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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: J Carbohydr Chem. 2012 Jul 2;31(4-6):384–419. doi: 10.1080/07328303.2012.658274

Solid-Phase Synthesis of 2-Aminoethyl Glucosamine Sulfoforms

Runhui Liu 1, Alexander Wei 1,✉
PMCID: PMC3501688  NIHMSID: NIHMS389075  PMID: 23180905

Abstract

Mono- and disaccharides of sulfonated glucosamines (GlcN sulfoforms) conjugated to 2-aminoethyl linkers were generated by solid-phase synthesis. Orthogonally protected intermediates were tethered onto tritylated polystyrene resin beads, subjected to a modular sequence of deprotection and sulfonation steps, then cleaved from solid support without degradation of N- or O-sulfate esters using solvolytic conditions, and finally purified by reverse-phase HPLC to afford the title compounds.

Introduction

Heparan sulfate proteoglycans (HSPGs) are well known for their many important roles in cellular function.1,2 HSPGs are recognized by a diverse range of heparin-binding proteins in key biological processes, such as angiogenesis (fibroblast growth factors, or FGFs),3 pathogenic infection (herpes simplex virus glycoprotein D),4 and inflammation (L-selectin).5 However, so far only a handful of HSPG sequences have been elucidated, due to the heterogeneity of structures that exist within heparin. Many studies have shown that the specificity of HS binding is defined in large part by sulfonation patterns, or sulfoforms. For example, FGF1 binding requires glucosamine-2,6-disulfate (GlcNS,6S), whereas FGF2 binding requires GlcNS and also iduronate-2-sulfate (IdoA-2S).6,7,8

The relevance of sulfoforms within HS and particularly glucosamine (GlcN) to protein binding suggests that even relatively simple sulfoforms are likely to exhibit sufficient binding avidity to serve as potential antagonists in HS-mediated signaling. Recent examples in the scientific literature supports this notion: a disaccharide sulfoform containing GlcN has been found to have significant binding to FGF2,6 and a clinical study showed that dendrimers presenting multiple GlcNAc-6S units can prevent scar tissue formation9 However, structure–affinity relationships between HS-like compounds and heparin-binding proteins remain rare, and are limited by challenges in identifying or producing structurally well-characterized ligands in sufficient quantities for study.

To address this bottleneck, we have introduced a method of generating GlcN sulfoforms using solid-phase synthesis.10 Performing deprotections and sulfonations on resin-immobilized intermediates greatly simplifies the purification process, and reduces the attrition in yield caused by multiple extraction and chromatography steps. Our previous study established proof of concept, but the glycol linker associated with these sulfoforms was not amenable to further conjugation. Here we further optimize the solid-phase synthesis approach in order to generate a number of mono- and disaccharide GlcN sulfoforms bearing a 2'-aminoethyl linker (Figure 1), and to facilitate the preparation of glycoconjugates for subsequent protein binding studies.

Figure 1.

Figure 1

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GlcN sulfoforms with 2'-aminoethyl linkers (1–4), prepared by solid-phase synthesis. R = H, Ac, or SO3−; for specific sulfoforms, see Table 2.

Results and Discussion

Synthesis of orthogonally protected GlcN monosaccharide and disaccharide

The preparation of orthogonally protected α-GlcN monosaccharides with ethanolamine linkers is described in Scheme 1. Phthalimide-protected GlcN derivative 5 was converted in 85% yield over three steps to 2-azido-2-deoxyglucoside,10 followed by reductive ring opening of the 4,6-benzylidene acetal using BH3/Bu2BOTf11 and formation of 6-O-TBDPS ether to afford 6 in 62% yield over two steps. Glycoside 6 was subjected to glycosylation with 9-fluorenylmethoxycarbonyl (Fmoc)-protected ethanolamine to yield 7 as a mixture of anomers (4.2:1 α:β ratio) in 99% yield. The major and minor isomers were carefully separated by silica gel chromatography, then treated with 20% piperidine in DMF to provide amine-terminated GlcN derivatives 8a and 8b respectively, each in 99% isolated yield. The selective cleavage of Fmoc in the presence of other protecting groups was essential for conducting our orthogonal deprotection/sulfonation strategy on a solid-phase support.

Scheme 1.

Scheme 1

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Synthesis of orthogonally protected GlcN with 2’-aminoethyl linker

Reaction conditions: (a) (i) NH2(CH2)2NH2, BuOH, 100 °C; (ii) TfN3, K2CO3, CuSO4, CH2Cl2/MeOH/H2O, 0 °C to rt; (iii) Ac2O/Py, 0 °C to rt (85% over three steps). (b) (i) BH3, Bu2BOTf, THF, 0 °C; (ii) TDBPS-Cl, imidazole, DMF, rt (62% over two steps). (c) NIS/TfOH, AE-Fmoc, Cl(CH2)2Cl/Et2O, −20 °C (80%). (d) 20% piperidine in DMF, rt (99%). Abbreviations: AE-Fmoc = 2'-aminoethyl-2-(9H-fluorenyl)methoxycarbamate; Phth = phthalimidyl; TBDPS = tert-butyldiphenylsilyl; Tol = p-tolyl.

In order to prepare β-aminoethyl-linked GlcN(β1→4)Glc derivative 12, the trichloroethoxycarbonyl (Troc) group was installed at the C2 position of GlcN to provide anchimeric assistance during glycosyl coupling (Scheme 2). Troc-protected GlcN derivative 9 was prepared from 6 in 94% overall yield, then subjected to NIS/TfOH-promoted glycosylation with 15 to yield β-linked disaccharide 10 in 90% yield, followed by selective deprotection with indium powder in aqueous NH4Cl12 and Cu2+-mediated diazo transfer to yield 11 in 91% yield over 2 steps. Selective Fmoc deprotection with 20% piperidine in DMF gave 2'-aminoethyl GlcN derivative 12 in 99% isolated yield.

Scheme 2.

Scheme 2

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Synthesis of GlcN(β1→4)Glc disaccharide with β-linked 2’-aminoethyl group

Reaction conditions: (a) (i) NaOMe, MeOH, rt; (ii) Bu3P, CH2Cl2, rt; (iii) H2O:CH2Cl2 1:1, rt; (iv) Troc-Cl, pyridine, rt; (v) Ac2O/Py, rt (94% over 5 steps). (b) 15 (see Scheme 3), NIS/TfOH, CH2Cl2, −30 °C (90%). (c) (i) In powder, NH4Cl, EtOH/H2O, 98 °C; (ii) TfN3, CuSO4, K2CO3, CH2Cl2/MeOH, 0 °C to rt (91% over 2 steps). (d) piperidine, DMF, rt (99%). Abbreviations: Troc = trichloroethoxycarbonyl.

The β-aminoethyl-linked GlcN(α1→4)Glcβ derivative 17 was synthesized as described in Scheme 3. 2'-Azidoethyl β-glucoside tetraacetate 13 was prepared in 46% yield from glucose pentaaceate by glycosylation with 2-bromoethanol and azide substitution. Saponification, 4,6-O-benzylidene acetal formation, benzylation of the C2 and C3 hydroxyls, and regioselective reductive ring opening with Et3SiH and TfOH yielded glycosyl acceptor 14 in 56% yield over 4 steps.13 The azide of 14 was converted into Fmoc derivative 15 in 54% yield over 3 steps, and NIS/TfOH-promoted glycosyl coupling of 15 and thioglucoside 6 yielded α-1,4-linked disaccharide 16 in 60% yield. The Fmoc group was efficiently cleaved to produce 2'-aminoethyl derivative 17 in 99% yield.

Scheme 3.

Scheme 3

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Synthesis of GlcN(α1→4)Glc disaccharide with β-linked 2’-aminoethyl group

Reaction conditions: (a) (i) NaOMe, MeOH, rt; (ii) PhCH(OMe)2, TsOH, THF, reflux; (iii) NaH, BnBr, DMF, 0 °C to rt (75% over 3 steps). (b) Et3SiH, TfOH, 4A mol sieves, CH2Cl2, −78 °C (75%). (c) (i) Bu3P, CH2Cl2, rt; (ii) H2O:CH2Cl2 1:1, rt; (iii) Fmoc-Cl, NaHCO3, CH2Cl2, 0 °C to rt (54% over 3 steps). (d) Donor 6, NIS, TfOH, Cl(CH2)2Cl/Et2O, −20 °C (60%). (e) piperidine, DMF, rt (99%).

Loading of orthogonally protected GlcN derivatives onto resins

The loading of 2'-aminoethyl GlcN derivatives onto trityl-PS resins was initially accompanied by multiple side reactions, presumably because of the nucleophilicity of the terminal amine. Moreover, the recovery and purification of excess aminoethyl-linked GlcNs from the reaction mixture was problematic. Using a lower concentration of 2'-aminoethyl GlcN derivatives in dry pyridine at 60 °C produced much cleaner results, but the resin loadings were commensurately lower.

Aminoethyl-linked GlcN derivatives 8a (GlcN-α), 8b (GlcN-β), 12 (GlcN(β1→4)Glc-β), and 17 (GlcN(α1→4)Glc-β) were each loaded onto trityl-PS resin in the presence of 0.4 equivalents of DMAP (Table 1). Introducing the amine-terminated derivatives 8a, 8b, and 12 at 50% loading capacity (0.75 mmol/g) and 17 at 33% loading capacity (0.5 mmol/g) produced resin-immobilized GlcN 18a, 18b, 19, and 20, with loading ratios of 0.38, 0.27, 0.18, and 0.18 mmol/g, respectively.

Table 1.

Loading of 2’-aminoethyl-linked derivatives onto trityl-PS resin

GlcN derivative product (loading ratio)
graphic file with name nihms389075t1.jpg 18a (0.38 mmol/g)
graphic file with name nihms389075t2.jpg 18b (0.27 mmol/g)
graphic file with name nihms389075t3.jpg 19 (0.18 mmol/g)
graphic file with name nihms389075t4.jpg 20 (0.18 mmol/g)
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Reaction conditions: Cl-Trityl–PS resin, DMAP, pyridine, 60°C, 48 h.

Generation of GlcN sulfoforms

We first investigated conditions for cleaving 2'-aminoethyl-linked glycosides from the trityl–PS resins. No detectable cleavage was found when using 0.3 M BF3∙Et2O in CH2Cl2 at −20 °C, the condition used to cleave glycol-linked GlcNs, and higher temperatures (above 0 °C) would result in the significant decomposition of N-sulfate esters.10 Fortunately, 30% hexafluoroisopropanol (HFIP) in CH2Cl2 at room temperature provided an ideal alternative: the cleavage was remarkably efficient and selective, yet mild enough to leave both O and N-sulfate esters unaffected.14 This condition was also found to be effective for cleaving glycol-linked sulfoforms (trityl ethers).

The solid-phase deprotection and sulfonation conditions developed in our earlier protocol10 was applied toward resin-immobilized monosaccharide 18a, to produce fully deprotected α-linked GlcN 1a in 87% yield after 6 operations, and sulfoforms 1b–1h in overall yields ranging from 76% (1b) to 44% (1f) after 8 operations (Table 2). This method was also used to generate GlcN sulfoforms 2(b,f), 3(b,f), and 4(b,f) in comparable yields from 18b, 19, and 20, respectively. The relatively low yields obtained for GlcNS,6S sulfoforms 1f–4f may be due to the premature removal of the 3-O-acetate or acetyl transfer from O3 to N2, with subsequent sulfonation of the C3 alcohol. This problem may be circumvented by protecting the C3 alcohol as a SEM ether, which previously demonstrated excellent orthogonality with several other protecting groups in solution-phase synthesis.15

Table 2.

Sulfoforms generated by orthogonal deprotection and sulfonation on trityl–PS resins

graphic file with name nihms389075t5.jpg
Sulfate patterna Product,b yield, no. opsc
graphic file with name nihms389075t6.jpg 1a, 87%, 6 ops (a, b, c, d, g, k)
graphic file with name nihms389075t7.jpg 1b, 76%, 8 ops (a, b, c, f, g, h, k, l)
2b, 72%, 8 ops (a, b, c, f, g, h, k, l)
3b, 67%, 8 ops (a, b, c, f, g, h, k, l)
4b, 69%, 8 ops (a, b, c, f, g, h, k, l)
graphic file with name nihms389075t8.jpg 1c, 64%, 8 ops (a, b, c, g, h, d, k, l)
graphic file with name nihms389075t9.jpg 1d, 69%, 7 ops (a, b, i, d, g, k, l)
graphic file with name nihms389075t10.jpg 1e, 68%, 8 ops (a, b, c, d, g, j, k, l)
graphic file with name nihms389075t11.jpg 1f, 44%, 8 ops (d, a, b, h, i, g, k, l)
2f, 42%, 8 ops (d, a, b, h, i, g, k, l)
3f, 41%, 8 ops (d, a, b, h, i, g, k, l)
4f, 42%, 8 ops (d, a, b, h, i, g, k, l)
graphic file with name nihms389075t12.jpg 1g, 68%, 7 ops (g, a, b, j, e, k, l)
graphic file with name nihms389075t13.jpg 1h, 64%, 7 ops (d, g, a, b, j, k, l)
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a

1: GlcN-α; 2 GlcN-β; 3 GlcN(β1→4)Glc-β; 4 GlcN(α1→4)Glc-β (see Figure 1)

b

All products were isolated as sodium salts.

c

Each step was conducted at rt for 24 h, unless otherwise noted. Reagents and conditions: (a) Bu3P, CH2Cl2, 5 h; (b) 95% aq THF; (c) Ac2O, pyridine; (d) TBAF (pH ≥ 10), THF; (e) TBAF (pH ≥ 10), THF/DMF; (f) TBAF (pH 7), THF; (g) NaOMe, MeOH/CH2Cl2; (h) SO3·Py, DMF, 10 h; (i) SO3·Py, Py/Et3N; (j) SO3·Py, Py/Et3N, 55 °C, 40 h; (k) 30% HFIP, CH2Cl2, 1 h; (l) Na ion-exchange chromatography.

It is worth mentioning that in our earlier studies, we performed ion exchange with tetrabutylammonium bromide before releasing sulfoforms from the resin, based on the belief that the hydrophobic Bu4N counterion (i) improved the stability of sulfate esters during cleavage, and (ii) aided in the swelling of resins in organic solvents.16 However, we have subsequently determined that sulfonation and cleavage does not require Bu4N counterions to enhance process chemistry. This was confirmed by subjecting resin-immobilized GlcNS-3S,6S 1h (containing a mixture of PyH+ and Et3NH+ counterions) to HFIP cleavage without prior treatment with Bu4N-Br, followed by passage through a Na ion-exchange column. Good yields of the Na salts were achieved after reverse-phase HPLC purification, and no degradation byproducts were observed. We thus do not need to perform Bu4N ion exchange in our optimized solid-phase synthesis protocol.

Our optimized solid-phase synthesis generated sulfoforms with minimal byproduct formation in nearly all cases. To take one example, the resin-immobilized GlcN 18a was subjected to 7 operations involving deprotection, sulfonation, and HFIP cleavage to generate GlcNAc-3S,6S (1e) with Et3NH+ counterions. The clean 1H NMR of the as-isolated product supports the high quality of each conversion (Figure 2), validating this method of generating sulfoforms from a single, orthogonally protected precursor.

Figure 2.

Figure 2

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1H NMR spectrum (400 MHz, CD3OD) of sulfoform 1e (3,6-di-O-sulfate, bis-triethylammonium salt) after cleavage from solid-phase support, prior to Na ion exchange and HPLC purification (color figure available online).

The modular deprotection/sulfonation steps can efficiently generate sulfoforms on trityl–PS resins with up to three sulfate esters per molecule, but generating derivatives with a larger number of sulfate esters is more challenging. The high charge density of such compounds creates a highly heterogeneous environment within the resin and can raise the barrier to ionic exchange. To test the limits of the solid-phase approach with trityl–PS resins, we attempted to generate a hexasulfate ester from a resin-immobilized GlcN disaccharide derivative (GlcNS,3S,6S(α1→4)GlcNS,3S,6S), but were unable to achieve complete sulfonation (unpublished results). This limitation might be addressed by using less hydrophobic substrates such as PEG-grafted PS resins, which have been used to assemble protected HS oligosaccharides.17 To our knowledge, on-resin deprotection and sulfonation steps for such derivatives have not yet been reported, although we note a case in which a sulfate ester was installed on a synthetic tetrasaccharide immobilized on a polymeric PEG support.18

In closing, we have demonstrated the generation of GlcN sulfoforms with 2'-aminoethyl linkers using solid-phase synthesis methods and orthogonally protected scaffolds. Each operation can be performed in high yields without the need for ion exchange, and the amine-terminated sulfoforms can be cleaved from trityl–PS resins under mild conditions.

Experimental details

All starting materials and reagents were obtained from commercial sources and used as received unless otherwise noted. All solvent were freshly distilled prior to use. All glucosamine sulfoforms were purified by HPLC using a Hydro-RP C18 reverse-phase column, with UV detection at 214 nm. IR spectra were acquired from NaCl plate, using a Thermo-Nicolet Nexus 670 FT-IR spectrometer. 1H NMR spectra were recorded at 300, 400, or 500 MHz; 13C NMR spectra were recorded at 75, 100, or 125 MHz. Chemical shifts were referenced to the solvent used (δ 7.27 and 77.00 for CDCl3, δ 3.31 and 49.15 for CD3OD, and δ 4.80 in D2O). Mass spectra were acquired using either a Hewlett-Packard 5989B or a Finnigan 40000 mass spectrometer. Optical rotations were measured by polarimetry at rt. Silica gel chromatography was performed with ICN SiliTech 32-63D.

Thiotolyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-β-d-glucopyranoside (5)

Glucosamine hydrochloride (50 g, 232 mmol) was mechanically agitated with 1M NaOMe in MeOH (232 mL) for 2 h at rt, followed by addition of finely powdered phthalic anhydride (19 g, 128 mmol) and Et3N (35.5 mL, 255 mmol). The mixture was stirred for 45 min, then treated with a second portion of phthalic anhydride (19 g, 128 mmol) and stirred for 24 h at rt. The mixture was cooled to −20 °C for 4 h, filtered, washed with cold MeOH and dried under reduced pressure to yield a lighter yellow solid. The solid was redispersed in pyridine (500 mL) followed by slow addition of Ac2O (330 mL) at 0 °C and continuous agitation at rt for 48 h. The crude was concentrated under reduced pressure and purified by recrystallization in 95% EtOH to afford 1,3,4,6-tetra-O-acetyl-2-phthalimido-2-deoxy-β-d-glucopyranoside as an amorphous white solid (76 g, 69% over 3 steps).19 1H NMR (300 MHz, CDCl3): δ 7.84 (dd, 2H, J =3, 6 Hz), 7.73 (dd, 2H, J =3, 5.7 Hz), 6.49 (d, 1H, J = 8.7 Hz), 5.86 (dd, 1H, J =9.3, 10.5 Hz), 5.19 (t, 1H, J = 9.3 Hz), 4.44 (dd, 1H, J =8.7, 10.5 Hz), 4.34 (dd, 1H, J =3.9, 12.3 Hz), 4.11 (dd, 1H, J =2.4, 12.3 Hz), 4.00 (ddd, 1H, J =2.4, 4.8, 10.8 Hz), 2.08 (s, 3H), 1.97 (s, 3H), 1.84 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 170.70, 170.08, 169.55, 168.71, 167.44, 134.57, 131.26, 123.87, 89.82, 72.67, 70.56, 68.32, 61.58, 53.55, 20.82, 20.68, 20.48.

A solution of above tetraacetate (20 g, 41.9 mmol) and p-thiocresol (6.8 g, 54.5 mmol) in CH2Cl2 (40 mL) was cooled to 0 °C and treated with BF3∙Et2O (12 mL). The mixture was stirred at rt for 24 h, quenched with saturated NaHCO3 solution (100 mL), extracted with CH2Cl2 (3 × 200 mL), washed with brine (50 mL), then dried over Na2SO4 and concentrated. The crude product was purified by recrystallization in 95% EtOH to afford thiotolyl 3,4,6-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranoside as an amorphous white solid (19.7 g, 87%).19 1H NMR (300 MHz, CDCl3): δ 7.86 (dd, 2H, J = 2.7, 5.1 Hz), 7.75 (dd, 2H, J = 3, 5.4 Hz), 7.29 (d, 2H, J = 7.2 Hz), 7.06 (d, 2H, J = 7.5 Hz), 5.77 (t, 1H, J = 9.9 Hz), 5.64 (d, 1H, J = 10.5 Hz), 5.11 (t, 1H, J = 9.6 Hz), 4.24–4.30 (m, 2H), 4.19 (dd, 1H, J = 2.4, 12.3 Hz), 3.87 (ddd, 1H, J = 3, 5.4, 10.8 Hz), 2.31 (s, 3H), 2.09 (s, 3H), 2.01 (s, 3H), 1.82 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 170.76, 170.22, 169.58, 167.95, 167.08, 138.89, 134.57, 134.43, 134.04, 131.71, 131.29, 129.77, 127.05, 123.82, 83.22, 75.95, 71.77, 68.79, 62.31, 53.69, 21.29, 20.90, 20.73, 20.54; IR (NaCl): 1749, 1719, 1384, 1227, 1037 cm−1; [α]20D = +40.3 (c 1.01, CH2Cl2); HRESI-MS: m/z calcd for C27H27NNaO9S [M+Na]+: 564.1304; found: 564.1301.

The triacetate above (15.8 g, 29.1 mmol) was dissolved in 2:3 CH2Cl2:MeOH (80 mL) and cooled to 0 °C, then treated with 1 M NaOMe solution in MeOH (10.5 mL, 10.5 mmol). The mixture was stirred at 0 °C for 3 h, neutralized with activated Dowex 50X-W-H+ ion-exchange resin, filtered, concentrated, and dried under reduced pressure. A solution of the crude triol in THF (97 mL) was treated with benzaldehyde dimethyl acetal (13 mL, 87.2 mmol) and TsOH∙H2O (554 mg, 2.91 mmol). The mixture was heated to reflux for 10 h, quenched with saturated NaHCO3 solution (30 mL), extracted with CH2Cl2 (3 × 150 mL), dried over Na2SO4 and concentrated under reduced pressure. Purification by recrystallization in 95% EtOH yielded 4,6-O-benzylidene acetal 5 as an amorphous white solid (12.5 g, 85% over 2 steps). 1H NMR (300 MHz, CDCl3): δ 7.82 (dd, 2H, J = 5.4, 9.9 Hz), 7.64–7.72 (m, 2H), 7.41–7.51 (m, 2H), 7.22–7.35 (m, 5H), 7.06 (d, 2H, J = 8.4 Hz), 5.58 (d, 1H, J = 10.5 Hz), 5.52 (s, 1H), 4.55 (dt, 1H, J = 3.6, 9.9 Hz), 4.33 (dd, 1H, J = 4.2, 10.2 Hz), 4.27 (t, 1H, J = 10.5 Hz), 3.77 (t, 1H, J = 9.9 Hz), 3.58 (dt, 1H, J = 4.5, 8.7 Hz), 3.51 (t, 1H, J = 9.3 Hz), 3.30 (d, 1H, J = 3 Hz), 2.30 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 168.31, 167.61, 138.39, 136.96, 134.23, 133.22, 131.54, 129.74, 129.35, 128.37, 127.95, 126.37, 123.85, 123.37, 101.87, 84.49, 81.79, 70.28, 69.61, 68.54, 55.71, 21,15; IR (NaCl): 3474, 1776, 1713, 1386, 1092 cm−1; [α]20D = +31.2 (c 1.10, CH2Cl2); HRESI-MS: m/z calcd for C28H25NNaO6S [M+Na]+: 526.1300; found: 526.1292.

Thiotolyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-β-d-glucopyranoside (6)

Preparation of TfN3: A solution of NaN3 (73 g, 1125 mmol) in water (30 mL) and CH2Cl2 (30 mL) at 0 °C was treated with Tf2O (38 mL, 225 mmol). The mixture was stirred for 2 h at 0 °C, extracted with CH2Cl2 (2 × 35 mL), washed with saturated aqueous Na2CO3 solution (75 mL), then brine (30 mL) and used immediately.

A solution of 5 (12.5 g, 24.9 mmol) and ethylene diamine (33 mL, 500 mmol) in n-butanol was stirred at 100 °C for 10 h in a pressure tube. The mixture was concentrated, azeotroped with toluene and dried under reduced pressure. This was redispersed in 66% aqueous MeOH (231 mL) and treated at 0 °C with K2CO3 (5.2 g, 37.5 mmol), CuSO4∙5 H2O (312 mg, 1.25 mmol) and freshly prepared TfN3 (70 mL, 3.2 M solution in CH2Cl2). An additional 30 mL of MeOH was added to homogenize the mixture, which was slowly warmed up to rt over 3 h and continually stirred for another 10 h at rt, concentrated and azeotroped with toluene, then dried under reduced pressure. The crude azide was dissolved in pyridine (150 mL) followed by addition of Ac2O (75 mL) at 0 °C. The mixture was warmed up to rt over 2 h and continually stirred for another 10 h, concentrated, and dried under reduced pressure. Purification by silica gel chromatography (6% EtOAc in hexanes) yielded thiotolyl 3-O-acetyl-2-azido-4,6-O-benzylidene-2-deoxy-β-D-glucopyranoside as an amorphous white solid (9.3 g, 85% over 3 steps). 1H NMR (300 MHz, CDCl3): δ 7.48 (d, 1H, J = 7.5 Hz), 7.37–7.45 (m, 2H), 7.33–7.36 (m, 2H), 7.19 (d, 2H, J = 7.5 Hz), 5.47 (s, 1H), 5.24 (t, 1H, J = 10.2 Hz), 4.55 (d, 1H, J = 10.2 Hz), 3.38 (dd, 1H, J = 3.9, 10.5 Hz), 3.77 (t, 1H, J = 10.5 Hz), 3.48–3.58 (m, 2H), 3.39 (t, 1H, J = 9.9 Hz), 2.39 (s, 3H), 2.12 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 169.72, 139.37, 136.82, 134.49, 130.11, 129.26, 128.37, 126.71, 126.23, 101.56, 87.10, 78.37, 73.12, 70.76, 68.51, 63.57, 21.38, 20.93; IR (NaCl): 2110, 1754, 1368, 1216, 1099 cm−1; [α]20D = −121.0 (c 2.00, CH2Cl2); HRESI-MS: m/z calcd for C22H33N3NaO5S [M+Na]+: 464.1256; found: 464.1246.

The intermediate above (5 g, 11.3 mmol) was dissolved in a 1 M solution of BH3 in THF (113 mL), then treated with a 1 M solution of Bu2BOTf in THF (11.9 mL) at 0 °C in the presence of 2,6-lutidine (2.63 mL, 22.6 mmol).11 The mixture was stirred for 10 h at 0 °C, then cooled to −78 °C and quenched with Et3N (3.14 mL, 22.6 mmol), followed by the slow addition of cold MeOH (60 mL). The mixture was slowly warmed to rt, concentrated, then purified by silica gel chromatography (20% EtOAc in hexanes) to yield the intermediate C6 alcohol (4.04 g, 10.68 mmol). This was dissolved in DMF (14 mL) and treated with imidazole (2.2 g, 32.04 mmol) and TBDPS-Cl (5.56 mL, 21.36 mmol). The mixture was stirred for 12 h at rt, quenched with saturated NaHCO3 solution (40 mL), extracted with CH2Cl2 (2 × 100 mL), washed with brine (40 mL), dried over Na2SO4 then concentrated under reduced pressure. Purification by silica gel chromatography (6.25% EtOAc in hexanes) yielded 6-O-TBDPS ether 6 as an amorphous white solid (5.04 g, 62% over 2 steps). 1H NMR (300 MHz, CDCl3): δ 7.82 (dd, 2H, J = 1.8, 7.5 Hz), 7.73 (dd, 2H, J = 1.2, 7.5 Hz), 7.54 (d, 2H, J = 8.4 Hz), 7.25–7.45 (m, 9H), 7.15–7.18 (m, 2H), 7.07 (d, 2H, J = 8.1 Hz), 5.18 (t, 1H, J = 9.3 Hz), 4.61 (dd, 2H, J = 11.1, 19.8 Hz), 4.47 (d, 1H, J = 10.2 Hz), 4.02 (dd, 1H, J = 1.2, 11.7 Hz), 3.94 (dd, 1H, J = 3, 11.7 Hz), 3.79 (t, 1H, J = 9.3 Hz), 3.42 (d, 1H, J = 9.3 Hz), 3.35 (t, 1H, J = 9.9 Hz), 2.34 (s, 3H), 2.02 (s, 3H), 1.14 (s, 9H); 13C NMR (75 MHz, CDCl3): δ 169.94, 138.81, 137.77, 135.97, 135.72, 134.26, 133.36, 132.83, 129.97, 128.62, 127.97, 127.89, 127.36, 86.14, 80.08, 76.32, 75.48, 74.91, 63.21, 62.37, 27.05, 21.35, 21.01, 19.547; IR (NaCl): 2030, 2857, 2109, 1753, 1218, 1112, 1045 cm−1; [α]20D = −60.1 (c 1.21, CH2Cl2); HRESI-MS: m/z calcd for C38H43N3NaO5SSi [M+Na]+: 704.2590; found: 704.2595.

2’-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-α-d-glucopyranoside (7a)

A solution of ethanolamine (130 µL, 2.13 mmol) in CH2Cl2 (6 mL) was treated at 0 °C with Fmoc-Cl (500 mg, 1.93 mmol) in the presence of NaHCO3 (974 mg, 11.6 mmol). The mixture was warmed up to rt over 2 h and continuously stirred for another 8 h, extracted with CH2Cl2 (3 × 100 mL), washed with water (30 mL), brine (30 mL), and dried over Na2SO4, then concentrated to afford Fmoc-protected ethanolamine in 96% yield as an amorphous white solid

A mixture of thioglycoside 6 (500 mg, 0.73 mmol) and 2’-hydroxyethyl 2-(9H-fluoren-9-yl)methylcarbamate (270 mg, 0.95 mmol) was azeotroped with toluene (3 × 4 mL), dried under reduced pressure for 1 h, then dissolved in 1:2 (CH2Cl)2:Et2O (6 mL) and stirred with activated 4A molecular sieves for 1 h at rt under Ar. The mixture was cooled to −20 °C and treated with N-iodosuccinimide (180 mg, 0.8 mmol) and TfOH (10 µL, 0.11 mmol). The mixture was stirred continuously for another 5 h at −20 °C, quenched with Et3N (35 µL, 0.22 mmol), warmed to rt, filtered through Celite, extracted with CH2Cl2 (3 × 70 mL), washed with brine (40 mL), dried over Na2SO4, then concentrated under reduced pressure to yield the corresponding Fmoc-protected 2'-aminoethyl glycoside as a 4.2:1 mixture of anomers. Purification by silica gel chromatography (11% EtOAc in hexanes) yielded α-GlcN derivative 7a (486 mg, 80%) and β-GlcN derivative 7b (116 mg, 19%) as colorless oils.

Major isomer 7a: 1H NMR (400 MHz, CDCl3): δ 7.77 (d, 2H, J = 7.5 Hz), 7.72 (t, 4H, J = 6.7 Hz), 7.61 (dd, 2H, J = 4.6, 7.1 Hz), 7.37–7.49 (m, 8H), 7.27–7.35 (m, 5H), 7.20 (dd, 2H, J = 2.5, 7.6 Hz), 5.63 (m, 1H), 5.39 (t, 1H, J = 5.4 Hz), 5.01 (d, 1H, J = 3.3 Hz), 4.67 (s, 2H), 4.37 (d, 2H, J = 7.3 Hz), 4.22 (t, 1H, J = 7.1 Hz), 3.90–3.98 (m, 2H), 3.77–3.86 (m, 3H), 3.58 (t, 2H, J = 8.4 Hz), 3.35 (m, 1H), 3.17 (dd, 1H, J = 3.4, 10.6 Hz), 2.09 (s, 3H), 1.12 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 170.00, 156.35, 143.95, 143.82, 141.22, 137.63, 135.74, 135.53, 133.27, 132.85, 129.75, 128.44, 127.84, 127.71, 127.62, 127.00, 125.16, 119.86, 98.39, 74.89, 72.10, 71.83, 67.65, 66.87, 62.37, 61.50, 47.10, 40.60, 26.82, 20.90, 19.29; IR (NaCl): 2929, 2108, 1726, 1517, 1226, 1114, 1041 cm−1; [α]20D = +80.1 (c 0.89, CH2Cl2); HRESI-MS: m/z calcd for C48H52N4NaO8Si [M+Na]+: 863.3452; found: 863.3462.

Minor isomer 7b: 1H NMR (400 MHz, CDCl3): δ 7.65–7.80 (m, 6H), 7.57 (dd, 2H, J = 7.5, 11.5 Hz), 7.26–7.46 (m, 13H), 7.20 (dd, 2H, J = 2.1, 7.5 Hz), 5.27 (t, 1H, J = 5.7 Hz), 5.07 (t, 1H, J = 9.9 Hz), 4.60–4.70 (m, 2H), 4.40 (dd, 2H, J = 4.1, 7.0 Hz), 4.32 (d, 2H, J = 8.0 Hz), 4.18 (t, 1H, J = 6.8 Hz), 3.89 (m, 2H), 3.81 (t, 1H, J = 9.5 Hz), 3.68 (m, 1H), 3.40–3.55 (m, 3H), 3.32 (d, 1H, J = 9.5 Hz), 2.05 (s, 3H), 1.09 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 170.01, 156.60, 144.03, 141.42, 137.79, 135.98, 135.65, 133.49, 132.89, 129.96, 128.66, 128.10, 127.95, 127.93, 127.85, 127.78, 127.17, 125.25, 125.17, 120.15, 102.30, 75.88, 75.04, 74.00, 69.67, 66.71, 64.44, 62.32, 47.39, 41.17, 26.99, 21.09, 19.47; found: 863.3461; IR (NaCl): 2932, 2110, 1752, 1726, 1225, 1050 cm−1; [α]20D = −5.3 (c 1.57, CH2Cl2); HRESI-MS: m/z calcd for C48H52N4NaO8Si [M+Na]+: 863.3452.

2’-Aminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-α-d-glucopyranoside (8a)

Fmoc-protected 7a (840 mg, 1 mmol) was dissolved in 1:4 piperidine:DMF (5 mL) and stirred at rt for 30 min. The mixture was concentrated and purified by silica gel chromatography (EtOAc) to afford 2'-aminoethyl α-GlcN derivative 8a as a colorless oil (612 mg, 99%). 1H NMR (400 MHz, CDCl3): δ 7.72 (dt, 4H, J = 1.1, 8.3 Hz), 7.36–7.46 (m, 6H), 7.25–7.36 (m, 3H), 7.20 (dd, 2H, J = 2.1, 7.7 Hz), 5.61 (m, 1H), 5.03 (d, 1H, J = 3.6 Hz), 4.66 (s, 2H), 3.90–4.03 (m, 2H), 3.83 (d, 2H, J = 5.6 Hz), 3.77 (m, 1H), 3.50 (m, 1H), 3.15 (dd, 1H, J = 3.5, 10.8 Hz), 2.80–2.95 (m, 2H), 2.06 (s, 3H), 1.11 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 167.93, 137.71, 135.76, 135.53, 133.35, 132.92, 129.71, 128.42, 127.79, 127.70, 127.63, 127.59, 98.26, 76.21, 74.80, 72.16, 71.58, 70.65, 62.40, 61.55, 41.56, 26.82, 20.89, 19.30; IR (NaCl): 2929, 2856, 2106, 1748, 1225, 1111, 1042 cm−1; [α]20D = +96.5 (c 1.33, CH2Cl2); HRESI-MS: m/z calcd for C33H43N4O6Si [M+H]+: 619.2952; found: 619.2955.

2’-Aminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-β-d-glucopyranoside (8b)

Fmoc-protected 7b (266 mg, 0.32 mmol) was dissolved in 1:4 piperidine:DMF (5 mL) and stirred at rt for 30 min. The mixture was concentrated and purified by silica gel chromatography (EtOAc) to afford 2'-aminoethyl β-GlcN derivative 8b as a colorless oil (194 mg, 99%). 1H NMR (400 MHz, CDCl3): δ 7.75 (dd, 2H, J = 1.6, 8.0 Hz), 7.70 (dd, 2H, J = 1.0, 7.5 Hz), 7.44 (t, 2H, J = 7.2 Hz), 7.38 (t, 4H, J = 7.1 Hz), 7.25–7.35 (m, 3H), 7.20 (dd, 2H, J = 2.1, 7.6 Hz), 5.08 (t, 1H, J = 9.8 Hz), 4.66 (q, 2H, J = 11.1 Hz), 4.42 (d, 1H, J = 8.0 Hz), 3.90–4.01 (m, 3H), 3.83 (t, 1H, J = 9.5 Hz), 3.62 (m, 1H), 3.46 (dd, 1H, J = 8.0, 10.3 Hz), 3.39 (td, 1H, J = 2.5, 9.6 Hz), 2.90–3.06 (m, 2H), 2.04 (s, 3H), 1.43 (br, 2H), 1.11 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 169.83, 137.60, 135.81, 135.46, 133.36, 132.79, 129.71, 128.44, 127.85, 127.72, 127.70, 127.54, 101.99, 75.75, 75.66, 74.79, 73.94, 72.46, 64.36, 62.24, 41.85, 26.78, 20.88, 19.29; IR (KBr): 2930, 2857, 2110, 1752, 1226, 1112, 1047 cm−1; [α]20D = −13.8 (c 0.72, CH2Cl2); HRESI-MS: m/z calcd for C33H43N4O6Si [M+H]+: 619.2952; found: 619.2949.

Thiotolyl 3-O-acetyl-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-2-(trichloro-ethylcarbonylamino)-β-d-glucopyranoside (9)

3-O-acetate 6 (600 mg, 0.88 mmol) was dissolved in anhydrous MeOH (20 mL) and treated at rt with 1 M NaOMe solution in MeOH (264 µL, 0.26 mmol). The mixture was stirred at rt for 3 h, neutralized with activated Dowex 50X-W-H+ ion-exchange resin, filtered, concentrated, and dried under reduced pressure. A solution of the crude alcohol in CH2Cl2 (10 mL) was treated at rt with Bu3P (241 µL, 0.97 mmol), followed by addition of H2O (10 mL) after 3 h. The mixture was stirred at rt for 1 day, concentrated, azeotroped with toluene (3 × 10 mL), and dried under reduced pressure. The crude amine was redispersed in pyridine (10 mL) and treated with Troc-Cl (127 µL, 0.92 mmol) at rt. The mixture was stirred at rt for 10 h and concentrated. The crude product was redispersed in pyridine (20 mL) and treated with Ac2O (10 mL) at rt. The mixture was stirred at rt for 10 h, concentrated and purified by silica gel chromatography (20% EtOAc in hexanes) to afford Troc-protected derivative 9 as an amorphous white solid (581 g, 94%). 1H NMR (400 MHz, CDCl3): δ 7.83 (dd, 2H, J = 1.2, 7.9 Hz), 7.75 (dd, 2H, J = 1.0, 7.9 Hz), 7.51–7.37 (m, 8H), 7.16–7.23 (m, 2H), 7.07 (d, 4H, J = 8.1 Hz), 5.70 (d, 1H, J = 9.9 Hz), 5.28 (t, 1H, J = 9.8 Hz), 4.89 (d, 1H, J = 12.1 Hz), 4.78 (d, 1H, J = 12.0 Hz), 4.64 (d, 1H, J = 12.7 Hz), 4.62 (s, 2H), 3.77–3.95 (m, 4H), 3.40 (d, 1H, J = 9.6 Hz), 3.34 (s, 3H), 2.01 (s, 3H), 1.15 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 171.06, 154.33, 137.99, 137.59, 135.93, 135.56, 133.43, 133.22, 132.68, 129.70, 129.63, 128.94, 128.44, 127.88, 127.77, 127.68, 95.65, 87.14, 79.56, 76.39, 75.50, 74.88, 74.45, 62.17, 55.15, 26.88, 21.16, 20.94, 19.27; IR (NaCl): 2930, 2858, 1749, 1728, 1242, 1112, 1085, 1035 cm−1; [α]20D = −24.8 (c 0.81, CH2Cl2); HRESI-MS: m/z calcd for C41H46Cl3NNaO7SSi [M+Na]+: 852.1728; found: 852.1723.

2’-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl 4-O-[3-O-acetyl-4-O-benzyl-2-deoxy-6-O-(tert-butyldiphenylsilyl)-2-(trichloroethylcarbonylamino)-β-d-glucopyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside (10)

A mixture of glycosyl donor 9 (560 mg, 0.68 mmol) and glycosyl acceptor 15 (580 mg, 0.81 mmol) was azeotroped with toluene (3 × 10 mL), dried under reduced pressure for 1 h, then redissolved in 1:2 (CH2Cl)2:Et2O (3 mL) with freshly activated 4A molecular sieves and stirred at rt for 1 h. NIS (198 mg, 0.88 mmol) was added and the mixture was cooled to −30 °C, followed by addition of TfOH (9 µL, 0.1 mmol). The mixture was stirred at −30 °C for 6 h, quenched with Et3N (32 µL, 0.2 mmol), filtered through Celite, treated with aqueous Na2S2O3 (30 mL), extracted with CH2Cl2 (3 × 100 mL), washed with brine (20 mL), then dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography (20% EtOAc in hexanes) to afford 1,4-β-linked disaccharide 10 as an amorphous white solid (860 mg, 90%). 1H NMR (400 MHz, CDCl3): δ 7.78–7.90 (m, 4H), 7.73 (d, 2H, J = 6.0 Hz), 7.64 (d, 2H, J = 7.4 Hz), 7.30–7.47 (m, 24H), 7.15–7.27 (m, 6H), 5.57 (t, 1H, J = 6.3 Hz), 4.97 (t, 1H, J = 10.4 Hz), 4.68–4.90 (m, 7H), 4.68 (s, 2H), 4.40–4.57 (m, 5H), 4.35 (d, 1H, J = 8.0 Hz), 4.24 (t, 1H, J = 5.6 Hz), 4.00 (t, 1H, J = 8.9 Hz), 3.58–3.95 (m, 9H), 3.33–3.53 (m, 4H), 3.18 (d, 1H, J = 9.7 Hz), 2.00 (s, 3H), 1.14 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 170.55, 156.53, 154.26, 144.00, 143.92, 141.30, 139.00, 138.28, 137.79, 137.44, 135.90, 135.65, 133.20, 132.90, 129.75, 128.72, 128.45, 128.30, 128.03, 128.00, 127.80, 127.64, 127.38, 127.04, 125.08, 125.03, 120.00, 103.83, 100.31, 95.64, 81.86, 77.30, 76.38, 75.84, 75.78, 74.82, 74.87, 74.82, 74.58, 74.33, 74.10, 73.60, 69.87, 68.20, 66.49, 62.21, 57.15, 47.27, 41.51, 26.93, 20.85, 19.22; IR (NaCl): 2941, 2858, 1748, 1729, 1520, 1452, 1236, 1109, 1052 cm−1; [α]20D = −15.5 (c 0.84, CH2Cl2); ESI-MS: m/z calcd for C78H83Cl3N2NaO15Si [M+Na]+: 1443.45; found: 1443.44.

2’-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-(tert-butyldiphenylsilyl)-β-d-glucopyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside (11)

Troc-protected 10 (282 mg, 0.2 mmol) was dissolved in 3:2 EtOH:H2O (2 mL), treated with indium metal powder (46 mg, 0.4 mmol) and NH4Cl (32 mg, 0.6 mmol), then stirred for 6 h at 98 °C in a high-pressure reaction vessel. The mixture was concentrated, redispersed in 2:1 MeOH:H2O (15 mL) with K2CO3 (41 mg, 0.3 mmol) and CuSO4∙5H2O (5 mg, 0.02 mmol), then treated at 0 °C with fresh prepared 0.03 M TfN3 solution in CH2Cl2 (60 mL). The mixture was warmed to rt slowly over 2 h and stirred at rt for another 8 h. The mixture was quenched with solution of NaHCO3 (50 mL), filtered through Celite, extracted with CH2Cl2 (3 × 50 mL), washed with brine (20 mL), then dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography (20% EtOAc in hexanes) to afford azide 11 as an amorphous white solid (230 mg, 91%). 1H NMR (400 MHz, CDCl3): δ 7.80 (dd, 4H, J = 2.7, 7.4 Hz), 7.72 (dd, 2H, J = 1.1, 7.9 Hz), 7.62 (d, 2H, J = 7.5 Hz), 7.46 (dd, 4H, J = 7.7, 9.0 Hz), 7.40 (t, 4H, J = 8.1 Hz), 7.27–7.38 (m, 15H), 7.25–7.25 (m, 7H), 5.58 (t, 1H, J = 5.4 Hz), 5.03 (t, 1H, J = 9.3 Hz), 4.89 (d, 2H, J = 11.1 Hz), 4.84 (d, 1H, J = 12.5 Hz), 4.76 (d, 1H, J = 11.1 Hz), 4.57–4.64 (m, 3H), 4.49 (d, 2H, J = 8.0 Hz), 4.46 (d, 1H, J = 4.5 Hz), 4.41 (dd, 2H, J = 6.7, 11.2 Hz), 4.23 (t, 1H, J = 6.7 Hz), 4.08 (t, 1H, J = 9.3 Hz), 3.60–3.92 (m, 8H), 3.38–3.55 (m, 4H), 3.32 (dd, 1H, J = 8.3, 10.4 Hz), 3.05 (d, 1H, J = 9.6 Hz), 2.08 (s, 3H), 1.12 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 169.84, 156.51, 144.00, 143.90, 141.28, 138.38, 138.24, 137.71, 137.56, 135.87, 135.60, 133.13, 132.75, 129.75, 128.44, 128.32, 128.03, 127.98, 127.87, 127.81, 127.65, 127.63, 127.10, 127.01, 125.09, 125.01, 119.94, 103.82, 100.39, 81.86, 81.74, 77.26, 75.75, 74.85, 74.49, 74.30, 74.17, 73.43, 69.98, 68.53, 66.45, 65.27, 61.94, 47.26, 41.52, 26.92, 20.90, 19.20; IR (NaCl): 2930, 2858, 2109, 1752, 1722, 1357, 1222, 1111, 1053 cm−1; [α]20D = −4.9 (c 0.71, CH2Cl2); MALDI-MS: m/z calcd for C75H80N4NaO13Si [M+Na]+: 1295.5389; found: 1295.4942.

2’-Aminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-(tert-butyldiphenyl-silyl)-β-d-glucopyranosyl]-2,3,6-O-benzyl-β-d-glucopyranoside (12)

Fmoc-protected 11 (184 mg, 0.14 mmol) was dissolved in 1:4 piperidine:DMF (5 mL) and stirred at rt for 30 min. The mixture was concentrated and purified by silica gel chromatography (5% MeOH in EtOAc) to afford 2'-aminoethyl β-glycoside 12 as a colorless oil (150 mg, 99%). 1H NMR (400 MHz, CDCl3): δ 7.80 (d, 2H, J = 4.9 Hz), 7.70 (d, 2H, J = 7.2 Hz), 7.20–7.52 (m, 17H), 7.17–7.25 (m, 5H), 5.02 (t, 1H, J = 9.8), 4.94 (d, 1H, J = 11.0 Hz), 4.85–4.90 (m, 2H), 4.79 (d, 1H, J = 10.9 Hz), 4.71 (d, 1H, J = 12.2 Hz), 4.64 (dd, 2H, J = 11.3, 22.9 Hz), 4.57 (d, 1H, J = 12.3 Hz), 4.50 (dd, 2H, J = 8.3, 17.9 Hz), 4.17 (t, 1H, J = 8.7 Hz), 3.98 (m, 1H), 3.85 (s, 2H), 3.60–3.85 (m, 5H), 3.55 (t, 2H, J = 8.7 Hz), 3.31 (t, 1H, J = 9.4 Hz), 3.07 (d, 1H, J = 8.7 Hz), 2.95 (br, 1H), 2.08 (s, 3H), 1.13 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 169.84, 138.89, 138.33, 137.74, 135.87, 135.61, 133.13, 132.75, 129.75, 128.44, 128.29, 128.01, 127.95, 127.83, 127.62, 127.06, 103.64, 100.26, 81.89, 81.66, 77.33, 75.78, 75.74, 75.53, 74.86, 74.47, 74.17, 73.44, 72.41, 68.54, 65.17, 61.99, 26.92, 20.91, 19.19; IR (NaCl): 2930, 2856, 2109, 1752, 1223, 1109, 1055 cm−1; [α]20D = −5.1 (c 0.55, CH2Cl2); ESI-MS: m/z calcd for C60H71N4O11Si [M+H]+: 1051.49; found: 1051.36.

2’-Azidoethyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside (13).20

To a solution of β-d-glucose pentaacetate (10 g, 25.6 mmol), 2-bromoethanol (5.5 mL, 76.9 mmol) and DIPEA (223 µL, 1.3 mmol) in CH2Cl2 (60 mL) at 0 °C was added a 1 M BF3∙Et2O solution in CH2Cl2 (4.9 mL, 4.9 mmol) slowly. The mixture was slowly warmed to rt over 2 h, stirred continually at rt for another 10 h, quenched with saturated NaHCO3 solution (100 mL), extracted with CH2Cl2 (3 × 200 mL), washed with brine (50 mL), then dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography (25% EtOAc in hexanes) to afford an amorphous white solid (5.6 g). This was redispersed with NaN3 (3.2 g, 49.5 mmol) in CH2Cl2 and heated to reflux for 12 h, extracted with CH2Cl2 (3 × 200 mL), washed with brine (50 mL), then dried over Na2SO4 and concentrated to afford 2’-azidoethyl β-glucoside 13 as an amorphous white solid (4.9 g, 46%). 1H NMR (400 MHz, CDCl3): δ 5.19 (t, 1H, J = 9.5 Hz), 5.08 (t, 1H, J = 7.2 Hz), 5.01 (dd, 1H, J = 8.0, 9.6 Hz), 4.58 (d, 1H, J = 7.8 Hz), 4.23 (dd, 1H, J = 4.6, 12.3 Hz), 4.13 (dd, 1H, J = 2.4, 12.3 Hz), 4.01 (ddd, 1H, J = 1.0, 4.7, 10.9 Hz), 3.65–3.72 (m, 2H), 3.47 (ddd, 1H, J = 3.6, 8.4, 12.8 Hz), 3.26 (ddd, 1H, J = 3.6, 4.8, 12.8 Hz), 2.07 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.98 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 170.55, 170.16, 169.29, 100.54, 72.65, 71.79, 70.92, 68.48, 68.16, 61.69, 50.39, 20.63, 20.58, 20.49; IR (NaCl): 2945, 2106, 1756, 1369, 1226, 1039 cm−1; [α]20D = −31.2 (c 1.14, CH2Cl2); ESI-MS: m/z calcd for C16H23N3NaO10[M+Na]+: 440.1281; found: 440.1290.

2’-Azidoethyl 2,3,6-tri-O-benzyl-β-d-glucopyranoside (14)

A solution of tetraacetate 13 (4.9 g, 11.8 mmol) was dissolved in anhydrous MeOH (40 mL) and treated at rt with 1 M NaOMe solution in MeOH (3.5 mL). The mixture was stirred at rt for 3 h, neutralized with activated Dowex 50X-W-H+ ion-exchange resin, filtered, concentrated, and dried under reduced pressure. A solution of the crude in THF (80 mL) was treated with benzaldehyde dimethyl acetal (5.3 mL, 35.4 mmol) and TsOH∙H2O (225 mg, 1.18 mmol). The mixture was heated to reflux for 10 h, quenched with saturated NaHCO3 solution (30 mL), extracted with CH2Cl2 (3 × 150 mL), dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (50% EtOAc in hexanes) to afford an amorphous white solid (3.7 g). This was redispersed with BnBr (5.1 mL, 42.3 mmol) in DMF and treated at 0 °C with NaH (1.3 g, 32.5 mmol). The mixture was slowly warmed to rt over 2 h and continuously stirred at rt for another 10 h, quenched with saturated NH4Cl solution (100 mL), extracted with CH2Cl2 (3 × 200 mL), washed with brine (50 mL), then dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography (14% EtOAc in hexanes) to afford the desired 2,3-di-O-benzyl glucoside as an amorphous white solid (4.5 g, 75%). 1H NMR (400 MHz, CDCl3): δ 7.52 (dd, 2H, J = 2.2, 7.9 Hz), 7.28–7.48 (m, 13H), 5.60 (s, 1H), 4.49 (dd, 2H, J = 1.7, 11.9 Hz), 4.82 (dd, 2H, J = 4.4, 11.4 Hz), 4.56 (d, 1H, J = 7.8 Hz), 4.38 (dd, 1H, J = 4.8, 10.4 Hz), 4.06 (ddd, 1H, J = 4, 6.4, 10.4 Hz), 3.62–3.86 (m, 2H), 3.38–3.55 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 138.37, 138.27, 137.19, 128.92, 128.31, 128.26, 128.20, 128.04, 127.97, 127.69, 127.60, 125.94, 103.99, 101.07, 82.03, 81.31, 80.75, 75.39, 75.07, 68.64, 68.49, 66.01, 50.91; IR (NaCl): 2883, 2104, 1094, 1072 cm−1; [α]20D = −33.5 (c 0.79, CH2Cl2); ESI-MS: m/z calcd for C29H30N3NaO6 [M+Na]+: 540.2111; found: 540.2102.

A solution of the protected intermediate above (4.4 g, 8.6 mmol) and Et3SiH (5.5 mL, 34.2 mmol) in CH2Cl2 (86 mL) was cooled to −78 °C and treated with TfOH (3 mL, 34.2 mmol) in the presence of 4A molecular sieve. The mixture was stirred at −78 °C for 6 h, quenched with Et3N (5.4 mL, 38.5 mmol) and MeOH (5 mL), warmed up to rt slowly over 1 h, filtered through Celite, extracted with CH2Cl2 (3 × 100 mL), washed with brine (20 mL), then dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography (20% EtOAc in hexanes) to afford 6-O-benzyl ether 14 as an amorphous white solid (3.3 g, 75%). 1H NMR (400 MHz, CDCl3): δ 7.34 (m, 15H), 4.96 (t, 2H, J = 10.9 Hz), 4.74 (dd, 2H, J = 3.8, 11.0 Hz), 4.59 (dd, 2H, J = 11.6, 17.2 Hz), 4.46 (dd, 1H, J = 2.4, 5.6 Hz), 4.06 (ddd, 1H, J = 3.6, 5.6, 10.4 Hz), 3.78 (dd, 1H, J = 3.5, 10.4 Hz), 3.67–3.79 (m, 2H), 3.59 (d, 1H, J = 8.8 Hz), 3.40–3.55 (m, 5H), 2.51 (s, 1H); 13C NMR (100 MHz, CDCl3): δ 138.47, 138.30, 137.80, 128.50, 128.37, 128.33, 128.06, 127.90, 127.81, 127.69, 127.65, 103.64, 83.85, 81.64, 75.21, 74.76, 74.10, 73.59, 71.21, 70.04, 68.12, 50.96; IR (NaCl): 3442, 2872, 2103, 1452, 1064 cm−1; [α]20D = −15.3 (c 0.90, CH2Cl2); ESI-MS: m/z calcd for C29H33N3NaO6 [M+Na]+: 542.2267; found: 542.2272.

2’-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl 2,3,6-tri-O-benzyl-β-d-glucopyranoside (15)

A solution of 2’-azidoethyl glucoside 14 (2.4 g, 4.6 mmol) in CH2Cl2 (30 mL) was treated at rt with Bu3P (1.3 mL, 5.1 mmol), followed by addition of H2O (30 mL) after 3 h. The mixture was stirred at rt for 1 day, concentrated, azeotroped with toluene (3 × 10 mL), and dried under reduced pressure to afford a yellowish oil. This was redispersed with NaHCO3 (2.3 g, 27.9 mmol) in CH2Cl2 (35 mL) and treated at 0 °C with Fmoc-Cl (1.2 g, 4.6 mmol) dissolved in CH2Cl2 (5 mL). The mixture was slowly warmed to rt over 2 h and stirred continually for another 10 h at rt, extracted with CH2Cl2 (3 × 100 mL), washed with brine (20 mL), then dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography (20% EtOAc in hexanes) to afford Fmoc-protected 15 as an amorphous white solid (1.6 g, 54%). 1H NMR (400 MHz, CDCl3): δ 7.77 (d, 2H, J = 7.5 Hz), 7.61 (d, 2H, J = 7.4 Hz), 7.20–7.55 (m, 19H), 5.61 (t, 1H, J = 5.0 Hz), 4.96 (dd, 2H, J = 11.5, 20.1 Hz), 4.78 (d, 2H, J = 11.4 Hz), 4.52 (dd, 2H, J = 12.1, 26.7 Hz), 4.35–4.45 (m, 3H), 4.19 (t, 1H, J = 6.7 Hz), 3.79–3.90 (m, 2H), 3.75 (dd, 1H, J = 3.2, 10.2 Hz), 3.66 (dd, 1H, J = 5.8, 10.1 Hz), 3.59 (t, 1H, J = 8.8 Hz), 3.25–3.52 (m, 5H), 2.45 (br, 1H); 13C NMR (100 MHz, CDCl3): δ 156.52, 144.01, 143.87, 141.26, 138.46, 138.21, 137.67, 128.55, 128.42, 128.36, 127.98, 127.96, 127.89, 127.77, 127.69, 127.64, 126.99, 125.03, 119.93, 103.96, 83.92, 81.65, 75.22, 74.79, 73.97, 73.53, 71.23, 70.13, 69.96, 66.43, 47.23, 41.56; IR (NaCl): 3430, 3031, 2874, 1711, 1452, 1251, 1062 cm−1; [α]20D = −10.9 (c 1.31, CH2Cl2); ESI–MS: m/z calcd for C44H45NNaO8 [M+Na]+: 738.3043; found: 738.3045.

2’-(9H-Fluoren-9-yl)methoxycarbonylaminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-(tert-butyldiphenylsilyl)-α-d-glucopyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside (16)

A mixture of glycosyl acceptor 15 (503 mg, 0.7 mmol) and glycosyl donor 6 (400 mg, 0.59 mmol) was azeotroped with toluene (3 × 10 mL), dried under reduced pressure for 1 h, then redispersed in 1:2 (CH2Cl)2:Et2O (3 mL) with freshly activated 4A molecular sieves and stirred at rt for 1 h. NIS (145 mg, 0.65 mmol) was added and the mixture was cooled to −20 °C, followed by addition of TfOH (16 µL, 0.18 mmol). The mixture was stirred at −20 °C for 10 h, quenched with Et3N (58 µL, 0.42 mmol), and filtered through Celite, then treatd with saturated Na2S2O3 (30 mL), extracted with CH2Cl2 (3 × 100 mL), washed with brine (20 mL), then dried over Na2SO4 and concentrated. The crude product was purified by silica gel chromatography (20% EtOAc in hexanes) to afford 1,4-α-linked disaccharide 16 as an amorphous white solid (447 mg, 60%). 1H NMR (400 MHz, CDCl3): δ 7.77 (dd, 2H, J = 3.1, 7.3 Hz), 7.62 (ddd, 4H, J = 0.9, 8.0, 12.3 Hz), 7.56 (t, 2H, J = 7.1 Hz), 7.25–7.50 (m, 24H), 7.21 (d, 2H, J = 7.4 Hz), 6.94–7.06 (m, 4H), 5.69 (d, 1H, J = 3.7 Hz), 5.65 (t, 1H, J = 10.3 Hz), 5.10 (d, 1H, J = 10.9 Hz), 4.90 (d, 1H, J = 11.0 Hz), 4.82 (d, 1H, J = 10.8 Hz), 4.69 (d, 1H, J = 11.0 Hz), 4.66 (s, 2H), 4.54 (m, 1H), 4.35 (dd, 1H, J = 6.1, 10.6 Hz), 4.25 (d, 1H, J = 7.6 Hz), 4.23 (d, 1H, J = 15.2 Hz), 4.15 (m, 1H), 3.74–3.90 (m, 4H), 3.69 (d, 2H, J = 11.6 Hz), 3.57 (d, 2H, J = 10.5 Hz), 3.21–3.57 (m, 7H), 3.06 (dd, 1H, J = 3.7, 10.8 Hz), 2.06 (s, 3H), 1.05 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 170.03, 156.58, 144.07, 143.74, 141.39, 141.33, 138.47, 138.18, 138.02, 137.49, 135.82, 135.56, 133.49, 132.87, 129.68, 128.42, 128.15, 128.04, 127.89, 127.70, 127.62, 127.45, 127.31, 127.03, 126.94, 125.03, 124.79, 119.98, 104.02, 97.96, 84.62, 82.63, 75.74, 74.81, 74.64, 74.59, 73.57, 73.21, 73.08, 72.06, 70.83, 68.77, 65.79, 61.68, 61.41, 47.44, 41.76, 26.91, 20.92, 19.32; IR (NaCl): 2929, 2858, 2108, 1749, 1730, 1227, 1043 cm−1; [α]20D = +42.6 (c 0.29, CH2Cl2); ESI-MS: m/z calcd for C75H80N4NaO13Si [M+Na]+: 1295.54; found: 1295.29.

2’-Aminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-(tert-butyl-diphenylsilyl)-α-d-glucopyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside (17)

Fmoc-protected 16 (427 mg, 0.24 mmol) was dissolved in 1:4 piperidine:DMF (5 mL) and stirred at rt for 30 min. The mixture was concentrated and purified by silica gel chromatography (EtOAc) to afford 2'-aminoethyl β-glycoside 17 as a colorless oil (349 mg, 99%). 1H NMR (400 MHz, CDCl3): δ 7.65 (dd, 4H, J = 6.9, 15.0 Hz), 7.41–7.47 (m, 2H), 7.25–7.40 (m, 18H), 7.21 (dd, 2H, J = 1.8, 8.1 Hz), 7.13 (dd, 2H, J = 1.8, 7.5 Hz), 7.06 (dd, 2H, J = 1.1, 5.9 Hz), 5.73 (d, 1H, J = 3.8 Hz), 5.49 (t, 1H, J = 9.5 Hz), 5.12 (d, 1H, J = 10.9 Hz), 4.97 (d, 1H, J = 11.0 Hz), 4.83 (d, 1H, J = 10.8 Hz), 4.75 (d, 1H, J = 11.0 Hz), 4.68 (d, 1H, J = 13.9 Hz), 4.68 (d, 2H, J = 2.6 Hz), 4.46 (d, 1H, J = 7.8 Hz), 4.42 (d, 1H, J = 5.7 Hz), 4.41 (d, 1H, J = 13.9 Hz), 3.77–3.95 (m, 4H), 3.57–3.75 (m, 6H), 3.40–3.55 (m, 2H), 3.07 (dd, 1H, J = 3.8, 10.8 Hz), 2.82–2.97 (m, 2H), 2.10 (br, 1H), 2.05 (s, 3H), 1.08 (s, 9H); 13C NMR (100 MHz, CDCl3): δ 169.94, 138.46, 138.14, 137.96, 137.73, 135.79, 135.52, 133.49, 132.88, 129.62, 128.39, 128.35, 128.15, 127.86, 127.70, 127.64, 127.60, 127.54, 127.45, 127.39, 127.25, 103.52, 97.48, 84.68, 82.57, 75.75, 74.73, 74.66, 74.50, 73.99, 73.24, 72.93, 72.20, 72.02, 71.97, 68.95, 61.76, 61.39, 41.89, 26.87, 20.87, 19.29; IR (NaCl): 3030, 2930, 2857, 2107, 1751, 1360, 1225, 1090, 1039 cm−1; [α]20D = +53.9 (c 1.21, CH2Cl2); ESI-MS: m/z calcd for C60H71N4O11Si [M+H]+: 1051.4889; found: 1051.4905.

2’-Aminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-α-d-glucopyranoside, bound to trityl–PS resin (18a)

Cl-Trityl–PS resin beads (1.29 g, 1.93 mmol) were suspended in dry pyridine (3 mL) for 5 min, followed by the addition of 2'-aminoethyl α-GlcN glycoside 8a (597 mg, 0.96 mmol) and DMAP (47 mg, 0.39 mmol). The mixture was shaken for 2 days at 60 °C then cooled to rt. Anhydrous MeOH (0.5 mL) was added and the mixture was shaken at rt for 1 h. The resin was filtered, washed with 5% iPr2NEt in 8:1 CH2Cl2:MeOH (2 × 4 mL), then alternatively with DMF (2 × 4 mL) and CH2Cl2 (2 × 4 mL), then finally with additional CH2Cl2 (3 × 4 mL). The resin was dried under reduced pressure in the presence of KOH pellets to afford fully resin-bound α-GlcN derivative 18a, with a loading ratio of 0.38 mmol/g. IR (KBr): 2925, 2107, 1749, 1644, 1491, 1447, 1223, 1074 cm−1. To determine the loading ratio, 50 mg of 18a was treated with 30% HFIP in CH2Cl2 (3 mL) at rt for 1 h. The combined washings were concentrated under reduced pressure for recovery of free α-GlcN derivative 8a.

2’-Aminoethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-β-d-glucopyranoside, bound to trityl–PS resin (18b)

Cl-Trityl–PS resin beads (386 mg, 0.55 mmol) were suspended in dry pyridine (3 mL) for 5 min, followed by addition of 2'-aminoethyl β-GlcN glycoside 8b (170 mg, 0.28 mmol) and DMAP (13 mg, 0.11 mmol). The mixture was shaken for 2 days at 60 °C, then cooled to rt. Anhydrous MeOH (0.5 mL) was added and the mixture was shaken at rt for 1 h. The resin was filtered, washed with 5% iPr2NEt in 8:1 CH2Cl2:MeOH (2 × 4 mL), then alternatively with DMF (2 × 4 mL) and CH2Cl2 (2 × 4 mL), then finally with additional CH2Cl2 (3 × 4 mL). The resin was dried under reduced pressure in the presence of KOH pellets to afford fully resin-bound β-GlcN derivative 18b, with a loading ratio of 0.27 mmol/g. To determine the loading ratio, 50 mg of 18b was treated with 30% HFIP in CH2Cl2 (3 mL) under standard cleavage conditions. The combined washings were then concentrated for recovery of free β-GlcN derivative 8b.

2’-Aminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-(tert-butyldiphenyl-silyl)-β-d-glucopyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside, bound to trityl–PS resin (19)

Cl-Trityl–PS resin beads (294 mg, 0.44 mmol) were suspended in dry pyridine (3 mL) for 5 min, followed by addition of GlcN(β1→4)-Glcβ derivative 12 (232 mg, 0.22 mmol) and DMAP (10.8 mg, 0.088 mmol). The mixture was shaken for 2 days at 60 °C, then cooled to rt. Anhydrous MeOH (0.3 mL) was added and the mixture was shaken at rt for 1 h. The resin was filtered, washed with 5% iPr2NEt in 8:1 CH2Cl2:MeOH (2 × 4 mL), then alternatively with DMF (2 × 4 mL) and CH2Cl2 (2 × 4 mL), then finally with additional CH2Cl2 (3 × 4 mL). The resin was dried under reduced pressure in the presence of KOH pellets to afford fully resin-bound GlcN(β1→4)-Glcβ derivative 19, with a ratio of 0.17–0.18 mmol/g. To determine the loading ratio, 50 mg of 19 was treated with 30% HFIP in CH2Cl2 (3 mL) under standard cleavage conditions. The combined washings were concentrated under reduced pressure for recovery of free GlcN(β1→4)-Glcβ derivative 12.

2’-Aminoethyl 4-O-[3-O-acetyl-2-azido-4-O-benzyl-2-deoxy-6-O-(tert-butyldiphenyl-silyl)-α-d-glucopyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside, bound to trityl–PS resin (20)

Cl-Trityl–PS resin beads (267 mg, 0.40 mmol) were suspended in dry pyridine (3 mL) for 5 min followed by addition of GlcN(α1→4)-Glcβ derivative 17 (140 mg, 0.13 mmol) and DMAP (6.5 mg, 0.053 mmol). The mixture was shaken for 2 days at 60 °C, then cooled to rt. Anhydrous MeOH (0.3 mL) was added and the mixture was shaken at rt for 1 h. The resin was filtered, washed with 5% iPr2NEt in 8:1 CH2Cl2:MeOH (2 × 4 mL), then alternatively with DMF (2 × 4 mL) and CH2Cl2 (2 × 4 mL), then finally with additional CH2Cl2 (3 × 4 mL). The resin was dried under reduced pressure in the presence of KOH pellets to afford resin-bound GlcN(α1→4)-Glcβ derivative 20, with a ratio of 0.17–0.18 mmol/g. To determine the loading ratio, 50 mg of 20 was treated with 30% HFIP in CH2Cl2 (3 mL) under standard cleavage conditions. The combined washings were concentrated under reduced pressure for recovery of free GlcN(α1→4)-Glcβ derivative 17.

2’-Aminoethyl 2-acetamido-4-O-benzyl-2-deoxy-α-d-glucopyranoside (1a)

Op. 1: Resin-bound α-GlcN derivative 18a (44 mg, 0.0167 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (25 µL, 0.1 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac2O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in CH2Cl2 (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (50 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound α-GlcN. Op. 6: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Purification by reverse-phase HPLC yielded 2'-aminoethyl α-GlcN 1a as an amorphous white solid (5.1 mg, 87% over 6 operations). 1H NMR (400 MHz, CD3OD): δ 7.23–7.44 (m, 5H), 4.95 (d, 1H, J = 10.8 Hz), 4.75 (d, 1H, J = 3.6 Hz), 4.65 (d, 1H, J = 11.0 Hz), 3.99 (dd, 1H, J = 3.5, 10.5 Hz), 3.84 (dd, 1H, J = 8.5, 10.4 Hz), 3.77 (dd, 1H, J = 2.0, 9.8 Hz), 3.71 (dd, 1H, J = 5.0, 19.2 Hz), 3.57–3.69 (m, 2H), 3.43 (t, 1H, J = 9.2 Hz), 3.33–3.42 (m, 2H), 2.76–2.87 (m, 2H), 1.99 (s, 3H); 13C NMR (100 MHz, CD3OD): δ 173.69, 139.94, 129.28, 129.07, 128.68, 98.86, 80.02, 76.05, 73.53, 73.05, 70.27, 62.32, 55.57, 42.05, 22.57; IR (NaCl): 3292, 2920, 1656, 1576, 1315, 1068, 1029 cm−1; [α]20D = +58.1 (c 0.31, MeOH); ESI-MS: m/z calcd for C17H26N2O6Na [M+Na]+: 377.1689; found: 377.1697.

2’-Aminoethyl 2-acetamido-4-O-benzyl-2-deoxy-6-O-sulfonato-α-d-glucopyranoside, sodium salt (1b)

Op. 1: Resin-bound α-GlcN derivative 18a (44 mg, 0.0167 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (25 µL, 0.1 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac2O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF adjusted to pH 7 with AcOH (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with 5:1 THF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (27 mg, 0.17 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 6: The resin was swollen in CH2Cl2 (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (50 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 6-O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl α-GlcN 6-O-sulfate 1b (sodium salt) as an amorphous white solid (5.5 mg, 76% over 8 operations). 1H NMR (400 MHz, CD3OD): δ 7.41 (d, 2H, J = 7.1 Hz), 7.23–7.34 (m, 3H), 4.92 (d, 1H, J = 10.8 Hz), 4.78 (d, 1H, J = 3.7 Hz), 4.72 (d, 1H, J = 10.5 Hz), 4.34 (dd, 1H, J = 1.8, 11.0 Hz), 4.12 (dd, 1H, J = 6.2, 10.9 Hz), 4.03 (dd, 1H, J = 3.8, 10.8 Hz), 3.83–3.97 (m, 2H), 3.81 (dd, 1H, J = 9.0, 10.7 Hz), 3.52 (m, 1H), 3.41 (t, 1H, J = 9.4 Hz), 3.03 (t, 2H, J = 4.8 Hz), 2.01 (s, 3H); 13C NMR (100 MHz, CD3OD): δ 173.63, 139.77, 129.47, 129.24, 128.67, 99.90, 79.87, 76.10, 73.59, 71.00, 68.88, 68.00, 55.40, 41.60, 22.63; IR (NaCl): 3449, 2925, 1659, 1543, 1223, 1222, 1068, 1013 cm−1; [α]20D = +64.7 (c 0.41, MeOH); ESI-MS: m/z calcd for C17H25N2O9S [M]−: 433.1281; found: 433.1279.

2’-Aminoethyl 2-acetamido-4-O-benzyl-2-deoxy-3-O-sulfonato-α-d-glucopyranoside, sodium salt (1c)

Op. 1: Resin-bound α-GlcN derivative 18a (44 mg, 0.0167 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (25 µL, 0.1 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac2O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in CH2Cl2 (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (50 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give to give the C3 alcohol. Op. 5: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (27 mg, 0.17 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 3-O-sulfate. Op. 6: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 3-O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl α-GlcN 3-O-sulfate 1c (sodium salt) as an amorphous white solid (4.6 mg, 64% over 8 operations). 1H NMR (400 MHz, CD3OD): δ 7.43 (d, 2H, J = 6.9 Hz), 7.22–7.31 (m, 3H), 5.17 (d, 1H, J = 10.3 Hz), 4.98 (d, 1H, J = 3.3 Hz), 4.72 (dd, 1H, J = 8.6, 11.0 Hz), 4.52 (d, 1H, J = 10.3 Hz), 3.97 (dd, 1H, J = 3.3, 11.0 Hz), 3.73–3.86 (m, 2H), 3.61–3.70 (m, 2H), 3.55 (t, 1H, J = 8.9 Hz), 3.49 (m, 1H), 2.99 (t, 2H, J = 4.7 Hz), 1.98 (s, 3H); 13C NMR (100 MHz, CD3OD): δ 173.72, 139.73, 129.99, 129.15, 128.69, 98.63, 79.10, 77.84, 76.09, 73.61, 67.69, 62.17, 55.09, 41.27, 22.90; IR (NaCl): 3359, 2925, 1664, 1651, 1225, 1122, 1052 cm−1; [α]20D = +59.3 (c 0.24, MeOH); ESI-MS: m/z calcd for C17H25N2O9S [M]−: 433.1281; found: 433.1284.

2’-Aminoethyl 2-amino-4-O-benzyl-2-deoxy-2-N-sulfonato-α-d-glucopyranoside, sodium salt (1d)

Op. 1: Resin-bound α-GlcN derivative 18a (120 mg, 0.0456 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (68 µL, 0.274 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 THF:H2O (3 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et3N (0.6 mL), SO3∙Py (73 mg, 0.46 mmol) and shaken for 1 day at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound N-sulfate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in CH2Cl2 (3 mL) for 5 min, treated with 1 M NaOMe in MeOH (140 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound N-sulfate. Op. 6: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.7: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reversed-phase HPLC to yield 2'-aminoethyl α-GlcN 2-N-sulfate 1d (sodium salt) as an amorphous white solid (13 mg, 69% over 7 operations). 1H NMR (400 MHz, CD3OD): δ 7.23–7.40 (m, 5H), 5.19 (d, 1H, J = 3.8 Hz), 4.97 (d, 1H, J = 10.9 Hz), 4.63 (d, 1H, J = 10.9 Hz), 3.89 (ddd, 1H, J = 3.5, 5.5, 11.6 Hz), 3.77 (dd, 1H, J = 1.3, 9.6 Hz), 3.74 (dd, 1H, J = 5.0, 7.0 Hz), 3.62 (m, 3H), 3.42 (t, 1H, J = 9.3 Hz), 3.17 (ddd, 1H, J = 3.0, 5.5, 13.5 Hz), 3.06 (ddd, 1H, J = 3.1, 7.7, 13.2 Hz); 13C NMR (100 MHz, CD3OD): δ 129.25, 129.15, 128.63, 98.92, 79.76, 75.82, 74.14, 72.77, 68.10, 62.48, 59.96, 41.24; IR (NaCl): 3390, 2920, 1207, 1309 cm−1; [α]20D = +89.3 (c 0.41, MeOH); ESI–MS: m/z calcd for C15H23N2O8S [M–H]−: 391.1175; found: 391.1180.

2’-Aminoethyl 2-acetamido-4-O-benzyl-2-deoxy-3,6-di-O-sulfonato-α-d-glucopyranoside, disodium salt (1e)

Op. 1: Resin-bound α-GlcN derivative 18a (120 mg, 0.0456 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (67 µL, 0.27 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac2O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in CH2Cl2 (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (137 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the corresponding 3,6-diol. Op. 6: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et3N (0.6 mL), SO3∙Py (362 mg, 2.28 mmol) and shaken for 2 day at 55 °C. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 3,6-di-O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl α-GlcN 3,6-di-O-sulfate 1e (disodium salt) as an amorphous white solid (17.3 mg, 68% over 8 operations). 1H NMR (400 MHz, CD3OD): δ 7.50 (dd, 2H, J = 1.4, 8.3 Hz), 7.22–7.33 (m, 3H), 5.13 (d, 1H, J = 10.1 Hz), 5.01 (d, 1H, J = 3.3 Hz), 4.71 (dd, 1H, J = 8.6, 10.8 Hz), 4.60 (d, 1H, J = 10.1 Hz), 4.34 (dd, 1H, J = 2.0, 11.1 Hz), 4.08 (dd, 1H, J = 6.1, 10.8 Hz), 3.89–4.03 (m, 3H), 3.57–3.62 (m, 1H), 3.54 (t, 1H, J = 9.1 Hz), 3.17 (t, 2H, J = 5.0 Hz), 1.98 (s, 3H); 13C NMR (100 MHz, CD3OD): δ 173.64, 139.47, 130.34, 129.13, 128.69, 98.59, 78.82, 77.81, 75.95, 71.48, 68.21, 65.27, 55.01, 40.62, 22.91; IR (NaCl): 2925, 1659, 1225, 1052, 1024 cm−1; [α]20D = +87.8 (c 0.13, MeOH); ESI-MS: m/z calcd for C17H25N2O12S2 [M+H]−: 513.0849; found: 513.0853.

2’-Aminoethyl 2-amino-4-O-benzyl-2-deoxy-2,6-di-N,O-sulfonato-α-d-gluco-pyranoside, disodium salt (1f)

Op. 1: Resin-bound α-GlcN derivative 18a (120 mg, 0.0456 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 2: The resin was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (67 µL, 0.27 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 3: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-amino alcohol. Op. 4: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (73 mg, 0.46 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 5: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et3N (0.6 mL), SO3∙Py (73 mg, 0.46 mmol) and shaken for 1 day at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-di-N,O-sulfate. Op. 6: The resin was swollen in CH2Cl2 (3 mL) for 5 min, treated with 1 M NaOMe in MeOH (137 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 2,6-di-N,O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl α-GlcN 2,6-di-N,O-sulfate 1f (disodium salt) as an amorphous white solid (10.3 mg, 44% over 8 operations). 1H NMR (400 MHz, CD3OD): δ 7.40 (d, 2H, J = 7.2 Hz), 7.20–7.32 (m, 3H), 5.15 (d, 1H, J = 3.8 Hz), 4.90 (d, 1H, J = 10.6 Hz), 4.71 (d, 1H, J = 10.6 Hz), 4.34 (dd, 1H, J = 2.0, 10.8 Hz), 4.07 (dd, 1H, J = 6.4, 10.8 Hz), 3.85–4.00 (m, 3H), 3.71 (dd, 1H, J = 8.9, 10.2 Hz), 3.61 (ddd, 1H, J = 3.2, 7.6, 11.2 Hz), 3.40 (m, 1H), 3.01–3.17 (m, 2H); ESI-MS: m/z calcd for C15H22N2Na2O11S2 [M+H+2 Na]+: 539.0358; found: 539.0354.

2’-Aminoethyl 2-amino-4-O-benzyl-2-deoxy-2,3-di-N,O-sulfonato-α-d-glucopyranoside, disodium salt (1g)

Op. 1: Resin-bound α-GlcN derivative 18a (120 mg, 0.0456 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, treated with 1 M NaOMe in MeOH (137 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the corresponding C3 alcohol. Op. 2: The resin was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (67 µL, 0.27 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 3: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,3-amino alcohol. Op. 4: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et3N (0.6 mL), SO3∙Py (362 mg, 2.28 mmol) and shaken for 2 day at 55 °C. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,3-di-N,O-sulfate. Op. 5: The resin was swollen in DMF (2 mL) for 5 min, treated with 1 M TBAF in THF (2 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 2,3-di-N,O-sulfate. Op. 6: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.7: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl α-GlcN 2,3-di-N,O-sulfate 1g (disodium salt) as an amorphous white solid (15.9 mg, 68% over 7 operations). 1H NMR (400 MHz, CD3OD): δ 7.43 (d, 2H, J = 6.8 Hz), 7.23–7.35 (m, 3H), 5.26 (d, 1H, J = 3.4 Hz), 5.15 (d, 1H, J = 10.4 Hz), 4.64 (dd, 1H, J = 8.6, 10.7 Hz), 4.53 (d, 1H, J = 10.5 Hz), 3.89 (m, 1H), 3.73 (d, 1H, J = 9.7 Hz), 3.52–3.68 (m, 4H), 3.46 (dd, 1H, J = 3.4, 10.7 Hz), 2.98–3.10 (m, 2H); 13C NMR (100 MHz, CD3OD): δ 138.27, 128.46, 127.63, 127.14, 97.45, 78.17, 76.52, 74.56, 71.73, 65.22, 60.74, 57.50, 39.21; IR (NaCl): 3389, 2919, 1207, 1039 cm−1; [α]20D = +65.9 (c 0.71, MeOH); ESI-MS: m/z calcd for C15H23N2O11S2 [M+H]−: 471.0743; found: 471.0741.

2’-Aminoethyl 2-amino-4-O-benzyl-2-deoxy-2,3,6-tri-N,O-sulfonato-α-d-glucopyranoside, trisodium salt (1h)

Op. 1: Resin-bound α-GlcN derivative 18a (120 mg, 0.0456 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 2: The resin was swollen in CH2Cl2 (3 mL) for 5 min, treated with 1 M NaOMe in MeOH (137 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the 3,6-diol. Op. 3: The resin was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (67 µL, 0.27 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 4: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound fully deprotected α-GlcN. Op. 5: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et3N (0.6 mL), SO3∙Py (362 mg, 2.28 mmol) and shaken for 2 day at 55 °C. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2-N-,3-O-,6-O-trisulfate. Op. 6: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.7: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl α-GlcN 2,3,6-trisulfate 1h (trisodium salt) as an amorphous white solid (18.1 mg, 64% over 7 operations). 1H NMR (400 MHz, CD3OD): δ 7.52 (dd, 2H, J = 1.4, 8.3 Hz), 7.17–7.36 (m, 3H), 5.24 (d, 1H, J = 3.1 Hz), 5.13 (d, 1H, J = 9.9 Hz), 4.64 (t, 1H, J = 8.9 Hz), 4.63 (d, 1H, J = 10.0 Hz), 4.26 (dd, 1H, J = 2.0, 10.8 Hz), 4.16 (dd, 1H, J = 5.1, 10.4 Hz), 3.86–4.09 (m, 2H), 3.61–3.75 (m, 2H), 3.48 (dd, 1H, J = 3.2, 10.5 Hz), 3.14 (ddd, 1H, J = 3.1, 6.4, 13.4 Hz), 3.02 (ddd, 1H, J = 3.5, 7.3, 13.2 Hz); 13C NMR (100 MHz, CD3OD): δ 139.60, 130.42, 129.05, 128.59, 98.93, 79.56, 77.84, 76.00, 71.10, 67.85, 66.49, 58.97, 40.65; IR (NaCl): 2925, 1225, 1047 cm−1; [α]20D = +50.3 (c 0.25, MeOH); ESI-MS: m/z calcd for C15H21N2O14S3Na2 [M+2 Na]−: 594.9950; found: 594.9959.

2’-Aminoethyl 2-acetamido-4-O-benzyl-2-deoxy-6-O-sulfonato-β-d-glucopyranoside, sodium salt (2b)

Op. 1: Resin-bound β-GlcN derivative 18b (180 mg, 0.0481 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (72 µL, 0.29 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac2O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF adjusted to pH 7 with AcOH (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with 5:1 THF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (115 mg, 0.72 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 6: The resin was swollen in CH2Cl2 (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (144 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 6-O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl β-GlcN 6-O-sulfate 2b (sodium salt) as an amorphous white solid (15.8 mg, 72% over 8 operations). 1H NMR (500 MHz, CD3OD): δ 7.36–7.46 (m, 2H), 7.27–7.36 (m, 2H), 7.24 (m, 1H), 4.91 (d, 1H, J = 10.7 Hz), 4.69 (d, 1H, J = 10.7 Hz), 4.40 (d, 1H, J = 8.4 Hz), 4.30 (dd, 1H, J = 2.0, 11.1 Hz), 4.15 (dd, 1H, J = 5.5, 11.0 Hz), 3.86–3.96 (m, 2H), 3.75 (dd, 1H, J = 8.4, 10.3 Hz), 3.62 (dd, 1H, J = 8.7, 10.3 Hz), 3.56 (m, 1H), 3.41 (t, 1H, J = 8.7 Hz), 3.08 (m, 1H), 2.97 (m, 1H), 1.98 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 172.60, 138.17, 127.88, 127.77, 127.22, 101.15, 77.89, 74.61, 74.42, 73.36, 66.38, 66.27, 55.74, 39.70, 21.46; [α]20D = −6.93 (c 1.05, MeOH); ESI-MS: m/z calcd for C17H25N2O9S [M]−: 433.1281; found: 433.1279.

2’-Aminoethyl 2-amino-4-O-benzyl-2-deoxy-2,6-di-N,O-sulfonato-β-d-glucopyranoside, disodium salt (2f)

Op. 1: Resin-bound β-GlcN derivative 18b (180 mg, 0.0481 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 2: The resin was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (72 µL, 0.29 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 3: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-amino alcohol. Op. 4: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (115 mg, 0.72 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 5: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et3N (0.6 mL), SO3∙Py (115 mg, 0.72 mmol) and shaken for 1 day at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-di-N,O-sulfate. Op. 6: The resin was swollen in CH2Cl2 (3 mL) for 5 min, treated with 1 M NaOMe in MeOH (144 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 2,6-di-N,O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl β-GlcN 2,6-di-N,O-sulfate 2f (disodium salt) as an amorphous white solid (10.4 mg, 42% over 8 operations). 1H NMR (400 MHz, CD3OD): δ 7.51 (dd, 2H, J = 1.4, 8.3 Hz), 7.20–7.33 (m, 3H), 5.12 (d, 1H, J = 10.2 Hz), 4.61 (d, 1H, J = 9.8 Hz), 4.41 (d, 1H, J = 7.9 Hz), 4.25 (d, 1H, J = 10.4 Hz), 4.18 (m, 1H), 4.10 (td, 1H, J = 4.1, 11.2 Hz), 3.70 (m, 1H), 3.61 (d, 2H, J = 6.5 Hz), 3.39–3.48 (m, 2H), 3.12 (m, 2H); 13C NMR (125 MHz, CD3OD): δ 138.03, 128.95, 127.54, 127.10, 103.05, 79.54, 75.96, 74.35, 73.18, 66.21, 65.30, 59.31, 39.12; [α]20D = +8.57 (c 0.93, MeOH); ESI-MS: m/z calcd for C15H22N2NaO11S2 [M+Na]−: 493.0563; found: 493.0565.

2’-Aminoethyl 4-O-[2-acetamido-4-O-benzyl-2-deoxy-6-O-sulfonato-β-d-glucopyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside, sodium salt (3b)

Op. 1: Resin-bound GlcN(β1→4)Glc derivative 19 (110 mg, 0.0187 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (28 µL, 0.11 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac2O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF adjusted to pH 7 with AcOH (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with 5:1 THF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (45 mg, 0.28 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 6: The resin was swollen in CH2Cl2 (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (56 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 6-O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl β-linked GlcN(β1→4)Glc 6-O-sulfate 3b (sodium salt) as an amorphous white solid (11.5 mg, 67% over 8 operations). 1H NMR (500 MHz, CD3OD): δ 7.15–7.48 (m, 20H), 5.00 (d, 1H, J = 11.4 Hz), 4.84 (d, 1H, J = 10.6 Hz), 4.78 (d, 1H, J = 11.5 Hz), 4.66–4.75 (m, 3H), 4.54–4.65 (m, 3H), 4.50 (d, 1H, J = 7.5 Hz), 4.22 (dd, 1H, J = 2.0, 11.6 Hz), 4.04 (dd, 1H, J = 5.3, 10.8 Hz), 3.80–3.99 (m, 4H), 3.67–3.76 (m, 3H), 3.57–3.65 (m, 2H), 3.34–3.48 (m, 2H), 2.95–3.12 (m, 2H), 1.89 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 172.13, 138.85, 138.43, 138.28, 137.99, 128.09, 128.00, 127.81, 127.74, 127.71, 127.40, 127.34, 127.14, 127.04, 126.79, 102.85, 99.33, 81.41, 81.22, 77.96, 75.34, 74.42, 74.28, 74.00, 73.84, 73.48, 72.80, 68.84, 66.42, 66.23, 57.21, 39.87, 21.64; [α]20D = +19.5 (c 0.49, MeOH); ESI-MS: m/z calcd for C44H54N2NaO14S [M+H+Na]+: 889.3194; found: 889.3197.

2’-Aminoethyl 4-O-[2-amino-4-O-benzyl-2-deoxy-2,6-di-N,O-sulfonato-β-d-glucopyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside, disodium salt (3f)

Op. 1: Resin-bound GlcN(β1→4)Glc derivative 19 (110 mg, 0.0187 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 2: The resin was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (28 µL, 0.11 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 3: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-amino alcohol. Op. 4: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (41 mg, 0.26 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 5: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et3N (0.6 mL), SO3∙Py (41 mg, 0.26 mmol) and shaken for 1 day at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-di-N,O-sulfate. Op. 6: The resin was swollen in CH2Cl2 (3 mL) for 5 min, treated with 1 M NaOMe in MeOH (56 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 2,6-di-N,O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl β-linked GlcN(β1→4)Glc 2,6-di-N,O-sulfate 3f (disodium salt) as an amorphous white solid (6.4 mg, 41% over 8 operations). 1H NMR (500 MHz, CD3OD): δ 7.49 (d, 2H, J = 7.1 Hz), 7.32–7.40 (m, 6H), 7.16–7.31 (m, 12H), 5.03 (d, 1H, J = 9.9 Hz), 4.94 (d, 1H, J = 11.4 Hz), 4.75 (dd, 2H, J = 5.3, 11.3 Hz), 4.51–4.64 (m, 5H), 4.46 (d, 1H, J = 8.1 Hz), 4.35 (dd, 1H, J = 8.7, 9.8 Hz), 4.22 (dd, 1H, J = 2.0, 11.0 Hz), 4.01–4.09 (m, 2H), 3.71–3.98 (m, 6H), 3.50 (t, 1H, J = 8.7 Hz), 3.43 (t, 1H, J = 8.1 Hz), 3.35 (m, 1H), 2.98–3.15 (m, 2H), 2.84 (dd, 1H, J = 8.1, 9.9 Hz); 13C NMR (125 MHz, CD3OD): δ 138.49, 138.40, 138.00, 137.70, 128.90, 128.11, 127.85, 127.73, 127.57, 127.55, 127.51, 127.37, 127.10, 127.04, 126.98, 109.23, 102.72, 99.56, 82.22, 81.41, 80.76, 75.49, 74.31, 74.11, 73.88, 73.85, 73.77, 73.40, 72.65, 68.54, 66.06, 65.68, 56.97, 39.69; [α]20D = +88.3 (c 0.12, MeOH); ESI-MS: m/z calcd for C42H50N2Na3O16S2 [M+3 Na]+: 971.2296; found: 971.2301.

2’-Aminoethyl 4-O-[2-acetamido-4-O-benzyl-2-deoxy-6-O-sulfonato-α-d-gluco-pyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside, sodium salt (4b)

Op. 1: Resin-bound GlcN(α1→4)Glc disaccharide derivative 20 (110 mg, 0.0187 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (28 µL, 0.11 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac2O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF adjusted to pH 7 with AcOH (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with 5:1 THF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (45 mg, 0.28 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 6: The resin was swollen in CH2Cl2 (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (56 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 6-O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl β-linked GlcN(α1→4)Glc disaccharide 6-O-sulfate 4b (sodium salt) as an amorphous white solid (11.4 mg, 69% over 8 operations). 1H NMR (500 MHz, CD3OD): δ 7.42–7.49 (m, 2H), 7.11–7.41 (m, 18H), 5.34 (d, 1H, J = 3.7 Hz), 4.52–4.73 (m, 6H), 4.46 (d, 1H, J = 11.8 Hz), 4.26 (dd, 1H, J = 1.5, 10.7 Hz), 4.11 (dd, 1H, J = 5.5, 10.6 Hz), 4.03 (dd, 1H, J = 3.8, 10.7 Hz), 3.86–3.97 (m, 2H), 3.70–3.84 (m, 5H), 3.66 (dd, 1H, J = 8.9, 10.8 Hz), 3.61 (m, 1H), 3.51 (t, 1H, J = 7.9 Hz), 3.40 (t, 1H, J = 10.0 Hz), 2.95–3.18 (m, 2H), 1.73 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 171.90, 138.30, 137.48, 137.41, 128.09, 128.03, 127.97, 127.86, 127.76, 127.67, 127.41, 127.34, 127.20, 102.89, 97.19, 83.69, 81.90, 78.37, 74.71, 74.03, 73.87, 73.64, 73.13, 72.84, 72.20, 70.55, 68.90, 66.61, 66.33, 53.71, 39.88, 21.03; [α]20D = +31.5 (c 0.26, MeOH); ESI-MS: m/z calcd for C44H54N2NaO14S [M+H+Na]+: 889.3194; found: 889.3190.

2’-Aminoethyl 4-O-[2-amino-4-O-benzyl-2-deoxy-2,6-di-N,O-sulfonato-α-d-gluco-pyranosyl]-2,3,6-tri-O-benzyl-β-d-glucopyranoside, disodium salt (4f)

Op. 1: Resin-bound GlcN(α1→4)Glc disaccharide derivative 20 (100 mg, 0.017 mmol sugar) was swollen in CH2Cl2 (3 mL) for 5 min, then treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 2: The resin was swollen in CH2Cl2 (3 mL) for 5 min, then treated with Bu3P (26 µL, 0.1 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 3: The resin was treated with 95:5 THF:H2O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-amino alcohol. Op. 4: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO3∙Py (41 mg, 0.26 mmol) and shaken for 10 h at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 5: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et3N (0.6 mL), SO3∙Py (41 mg, 0.26 mmol) and shaken for 1 day at rt. The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-di-N,O-sulfate. Op. 6: The resin was swollen in CH2Cl2 (3 mL) for 5 min, treated with 1 M NaOMe in MeOH (51 µL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH2Cl2 (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 2,6-di-N,O-sulfate. Op. 7: The resin was treated with 30% HFIP in CH2Cl2 under standard cleavage conditions. Op.8: The crude was passed through an ion-exchange column (Dowex Marathon MSC, Na form) and purified by reverse-phase HPLC to yield 2'-aminoethyl β-linked GlcN(α1→4)Glc disaccharide 2,6-di-N,O-sulfate 4f (disodium salt) as an amorphous white solid (6.7 mg, 42% over 8 operations). 1H NMR (400 MHz, CD3OD): δ 7.10–7.48 (m, 20H), 5.54 (d, 1H, J = 3.6 Hz), 4.93–5.03 (m, 3H), 4.53–4.76 (m, 5H), 4.44 (d, 1H, J = 12.2 Hz), 4.30 (dd, 1H, J = 1.6, 10.8 Hz), 3.60–4.05 (m, 9H), 3.52 (t, 1H, J = 6.8 Hz), 3.36–3.45 (m, 3H), 2.87–3.12 (m, 2H); 13C NMR (125 MHz, CD3OD): δ 138.51, 138.33, 137.69, 137.63, 128.39, 128.03, 127.97, 127.89, 127.80, 127.70, 127.44, 127.26, 127.10, 102.36, 97.52, 82.00, 81.43, 78.15, 74.15, 73.85, 73.54, 73.32, 73.19, 73.12, 72.80, 69.89, 68.80, 66.96, 66.03, 58.61, 39.84; [α]20D = +53.3 (c 0.30, MeOH); ESI-MS: m/z calcd for C42H53N2NaO16S2 [M+2H+Na]+: 905.2837; found: 905.2828.

Acknowledgments

This work was supported by the National Institutes of Health under grant GM-06982. The authors also gratefully acknowledge NMR and MS support from the Purdue University Center for Cancer Research.

Footnotes

Supporting Information

1H and 13C NMR spectra of all compounds are available directly from the corresponding author.

References

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