Synthesis of a Lewis b hexasaccharide thioglycoside donor and its use towards an extended mucin core Tn heptasaccharide structure and a photoreactive biotinylated serine linked hexasaccharide

Abstract

Investigation into Heliobacter pylori binding to Lewis b (Le^b) antigens through the blood group antigen binding adhesion protein (BabA) requires structurally well-defined tools. A Le^b hexasaccharide thioglycoside donor was chemically prepared through a linear approach starting from D-lactose. This donor can be used to attach reducing end linkers providing a range of options for conjugation techniques or to further extend the oligosaccharide structure. To evaluate its efficiency as a donor, it was coupled to a 6-OH GalNAc acceptor, producing an extended Le^b-containing Tn mucin core structure in 84% yield, and to L-serine in 72% yield. The latter compound was subsequently functionalized with a photolabile diazirine linker and biotin, creating a Le^b hexasaccharide structure–function tool suitable for lectin tagging interaction studies. This donor opens a wide range of possibilities for conjugation of Le^b structures to produce a variety of chemical biology tools to assist in the study of these interactions.

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Introduction

Heliobacter pylori infections of the gastric epithelium can lead to gastric inflammation and may progress to gastroduodenal diseases such as peptic ulceration and gastric adenocarcinoma.^1–5H. pylori binds to Lewis b-antigen (Le^b) structures present in the gastric epithelium. This binding is mediated by its blood group antigen binding adhesion protein (BabA).^6–8 Chemically defined structures have proved to be an invaluable tool for probing the binding of H. pylori BabA to Le^b antigens.^8–11 In our first synthesis of the Le^b hexasaccharide, we constructed and utilized a Le^b tetrasaccharide thioglycoside donor that was coupled to an azide-propyl lactose acceptor to afford the hexasaccharide.¹² However, the donor was prone to elimination rather than glycosylation reaction. More effective routes to the hexasaccharide were developed using a linear synthesis of the Le^b hexasaccharide starting from the same lactose acceptor;^13–15 these syntheses are high-yielding and can be performed on a large scale but give little flexibility for changes at the reducing end of the molecule. Therefore, a more adaptable approach involving a Le^b hexasaccharide donor was investigated, allowing the construction of a variety of Le^b-containing chemical biology tools. Two earlier examples of Le^b hexasaccharide donors have been reported, a fluoride donor by Sato et al.¹⁶ and a 1,2-epoxide donor by Danishefsky et al.¹⁷ However, a very low yield¹⁶ and no stereoselectivity¹⁷ were experienced in initial glycosylations with these two donors limiting their use in further applications. Considering our earlier good experiences with large block thioglycoside donors,^18–22 a Le^b hexasaccharide thioglycoside 1 (Fig. 1) was designed as the target donor.

Fig. 1 Target Le^b hexasaccharide thioglycoside donor.

Results and discussion

Apart from the orthogonal glycosylations required, the major difference from our earlier syntheses was the design and synthesis of the lactose acceptor, in particular, its protecting group pattern. Benzyl protecting groups were used on the galactose residue to ensure good reactivity of the 3′-OH acceptor, and acyl protecting groups on the glucose residue, to ensure β-selectivity in the glycosylations with the hexasaccharide donor 1. This protecting group pattern could be introduced starting from known compound 2 (Scheme 1).^23,24 The Catelani group published procedure to 2 can effectively be performed on a large scale, the triisopropylidenelactose intermediate has been synthesised on a 250 kg scale²⁵ and we synthesized compound 2 on a 30 g scale. Regioselective introduction of a naphtylmethyl ether (Nap) in the 3′-position using tin activation (→3) followed by benzylation afforded compound 4, with the desired protecting group pattern in the galactose moiety. Acidic acetal hydrolysis followed by acetylation gave compound 5 with an acylated glucose. Due to the presence of the benzyl protecting groups, the Lewis acid-promoted formation of the ethyl thiodisaccharide 6, had to be monitored very carefully by TLC (R_f 0.44 in toluene : EtOAc 4 : 1, v/v) to avoid decomposition. Attempts to use TMSOTf for the introduction of the thioethyl group were not successful, but with BF₃-etherate as promoter compound 6 was obtained in an 81% yield based on recovered starting material predominantly consisting of the less reactive α-acetate. The Nap group of disaccharide 6 was then cleaved using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to give lactose acceptor 7 in a 79% yield.

Scheme 1 Reagents and conditions: (a) dibutyltin oxide, tetra-butyl ammonium bromide, 2-(bromomethyl)naphthalene, toluene, reflux, 81%; (b) NaH, BnBr, 0 °C → rt, 89%; (c) 80% AcOH, 70 °C; (d) sodium acetate, Ac₂O, reflux, 83% (over 2 steps, α/β mixture (3 : 1)); (e) EtSH, BF₃·OEt₂, ClCH₂CH₂Cl, 0 °C, 81% (based on recovered starting material); (f) DDQ, CH₂Cl₂–MeOH 4 : 1, rt, 79%.

An orthogonal glycosylation strategy was needed to build the hexasaccharide without activating the reducing end thioglycoside donor. It was decided to use trichloroacetimidate (TCA) donors for the introduction of the glucosamine and galactose residues and glycosyl bromides with halide-assisted glycosylation conditions for the introduction of the two fucose moieties, the latter being reproducibly high yielding in earlier synthesis.^13–15 When performing orthogonal glycosylations with thioglycoside acceptors, especially with non-bulky thio aglycons, there is always the risk of intramolecular aglycon transfer, the extent is hard to predict and very much dependent on the acceptor structure and of the thioaglycone used.²⁶ In this synthesis we found that acceptor 7, although being an ethyl thiosaccharide, showed no tendency at all for aglycon transfer reactions.

For the introduction of the glucosamine moiety a suitable donor with a β-directing participating group and orthogonal protecting groups at the 3- and 4-position was needed to be able to selectively glycosylate these positions at a later stage, N-phthalimido derivative 8^27,28 (Scheme 2) was selected. Compound 8 possesses an orthogonal para-methoxybenzyl group (PMB) at the 3-position, and a benzylidene acetal in the 4,6-positions, which can be selectively opened to free the 4-position at a later stage. The thioethyl donor 8 was converted into its trichloroacetimidate 9 by hydrolysis of the thioethyl group using N-iodosuccinimide (NIS), followed by formation of the imidate with trichloroacetonitrile and 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (92% over 2 steps after silica gel column chromatography).

Scheme 2 Reagents and conditions: (a) NIS, CH₃CN–H₂O 10 : 1, rt; (b) Cl₃CCN, DBU, ClCH₂CH₂Cl, 0 °C, 92% (over 2 steps); (c) TMSOTf, CH₂Cl₂, −20 °C, 78%; (d) sodium methoxide, MeOH, rt; (e) EtOH, hydrazine hydrate, reflux; (f) Ac₂O, MeOH, rt.; (g) BzCl, pyridine, 0 °C, 83% (over 4 steps); (h) DDQ, CH₂Cl₂/iso-butanol (4 : 1), 78%.

Donor 9 was employed in a TMSOTf-catalyzed glycosylation reaction with acceptor 7 affording the desired trisaccharide 10 in a 78% yield. Due to earlier findings, in which the removal of the phthalimido group in a fully assembled hexasaccharide was problematic and low yielding,¹³ we decided to remove this protecting group and introduce the acetamide moiety prior to further glycosylation steps, despite being aware of problematic reports on the use of acetamido-containing acceptors.^29–31 The acetyl groups in trisaccharide 10 were removed using Zemplén conditions and the phthalimido group cleaved using hydrazine hydrate in ethanol. The amino group was then chemoselectively acetylated with acetic anhydride in methanol followed by benzoylation of the hydroxyl groups to give compound 11 in a yield of 83% over 4 steps. The benzoyl groups were preferred over acetyl protecting groups to enhance lipophilicity and therefore solubility of subsequent intermediates but also to improve glycosylation properties of the target hexasaccharide donor.³² In contrast to the removal of the Nap group in compound 6, the removal of the PMB group to obtain derivative 12 was challenging due to persistent side reactions resulting in poor yields when carrying out the reaction with DDQ in CH₂Cl₂–MeOH 4 : 1. By varying the solvent mixture to CH₂Cl₂–isobutanol, yields were significantly improved and trisaccharide acceptor 12 obtained in 78% yield.³³

For the introduction of the galactose moiety, a donor with an orthogonal β-selective participating group at the 2-position was needed. Since benzoyl groups were present in trisaccharide 12, and it was known that removal of a 2′′′-O-acetyl group in a lacto-N-tetraose (LNT) tetrasaccharide requires unusually strong basic conditions,¹⁵ we chose a chloroacetyl group to protect this position (Scheme 3). The acetyl group of the known galactose derivative 13³⁴ was removed using Zemplén conditions and replaced with a chloroacetyl group (→14, 88%) followed by conversion into the corresponding trichloroacetimidate donor 15. This time, NBS was used to hydrolyse the thioethyl group whereafter treatment with DBU and trichloroacetonitrile afforded compound 15 as a α/β mixture (10 : 1). It was possible to separate the anomers using silica gel chromatography. However, the high reactivity of the compound³⁵ resulting in a rather low yield of 47% after chromatography.

Scheme 3 Reagents and conditions: (a) sodium methoxide, MeOH, rt, 99%; (b) chloroacetyl chloride, CH₂Cl₂–pyridine 14 : 1, 0 °C → rt, 89%; (c) NBS, CH₃CN–H₂O 10 : 1, rt; (d) trichloroacetonitrile, DBU, ClCH₂CH₂Cl, 0 °C, 47% (over 2 steps, α/β mixture (10 : 1)). (e) TMSOTf, CH₂Cl₂, −30 °C → −5 °C; (f) lutidine, thiourea, MeOH, 70 °C, 47% (over 2 steps); (g) NaCNBH₃, HCl (1 M in Et₂O), 71%; (h) Br₂, CH₂Cl₂, rt; (i) tetraethylammonium bromide, CH₂Cl₂–DMF 3.5 : 1, rt, 89%.

In the next step, donor 15 was employed in a TMSOTf-catalyzed glycosylation reaction with trisaccharide acceptor 12 (Scheme 3). The reaction had to be monitored carefully by TLC (toluene : EtOAc 1 : 1) as an intermediate (possibly the orthoester) was initially formed which was slowly converted into the desired tetrasaccharide. Due to difficulties in the isolation of the product, the crudely worked-up reaction mixture was directly treated with thiourea and lutidine in methanol to remove the chloroacetyl group affording the 2′′′-OH tetrasaccharide 16 (47% over two steps). The benzylidene group of the GlcNAc moiety of compound 16 was then selectively opened to free the 4′′-position using NaCNBH₃, affording tetrasaccharide 2′′′,4′′-diol acceptor 17. In the final step towards the hexasaccharide donor, fucose thioglycoside 18³⁶ was converted into its bromide 19 and then directly employed in a completely α-selective halide-assisted glycosylation with tetrasaccharide acceptor 17 to give the target Lewis b donor 1 in an 89% yield. In summary, hexasaccharide donor 1 (600 mg) was synthesized from compound 2 in a ten step sequence and in a 15% overall yield.

The donor properties of hexasaccharide donor 1 were then investigated. We were interested in Lewis b extended mucin core structures so GalNAc derivative 21 was chosen as a trial acceptor. Acceptor 21 was prepared from compound 20³⁷via introduction of a benzylidene group at the 4,6-positions, followed by subsequent benzoylation and final removal of the acetal (Scheme 4). Glycosylation of the 4,6-diol acceptor 21 with hexasaccharide donor 1, using NIS and AgOTf as promoters, afforded exclusively the β-(1 → 6)-linked heptasaccharide 22 in an 84% yield. Heptasaccharide 22 was then deprotected, the benzoyl groups were removed under Zemplén conditions, followed by hydrogenolysis of the benzyl and azido groups to afford globally deprotected heptasaccharide 23 in a 92% yield over the 2 steps.

Scheme 4 Reagents and conditions: (a) benzaldehyde dimethylacetal, p-TsOH·H₂O, DMF, rt; (b) benzoyl chloride, pyridine, rt; (c) AcOH (80% in H₂O), 85 °C, 34% (over 3 steps); (d) AgOTf, NIS, CH₂Cl₂, −40 °C → 0 °C, 84%; (e) sodium methoxide, MeOH, rt; (f) H₂ (5 bar), Pd/C, HCl (1 N in H₂O), THF–EtOH–H₂O, 92% (over 2 steps).

We were further interested in photo-active tools to enable cross-linking of Lewis b-structures to neighbouring interacting structures. Hexasaccharide donor 1 was therefore glycosylated with Boc-Ser-OBn using the NIS/AgOTf promotor system producing the serine linked hexasaccharide 24 in 72% yield (Scheme 5). Hydrogenolysis with Pd/C and H₂ followed by saponification using Zemplén conditions produced the globally deprotected compound (88% over 2 steps) which was carried through as crude material to the following two-step bifunctionalising process. A photolabile diazirine linker was introduced to the free amine of the serine residue using an activated N-hydroxysuccinimide ester of the corresponding diazirine linker, subsequently the carboxylic acid group of the serine was coupled to biotin hydrazate, using HOBt and EDCI as coupling reagents, producing the photoreactive biotinylated hexasaccharide 25.

Scheme 5 Reagents and conditions (a) NIS, AgOTf, −40 °C, 72%; (b) Pd/C, H₂; (c) NaOMe, MeOH, 88% (over 2 steps); (d) N-succinimidyl-diazirine (SDA), PBS buffer; (e) HOBt, EDCl, biotin hydrazate.

Conclusions

We have successfully synthesized a novel Le^b block donor via a linear approach using a lactose acceptor that can be produced on a large scale and two new selectively protected trichloroacetimidate donors. The synthesized Le^b hexasaccharide block donor gave high yields and complete stereoselectivity in a glycosylation reaction to obtain a Le^b-extended Tn mucin core structure. Coupling of the donor to serine and additional extension to a bifunctionalised diazirine-biotin linked hexasaccharide further showed the versatility of the donor and its potential use in carbohydrate–protein structure–function interaction elucidation. This donor opens a wide range of possibilities for conjugation of Le^b structures to produce alternative targets.

Experimental

General methods

Please see ESI† for NMR spectra and experimental for compounds 3–7, 9, 14–15 and 21.

General methods

The ¹H/¹³C NMR spectra (δ in ppm, relative to TMS in CDCl₃ and relative to solvent peak in D₂O) were recorded with Varian spectrometers (400/100 MHz, 500/125 MHz or 600/150 MHz) at 25 °C. Assignments were aided by ¹H–¹H and ¹H–¹³C correlation experiments. HRMS spectra were recorded on a micromass LCT instrument from Waters. Optical rotations were measured on a PerkinElmer polarimeter with a Na lamp (589 nm) at 20 °C and are not corrected. TLC was carried out on precoated 60 F254 silica gel alumina plates (Merck) using UV-light, H₂SO₄ (10% in ethanol) and/or ninhydrin-solution (ninhydrin/CH₃COOH/ethanol [0.3 : 3 : 100 w/v/v]. Flash chromatography was performed on silica gel (Apollo scientific, pore size 60 Å, particle size 40–63 μm) or via pre-packed columns (Biotage AB, particle size 50 μm) on a Biotage SP4 system. Size exclusion chromatography was performed using Biogel P2 (Biorad, <45 μm bead size, 100–1800 MW) with H₂O-n-butanol (99 : 1) as eluent.

Ethyl (4,6-O-benzylidene-3-O-p-methoxybenzyl-2-deoxy-2-phthalamido-β-D-glucopyranosyl)-(1 → 3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-acetyl-1-thio-β-D-glucopyranoside (10). Acceptor 7 (1.90 g, 2.43 mmol) and donor 9 (2.41 g, 3.64 mmol) were dissolved in CH₂Cl₂ (50 mL), MS 4 Å were added and the mixture was stirred for 15 min. The suspension was cooled to −20 °C, TMSOTf (14 μL, 0.08 mmol) added and the mixture stirred for 30 min. After completion of the reaction Et₃N (50 μL) was added and the mixture concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (toluene–EtOAc) to afford 10 (2.43 g, 78%) as a colourless solid. R_f 0.45 (toluene–EtOAc 4 : 1); [α]²⁰_D –1.7° (c 1.33, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.70–7.63 (m, 1H, Ar), 7.56–7.50 (m, 3H, Ar), 7.43–7.26 (m, 14H, Ar), 7.19–7.10 (m, 4H, Ar), 6.90–6.84 (m, 4H, Ar) and 6.36–6.31 (m, 2H, Ar), 5.62 (s, 1H, CHPh), 5.46 (d, J_1,2 = 8.4 Hz, 1H, H-1^III), 5.00–4.91 (m, 2H), 4.88–4.82 (m, 1H), 4.68 (d, J = 12.1 Hz, 1H), 4.52–4.35 (m, 7H), 4.29–4.20 (m, 3H), 4.07 (d, J_1,2 = 7.6 Hz, 1H, H-1^II), 4.04–3.98 (m, 2H), 3.88–3.75 (m, 4H), 3.70–3.59 (m, 3H), 3.57 (s, 3H, C₆H₄OCH₃), 3.54–3.44 (m, 3H), 3.37 (dd, J = 9.8, 7.6 Hz, 1H), 3.11 (ddd, J = 9.8, J = 5.4, J = 1.7 Hz, 1H), 2.63–2.50 (m, 2H, CH₂CH₃), 2.00, 1.88 and 1.78 (9H, COCH₃), 1.17 (t, J = 7.4 Hz, 3H, CH₂CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 170.5, 170.2 and 169.6 (COCH₃), 167.37 (NCO(Phth)), 158.8, 138.9, 138.5, 137.8, 137.3, 133.5, 131.2, 130.1, 129.5, 129.1, 128.5, 128.3, 128.2, 127.9, 127.9, 127.7, 127.4, 126.7, 126.3, 126.1, 123.1 and 113.3 (Ar), 102.5 (C-1^II), 101.4 (CHPh), 100.1 (C-1^III), 83.2 (C-1^I), 83.1 (2C, C-4^III, C-3^II), 79.0 (C-2^II), 77.1 (C-5^I (HSQC)), 76.1 (C-4^II), 74.8 (CH₂Ph), 74.2 (2C, CH₂Ph, C-3^III), 73.8 (2C, CH₂PhOCH₃, C-4^I), 73.5 (2C, C-3^I, CH₂Ph), 73.4 (C-5^II), 69.8 (C-2^I), 68.8 (C-6^III), 68.6 (C-6^II), 65.9 (C-5^III), 62.3 (C-6^I), 56.4 (C-2^III), 54.9 (PhOCH₃), 24.1 (CH₂CH₃), 20.8, 20.7, 20.6 (COCH₃), 14.9 (CH₂CH₃); ES-HRMS calcd for C₇₀H₇₅NO₂₀S [Na]⁺ 1304.4501 found 1304.4436.

Ethyl (4,6-O-benzylidene-3-O-p-methoxybenzyl-2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside (11). A solution of sodium methoxide (1 M in MeOH) was added dropwise to a solution of compound 10 (4.90 g, 3.82 mmol) in dry MeOH (200 mL) and the mixture stirred for 3 h. After completion of the reaction (TLC toluene–EtOAc 1 : 1) the solution was neutralised with Dowex 50 W+ ion exchange resin, filtered, concentrated under reduced pressure and directly used for the next step. The residue was dissolved in EtOH (150 mL), hydrazine hydrate (5.54 mL, 114 mmol) added and the mixture refluxed for 36 h. After completion of the reaction the solvent was evaporated and the residue co-evaporated with toluene (2 × 30 mL). The crude material was dissolved in EtOAc (150 mL), washed with saturated aq NaHCO₃ (100 mL) and the aqueous layer extracted with EtOAc (2 × 50 mL). The combined organic fractions were dried over MgSO₄, filtered, concentrated under reduced pressure, and the crude was dried in vacuo. The residue was dissolved in MeOH (150 mL) and the resulting solution cooled to 0 °C before acetic anhydride (0.467 mL, 4.95 mmol) was added. The reaction mixture was stirred for 3 h while allowing to warm to room temperature. Then the mixture was concentrated, co-evaporated with toluene (3 × 20 mL) and dried in vacuo. The crude material was dissolved in pyridine (40 mL), benzoyl chloride (1.55 mL, 13.3 mmol) added at 0 °C and the mixture stirred for 2 h. After completion of the reaction, MeOH (2 mL) was added and the mixture concentrated and co-evapoarted with toluene. The residue was dissolved in CH₂Cl₂ (200 mL), washed with saturated aq NaHCO₃ (100 mL) and the organic layer dried over MgSO₄, filtered and concentrated. Purification by flash chromatography on silica gel (toluene–EtOAc) gave 11 (4.36 g, 83%) as a colourless solid. R_f 0.62 (toluene–EtOAc 2 : 1); [α]²⁰_D +2.7° (c 1.79, CHCl₃); ¹H NMR (500 MHz, CDCl₃) 8.01–7.81 (m, 6H, Ar), 7.60–7.08 (m, 31H, Ar), 6.82–6.76 (m, 2H, Ar), 5.68–5.61 (m, 1H), 5.53 (s, 1H, CHPh), 5.46–5.40 (m, 1H), 4.91–4.84 (m, 2H), 4.77 (d, J = 11.8 Hz, 1H), 4.73–4.66 (m, 4H), 4.61 (d, J = 12.3 Hz, 1H), 4.47 (d, J = 11.5 Hz, 1H), 4.42 (dd, J = 12.1, J = 5.2 Hz, 1H), 4.35–4.26 (m, 3H), 4.15–4.06 (m, 3H), 3.80–3.49 (m, 11H), 3.44–3.37 (m, 1H), 3.31–3.26 (m, 1H), 2.91 (dd, J = 9.0, J = 5.1 Hz, 1H), 2.85–2.79 (m, 1H), 2.73–2.59 (m, 2H, CH₂CH₃), 1.46 (s, 3H, NCOCH₃), 1.19 (t, J = 7.4 Hz, 3H, CH₂CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 169.9 (NCOCH₃), 165.9, 165.7 and 165.4 (COPh), 159.2, 139.2, 138.8, 138.0, 137.4, 133.2, 133.1, 132.6, 130.5, 130.3, 129.9, 129.8, 129.6, 129.3, 129.0, 128.5, 128.4, 128.3, 128.0, 127.8, 127.6, 127.5, 127.1, 126.6, 126.1 and 113.7 (Ar), 103.1 (C-1^II), 101.9 (C-1^III), 101.2 (CHPh), 83.6 (C-1^I), 82.4 (C-4^III), 80.7 (C-3^II), 79.9 (C-2^II), 77.6 (C-5^I), 77.0 (C-3^III (HSQC)) 75.6 (C-4^II), 75.3 (C-4^I), 74.9 (CH₂Ph), 74.4 (CH₂Ph), 74.2 (C-3^I), 73.6 (CH₂PhOCH₃), 73.1 (2C, CH₂Ph, C-5^II), 70.6 (C-2^I), 68.8 (C-6^II), 67.7 (C-6^III), 65.9 (C-5^III), 63.0 (C-6^I), 56.7 (C-2^III), 55.3 (PhOCH₃), 24.3 (CH₂CH₃), 23.1 (NCOCH₃), 14.9 (CH₂CH₃); ES-HRMS calcd for C₇₉H₈₁NO₁₉S [Na]⁺ 1402.5021 found 1402.4957.

Ethyl (4,6-O-benzylidene-2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside (12). DDQ (0.438 g, 1.93 mmol) was added to a solution of 11 (0.761 g, 0.511 mmol) in dry CH₂Cl₂ (12 mL) and isobutanol (3 mL) at 0 °C and the mixture stirred for 2 h while allowing to warm to room temperature. The solvent was diluted with CH₂Cl₂ (50 mL) and washed with saturated aq NaHCO₃ (2 × 100 mL) and the organic layer dried over MgSO₄, filtered and concentrated. Flash chromatography (toluene–EtOAc) afforded 12 (0.539 g, 78%) as a white solid. R_f 0.38 (toluene–EtOAc 1 : 1); [α]²⁰_D –10.5° (c 1.00, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.01–7.92 (m, 4H), 7.90–7.85 (m, 2H, Ar), 7.60–7.54 (m, 1H, Ar), 7.53–7.26 (m, 21H, Ar), 7.23–7.10 (m, 7H, Ar), 5.71–5.63 (m, 1H), 5.53 (s, 1H), 5.49–5.43 (m, 1H), 5.30 (d, J = 5.2 Hz, 1H), 5.08 (d, J = 12.7 Hz, 1H), 4.81–4.74 (m, 1H), 4.68 (m, 5H), 4.45 (dd, J = 12.1, J = 5.1 Hz, 1H), 4.38–4.28 (m, 3H), 4.19–4.09 (m, 3H), 3.84–3.77 (m, 1H), 3.77–3.68 (m, 4H), 3.62–3.49 (m, 3H), 3.40 (m, 1H), 3.36–3.30 (m, 1H), 2.97 (dd, J = 9.0, J = 5.3 Hz, 1H), 2.86–2.80 (m, 1H), 2.75–2.61 (m, 2H, CH₂CH₃), 1.48 (s, 3H, COCH₃), 1.20 (t, J = 7.4 Hz, 3H, CH₂CH₃) (OH was not observed); ¹³C NMR (125 MHz, CDCl₃) 172.4 (NCOCH₃), 165.9, 165.8 and 165.3 (COPh), 138.9, 138.3, 137.8, 137.0, 133.3, 132.6, 130.2, 129.9, 129.7, 129.6, 129.3, 129.1, 128.8, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.3, 126.4 and 126.1 (Ar), 103.0 (C-1^II), 102.3 (C-1^III), 101.9 (CHPh), 83.6 (C-1^I), 81.5 (C-4^III), 81.1 (C-3^II), 79.6 (C-2^II), 77.5 (C-5^I), 75.7 (C-4^II), 75.0 (C-4^I), 74.6 (CH₂Ph), 74.5 (CH₂Ph), 74.1 (C-3^I), 73.2 (2C, CH₂Ph, C-5^II), 72.7 (C-3^III), 70.5 (C-2^I), 68.5 (C-6^III), 67.5 (C-6^II), 66.3 (C-5^III), 63.1 (C-6^I), 59.1 (C-2^III), 24.3 (CH₂CH₃), 22.9 (NHCOCH₃), 14.9 (CH₂CH₃); ES-HRMS calcd for C₇₁H₇₃NO₁₈S [Na]⁺ 1282.4446 found 1282.4392.

Ethyl (3,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 3)-(4,6-O-benzylidene-2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside (16). Acceptor 12 (1.095 g, 0.869 mmol) and imidate 15 (0.875 g, 1.30 mmol) were dissolved in CH₂Cl₂ (43.5 mL), molecular sieves 4 Å were added and the mixture was stirred for 15 min. The mixture was then cooled to −30 °C and TMSOTf (55 μL, 0.30 mmol) added, and the reaction mixture stirred for 3 h while allowing to warm to −5 °C. The reaction was quenched by addition of Et₃N (0.05 mL), and the mixture filtered through a pad of Celite and concentrated. Purification by flash chromatography (toluene–EtOAc) afforded a mixture of the tetrasaccharide together with inseparable impurities (1.188 g) and recovered acceptor 12 (0.090 g). 2,6-Lutidine (1.60 mL, 13.4 mmol) and thiourea (1.02 g, 13.4 mmol) were subsequently added to a solution of the obtained crude material (1.188 g) in MeOH (100 mL) and the mixture was stirred at 70 °C overnight. When all starting material was consumed the mixture was concentrated, dissolved in CH₂Cl₂ (50 mL) and subsequently washed with 1 N aq HCl (30 mL) and saturated aq NaHCO₃ (30 mL). The organic layer was dried over MgSO₄, filtered and concentrated. Flash chromatography (toluene–EtOAc) afforded 16 (0.640 g, 47%) over 2 steps (based on recovered starting material in the first step) as a colourless solid. R_f 0.36 (toluene–EtOAc 2 : 1); [α]²⁰_D –9.6° (c 1.88, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.01–7.92 (m, 4H, Ar), 7.89–7.83 (m, 2H, Ar), 7.58–7.53 (m, 1H, Ar), 7.52–7.08 (m, 43H, Ar), 5.67–5.61 (m, 1H), 5.50 (s, 1H), 5.48–5.40 (m, 2H), 4.90 (d, J = 11.5 Hz, 1H), 5.67–5.61 (m, 1H), 5.50 (s, 1H), 5.48–5.40 (m, 2H), 4.90 (d, J = 11.5 Hz, 1H), 4.86–4.77 (m, 3H), 4.70 (d, J_1,2 = 9.9 Hz, 1H, H-1^I), 4.66 (m, 2H), 4.59 (m, 3H), 4.40 (dd, J = 12.0, J = 5.3 Hz, 1H), 4.36 (d, J = 12.0 Hz, 1H), 4.33–4.24 (m, 4H), 4.22 (d, J_1,2 = 7.9 Hz, 1H, H-1^IV), 4.14–4.04 (m, 3H), 4.01–3.95 (m, 1H), 3.91–3.87 (m, 1H), 3.86–3.84 (m, 1H), 3.74 (m, 1H), 3.71–3.63 (m, 4H), 3.60–3.54 (m, 2H), 3.52 (dd, J = 10.7, J = 5.5 Hz, 1H), 3.49–3.44 (m, 1H), 3.39 (dd, J = 9.7, J = 4.9 Hz, 1H), 3.35 (dd, J = 8.4, J = 4.9 Hz, 1H), 3.31–3.25 (m, 2H), 2.96–2.75 (m, 3H), 2.73–2.59 (m, 2H, SCH₂CH₃), 1.53 (s, J = 11.6 Hz, 3H, NCOCH₃), 1.18 (t, J = 7.4 Hz, 3H, CH₂CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 170.0 (NCOCH₃), 164.8, 164.7 and 164.3 (COPh), 138.2, 137.8, 137.7, 137.5, 137.0, 136.7, 135.8, 132.2, 132.1, 131.5, 129.3, 128.8, 128.6, 128.3, 128.1, 127.4, 127.3, 127.2, 127.0, 126.9, 126.8, 126.7, 126.6, 126.5, 126.4, 126.1, 125.8 and 125.2 (Ar), 102.2 (C-1^IV), 102.1 (C-1^II), 101.6 (C-1^III), 100.6 (CHPh), 82.5 (C-1^I), 80.5 (C-3^IV), 80.0 (C-3^II), 79.2 (C-4^III), 78.6 (C-2^II), 76.6 (C-5^I), 74.8 (C-3^III), 74.6 (C-4^II), 74.3 (C-4^I), 73.9 (CH₂Ph), 73.7 (CH₂Ph), 73.4 (CH₂Ph), 73.2 (C-3^I), 72.7 (C-5^IV), 72.4 (2C, C-4^IV, CH₂Ph), 72.1 (2C, CH₂Ph, C-5^II), 71.6 (CH₂Ph), 69.5 (2C, C-2^I, C-2^IV), 67.7 (C-6^III), 67.3 (C-6^IV), 66.7 (C-6^II), 65.2 (C-5^III), 62.0 (C-6^I), 55.2 (C-2^III), 23.2 (CH₂CH₃), 22.2 (NCOCH₃), 13.9 (CH₂CH₃); ES-HRMS calcd for C₉₈H₁₀₁NO₂₃S [Na]⁺ 1714.6383 found 1714.6360.

Ethyl (3,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 3)-(6-O-benzyl-2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside (17). A solution of HCl (1 M in Et₂O) was added drop-wise to a mixture 16 (0.170 g, 0.100 mmol) and NaCNBH₃ (158 mg, 2.51 mmol) in dry THF (6 mL) until a pH 1–2 was obtained. The reaction mixture was stirred for 2 h and frequently monitored by TLC. The mixture was neutralized with Et₃N and filtered through a Celite pad. Concentration and co-evaporation with MeOH (3 × 10 mL), followed by flash chromatography (toluene–EtOAc) afforded 17 (0.121 g, 71%) as a colourless solid. R_f 0.60 (toluene–EtOAc 1 : 1); [α]²⁰_D +4.1° (c 0.38, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 7.99–7.92 (m, 4H, Ar), 7.89–7.84 (m, 2H, Ar), 7.56–7.52 (m, 1H, Ar), 7.52–7.48 (m, 1H, Ar), 7.41–7.10 (m, 42H, Ar), 5.68–5.63 (m, 1H), 5.46–5.40 (m, 1H), 5.06 (d, J = 8.2 Hz, 1H, NHCOCH₃), 4.95 (d, J = 12.4 Hz, 1H), 4.87 (d, J = 11.6 Hz, 1H), 4.81–4.75 (m, 2H), 4.73–4.63 (m, 5H), 4.53 (d, J = 11.7 Hz, 1H), 4.49–4.46 (m, 2H), 4.44–4.39 (m, 2H), 4.37–4.30 (m, 4H), 4.13–4.05 (m, 3H), 4.01 (d, J_1,2 = 7.7 Hz, 1H, H-1^IV), 3.92–3.88 (m, 2H), 3.83–3.75 (m, 4H), 3.68–3.63 (m, 1H), 3.62–3.53 (m, 4H), 3.48–3.42 (m, 3H), 3.40 (dd, J = 8.8, J = 5.2 Hz, 1H), 3.34 (dd, J = 9.8, J = 2.9 Hz, 1H), 3.32–3.28 (m, 1H), 2.93–2.92 (m, 1H), 2.90 (dd, J = 9.0, J = 5.4 Hz, 1H), 2.80 (dd, J = 8.9, J = 7.8 Hz, 1H), 2.72–2.60 (m, 2H, SCH₂CH₃), 1.48 (s, 3H, NCOCH₃), 1.20–1.16 (m, 3H, SCH₂CH₃); ¹³C NMR (150 MHz, CDCl₃) δ 171.7 (NHCOCH₃), 165.9, 165.8 and 165.3 (COPh), 139.1, 138.9, 138.4, 138.3, 138.2, 138.0, 137.5, 130.2, 129.9, 129.8, 129.6, 129.3, 128.6, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.1 and 126.2 (Ar), 104.8 (C-1^IV), 103.0 (C-1^II), 101.6 (C-1^III), 87.1 (C-3^III), 83.5 (C-1^I), 81.4 (2C, C-3^II, C-3^IV), 79.7 (C-2^II), 77.6 (C-5^I), 75.7 (C-4^II), 75.3 (C-4^III), 75.2 (C-4^I), 74.7 (CH₂Ph), 74.6 (CH₂Ph), 74.5 (CH₂Ph), 74.1 (2C, C-3^I, C-5^IV), 73.7 (CH₂Ph), 73.4 (CH₂Ph), 73.3 (2C, C-4^IV, C-5^II), 73.1 (CH₂Ph), 72.9 (CH₂Ph), 71.2 (C-2^IV), 70.5 (C-2^I), 69.8 (C-6^III), 69.3 (C-5^III), 68.8 (C-6^IV), 67.8 (C-6^II), 63.0 (C-6^I), 55.6 (C-2^III), 24.2 (SCH₂CH₃), 23.1 (NHCOCH₃), 14.9 (SCH₂CH₃); ES-HRMS calcd for C₉₈H₁₀₃NO₂₃S [Na]⁺ 1716.6539 found 1716.6548.

Ethyl (2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1 → 2)-(3,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 3)-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)]-(1 → 4)-(6-O-benzyl-2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranoside (1). To an ice-cold solution of 18³⁶ (0.635 g, 1.33 mmol) in CH₂Cl₂ (6 mL) was added bromine (75.0 μL, 1.46 mmol) and the solution stirred for 10 min. Cyclohexene (50 μL) was then added and the mixture concentrated and dried in-vacuo. The residue was dissolved in CH₂Cl₂ (4 mL) and added to a mixture of 17 (450 mg, 0.265 mmol) and tetraethylammonium bromide (0.084 g, 0.398 mmol) in CH₂Cl₂ (6 mL) and DMF (2.8 mL) which had been stirred with molecular sieves 4 Å at room temperature for 40 min. The resulting reaction mixture was stirred under a N₂ – atmosphere for 3 d and excluded from light. Afterwards the mixture was filtered through a pad of Celite, the solution concentrated, and the mixture purified by two successive flash chromatography columns (column 1: pentane–EtOAc 5 : 1 to 2 : 1, column 2: toluene–acetonitrile 7 : 1 to 4 : 1) to obtain 1 (0.600 g, 89%) as a colourless solid. R_f 0.72 (toluene–EtOAc 2 : 1), [α]²⁰_D –36.9° (c 0.73, CHCl₃); ¹³C NMR (125 MHz, CD₂Cl₂) δ 169.63 (NHCOCH₃), 166.3, 166.1 and 165.7 (COPh), 140.3, 140.0, 139.8, 139.7, 139.5, 139.3, 139.0, 138.9, 138.8, 138.6, 133.9, 133.7, 133.3, 130.8, 130.5, 130.3, 130.2, 130.1, 129.9, 129.2, 129.1, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.5, 127.4 and 127.0 (Ar), 104.0 (C-1^II), 102.7 (C-1^III), 102.0 (C-1^IV), 99.2 (C-1^VI), 98.2 (C-1^V), 84.3 (C-3^IV), 84.1 (C-1^I), 81.1 (C-2^II), 80.8 (C-3^V), 80.5 (C-3^II), 79.5 (C-3^VI), 79.1 (C-4^VI), 78.8 (C-4^V), 78.1 (C-5^I), 76.6 (2C, C-2^V, C-4^II), 76.5 (2C, C-2^VI, C-4^I), 76.2 (CH₂Ph), 76.0 (C-5^III), 75.8 (C-3^III), 75.5 and 75.3 (CH₂Ph), 75.2 (2C, CH₂Ph), 74.8 (2C, C-3^I, C-2^IV), 74.3 (C-4^IV), 74.1 (CH₂Ph), 73.8 (C-5^II), 73.6 (CH₂Ph), 73.5 (2C, C-5^IV, CH₂Ph) 73.3 (C-4^III), 73.2 (CH₂Ph), 72.2 (2C, CH₂Ph), 71.9 (CH₂Ph), 71.3 (C-2^I), 69.2 (C-6^IV), 68.2 (2C, C-6^III, C-6^II), 67.3 (C-5^V), 66.9 (C-5^VI), 63.5 (C-6^I), 56.5 (C-2^III), 24.9 (CH₂CH₃), 23.8 (NHCOCH₃), 16.5 (2C, C-6^V, C-6^VI), 15.4 (CH₂CH₃); ES-HRMS calcd for C₁₅₂H₁₅₉NO₃₁S [Na]⁺ 2549.0515 found 2549.0415.

2-Azidoethyl (2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1 → 2)-(3,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 3)-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)]-(1 → 4)-(6-O-benzyl-2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-(2,3,6-tri-O-benzoyl-1-thio-β-D-glucopyranosyl)-(1 → 6)-3-O-benzoyl-2-deoxy-2-acetamido-α-D-galactopyranoside (22). A mixture of donor 1 (90 mg, 0.036 mmol), acceptor 21 (21 mg, 0.053 mmol) and molecular sieves 4 Å in CH₂Cl₂ (3 mL) was stirred for 20 min. The mixture was cooled to −40 °C, NIS (16 mg, 0.071 mmol) and AgOTf (cat) added and the mixture stirred for 2 h while gradually warming to 0 °C. Upon completion of the reaction, Et₃N (20 μL) was added and the mixture diluted with CH₂Cl₂ (20 mL) and filtered through a pad of Celite. The mixture was washed with 10% aq Na₂SO₃ and the organic layer dried over MgSO₄, filtered and concentrated. Purification by flash chromatography (toluene–EtOAc) gave 22 (0.086 g, 84%) as a colourless solid. R_f 0.35 (toluene–EtOAc 1 : 1); [α]²⁰_D –21.1° (c 2.33, CHCl₃); ¹³C NMR (150 MHz, CDCl₃) δ 170.1 and 169.1 (NHCOCH₃), 166.4, 166.0, 165.7 and 165.3 (COPh), 139.5, 139.3, 139.2, 138.9, 138.6, 138.4, 138.2, 138.0, 137.7, 133.4, 133.3, 133.2, 132.6, 130.1, 130.0, 129.8, 129.6, 129.4, 128.6, 128.4, 128.3, 128.2, 128.0, 127.8, 127.7, 127.6, 127.5, 127.3, 127.1, 127.0, 126.9, 126.5 and 126.2 (Ar), 103.0 (C-1^II), 101.8 (C-1^III), 101.6 (C-1^IV), 101.2 (C-1^I), 98.4 (C-1^VI), 97.8 (2C, C-1^V, C-1^VII), 83.8 (C-3^IV), 80.6 (C-2^II), 80.3 (C-3^V), 79.7 (C-3^II), 79.1 (C-3^VI), 78.1 (C-4^V), 78.1 (C-4^VI), 75.8 (C-4^II), 75.7 (C-2^V), 75.4 (3C, C-2^VI, C-5^III, CH₂Ph), 75.1 (C-3^III), 75.0 (C-4^I), 74.9 and 74.8 (CH₂Ph), 74.7 (2C, CH₂Ph), 74.5 (CH₂Ph), 74.0 (C-2^IV), 73.8 (C-5^I), 73.6 (CH₂Ph), 73.5 (C-5^II), 73.2 (CH₂Ph), 73.1 (2C, C-4^IV, C-4^III), 72.9 (2C, CH₂Ph, C-5^IV), 72.6 (2C, C-3^I, CH₂Ph), 71.7 (4C, C-2^I, C-3^VII, 2 x CH₂Ph) 71.2 (CH₂Ph), 69.4 (C-5^VII), 68.9 (C-6^VII), 68.5 (C-6^IV), 67.9 (C-6^II), 67.3 (C-6^III), 67.1 (C-4^VII), 67.0 (CH₂CH₂N₃), 66.8 (C-5^V), 66.6 (C-5^VI), 62.2 (C-6^I), 56.4 (C-2^III), 50.2 (CH₂CH₂N₃), 47.3 (C-2^VII), 23.4 and 23.2 (NHCOCH₃), 16.3 and 16.2 (C-6^V and C-6^VI); ES-HRMS calcd for C₁₆₇H₁₇₅N₅O₃₈ [Na]⁺ 2881.1813 found 2881.1875.

2-Aminoethyl (α-L-fucopyranosyl)-(1 → 2)-(β-D-galactopyranosyl)-(1 → 3)-[(α-L-fucopyranosyl)]-(1 → 4)-(2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(β-D-galactopyranosyl)-(1 → 4)-(β-D-glucopyranosyl)-(1 → 6)-2-deoxy-2-acetamido-α-D-galactopyranoside (23). Sodium methoxide was added to a solution of 22 (35 mg, 0.012 mmol) in dry MeOH (5 mL) until a pH of 13 was reached, the solution was stirred overnight. Upon completion of the reaction the mixture was neutralized using Dowex 50 W+ ion exchange resin, filtered, concentrated and dried in vacuo. The residue was dissolved in a mixture of THF (2 mL), EtOH (2 mL) and H₂O (55 μL). HCl (aq, 1 N, 20 μL) and Pd/C (35 mg) was added and the suspension was stirred under H₂ atmosphere (5 bar) for 18 h. Additional H₂O (3 mL), HCl (aq, 1 N, 10 μL) and Pd/C (35 mg) were added and the mixture was stirred under a H₂ atmosphere (5 bar) for another 18 h. Upon completion of the reaction the mixture was neutralized with saturated aq NaHCO₃, filtered through frits (20 μm, 10 μm, 5 μm), and the solution concentrated. The crude material was purified on a P2 Biogel column to obtain 23 (14 mg, 92%) after freeze drying as a colourless solid. R_f 0.23 (EtOAc–MeOH–H₂O 2 : 2 : 2 +0.5% AcOH); [α]²⁰_D –12.8° (c 0.67, H₂O); ¹³C NMR (150 MHz, D₂O) δ 174.6 and 174.1 (NHCOCH₃), 103.2 (C-1^III), 102.9 (C-1^II), 102.4 (C-1^V), 100.6 (C-1^IV), 99.5 (C-1^VI), 97.7 (C-1^I), 97.6 (C-1^VII), 81.5, 78.1, 76.4, 75.1, 74.8, 74.7 (2C), 74.4, 74.2, 73.6, 72.7, 71.9 (2C), 71.7, 70.2, 70.1, 69.4, 69.2, 69.0, 68.7, 68.5, 68.4, 68.2, 67.7, 67.4, 67.0, 66.2, 64.5, 61.5, 60.9, 59.9, 59.4, 55.7, 49.5, 39.2 (CH₂CH₂N₃), 22.1, 21.9 (NHCOCH₃), 15.3, 15.2 (C-6^VI, C-6^VII); ES-HRMS calcd for C₄₈H₈₃N₃O₃₄ [Na]⁺ 1268.4756 found 1268.4805.

N-[(tert-Butoxy)carbonyl] O-(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)-(1 → 2)-(3,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 3)-[(2,3,4-tri-O-benzyl-α-L-fucopyranosyl)]-(1 → 4)-(6-O-benzyl-2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(2,4,6-tri-O-benzyl-β-D-galactopyranosyl)-(1 → 4)-O-(2,3,6-tri-O-benzoyl-β-D-glucopyranosyl)-L-serine benzyl ester (24). Dichloromethane (6 mL) was added to a mixture of 1 (150 mg, 0.059 mmol), Boc-Ser-OBn (27 mg, 0.089 mmol) and 4 Å molecular sieves. The suspension was cooled to −40 °C before adding NIS (27 mg, 0.119 mg) and AgOTf (catalytic). The mixture was stirred for 2 h while gradually warming to 0 °C. The reaction was quenched with Et₃N (20 μL) and diluted with dichloromethane (20 mL) before filtering through a Celite pad. The mixture was washed with 1-% aqueous Na₂S₂O₃ and the organic layer dried over MgSO₄, filtered and concentrated. Purification by silica gel via Biotage (column 1 gradient elution toluene/ethyl acetate, column 2 gradient toluene/acetonitrile) gave 24 (118 mg, 72%) as a colourless solid. ¹H NMR (500 MHz, MeOD) δ 7.91–7.86 (m, 3H, ArH), 7.79–7.77 (dd, J = 8.3, 1.2 Hz, 2H, ArH), 7.50–7.43 (m, 2H, ArH), 7.36–7.00 (ArH), 6.91–6.89 (d, J = 7.0 Hz, ArH), 5.56–5.53 (m, 2H), 5.41–5.40 (d, J = 3.9 Hz, 1H), 5.25–5.22 (m, 1H), 5.20 (t, 3H), 5.14–5.13 (d, 1 H), 4.97–4.92 (m, 2H), 4.86–4.86 (d, J = 3.5 Hz, 1H) 4.84–4.77 (m, 3H), 4.71–4.58 (m 7H), 4.54–4.38 (m, 9H), 4.35–4.24 (m, 8H), 4.21–4.19 (m, 2H), 4.16–4.14 (m, 1H), 4.09–3.99 (m, 4H), 3.90–3.85 (m, 3H), 3.81–3.76 (m, 3H), 3.74–3.64 (m, 7H), 3.57–3.54 (dd, J = 9.9, 3.0 Hz, 2H), 3.50–3.42 (m, 4H), 3.27–3.22 (m, 2H), 3.08 (bs, 1H), 2.85–2.80 (m, 2H), 1.55 (s, 3H, COCH₃), 1.21 (s, 9H, ^tBu), 1.08–1.07 (d, J = 6.5 Hz, 6H, CH^IV,V₃); ¹³C NMR (126 MHz, MeOD) δ 165.3 (COOPh), 164.6 (NHCOCH₃), 161.3, 161.0, 160.7 (COPh), 150.7 (HNCOO^tBu), 135.3, 135.1, 135.0, 134.8, 134.6, 134.5, 134.5, 134.2, 133.9, 133.9, 133.8, 133.6, 131.1, 128.8, 128.6, 128.3 (17 qC), 125.6, 125.4, 125.3, 125.2, 125.1, 124.9, 124.2, 124.1, 124.0, 124.0, 124.0, 123.9, 123.9, 123.9, 123.8, 123.8, 123.8, 123.8, 123.7, 123.7, 123.6, 123.6, 123.5, 123.5, 123.5, 123.4, 123.3, 123.2, 123.1, 123.1, 123.0, 122.9, 122.9, 122.8, 122.8, 122.7, 122.7, 122.6, 122.6, 122.5, 122.4, 122.0, 122.0 (ArC), 98.9, 97.6, 97.0, 96.5, 94.1, 93.1 (C1^I−VI), 79.2, 76.1, 75.8, 75.1, 74.5, 74.1, 73.8, 71.6, 71.5, 71.4, 71.2, 71.2, 71.0, 70.7, 70.5, 70.3, 70.2, 69.8, 69.2, 69.1, 69.1, 68.8, 68.6, 68.4, 68.4, 68.4, 68.3, 68.1, 67.3, 67.2, 66.9, 65.1, 64.2, 63.2, 63.1, 62.5, 62.3, 61.9, 58.2, 49.5, 23.4 (^tBu), 18.8 (NCOCH₃), 11.5 (C6^V,VI); ES-HRMS calcd for C₁₆₅H₁₇₄N₂O₃₆Na₂ 1402.5821 found 1402.5828.

N-[((3-Methyl-3H-diazirine-3-yl)propanoxy)carbonyl] O-(α-L-fucopyranosyl)-(1 → 2)-(β-D-galactopyranosyl)-(1 → 3)-[(α-L-fucopyranosyl)]-(1 → 4)-(2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(β-D-galactopyranosyl)-(1 → 4)-O-(β-D-glucopyranosyl)-L-serine D-biotin hydrazide (25). To a solution of compound 24 (115 mg, 0.042 mmol) in dry THF, Pd/C (20 mg) was added and the mixture was stirred for 1 h. Pd/C was filtered off and the solvent was removed. The residue was taken up in a mixture of THF (4 mL), EtOH (4 mL), H₂O (110 μL) and 1N aqueous HCl (40 μL). Pd/C was added, and the mixture was stirred under a H₂ atmosphere (5 bar) for 4 h. Additional H₂O (4 mL) was added, and the mixture was stirred under a H₂ atmosphere overnight. Upon completion of the reaction, the mixture was neutralised with saturated NaHCO₃, filtered through frits (20 μm, 10 μm and 5 μm) and reduced to dryness. The residue was dissolved in dry methanol (5 mL) and sodium methoxide (cat.) was added until a pH of 12–13 was reached. The reaction was stirred for 4 h then neutralised with Dowex W50⁺ ion exchange resin. The resin was filtered off and the liquors were concentrated. Crude material was passed through a short C18 column to afford 40 mg (88%) of O-(α-L-fucopyranosyl)-(1 → 2)-(β-D-galactopyranosyl)-(1 → 3)-[(α-L-fucopyranosyl)]-(1 → 4)-(2-deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 3)-(β-D-galactopyranosyl)-(1 → 4)-O-(β-D-glucopyranosyl)-L-serine ES-HRMS: calcd for C₄₁H₆₉N₂O₃₁ [M − H]⁻ 1085.3884, found 1085.3845. This crude compound was dissolved in PBS buffer (2.00 mL, 100 mM sodium phosphate, pH 7.4) and the N-succinimidyl-diazirine (SDA) (10.7 mg), dissolved in DMF (100 μL) was added. The mixture was stirred overnight then concentrated. NMR analysis showed incorporation of the diazirine linker. ¹H NMR (500 MHz, D₂O) δ 5.16–5.15 (d, J = 3.7 Hz, 1H), 5.04–5.03 (d, J = 3.7 Hz 1H), 4.89–4.85(m, 1H), 4.67–4.66 (d, J = 7.5 Hz, 1H), 4.62–4.60 (d, J = 8.6 Hz, 1H), 4.49–4.47(d, J = 7.9 Hz, 1H), 4.44–4.41(m, 2H), 4.37–4.33 (dd, J = 12.5, 5.9 Hz, 1H), 4.28–4.25 (dd, J = 10.6, 5.1 Hz, 1H), 4.16–4.12 (t, J = 9.8 Hz, 1H), 3.99–3.85 (m, 7H), 3.83–3.68 (m, 13H), 3.65–3.47 (m, 8H), 3.35(t, J = 8.4 Hz, 1H), 2.73 (s, 1H), 2.27 (t, J = 7.5 Hz, 2H, CH₂), 2.07 (s, 3H, COCH₃), 1.73–1.69 (m, J = 14.8, 7.4 Hz, 2H, CH₂), 1.27 (t, J = 7.1 Hz, 6H, CH^VI,V₃), 1.04 (CH₃) The crude was dissolved in water, biotin hydrazate (13.0 mg, 0.051 mmol), EDCl (9.70 mg, 0.051 mmol) and HOBt·H₂O (2.60 mg, 0.017 mmol) were added. The reaction was stirred overnight and then concentrated. The residue was purified by C18 column chromatography (H₂O/MeOH 90 : 10 to 50 : 50) followed by a Biogel P2 size exclusion column to give 25 (6.5 mg, 0.0045 mmol, 11% over four steps). ¹H NMR (600 MHz, D₂O) δ 5.16 (d, J = 4.1 Hz, 1H), 5.04 (d, J = 3.9 Hz, 1H), 4.88 (d, J = 6.8 Hz, 1H), 4.75 (d, J = 5.1 Hz,1H), 4.65–4.59 (m, 2H), 4.49–4.40 (m, 2H), 4.35 (q, J = 6.6 Hz, 1H), 4.25 (dd, J = 10.7, 5.3 Hz, 1H), 4.15 (m, 3H), 4.00–3.50 (m, 28H), 3.36 (m, 2H), 3.02 (dd, J = 13.1, 5.0 Hz, 1H, biotin h), 2.80 (d, J = 13.0 Hz, 1H, biotin h), 2.35 (t, J = 7.3 Hz, 2H, biotin a), 2.29 (t, J = 7.4 Hz, 2H, diazirine i), 2.07 (s, 3H, COOCH₃), 1.78–1.67 (m, 4H, biotin d′, b and diazirine j), 1.62 (dd, J = 14.1, 7.6 Hz, 1H, biotin d′′), 1.48 (q, J = 7.6 Hz, 2H, biotin c), 1.28 (dd, J = 9.2, 6.6 Hz, 6H, CH^V,VI₃), 1.04 (s, 3H, diazirine CH₃). ¹³C NMR (151 MHz, D₂O) δ 175.5, 174.1, 165.3 (C=O), 103.17, 102.93, 102.08, 100.57, 99.49, 97.71 (C-1^I−VI), 81.48, 78.12, 76.42, 75.11, 74.72, 74.40, 74.06, 73.56, 72.62, 71.91, 71.71, 70.07, 69.37, 69.04, 68.67, 68.55 (CH₂ Serine), 68.20, 67.73, 66.96, 66.17, 61.90, 61.52, 60.88, 59.9, 59.4 (C6^I−IV), 60.19, 55.68, 55.15, 52.33, 39.63 (CH₂ biotin h), 33.09 (CH₂ biotin a), 29.81 (CH₂ diazirine i), 29.42 (CH₂ biotin b), 27.59 (CH₂ biotin c), 27.45 (CH₂ biotin d), 24.59 (CH₂ diazirine j), 22.10 (COOCH₃), 18.49 (CH₃ diazirine k), 15.29 (CH^V,VI₃). MALDI-TOF MS: C₅₆H₉₂N₈NaO₃3S [M + Na]⁺ calcd 1459.5385, found 1459.49.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Financial support from Science Foundation Ireland (Grants 08/IN.1/B2067, 13/IA/1959, and 16/RC/3889) is gratefully acknowledged.

References

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d2ob00477a

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