 4.52–4.59 (m, 4H), 4.68–4.75 (m, 5H), 4.85–4.87 (m, 2H), 5.07 (s, 1H, PhCH), 5.17 (d, ³J(H,H) = 8.4 Hz, 1H), 5.25 (d, ³J(H,H) = 8.4 Hz, 1H), 5.31 (s, 1H, PhCH), 6.81–7.85 (m, 60H, ArH); ¹³C NMR (150 MHz, CD₂Cl₂): δ = −3.8, −4.3, 18.0, 25.8, 55.4, 55.4, 55.9, 56.0, 56.9, 66.1, 66.3, 67.3, 68.1, 68.6, 69.0, 71.0, 72.3, 72.4, 73.1, 73.5, 74.1, 74.2, 74.3, 74.4, 74.5, 74.6, 74.8, 74.9, 75.5, 75.8, 76.1, 76.2, 80.5, 81.3, 81.5, 81.8, 82.7, 83.4, 97.3, 97.8, 99.4, 100.3, 101.4, 101.6, 104.6, 113.7–139.6 (aromatic carbons), 159.4, 159.5, 164.6, 164.7 (carbonyl groups); HRMS: m/z: calcd for C₁₃₃H₁₃₈N₂NaO₃₃Si: 2341.8849, found: 2341.8850 [M+Na]⁺.

Methyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (30)

Hexasaccharide 30 (327 mg, 0.12 mmol) was synthesized in 54–60% yield using building blocks 11 (178 mg, 0.249 mmol), 2 (113 mg, 0.224 mmol), 21 (198 mg, 0.201 mmol) and 22 (178 mg, 0.201 mmol) following protocol B for one-pot synthesis. ¹H NMR (600 MHz, CDCl₃): δ = −0.17 (s, 3H, CH₃Si), −0.08 (s, 3H, CH₃Si), 0.75 (s, 9H, (CH₃)₃CSi), 2.56–2.61 (m, 2H), 2.67 (dd, ³J(H,H) = 3.0, 10.2 Hz, 1H), 2.71 (dd, ³J(H,H) = 3.0, 10.2 Hz, 1H), 2.90 (d, ³J(H,H) = 9.6 Hz, 1H), 2.95 (d, ³J(H,H) = 9.6 Hz, 1H), 3.03 (m, 1H), 3.18 (m, 1H), 3.21 (m, 1H), 3.28 (s, 3H, CH₃O), 3.31–3.34 (m, 4H), 3.36–3.40 (m, 2H), 3.42 (m, 2H), 3.47 (m, 1H), 3.55–3.60 (m, 2H), 3.63–3.66 (m, 2H), 3.69 (s, 3H, p-CH₃OPh), 3.72 (s, 3H, p-CH₃OPh), 3.74 (s, 3H, p-CH₃OPh), 3.78 (t, ³J(H,H) = 9.6 Hz, 1H), 3.90–3.96 (m, 3H), 4.02 (dd, ³J(H,H) = 4.8, 10.8 Hz, 1H), 4.04–4.16 (m, 4H), 4.22 (dd, ³J(H,H) = 4.8, 10.2 Hz, 1H), 4.26–4.33 (m, 6H), 4.39–4.52 (m, 6H), 4.56 (dd, ³J(H,H) = 9.0, 10.2 Hz, 1H), 4.62–4.74 (m, 5H), 4.84 (dd, ³J(H,H) = 8.4, 9.0 Hz, 1H), 4.93 (d, ³J(H,H) = 8.4 Hz, 1H), 5.01 (s, 1H, PhCH), 5.07 (s, 1H, PhCH), 5.11 (d, J = 8.4 Hz, 1H), 5.14 (d, J = 8.4 Hz, 1H), 5.30 (s, 1H, PhCH), 6.80–7.82 (m, 69H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ = −4.8, −3.7, 18.1, 26.0, 55.1, 55.4, 55.5, 57.0, 65.9, 66.2, 66.4, 68.6, 68.8, 71.0, 72.2, 72.3, 73.1, 73.8, 73.9, 74.0, 74.1, 74.3, 74.5, 75.0, 75.1, 75.4, 75.8, 75.9, 76.1, 76.7, 76.8, 80.8, 81.3, 81.4, 81.5, 83.4, 97.4, 97.5, 99.4, 99.7, 100.2, 100.3, 101.4, 101.4, 101.6, 113.9–138.6 (aromatic carbons), 159.4, 159.5, 164.4, 164.6 (carbonyl groups); HRMS: m/z: calcd for C₁₅₄H₁₅₃N₃NaO₄₀Si: 2734.9697, found: 2734.9697 [M+Na]⁺.

Methyl 2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-3,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (31)

Compound 31 was obtained from compound 30 in 90% yield following the general procedure for TBS removal. ¹H NMR (600 MHz, CDCl₃): δ = 2.59–2.69 (m, 5H), 2.87 (d, ³J(H,H) = 9.6 Hz, 1H), 2.92 (d, ³J(H,H) = 10.2 Hz, 1H), 3.01–3.04 (m, 2H), 3.10 (m, 1H), 3.17 (t, ³J(H,H) = 10.2 Hz, 1H), 3.28–3.37 (m, 8H), 3.43–3.47 (m, 3H), 3.58–3.63 (m, 7H), 3.65 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 3.83–3.91 (m, 4H), 3.96 (d, ³J(H,H) = 12 Hz, 1H), 4.03–4.21 (m, 8H), 4.27–4.40 (m, 8H), 4.45 (d, ³J(H,H) = 12 Hz, 1H), 4.47 (t, ³J(H,H) = 10.2 Hz, 1H), 4.50–4.61 (m, 4H), 4.81 (t, ³J(H,H) = 8.4 Hz, 1H), 4.84 (d, ³J(H,H) = 8.4 Hz, 1H), 4.86 (d, ³J(H,H) = 9.0 Hz, 1H), 4.90–4.94 (m, 3H), 5.09 (s, 1H, PhCH), 5.11 (s, 1H, PhCH), 5.25 (s, 1H, PhCH), 5.35 (d, ³J(H,H) = 10.2 Hz, 1H), 6.78–7.82 (m, 69H); ¹³C NMR (100 MHz, CDCl₃): δ = 55.1, 55.4, 55.46, 55.48, 55.8, 55.9, 57.1, 60.6, 64.6, 65.9, 66.0, 66.3, 66.7, 67.1, 68.6, 68.8, 70.5, 72.1, 72.3, 72.8, 72.9, 73.5, 73.6, 73.7, 73.8, 73.8, 74.0, 74.2, 74.9, 75.1, 75.7, 75.8, 75.9, 80.7, 80.7, 81.3, 81.4, 81.5, 82.3, 97.3, 97.5, 99.6, 100.0, 100.2, 100.3, 101.4, 101.5, 101.6, 113.9–138.6 (aromatic carbons), 159.37, 159.39, 159.6, 164.5, 164.6 (carbonyl groups); ESI-MS: m/z: calcd for C₁₄₈H₁₃₉N₃NaO₄₀: 2622.6, found: 2622.7 [M+Na]⁺.

Methyl 2-O-acetyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-2-deoxy-2-N-acetyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→4)-2-O-acetyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-2-deoxy-2-N-acetyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→4)-2-O-acetyl-3-O-benzyl-6-O-p-methoxybenzyl-β-D-glucopyranosyl-(1→3)-2-deoxy-2-N-acetyl-3,6-O-benzylidene-β-D-glucopyranoside (34)

Acetic anhydride (0.3 mL) and DMAP (50 mg) was added to a solution of compound 31 (100 mg) in anhydrous pyridine (5 mL). The reaction mixture was stirred for 1 h at room temperature, diluted with dichloromethane and washed with aqueous solution of 1N HCl. The organic layer was dried over Na₂SO₄ and the desired compound 32 (102 mg) was obtained from flash chromatography (hexanes/EtOAc/CH₂Cl₂ 1:1:1) in 100% yield. A solution of compound 32 (50 mg) and ethylenediamine (0.5 mL) in n-butanol (5 mL) was heated under reflux for 1.5 h until no starting material was observed by TLC. The solution was dried under reduced pressure followed by chromatography to afford free amine. The free amine was dissolved in anhydrous pyridine (3 mL) followed by addition of acetic anhydride (0.2 mL) and DMAP (100 mg). The reaction was stirred for 1 h and diluted with dichloromethane followed by extraction and flash chromatography (CH₂Cl₂/MeOH 30:1) to provide the desired product 34 (40 mg, 50%). ¹H NMR (600 MHz, CD₂Cl₂): δ = 1.74 (s, 3H), 1.76 (s, 3H), 1.88 (s, 3H), 1.89 (s, 3H), 1.91 (s, 3H), 1.95 (s, 3H), 1.96 (s, 3H), 3.09 (m, 1H), 3.17–3.22 (m, 3H), 3.24–3.33 (m, 4H), 3.45 (s, 3H, OCH₃), 3.37–3.49 (m, 8H), 3.52–3.57 (m, 4H), 3.65 (t, ³J(H,H) = 9.0 Hz, 1H), 3.69–3.71 (m, 3H), 3.75 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.86–3.97 (m, 4H), 4.09 (m, 1H), 4.22–4.29 (m, 4H), 4.36–4.39 (m, 2H), 4.43–4.50 (m, 6H), 4.54–4.57 (m, 4H), 4.65 (d, ³J(H,H) = 7.8 Hz, 1H), 4.69–4.75 (m, 3H), 4.78–4.95 (m, 4H), 4.98 (t, ³J(H,H) = 9.0 Hz, 1H), 5.37 (s, 1H, PhCH), 5.40 (s, 1H, PhCH), 5.44 (s, 1H, PhCH), 6.81–7.44 (m, 42H); ESI-MS: m/z: calcd for C₁₁₇H₁₃₆N₃O₃₈: 2190.9, found: 2190.3 [M+H]⁺.

Methyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl-(1→4)-2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranoside (36)

Compound 36 (103 mg, 0.437 mmol) was synthesized in 74% yield from compound 30 (160 mg, 0.059 mmol) following the general procedure for PMB removal. ¹H NMR (600 MHz, CDCl₃): δ = −0.19 (s, 3H, CH₃Si), −0.07 (s, 3H, CH₃Si), 0.77 (s, 9H, (CH₃)₃CSi), 2.67–2.72 (m, 2H), 2.85–2.86 (m, 2H), 3.03 (m, 1H), 3.11–3.16 (m, 2H), 3.22 (t, ³J(H,H) = 10.2 Hz, 1H), 3.28 (s, 3H, OCH₃), 3.29–3.42 (m, 4H), 3.47–3.57 (m, 3H), 3.63 (t, ³J(H,H) = 9.0 Hz, 1H), 3.67 (t, ³J(H,H) = 9.0 Hz, 1H), 3.73–3.79 (m, 3H), 3.94 (dd, ³J(H,H) = 4.2, 10.2 Hz, 1H), 4.01 (dd, ³J(H,H) = 4.8, 10.2 Hz, 1H), 4.03–4.08 (m, 1H), 4.14–4.18 (m, 1H), 4.28–4.37 (m, 4H), 4.42–4.50 (m, 3H), 4.5–4.65 (m, 4H), 4.83 (t, ³J(H,H) = 8.4 Hz, 1H), 4.85 (t, ³J(H,H) = 9.0 Hz, 1H), 4.91 (d, ³J(H,H) = 9.0 Hz, 1H), 4.97 (t, ³J(H,H) = 8.4 Hz, 1H), 5.08 (d, ³J(H,H) = 8.4 Hz, 1H), 5.12 (d, ³J(H,H) = 8.4 Hz, 1H), 5.22 (s, 1H, PhCH), 5.39 (s, 1H, PhCH), 5.41 (s, 1H, PhCH), 6.83–7.62 (m, 57H, ArH); ¹³C NMR (100 MHz, CDCl₃): δ = −4.7, −3.8, 18.1, 26.0, 55.5, 56.1, 56.3, 57.1 (2C), 60.82, 60.83, 62.0, 66.1, 66.2, 66.5, 68.6, 68.7, 68.9, 70.6, 73.8 (2C), 74.2, 74.3, 74.5, 74.6, 74.7, 74.9, 75.3 (2C), 75.4, 75.6, 76.7, 80.7 (2C), 80.8, 83.3, 98.1 (2C), 99.76, 99.83, 100.0 (2C), 101.8, 101.9, 102.0, 123.6–138.4 (aromatic carbons), 164.8, 164.8, 164.4, 164.9 (carbonyl groups); HRMS: m/z: calcd for C₁₃₀H₁₂₉N₃NaO₃₇Si: 2374.7972, found: 2374.7881 [M+Na]⁺.

Methyl (benzyl 2-O-benzoyl-3-O-benzyl-4-O-tert-butyldimethylsilyl-β-D-glucopyranosyluronate)-(1→3)-(2-deoxy-2-N-phthalimido-4,6-O-benzylidene-β-D-glucopyranosyl)-(1→4)-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-(1→3)-(4,6-O-benzylidene-2-deoxy-2-N-phthalimido-β-D-glucopyranosyl)-(1→4)-(benzyl 2-O-benzoyl-3-O-benzyl-β-D-glucopyranosyluronate)-(1→3)-(2-deoxy-2-N-phthalimido-4,6-O-benzylidene-β-D-glucopyranoside) (37)

Compound 37 (238 mg, 0.089 mmol) was synthesized in 82% yield from compound 36 (258 mg, 0.109 mmol) following the general procedure for oxidation and benzyl ester formation. ¹H NMR (600 MHz, CD₂Cl₂): δ = −0.14 (s, 3H, CH₃Si), −0.26 (s, 3H, CH₃Si), 0.72 (s, 9H, (CH₃)₃CSi), 3.04 (t, ³J(H,H) = 10.2 Hz, 1H), 3.11–3.15 (m, 2H), 3.21–3.31 (m, 5H), 3.27 (s, 3H, OCH₃), 3.38–3.47 (m, 6H), 3.56–3.61 (m, 2H), 3.67 (t, ³J(H,H) = 8.4 Hz, 1H), 3.84 (dd, ³J(H,H) = 4.2, 10.2 Hz, 1H), 3.93–4.04 (m, 6H), 4.07–4.12 (m, 2H), 4.18 (dd, ³J(H,H) = 4.2, 9.6 Hz, 1H), 4.27–4.38 (m, 6H), 4.45 (d, ³J(H,H) = 12 Hz, 1H), 4.48 (dd, ³J(H,H) = 8.4, 10.2 Hz, 1H), 4.52–4.58 (m, 3H), 4.62–4.68 (m, 3H), 4.97 (d, ³J(H,H) = 8.4 Hz, 1H), 4.99 (s, 1H, PhCH), 5.01 (s, 1H, PhCH), 5.03 (d, ³J(H,H) = 8.4 Hz, 1H), 5.10 (d, ³J(H,H) = 12 Hz, 1H), 5.12 (s, 1H, PhCH), 5.17 (d, ³J(H,H) = 12 Hz, 1H), 4.91–4.93 (m, 3H), 6.86–7.73 (m, 72H, ArH); ¹³C NMR (100 MHz, CD₂Cl₂): δ = −5.2, −4.3, 17.9, 25.7, 53.6, 53.9, 54.1, 55.1, 55.6, 55.7, 57.0 (2C), 65.7, 65.9, 66.3, 67.28, 67.31, 68.2, 68.4, 68.5, 71.9, 72.9, 73.9, 72.2, 74.2, 74.6, 74.7, 75.8 (2C), 76.1, 77.0, 77.5, 79.8, 79.9, 80.7, 81.0, 81.152, 82.2, 98.2 (2C), 99.5, 99.6, 99.7, 99.8, 101.0, 101.1, 101.5, 123.2–138.3 (aromatic carbons), 164.5, 164.6, 164.6, 166.9, 166.9, 168.1 (carbonyl groups); HRMS: calcd for C₁₅₁H₁₄₁N₃NaO₄₀Si: 2686.8758, found: 2686.8855 [M+Na]⁺.

Methyl (β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-acetyl-β-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-acetyl-β-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-acetyl-β-D-glucopyranoside) (38)

Compound 38 (27 mg, 0.023 mmol) was synthesized in 53% yield from compound 37 (102 mg, 0.043 mmol) following the general procedures for hydrogenation, benzoyl/phthalimide removal and acetylation. ¹H NMR (400 MHz, D₂O): δ = 2.01 (s, 3H, AcNH), 2.02 (s, 3H, AcNH), 2.03 (s, 3H, AcNH), 3.31–3.39 (m, 3H), 3.49–3.59 (m, 10H), 3.51 (s, 3H, MeO), 3.70–3.94 (m, 17H), 4.26 (d, ³J(H,H) = 8.4 Hz, 1H), 4.29–4.31 (m, 3H), 4.36 (d, ³J(H,H) = 8.4 Hz, 1H), 4.36 (d, ³J(H,H) = 8.0 Hz, 1H); ¹³C NMR (100 MHz, D₂O): δ = 22.4, 22.64, 22.64, 54.4, 54.4, 54.5, 57.3, 60.6, 60.7, 60.8, 68.6, 68.7, 68.8, 71.9, 72.6, 72.6, 72.9, 73.7, 73.8, 75.45, 75.49, 75.5, 75.9, 76.45, 76.54, 80.08, 80.14, 82.5, 82.6, 83.2, 100.7, 100.7, 101.9, 103.1, 103.3, 103.3, 174.38, 174.32, 174.9, 175.08, 175.08, 175.6 (carbonyl groups); HRMS: m/z: calcd for C₄₃H₆₇N₃NaO₃₄: 1192.3504, found: 1192.3550 [M+Na]⁺.

Methyl (β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-acetyl-β-D-glucopyranoside) (39)

Compound 39 (2.6 mg, 6 μmol) was obtained from disaccharide 15 (13 mg, 14.6 μmol) following the general procedures for deprotection and oxidation-state adjustment in 41% overall yield. ¹H NMR (600 MHz, D₂O): δ = 2.47 (s, 3H, AcNH), 3.80 (m, 1H), 3.91–4.03 (m, 4H), 3.96 (s, 3H, OCH₃), 4.18 (t, ³J(H,H) = 8.4 Hz, 1H), 4.22 (dd, ³J(H,H) = 6.0, 12.6 Hz, 1H), 4.23–4.31 (m, 2H), 4.38 (dd, ³J(H,H) = 1.8, 12.6 Hz, 1H), 4.91 (d, ³J(H,H) = 8.4 Hz, 1H), 4.94 (d, ³J(H,H) = 7.8 Hz, 1H); HRMS: calcd for C₁₅H₂₅NNaO₁₂ : 434.1274, found:434.1266 [M+Na]⁺.

Methyl (β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-Acetyl-β-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyluronic acid)-(1→3)-2-deoxy-2-N-acetyl-β-D-glucopyranoside (40)

Compound 40 (32 mg, 39 μmol) was obtained from tetrasaccharide 19 (210 mg, 0.112 mmol) following the general procedures for deprotection and oxidation-state adjustment in 35% overall yield. ¹H NMR (600 MHz, D₂O): δ = 1.98 (s, 3H, AcNH), 1.99 (s, 3H, AcNH), 3.11–3.15 (m, 2H), 3.25–3.48 (m, 8H), 3.47 (s, 3H, CH₃O), 3.48–3.63 (m, 8H), 4.68–4.71 (m, 2H), 4.24 (d, ³J(H,H) = 9.0 Hz, 1H), 4.26 (d, ³J(H,H) = 7.8 Hz, 2H), 4.33 (d, ³J(H,H) = 8.4 Hz, 1H); ¹³C NMR (150 MHz, D₂O): δ = 22.3, 22.6, 54.3, 54.5, 57.2, 60.6, 60.8, 68.6, 68.7, 71.8, 72.5, 72.8, 73.7, 75.4, 75.45, 75.49, 75.9, 76.5, 80.1, 82.4, 83.1, 100.7, 101.9, 103.1, 103.2, 174.3, 174.9, 175.0, 175.6; HRMS: m/z: calcd for C₂₉H₄₆N₂NaO₂₃: 813.2389, found: 813.2351 [M+Na]⁺.

Methyl (2-deoxy-2-N-acetyl-β-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-acetyl-β-D-glucopyranosyl)-(1→ 4)-(β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-acetyl-β-D-glu-copyranoside) (41)

Compound 41 (18 mg, 0.018 mmol) was obtained from pentasaccharide 28 (130 mg, 0.058 mmol) following the general procedures for deprotection and oxidation-state adjustment in 31% overall yield. ¹H NMR (600 MHz, D₂O): δ = 1.96 (s, 3H, AcNH), 1.97 (s, 3H, AcNH), 1.99 (s, 3H, AcNH), 3.29–3.31 (m, 2H), 3.40–3.55 (m, 9H), 3.46 (s, 3H, CH₃O), 3.65–3.72 (m, 10H), 3.78–3.80 (m, 2H), 3.86–3.88 (m, 3H), 4.39 (d, ³J(H,H) = 8.4 Hz, 1H), 4.42–4.44 (m, 2H), 4.47 (d, ³J(H,H) = 8.4 Hz, 1H), 4.50 (d, ³J(H,H) = 8.4 Hz, 1H); ¹³C NMR (150 MHz, D₂O): δ = 22.3, 22.5, 22.6, 54.4, 54.5, 55.5, 57.2, 60.59, 60.60, 60.8, 68.45, 68.7, 69.8, 72.5, 72.6, 73.6, 73.7, 74.0, 75.4, 75.5, 76.0, 76.4, 76.5, 79.9, 80.1, 82.4, 82.6, 100.7, 100.8, 101.9, 103.23, 103.24, 173.3, 174.4, 174.9, 175.0, 175.1; HRMS: calcd for C₃₇H₅₉N₃NaO₂₈ : 1016.3183, found: 1016.3179 [M+Na]⁺.

Methyl (β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-acetyl-β-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyluronic acid)-(1→3)-(2-deoxy-2-N-acetyl-β-D-glucopyranosyl)-(1→4)-(β-D-glucopyranosyluronic acid) (42)

Compound 42 (21 mg, 0.021 mmol) was obtained from pentasaccharide 29 (180 mg, 0.077 mmol) following the general procedures for de-protection and oxidation-state adjustment in 27% overall yield. ¹H NMR (600 MHz, D₂O): δ = 1.97 (s, 3H, AcNH), 1.99 (s, 3H, AcNH), 3.26–3.31 (m, 3H), 3.43–3.57 (m, 8H), 3.36 (s, 3H, MeO), 3.65–3.74 (m, 9H), 3.75–3.80 (m, 2H), 3.85–3.87 (m, 2H), 4.34 (d, ³J(H,H) = 8.4 Hz, 1H), 4.44 (d, ³J(H,H) = 7.8 Hz, 2H), 4.50 (d, ³J(H,H) = 7.8 Hz, 2H); ¹³C NMR (150 MHz, D₂O): δ = 22.43, 22.44, 54.3, 54.4, 57.6, 60.5, 68.2, 68.3, 71.2, 72.2, 72.4, 72.5, 73.3, 73.5, 73.7, 73.9, 74.2, 75.1, 75.4, 80.2, 80.3, 82.4, 82.7, 101.32, 101.32, 102.7, 102.9, 103.6, 171.11, 171.11, 172.6, 174.79, 174.84 (carbonyl groups); HRMS: m/z: calcd for C₃₅H₅₄N₂NaO₂₉ : 989.2710, found: 989.2687 [M+Na]⁺.