Synthesis of an aminooxy derivative of the GM3 antigen and its application in oxime ligation

Abstract

The anomeric aminooxy GM3 trisaccharide cancer antigen (Neu5Acα2,3Galβ1,4Glcβ-ONH₂) has been chemically synthesized using a linear glycosylation approach. The key step involves a highly α(2,3)-stereoselective sialylation to a galactose acceptor. The Neu5Acα2,3Gal intermediate was functionalized as a donor for a [2 + 1] glycosylation including a glucose acceptor that featured an O-succinimidyl group on the reducing end as an aminooxy precursor. The fully deprotected anomeric aminooxy GM3 trisaccharide was then conjugated to the immunologically relevant zwitterionic polysaccharide PS A1 via an oxime link.

Graphical Abstract

Oxime ligation has emerged as an attractive conjugation method in vaccine formulation for a number of reasons: 1) oxime bonds are hydrolytically stable in physiological conditions,^1–3 2) the oxime bond is immunologically “silent” as compared to other conjugation linkages,^4–5 3) the condensation of aminooxy functions are fast, quantitative, and only produce water as a byproduct.^6–7 Our group has used the oxime ligation strategy to create entirely carbohydrate immunogens capable of generating protective immune responses against tumor associated carbohydrate antigens (TACAs).^8–11 These glycoconjugates feature the unique zwitterionic polysaccharide PS A1 which is isolated from the commensal organism Bacteroides fragilis (ATCC 25285/NCTC 9343). The alternating charge character on the repeating unit of PS A1 provides distinct properties that permit binding to the MHC II complex on antigen presenting cells and downstream activation of CD4+ T cells through the MHC II:αβTCR complex pathway.^12–13 Formation of these immunogens is materialized by the oxime condensation of a chemically derived aldehyde on PS A1 and with an aminooxy derivative of a chosen TACA (Figure 1). This vaccine platform has been successfully demonstrated with Tn-PS A1^{8, 14} and STn-PS A1¹⁰ where we observed robust IgG antibody production, cellular immunity, and tumor cell lysis.

Figure 1.

General structure of TACA-PS A1 glycoconjugates and aminooxy derivatives of TACAs.

Progress of these initial developments led us to expand our TACA toolbox to incorporate the ganglioside GM3 (Neu5Acα2,3Galβ1,4Glcβ-Cer). GM3 is overexpressed in carcinomas of the breast and skin and has implications in tumor cell proliferation and metastasis.^15–16 GM3 overexpression is also positively correlated with tumor malignancy leading to poor prognosis.¹⁶ Due to these characteristics, GM3 has been prioritized within the top 50 cancer antigens for immune intervention.¹⁷

The biological relevance of the GM3 antigen implicates necessity for preparative ease and availability for immunological experiments. Indeed, both chemo-enzymatic and chemical methods exist for the production of the GM3 antigen, but they have limitations.^18–25 Chemo-enzymatic methods offer excellent regio- and stereo-selective control of glycan production but are typically demonstrated on smaller scales.^{19, 26} On the other hand, synthetic reports of the GM3 glycan relay poor stereoselectivity forming the challenging α(2→3) glycosidic bond. Up to date, only one synthesis of the aminooxy GM3 glycan has been reported through a sialyltransferase catalyzed glycosylation.¹⁹ Herein, we describe our chemical synthetic platform to produce the aminooxy GM3 glycan by using a linear approach that features a high α-selective sialylation in good yield. The final step involves a microwave assisted global deprotection of -OAc/-OBz protecting groups and an O-succinimidyl (OSu) protecting group to achieve the corresponding alcohol and aminooxy functions, respectively. The fully deprotected aminooxy GM3 glycan was then conjugated to PS A1 by oxime ligation to form the GM3-PS A1 glycoconjugate.

The synthetic route to the aminooxy GM3 glycan (1) must feature high α-selectivity in the Neu5Ac2,3Gal bond forming step. To achieve this, we elected to use the α-directing transfused 4O,5N-acetyl oxazolidinone ring system on the sialic acid donor.^{10, 27–28} This moiety exhibits strain on the cyclic system which destabilizes the formation of the glycosyl oxocarbenium ion intermediate, and instead a β-triflate intermediate is more likely formed. This pushes the mechanism of glycosylation towards more of an S_N2-like associative mechanism as opposed to an unselective S_N1 dissociative mechanism.²⁹ Additionally, a highly reactive anomeric leaving group, such as a phosphate ester,^30–31 was desired based on our previous experiences.¹⁰ These two features led us to adopt the sialyl phosphate donor 3 (Scheme 1, Scheme S1). The synthetic strategy can be further broken down through pathway A or pathway B (Scheme 1). Reports that describe pathway A in the synthesis of the GM3 glycan discuss high yields and α-selectivity in the α(2→3) bond forming step, but feature a greater number of overall synthetic steps than pathway B.²⁴ Contemporary reports of GM3 synthesis describe pathway B with the use of lactosyl acceptors, but these reports describe low yields in the α(2→3) bond forming step.²⁵ We must also account for the introduction of -OSu on the reducing end of the protected trisaccharide as the precursor for an aminooxy group.^9–10 This requires limited use of benzyl ether protecting groups, as long exposure times of hydrogenation can cause undesired cleavage of the -OSu N-O bond.³² We proceeded to screen various glycosyl acceptors for the α(2→3) bond forming step using sialyl phosphate 3 as the donor (Table 1).

Scheme 1.

Retrosynthetic Analysis of the Aminooxy GM3 Glycan

Table 1.

Optimization of α(2,3)-Glycosylation^a

entry	acceptor	product	yieldb	α:β ratioc
1			68%	1:5
2			81%	1:4
3			52%	1:1
4			89%	8:1
5			87%	16:1

^aTypical conditions: 1.5 equivalents of donor, 1.0 equivalent of acceptor, 1.5 equivalents of TMSOTf, in dry CH₂Cl₂ at −78 °C for 1 hour.

^bIsolated yield.

^cDetermined by ¹H NMR spectroscopic analysis of the unpurified reaction mixture.

We commenced with screening lactose acceptors 4 (Table 1, entry 1) and 5 (Table 1, entry 2, Scheme S2). Both acceptors feature a 3’,4’-diol and differ on their reducing ends with 4 containing thiophenol and 5 containing an O-succinimidyl. Both acceptors 4 and 5 provided the β-anomer as the major product with α:β ratios of 1:5 and 1:4 respectively. Poor selectivity is in line with previous reports and is a consequence of the disarming effect of acetyl group protections.^{25, 33} We then looked toward the 3, 2’, 3’, 4’-unprotected lactoside 6 (Table 1, entry 3) which has been described to provide improved α-formation as compared to 4 and 5 due to the relatively weaker disarming effect of pivaloyl groups.³⁴ Acceptor 6 resulted in an improved α:β ratio at 1:1, although this ratio remained unsatisfactory. Based on these observed limitations of lactose acceptors, we investigated the use of galactose acceptor 7³⁵ which featured a 3,4-diol with a 2-O-benzoyl-6-O-benzyl protection strategy (Table 1, entry 4). The resulting α:β ratio was drastically improved to provide the α-anomer in an 8:1 ratio in 89% yield. The greatly improved α:β ratio led us to explore galactose acceptor 8³¹ which contains the conformation restricting 4,6-O-benzylidene acetal (Table 1, entry 5). Galactose acceptor 8 provided excellent α-selectivity with an α:β ratio of 16:1 in 87% yield. It was logical at this point to proceed through pathway A using galactosyl acceptor 8.

With disaccharide 13 in hand, we turned our attention to the D-glucose acceptor 16 as shown in Scheme 2. Here, thio-donor 14 was activated using N-iodosuccinimide (NIS) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) in the presence of N-hydroxysuccinimide (NHS) to afford compound 15 in 84% yield as a pure β-anomer. Selective ring opening of the 4,6-benzylidene acetal, on compound 15, was performed with triethylsilane (Et₃SiH) as the hydride source and boron trifluoride diethyl etherate (BF₃·Et₂O) as the catalyst. Successful regioselective ring opening to afford 16 was confirmed using HMBC as the C-6 position nicely correlated with the benzylic protons on the benzyl ether.

Scheme 2.

Synthesis of Glc-OSu Acceptor 16

Disaccharide 13 (Table 1, entry 5) was subjected to Zemplén sodium methoxide conditions to remove the oxazolidinone, and the resulting free hydroxyls were acetylated to provide compound 17 as shown in Scheme 3. Interestingly, the C-2 O-benzoylated protection on compound 13 was not removed during the Zemplén method. This is likely due to the steric hinderance of this position, especially within disaccharide systems.²¹ With disaccharide donor 17 and glucose acceptor 16 in hand, we sought to perform this [2 + 1] glycosylation under typical

Scheme 3.

Attempted 2 + 1 Glycosylation Through Intermediate 13

NIS/TMSOTf activation conditions

TLC analysis of the reaction mixture revealed partial consumption of the donor (Rf = 0.4) accompanied by the presence of a new spot (Rf = 0.2). Investigation of the reaction revealed the new spot to be the glycosyl succinimide product (S6). Indeed, this activation system produces the weakly nucleophilic succinimide as a by-product, which can outcompete weakly nucleophilic or sterically hindered alcohols on glycosyl acceptors. This phenomenon has been described for donor/acceptor pairs with mismatched reactivities.³⁶ Notably, we also discovered partial proton exchange with trimethyl silane on acceptor 16. We attempted the [2 + 1] coupling using different activation systems such as NIS/trifluoromethanesulfonic acid (TfOH), dimethyl disulfidetriflic anhydride (Me₂S₂-Tf₂O), and a trichloroacetamidate (TCA) donor (18). The results led to a glycosyl succinimide by-product, a complex mixture, and the Chapman rearrangement³⁷ respectively (Scheme 3). These experiments offer further evidence of the torsional disarming effect from 4,6-O-acetal bicyclic donors.³⁸

We sought to deviate the synthetic route through the more armed disaccharide donor compound 12 (Table 1, entry 4). As illustrated in Scheme 4, preparation of the disaccharide donor 20 mirrored the preparation of donor 17, with the C-2-O-benzoyl protection remaining intact. Donor 20 was then activated with the NIS/TfOH system in the presence of acceptor 16 and provided the desired, fully protected trisaccharide 2 in 87% yield as the β-anomer. No evidence of the glycosyl succinimide was present.

Scheme 4.

Synthesis of the Aminooxy GM3 Glycan

Deprotection of key intermediate 2 began with the cleavage of the benzyl ethers using hydrogen gas at 1 atm and a suspension of Pearlman’s catalyst. TLC indicated full consumption of starting material after 1 hour of mixing and the reaction was purged with argon gas to remove dissolved hydrogen gas. The reaction was filtered, concentrated, and passed through a short silica column to afford the debenzylated product S7 in 89% yield with no evidence of -OSu N-O cleavage. Deprotection of the Neu5Ac C-1 methyl ester was accomplished using the Krapcho-like demethylation by lithium iodide in pyridine.¹⁰ This mixture was refluxed at 110 °C for 3 hours in the absence of light. Pyridine was then removed in vacuo and the crude material was purified by silica flash chromatography to afford compound 21 in 68% yield.

In the final deprotection step, we utilized excess hydrazinehydrate to remove the remaining -OAc/-OBz protecting groups and to remove -OSu to expose the aminooxy functionality. However, based on the results of Zemplén sodium methoxide conditions as described for 12 and 13 we did not expect deprotection of the C-2-O-benzoylatd positions using hydrazine hydrate at room temperature. Additionally, literature precedent suggested long reaction times²⁴ and elevated temperatures²⁵ in hydroxide or methoxide conditions for C-2-O-benzoyl removal, which would then cause an undesirable ring opening of the installed -OSu group and failure of aminooxy deprotection. To overcome this limitation, we turned to microwave assistance in the final deprotection step. Compound 22 was dissolved in ethanol followed by the addition of excess hydrazine hydrate. The mixture was then stirred for 1 hour at room temperature and MS analysis did not detect any formation of product. The reaction was then subjected to microwave irradiation at 200 Watts with constant temperature of 40 °C for 20 minutes. Once again, MS analysis indicated little-to-no deprotection of compound 21. We then increased the temperature to a constant 90 °C for 30 minutes with no change in maximum Watts. Following this procedure, MS analysis of the reaction mixture revealed partial deprotection of 21. Repetitive cycling (flow-chemistry techniques) of the aforementioned conditions led to MS analysis clearly depicting fully deprotected 21. The reaction mixture was then concentrated, resuspended in water, and passed through a P2 BioGel® column using water as the eluent. Fractions identified to contain compound 1 by TLC staining were collected, frozen, and lyophilized. The resulting white solid was characterized and compound 1 was definitively confirmed by NMR and HRMS (See supporting information).

With the aminooxy GM3 glycan complete, we pursued oxime ligation with PS A1 (Scheme 5, 22). The repeating unit of PS A1 contains the side chain D-galactofuranose with a 1^o and 2^o vicinal diol that can be chemo- and regio-selectively oxidized by sodium periodate to afford an aldehyde.^8–11 After PS A1 oxidation, the aldehyde moiety was then subjected to four equivalents of 1 and subsequently stirred for 18 hours in a 0.1 M sodium acetate buffer at a pH of 5. The reaction mixture was then filtered and rinsed through a 10 kDa molecular weight cutoff filter. The filtered material was then resuspended in water, frozen, and lyophilized to produce 23 as a white (cotton-like) substance. NMR analysis of this material at 60 °C provided the evidence we needed to note successful conjugation with PS A1. The defining peaks included an oxime proton peak (δ = 7.99–8.00), an additional anomeric proton peak (δ = 5.37), an additional N-acetyl peak (δ = 2.31), and the presence of the Neu5Ac H-3eq (δ = 3.03–3.06) and H-3ax peaks (δ = 2.04–2.08). Based on integration analysis of the Neu5Ac H-3eq and PS A1 AAT methyl peak, we determined the relative ratio of GM3:PS A1 to be 1:2.33, which translates into approximately 51.5 GM3 conjugation sites per PS A1.

Scheme 5.

Synthesis of GM3-PS A1

In conclusion, we have discussed the first fully chemical synthetic method to provide the aminooxy GM3 glycan. This synthesis features highly α-selective chemistries in the formation of the Neu5Ac(2→3)Gal bond. In these experiments we showed the benefit of using the armed galactose acceptors as opposed to the disarmed lactose acceptors, which gave undesirable α:β ratios. This synthesis also features a 3-step deprotection strategy of the fully protected trisaccharide. Notably, the final step utilized microwave assisted chemistry to deprotect the sterically hindered C-2 O-benzoylations in excellent yield, after reiterations. Lastly, we highlighted the utility of the aminooxy functionality by conjugating the GM3 glycan to the immunologically relevant PS A1 to generate the GM3-PS A1 glycoconjugate. The GM3-PS A1 vaccine candidate will be investigated for its immunological characteristics in due time.

EXPERIMENTAL SECTION

General Information

Relevant reagents and solvents were purchased from commercial sources and used without further purification. Molecular sieves (4 Å) were dried overnight in a vacuum oven set to 140 °C, and further dried under high vacuum using a heat gun for 5 minutes and cooled to room temperature under high vacuum. Synthesized compounds were purified using flash chromatography with SiliCycle Inc. 60 Å 230–400 mesh silica gel or size exclusion chromatography with Bio-Gel P-2 (Bio-Rad Laboratories, Inc). Thin layer chromatography (TLC) was performed with SiliCycle Inc. silica gel TLC 250 μm w/h F-254. Proton and carbon NMR spectra were recorded using a Bruker Avance III 600 Ultrafield Cryoprobe spectrometer. CDCl₃, CD₃OD, and D₂O were used as solvents, and the chemical shifts were referenced relative to residual solvent. Low resolution mass spectrometry data were taken on an LCQ Deca ESI-MS machine. High resolution mass spectrometry data were collected using an Orbitrap Fusion Tribrid Mass Spectrometer.

Experimental Procedures

Methyl (phenyl-5-acetamido-7,8,9-tri-O-acetyl-5-N,4-Ocarbonyl-3,5-dideoxy-2-thio-d-glycero-α,β-d-galacto-non-2ulopyranoside)onate (S2)

Compound S1²⁸ (5.20 g, 8.91 mmol, 1.0 equiv) was suspended in 75 mL of MeOH with vigorous stirring. To the suspension was added MeSO₃H (2.90 mL, 44.6 mmol, 5.0 equiv) at room temperature and the suspension was refluxed for 24 hours. The mixture was then cooled to room temperature and the reaction quenched with excess TEA. The solution was then concentrated under reduced pressure and dried under vacuum. The resulting crude concentrate was then dissolved in $5\overline{0}$ mL MeCN and $10\overline{0}$ mL H₂O followed by the addition of NaHCO₃ (3.74 g, 44.6 mmol, 5.0 equiv). The mixture was stirred vigorously to a uniform suspension and cooled to 0 °C. A separate solution of triphosgene (7.93 g, 26.7 mmol, 3.0 equiv) dissolved in $5\overline{0}$ mL MeCN was then added to the vigorously stirred suspension via dropping funnel at a slow rate of ~1 drop/5 seconds. The reaction was maintained at 0 °C for 5 hours. EtOAc ($1\overline{0}0$ mL) was then added to the mixture and vigorously stirred for 10 minutes. The organic and aqueous layers were separated, and the aqueous layer was extracted with EtOAc ($1\overline{0}0$ mL, 2X). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The resulting residue was passed through a short, thick bed of silica eluting with EtOAc:MeOH (9:1). Fractions containing the intermediate were pooled and concentrated under reduced pressure and then set under high vacuum overnight to give the crude intermediate (3.02 g, 7.56 mmol, 85% yield of crude). The crude material was then dissolved in pyridine (35 mL) followed by the addition of DMAP (0.18 g, 1.5 mmol, 0.2 equiv). Gas was exchanged with Ar and the reaction mixture was cooled to 0 °C with vigorous stirring. Ac₂O (14.3 mL, 151 mmol, 20 equiv) was then injected into the reaction mixture and the reaction was stirred for 12 hours while the reaction was allowed to slowly warm to room temperature. The reaction was then quenched with MeOH at 0 °C and then concentrated under reduced pressure followed by co-distillation with toluene (3X). The crude material was then dissolved in CH₂Cl₂ (50 mL) and washed with saturated NaHCO₃ (50 mL, 3X). The resulting organic layer was washed with brine, dried with Na₂SO₄, filtered, and concentrated under reduced pressure. The crude material was then purified by silica flash chromatography eluting with hexanes:EtOAc (1:1) to give compound S2 as a white solid (4.00 g, 7.05 mmol, 79% yield over 3 steps). ¹H NMR (CDCl₃, 600 MHz): δ = 1.96 (3H, s), 2.09 (3H, s), 2.15 (3H, s), 2.34 (1H, t, J = 13.0 Hz), 2.53 (3H, s), 2.91 (1H, dd, J = 3.7, 13.1 Hz), 3.63 (3H, s), 3.76 (1H, dd, J = 9.2, 11.3 Hz), 3.90 (1H, dd, J = 8.2, 12.1 Hz), 4.37 (1H, dd, J = 2.5, 12.0 Hz), 4.78 (1H, td, J = 4.3, 15.0 Hz), 4.89 (1H, dd, J = 11.7, 6.6 Hz), 5.00 (1H, td, J = 2.2, 8.2 Hz), 5.56 (1H, t, J = 2.3 Hz), 7.36 (2H, m), 7.43 (1H, m), 7.50 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.7, 20.8, 21.1, 24.7, 36.0, 52.8, 59.6, 62.9, 72.7, 73.9, 75.1, 75.7, 88.4, 128.2, 129.2, 130.2, 136.8, 153.6, 167.8, 169.7, 170.4, 171.2, 172.5. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₅H₂₉NO₁₂SNa 590.1; Found 590.2.

Methyl (5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl2-(dibutylphosphoryl)-3,5-dideoxy-d-glycero-α,β-d-galactonon-2-ulopyranoside)onate (3)

Compound S2 (1.00 g, 1.76 mmol, 1.0 equiv) was dried by co-distillation with toluene (3X) followed by high vacuum for 3 hours. The dry compound was then dissolved in $2\overline{0}$ mL of dry CH₂Cl₂ followed by the addition of 4 Å molecular sieves (4 g) and dibutyl phosphate (1.75 mL, 8.80 mmol, 5.0 equiv). Gas in the reaction vessel was exchanged with Ar and this solution was then stirred for 30 minutes at room temperature, followed by the addition of NIS (1.19 g, 5.28 mmol, 3.0 equiv). Gas was then exchanged with Ar again and the reaction was stirred at room temperature for 12 hours. After 12 hours, the reaction was diluted with CH₂Cl₂ (30 mL) and quenched with a saturated solution of Na₂S₂O₃ and NaHCO₃ (30 mL). The quenched reaction was then filtered, and the collected organic layer was washed with saturated Na₂S₂O₃ and NaHCO₃ (30 mL, 2X). The resulting organic layer was washed with brine, dried over Na₂SO₄, filtered, and then concentrated under reduced pressure. The crude compound was then purified via flash chromatography using hexanes:EtOAc (1:1) to give sialyl donor 3 as an anomeric mixture and as a yellow oil (1.05 g, 1.57 mmol, 89% yield). (3α) ¹H NMR (CDCl₃, 600 MHz): δ = 0.96 (6H, t, J = 7.4 Hz), 1.43 (4H, m), 1.68 (4H, m), 2.06 (3H, s), 2.13 (3H, s), 2.17 (3H, s), 2.52 (3H, s), 2.71 (1H, t, J = 12.7 Hz), 3.03 (1H, dd, J = 4.0, 12.2 Hz), 3.86 (3H, s), 3.89 (1H, t, J = 10.1 Hz), 4.14 (6H, m), 4.43 (1H, dd, J = 2.8, 12.3 Hz), 4.78 (1H, dd, J = 1.4, 9.5 Hz), 5.34 (1H, m), 5.69 (1H, dd, J = 1.4, 7.5 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 13.6, 18.6, 18.7, 20.8, 20.8, 21.0, 24.7, 32.1, 32.1, 32.1, 35.9, 35.9, 53.5, 58.3, 62.5, 68.0, 68.1, 68.1, 68.2, 69.8, 71.5, 74.2, 98.2, 98.2, 153.6, 167.3, 167.3, 169.9, 170.0, 170.7, 171.9. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₇H₄₂NO₁₆PNa 690.2; Found 690.4. (3β) ¹H NMR (CDCl₃, 600 MHz): δ = 0.96 (6H, dt, J = 4.2, 7.4 Hz), 1.42 (4H, m), 1.68 (4H, m), 2.05 (3H, s), 2.12 (3H, s), 2.15 (3H, s), 2.32 (1H, dt, J = 2.8, 12.7 Hz), 2.52 (3H, s), 2.91 (1H, dd, J = 3.7, 12.8 Hz), 3.79 (1H, dd, J = 9.5, 11.3 Hz), 3.88 (3H, s), 4.13 (5H, m), 4.56 (1H, dd, J = 2.6, 12.2 Hz), 4.60 (1H, td, J = 3.7, 11.4, 12.6 Hz), 4.75 (1H, dd, J = 2.0, 9.5 Hz), 5.28 (1H, m), 5.64 (1H, dd, J = 2.0, 4.0 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 13.6, 18.6, 18.6, 20.8, 21.0, 24.7, 32.1, 32.1, 32.1, 32.2, 36.1, 36.1, 53.5, 58.9, 62.9, 68.4, 68.4, 68.5, 68.6, 71.8, 72.6, 74.0, 76.7, 98.8, 98.9, 153.5, 165.6, 169.8, 170.6, 170.7, 172.2. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₇H₄₂NO₁₆PNa 690.2; Found 690.4.

Phenyl (2,6-di-O-acetyl-β-d-galactopyranosyl)-(1→4)2,3,6-tri-O-acetyl-1-thio-β-d-glucopyranoside (4)

Acceptor 4 was prepared as previously described.³⁹ Acceptor 4 was purified by silica flash chromatography using 2% MeOH in CH₂Cl₂ as the eluent to provide 4 as a white solid (0.50 g, 0.78 mmol). ¹H NMR (CDCl₃, 600 MHz): δ = 2.03 (3H, s), 2.08 (3H, s), 2.09 (3H, s), 2.10 (3H, s), 2.11 (3H, s), 3.59 (1H, dd, J = 3.5, 9.8 Hz), 3.62 (1H, t, J = 6.5 Hz), 3.69 (2H, m), 3.86 (1H, d, J = 3.0 Hz), 4.16 (1H, dd, J = 5.5, 11.8 Hz), 4.22 (1H, dd, J = 6.6, 11.4 Hz), 4.32 (2H, m), 4.51 (1H, dd, J = 1.9, 12.0 Hz), 4.69 (1H, d, J = 10.1 Hz), 4.89 (1H, dd, J = 8.0, 9.8 Hz), 4.93 (1H, t, J = 9.7 Hz), 5.19 (1H, t, J = 8.9 Hz), 7.31 (3H, m), 7.47 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 21.1, 21.1, 21.2, 21.2, 62.7, 68.8, 70.4, 72.6, 73.0, 73.7, 74.1, 76.5, 77.1, 85.9, 101.2, 128.5, 129.2, 133.0, 169.9, 170.8, 170.8, 171.3, 171.7. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₈H₃₆O₁₅SNa 667.1667; Found 667.1769.

Trichloroacetimidatyl (2,6-di-O-acetyl-3,4-O-isopropylidene-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-d-glucopyranoside (S4)

Compound S3³⁹ (0.67 g, 0.98 mmol, 1.0 equiv) was dissolved in an acetone:H₂O (4:1, $1\overline{0}$ mL) mixture and then cooled to 0 °C. While stirring the mixture, NBS (0.52 g, 2.9 mmol, 3.0 equiv) was added to the reaction gradually over the duration of 30 minutes. The reaction was then stirred for an additional 30 minutes at room temperature. The solution was then concentrated under reduced pressure until the solution became turbid. The turbid mixture was diluted with CH₂Cl₂ (20 mL) and then quenched and washed with saturated Na₂S₂O₃ and NaHCO₃ (15 mL, 3X). The organic layer was then washed with brine, dried with Na₂SO₄, filtered, and then concentrated under reduced pressure to give the crude hemiacetal intermediate as a white solid (0.51 g, 0.86 mmol, 88% yield of crude). The crude hemiacetal was then co-distilled with toluene under reduced pressure (3X) and set under high vacuum for 3 hours. The crude hemiacetal was then dissolved in dry CH₂Cl₂ ($1\overline{0}$ mL) followed by the addition of 4 Å molecular sieves (2 g) and trichloroacetonitrile (345 μL, 3.44 mmol, 4.0 equiv), and then gas was exchanged with Ar. The solution was stirred at room temperature for 30 minutes and was then cooled to 0 °C followed by the addition of DBU (26 μL, 0.17 mmol, 0.2 equiv). The reaction was then stirred for 1 hour at 0 °C. The reaction was filtered and then concentrated under reduced pressure to provide the crude lactosyl donor. The crude material was then purified by silica flash chromatography using toluene:acetone (3:2) as the eluent to provide lactosyl donor S4 as a white solid (0.44 g, 0.60 mmol, 62% yield over 2 steps). ¹H NMR (CDCl₃, 600 MHz): δ = 1.31 (3H, s), 1.54 (3H, s), 2.01 (3H, s), 2.07 (3H, s), 2.08 (3H, s), 2.09 (3H, s), 2.12 (3H, s), 3.83 (1H, t, J = 9.8 Hz), 3.93 (1H, m), 4.14 (3H, m), 4.19 (1H, dd, J = 4.5, 12.2 Hz), 4.31 (2H, m), 4.41 (1H, d, J = 7.3 Hz), 4.43 (1H, dd, J = 2.0, 12.2 Hz), 4.85 (1H, dd, J = 6.1, 7.3 Hz), 5.06 (1H, dd, J = 3.8, 10.1 Hz), 5.55 (1H, t, J = 9.8 Hz), 6.48 (1H, d, J = 3.8 Hz), 8.64 (1H, s). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.7, 21.0, 21.0, 21.0, 21.1, 26.2, 27.4, 61.9, 63.3, 69.3, 70.1, 71.0, 71.2, 72.9, 73.1, 75.8, 77.0, 90.8, 93.1, 100.8, 111.0, 161.1, 169.4, 169.8, 170.3, 170.5, 170.9. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₇H₃₆Cl₃NO₁₆Na 758.1; Found 758.3.

Succinimidyl (2,6-di-O-acetyl-3,4-O-isopropylidene-β-d-galactopyranosyl)-(1→4)-2,3,6-tri-O-acetyl-β-d-glucopyranoside (S5)

Lactosyl donor S4 ($8\overline{0}$ mg, 0.11 mmol, 1.0 equiv) and NHS (63 mg, 0.55 mmol, 5.0 equiv) were co-distilled with toluene under reduced pressure (3X) followed by high vacuum overnight. The mixture was then dissolved in 5.0 mL of dry CH₂Cl₂ and then 4 Å molecular sieves (0.2 g) were added to the solution. Gas was then exchanged with Ar and the solution was stirred at room temperature for 30 minutes. TMSOTf (4.0 μL, 0.022 mmol, 0.2 equiv) was then introduced into the mixture at room temperature and the reaction was stirred for 30 minutes. The reaction was then quenched with TEA, filtered, and then concentrated under reduced pressure. The crude material was then purified by silica flash chromatography using 1% MeOH in CH₂Cl₂ to provide compound S5 as a white solid (69 mg, 0.10 mmol, 90% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 1.31 (3H, s), 1.53 (3H, s), 2.08 (3H, s), 2.08 (3H, s), 2.10 (3H, s), 2.12 (3H, s), 2.12 (3H, s), 2.73 (4H, s), 3.74 (1H, m), 3.94 (1H, m), 4.10 (1H, dd, J = 8.5, 10.0 Hz), 4.14 (2H, m), 4.19 (1H, dd, J = 5.8, 12.0 Hz), 4.27 (1H, dd, J = 7.5, 11.7 Hz), 4.33 (1H, dd, J = 4.7, 11.7 Hz), 4.39 (1H, d, J = 7.7 Hz), 4.41 (1H, dd, J = 2.3, 12.0 Hz), 4.86 (1H, dd, J = 6.4, 7.6 Hz), 5.13 (1H, d, J = 6.3 Hz), 5.17 (1H, t, J = 6.8 Hz), 5.20 (1H, t, J = 7.9 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.8, 20.9, 21.0, 21.0, 25.5, 26.3, 27.5, 62.1, 63.3, 70.2, 71.1, 72.5, 72.8, 73.1, 73.2, 75.5, 77.0, 100.7, 102.7, 111.0, 169.4, 169.6, 169.9, 170.3, 170.6, 171.0. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₉H₃₉NO₁₈Na 712.2; Found 712.1.

Succinimidyl (2,6-di-O-acetyl-β-d-galactopyranosyl)(1→4)-2,3,6-tri-O-acetyl-β-d-glucopyranoside (5)

Compound S5 (69 mg, 0.10 mmol, 1.0 equiv) was dissolved in 5 mL of an AcOH:H₂O (4:1) mixture and was heated at 80 °C for 1 hour with a condenser attached to the reaction flask. The reaction was then concentrated under reduced pressure followed by co-distillation with toluene under reduced pressure (3X). The crude was then purified by silica flash chromatography using 2% MeOH in CH₂Cl₂ as the eluent to provide lactosyl acceptor 5 as a white solid (55 mg, 0.084 mmol, 84% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 2.08 (3H, s), 2.11 (3H, s), 2.11 (3H, s), 2.12 (3H, s), 2.13 (3H, s), 2.73 (4H, s), 3.00 (1H, br), 3.28 (1H, br), 3.61 (1H, dd, J = 2.7, 9.4 Hz), 3.64 (1H, t, J = 6.5 Hz), 3.75 (1H, m), 3.84 (1H, br), 4.10 (1H, t, J = 9.4 Hz), 4.21 (2H, m), 4.36 (1H, dd, J = 6.4, 11.5 Hz), 4.40 (1H, d, J = 7.9 Hz), 4.47 (1H, dd, J = 2.3, 12.0 Hz), 4.86 (1H, dd, J = 8.0, 9.6 Hz), 5.13 (1H, d, J = 6.4 Hz), 5.19 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.8, 21.0, 21.0, 21.0, 21.0, 25.5, 62.1, 62.4, 68.4, 70.1, 72.3, 72.7, 72.9, 73.1, 73.9, 75.7, 101.0, 102.7, 169.6, 170.2, 170.4, 170.7, 171.2, 171.8. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₆H₃₅NO₁₈Na 672.1746; Found 672.1750.

Phenyl (6-O-pivaloyl-β-d-galactopyranosyl)-(1→4)-2,6-diO-pivaloyl-1-thio-β-d-glucopyranoside (6)

Acceptor 6 was prepared as previously described.³⁴ Acceptor 6 was purified by silica flash chromatography using toluene:acetone (1:1) as the eluent to provide 6 as a white solid (0.31 g, 0.45 mmol). ¹H NMR (CDCl₃, 600 MHz): δ = 1.19 (9H, s), 1.21 (9H, s), 1.26 (9H, s), 3.46 (1H, t, J = 9.2 Hz), 3.60 (1H, m), 3.66 (1H, m), 3.77 (4H, m), 3.91 (1H, t, J = 3.3 Hz), 4.16 (2H, m), 4.27 (3H, m), 4.36 (1H, dd, J = 4.1, 11.9 Hz), 4.50 (1H, d, J = 3.8 Hz), 4.69 (1H, d, J = 10.9 Hz), 4.72 (1H, d, J = 10.2 Hz), 4.90 (1H, t, J = 9.7 Hz), 7.28 (3H, m), 7.50 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 27.1, 27.1, 27.2, 38.7, 38.8, 38.9, 63.4, 63.7, 68.6, 70.7, 70.9, 73.2, 73.3, 74.4, 76.8, 81.5, 86.2, 104.2, 127.8, 128.9, 132.2, 133.3, 176.8, 178.8, 179.2. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₃₃H₅₀O₁₃SNa 709.3; Found 709.2.

Phenyl (6-O-benzyl-2-O-benzoyl-1-thio-β-d-galactopyranoside (7)

Acceptor 7 was prepared as previously described.³⁵ Acceptor 7 was purified by silica flash chromatography using hexanes:EtOAc (1:1) as the eluent to provide 7 as a white solid (0.80 g, 1.7 mmol). ¹H NMR (CDCl₃, 600 MHz): δ = 2.95 (1H, d, J = 3.6 Hz), 3.21 (1H, d, J = 7.6 Hz), 3.78 (1H, t, J = 4.8 Hz), 3.83 (1H, m), 3.88 (2H, d, J = 5.10 Hz), 4.14 (1H, t, J = 3.5 Hz), 4.64 (2H, br), 4.84 (1H, d, J = 10.0 Hz), 5.23 (1H, t, J = 9.6 Hz), 7.28 (3H, m), 7.37 (5H, m), 7.50 (4H, m), 7.63 (1H, m), 8.11 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 69.7, 69.8, 72.2, 73.8, 74.1, 77.2, 86.0, 127.8, 127.9, 128.0, 128.5, 128.5, 128.9, 129.5, 130.1, 132.6, 132.6, 133.5, 137.7, 167.0. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₆H₂₆O₆SNa 489.1; Found 489.0.

Phenyl (4,6-O-benzylidene-2-O-benzoyl-1-thio-β-d-galactopyranoside) (8)

Acceptor 8 was prepared as previously described.³¹ Acceptor 8 was purified by silica flash chromatography using hexanes:EtOAc (1:1) as the eluent to provide 8 as a white solid (1.70 g, 3.66 mmol). ¹H NMR (CDCl₃, 600 MHz): δ = 2.65 (1H, d, J = 10.8 Hz), 3.61 (1H, s), 3.91 (1H, td, J = 10.1, 3.6 Hz), 4.06 (1H, d, J = 12.3 Hz), 4.26 (1H, d, J = 3.5 Hz), 4.43 (1H, d, J = 12.3 Hz), 4.84 (1H, d, J = 9.8 Hz), 5.28 (1H, t, J = 9.7 Hz), 5.55 (1H, s), 7.28 (2H, m), 7.34 (1H, m), 7.45 (7H, m), 7.60 (3H, m), 8.09 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 69.2, 70.1, 70.7, 73.0, 75.7, 84.9, 101.5, 126.6, 128.3, 128.3, 128.5, 128.9, 129.5, 129.9, 130.0, 131.2, 133.3, 134.0, 137.5, 166.0. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₆H₂₄O₆SNa 487.1; Found 487.1.

General procedure for sialylations in Table 1

Acceptors (1.0 equiv) were co-distilled with toluene under reduced pressure (3X) and then were combined with freshly prepared sialyl phosphate donor 3 (1.5 equiv) followed by high vacuum for 3 hours. The donor acceptor pair were then dissolved in dry CH₂Cl₂ (volume calculated to make 20 mM solution of acceptor), 4 Å molecular sieves were added (mass = 4 × acceptor mass), gas was exchanged with Ar, and the resulting solution was stirred at room temperature for 1 hour. The reaction mixture was cooled to −78 °C and TMSOTf (1.5 equiv) was added to initiate the reaction. The reaction was stirred at −78 °C for 1 hour and the reaction was quenched with saturated NaHCO₃ with vigorous stirring as the reaction slowly warmed to room temperature. The quenched reaction mixture was then diluted with CH₂Cl₂ (10 mL) and the organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was then analyzed by ¹H NMR to determine α:β ratios based on the integrals of the Neu5Ac C3 equatorial protons. Crudes were then purified by silica flash chromatography using appropriate solvent mixtures to isolate α:β mixtures of the products to obtain the isolated yield. Isolated and pure α anomers were then characterized.

Phenyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-(2,6-di-O-acetyl-β-d-galactopyranosyl)(1→4)-2,3,6-tri-O-acetyl-1-thio-β-d-glucopyranoside (9)

Compound 9 was purified by silica flash chromatography using toluene:acetone (1:1) as the eluent to provide 9 as a white solid (0.29 g, 0.26 mmol, 68% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 2.01 (3H, s), 2.02 (3H, s), 2.03 (3H, s), 2.07 (6H, s), 2.08 (3H, s), 2.10 (3H, s), 2.12 (1H, m), 2.16 (3H, s), 2.49 (3H, s), 2.70 (1H, d, J = 5.0 Hz), 2.83 (1H, dd, J = 3.5, 12.1 Hz), 3.67 (3H, m), 3.77 (2H, m), 3.81 (4H, m), 4.01 (2H, m), 4.09 (1H, t, J = 3.8 Hz), 4.14 (1H, dd, J = 6.3, 11.91 Hz), 4.40 (1H, dd, J = 2.8, 12.2 Hz), 4.51 (2H, m), 4.59 (1H, dd, J = 1.5, 9.4 Hz), 4.68 (1H, d, J = 10.1 Hz), 4.87 (2H, m), 5.16 (1H, dd, J = 7.9, 10.2 Hz), 5.20 (1H, t, J = 9.2 Hz), 5.36 (1H, dt, J = 2.8, 7.6 Hz), 5.56 (1H, dd, J = 1.6, 7.7 Hz), 7.29 (3H, m), 7.46 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.8, 20.9, 20.9, 21.0, 21.0, 21.0, 21.2, 21.6, 24.8, 35.8, 53.5, 53.6, 59.0, 62.5, 63.0, 63.5, 66.4, 69.3, 69.7, 70.4, 71.9, 72.8, 73.5, 73.9, 74.8, 76.0, 76.1, 85.4, 99.3, 100.9, 128.3, 129.0, 132.0, 133.0, 153.6, 168.4, 169.4, 169.8, 169.9, 170.2, 170.3, 170.4, 170.5, 171.2, 172.3. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₇H₅₉NO₂₇SNa 1124.2887; Found 1124.2898.

Succinimidyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4O-carbonyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-(2,6-di-O-acetyl-β-d-galactopyranosyl)(1→4)-2,3,6-tri-O-acetyl-β-d-glucopyranoside (10)

Compound 10 was purified by silica flash chromatography using toluene:acetone (1:1) as the eluent to provide 10 as a white solid (0.13 g, 0.12 mmol, 81% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 2.00 (1H, t, J = 12.8 Hz), 2.07 (3H, s), 2.09 (3H, s), 2.11 (3H, s), 2.11 (3H, s), 2.14 (9H, s), 2.23 (3H, s), 2.50 (3H, s), 2.74 (4H, s), 2.97 (1H, dd, J = 3.2, 12.0 Hz), 3.47 (1H, br), 3.73 (3H, m), 3.81 (3H, s), 3.89 (2H, m), 3.98 (1H, dd, J = 7.1, 12.3 Hz), 4.18 (1H, t, J = 9.3 Hz), 4.23 (1H, dd, J = 6.3, 11.9 Hz), 4.27 (2H, d, J = 6.2 Hz), 4.36 (1H, dd, J = 3.0, 10.0 Hz), 4.42 (1H, dd, J = 2.0, 11.8 Hz), 4.48 (1H, dd, J = 2.4, 12.2 Hz), 4.53 (1H, dd, J = 1.7, 9.4 Hz), 4.64 (1H, d, J = 7.9 Hz), 5.05 (1H, dd, J = 8.1, 9.9 Hz), 5.12 (1H, t, J = 6.6 Hz), 5.17 (1H, t, J = 7.2 Hz), 5.24 (1H, t, J = 8.1 Hz), 5.60 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.8, 20.8, 20.9, 20.9, 20.9, 21.0, 21.2, 24.9, 25.5, 36.8, 53.1, 59.3, 62.1, 63.2, 68.9, 69.8, 69.8, 71.0, 72.2, 72.5, 72.9, 73.7, 74.5, 74.9, 75.9, 76.4, 79.3, 99.8, 101.6, 103.3, 154.5, 166.7, 168.9, 169.5, 170.0, 170.2, 170.7, 170.7, 170.8, 171.0, 171.3, 173.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₅H₅₈N₂O₃₀Na 1129.2967; Found 1129.2948.

Phenyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-(6-O-pivaloyl-β-d-galactopyranosyl)(1→4)-2,6-di-O-pivaloyl-1-thio-β-d-glucopyranoside (11)

Compound 11 was purified by silica flash chromatography using toluene:acetone (1:1) as the eluent to provide 11 as a white solid (0.16 g, 0.14 mmol, 52% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 1.20 (9H, s), 1.21 (9H, s), 1.26 (9H, s), 2.05 (3H, s), 2.13 (3H, s), 2.16 (3H, s), 2.21 (1H, t, J = 13.0 Hz), 2.49 (1H, d, J = 2.64Hz), 2.52 (3H, s), 3.00 (1H, dd, J = 3. 5, 12.3 Hz), 3.20 (1H, d, J = 1.7 Hz), 3.52 (1H, t, J =9.2 Hz), 3.70 (2H, m), 3.78 (4H, m), 3.84 (3H, s), 4.04 (2H, m), 4.09 (1H, dd, J = 7.9, 12.1 Hz), 4.15 (1H, dd, J = 3.3, 9.6 Hz), 4.23 (1H, dd, J = 8.1, 11.9 Hz), 4.31 (1H, d, J = 1.0 Hz), 4.40 (1H, dd, J = 4.2, 11.9 Hz), 4.47 (1H, dd, J = 2.6, 12.3 Hz), 4.51 (1H, d, J = 7.9 Hz), 4.65 (1H, dd, J = 1.1, 9.5 Hz), 4.71 (1H, d, J = 10.3 Hz), 4.88 (1H, dd, J = 1.5, 12.1 Hz), 4.94 (1H, dd, J = 9.4, 10.1 Hz), 5.55 (1H, td, J = 4.1, 10.7 Hz), 5.70 (1H, dd, J = 1.1, 8.5 Hz), 7.28 (3H, m), 7.51 (2H, m). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.7, 20.9, 21.2, 24.7, 27.1, 27.1, 27.2, 36.6, 38.7, 38.8, 53.5, 58.8, 63.2, 63.3, 63.6, 67.9, 68.8, 68.9, 70.7, 71.2, 72.6, 74.4, 74.7, 76.7, 76.9, 77.2, 82.2, 86.6, 97.9, 104.2, 127.6, 128.9, 131.8, 134.0, 153.3, 168.3, 169.8, 170.4, 170.7, 172.0, 176.7, 177.9, 178.6. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₅₂H₇₃NO₂₅SNa 1166.4085; Found 1166.4072.

Phenyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-2-O-benzoyl-6-O-benzyl-1-thio-β-d-galactopyranoside (12)

Compound 12 was purified by silica flash chromatography using hexanes:EtOAc (1:1) as the eluent to provide 12 as a white solid (1.30 g, 1.41 mmol, 89% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 1.48 (3H, s), 2.02 (3H, s), 2.07 (1H, t, J = 6.3 Hz), 2.13 (3H, s), 2.44 (3H, s), 2.74 (1H, br), 2.86 (1H, dd, J = 3.4, 12.0 Hz), 3.55 (1H, dd, J = 9.4, 11.2 Hz), 3.73 (3H, s), 3.85 (5H, m), 3.95 (1H, dd, J = 7.4, 12.2 Hz), 4.43 (1H, dd, J = 2.5, 12.3 Hz), 4.49 (1H, dd, J = 1.8, 9.4 Hz), 4.52 (1H, dd, J = 3.1, 9.5 Hz), 4.61 (2H, m), 4.96 (1H, d, J = 10.0 Hz), 5.43 (1H, t, J = 9.7 Hz), 5.49 (1H, dd, J = 1.9, 8.7 Hz), 5.55 (1H, td, J = 2.4, 8.7 Hz), 7.25 (3H, m), 7.35 (5H, m), 7.49 (4H, m), 7.61 (1H, t, J = 7.4 Hz), 8.18 (2H, dd, J = 1.0, 8.1 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.2, 20.8, 21.3, 24.6, 35.9, 53.3, 58.8, 63.4, 67.5, 68.5, 68.9, 69.1, 71.5, 73.6, 74.9,75.2, 75.5, 76.5, 86.7, 97.5, 127.7, 127.7, 127.8, 128.4, 128.5, 128.8, 130.1, 130.2, 132.6, 132.9, 133.3, 138.1, 153.4, 165.3, 168.5, 170.0, 170.5, 170.8, 171.8. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₅H₄₉NO₁₈SNa 946.3; Found 946.2.

Phenyl (methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-carbonyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-2-O-benzoyl-4,6-O-benzylidene-1-thio-βD-galactopyranoside (13)

Compound 13 was purified by silica flash chromatography using hexanes:EtOAc (1:1) as the eluent to provide 13 as a white solid (0.96 g, 1.0 mmol, 87% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 1.76 (1H, dd, J = 12.3, 13.5 Hz), 1.82 (3H, s), 2.05 (3H, s), 2.21 (3H, s), 2.46 (3H, s), 2.92 (1H, dd, J = 3.2, 12.1 Hz), 3.48 (3H, s), 3.53 (1H, dd, J = 9.5, 11.2 Hz), 3.73 (1H, br), 3.75 (1H, m), 4.01 (1H, m), 4.12 (1H, d, J = 3.2 Hz), 4.17 (1H, dd, J = 1.4, 12.0 Hz), 4.39 (1H, dd, J = 1.4, 12.1 Hz), 4.47 (2H, m), 4.62 (1H, dd, J = 3.5, 9.7 Hz), 5.00 (1H, d, J = 9.8 Hz), 5.38 (1H, s), 5.44 (1H, t, J = 9.7 Hz), 5.57 (2H, m), 7.25 (2H, m), 7.31 (1H, m), 7.38 (3H, m), 7.43 (2H, m), 7.50 (2H, t, J = 7.8 Hz), 7.61 (3H, m), 8.16 (2H, d, J = 7.1 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.7, 21.0, 21.6, 24.8, 37.2, 53.0, 58.9, 63.9, 68.1, 68.3, 69.3, 69.6, 71.6, 73.0, 73.9, 75.0, 75.1, 85.2, 96.9, 101.0, 126.7, 128.1, 128.3, 128.6, 128.8, 129.2, 130.1, 130.5, 131.4, 133.3, 134.1, 137.9, 153.5, 165.0, 168.8, 170.3, 170.6, 171.1, 172.1. LRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₅H₄₇NO₁₈SNa 944.2; Found 944.1.

Succinimidyl (4,6-O-benzylidene-2,3-di-O-benzoyl-β-d-glucopyranoside (15)

Compound 14⁴⁰ (1.58 g, 2.78 mmol, 1.0 equiv) and NHS (1.60 g, 13.9 mmol, 5.0 equiv) were co-distilled with toluene under reduced pressure (3X) followed by high vacuum for 3 hours. The mixture was then dissolved in $1\overline{0}$ mL of dry CH₂Cl₂ and 4 Å molecular sieves (5 g) were added to the solution. Gas was then exchanged with Ar and the solution was stirred at room temperature for 30 minutes. The mixture was then cooled to −10 °C using a salted ice bath and then NIS (1.88 g, 8.34 mmol, 3.0 equiv) was quickly added to the reaction vessel. Gas was exchanged with Ar again, and TMSOTf (0.10 mL, 0.56 mmol, 0.2 equiv) was then introduced into the mixture and the reaction was stirred at −10 °C for 30 minutes and then at 0 °C for 1 hour. The reaction was then diluted with CH₂Cl₂ (10 mL) and quenched with a saturated solution of Na₂S₂O₃ and NaHCO₃ (20 mL). The quenched reaction was then filtered, and the resulting organic layer was washed with saturated Na₂S₂O₃ and NaHCO₃ (20 mL, 2X). The collected organic layer was then washed with brine, dried over Na₂SO₄, filtered, and then concentrated under reduced pressure. The crude material was then purified by silica flash chromatography using hexanes:EtOAc (4:1) as the eluent with 10% CH₂Cl₂ to ensure compound solubility on the column. Compound 15 was isolated as a white solid (1.34 g, 2.34 mmol, 84% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 2.72 (4H, s), 3.91 (1H, m), 4.02 (1H, t, J = 10.3 Hz), 4.41 (1H, dd, J = 4.9, 10.7 Hz), 4.53 (1H, t, J = 9.3 Hz), 5.49 (1H, d, J = 5.9 Hz), 5.65 (1H, s), 5.76 (2H, m), 7.35 (3H, m), 7.44 (6H, m), 7.56 (2H, m), 8.04 (2H, d, J = 7.1 Hz), 8.10 (2H, d, J = 7.1 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 25.4, 66.6, 68.3, 70.9, 72.3, 77.3, 101.5, 103.1, 126.2, 128.3, 128.4, 128.5, 128.9, 129.2, 129.2, 130.0, 130.1, 133.4, 133.6, 136.7, 165.0, 165.5, 170.4. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₃₁H₂₇NO₁₀Na 596.1527; Found 596.1529.

Succinimidyl (2,3-di-O-benzoyl-6-O-benzyl-β-d-glucopyranoside (16)

Compound 15 (0.80 g, 1.4 mmol, 1.0 equiv) was co-distilled with toluene under reduced pressure (3X), set under high vacuum for 3 hours, and was then dissolved in $1\overline{0}$ mL of dry CH₂Cl₂. Gas was then exchanged with Ar and the solution was cooled to 0 °C followed by the addition of Et₃SiH (0.67 mL, 4.2 mmol, 3.0 equiv) into the reaction vessel. This mixture was then stirred for 10 minutes at 0 °C and then BF₃ Et₂O (0.69 mL, 5.6 mmol, 4.0 equiv) was introduced into the reaction dropwise. The reaction was then stirred for 1.5 hours at 0 °C. The reaction was then quenched with saturated NaHCO₃ (20 mL). The aqueous layer was extracted with CH₂Cl₂ (20 mL, 3X) and the combined organic layers were washed with brine, dried with Na₂SO₄, filtered, and concentrated under reduced pressure. The crude was then purified by silica flash chromatography using toluene:EtOAc (4:1) as the eluent with 10% CH₂Cl₂ to ensure compound solubility. Compound 16 was isolated as a white solid (0.65 g, 1.1 mmol, 81% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 2.67 (4H, m), 3.41 (1H, br), 3.80 (1H, m), 3.85 (1H, m), 3.95 (1H, dd, J = 3.9, 10.6 Hz), 4.06 (1H, t, J = 9.2 Hz), 4.65 (2H, q, J = 11.5 Hz), 5.33 (1H, d, J = 7.8 Hz), 5.5 (1H, t, J = 9.1 Hz), 5.70 (1H, dd, J = 7.9, 9.1 Hz), 7.33 (1H, m), 7.40 (8H, m), 7.55 (2H, m), 8.02 (2H, d, J = 7.1 Hz), 8.08 (2H, d, J = 7.1 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 25.4, 69.7, 69.9, 70.6, 73.8, 75.5, 76.6, 103.5, 127.7, 127.9, 128.4, 128.5, 128.5, 128.8, 129.1, 130.0, 130.1, 133.4, 133.7, 137.8, 165.4, 167.2, 170.1. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₃₁H₂₉NO₁₀Na 598.1684; Found 598.1680.

Phenyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)2-O-benzoyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (17)

Compound 13 (0.66 g, 0.72 mmol, 1.0 equiv) was dissolved in MeOH (9 mL) and cooled to 0 °C. Freshly prepared 1.0 M NaOMe in MeOH (1 mL) was then introduced into the reaction mixture to generate a 0.1 M NaOMe solution with the substrate, and was stirred for 15 minutes at 0 °C. The reaction was stirred at room temperature for 1 hour and then quenched with Amberlite® IR120 acidic resin, filtered, and concentrated under reduced pressure to reveal a white solid in quantitative yield. The solid was dried by co-distillation with toluene under reduced pressure (3X) followed by high vacuum for 3 hours. Dry intermediate was then dissolved in pyridine ($1\overline{0}$ mL) followed by the addition of DMAP (18 mg, 0.14 mmol, 0.2 equiv). Gas was exchanged with Ar and then the mixture was cooled to 0 °C. Once cool, Ac₂O (1.4 mL, 14 mmol, 20 equiv) was introduced and the reaction was stirred for 12 hours while gradually warming to room temperature. The reaction was then cooled to 0 °C again and quenched with excess MeOH and stirred for 10 minutes. The crude was concentrated under reduced pressure, co-distilled with toluene under reduced pressure (3X) and was reconstituted in CH₂Cl₂ (20 mL). The crude was then washed with saturated NaHCO₃ (20 mL, 3X) and the resulting organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude was then purified by silica flash chromatography using toluene:acetone (3:1) as the eluent to reveal 17 as a white solid (0.51 g, 0.55 mmol, 76% yield over 2 steps). ¹H NMR (CDCl₃, 600 MHz): δ = 1.69 (1H, t, J = 12.7 Hz), 1.82 (3H, s), 1.83 (3H, s), 1.95 (3H, s), 2.07 (3H, s), 2.23 (3H, s), 2.56 (1H, dd, J = 4.5, 12.9 Hz), 3.56 (3H, s), 3.67 (1H, br), 3.90 (2H, m), 4.00 (1H, dd, J = 6.2, 12.2 Hz), 4.05 (1H, d, J = 3.4 Hz), 4.13 (1H, d, J = 11.2 Hz), 4.35 (1H, dd, J = 2.5, 12.4 Hz), 4.39 (1H, d, J = 12.0 Hz), 4.61 (1H, dd, J = 3.4, 9.7 Hz), 4.71 (1H, m), 4.99 (1H, d, J = 9.8 Hz), 5.06 (1H, br), 5.25 (1H, d, J = 9.5 Hz), 5.38 (1H, s), 5.43 (1H, t, J = 9.8 Hz), 5.52 (1H, m), 7.25 (3H, m), 7.38 (3H, m), 7.44 (2H, m), 7.51 (2H, t, J = 7.7 Hz), 7.60 (3H, m), 8.16 (2H, d, J = 7.1 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.6, 20.8, 20.9, 21.5, 23.2, 38.2, 49.1, 52.8, 62.6, 66.9, 67.6, 68.1, 68.8, 69.3, 69.6, 72.3, 73.3, 73.6, 85.3, 96.7, 101.0, 126.6, 127.9, 128.1, 128.4, 128.7, 129.1, 130.0, 130.4, 131.6, 133.1, 133.7, 137.8, 164.9, 168.8, 170.1, 170.2, 170.3, 170.8, 170.9. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₆H₅₁NO₁₈SNa 960.2719; Found 960.2715.

Succinimidyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)(2→3)-2-O-benzoyl-4,6-O-benzylidene-1-amino-β-d-galactopyranoside (S6)

Donor 17 ($5\overline{0}$ mg, 0.053 mmol, 1.5 equiv) and acceptor 16 (21 mg, 0.036 mmol, 1.0 equiv) were combined and co-distilled with toluene under reduced pressure (3X) and set under high vacuum for 3 hours. The donor and acceptor pair were dissolved in dry CH₂Cl₂ (5 mL) followed by the addition of 4 Å molecular sieves (0.2 g) and gas was exchanged with Ar. The mixture was stirred at room temperature for 1 hour before cooling the mixture to 0 °C. NIS (36 mg, 0.16 mmol, 4.5 equiv) was then introduced into the reaction mixture and gas was exchanged with Ar again followed by the addition of TMSOTf (2.0 μL, 0.011 mmol, 0.3 equiv). The reaction was then stirred at 0 °C for 1.5 hours. The reaction was then diluted with CH₂Cl₂ (5 mL) and quenched with a saturated solution of Na₂S₂O₃ and NaHCO₃ (10 mL). The quenched reaction was then filtered, and the resulting organic layer was washed with saturated Na₂S₂O₃ and NaHCO₃ (10 mL, 2X). The resulting organic layer was washed with brine, dried with Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude solid was then purified by silica flash chromatography using toluene:acetone (3:1) as the eluent to reveal compound S6, and not desired compound. This procedure was repeated using TfOH as the acid catalyst and resulted in the same product. ¹H NMR (CDCl₃, 600 MHz): δ = 1.68 (1H, t, J = 12.8 Hz), 1.84 (3H, s), 1.93 (3H, s), 1.97 (3H, s), 2.09 (3H, s), 2.24 (3H, s), 2.53 (1H, br), 2.56 (1H, dd, J = 4.4, 12.9 Hz), 2.65 (3H, m), 3.49 (3H, s), 3.72 (1H, s), 3.97 (2H, m), 4.04 (1H, dd, J = 6.2, 12.4 Hz), 4.15 (2H, m), 4.24 (1H, dd, J = 1.4, 12.3 Hz), 4.34 (1H, dd, J = 2.6, 12.4 Hz), 4.65 (1H, dd, J = 3.3, 9.9 Hz), 4.70 (1H, m), 5.06 (1H, br), 5.28 (1H, d, J = 10.2 Hz), 5.37 (1H, s), 5.50 (1H, td, J = 3.0, 12.2 Hz), 5.53 (1H, d, J = 9.2 Hz), 6.36 (1H, t, J = 9.6 Hz), 7.34 (1H, m), 7.39 (2H, t, J = 7.3 Hz), 7.47 (2H, t, J = 7.8 Hz), 7.58 (1H, t, J = 7.4 Hz), 7.66 (2H, d, J = 7.0 Hz), 8.02 (2H, d, J = 7.1 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.7, 20.8, 20.9, 21.5, 23.2, 27.9, 38.6, 49.3, 52.8, 62.6, 66.8, 67.0, 67.6, 68.2, 68.7, 68.7, 72.5, 72.7, 72.9, 78.7, 96.7, 101.7, 127.1, 128.3, 128.6, 129.2, 129.6, 129.9, 133.4, 137.9, 165.4, 168.8, 170.1, 170.3, 170.8, 171.0, 174.8, 176.2. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₄H₅₀N₂O₂₀Na 949.2849; Found 949.2838.

Trichloroacetimidatyl (methyl 5-acetamido-4,7,8,9-tetra-Oacetyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-2-O-benzoyl-4,6-O-benzylidene-β-d-galactopyranoside (18)

Compound 17 (0.41 g, 0.44 mmol, 1.0 equiv) was dissolved in an acetone:H₂O (4:1) mixture ($1\overline{0}$ mL) and then cooled to 0 °C. While stirring, NBS (94 mg, 0.53 mmol, 1.2 equiv) was added gradually to the reaction and the mixture was stirred for 30 minutes. After 30 minutes of mixing, NBS (78 mg, 0.44 mmol, 1.0 equiv) was added and the mixture was stirred at 0 °C for another 1 hour. The reaction was then concentrated under reduced pressure until the solution became turbid. The turbid mixture was diluted with CH₂Cl₂ (20 mL) and then quenched and washed with saturated Na₂S₂O₃ and NaHCO₃ (15 mL, 3X). The organic layer was then washed with brine, dried with Na₂SO₄, filtered, and then concentrated under reduced pressure. The crude hemiacetal was passed through a short bed of silica using toluene:acetone (2:1) as the eluent to give the crude hemiacetal intermediate as a white solid (0.31 g, 0.36 mmol, 82% crude). The crude hemiacetal was then co-distilled with toluene under reduced pressure (3X) and set under high vacuum for 3 hours. The crude hemiacetal was then dissolved in dry CH₂Cl₂ (5 mL) followed by the addition of 4 Å molecular sieves (1 g), trichloroacetonitrile (144 μL, 1.44 mmol, 4.0 equiv), and the gas was exchanged with Ar. The solution was stirred at room temperature for 30 minutes. The mixture was then cooled to 0 °C followed by the addition of DBU ($1\overline{0}$ μL, 0.072 mmol, 0.2 equiv) and the reaction was then stirred for 1.5 hours at 0 °C. The reaction was filtered and then concentrated under reduced pressure to provide the crude donor. The crude material was then purified by silica flash chromatography using toluene:acetone (3:1) as the eluent to provide donor 18 as a white solid (0.32 g, 0.32 mmol, 73% yield over 2 steps). ¹H NMR (CDCl₃, 600 MHz): δ = 1.79 (1H, t, J = 12.7 Hz), 1.90 (3H, s), 1.96 (3H, s), 2.06 (3H, s), 2.16 (3H, s), 2.21 (3H, s), 2.59 (1H, dd, J = 4.4, 12.9 Hz), 3.52 (3H, s), 4.08 (3H, m), 4.14 (1H, br), 4.21 (1H, d, J = 12.5 Hz), 4.26 (1H, dd, J = 2.7, 12.5 Hz), 4.32 (1H, d, J = 12.6 Hz), 4.42 (1H, d, J = 3.1 Hz), 4.78 (1H, m), 5.11 (1H, d, J = 9.4 Hz), 5.18 (1H, dd, J = 3.3, 10.7 Hz), 5.37 (1H, dd, J = 1.5, 9.8 Hz), 5.47 (1H, s), 5.49 (1H, m), 5.70 (1H, dd, J = 3.3, 10.7 Hz), 6.79 (1H, d, J = 3.3 Hz), 7.37 (3H, m), 7.47 (2H, t, J = 7.8 Hz), 7.54 (2H, d, J = 6.7 Hz), 7.59 (1H, t, J = 7.4 Hz), 8.07 (2H, d, J = 7.1 Hz), 8.49 (1H, s). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.8, 20.9, 21.4, 23.2, 38.7, 49.5, 52.9, 62.5, 65.2, 67.0, 67.4, 68.0, 68.6, 69.0, 72.7, 73.5, 91.2, 95.1, 96.6, 100.6, 126.3, 128.2, 128.3, 128.5, 129.0, 129.1, 129.6, 129.8, 133.4, 137.7, 160.4, 165.5, 169.0, 169.8, 170.0, 170.3, 170.7, 171.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₂H₄₇Cl₃N₂O₁₉Na 1011.2; Found 1011.1.

Phenyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)4-O-acetyl-2-O-benzoyl-6-O-benzyl-1-thio-β-d-galactopyranoside (20)

Compound 12 (1.3 g, 1.4 mmol, 1.0 equiv) was dissolved in MeOH (18 mL) and cooled to 0 °C. Freshly prepared 1.0 M NaOMe in MeOH (2.0 mL) was then introduced to the reaction mixture to generate a 0.1 M NaOMe solution with the substrate, and was stirred for 15 minutes at 0 °C. The reaction was then stirred at room temperature for 1 hour and quenched with Amberlite® IR120 acidic resin, filtered, and concentrated under reduced pressure to reveal a white solid in quantitative yield. The solid was dried by co-distillation with toluene under reduced pressure (3X) followed by high vacuum for 3 hours. Dry intermediate was then dissolved in pyridine ($2\overline{0}$ mL) followed by the addition of DMAP (35 mg, 0.28 mmol, 0.2 equiv). Gas was exchanged with Ar and the mixture was cooled to 0 °C. Once cool, Ac₂O (3.3 mL, 35 mmol, 25 equiv) was introduced and the reaction was stirred for 12 hours while gradually warming to room temperature. The reaction was then cooled to 0 °C again and quenched with excess MeOH and stirred for 10 minutes. The crude was concentrated under reduced pressure, co-distilled with toluene under reduced pressure (3X) and was reconstituted in CH₂Cl₂ (20 mL). The crude was then washed with saturated NaHCO₃ (20 mL, X 3) and the resulting organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude was then purified by silica flash chromatography using toluene:acetone (3:1) as the eluent to reveal 20 as a white solid (1.02 g, 1.04 mmol, 74% yield over 2 steps). ¹H NMR (CDCl₃, 600 MHz): δ = 1.43 (3H, s), 1.74 (1H, t, J = 12.4 Hz), 1.80 (3H, s), 1.99 (3H, s), 2.02 (3H, s), 2.08 (3H, s), 2.17 (3H, s), 2.56 (1H, dd, J = 4.6, 12.6 Hz), 3.51 (1H, dd, J = 6.1, 10.0 Hz), 3.61 (2H, m), 3.82 (1H, m), 3.86 (3H, s), 3.90 (1H, dd, J = 5.7, 12.5 Hz), 3.98 (1H, t, J = 6.2 Hz), 4.27 (1H, dd, J = 2.4, 12.5 Hz), 4.48 (1H, d, J = 11.6 Hz), 4.56 (1H, d, J = 11.6 Hz), 4.76 (1H, dd, J = 3.2, 9.6 Hz), 4.85 (1H, m), 4.96 (1H, d, J = 10.3 Hz), 5.10 (2H, m), 5.19 (1H, dd, J = 2.8, 9.5 Hz), 5.32 (1H, t, J = 9.8 Hz), 5.57 (1H, m), 7.25 (3H, m), 7.35 (5H, m), 7.50 (4H, m), 7.59 (1H, t, J = 7.4 Hz), 8.19 (2H, d, J = 7.1 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.2, 20.8, 20.8, 21.5, 23.2, 37.4, 48.8, 53.2, 62.3, 66.5, 67.4, 68.4, 68.5, 69.3, 69.4, 71.8, 72.4, 73.5, 75.8, 86.5, 96.8, 127.7, 127.7, 127.7, 128.3, 128.4, 128.8, 130.2, 130.4, 132.3, 133.0, 133.2, 138.0, 165.3, 168.0, 170.1, 170.3, 170.3, 170.5, 170.7, 170.9. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₄₈H₅₅NO₁₉SNa 1004.2981; Found 1004.2967.

Succinimidyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)(2→3)-(4-O-acetyl-2-O-benzoyl-6-O-benzyl-β-d-galactopyranosyl)-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-d-glucopyranoside (2)

Donor 20 (0.36 g, 0.37 mmol, 1.5 equiv) and acceptor 16 (0.14 g, 0.25 mmol, 1.0 equiv) were combined and co-distilled with toluene under reduced pressure (3X) and set under high vacuum for 3 hours. The donor acceptor pair were dissolved in dry CH₂Cl₂ (5 mL) followed by the addition of 4 Å molecular sieves (1 g) and gas was exchanged with Ar. The mixture was stirred at room temperature for 1 hour before cooling the mixture to 0 °C. NIS (0.25 g, 1.1 mmol, 4.5 equiv) was then introduced into the reaction mixture and gas was exchanged with Ar again followed by the addition of TfOH (6 μL, 0.07 mmol, 0.3 equiv). The reaction was then stirred at 0 °C for 1.5 hours and the reaction was diluted with CH₂Cl₂ (15 mL) and then quenched with saturated Na₂S₂O₃ and NaHCO₃ (20 mL). The organic layer was separated, filtered, and washed with saturated Na₂S₂O₃ and NaHCO₃ (20 mL, 2X). The resulting organic layer was washed with brine, dried with Na₂SO₄, filtered, and concentrated under reduced pressure. The resulting crude solid was then purified by silica flash chromatography using toluene:acetone (3:1) as the eluent to afford compound 2 as a white solid (0.32 g, 0.22 mmol, 87% yield). Only β anomer was observed due to anchimeric assistance. ¹H NMR (CDCl₃, 600 MHz): δ = 1.41 (3H, s), 1.71 (1H, t, J = 12.4 Hz), 1.78 (3H, s), 1.94 (3H, s), 1.97 (3H, s), 2.09 (3H, s), 2.13 (3H, s), 2.56 (5H, m), 2.80 (1H, t, J = 9.2 Hz), 2.91 (1H, dd, J = 4.8, 9.5 Hz), 3.46 (2H, m), 3.55 (1H, dd, J = 2.8, 10.7 Hz), 3.65 (1H, d, J = 9.2 Hz), 3.71 (1H, t, J = 7.6 Hz), 3.78 (3H, s), 3.81 (1H, q, J = 10.5 Hz), 3.96 (1H, dd, J = 6.1, 12.5 Hz), 4.02 (1H, d, J = 11.7 Hz), 4.10 (1H, d, J = 11.8 Hz), 4.24 (2H, br), 4.34 (1H, t, J = 9.3 Hz), 4.38 (1H, dd, J = 2.28, 12.42 Hz), 4.58 (1H, dd, J = 3.2, 10.1 Hz), 4.83 (1H, td, J = 4.6, 11.9 Hz), 4.94 (1H, d, J = 3.0 Hz), 4.98 (2H, t, J = 8.9 Hz), 5.19 (2H, dd, J = 7.6, 10.0 Hz), 5.24 (1H, d, J = 7.4 Hz), 5.56 (1H, t, J = 7.6 Hz), 5.67 (2H, t, J = 8.6 Hz), 7.18 (2H, t, J = 7.3 Hz), 7.27 (3H, m), 7.32 (7H, m), 7.39 (2H, t, J = 7.8 Hz), 7.50 (4H, m), 7.57 (1H, t, J = 7.3 Hz), 8.02 (4H, t, J = 6.7 Hz), 8.26 (2H, d, J = 7.2 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.3, 20.7, 20.8, 20.9, 21.5, 23.1, 25.3, 37.2, 48.7, 53.2, 62.6, 66.4, 66.6, 67.2, 67.3, 68.7, 69.5, 70.4, 71.0, 71.7, 71.7, 72.8, 72.9, 74.0, 75.0, 75.7, 96.9, 101.3, 102.9, 127.4, 127.4, 127.4, 127.5, 128.2, 128.3, 128.3, 128.3, 128.6, 129.1, 129.2, 129.8, 129.9, 130.0, 130.1, 130.5, 133.0, 133.2, 138.0, 138.4, 164.9, 165.0, 165.2, 167.9, 170.0, 170.0, 170.2, 170.3, 170.4, 170.8, 170.8. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₇₃H₇₈N₂O₂₉Na 1469.4582; Found 1469.4562.

Succinimidyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonate)-(2→3)-(4-O-acetyl-2-O-benzoyl-β-d-galactopyranosyl)(1→4)-2,3-di-O-benzoyl-β-d-glucopyranoside (S7)

Compound 2 (0.32 g, 0.22 mmol, 1.0 equiv) was dissolved in a mixture of MeOH:EtOAc (1:3, $1\overline{0}$ mL) followed by the addition of palladium hydroxide on carbon (55 mg, 0.25 g/mmol substrate) at room temperature. The reaction was then stirred for 1 hour under an atmosphere of H₂ gas (balloon, 1 atm). After 1 hour the reaction was diluted with 10 mL of the same MeOH:EtOAc mixture followed by bubbling Ar gas into the reaction solution. The reaction was then filtered over a bed a Celite® followed by a short bed of silica using 1% MeOH in CH₂Cl₂ as the eluent. Compound S7 was then concentrated under reduced pressure to a white solid (0.25 g, 0.20 mmol, 89% yield). ¹H NMR (CDCl₃, 600 MHz): δ = 1.46 (3H, s), 1.74 (1H, t, J = 12.5 Hz), 1.80 (3H, s), 1.98 (3H, s), 2.02 (3H, s), 2.13 (3H, s), 2.18 (3H, s), 2.52 (1H, dd, J = 4.5, 12.6 Hz), 2.68 (4H, s), 2.73 (1H, dd, J = 6.8, 12.0 Hz), 2.97 (1H, dd, J = 6.4, 12.0 Hz), 3.50 (1H, t, J = 6.7 Hz), 3.56 (1H, td, J = 3.1, 9.3 Hz), 3.69 (1H, dd, J = 2.7, 10.7 Hz), 3.76 (5H, m), 3.83 (1H, q, J = 10.4 Hz), 3.90 (1H, dd, J = 7.9, 12.2 Hz), 4.37 (1H, t, J = 8.8 Hz), 4.46 (1H, dd, J = 2.5, 12.2 Hz), 4.55 (1H, dd, J = 3.4, 10.1 Hz), 4.69 (1H, d, J = 3.2 Hz), 4.73 (1H, td, J = 6.1, 14.9 Hz), 4.91 (1H, d, J = 7.9 Hz), 4.96 (1H, d, J = 10.3 Hz), 5.13 (1H, dd, J = 2.6, 9.6 Hz), 5.22 (2H, m), 5.62 (2H, m), 5.70 (1H, td, J = 3.9, 12.1 Hz), 7.41 (4H, q, J = 8.2 Hz), 7.53 (4H, m), 7.60 (1H, t, J = 7.3 Hz), 8.02 (4H, d, J = 8.3 Hz), 8.26 (2H, d, J = 7.1 Hz). ¹³C{¹H} NMR (CDCl₃, 150 MHz): δ = 20.3, 20.6, 20.8, 20.8, 21.4, 23.1, 25.3, 37.4, 48.7, 53.3, 59.9, 60.5, 63.3, 67.0, 67.1, 68.1, 69.2, 70.1, 71.1, 71.1, 71.9, 73.0, 74.5, 76.3, 96.8, 100.9, 104.1, 128.2, 128.3, 128.4, 128.5, 129.1, 129.1, 129.8, 129.9, 130.0, 130.4, 133.2, 133.2, 133.3, 164.9, 165.2, 165.2, 168.1, 170.2, 170.3, 170.4, 170.6, 170.8, 171.7, 171.9. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₅₉H₆₆N₂O₂₉Na 1289.3643; Found 1289.3627.

Succinimidyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-α-d-galacto-non-2-ulopyranosylonic acid)(2→3)-(4-O-acetyl-2-O-benzoyl-β-d-galactopyranosyl)(1→4)-2,3-di-O-benzoyl-β-d-glucopyranoside (21)

Compound S7 (0.25 g, 0.20 mmol, 1.0 equiv) was dissolved in $1\overline{0}$ mL pyridine followed by the addition of LiI (0.54 g, 4.0 mmol, 20 equiv) in the dark. This solution was then refluxed in the dark for 3 hours, after which pyridine was removed under reduced pressure followed by co-distillation with toluene under reduced pressure (3X). This crude was then purified by silica flash chromatography using 5% MeOH in CH₂Cl₂ as the eluent to afford compound 21 as a white solid (0.18 g, 0.14 mmol, 68% yield). Note: The white solid pertains to the protonated form of the compound. A yellow oily material may result as the lithiated species. This species has identical NMR characterization as the protonated form but exhibits the lithiated form in MS analysis i.e. [M➔Li + Na]⁺. ¹H NMR (CD₃OD, 600 MHz): δ = 1.58 (1H, br), 1.70 (3H, br), 1.78 (3H, s), 1.92 (3H, s), 2.04 (3H, s), 2.08 (3H, s), 2.10 (3H, s), 2.51 (1H, br), 2.66 (4H, s), 3.52 (1H, br, J = 9.6 Hz), 3.62 (2H, m), 3.69 (4H, m), 3.90 (1H, br), 3.94 (1H, dd, J = 5.6, 11.0 Hz), 4.04 (1H, dd, J = 6.2, 12.3 Hz), 4.21 (1H, br), 4.32 (1H, t, J = 9.4 Hz), 4.39 (1H, dd, J = 2.3, 12.4 Hz), 4.46 (1H, br, J = 9.1 Hz), 4.94 (1H, d, J = 8.00 Hz), 5.23 (2H, br), 5.33 (1H, d, J = 8.00 Hz), 5.44 (1H, t, J = 8.6 Hz), 5.54 (1H, br), 5.66 (1H, t, J = 9.2 Hz), 7.43 (4H, m), 7.56 (4H, m), 7.65 (1H, t, J = 7.4 Hz), 7.99 (4H, m), 8.17 (2H, d, J = 7.5 Hz). ¹³C{¹H} NMR (CD₃OD, 150 MHz): δ = 19.4, 19.5, 20.1, 21.2, 25.0, 48.2, 49.1, 59.7, 62.4, 62.8, 67.5, 68.1, 70.7, 71.0, 71.2, 72.3, 73.0, 73.5, 74.5, 76.1, 101.0, 103.3, 128.0, 128.1, 128.4, 129.3, 129.5, 129.5, 129.5, 129.7, 129.8, 130.0, 133.0, 133.0, 165.5, 165.8, 170.3, 170.4, 170.5, 170.9, 170.9, 171.3, 171.9, 172.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₅₈H₆₃N₂O₂₉LiNa 1281.3569; Found 1281.3561. LRMS (ESI): [M]^- calcd for C₅₈H₆₄N₂O₂₉ 1252.4; Found 1252.1.

Aminooxy (5-acetamido-3,5-dideoxy-d-glycero-α-d-galactonon-2-ulopyranosylonic acid)-(2→3)-(β-d-galactopyranosyl)(1→4)-β-d-glucopyranoside (1)

Compound 21 ($2\overline{0}$ mg, 0.016 mmol, 1.0 equiv) was dissolved in EtOH (1 mL) in a microwave reaction tube. Hydrazine hydrate ($3\overline{0}$ μL, 0.96 mmol, 60 equiv) was then introduced into the vessel which was then capped and stirred at room temperature for 1 hour. Compound 21 was then subjected to microwave conditions (200 W, 90 °C) for 1 hour total in two 30 minute repetitive cycles. MS analysis revealed a single peak at 687.6 [M]⁻. This peak is the acetone adduct of the aminooxy GM3 which spontaneously forms with residual acetone in the injector of the MS instrument. The reaction mixture was then concentrated under reduced pressure and then passed through a P2 biogel column using H₂O as the eluent. Fractions were found to contain final compound by staining silica gel plates. These fractions were frozen and the lyophilized to reveal compound 1 as a white solid (8.0 mg, 0.012 mmol, 77% yield). ¹H NMR (D₂O, 600 MHz): δ = 1.70 (1H, t, J = 12.1 Hz), 1.94 (3H, s), 2.66 (1H, dd, J =4.6, 12.4 Hz), 3.26 (1H, m), 3.57 (12H, m), 3.77 (4H, m), 3.86 (1H, d, J = 3.1 Hz), 3.92 (1H, dd, J = 2.1, 12.2 Hz), 4.02 (1H, dd, J = 3.2, 9.9 Hz), 4.43 (1H, d, J = 7.9 Hz), 4.51 (1H, d, J = 8.3 Hz). ¹³C{¹H} NMR (D₂O, 150 MHz): δ = 22.0, 39.6, 51.6, 59.9, 61.0, 62.5, 67.4, 68.1, 68.3, 69.3, 71.3, 71.7, 72.8, 74.3, 74.7, 75.1, 75.4, 78.0, 99.8, 102.6, 104.8, 173.9, 175.0. HRMS (ESI) m/z: [M + Na]⁺ calcd for C₂₃H₄₀N₂O₁₉Na 671.2117; Found 671.2106.

GM3-PS A1 (23)

Polysaccharide PS A1 (22) was isolated and purified as previously described.^{8, 41} PS A1 (1.0 mg, 9.1 X 10⁻⁹ mol) was dissolved in NaOAc buffer (1.0 mL, 0.1 M, pH = 5). To this solution was added 55 μL of 10 mM NaIO₄ solution (5.5 × 10⁻⁷ mol). The reaction was allowed to shake gently for 90 min in the dark at room temperature. Excess NaIO₄ was quenched by adding ethylene glycol and the mixture was continually shaken for another 20 min in the dark. The oxidized PS A1 was purified with a centrifugal filter (Vivaspin, MWCO = 30 kDa). PS A1 was redissolved in NaOAc buffer (1.0 mL, 0.1 M, pH = 5). Subsequently, 2.9 mg (4.4 × 10⁻⁶ mol) of β-aminooxy GM3 (1) was added and the reaction was gently shaken for 18 hours in the dark at room temperature. Unreacted β-aminooxy GM3 (1) was removed using a 30 kDa centrifugal filter and purified GM3-PS A1 (23) conjugate was obtained as a lyophilized white foam (1.0–1.1 mg).

GM3-PS A1 NMR Analysis

NMR analysis was performed using a Bruker Avance III 600 MHz spectrometer and data processed with Bruker TopSpin 4.0.3. GM3-PS A1 ¹H spectra were obtained with a probe temperature of 60 °C using the zg30 pulse program (D1 = 3.0 sec, AQ = 2.7 sec, NS = 128). Integrations of the key “diagnostic” peaks were obtained using an automated baseline correction. These integrations suggested a 1:2.33 ratio between GM3:PSA1 repeating unit. On average, PS A1 has at least 120 repeating units¹² which translates to approximately 51.5 GM3 conjugation sites per PS A1 molecule.