Synthesis of propargyl glycosides of Streptococcus pneumoniae serotypes 6A and 6B for glycoconjugate vaccines

Abstract

We developed a method for making immune responses to bacterial glycans T cell-dependent, which involves attachment of short, synthetic glycans to a virus-like nanoparticle (VLP). This strategy enhances immune responses to glycans by facilitating cognate T cell help of B cells, leading to antibody class switching and affinity maturation yielding high-affinity, anti-glycan antibodies. This method requires synthesis of bacterial glycans as propargyl glycosides for covalent attachment to VLPs, and the resulting short linker between the VLP and glycan is important for optimal T cell receptor recognition. In this work, glycans that are part of the capsular polysaccharides (CPS) produced by Streptococcus pneumoniae serotypes Sp6A and Sp6B were synthesized as disaccharides and trisaccharides. The optimal glycan epitope for antibody binding to the CPS from these serotypes is unknown, and differing “frames” of disaccharides and trisaccharides were prepared to elucidate the optimal antigen for antibody binding.

Graphical Abstract

1. Introduction

Streptococcus pneumoniae (Spn) is a globally prevalent, Gram-positive human pathogen that causes invasive pneumococcal diseases (IPD), such as bacteremia, meningitis, and other life-threatening diseases, especially in the elderly, children, and immunocompromised individuals [1]. In 2019, 740,180 children died from pneumonia, which accounts for 14% of all deaths in children under the age of five, according to the World Health Organization (WHO) [2]. Spn pneumococci are shielded by structurally diverse capsular polysaccharides (CPS) from recognition and attack by the host immune system, including phagocytosis, which allow the bacteria to invade and propagate in the host [3,4]. Currently, over 90 serotypes have been discovered, and each produces a CPS with unique chemical structure and immunologic properties [5–7]. Because the first molecular patterns on these bacteria that are encountered by the host immune system are the CPSs, these constitute attractive targets for vaccine development.

Spn serotypes 6A (Sp6A) and 6B (Sp6B) are two of the most common causes of IPD around the world and are more frequently isolated from infections than most of the other serotypes [1,8–10]. Both serotypes produce capsular polysaccharides with repeating units of galactose-glucose-rhamnose-ribitol-phosphate, which only vary in the position of the trisaccharide bonded to ribitol (Figure 1). Due to this structural similarity, multi-valent pneumococcal vaccines containing either Sp6A or Sp6B can generate serotype-specific antibodies that cross-react with the other serotype after vaccination [11]. In the 7-valent pneumococcal conjugate vaccine (PCV) Prevnar^® (PCV7, Pfizer, approved in 2000), Sp6B was selected because of its better hydrolytic stability, thus being easier to acquire from natural sources [12]. After the introduction of PCV7, Sp6A and Sp6B pneumococci were less prevalent; however, carriage and infection of non-vaccine serotypes increased. For instance, less well-known serotype Sp6C began to be more prevalent [13–16].

Figure 1.

Repeating units of Streptococcus pneumoniae serotypes 6A (Sp6A) and 6B (Sp6B).

Notably, Sp6B-based vaccines are not always able to elicit functional antibodies against Sp6A, even though the post-PCV7 prevalence of 6A was reduced [15–18]. In 2010, Sp6A was included in PCV13 (Prevnar 13^®, Pfizer) to offer effective cross-protection against Sp6C, and the valency of PCV was expanded to 20 (PCV20, Prevnar 20^®, Pfizer, approved in 2021) afterward with additional pathogenic serotypes [13,19–22]. Based on our previous studies on immune responses of children and adults to PCV13, the protection acquired through vaccination was poor, especially to serotypes Sp6A and Sp6B [23]. Relatively low titers of IgM were identified, and higher affinity IgG antibodies for these glycans were not generated. This lack of high-affinity antibodies highlights the need for improved approaches for vaccine development for pneumococcal bacteria.

Our group has been working with conjugate vaccines for pneumococcal bacteria, and we have developed a method for making immune responses to bacterial glycans T cell dependent. This method involves attachment of short, synthetic glycans to a virus-like nanoparticle (VLP), and in combination with a natural killer T cell (NKT cell) adjuvant, the resultant vaccines generate high-affinity, anti-glycan antibody production in mice. In this design, immune responses are enhanced through T cell help of B cells leading to antibody class switching and affinity maturation. Effectiveness of this method was initially demonstrated with two serotypes of Spn (Sp3 and Sp14) [23]. From previous studies, we found that Spn serotype 3 disaccharide and tetrasaccharide induced comparable immune responses, suggesting that the glycan antigen may be relatively small while remaining effective.

The repeating carbohydrate pattern in Sp6A and Sp6B CPS is a tetrasaccharide, and the optimized epitope for antibody recognition of this glycan is not known. Jansen et al. reported that the Sp6B disaccharide (rhamnose-ribitol-phosphate) could induce effective Sp6B-specific antibodies [18]; however, systematic exploration of immune responses to varied portions or “frames” of Sp6A/B CPS has not been reported. An attractive strategy to identify the best epitopes for generation of high-affinity antibodies is use of “frame-shifting” along the CPS. Starting from different points of the repeating unit, a series of truncated glycan targets are generated. This approach allows detailed characterization of the truncated fragments and a deeper understanding of their potential application in vaccine development and therapeutics. Herein, we present syntheses of frame-shifted Sp6A and Sp6B disaccharides and trisaccharides as propargyl glycosides for coupling onto VLP to generate effective vaccines for antibody production. These glycans from Sp6A and Sp6B (1–6) are shown in Figure 2.

Figure 2.

Glycans of Sp6A and Sp6B synthesized in this work.

2. Results and discussion

The CPS of Sp6A and Sp6B consist of repeating units of structurally similar tetrasaccharides, which are connected through a phosphate bridge. By shifting the frame from the reducing end, disaccharides containing rhamnose-ribitol-phosphate and trisaccharides containing glucose-rhamnose-ribitol-phosphate (1–4, Figure 2) were selected, allowing the unique linkage between L-rhamnose and D-ribitol to be included in the truncated glycans. Also, the common unit of galactose-glucose-rhamnose (6, Figure 2) shared by both serotypes, as well as a disaccharide of galactose-glucose (5, Figure 2) were synthesized to explore minimal epitopes capable of inducing serotype-specific and cross-reactive, high-affinity antibodies. As noted, the glycans were prepared as propargyl glycosides to yield a short linker to the VLPs through the Huisgen reaction with the azidohomoalanine (AHA), which was incorporated using mutagenesis to modify the side chain of methionine by replacing the S-methyl thioether with azide. Upon intracellular processing of the glycosylated VLPs to peptide fragments, the resulting glycosylated peptides can be presented in major histocompatibility complex II and recognized by T cells, and facilitate better T cell help. While chemical syntheses of Sp6A and Sp6B oligosaccharides have been reported [24–35], none have included the required proparglycoside groups. Syntheses of the targeted Sp6A and Sp6B glycans had to be designed to avoid use of reagents that would react with these alkynes.

2.1. Description of building blocks

Oligosaccharides were assembled from the non-reducing end to the reducing end, and key intermediates are shown in Scheme 1．To afford the α-linkages, a non-participating group at C2 (Nap or PMB ether), was needed to form the 1,2-cis-linked galactoside and glucoside, while a participating group (Ac or Bz) at C2 on the rhamnose unit was required to assist the formation of the 1,2-trans glycosidic linkage. A propargyl linker was installed on each oligosaccharide at one of the sites from which the neighboring glycan or phosphate unit groups are bonded in the intact CPSs. An appropriate protecting group (PMB or TBDPS ether) was used to selectively protect alcohols involved in phosphate formation until the need arose for installation of the terminal phosphate unit.

Scheme 1．.

Key intermediates required for the synthesis of Sp6A and Sp6B oligosaccharides.

2.2. Synthesis of building blocks

2.2.1. Synthesis of ribose and ribitol acceptors 9 and 16

The D-ribitol unit was synthesized from commercially available D-ribose tetra-acetate (Scheme 2). It can be challenging to trap D-ribose in the furanose form at equilibrium, and use of the pyranose form may lead to incorrect regio- and stereochemistry after reductive ring opening. Thus, fully protected D-ribose in the furanose form was used as the starting material. The anomeric carbon was first protected by thiophenol before reduction to keep the furanose ring in the proper ring size (Scheme 2). For Sp6A, 3-OH and 5-OH were protected with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSiCl₂) followed by installation of a Nap group at 2-OH and a subsequent deprotection of the silyl group to give diol 8. Then, the primary 5-OH was selectively protected with a TBDPS group to afford acceptor 9. For Sp6B, the 5-OH was protected by a trityl group, which was switched to a PMB group after both 2-OH and 3-OH were protected by the Nap group to avoid troublesome deprotection before and unwanted deprotection during the later coupling. Thioglycoside 13 was hydrolyzed before reduction using NaBH₄ to afford the ribitol 15. The primary 1-OH was protected by a TBDPS group giving ribitol acceptor 16.

Scheme 2．.

Synthesis of acceptors 9 and 16. Reagents and conditions: (a) (i) PhSH, BF₃·OEt₂, DCM, 0 °C, 2.5 h, 94%; (ii) NaOMe, MeOH/THF, rt, 30 min, 92%; (b) (i) TIPDSiCl₂, imidazole, THF, 0 °C to rt, 1 h; (ii) NaH, TBAI, NapBr, THF, 0 °C to rt, 5 h; (iii) HF·pyridine, pyridine/THF, 0 °C, 2 h, 56%, over 3 steps; (c) TBDPSCl, imidazole, THF, −20 °C to rt, 16 h, 85%; (d) TrCl, pyridine, rt, 16 h, 99%; (e) NaH, TBAI, NapBr, DMF, 0 °C to rt, 4 h, 94%; (f) p-TsOH, DCM/MeOH, rt, 16 h, 79%; (g) NaH, TBAI, PMBCl, THF, 0 °C, rt, 16 h, 99%; (h) NBS, acetone/H₂O, rt, 3 h; (i) NaBH₄, EtOH, rt, 16 h, 96%, over 2 steps; (j) TBDPSCl, imidazole, THF, 0 °C to rt, 16 h, 95%.

2.2.2. Synthesis of rhamnose acceptors 21, 24 and 27

Intermediate 19 (Scheme 3) was synthesized as described by Frihed et al. [36] The acetonide was removed followed by selective protection on 2-OH with a Bz group to afford acceptor 21 using the method reported by Pozsgay et al. [37] The participating Bz group at 2-OH was expected to provide good stereoselectivity (α favored) in the glycosylation due to anchimeric assistance and the kinetic anomeric effect [38,39]. A trichloroethoxycarbonyl (Troc) group was used to temporarily protect exposed 3-OH, allowing a switch of 1-OAc from 1-SPh via coupling of compound 22 and acetic acid. Rhamnose acceptor 24 was obtained after the removal of the Troc group through reductive elimination with Zn in acetic acid. For generating Sp6A/6B di- and trisaccharide, a propargyl glycoside acceptor was prepared from intermediate 19. The alkyne linker was first installed at the reducing end of the rhamnose acceptor through the coupling of thioglycoside 19 and propargyl alcohol to form intermediate 25. The acetonide was then removed followed by selective protection on 2-OH with a Bz group to afford acceptor 27.

Scheme 3．.

Synthesis of acceptors 21, 24 and 27. Reagents and conditions: (a) p-TsOH, MeOH/THF, rt, 16 h, 81%; (b) (i) PhC(OMe)₃, CSA, DCM, rt, 3.5 h; (ii) 80% AcOH, rt, 1 h, 85%; (c) TrocCl, pyridine, rt, 3.5 h, 71%; (d) AcOH, NIS, TMSOTf, 4Å MS, DCM, −20 °C to rt, 1 h; (e) Zn, THF/AcOH, rt, 45 min, 89%; (f) CHCCH₂OH, NIS, TMSOTf, 4Å MS, DCM, −20 °C, 40 min, 81%; (g) p-TsOH, MeOH/THF, rt, 16 h, 76%; (h) (i) PhC(OMe)₃, CSA, DCM, rt, 4 h; (ii) 80% AcOH, rt, 1 h, 93%.

2.2.3. Synthesis of glucose donors 34 and 38, and acceptor 35

In Sp6A and Sp6B CPS, glucose is linked through the C3 oxygen; consequently, this site was chosen for conjugation to the VLP. Thus, a propargyl ether was generated from known intermediate 28 [40] to give 29 (Scheme 4). The common method of using dibutyltin oxide and refluxing in toluene (110 °C) failed in this reaction due to the low boiling point of propargyl bromide (89 °C). By using dibutyltin dichloride (Bu₂SnCl₂) in acetonitrile at refluxing temperature (82 °C), compound 29 was formed in 51% yield with the method reported by Xu et al. [41] The exposed 2-OH was protected with a Nap group, and then the 4, 6-O-benzylidene was replaced with acetyl groups. Via hydrolysis of 1-SPh followed by installation of the diphenyl phosphoryl group, phosphoryl donor 34 was generated as the α anomer. Donor 38 was prepared from known intermediate 36 [42] by replacing the benzylidene group with acetyl groups. Acceptor 35 was afforded via selective protection of a Nap group at 2-OH from intermediate 28 using the method reported by Matsuo et al. [43]

Scheme 4．.

Synthesis of donors 34 and 38, and acceptor 35. Reagents and conditions: (a) K₂CO₃, Bu₂SnCl₂, CHCCH₂Br, CH₃CN, reflux, 2 d, 51%; (b) NaH, TBAI, NapBr, THF, 0 °C to rt, 16 h; (c) p-TsOH, MeOH/THF, rt, 2 d, 96%, over 2 steps; (d) Ac₂O, pyridine, rt, 16 h, 95%; (e) NBS, acetone/H₂O, rt, 15 min, 98%; (f) (PhO)₂POCl, DMAP, DCM, 0 °C, 1 h, 75%; (g) NapBr, Bu₄NHSO₄, aq. NaOH, DCM, 40 °C, 16 h, 44%; (h) p-TsOH, MeOH/THF, rt, 30 h; (i) Ac₂O, pyridine, rt, 16 h, 60%, over 2 steps.

2.2.4. Synthesis of galactose donor 42

Triol 39 (Scheme 5) was synthesized as described by Mancuso et al. [44] The cis-diol (3-OH and 4-OH) was protected by acetonide, and then the 2-OH was temporally protected with a PMB group before introducing the phosphate, followed by hydrolysis of the 1-SPh to give donor 42.

Scheme 5．.

Synthesis of donor 42. Reagents and conditions: (a) (CH₃)₂C(OCH₃)₂, PPTS, 4Å MS, 55 °C, 2 d; (b) NaH, TBAI, PMBCl, THF, 0 °C to rt, 16 h, 64%, over 2 steps; (c) NBS, acetone/H₂O, 0 °C, 30 min, 89%.

2.2.5. Synthesis of Sp6A-disaccharide 1

Disaccharide 43 (Scheme 6) was obtained from glycosylation of known Schmidt’s donor 17 [45] and acceptor 9. The 2-OBz on donor 17 assisted the formation of the α anomer through the neighboring group effect of the ester group at C2. The TBDPS ether at C5 on the ribose unit was then hydrolyzed by HF·pyridine before installing the terminal phosphate with an alkyne linker. The reduction of D-ribose was reserved until the attachment of the phosphodiester at C5 on the ribose unit to drastically reduce the number of steps involving protecting groups within the synthetic route, and improve the overall yield. A commercially available phosphorodiamidite reagent 81 was used to introduce the phosphate via a two-stage phosphorylation and subsequent oxidation with hydrogen peroxide to afford phosphodiester 45. The 2-ONap on the ribose unit was replaced with an acetyl group for easier purification. Thioglycoside 46 was hydrolyzed before reduction to afford the ribitol, followed by global deprotection to give Sp6A disaccharide 1.

Scheme 6．.

Synthesis of Sp6A disaccharide 1. Reagents and conditions: (a) TMSOTf, DCM, 4Å MS, −20 °C, 45 min, 60%; (b) HF·pyridine, pyridine/THF, 0 °C, 3 h, 69%; (c) (i) 81, 5-phenyl-1H-tetrazole, DCM, 0 °C, 3 h; (ii) propargyl alcohol, 5-phenyl-1H-tetrazole, DCM, rt, 1.5 h; (iii) H₂O₂, CH₃CN, rt, 1 h, 60%, over 3 steps; (d) (i) DDQ, DCM/MeOH, rt, 1 h; (ii) Ac₂O, pyridine, rt, 4 h, 81%, over 2 steps; (e) NBS, acetone/H₂O, rt, 15 min, 59%; (f) (i) TEA, DCM, rt, 16 h; (ii) NaBH₄, EtOH, rt, 4 h; (iii) NH₄OH, EtOH, 35 °C, 16 h, 69%, over 3 steps.

2.2.6. Synthesis of Sp6B-disaccharide 3

Disaccharide 48 (Scheme 7) was obtained from glycosylation of known donor 18 [46] and acceptor 16. The 2-OAc on donor 18 assisted the formation of the α anomer through the neighboring group effect of the ester group at C2. The PMB ether at C5 on the ribitol unit was then hydrolyzed by trifluoroacetic acid (TFA) before installing the terminal phosphate with an alkyne linker, without affecting the Nap group. Phosphodiester 50 was afforded through similar procedures as compound 45. The protecting groups Nap, TBDPS, cyanoethyl, and acetyl were removed with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), HF·pyridine, triethylamine (TEA), and ammonium hydroxide (NH₄OH), respectively. The Nap groups were removed before TBDPS to avoid ring closure with a free cis-hydroxyl group during the deprotection. The yield of the TBDPS deprotection with HF·pyridine was only 19% due to the formation of the phosphomonoester by-product (without the cyanoethyl group). Acetyl residues were observed after a 3-day deprotection when removed simultaneously with the cyanoethyl group. By removing the cyanoethyl group first with TEA, and then the acetyl groups, Sp6B disaccharide 3 was generated in good yield.

Scheme 7．.

Synthesis of Sp6B disaccharide 3. Reagents and conditions: (a) NIS, TMSOTf, 4Å MS, DCM, −40 °C, 30 min, 82%; (b)TFA, DCM, 0 °C, 30 min, 77%; (c) (i) 81, 5-phenyl-1H-tetrazole, DCM, 0 °C to rt, 1 h; (ii) propargyl alcohol, 5-phenyl-1H-tetrazole, DCM, rt, 1 h; (iii) H2O2, CH₃CN/DCM, rt, 16 h, 82%, over 3 steps; (d) DDQ, DCM/MeOH, rt, 16 h, 97%; (e) HF·pyridine, pyridine/THF, 0 °C to rt, 16 h, 17%; (f) TEA, DCM, rt, 16 h, 88%; (g) NH₄OH, EtOH, rt, 16 h, 63%.

2.2.7. Synthesis of Sp6A-trisaccharide 2

Disaccharide 54 (Scheme 8) was obtained from glycosylation of phosphoryl donor 34 and acceptor 21. The yield of the coupling was only 25% due to the formation of a trisaccharide by-product (glucose-rhamnose-rhamnose). The Nap ethers were replaced with acetyl groups for better purification. Schmidt’s donor 57 was prepared from hydrolysis of 1-SPh on the rhamnose unit followed by installation of the trichloroacetimidate group. This disaccharide donor was then coupled with acceptor 9 to afford trisaccharide 58 using the same strategy as Scheme 6 to achieve a higher overall yield in fewer steps. The Sp6A trisaccharide 2 was generated through similar procedures as compound 1 (Scheme 6).

Scheme 8．.

Synthesis of Sp6A trisaccharide 2. Reagents and conditions: (a) TMSOTf, 4Å MS, DCM, −45 °C, 30 min; (b) (i) DDQ, DCM/MeOH, rt, 2 d; (ii) Ac₂O, pyridine, rt, 6 h, 18%, over 3 steps; (c) NIS, acetone/H₂O, rt, 5 h, 58%; (d) CCl₃CN, K₂CO₃, rt, 16 h, 61%; (e) 9, TMSOTf, 4Å MS, DCM, −30 °C, 1 h, 66%; (f) HF·pyridine, pyridine/THF, 0 °C, 2 h, 83%; (g) (i) 81, 5-phenyl-1H-tetrazole, DCM, 0 °C to rt, 4 h; (ii) MeOH, 5-phenyl-1H-tetrazole, DCM, rt, 3 h; (iii) H₂O₂, CH₃CN, rt, 30 min, 90%, over 3 steps; (h) (i) DDQ, DCM/MeOH, rt, 4 h; (ii) Ac₂O, pyridine, rt, 16 h, 69%, over 2 steps; (i) NBS, acetone/H₂O, rt, 45 min, 74%; (j) (i) TEA, DCM, rt, 1 d; (ii) NaBH₄, EtOH, rt, 4 h; (iii) NH₄OH, EtOH, 35 °C, 2 d, 59%, over 3 steps.

2.2.8. Synthesis of Sp6B-trisaccharide 4

Disaccharide 63 (Scheme 9) was obtained from glycosylation of phosphoryl donor 34 and acceptor 24. The use of 1-OAc on acceptor 24 avoids the formation of the trisaccharide by-product as observed when synthesizing disaccharide 54. Schmidt’s donor 65 was prepared from hydrolysis of 1-OAc on the rhamnose unit followed by installation of the trichloroacetimidate group. It was then coupled with acceptor 16 giving trisaccharide 66. The Sp6B trisaccharide 4 was afforded through similar procedures as compound 3 except for the TBDPS deprotection. Instead of HF·pyridine, tetra-n-butylammonium fluoride (TBAF) with acetic acid was used to remove the TBDPS group. Simultaneous removal of the TBDPS and the cyanoethyl groups was achieved without observing any remaining of cyanoethyl groups.

Scheme 9．.

Synthesis of Sp6B trisaccharide 4. Reagents and conditions: (a) TMSOTf, DCM, 4Å MS, −45 °C, 1.5 h, 58%; (b) NH₂NH₂·AcOH, THF/MeOH, rt, 2 d, 74%; (c) CCl₃CN, K₂CO₃, DCM, rt, 16 h, 65%; (d) 16, TMSOTf, DCM, 4Å MS, −10 °C to rt, 1 h, 68%; (e)TFA, DCM, 0 °C, 1h, 88%; (f) (i) 81, 5-phenyl-1H-tetrazole, DCM, 0 °C to rt, 4 h; (ii) MeOH, 5-phenyl-1H-tetrazole, DCM, rt, 2.5 h; (iii) H₂O₂, THF/DCM, rt, 30 min, 63%, over 3 steps; (g) DDQ, DCM/MeOH, rt, 16 h; (h) TBAF, AcOH, THF, rt, 16 h; (i) NH₄OH, EtOH, rt, 16 h, 53%, over 3 steps.

2.2.9. Synthesis of Sp6A/6B-disaccharide 5

Disaccharide 71 (Scheme 10) was obtained via glycosylation of thioglycoside donor 38 and acceptor 27. The acetyl group was used to protect the free hydroxyl groups after the Nap deprotection giving compound 72 for easier purification. Disaccharide 5, shared by Spn serotypes 6A, 6B, 18C, and 19A was afforded by ester removal using sodium methoxide (NaOMe).

Scheme 10．.

Synthesis of Sp6A/6B disaccharide 5. Reagents and conditions: (a) TTBP, Ph₂SO, Tf₂O, 4Å MS, DCM, −78 °C to rt, 4 h, 58%; (b) (i) DDQ, DCM/MeOH, rt, 16 h; (ii) Ac₂O, pyridine, rt, 16 h, 77%, over 2 steps; (c) NaOMe, MeOH/THF, rt, 3 h, 91%.

2.2.10. Synthesis of Sp6A/6B-drisaccharide 6

Disaccharide 73 (Scheme 11) was afforded through Crich coupling of acceptor 35 and thioglycoside donor 41 or hydrolyzed donor 42 with a trace amount of product formed versus a yield of 33%, respectively. The acetonide and benzylidene groups were removed simultaneously with camphorsulfonic acid (CSA) and re-protected with acetyl groups to give intermediate 74. The hydrolyzed donor 75 was used to couple with acceptor 27 through Crich’s method to afford trisaccharide 76. The trisaccharide 6 shared by Spn serotypes 6A and 6B was prepared using similar approaches as compound 4.

Scheme 11．.

Synthesis of Sp6A/6B trisaccharide 6. Reagents and conditions: (a) TTBP, Ph₂SO, Tf₂O, 4Å MS, DCM, −60 °C to −40 °C to rt, 1.5 h, 33%; (b) (i) CSA, MeOH/DCM, rt, 3 d; (ii) Ac₂O, pyridine, rt, 16 h, 40%, over 2 steps; (c) NBS, acetone/H₂O, 0 °C, 30 min, 85%; (d) 27, TTBP, Ph₂SO, Tf₂O, 4Å MS, DCM, −60 °C to −40 °C to rt, 1 h, 33%; (e) TFA, DCM, 0 °C, 1.5 h, 90%; (f) (i) 81, 5-phenyl-1H-tetrazole, DCM, 0 °C to rt, 16 h; (ii) MeOH, 5-phenyl-1H-tetrazole, DCM, rt, 5 h; (iii) H₂O₂, THF/DCM, rt, 1 min, 57%, over 3 steps; (g) (i) DDQ, DCM/MeOH, rt, 5 d; (ii) Ac₂O, pyridine, rt, 16 h; (h) TEA, DCM, rt, 16 h; (i) NH₄OH, EtOH, rt, 4 d, 19%, over 4 steps.

3. Conclusion

We successfully prepared the disaccharides and trisaccharides of CPS from Spn serotypes 6A and 6B as propargyl glycosyl antigens for future immunological studies with our vaccine model. The targets were selected through “frame-shifting” along the CPS. This strategy will enable us to find the minimal epitopes capable of inducing high-affinity antibodies. Mouse immunization was performed with vaccines derived from Sp6A-disaccharide 1 and Sp6B-disaccharide 3. Immune responses were observed with vaccines from both disaccharides, however, only monoclonal antibodies from Sp6B-disaccharide 3 were isolated successfully. Immunogenicity studies on the remaining synthetic glycans are ongoing.

4. Experimental

4.1. General

All reactions were carried out under nitrogen with anhydrous solvents and oven-dried glassware unless mentioned otherwise. All reagents and chemicals were purchased from commercial companies and used without further purification. TLC was performed to monitor the progress of all reactions using pre-coated silica gel (60 F₂₅₄, Merck) aluminum or glass plates. Column chromatography was carried out with silica gel 60 (230–240 Mesh). NMR spectra were recorded with Varian 300 MHz, Varian 500 MHz, or Bruker 500 MHz spectrometer at 25 °C. Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), and coupling constant J (measured in Hz). Mass spectra were obtained using an Agilent Technologies LC/Q-TOF mass spectrometer.

4.2. Synthesis and characterization data

4.2.1. Phenyl 1-thio-β-D-ribofuranoside (7)

To a solution of tetra-O-acetyl-D-ribofuranoside (9.91 g, 31.1 mmol) in DCM (30 mL), PhSH (5.4 mL, 52.6 mmol) and BF₃·OEt₂ (8 mL, 64.8 mmol) were added, and the mixture was stirred at 0 °C for 2.5 h. The reaction mixture was then diluted with DCM and washed with water, sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10% EtOAc/Hexanes), and a light yellow syrup was collected (10.73 g, 94%). HRMS (ESI) (m/z) calculated for C₁₇H₂₀O₇S, [M+NH₄]⁺ 386.1268, found 386.1243. The syrup (5.79 g, 15.7 mmol) was then dissolved in MeOH (15 mL) and THF (10 mL), NaOMe (2.0 mL of a 1.74 M solution, 3.48 mmol) was added. The mixture was stirred at room temperature for 30 min before the reaction was quenched with AcOH (1 mL). The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (5% MeOH/DCM) to afford compound 7 (3.48 g, 92%) as a white solid. ¹H NMR (500 MHz, MeOD) δ 7.52 (d, J = 7.3 Hz, 2H), 7.34 – 7.24 (m, 3H), 5.20 (d, J = 4.4 Hz, 1H), 4.00 – 3.92 (m, 3H), 3.65 – 3.53 (m, 2H). ¹³C NMR (126 MHz, MeOD) δ 135.5, 132.9, 123.0, 128.4, 91.6, 86.5, 76.5, 72.6, 63.9. HRMS (ESI) (m/z) calculated for C₁₁H₁₄O₄S, [M+NH₄]⁺ 260.0951, found 260.0969.

4.2.2. Phenyl 2-O-(2-naphthalenylmethyl)-1-thio-β-D-ribofuranoside (8)

Compound 7 (11.1 g, 46.0 mmol) was dissolved in THF (100 mL) with imidazole (10.0 g, 184 mmol). This was followed by the dropwise addition of TIPDSiCl₂ (11.9 mL, 46.0 mmol) at room temperature. After 1 h, the reaction was complete. It was then quenched with methanol at 0 °C and washed with an aqueous solution of 5% HCl. The compound was extracted from this solution using DCM, dried over Na₂SO₄, and concentrated. HRMS (ESI) (m/z) calculated for C₂₃H₄₀O₅SSi₂ [M+NH₄]⁺ 502.2479, found 502.2470. The concentrate was then dissolved in THF (20 mL) and brought to 0 °C. Next, NaH (3.20 g, 60% dispersion in mineral oil, 184 mmol) was then slowly added to the flask. After allowing the NaH to react for 5 min, 2-(Bromomethyl)naphthalene (NapBr) (4.4 g, 46.02 mmol) was then added to the flask, immediately followed by tetrabutylammonium iodide (TBAI) (1.60 g, 11.5 mmol). The reaction stirred for five hours before completion. It was then quenched with MeOH at 0 °C and washed with an aqueous solution of 5% HCl. The product was extracted from this solution using DCM, dried over Na₂SO₄, and concentrated. HRMS (ESI) (m/z) calculated for C₃₄H₄₈O₅SSi₂ [M+H]⁺ 625.2839, found 625.2889. The concentrate was then dissolved in THF (100 mL) and pyridine (50 mL) and stirred at 0 °C within a polyethylene bottle. Next, HF·pyridine (15.0 mL, 70% HF in pyridine mixture, 575 mmol) was added to the flask. The reaction completed after stirring for 2 h. It was then quenched with NaHCO₃, and then washed with sat. NaHCO₃. EtOAc was used to extract the compound. The extracts were then followed by another wash in an aqueous solution of 5% HCl. The compound was once again extracted with EtOAc, dried over Na₂SO₄, and concentrated. The residue was then purified by column chromatography (10–50% EtOAc/Hexanes) to afford compound 8 as a white solid (9.79 g, 56%, 3 steps). ¹H NMR (500 MHz, CDCl₃) δ 7.89 – 7.75 (m, 4H), 7.54 – 7.26 (m, 8H), 5.41 (d, J = 4.1 Hz, 1H), 4.89 (d, J = 11.8 Hz, 1H), 4.79 (d, J = 11.8 Hz, 1H), 4.20 (dd, J = 10.9, 5.4 Hz, 1H), 4.09 – 4.02 (m, 2H), 3.80 (d, J = 12.1 Hz, 1H), 3.62 (d, J = 9.5 Hz, 1H), 2.69 (d, J = 6.4 Hz, 1H), 1.83 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 134.2, 133.4, 133.3, 133.0, 132.3, 129.3, 128.8, 128.1, 128.0, 127.9, 127.4, 126.6, 126.5, 125.9, 88.6, 86.1, 82.5, 73.2, 70.8, 62.4. HRMS (ESI) (m/z) calculated for C₂₂H₂₂O₄S, [M+NH₄]⁺ 400.1583, found 400.1594.

4.2.3. Phenyl 5-O-[(1,1-Dimethylethyl)diphenylsilyl]-2-O-(2-naphthalenylmethyl)-1-thio-β-D-ribofuranoside (9)

Compound 8 (9.79 g, 25.6 mmol) was dissolved in THF (30 mL) with imidazole (3.50 g, 128 mmol) at −20 °C. Next, tert-butyl-diphenyl-disiloxane (TBDPSCl) (2.70 mL, 30.8 mmol) was added to the flask. After the reaction was completed, the mixture was then quenched with MeOH and washed with an aqueous solution of 5% HCl. The compound was then extracted from the wash using DCM and dried over Na₂SO₄. The residue was then concentrated and purified by column chromatography (4–8% EtOAc/Hexanes) to afford compound 9 as a clear oil (13.5 g, 85%). ¹H NMR (500 MHz, CDCl₃) δ 7.85 (d, J = 8.1 Hz, 2H), 7.81 (dd, J = 5.8, 3.5 Hz, 1H), 7.74 (s, 1H), 7.67 (dd, J = 15.9, 6.9 Hz, 4H), 7.52 – 7.45 (m, 6H), 7.43 – 7.29 (m, 7H), 7.23 (d, J = 1.5 Hz, 1H), 5.44 (d, J = 5.0 Hz, 1H), 4.91 (d, J = 11.9 Hz, 1H), 4.77 (d, J = 11.9 Hz, 1H), 4.25 – 4.19 (m, 1H), 4.12 – 4.04 (m, 2H), 3.72 (qd, J = 11.3, 3.8 Hz, 2H), 2.64 (d, J = 5.2 Hz, 1H), 1.00 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 135.8, 135.7, 134.4, 134.3, 133.3, 133.2, 133.0, 131.7, 129.8, 129.7, 129.0, 128.6, 128.0, 127.8, 127.8, 127.7, 127.4, 127.2, 126.4, 126.3, 125.8, 88.5, 88.4, 85.8, 82.2, 73.0, 70.9, 64.0, 26.9, 19.2. HRMS (ESI) (m/z) calculated for C₃₈H₄₀O₄SSi [M+NH₄]⁺ 638.2760, found 638.2746.

4.2.4. Phenyl 2,3-bis-O-(2-naphthalenylmethyl)-1-thio-5-O-(triphenylmethyl)-β-D-ribofuranoside (11)

To a solution of 7 (0.86 g, 3.55 mmol) in pyridine (35 mL), trityl chloride (TrCl) (3.00 g, 10.8 mmol) was added. The mixture was stirred at room temperature for 16 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30% EtOAc/Hexanes) to afford compound 10 (1.71 g, 99%) as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₃₀H₂₈O₄S, [M+Na]⁺ 507.1601, found 507.1630. To a solution of 10 (5.26 g, 10.9 mmol) in DMF (120 mL), NaH (60% dispersion in mineral oil, 3.18 g, 133 mmol) and TBAI (0.86 g, 2.33 mmol) were added and stirred at 0 °C for 30 min. NapBr (6.45 g, 29.2 mmol) was then added to the mixture. The mixture was allowed to warm to room temperature while stirring for 4 h before the reaction was quenched with MeOH. After diluting with Et₂O, the mixture was washed with water and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10% EtOAc/Hexanes) to afford compound 11 (7.84 g, 94%) as a yellow syrup. ¹H NMR (300 MHz, CDCl₃) δ 7.96 – 7.84 (m, 3H), 7.83 – 7.74 (m, 3H), 7.70 (dd, J = 9.8, 7.5 Hz, 2H), 7.56 (td, J = 5.6, 1.8 Hz, 13H), 7.43 (dd, J = 8.4, 1.3 Hz, 1H), 7.34 – 7.25 (m, 12H), 5.72 (d, J = 3.5 Hz, 1H), 4.94 (d, J = 12.3 Hz, 1H), 4.81 (d, J = 12.3 Hz, 1H), 4.77 – 4.64 (m, 2H), 4.47 (dd, J = 8.5, 4.0 Hz, 1H), 4.14 (p, J = 4.9 Hz, 2H), 3.41 (dd, J = 10.3, 4.1 Hz, 1H), 3.21 (dd, J = 10.3, 4.0 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 143.9, 135.1, 135.0, 133.8, 133.3, 133.2, 133.2, 133.1, 132.2, 129.0, 128.8, 128.7, 128.3, 128.3, 128.1, 128.0, 127.9, 127.8, 127.8, 127.5, 127.1, 127.0, 126.9, 126.2, 126.2, 126.1, 126.0, 126.0, 88.8, 86.8, 82.1, 80.6, 77.3, 72.5, 72.3, 63.5. HRMS (ESI) (m/z) calculated for C₅₂H₄₄O₄S, [M+NH₄]⁺ 782.3299, found 782.3348.

4.2.5. Phenyl 5-O-[(4-methoxyphenyl)methyl]-2,3-bis-O-(2-naphthalenylmethyl)-1-thio-β-D-ribofuranoside (13)

To a solution of 11 (7.84 g, 10.2 mmol) in DCM (60 mL) and MeOH (60 mL), p-toluenesulfonic acid (p-TsOH) (2.41 g, 12.7 mmol) was added and stirred at room temperature for 16 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20% EtOAc/Hexanes) to afford compound 12 (4.22 g, 79%) as a yellow syrup. HRMS (ESI) (m/z) calculated for C₃₃H₃₀O₄S, [M+NH₄]⁺ 540.2203, found 540.2213. To a solution of 12 (1.98 g, 3.79 mmol) in THF (30 mL), NaH (60% dispersion in mineral oil, 0.32 g, 13.3 mmol) and TBAI (0.71 g, 1.92 mmol) was added and stirred at 0 °C for 1 h. PMBCl (0.65 mL, 4.77 mmol) was then added to the mixture. The mixture was allowed to warm to room temperature while stirring for 16 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10% EtOAc/Hexanes) to afford compound 13 (2.42 g, 99%) as a yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.93 – 7.76 (m, 5H), 7.76 – 7.67 (m, 3H), 7.61 – 7.44 (m, 8H), 7.30 – 7.21 (m, 5H), 6.86 (d, J = 8.6 Hz, 2H), 5.59 (d, J = 3.9 Hz, 1H), 4.86 (d, J = 12.2 Hz, 1H), 4.73 (dd, J = 26.3, 12.9 Hz, 3H), 4.55 (d, J = 11.7 Hz, 1H), 4.48 (d, J = 11.7 Hz, 1H), 4.44 (q, J = 4.6 Hz, 1H), 4.04 (dq, J = 10.4, 5.1 Hz, 2H), 3.82 (s, 3H), 3.60 (d, J = 4.6 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 159.2, 135.2, 135.0, 133.5, 133.3, 133.3, 133.2, 133.1, 132.5, 130.3, 129.3, 128.9, 128.3, 128.2, 128.0, 128.0, 127.8, 127.8, 127.6, 127.0, 126.9, 126.2, 126.2, 126.1, 126.1, 126.0, 126.0, 113.8, 88.9, 82.1, 80.5, 77.5, 73.1, 72.3, 72.3, 70.0, 55.3. HRMS (ESI) (m/z) calculated for C₄₁H₃₈O₅S, [M+NH₄]⁺ 660.2778, found 660.2687.

4.2.6. 5-O-[(4-Methoxyphenyl)methyl]-2,3-bis-O-(2-naphthalenylmethyl)-D-ribitol (15)

To a solution of 13 (3.80 g, 5.91 mmol) in acetone (50 mL) and water (10 mL), N-bromosuccinimide (NBS) (2.10 g, 11.8 mmol) was added. The mixture was stirred at room temperature for 3 h before the reaction mixture was concentrated and re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. HRMS (ESI) (m/z) calculated for C₃₅H₃₄O₆, [M+NH₄]⁺ 568.2694, found 568.2703. The crude product 14 was dissolved in EtOH (100 mL). NaBH₄ (0.22 g, 5.82 mmol) was added to the solution and stirred at room temperature for 16 h. The pH of the reaction solution was adjusted to 4 by adding AcOH dropwise. The reaction mixture was then concentrated and re-dissolved in DCM. After washing with brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30%, 40%, 50% EtOAc/Hexanes) to afford compound 15 (3.14 g, 96%, 2 steps) as a light yellow syrup. ¹H NMR (300 MHz, CDCl₃) δ 7.94 – 7.81 (m, 7H), 7.77 (s, 1H), 7.55 (dd, J = 6.2, 3.3 Hz, 5H), 7.48 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H), 4.90 (dt, J = 20.1, 11.2 Hz, 4H), 4.48 (q, J = 11.5 Hz, 2H), 4.17 (d, J = 5.3 Hz, 1H), 4.00 (td, J = 12.7, 6.9 Hz, 4H), 3.80 (s, 3H), 3.68 (d, J = 4.4 Hz, 2H), 3.19 (d, J = 18.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 159.3, 135.6, 135.5, 133.2, 133.2, 133.0, 129.9, 129.6, 128.3, 128.2, 127.9, 127.7, 127.7, 126.8, 126.6, 126.2, 126.1, 126.1, 126.0, 125.9, 113.8, 79.5, 79.4, 73.9, 73.0, 72.1, 70.7, 70.5, 61.1, 55.2. HRMS (ESI) (m/z) calculated for C₃₅H₃₆O₆, [M+NH₄]⁺ 570.2850, found 570.2857.

4.2.7. 1-O-[(1,1-Dimethylethyl)diphenylsilyl]-5-O-[(4-methoxyphenyl)methyl]-2,3-bis-O-(2-naphthalenylmethyl)-D-ribitol (16)

To a stirring solution of 15 (3.14 g, 5.68 mmol) in THF (50 mL), imidazole (0.79 g, 11.6 mmol) and TBDPSCl (2.2 mL, 8.46 mmol) were added at 0 °C. The mixture was allowed to warm to room temperature while stirring for 16 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (15% EtOAc/Hexanes) to afford compound 16 (4.28 g, 95%) as a light yellow syrup. ¹H NMR (300 MHz, CDCl₃) δ 7.89 – 7.69 (m, 11H), 7.63 (s, 1H), 7.54 – 7.38 (m, 7H), 7.34 (dd, J = 11.9, 5.1 Hz, 5H), 7.20 (d, J = 8.5 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H), 4.93 – 4.69 (m, 4H), 4.43 (q, J = 11.6 Hz, 2H), 4.14 – 3.99 (m, 3H), 3.95 (app t, J = 6.7 Hz, 2H), 3.77 (s, 3H), 3.66 – 3.59 (m, 2H), 1.12 (s, 9H). ¹³C NMR (75 MHz, CDCl₃) δ 159.3, 136.0, 136.0 135.9, 135.8, 133.5, 133.4, 133.3, 133.1, 130.3, 129.8, 129.6, 128.2, 128.1, 128.1, 127.8, 127.8, 127.8, 126.7, 126.5, 126.2, 126.1, 126.0, 125.9, 113.9, 80.7, 79.0, 73.9, 73.1, 72.7, 71.2, 70.9, 63.5, 55.3, 27.0, 19.3. HRMS (ESI) (m/z) calculated for C₅₁H₅₄O₆Si, [M+H]⁺ 791.3762, found 791.3693.

4.2.8. Phenyl 4-O-(2-naphthalenylmethyl)-1-thio-α-L-rhamnopyranoside (20)

To a solution of 19 (4.49 g, 10.3 mmol) in THF (20 mL) and MeOH (50 mL), p-TsOH (0.38 g, 2.00 mmol) was added and stirred at room temperature for 16 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30% EtOAc/Hexanes) to afford compound 20 (3.31 g, 81%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.81 (d, J = 7.1 Hz, 4H), 7.58 – 7.42 (m, 5H), 7.27 (d, J = 7.4 Hz, 3H), 5.46 (s, 1H), 4.88 (s, 2H), 4.22 (dd, J = 18.7, 12.2 Hz, 2H), 3.95 (d, J = 7.1 Hz, 1H), 3.46 (t, J = 9.2 Hz, 1H), 2.89 (s, 1H), 2.66 (s, 1H), 1.37 (d, J = 6.0 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 135.6, 134.2, 133.4, 133.2, 131.5, 129.2, 128.6, 128.1, 127.9, 127.5, 126.6, 126.4, 126.2, 125.9, 115.2, 87.5, 81.9, 75.3, 72.7, 72.1, 68.7, 18.1. HRMS (ESI) (m/z) calculated for C₂₃H₂₄O₄S, [M+NH₄]⁺ 414.1734, found 414.1736.

4.2.9. Phenyl 2-O-benzoyl-4-O-(2-naphthalenylmethyl)-1-thio-α-L-rhamnopyranoside (21)

To a solution of 20 (1.40 g, 3.53 mmol) in DCM (15 mL), trimethyl orthobenzoate (PhC(OMe)₃) (1.5 mL, 8.73 mmol) and CSA (0.0817 g, 0.352 mmol) were added and stirred at room temperature for 3.5 h. The solvent was evaporated and the residue was dissolved in AcOH (80% in H₂O, v/v, 10 mL). After stirring at room temperature for 1 h, the reaction mixture was diluted with DCM and washed with water, sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10% EtOAc/Hexanes) to afford compound 21 (1.50 g, 85%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.07 – 7.95 (m, 2H), 7.83 (app t, J = 6.8 Hz, 4H), 7.63 – 7.24 (m, 11H), 5.62 (dd, J = 3.2, 1.5 Hz, 1H), 5.56 (d, J = 1.0 Hz, 1H), 4.98 (dd, J = 27.6, 11.4 Hz, 2H), 4.39 – 4.30 (m, 1H), 4.29 – 4.21 (m, 1H), 3.61 (t, J = 9.4 Hz, 1H), 2.38 (d, J = 4.9 Hz, 1H), 1.43 (d, J = 6.2 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 166.3, 135.6, 134.0, 133.5, 133.4, 133.2, 131.9, 130.0, 129.7, 129.2, 128.6, 128.6, 128.1, 127.9, 127.8, 127.0, 126.3, 126.2, 126.1, 86.1, 81.8, 75.4, 75.0, 71.3, 69.0, 18.3. HRMS (ESI) (m/z) calculated for C₃₀H₂₈O₅S, [M+NH₄]⁺ 518.1996, found 518.1995.

4.2.10. 1-O-Acetyl-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-3-O-(2,2,2-trichloroethoxycarbony-α-L-rhamnopyranoside (23)

To a solution of 21 (0.85 g, 1.70 mmol) in pyridine (20 mL), 2,2,2-trichloroethyl chloroformate (TrocCl) (0.3 mL, 2.18 mmol) was added. The mixture was stirred at room temperature for 3.5 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (7.5% EtOAc/Hexanes) to afford compound 22 (0.82 g, 71%) as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₃₃H₂₉Cl₃O₇S, [M+NH₄]⁺ 692.1038, found 692.1097. To a solution of 22 (0.82 g, 1.21 mmol) in DCM (20 mL), AcOH (0.15 mL, 2.62 mmol) and ground 4Å molecular sieves (0.93 g) were added and stirred at room temperature for 16 h. After cooling down to −20 °C, N-iodosuccinimide (NIS) (0.60 g, 2.67 mmol) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) (25 μL, 0.138 mmol) were added. The mixture was allowed to warm to 0 °C while stirring for 1 h before the molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. Na₂S₂O₃, sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10% EtOAc/Hexanes) to afford compound 23 (0.47 g, 62%) as a light yellow syrup. ¹H NMR (300 MHz, CDCl₃) δ 8.12 – 8.00 (m, 2H), 7.90 – 7.74 (m, 4H), 7.63 (t, J = 7.4 Hz, 1H), 7.55– 7.40 (m, 5H), 6.19 (d, J = 1.8 Hz, 1H), 5.65 (dd, J = 3.2, 2.1 Hz, 1H), 5.35 (dd, J = 9.7, 3.4 Hz, 1H), 5.00 (d, J = 11.0 Hz, 1H), 4.93 – 4.78 (m, 2H), 4.70 (d, J = 11.9 Hz, 1H), 4.02 (dq, J = 12.2, 6.1 Hz, 1H), 3.79 (t, J = 9.6 Hz, 1H), 2.17 (s, 3H), 1.46 (d, J = 6.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 168.6, 165.5, 153.3, 134.9, 133.8, 133.3, 133.2, 130.1, 129.2, 128.7, 128.4, 128.1, 127.8, 127.0, 126.4, 126.2, 126.0, 94.4, 90.8, 78.0, 77.2, 77.1, 75.8, 70.2, 69.2, 21.1, 18.3. HRMS (ESI) (m/z) calculated for C₂₉H₂₇Cl₃O₉, [M+NH₄]⁺ 642.1059, found 642.1053.

4.2.11. 1-O-Acetyl-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranoside (24)

To a solution of THF (4 mL) and AcOH (6 mL), zinc dust (< 10 micrometers, 5.32 g, 81.4 mmol) was added and stirred at room temperature for 1 h. Next, compound 23 (0.47 g, 0.751 mmol) in THF (12 mL) was added to the mixture and sonicated at room temperature for 45 min before the zinc dust was filtered through a celite pad. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30% EtOAc/Hexanes) to afford compound 24 (0.30 g, 89%) as a colorless syrup. ¹H NMR (300 MHz, CDCl₃) δ 8.11 – 7.98 (m, 2H), 7.91 – 7.76 (m, 4H), 7.60 (t, J = 7.4 Hz, 1H), 7.54 – 7.39 (m, 5H), 6.15 (d, J = 1.6 Hz, 1H), 5.39 (dd, J = 3.4, 1.9 Hz, 1H), 5.04 (d, J = 11.2 Hz, 1H), 4.92 (d, J = 11.2 Hz, 1H), 4.30 (dd, J = 9.4, 3.4 Hz, 1H), 3.92 (dq, J = 12.5, 6.2 Hz, 1H), 3.58 (t, J = 9.4 Hz, 1H), 2.51 (s, 1H), 2.11 (s, 3H), 1.43 (d, J = 6.2 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 168.8, 166.2, 135.4, 133.6, 130.0, 128.6, 128.5, 128.0, 127.8, 127.0, 126.3, 126.2, 126.1, 113.7, 91.0, 81.0, 77.2, 75.6, 72.2, 70.6, 70.0, 21.0, 18.4. HRMS (ESI) (m/z) calculated for C₂₆H₂₆O₇, [M+NH₄]⁺ 468.2017, found 468.1930.

4.2.12. 2-Propyn-1-yl 2,3-O-(1-methylethylidene)-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranoside (25)

To a solution of 19 (1.01 g, 2.31 mmol) in DCM (25 mL), propargyl alcohol (0.2 mL, 3.44 mmol) and ground 4Å molecular sieves (5.08 g) were added and stirred at room temperature for 3 h. After cooling down to −20 °C, NIS (1.36 g, 6.04 mmol) and TMSOTf (22 μL, 0.122 mmol) were added. The mixture was stirring for 40 min before the molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. Na₂S₂O₃, sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (5% EtOAc/Hexanes) to afford compound 25 (0.72 g, 81%) as a light yellow syrup. ¹H NMR (300 MHz, CDCl₃) δ 7.83 (dd, J = 8.2, 3.7 Hz, 4H), 7.56 – 7.42 (m, 3H), 5.19 (s, 1H), 5.07 (d, J = 11.8 Hz, 1H), 4.81 (d, J = 11.8 Hz, 1H), 4.36 – 4.30 (m, 1H), 4.24 (d, J = 2.2 Hz, 2H), 4.20 (d, J = 5.8 Hz, 1H), 3.73 (dq, J = 10.0, 6.2 Hz, 1H), 3.28 (dd, J = 9.8, 7.1 Hz, 1H), 2.46 (t, J = 2.4 Hz, 1H), 1.52 (s, 3H), 1.40 (s, 3H), 1.32 (d, J = 6.2 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 135.8, 133.3, 133.1, 128.2, 128.0, 127.8, 126.9, 126.2, 126.2, 126.0, 109.4, 95.7, 80.9, 78.8, 78.7, 76.0, 75.0, 73.1, 65.2, 54.2, 28.1, 26.5, 17.9. HRMS (ESI) (m/z) calculated for C₂₃H₂₆O₅, [M+NH₄]⁺ 400.2118, found 400.2119.

4.2.13. 2-Propyn-1-yl 4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranoside (26)

To a solution of 25 (0.48 g, 1.26 mmol) in MeOH (10 mL) and THF (10 mL), p-TsOH (0.0467, 0.246 mmol) was added, and the mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (50% EtOAc/Hexanes) to afford compound 26 (0.33 g, 76%) as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ 7.91 – 7.75 (m, 4H), 7.57 – 7.40 (m, 3H), 4.96 (s, 1H), 4.89 (s, 2H), 4.26 – 4.17 (m, 2H), 3.96 (s, 2H), 3.77 (dq, J = 9.6, 6.2 Hz, 1H), 3.42 (t, J = 9.2 Hz, 1H), 2.59 (s, 2H), 2.44 (t, J = 2.4 Hz, 1H), 1.38 (d, J = 6.3 Hz, 3H)). ¹³C NMR (75 MHz, CDCl₃) δ 135.7, 133.4, 133.1, 128.6, 128.1, 127.8, 126.8, 126.4, 126.2, 125.9, 98.0, 81.6, 78.9, 75.3, 74.9, 71.5, 71.1, 67.9, 54.4, 18.1. HRMS (ESI) (m/z) calculated for C₂₀H₂₂O₅, [M+NH₄]⁺ 360.1805, found 360.1786.

4.2.14. 2-Propyn-1-yl 2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranoside (27)

To a solution of 26 (0.33 g, 0.964 mmol) in DCM (9 mL), PhC(OMe)₃ (0.4 mL, 2.33 mmol) and CSA (0.0222 g, 0.0956 mmol) were added and stirred at room temperature for 4 h. The solvent was evaporated and the residue was dissolved in AcOH (80% in H₂O, v/v, 6 mL). After stirring at room temperature for 1 h, the reaction mixture was diluted with DCM and washed with water, sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20% EtOAc/Hexanes) to afford compound 27 (0.40 g, 93%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 8.08 – 8.01 (m, 2H), 7.88 – 7.79 (m, 4H), 7.59 (app t, J = 7.4 Hz, 1H), 7.51 – 7.47 (m, 3H), 7.44 (app t, J = 7.8 Hz, 2H), 5.40 (dd, J = 3.4, 1.7 Hz, 1H), 5.05 (dd, J = 16.5, 6.4 Hz, 2H), 4.92 (d, J = 11.3 Hz, 1H), 4.29 (dd, J = 9.4, 3.5 Hz, 1H), 4.26 (t, J = 2.3 Hz, 2H), 3.88 (dq, J = 9.5, 6.2 Hz, 1H), 3.55 (t, J = 9.4 Hz, 1H), 2.46 (t, J = 2.4 Hz, 1H), 2.24 (s, 1H), 1.43 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 135.7, 133.5, 133.4, 133.2, 130.0, 129.7, 128.6, 128.5, 128.1, 127.9, 127.0, 126.3, 126.1, 126.1, 96.3, 81.5, 78.7, 75.4, 75.1, 73.2, 70.7, 68.2, 54.7, 18.3. HRMS (ESI) (m/z) calculated for C₂₇H₂₆O₆, [M+Na]⁺ 469.1622, found 469.1523.

4.2.15. Phenyl 4,6-O-benzylidene-3-O-2-propyn-1-yl-1-thio-β-D-glucopyranoside (29)

To a solution of diol 28 [40] (6.74 g, 18.7 mmol) in CH₃CN (200 mL), K₂CO₃ (12.93 g, 93.6 mmol), dibutyltin dichloride (Bu₂SnCl₂) (0.57 g, 1.88 mmol) and propargyl bromide (2.5 mL, 23.0 mmol) were added and stirred at 80 °C using an oil bath for 2 days before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (2%, 3%, 4% EtOAc/Toluene) to afford compound 29 (3.81 g, 51%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.58 – 7.40 (m, 4H), 7.38 – 7.26 (m, 6H), 5.50 (s, 1H), 4.65 (d, J = 9.7 Hz, 1H), 4.47 (qd, J = 15.7, 2.4 Hz, 2H), 4.35 (dd, J = 10.5, 4.8 Hz, 1H), 3.80 – 3.68 (m, 2H), 3.58 (t, J = 9.2 Hz, 1H), 3.49 (dt, J = 16.4, 7.0 Hz, 2H), 2.87 (d, J = 2.0 Hz, 1H), 2.48 (t, J = 2.3 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 137.0, 133.2, 131.4, 129.1, 129.1, 128.4, 128.3, 128.3, 126.1, 113.6, 101.2, 88.2, 81.1, 80.8, 80.0, 75.1, 71.7, 70.5, 68.6, 60.0. HRMS (ESI) (m/z) calculated for C₂₂H₂₂O₅S, [M+H]⁺ 399.1261, found 399.1242.

4.2.16. Phenyl 4,6-di-O-acetyl-2-O-(2-naphthalenylmethyl)-3-O-2-propyn-1-yl-1-thio-β-D-glucopyranoside (32)

To a solution of 29 (3.63 g, 9.11 mmol) in THF (150 mL), NaH (60% dispersion in mineral oil, 0.92 g, 38.3 mmol) and TBAI (0.67 g, 1.81 mmol) were added and stirred at 0 °C for 20 min. NapBr (2.82 g, 12.8 mmol) was then added to the mixture. The mixture was allowed to warm to room temperature while stirring for 16 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. HRMS (ESI) (m/z) calculated for C₃₃H₃₀O₅S, [M+H]⁺ 539.1887, found 539.1890. The crude compound 30 was dissolved in MeOH (75 mL) and THF (75 mL). Next, p-TsOH (0.87 g, 4.57 mmol) was added and stirred at room temperature for 2 d. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (40%, 50% EtOAc/Hexanes) to afford compound 31 (3.93 g, 96%, 2 steps) as a yellow syrup. HRMS (ESI) (m/z) calculated for C₂₆H₂₆O₅S, [M+NH₄]⁺ 468.1839, found 468.1846. To a solution of 31 (3.93 g, 8.72 mmol) in pyridine (30 mL), Ac₂O (15 mL) was added and stirred at room temperature for 16 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20% EtOAc/Hexanes) to afford compound 32 (4.45 g, 95%) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 7.87 – 7.80 (m, 4H), 7.61 – 7.54 (m, 3H), 7.48 (dd, J = 5.8, 3.7 Hz, 2H), 7.34 – 7.28 (m, 3H), 5.09 – 4.94 (m, 2H), 4.86 (d, J = 10.5 Hz, 1H), 4.67 (d, J = 9.7 Hz, 1H), 4.36 (d, J = 2.3 Hz, 2H), 4.19 (qd, J = 12.2, 4.0 Hz, 2H), 3.75 – 3.52 (m, 3H), 2.39 (t, J = 2.3 Hz, 1H), 2.10 (d, J = 6.3 Hz, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 170.8, 169.8, 135.2, 133.4, 133.3, 132.4, 129.1, 128.4, 128.1, 128.0, 127.8, 127.3, 126.4, 126.3, 126.2, 87.7, 83.0, 80.7, 79.9, 76.0, 75.8, 74.7, 69.3, 62.8, 60.4, 21.2, 21.0. HRMS (ESI) (m/z) calculated for C₃₀H₃₀O₇S, [M+NH₄]⁺ 552.2050, found 552.2029.

4.2.17. Diphenyl 4,6-di-O-acetyl-2-O-(2-naphthalenylmethyl)-3-O-2-propyn-1-yl-α,β-D-glucopyranosyl phosphate (34)

To a solution of 32 (0.76 g, 1.43 mmol) in acetone (15 mL) and water (1 mL), NBS (1.01 g, 5.67 mmol) was added. The mixture was stirred at room temperature for 15 min before the reaction mixture was concentrated and re-dissolved in DCM. After washing with sat. Na₂SO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30%, 40% EtOAc/Hexanes) to afford compound 33 (0.62 g, 98%) as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₂₄H₂₆O₈, [M+NH₄]⁺ 460.1966, found 460.1933. To a solution of 33 (0.14 g, 0.316 mmol) in DCM (3 mL), 4-(dimethylamino)pyridine (DMAP) (0.0752 g, 0.616 mmol) and diphenyl phosphoryl chloride (0.08 mL, 0.386 mmol) were added and stirred at 0 °C for 1 h. The reaction mixture was concentrated and then re-dissolved in EtOAc. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20%, 30% EtOAc/Hexanes) to afford compound 34 (0.16 g, 75%) as a colorless syrup. α-isomer (major): ¹H NMR (500 MHz, CDCl₃) δ 7.84 – 7.71 (m, 4H), 7.50 – 7.43 (m, 2H), 7.40 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 7.8 Hz, 2H), 7.26 (dd, J = 12.7, 8.3 Hz, 4H), 7.19 (app t, J = 7.9 Hz, 3H), 7.08 (t, J = 7.3 Hz, 1H), 6.02 (dd, J = 6.7, 3.1 Hz, 1H), 4.99 (t, J = 10.0 Hz, 1H), 4.83 (d, J = 11.6 Hz, 1H), 4.74 (d, J = 11.6 Hz, 1H), 4.39 (qd, J = 16.0, 2.3 Hz, 2H), 4.15 (dd, J = 12.5, 4.1 Hz, 1H), 3.95 (ddd, J = 19.0, 8.4, 5.6 Hz, 2H), 3.79 (dd, J = 12.5, 1.7 Hz, 1H), 3.67 (dt, J = 9.5, 3.1 Hz, 1H), 2.46 (s, 1H), 2.08 (s, 3H), 1.97 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.6, 169.5, 150.4, 150.4, 150.3, 134.4, 133.1, 133.1, 129.9, 129.7, 128.4, 128.0, 127.7, 127.2, 126.2, 126.2, 126.0, 125.6, 125.5, 125.5, 120.5, 120.4, 120.2, 120.2, 96.3, 96.3, 80.0, 78.7, 78.6, 74.5, 73.3, 70.0, 67.8, 61.4, 60.2, 21.0, 20.6. ³¹P NMR (202 MHz, CDCl₃) δ −13.24. HRMS (ESI) (m/z) calculated for C₃₆H₃₅O₁₁P, [M+NH₄]⁺ 692.2255, found 692.2196.

4.2.18. Phenyl 4,6-O-benzylidene-2-O-(2-naphthalenylmethyl)-1-thio-α,β-D-glucopyranoside (35)

To a solution of 28 (4.40 g, 12.2 mmol) in DCM (35 mL), NaOH (5% in water, 0.85 g, 21.3 mmol, 18 mL), tetrabutylammonium hydrogen sulfate (Bu₄NHSO₄) (2.07 g, 6.10 mmol) and NapBr (3.24 g, 14.7 mmol) were added, and the mixture was stirred at 40 °C using an oil bath for 16 h. The reaction mixture was then diluted with DCM and washed with water and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (1%, 2% EtOAc/toluene) to afford compound 35 (2.67 g, 44%) as a white solid. β-isomer (major): ¹H NMR (500 MHz, CDCl₃) δ 7.84 – 7.78 (m, 4H), 7.56 – 7.53 (m, 2H), 7.48 – 7.42 (m, 5H), 7.35 – 7.29 (m, 6H), 5.49 (s, 1H), 5.07 (d, J = 11.1 Hz, 1H), 4.96 (d, J = 11.0 Hz, 1H), 4.75 (d, J = 9.7 Hz, 1H), 4.34 (dd, J = 10.5, 4.9 Hz, 1H), 3.90 (t, J = 8.8 Hz, 1H), 3.71 (dp, J = 18.3, 9.6 Hz, 2H), 3.50 – 3.43 (m, 2H), 2.62 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 137.1, 135.6, 133.4, 133.3, 133.3, 133.2, 132.3, 129.4, 129.2, 129.2, 128.5, 128.4, 128.4, 128.1, 128.0, 127.8, 127.2, 126.4, 126.3, 126.2, 126.2, 126.1, 101.9, 88.1, 80.8, 80.4, 75.6, 75.6, 70.2, 68.7. HRMS (ESI) (m/z) calculated for C₃₀H₂₈O₅S, [M+NH₄]⁺ 518.1996, found 518.2011.

4.2.19. Phenyl 4,6-di-O-acetyl-2,3-bis-O-[(4-methoxyphenyl)methyl]-1-thio-β-D-glucopyranoside (38)

The intermediate 36 [42] (0.55 g, 0.916 mmol) was dissolved in MeOH (10 mL) and THF (10 mL). Next, p-TsOH (0.70 g, 3.68 mmol) was added and stirred at room temperature for 2 d before the reaction was quenched with TEA. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. HRMS (ESI) (m/z) calculated for C₂₈H₃₂O₇S, [M+NH₄]⁺ 530.2207, found 530.2238. The crude compound 37 was dissolved in pyridine (5 mL), Ac₂O (3 mL) was added and stirred at room temperature for 16 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30% EtOAc/Hexanes) to afford compound 38 (0.33 g, 60%, 2 steps) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.61 – 7.54 (m, 2H), 7.36 – 7.27 (m, 5H), 7.17 (d, J = 8.6 Hz, 2H), 6.91 – 6.83 (m, 4H), 5.01 (t, J = 9.7 Hz, 1H), 4.81 (d, J = 9.9 Hz, 1H), 4.76 (d, J = 11.1 Hz, 1H), 4.65 (dd, J = 9.8, 6.2 Hz, 2H), 4.58 (d, J = 11.1 Hz, 1H), 4.20 (dd, J = 12.2, 5.8 Hz, 1H), 4.11 (dd, J = 12.2, 2.3 Hz, 1H), 3.80 (d, J = 8.0 Hz, 6H), 3.63 (t, J = 9.1 Hz, 1H), 3.57 (ddd, J = 10.0, 5.7, 2.3 Hz, 1H), 3.55 – 3.50 (m, 1H), 2.07 (s, 3H), 1.94 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.8, 169.7, 159.6, 159.4, 133.4, 132.3, 130.3, 130.1, 130.1, 129.5, 129.0, 127.9, 114.0, 113.9, 87.7, 83.5, 80.4, 76.0, 75.3, 75.2, 69.9, 62.8, 55.4, 55.4, 20.9, 20.9. HRMS (ESI) (m/z) calculated for C₃₂H₃₆O₉S, [M+NH₄]⁺ 614.2418, found 614.2503.

4.2.20. Phenyl 2-O-[(4-methoxyphenyl)methyl]-3,4-O-(1-methylethylidene)-6-O-(2-naphthalenylmethyl)-1-thio-β-D-galactopyranoside (41)

To a solution of 39 [44] (1.03 g, 2.50 mmol) in 2,2-dimethoxypropane (35 mL), ground 4Å molecular sieves (1.00 g) and pyridinium p-toluenesulfonate (PPTS) (2.54 g, 10.1 mmol) were added. The mixture was stirred at 55 °C using an oil bath for 2 days before the molecular sieves were filtered through a celite pad. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. HRMS (ESI) (m/z) calculated for C₂₆H₂₈O₅S, [M+NH₄]⁺ 470.1996, found 470.1967. The crude compound 40 was dissolved in THF (30 mL). Next, NaH (60% dispersion in mineral oil, 0.30 g, 7.50 mmol) and TBAI (0.18 g, 0.487 mmol) were added and stirred at 0 °C for 30 min. PMBCl (0.45 mL, 3.30 mmol) was then added and the reaction was allowed to warm to room temperature while stirring for 16 h before quenching with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (5%, 10% EtOAc/Hexanes) to afford compound 41 (0.92 g, 64%, 2 steps) as a yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.89 – 7.82 (m, 3H), 7.81 (s, 1H), 7.61 (dd, J = 7.9, 1.6 Hz, 2H), 7.53 – 7.47 (m, 3H), 7.40 (d, J = 8.6 Hz, 2H), 7.27 – 7.22 (m, 3H), 6.92 (d, J = 8.6 Hz, 2H), 4.82 – 4.77 (m, 2H), 4.74 – 4.65 (m, 3H), 4.29 (t, J = 5.9 Hz, 1H), 4.24 (dd, J = 5.7, 1.9 Hz, 1H), 4.02 – 3.98 (m, 1H), 3.91 – 3.84 (m, 2H), 3.82 (s, 3H), 3.58 (dd, J = 9.5, 6.2 Hz, 1H), 1.46 (s, 3H), 1.39 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 159.4, 135.8, 134.1, 133.3, 133.0, 131.7, 130.0, 129.9, 128.9, 128.2, 127.9, 127.7, 127.3, 126.4, 126.1, 125.9, 125.8, 113.8, 110.1, 86.3, 79.7, 77.9, 75.8, 73.9, 73.6, 73.1, 69.8, 55.3, 27.9, 26.4. HRMS (ESI) (m/z) calculated for C₃₄H₃₆O₆S, [M+NH₄]⁺ 590.2571, found 590.2547.

4.2.21. 2-O-[(4-Methoxyphenyl)methyl]-3,4-O-(1-methylethylidene)-6-O-(2-naphthalenylmethyl)-α,β-D-galactopyranoside (42)

To a solution of 41 (0.75 g, 1.31 mmol) in acetone/water 99:1 (45 mL), NBS (0.28 g, 1.57 mmol) was added. The mixture was stirred at 0 °C for 30 min before the reaction mixture was concentrated and re-dissolved in DCM. After washing with sat. Na₂SO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30% EtOAc/Hexanes) to afford compound 42 (0.56 g, 89%) as a white solid. α-isomer (major): ¹H NMR (500 MHz, CDCl₃) δ 7.80 – 7.76 (m, 4H), 7.46 – 7.44 (m, 3H), 7.26 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 8.5 Hz, 2H), 5.18 (t, J = 4.2 Hz, 1H), 4.79 (d, J = 12.3 Hz, 1H), 4.68 (d, 1H), 4.66 (d, J = 12.3 Hz, 1H), 4.60 (d, J = 11.7 Hz, 1H), 4.42 – 4.35 (m, 2H), 4.09 (dd, J = 5.9, 2.3 Hz, 1H), 3.77 (s, 3H), 3.69 (d, J = 6.5 Hz, 2H), 3.56 (dd, J = 6.2, 3.6 Hz, 1H), 1.38 (s, 3H), 1.28 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 159.5, 135.7, 133.3, 133.1, 129.9, 129.7, 128.2, 128.0, 127.8, 126.7, 126.2, 126.0, 126.0, 90.9, 75.5, 74.8, 73.6, 73.4, 72.5, 69.7, 67.4, 55.4, 27.7, 26.0. HRMS (ESI) (m/z) calculated for C₂₈H₃₂O₇, [M+NH₄]⁺ 498.2486, found 498.2453.

4.2.22. Phenyl 2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-5-O-[(1,1-Dimethylethyl)diphenylsilyl]-2-O-(2-naphthalenylmethyl)-1-thio-β-D-ribofuranoside (43)

Acceptor 9 (4.15 g, 6.69 mmol) and donor 17 [45] (6.22 g, 10.1 mmol) were dissolved in DCM (50 mL) with ground 4Å molecular sieve (11.0 g). The mixture was allowed to stir with the sieves overnight to ensure dryness. The next morning, the mixture was cooled down to −20 °C, and a catalytic amount of TMSOTf (40 μL, 0.221 mmol) was slowly added to the flask. The reaction was completed in 45 min, and quenched with TEA. The molecular sieves were filtered off through a celite pad. The filtrate was then concentrated and purified using column chromatography (10% Et₂O/cyclohexane) to afford compound 43 as a clear oil (4.32 g, 60%). ¹H NMR (500 MHz, CDCl₃) δ 8.08 (d, J = 7.6 Hz, 2H), 7.88 – 7.78 (m, 5H), 7.77 – 7.64 (m, 8H), 7.64 – 7.54 (m, 3H), 7.53 – 7.47 (m, 4H), 7.46 – 7.29 (m, 12H), 7.27 (s, 1H), 7.18 (t, J = 7.7 Hz, 2H), 5.89 (dd, J = 10.1, 3.1 Hz, 1H), 5.69 (s, 1H), 5.65 – 5.59 (m, 2H), 5.16 (s, 1H), 4.93 (d, J = 12.0 Hz, 1H), 4.80 (d, J = 12.0 Hz, 1H), 4.55 (t, J = 4.4 Hz, 1H), 4.41 (dd, J = 9.6, 6.3 Hz, 1H), 4.33 (d, J = 3.8 Hz, 1H), 4.11 (t, J = 4.8 Hz, 1H), 3.76 (dd, J = 11.3, 3.1 Hz, 1H), 3.68 (dd, J = 11.4, 4.6 Hz, 1H), 1.21 (d, J = 6.1 Hz, 3H), 0.99 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 165.9, 165.5, 135.9, 135.8, 135.0, 134.0, 133.6, 133.4, 133.3, 133.2, 133.2, 133.1, 133.0, 132.1, 130.1, 129.9, 129.8, 129.8, 129.5, 129.4, 129.2, 129.1, 128.7, 128.5, 128.5, 128.4, 128.2, 127.9, 127.8, 127.8, 127.6, 126.4, 126.3, 126.1, 125.6, 97.7, 88.2, 83.5, 81.5, 76.7, 72.5, 71.8, 71.1, 70.0, 67.3, 63.3, 26.9, 19.3, 17.9. HRMS (ESI) (m/z) calculated for C₆₅H₆₂O₁₁SSi [M+NH₄]⁺ 1096.4126, found 1096.4128.

4.2.23. Phenyl 2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-2-O-(2-naphthalenylmethyl)-1-thio-β-D-ribofuranoside (44)

Compound 43 (1.76 g, 1.63 mmol) was dissolved in THF (10 mL) and pyridine (8 mL) and stirred at 0 °C within a polyethylene bottle. Next, HF·pyridine (2.12 mL, 70% HF in pyridine mixture, 81.5 mmol) was added. The reaction was completed in 3 h and washed with sat. NaHCO_3. The EtOAc extracts from this wash were next washed in an aqueous solution of 5% HCl. The compound was then dried over Na₂SO₄, concentrated, and subjected to column chromatography (0–10% EtOAc/DCM) to afford compound 44 as a clear oil (1.00 g, 69%). ¹H NMR (500 MHz, CDCl₃) δ 8.08 (d, J = 7.3 Hz, 2H), 7.87 (s, 1H), 7.85 – 7.79 (m, 4H), 7.75 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 7.4 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.58 – 7.54 (m, 1H), 7.51 – 7.39 (m, 8H), 7.31 – 7.28 (m, 3H), 7.26 – 7.24 (m, 2H), 7.17 (t, J = 7.8 Hz, 2H), 5.88 (dd, J = 10.2, 3.4 Hz, 1H), 5.72 (dd, J = 3.0, 1.5 Hz, 1H), 5.65 – 5.59 (m, 2H), 5.17 (s, 1H), 4.94 (d, J = 12.0 Hz, 1H), 4.82 (d, J = 12.0 Hz, 1H), 4.49 – 4.36 (m, 3H), 4.22 – 4.16 (m, 1H), 3.89 (d, J = 12.5 Hz, 1H), 3.75 – 3.65 (m, 1H), 1.89 (dd, J = 9.2, 3.0 Hz, 1H), 1.20 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.8, 165.7, 165.6, 134.8, 133.7, 133.4, 133.3, 133.3, 133.2, 132.1, 130.1, 129.8, 129.7, 129.4, 129.3, 129.3, 129.2, 128.7, 128.6, 128.5, 128.4, 128.2, 128.0, 127.8, 126.4, 126.4, 126.2, 125.5, 98.5, 88.6, 83.1, 82.4, 77.3, 72.4, 71.6, 70.9, 70.0, 67.5, 61.6, 17.9. HRMS (ESI) (m/z) calculated for C₄₉H₄₄O₁₁S [M+NH₄]⁺ 858.2948, found 858.2892.

4.2.24. Phenyl 2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-2-O-(2-naphthalenylmethyl)-5-O-[2-propyn-1-yloxy-(2-cyanoethoxy)-phosphono]-1-thio-β-D-ribofuranoside (45)

To a solution of 44 (1.00 g, 1.19 mmol) in DCM (160 mL), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (950 μL, 2.98 mmol) and 5-phenyl-1H-tetrazole (0.348 g, 2.38 mmol) were added at 0 °C. The mixture was stirring for 3 h to afford the phosphoramidite product. HRMS (ESI) (m/z) calculated for C₅₈H₆₁N₂O₁₂PS, [M+H]⁺ 1041.3761, found 1041.3775. Propargyl alcohol (4.2 mL, 71.4 mmol) and 5-phenyl-1H-tetrazole (0.522 g, 3.57 mmol) were added to the reaction mixture and stirred at room temperature for 1.5 h to afford the phosphite. HRMS (ESI) (m/z) calculated for C₅₅H₅₀NO₁₃PS, [M+NH₄]⁺ 1013.3084, found 1013.3041. The crude product was concentrated and re-dissolved in CH₃CN (160 mL), and H₂O₂ (30% in H₂O, 465 μL, 5.95 mmol) were added to the mixture and stirred at room temperature for 1 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20–80% EtOAc/Hexanes) to afford compound 45 (0.723 g, 60%, 3 steps) as a clear oil. ¹H NMR (500 MHz, CDCl₃) δ 8.08 (d, J = 7.5 Hz, 2H), 7.87 – 7.78 (m, 5H), 7.75 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 7.7 Hz, 2H), 7.62 (t, J = 7.3 Hz, 1H), 7.55 – 7.40 (m, 9H), 7.31 (app t, J = 7.5 Hz, 3H), 7.25 (app t, J = 6.4 Hz, 2H), 7.16 (t, J = 7.6 Hz, 2H), 5.85 (d, J = 10.1 Hz, 1H), 5.71 (s, 1H), 5.66 – 5.59 (m, 2H), 5.17 (s, 1H), 4.92 (d, J = 11.9 Hz, 1H), 4.79 – 4.68 (m, 3H), 4.50 (s, 1H), 4.42 – 4.16 (m, 7H), 2.73 – 2.59 (m, 3H), 1.19 (d, J = 6.0 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.7, 165.6, 165.6, 165.5, 165.5, 134.5, 134.4, 133.6, 133.2, 133.2, 133.2, 133.1, 133.1, 133.0, 132.1, 132.0, 129.9, 129.7, 129.6, 129.2, 129.2, 129.2, 129.1, 129.0, 128.6, 128.4, 128.3, 128.3, 128.0, 127.9, 127.9, 127.7, 126.3, 126.3, 126.1, 125.3, 116.3, 116.3, 98.7, 98.7, 88.2, 88.2, 81.8, 80.0, 79.9, 72.1, 72.1, 71.3, 70.6, 69.9, 69.9, 67.5, 62.3, 62.2, 62.2, 55.8, 55.8, 55.8, 55.7, 19.5, 19.5, 19.4, 19.4, 17.8. HRMS (ESI) (m/z) calculated for C₅₅H₅₀NO₁₄PS [M+NH₄]⁺ 1029.3033, found 1029.2984.

4.2.25. Phenyl 2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-2-O-acetyl-5-O-[2-propyn-1-yloxy-(2-cyanoethoxy)-phosphono]-1-thio-β-D-ribofuranoside (46)

Compound 45 (121 mg, 0.120 mmol) was dissolved in MeOH/DCM (1/1, v/v, 1 mL) and stirred at room temperature. Next, DDQ (109 mg, 0.479 mmol) was added. After 4 h, DCM (10 mL) was added to the reaction mixture, along with pyridine (5 mL) and Ac₂O (3 mL), and stir at room temperature for 4 h. When complete, the reaction was quenched with MeOH and washed with sat. NaHCO₃. The EtOAc extracts of this wash were then washed with an aqueous solution of 5% HCl. The product was extracted again with EtOAc, dried over Na₂SO₄, concentrated, and subjected to column chromatography (20–60% EtOAc/Hexanes), affording compound 46 as a clear oil (98.0 mg, 81%). ¹H NMR (500 MHz, CDCl₃) δ 8.11 – 8.06 (m, 2H), 7.99 – 7.94 (m, 2H), 7.85 – 7.79 (m, 2H), 7.63 (t, J = 7.4 Hz, 1H), 7.53 (m, 5H), 7.47 – 7.30 (m, 6H), 7.27 (t, J = 7.7 Hz, 2H), 5.76 – 5.71 (m, 1H), 5.67 – 5.64 (m, 2H), 5.50 (d, J = 3.4 Hz, 1H), 5.30 (q, J = 3.9, 3.4 Hz, 1H), 5.13 (s, 1H), 4.74 (d, J = 11.2 Hz, 2H), 4.50 (dt, J = 8.8, 5.6 Hz, 1H), 4.42 – 4.38 (m, 1H), 4.37 – 4.25 (m, 5H), 4.18 (dq, J = 12.2, 6.3 Hz, 1H), 2.72 (td, J = 6.5, 2.0 Hz, 2H), 2.65 (t, J = 2.5 Hz, 1H), 2.30 (s, 3H), 1.35 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.1, 165.6, 165.6, 165.6, 165.6, 133.7, 133.4, 133.3, 132.7, 132.5, 132.3, 132.2, 129.9, 129.7, 129.2, 129.2, 129.1, 129.0, 128.7, 128.5, 128.3, 128.2, 128.2, 116.3, 116.3, 97.3, 97.3, 88.7, 88.6, 80.2, 80.2, 80.2, 80.2, 77.2, 77.2, 77.2, 77.1, 76.9, 75.8, 75.8, 75.1, 74.9, 71.3, 70.5, 69.6, 67.8, 67.0, 66.9, 66.8, 66.7, 62.6, 62.3, 62.3, 55.9, 55.8, 55.8, 55.8, 20.9, 19.5, 19.5, 19.5, 19.4, 17.6. HRMS (ESI) (m/z) calculated for C₄₆H₄₄NO₁₅PS [M+NH₄]⁺ 931.2513, found 931.2491.

4.2.26. α-L-rhamnopyranosyl-(1→3)-5-O-(2-propyn-1-yl-hydrogen phosphate)-D-ribitol (1)

Compound 46 (67.0 mg, 0.073 mmol) was dissolved in acetone (3 mL) with one drop of water. While stirring at room temperature, NBS (26.0 mg, 0.147 mmol) was added. After 15 min, the reaction mixture was washed with sat. Na₂SO₃. The compound was extracted from this wash using EtOAc, dried over Na₂SO₄, concentrated, and subjected to column chromatography (1/2–2/1 EtOAc/Hexanes) to afford compound 47 as a clear oil (36.0 mg, 59% yield). HRMS (ESI) (m/z) calculated for C₄₀H₄₀NO₁₆P [M+NH₄]⁺ 839.2428, found 839.2442. Compound 47 (23.0 mg, 0.028 mmol) was dissolved in TEA/DCM (1/1, v/v, 12 mL) and stirred at room temperature overnight to deprotect the phosphate. When the reaction was complete, the solvent was removed via rotavapor. HRMS (ESI) (m/z) calculated for C₃₇H₃₇O₁₆P [M−H]⁻ 767.1746, found 767.1702. The crude product was re-dissolved in EtOH (25 mL). Next, sodium NaBH₄ (2.6 mg, 0.067 mmol) was added over the course of 3 h and the reaction was stirred at room temperature for 4 h. After quenching with AcOH (30 μL), the solvent was removed via rotavapor. HRMS (ESI) (m/z) calculated for C₃₇H₃₉O₁₆P [M−H]− 769.1903, found 769.1758. The crude product was re-dissolved in EtOH (5 mL), and NH₄OH (25 mL) was added. The mixture was stirred at 35 °C overnight. After completion, the solvent was blown off, and the concentrate was subjected to column chromatography (60/5/1.5, 60/10/2.5, 60/20/5 EtOAc/MeOH/H₂O) to afford compound 1 (8 mg, 69%, 3 steps) as a clear oil. ¹H NMR (500 MHz, MeOD) δ 4.97 (s, 1H), 4.51 (d, J = 6.2 Hz, 2H), 4.13 – 4.02 (m, 2H), 4.01 – 3.92 (m, 2H), 3.89 – 3.82 (m, 1H), 3.81 – 3.70 (m, 3H), 3.67 – 3.62 (m, 1H), 3.59 (dd, J = 11.3, 6.9 Hz, 1H), 3.38 (t, J = 9.5 Hz, 1H), 2.86 (s, 1H), 1.26 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, MeOD) δ 101.7, 80.6, 79.4, 74.1, 72.4, 72.0, 70.8, 70.7, 70.3, 69.2, 66.7, 63.1, 52.9, 16.5. HRMS (ESI) calculated for C₁₄H₂₅O₁₂P [M−H]⁻ 415.1011, found 415.0947.

4.2.27. 2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl-(1→4)-1-O-[(1,1-Dimethylethyl)diphenylsilyl]-5-O-[(4-methoxyphenyl)methyl]-2,3-bis-O-(2-naphthalenylmethyl)-D-ribitol (48)

To a solution of 16 (1.44 g, 1.82 mmol) and 18 [46] (0.87 g, 2.27 mmol) in DCM (30 mL), ground 4Å molecular sieves (2.63 g) was added and stirred at room temperature for 16 h. After cooling down to −40 °C, NIS (1.05 g, 4.67 mmol) and TMSOTf (0.1 mL, 0.553 mmol) were added and stirred for 30 min before the molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. Na₂S₂O₃, sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (5%, 10%, 15%, 20% EtOAc/Hexanes) to afford compound 48 (1.58 g, 82%) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.88 – 7.81 (m, 4H), 7.80 – 7.77 (m, 3H), 7.76 – 7.72 (m, 5H), 7.52 – 7.45 (m, 5H), 7.43 – 7.36 (m, 3H), 7.30 (q, J = 7.7 Hz, 4H), 7.12 (d, J = 8.5 Hz, 2H), 6.78 (d, J = 8.6 Hz, 2H), 5.48 (dd, J = 3.1, 1.7 Hz, 1H), 5.43 (dd, J = 10.1, 3.4 Hz, 1H), 5.27 (s, 1H), 5.13 (t, J = 10.0 Hz, 1H), 4.97 (d, J = 11.3 Hz, 1H), 4.83 (d, J = 11.7 Hz, 1H), 4.78 (d, J = 11.3 Hz, 1H), 4.64 (d, J = 11.7 Hz, 1H), 4.52 (dd, J = 4.1, 2.5 Hz, 1H), 4.32 (q, J = 11.6 Hz, 2H), 4.13 – 3.99 (m, 4H), 3.77 (s, 3H), 3.75 – 3.71 (m, 2H), 3.68 (dd, J = 10.5, 3.2 Hz, 1H), 2.19 (s, 3H), 2.05 (s, 3H), 2.02 (s, 3H), 1.12 (s, 9H), 1.09 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.2, 170.1, 170.1, 159.1, 136.0, 135.9, 135.8, 135.7, 133.7, 133.4, 133.3, 133.0, 133.0, 130.3, 129.8, 129.8, 129.3, 128.1, 128.1, 127.8, 127.8, 127.7, 126.6, 126.4, 126.1, 126.1, 126.0, 125.9, 125.8, 113.7, 97.2, 79.4, 79.0, 76.1, 73.7, 73.0, 72.4, 71.1, 70.8, 70.1, 69.5, 66.9, 63.0, 55.3, 31.0, 27.0, 21.1, 20.9, 20.9, 19.4, 17.5, 14.3. HRMS (ESI) (m/z) calculated for C₆₃H₇₀O₁₃Si, [M+NH₄]⁺ 1080.4924, found 1080.4865.

4.2.28. 2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl-(1→4)-1-O-[(1,1-Dimethylethyl)diphenylsilyl]-2,3-bis-O-(2-naphthalenylmethyl)-D-ribitol (49)

To a solution of 48 (0.30 g, 0.282 mmol) in DCM (10 mL), TFA (10% in DCM, v/v, 10 mL) was added, and the mixture was stirred at 0 °C for 30 min. After quenching with TEA, the reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10%, 20%, 30% EtOAc/Hexanes) to afford compound 49 (0.2060 g, 77%) as a colorless syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.86 – 7.74 (m, 7H), 7.74 – 7.68 (m, 5H), 7.48 (dd, J = 6.6, 2.7 Hz, 4H), 7.46 – 7.34 (m, 4H), 7.30 (app t, J = 7.5 Hz, 4H), 5.45 – 5.39 (m, 2H), 5.15 – 5.08 (m, 2H), 4.97 (d, J = 11.2 Hz, 1H), 4.85 (d, J = 11.7 Hz, 1H), 4.79 (d, J = 11.3 Hz, 1H), 4.67 (d, J = 11.7 Hz, 1H), 4.25 (s, 1H), 4.15 – 4.11 (m, 1H), 4.02 (qd, J = 11.2, 3.6 Hz, 3H), 3.84 (dd, J = 23.5, 10.1 Hz, 2H), 3.78 (dd, J = 6.7, 3.5 Hz, 1H), 2.59 (s, 1H), 2.17 (s, 3H), 2.02 (d, J = 7.1 Hz, 6H), 1.13 – 1.07 (m, 12H). ¹³C NMR (126 MHz, CDCl₃) δ 170.2, 170.1, 170.1, 135.9, 135.7, 135.5, 135.4, 133.4, 133.3, 133.3, 133.2, 133.1, 133.0, 132.5, 130.1, 129.9, 129.5, 128.3, 128.2, 128.1, 128.0, 127.8, 127.8, 127.8, 127.8, 126.8, 126.6, 126.2, 126.1, 126.1, 126.0, 125.9, 96.3, 79.9, 79.3, 76.6, 74.2, 72.7, 71.0, 70.0, 69.4, 67.2, 62.9, 61.4, 60.5, 28.7, 27.0, 21.2, 21.0, 20.9, 20.9, 19.4, 17.5, 14.3. HRMS (ESI) (m/z) calculated for C₅₅H₆₂O₁₂Si, [M+NH₄]⁺ 960.4349, found 960.4255.

4.2.29. 2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl-(1→4)-1-O-[(1,1-dimethylethyl)diphenylsilyl]-5-O-[2-propyn-1-yloxy-(2-cyanoethoxy)-phosphono]-D-ribitol (51)

To a solution of 49 (0.10 g, 0.106 mmol) in DCM (3 mL), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (65.5 μL, 0.206 mmol) and 5-phenyl-1H-tetrazole (0.0209 g, 0.143 mmol) were added at 0 °C. The mixture was allowed to warm to room temperature while stirring for 1 h to afford the phosphoramidite. HRMS (ESI) (m/z) calculated for C₆₄H₇₉N₂O₁₃PSi, [M+H]⁺ 1143.5162, found 1143.5108. Propargyl alcohol (0.75 mL, 12.9 mmol) and 5-phenyl-1H-tetrazole (0.0223 g, 0.153 mmol) were added to the reaction mixture and stirred at room temperature for 1 h to afford the phosphite. HRMS (ESI) (m/z) calculated for C₆₁H₆₈NO₁₄PSi, [M+NH₄]⁺ 1115.4485, found 1115.4646. CH₃CN (6 mL) and H₂O₂ (30% in H₂O, 25 μL, 0.320 mmol) were added to the mixture and stirred at room temperature for 16 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30%, 40%, 50% EtOAc/Hexanes) to afford compound 50 (0.0969 g, 82%, 3 steps) as a colorless syrup. HRMS (ESI) (m/z) calculated for C₆₁H₆₈NO₁₅PSi, [M+NH₄]⁺ 1131.4434, found 1131.4404. To a solution of 50 (0.2341 g, 0.210 mmol) in DCM (8 mL) and MeOH (2 mL), DDQ (0.19 g, 0.837 mmol) was added and stirred at room temperature for 16 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. Na₂SO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (2%, 4%, 6%, 8%, 10% acetone/DCM) to afford compound 51 (0.17 g, 97%) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.70 – 7.63 (m, 4H), 7.47 – 7.37 (m, 6H), 5.33 (dd, J = 4.9, 3.7 Hz, 1H), 5.23 (ddd, J = 10.1, 3.0, 1.5 Hz, 1H), 5.07 (t, J = 9.9 Hz, 1H), 4.98 (d, J = 2.4 Hz, 1H), 4.70 (dd, J = 11.3, 2.3 Hz, 2H), 4.39 (dt, J = 13.1, 6.6 Hz, 2H), 4.33 – 4.25 (m, 2H), 4.16 – 4.11 (m, 1H), 3.96 – 3.81 (m, 4H), 3.69 (s, 1H), 2.89 (s, 1H), 2.82 – 2.77 (m, 2H), 2.62 (s, 1H), 2.16 (s, 1H), 2.14 (d, J = 1.4 Hz, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.12 (dd, J = 6.0, 4.2 Hz, 3H), 1.07 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 170.3, 170.3, 170.0, 135.7, 132.8, 132.7, 132.7, 130.2, 130.2, 128.0, 128.0, 116.9, 116.8, 98.4, 98.2, 79.0, 79.0, 78.8, 78.8, 76.8, 76.8, 72.6, 72.5, 70.8, 70.7, 69.7, 69.2, 67.5, 67.4, 67.4, 67.3, 65.3, 65.2, 62.6, 62.5, 62.5, 55.9, 55.8, 31.1, 27.0, 22.3, 21.0, 20.9, 20.9, 19.7, 19.7, 19.7, 19.6, 19.4, 17.4. ³¹P NMR (202 MHz, CDCl₃) δ −1.49, −1.61. HRMS (ESI) (m/z) calculated for C₃₉H₅₂NO₁₅PSi, [M+Na]⁺ 856.2736, found 856.2806.

4.2.30. 2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl-(1→4)-5-O-[2-propyn-1-yloxy-(2-cyanoethoxy)-phosphono]-D-ribitol (52)

To a solution of 51 (0.17 g, 0.204 mmol) in THF (3 mL), hydrogen fluoride pyridine (~70%, 0.4 mL, 15.4 mmol) was added at 0 °C. The reaction was allowed to warm to room temperature while stirring for 16 h before quenching with sat. NaHCO₃. The mixture was then extracted with EtOAc. The organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (2%, 4% MeOH/DCM) to afford compound 52 (21 mg, 17%) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 5.29 (ddd, J = 6.1, 3.3, 1.7 Hz, 1H), 5.22 (dd, J = 10.2, 3.3 Hz, 1H), 5.08 (t, J = 10.0 Hz, 1H), 5.01 (dd, J = 7.4, 1.3 Hz, 1H), 4.72 (ddd, J = 6.4, 4.1, 3.1 Hz, 2H), 4.50 – 4.35 (m, 2H), 4.35 – 4.25 (m, 2H), 4.11 (s, 1H), 4.01 – 3.92 (m, 1H), 3.92 – 3.85 (m, 1H), 3.85 – 3.74 (m, 3H), 3.29 (s, 3H), 2.89 – 2.75 (m, 2H), 2.71 (dt, J = 6.6, 2.4 Hz, 1H), 2.14 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H), 1.21 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.5, 170.4, 170.1, 117.0, 97.7, 97.5, 78.4, 78.3, 78.2, 78.1, 77.3, 77.3, 77.3, 77.3, 77.1, 72.4, 72.3, 71.1, 70.7, 70.7, 69.8, 69.3, 69.3, 67.6, 67.3, 63.8, 62.8, 62.8, 62.7, 62.6, 56.1, 56.1, 56.0, 56.0, 29.8, 21.1, 21.0, 20.9, 19.8, 19.7, 19.7, 19.7, 17.5. ³¹P NMR (202 MHz, CDCl₃) δ −1.44, −1.52. HRMS (ESI) (m/z) calculated for C₂₃H₃₄NO₁₅P, [M+NH₄]⁺ 613.2004, found 613.2022.

4.2.31. 2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl-(1→4)-5-O-(2-propyn-1-yl-hydrogen phosphate)-D-ribitol (53)

To a solution of 52 (29.5 mg, 0.0495 mmol) in dry DCM (6 mL), dry TEA (8 mL) was added and stirred at room temperature for 16 h. The reaction mixture was concentrated and then the crude product was purified by column chromatography on silica gel (12/9/1, 12/9/2, 12/9/3 CHCl₃/MeOH/AcOH) to afford compound 53 (23.5 mg, 88%) as a light yellow syrup. ¹H NMR (500 MHz, CD₃OD) δ 5.35 (dd, J = 3.3, 1.7 Hz, 1H), 5.28 (dd, J = 10.1, 3.4 Hz, 1H), 5.19 (d, J = 1.1 Hz, 1H), 5.01 (t, J = 10.0 Hz, 1H), 4.53 – 4.47 (m, 2H), 4.25 – 4.16 (m, 2H), 4.14 – 4.07 (m, 2H), 3.82 – 3.76 (m, 2H), 3.72 – 3.68 (m, 1H), 3.64 (dd, J = 11.1, 6.0 Hz, 1H), 2.83 (t, J = 2.4 Hz, 1H), 2.13 (s, 3H), 2.04 (s, 3H), 1.95 (s, 3H), 1.18 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 171.8, 171.7, 171.7, 98.5, 79.0, 78.9, 75.5, 74.1, 72.8, 72.3, 71.2, 71.0, 67.9, 66.0, 66.0, 64.8, 54.3, 54.2, 20.7, 20.7, 20.6, 17.7. HRMS (ESI) (m/z) calculated for C₂₀H₃₁O₁₅P, [M+Na]⁺ 565.1293, found 565.1320.

4.2.32. α-L-rhamnopyranosyl-(1→4)-5-O-(2-propyn-1-yl-hydrogen phosphate)-D-ribitol (3)

To a solution of 53 (20.7 mg, 0.0382 mmol) in EtOH (3 mL), NH₄OH (7 mL) was added, and the mixture was stirred at room temperature for 16 h. The solvent was blown off, and then the crude product was purified by column chromatography on silica gel (60/20/1, 60/20/2.5, 60/20/5, 60/30/5 EtOAc/MeOH/H₂O) to afford compound 3 (10 mg, 63%) as a white solid. ¹H NMR (500 MHz, D₂O) δ 5.09 (s, 1H), 4.54 (dd, J = 10.0, 2.3 Hz, 2H), 4.21 – 4.16 (m, 1H), 4.11 – 4.05 (m, 3H), 3.86 – 3.75 (m, 5H), 3.64 (dd, J = 11.5, 6.6 Hz, 1H), 3.46 (t, J = 9.7 Hz, 1H), 2.96 – 2.92 (m, 1H), 1.29 (d, J = 6.3 Hz, 3H). ¹³C NMR (126 MHz, D₂O) δ 100.2, 79.2, 77.0, 75.8, 72.0, 71.9, 71.3, 70.2, 70.0, 69.2, 64.4, 62.5, 53.6, 16.6. HRMS (ESI) (m/z) calculated for C₁₄H₂₅O₁₂P, [M−H]⁻ 415.1011, found 415.1041.

4.2.33. Phenyl 2,4,6-tri-O-acetyl-3-O-2-propyn-1-yl-α-D-glucopyranosyl-(1→3)-4-O-acetyl-2-O-benzoyl-1-thio-α-L-rhamnopyranoside (55)

To a solution of 34 (2.86 g, 4.24 mmol) and 21 (2.55 g, 5.09 mmol) in DCM (50 mL), ground 4Å molecular sieves (5.61 g) was added and stirred at room temperature for 16 h. After cooling down to −45 °C, TMSOTf (0.85 mL, 4.70 mmol) was added and stirred for 30 min before the molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10%, 15%, 20% EtOAc/Hexanes) to afford compound 54 as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₅₄H₅₂O₁₂S, [M+NH₄]⁺ 942.3523, found 942.3518. To a solution of 54 in DCM (20 mL) and MeOH (5 mL), DDQ (3.83 g, 16.9 mmol) was added and stirred at room temperature for 2 d. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. Na₂SO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. HRMS (ESI) (m/z) calculated for C₃₂H₃₆O₁₂S, [M+NH₄]⁺ 662.2271, found 662.2244. The crude compound was dissolved in pyridine (10 mL), Ac₂O (6 mL) was added and stirred at room temperature for 6 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30% EtOAc/Hexanes) to afford compound 55 (0.556 g, 18%, 3 steps) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 7.3 Hz, 1H), 7.46 – 7.35 (m, 4H), 7.29 – 7.20 (m, 3H), 5.70 (s, 1H), 5.52 (s, 1H), 5.30 (t, J = 9.8 Hz, 1H), 5.21 (d, J = 3.0 Hz, 1H), 4.91 (t, J = 9.8 Hz, 1H), 4.74 (dd, J = 10.0, 3.1 Hz, 1H), 4.31 (dd, J = 9.5, 6.2 Hz, 1H), 4.20 – 4.03 (m, 5H), 3.98 – 3.92 (m, 1H), 3.83 (t, J = 9.6 Hz, 1H), 2.32 (s, 1H), 2.16 (s, 3H), 2.11 (s, 3H), 2.02 (s, 3H), 1.58 (s, 3H), 1.27 (d, J = 6.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.5, 169.6, 169.1, 165.2, 133.5, 133.0, 131.6, 129.9, 129.1, 129.1, 128.5, 127.9, 92.8, 85.8, 79.5, 75.7, 74.4, 72.3, 72.1, 71.6, 69.7, 69.0, 68.2, 67.9, 62.1, 59.6, 20.8, 20.8, 20.7, 20.2, 17.5. HRMS (ESI) (m/z) calculated for C₃₆H₄₀O₁₄S, [M+NH₄]⁺ 746.2483, found 746.2471.

4.2.34. Phenyl 2,4,6-tri-O-acetyl-3-O-2-propyn-1-yl-α-D-glucopyranosyl-(1→3)-4-O-acetyl-2-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-5-O-[(1,1-Dimethylethyl)diphenylsilyl]-2-O-(2-naphthalenylmethyl)-1-thio-β-D-ribofuranoside (58)

To a solution of 55 (0.556 g, 0.763 mmol) in acetone (28 mL) and water (1 mL), NBS (0.688 g, 3.05 mmol) was added. The mixture was stirred at room temperature for 5 h before the reaction mixture was concentrated and re-dissolved in DCM. After washing with sat. Na₂SO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20–60% EtOAc/Hexanes) to afford compound 56 (0.283 g, 58%) as a light yellow syrup. HRMS (ESI) calculated for C₃₀H₃₆O₁₅ [M+NH₄]⁺ 654.2398, found 654.2370. Compound 56 (0.283 g, 0.445 mmol) was dissolved in DCM (5 mL) and stirred at room temperature. Next, K₂CO₃ (3.0 g) was added to the solution. The K₂CO₃ was filtered off through a celite pad after the reaction, the filtrate was then concentrated and purified by column chromatography on silica gel (10–50% EtOAc/Hexanes) to afford compound 57 as a clear oil (0.337 g, 61%). HRMS (ESI) calculated for C₃₂H₃₆C_l3NO₁₅ [M+Na]⁺ 802.1048, found 802.0972. Compound 57 (0. 337 g, 0.445 mmol) was dissolved in DCM (8 mL) along with compound 9 (0.402 g, 0.667 mmol) and ground 4Å molecular sieves (1.5 g). The mixture was stirred overnight at room temperature for drying. The next day, after cooling down to −30 °C, TMSOTf (5 μL, 0.018 mmol) was added to the mixture and stirred for 1 h. The reaction was then quenched with TEA, followed by removing the molecular sieves through a celite pad. The filtrate was concentrated and purified using column chromatography (2–14% EtOAc/toluene) to afford compound 58 as a clear oil (0.354 g, 66%). ¹H NMR (500 MHz, CDCl₃) δ 8.12 (d, J = 5.3 Hz, 2H), 7.91 – 7.83 (m, 3H), 7.80 (s, 1H), 7.74 – 7.60 (m, 5H), 7.52 (d, J = 1.6 Hz, 6H), 7.56 – 7.48 (m, 6H), 7.27 – 7.25 (m, 1H), 7.23 – 7.16 (m, 2H), 5.54 (s, 1H), 5.48 (s, 1H), 5.27 – 5.16 (m, 2H), 5.05 (s, 1H), 4.98 (app t, J = 8.2 Hz, 1H), 4.90 (d, J = 11.6 Hz, 1H), 4.80 (d, J = 9.9 Hz, 1H), 4.74 (d, J = 11.7 Hz, 1H), 4.48 (d, J = 3.4 Hz, 1H), 4.26 (s, 1H), 4.18 (d, J = 12.5 Hz, 2H), 4.15 – 4.05 (m, 4H), 4.01 (d, J = 12.2 Hz, 1H), 3.91 – 3.82 (m, 2H), 3.72 (d, J = 11.2 Hz, 1H), 3.65 (d, J = 11.0 Hz, 1H), 2.38 (s, 1H), 2.06 (d, J = 2.4 Hz, 3H), 2.03 (d, J = 2.5 Hz, 3H), 1.90 (d, J = 2.3 Hz, 3H), 1.67 (d, J = 2.4 Hz, 3H), 1.16 (s, 3H), 1.00 (s, 8H). ¹³C NMR (126 MHz, CDCl₃) δ 170.8, 169.9, 169.9, 169.2, 165.5, 135.8, 135.7, 134.9, 133.8, 133.6, 133.3, 133.2, 133.0, 132.9, 132.1, 130.1, 129.9, 129.8, 129.5, 129.1, 129.1, 128.7, 128.4, 128.3, 128.0, 127.9, 127.8, 127.8, 127.6, 126.5, 126.4, 126.2, 125.6, 125.4, 97.5, 93.8, 88.2, 83.6, 81.4, 79.7, 76.5, 75.9, 74.3, 72.7, 72.5, 72.2, 72.0, 69.1, 69.0, 68.3, 67.1, 63.3, 61.8, 59.8, 26.9, 21.0, 20.8, 20.7, 20.4, 19.2, 17.8. HRMS (ESI) calculated for C₆₈H₇₄O₁₈SSi [M+NH₄]⁺ 1256.4709, found 1256.4689.

4.2.35. Phenyl 2,4,6-tri-O-acetyl-3-O-2-propyn-1-yl-α-D-glucopyranosyl-(1→3)-4-O-acetyl-2-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-2-O-(2-naphthalenylmethyl)-5-O-[2-propyn-1-yloxy-(2-cyanoethoxy)-phosphono]-1-thio-β-D-ribofuranoside (60)

Compound 58 (0.354 g, 0.286 mmol) was dissolved in THF (2.5 mL) and pyridine (2 mL) and stirred at 0 °C within a polyethylene bottle. Next, HF·pyridine (0.53 mL, 70% HF in pyridine mixture, 20.0 mmol) was added. The reaction was completed in 2 h and washed with sat. NaHCO_3. The EtOAc extracts from this wash were next washed in an aqueous solution of 5% HCl. The compound was then dried over Na₂SO₄, concentrated, and subjected to column chromatography (10–50% EtOAc/Hexanes) to afford compound 59 as a clear oil (0.236 g, 83%). HRMS (ESI) (m/z) calculated for C₅₂H₅₆O₁₈S [M+NH₄]⁺ 1018.3531, found 1018.3543. To a solution of 59 (0.236 g, 0.236 mmol) in DCM (35 mL), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (188 μL, 0.590 mmol) and 5-phenyl-1H-tetrazole (0.0690 g, 0.472 mmol) were added at 0 °C. The mixture was stirring for 4 h to afford the phosphoramidite product. HRMS (ESI) (m/z) calculated for C₆₁H₇₃N₂O₁₉PS, [M+H]⁺ 1201.4344, found 1201.4352. Dry MeOH (0.6 mL, 14.2 mmol) and 5-phenyl-1H-tetrazole (0.103 g, 0.708 mmol) were added to the reaction mixture and stirred at room temperature for 3 h to afford the phosphite. HRMS (ESI) (m/z) calculated for C₅₆H₆₂NO₂₀PS, [M+H]⁺ 1132.3402, found 1132.3446. The crude product was concentrated and re-dissolved in CH₃CN (35 mL), and H₂O₂ (30% in H₂O, 92 μL, 1.18 mmol) were added to the mixture and stirred at room temperature for 30 min. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20–80% EtOAc/Hexanes) to afford compound 60 (0.244 g, 90%, 3 steps) as a clear oil. ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 7.4 Hz, 2H), 7.81 (d, J = 7.6 Hz, 3H), 7.72 (s, 1H), 7.57 (t, J = 7.1 Hz, 1H), 7.45 (d, J = 8.2 Hz, 7H), 7.30 – 7.20 (m, 3H), 5.50 (s, 1H), 5.42 (s, 1H), 5.15 (t, J = 9.6 Hz, 2H), 4.98 (s, 1H), 4.91 (t, J = 9.7 Hz, 1H), 4.84 (d, J = 11.7 Hz, 1H), 4.71 (dd, J = 9.8, 2.2 Hz, 1H), 4.65 (d, J = 11.7 Hz, 1H), 4.33 (s, 1H), 4.27 (d, J = 4.2 Hz, 1H), 4.22 – 3.99 (m, 13H), 3.95 (d, J = 12.1 Hz, 1H), 3.84 – 3.68 (m, 5H), 2.67 – 2.58 (m, 2H), 2.26 (s, 1H), 1.99 (s, 6H), 1.82 (s, 3H), 1.58 (s, 3H), 1.08 (d, J = 5.8 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 171.0, 170.5, 169.8, 169.6, 169.1, 165.4, 165.4, 134.5, 134.4, 133.6, 133.1, 133.0, 132.0, 129.9, 129.2, 129.1, 128.6, 128.3, 127.8, 127.7, 126.5, 126.5, 126.3, 126.2, 125.5, 116.5, 98.3, 93.2, 88.2, 81.7, 79.5, 75.8, 74.3, 72.4, 72.3, 71.7, 71.4, 68.8, 68.5, 68.1, 67.3, 62.0, 61.6, 60.3, 59.6, 21.0, 20.8, 20.6, 20.5, 20.2, 19.5, 19.5, 19.5, 19.4, 17.7, 14.1. HRMS (ESI) (m/z) calculated for C₅₆H₆₂NO₂₁PS [M+NH₄]⁺ 1165.3616, found 1165.3680.

4.2.36. Phenyl 2,4,6-tri-O-acetyl-3-O-2-propyn-1-yl-α-D-glucopyranosyl-(1→3)-4-O-acetyl-2-O-benzoyl-α-L-rhamnopyranosyl-(1→3)-2-O-acetyl-5-O-[2-propyn-1-yloxy-(2-cyanoethoxy)-phosphono]-1-thio-β-D-ribofuranoside (61)

Compound 60 (244 mg, 0.213 mmol) was dissolved in MeOH/DCM (1/1, v/v, 1 mL) and stirred at room temperature. Next, DDQ (193 mg, 0.851 mmol) was added and stirred for 4 h. HRMS (ESI) calculated for C₄₅H₅₄NO₂₁PS [M+NH₄]⁺ 1025.2990, found 1025.2996. The crude compound was concentrated and re-dissolved in DCM (20 mL) and pyridine (10 mL). Ac₂O (6 mL) was added and stirred at room temperature overnight. The reaction was then quenched with MeOH and washed with sat. NaHCO₃. The EtOAc extracts of this wash were then washed with an aqueous solution of 5% HCl. The product was extracted again with EtOAc, dried over Na₂SO₄, concentrated, and purified by column chromatography (40–100% EtOAc/Hexanes), affording compound 61 as a clear oil (154 mg, 69%). ¹H NMR (500 MHz, CDCl₃) δ 8.05 (d, J = 7.5 Hz, 2H), 7.59 (t, J = 7.1 Hz, 1H), 7.51 – 7.44 (m, 4H), 7.34 – 7.26 (m, 3H), 5.38 (s, 2H), 5.27 – 5.13 (m, 3H), 5.00 – 4.90 (m, 2H), 4.70 (dd, J = 9.8, 2.6 Hz, 1H), 4.39 – 4.31 (m, 1H), 4.27 – 4.02 (m, 11H), 3.92 (d, J = 10.0 Hz, 1H), 3.84 – 3.72 (m, 5H), 2.66 (t, J = 5.8 Hz, 2H), 2.28 (s, 1H), 2.12 (s, 6H), 2.06 (s, 3H), 2.02 (s, 3H), 1.58 (s, 3H), 1.21 (d, J = 6.3 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.6, 170.0, 169.8, 169.6, 169.2, 165.5, 165.5, 133.8, 132.5, 132.4, 132.2, 132.2, 130.0, 129.2, 129.0, 128.7, 128.2, 116.5, 116.5, 97.2, 93.2, 88.6, 88.6, 80.1, 80.0, 79.5, 75.9, 75.9, 75.8, 74.9, 74.4, 72.5, 71.5, 71.1, 68.8, 68.3, 68.2, 67.8, 66.8, 66.7, 62.2, 62.1, 62.1, 61.7, 60.3, 59.7, 55.0, 54.9, 21.0, 20.9, 20.9, 20.7, 20.7, 20.3, 19.6, 19.5, 17.5, 14.2. HRMS (ESI) calculated for C₄₇H₅₆NO₂₂PS [M+NH₄]⁺ 1067.3096, found 1067.3128.

4.2.37. 3-O-2-Propyn-1-yl-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-5-O-(methyl-hydrogen phosphate)-D-ribitol (2)

Compound 61 (154 mg, 0.147 mmol) was dissolved in acetone (6.5 mL) with 5 drops of water. While stirring at room temperature, NBS (39.0 mg, 0.220 mmol) was added. After 45 min, the reaction mixture was washed with sat. Na₂SO₃. The compound was extracted from this wash using EtOAc, dried over Na₂SO₄, concentrated, and subjected to column chromatography (1%, 2% MeOH/DCM) to afford compound 62 as a clear oil (104 mg, 74% yield). HRMS (ESI) (m/z) calculated for C₄₁H₅₂NO₂₃P [M+NH₄]⁺ 975.3011, found 975.3048. Compound 62 (22.0 mg, 0.028 mmol) was dissolved in TEA/DCM (1/1, v/v, 12 mL) and stirred at room temperature for a day to deprotect the phosphate. When the reaction was complete, the solvent was removed via rotavapor. HRMS (ESI) (m/z) calculated for C₃₈H₄₉O₂₃P [M−H]⁻ 903.2329, found 903.2293. The crude product was re-dissolved in EtOH (25 mL). Next, sodium NaBH₄ (2.1 mg, 0.055 mmol) was added and the reaction was stirred at room temperature for 16 h. After quenching with AcOH (30 μL), the solvent was removed via rotavapor. HRMS (ESI) (m/z) calculated for C₃₈H₅₁O₂₃P [M−H]⁻ 905.2486, found 905.2468. The crude product was re-dissolved in EtOH (5 mL), and NH₄OH (25 mL) was added. The mixture was stirred at rt for 2 d. After completion, the solvent was blown off, and the concentrate was subjected to column chromatography (60/5/1.25, 60/10/2.5, 60/20/5, 50/20/5, 45/20/5 EtOAc/MeOH/H₂O) to afford compound 2 (8 mg, 59%, 3 steps) as a clear oil. ¹H NMR (500 MHz, CD₃OD) δ 5.03 (s, 1H), 4.96 (d, J = 3.7 Hz, 1H), 4.50 (t, J = 2.4 Hz, 2H), 4.12 (s, 1H), 4.06 – 3.99 (m, 2H), 3.99 – 3.89 (m, 2H), 3.86 – 3.82 (m, 1H), 3.81 – 3.72 (m, 5H), 3.69 – 3.63 (m, 2H), 3.62 – 3.55 (m, 4H), 3.50 (dd, J = 15.5, 6.1 Hz, 2H), 3.41 (t, J = 9.4 Hz, 1H), 2.80 (s, 1H), 1.27 (d, J = 6.0 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 102.6, 97.7, 82.8, 82.0, 81.5, 78.3, 75.4, 73.4, 73.3, 72.0, 71.6, 71.0, 70.8, 68.9, 67.9, 64.5, 62.2, 60.9, 53.2, 18.0. HRMS (ESI) calculated for C₂₁H₃₇O₁₇P [M−H]⁻ 591.1696, found 591.1633.

4.2.38. 4,6-Di-O-acetyl-2-O-(2-naphthalenylmethyl)-3-O-2-propyn-1-yl-α-D-glucopyranosyl-(1→3)-1-O-Acetyl-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranoside (63)

To a solution of 34 (0.4 g, 0.593 mmol) and 24 (0.30 g, 0.666 mmol) in DCM (10 mL), ground 4Å molecular sieves (0.81 g) was added and stirred at room temperature for 3 h. After cooling down to −45 °C, TMSOTf (54 μL, 0.298 mmol) was added and stirred for 1.5 h before the molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20%, 30% EtOAc/Hexanes) to afford compound 63 (0.30 g, 58%) as a light yellow syrup. ¹H NMR (300 MHz, CDCl₃) δ 8.03 (d, J = 7.2 Hz, 2H), 7.93 – 7.84 (m, 5H), 7.74 – 7.68 (m, 2H), 7.55 – 7.46 (m, 7H), 7.30 (dd, J = 15.0, 7.3 Hz, 3H), 6.23 (d, J = 1.7 Hz, 1H), 5.76 – 5.70 (m, 1H), 5.37 (d, J = 3.3 Hz, 1H), 5.26 (s, 1H), 5.04 (d, J = 11.7 Hz, 1H), 4.94 (t, J = 9.9 Hz, 1H), 4.75 (d, J = 12.1 Hz, 1H), 4.60 (d, J = 12.1 Hz, 1H), 4.42 (dd, J = 9.6, 3.1 Hz, 1H), 4.33 (t, J = 2.6 Hz, 2H), 4.30 – 4.22 (m, 1H), 4.11 – 4.00 (m, 4H), 3.87 (t, J = 9.6 Hz, 1H), 3.67 (dd, J = 9.6, 3.4 Hz, 1H), 2.29 (t, J = 2.2 Hz, 1H), 2.17 (s, 3H), 2.08 (s, 3H), 1.68 (s, 3H), 1.50 (d, J = 6.1 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 170.6, 169.6, 168.6, 165.7, 135.3, 135.1, 133.4, 133.3, 133.1, 133.0, 132.9, 130.0, 129.2, 128.4, 128.1, 128.0, 127.8, 127.8, 127.6, 126.4, 126.3, 126.1, 126.0, 125.9, 125.5, 125.4, 92.5, 91.0, 80.3, 78.9, 77.7, 75.6, 74.2, 72.7, 72.2, 70.4, 68.5, 67.8, 67.1, 62.0, 59.9, 53.5, 21.0, 20.8, 20.5, 18.2. HRMS (ESI) (m/z) calculated for C₅₀H₅₀O₁₄, [M+NH₄]⁺ 892.3539, found 892.3504.

4.2.39. 4,6-Di-O-acetyl-2-O-(2-naphthalenylmethyl)-3-O-2-propyn-1-yl-α-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranosyl-(1→4)-1-O-[(1,1-dimethylethyl)diphenylsilyl]-5-O-[(4-methoxyphenyl)methyl]-2,3-bis-O-(2-naphthalenylmethyl)-D-ribitol (66)

Hydrazine monohydrate (0.15 mL) and AcOH (0.20 mL) were added to THF (5 mL). The mixture was stirred at 0 °C until white precipitate formed. The precipitate was washed with THF and collected to afford NH₂NH₂·AcOH as a white crystal. To a solution of 63 (0.30 g, 0.343 mmol) in THF (7 mL) and MeOH (0.7 mL), NH₂NH₂·AcOH (0.0383 mL, 0.416 mmol) was added, and the mixture was stirred at room temperature for 2 days. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with water, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (30% EtOAc/Hexanes) to afford compound 64 (0.21 g, 74%) as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₄₈H₄₈O₁₃, [M+NH₄]⁺ 850.3433, found 850.3405. To a solution of 64 (0.21 g, 0.252 mmol) in DCM (10 mL), trichloroacetonitrile (0.5 mL, 4.99 mmol) and dry K₂CO₃ (0.55 g, 3.98 mmol) were added, and the mixture was stirred at room temperature for 16 h. After filtering the molecular sieves through a celite pad, the reaction mixture was concentrated, and the crude product was purified by column chromatography on silica gel (15%, 20% EtOAc/Hexanes) to afford compound 65 (0.16 g, 65%) as a colorless syrup. HRMS (ESI) (m/z) calculated for C₅₀H₄₈C_l3NO₁₃, [M+Na]⁺ 998.2083, found 998.2056. To a solution of 65 (0.16 g, 0.164 mmol) and 16 (0.15 g, 0.190 mmol) in DCM (4 mL), ground 4Å molecular sieves (0.32 g) was added and stirred at room temperature for 3 h. After cooling down to −10 °C, TMSOTf (3 μL, 0.0166 mmol) was added. The mixture was allowed to warm to room temperature while stirring for 1 h before the molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (20% EtOAc/Hexanes) to afford compound 66 (0.18 g, 68%) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 8.06 (d, J = 7.6 Hz, 2H), 7.90 (s, 1H), 7.89 – 7.78 (m, 8H), 7.77 – 7.67 (m, 8H), 7.59 – 7.55 (m, 2H), 7.53 – 7.46 (m, 8H), 7.42 (dd, J = 13.3, 6.8 Hz, 2H), 7.40 – 7.25 (m, 9H), 7.20 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.5 Hz, 2H), 5.91 (s, 1H), 5.49 – 5.39 (m, 2H), 5.31 – 5.23 (m, 1H), 5.07 – 4.96 (m, 2H), 4.92 (t, J = 9.8 Hz, 1H), 4.85 (d, J = 11.8 Hz, 1H), 4.78 (dd, J = 11.6, 4.6 Hz, 2H), 4.67 – 4.52 (m, 4H), 4.43 (d, J = 11.6 Hz, 1H), 4.39 – 4.27 (m, 3H), 4.27 – 4.21 (m, 2H), 4.18 – 4.14 (m, 1H), 4.12 – 3.99 (m, 4H), 3.88 (t, J = 9.5 Hz, 1H), 3.84 – 3.77 (m, 2H), 3.76 – 3.72 (m, 2H), 3.71 (s, 3H), 3.66 (dd, J = 9.6, 3.3 Hz, 1H), 2.27 (s, 1H), 1.78 (s, 3H), 1.67 (s, 3H), 1.43 (d, J = 6.1 Hz, 3H), 1.12 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 170.4, 169.7, 165.9, 159.1, 136.0, 135.9, 135.8, 135.7, 135.3, 133.6, 133.4, 133.4, 133.3, 133.3, 133.2, 133.0, 133.0, 130.4, 130.0, 129.8, 129.8, 129.3, 128.4, 128.2, 128.1, 128.0, 127.9, 127.8, 127.8, 127.8, 127.8, 127.7, 127.7, 126.6, 126.4, 126.3, 126.3, 126.2, 126.1, 126.0, 126.0, 125.9, 125.9, 125.8, 125.8, 125.7, 113.7, 97.8, 92.0, 80.5, 79.7, 79.4, 79.4, 79.2, 77.8, 76.7, 75.6, 74.1, 73.0, 72.5, 72.3, 70.6, 68.9, 68.4, 68.2, 67.7, 62.9, 61.6, 60.5, 60.0, 55.2, 27.0, 20.6, 20.5, 19.4, 18.4. HRMS (ESI) (m/z) calculated for C₉₉H₁₀₀O₁₈Si, [M+NH₄]⁺ 1622.7017, found 1622.7026.

4.2.40. 4,6-Di-O-acetyl-2-O-(2-naphthalenylmethyl)-3-O-2-propyn-1-yl-α-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranosyl-(1→4)-1-O-[(1,1-dimethylethyl)diphenylsilyl]-2,3-bis-O-(2-naphthalenylmethyl)-D-ribitol (67)

To a solution of 66 (0.16 g, 0.0996 mmol) in DCM (5 mL), TFA (10% in DCM, v/v, 5 mL) was added, and the mixture was stirred at 0 °C for 1 h. After quenching with TEA, the reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (25% EtOAc/Hexanes) to afford compound 67 (0.13 g, 88%) as a colorless syrup. ¹H NMR (500 MHz, CDCl₃) δ 8.01 (d, J = 7.4 Hz, 2H), 7.88 (s, 1H), 7.87 – 7.56 (m, 7H), 7.76 – 7.63 (m, 10H), 7.56 – 7.50 (m, 2H), 7.50 – 7.41 (m, 8H), 7.41 – 7.32 (m, 4H), 7.32 – 7.27 (m, 5H), 7.25 (d, J = 8.6 Hz, 1H), 5.81 (s, 1H), 5.36 (d, J = 3.2 Hz, 1H), 5.29 – 5.22 (m, 2H), 4.99 (d, J = 11.0 Hz, 2H), 4.91 – 4.84 (m, 2H), 4.78 (d, J = 11.1 Hz, 1H), 4.71 (d, J = 12.0 Hz, 1H), 4.66 (d, J = 11.8 Hz, 1H), 4.57 – 4.49 (m, 2H), 4.35 – 4.25 (m, 3H), 4.21 (d, J = 10.2 Hz, 1H), 4.18 – 4.11 (m, 2H), 4.07 – 3.95 (m, 4H), 3.91 (dd, J = 12.1, 4.0 Hz, 1H), 3.85 (dd, J = 10.9, 6.9 Hz, 3H), 3.80 – 3.75 (m, 1H), 3.63 (dd, J = 9.6, 3.3 Hz, 1H), 2.24 (t, J = 2.1 Hz, 1H), 1.79 (s, 3H), 1.67 (s, 3H), 1.39 (d, J = 6.2 Hz, 3H), 1.08 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 170.5, 169.7, 166.0, 135.9, 135.7, 135.7, 135.5, 135.4, 135.2, 133.4, 133.4, 133.3, 133.3, 133.2, 133.2, 133.1, 133.0, 133.0, 130.0, 129.8, 129.6, 128.4, 128.3, 128.2, 128.1, 128.1, 128.1, 128.0, 127.9, 127.9, 127.8, 127.8, 127.7, 126.8, 126.5, 126.4, 126.4, 126.2, 126.2, 126.1, 126.0, 126.0, 126.0, 125.9, 125.9, 125.7, 125.6, 96.9, 92.2, 80.5, 80.3, 79.5, 79.5, 79.1, 77.8, 77.5, 75.6, 74.5, 74.1, 72.6, 72.5, 69.1, 68.5, 68.3, 67.9, 62.9, 61.7, 61.5, 60.0, 27.0, 20.7, 20.6, 19.3, 18.4. HRMS (ESI) (m/z) calculated for C₉₁H₉₂O₁₇Si, [M+NH₄]⁺ 1502.6442, found 1502.6408.

4.2.41. 4,6-Di-O-acetyl-2-O-(2-naphthalenylmethyl)-3-O-2-propyn-1-yl-α-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranosyl-(1→4)-1-O-[(1,1-dimethylethyl)diphenylsilyl]-5-O-[methoxy-(2-cyanoethoxy)-phosphono]-2,3-bis-O-(2-naphthalenylmethyl)-D-ribitol (68)

To a solution of 67 (0.1240 g, 0.0835 mmol) in DCM (6 mL), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (66 μL, 0.208 mmol) and 5-phenyl-1H-tetrazole (0.0244 g, 0.167 mmol) were added at 0 °C. The mixture was allowed to warm to room temperature while stirring for 4 h to afford the phosphoramidite product. HRMS (ESI) (m/z) calculated for C₁₀₀H₁₀₉N₂O₁₈PSi, [M+H]⁺ 1685.7255, found 1685.7199. Dry MeOH (0.1 mL, 2.47 mmol) and 5-phenyl-1H-tetrazole (0.0366 g, 0.250 mmol) were added to the reaction mixture and stirred at room temperature for 2.5 h to afford the phosphite. HRMS (ESI) (m/z) calculated for C₉₅H₉₈NO₁₉PSi, [M+Na]⁺ 1638.6132, found 1638.6116. THF (3 mL) and H₂O₂ (30% in H₂O, 7.8 μL, 0.333 mmol) were added to the mixture and stirred at room temperature for 30 min. The reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10%, 20%, 30%, 40%, 50% EtOAc/Hexanes) to afford compound 68 (85.8 mg, 63%, 3 steps) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.96 (d, J = 7.5 Hz, 2H), 7.85 – 7.72 (m, 9H), 7.72 – 7.58 (m, 9H), 7.52 – 7.18 (m, 21H), 5.76 (s, 1H), 5.32 (dd, J = 7.2, 3.4 Hz, 1H), 5.24 – 5.17 (m, 2H), 4.96 (t, J = 11.9 Hz, 2H), 4.90 – 4.83 (m, 1H), 4.78 (dd, J = 19.1, 11.5 Hz, 2H), 4.67 (d, J = 12.0 Hz, 1H), 4.59 (dd, J = 11.8, 3.9 Hz, 1H), 4.54 – 4.49 (m, 2H), 4.45 (d, J = 9.6 Hz, 1H), 4.37 (dd, J = 5.7, 3.4 Hz, 2H), 4.26 (tt, J = 16.0, 8.0 Hz, 2H), 4.20 – 4.07 (m, 4H), 4.05 – 3.90 (m, 6H), 3.80 (dd, J = 21.3, 11.4 Hz, 2H), 3.71 – 3.56 (m, 5H), 2.04 (s, 1H), 1.74 (s, 3H), 1.66 (s, 3H), 1.32 (dd, J = 5.9, 2.0 Hz, 3H), 1.04 (s, 9H). ¹³C NMR (126 MHz, CDCl₃) δ 170.4, 169.7, 166.0, 135.9, 135.8, 135.7, 135.6, 135.4, 135.3, 133.4, 133.4, 133.3, 133.2, 133.2, 133.1, 133.0, 130.0, 129.9, 129.6, 128.5, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9, 127.8, 127.8, 127.7, 126.6, 126.5, 126.4, 126.2, 126.2, 126.0, 126.0, 126.0, 125.9, 125.8, 125.7, 125.7, 125.6, 116.5, 98.2, 92.3, 80.5, 79.5, 79.4, 79.1, 77.9, 77.4, 75.7, 74.1, 72.6, 72.4, 69.2, 68.5, 68.2, 67.9, 62.4, 62.0, 61.9, 61.9, 61.7, 60.5, 60.0, 54.8, 54.8, 54.7, 27.0, 20.7, 20.6, 18.4. HRMS (ESI) (m/z) calculated for C₉₅H₉₈NO₂₀PSi, [M+NH₄]⁺ 1649.6527, found 1649.6497.

4.2.42. 3-O-2-Propyn-1-yl-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→4)-5-O-(methyl-hydrogen phosphate)-D-ribitol (4)

To a solution of 68 (0.0675 g, 0.0413 mmol) in DCM (4 mL) and MeOH (1 mL), DDQ (0.0731 g, 0.322 mmol) was added and stirred at room temperature for 16 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. Na₂SO₃, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. HRMS (ESI) (m/z) calculated for C₅₁H₆₆NO₂₀PSi, [M+NH₄]⁺ 1089.4023, found 1089.4020. The crude compound 69 was dissolved in THF (2 mL). Next, TBAF (0.2 mL, 1.0 M in THF) and AcOH (0.02 mL) were added and stirred at room temperature for 16 h before the reaction was quenched with TEA. The reaction mixture was concentrated and then the crude product was purified by column chromatography on silica gel (60/5/1, 60/10/2, 60/20/5 EtOAc/MeOH/H₂O) to afford compound 70 as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₃₂H₄₅O₂₀P, [M−H]⁻ 779.2169, found 779.2122. To a solution of 70 in EtOH (1 mL), NH₄OH (2 mL) was added, and the mixture was stirred at room temperature for 3 d. The solvent was blown off, and then the crude product was purified by column chromatography on silica gel (60/20/1, 60/20/2.5, 60/20/5 EtOAc/MeOH/H₂O) to afford compound 4 (12.9 mg, 53%, 3 steps) as a white solid. ¹H NMR (500 MHz, CD₃OD) δ 5.11 (s, 1H), 4.97 (d, J = 3.5 Hz, 1H), 4.55 – 4.47 (m, 2H), 4.21 – 4.10 (m, 2H), 4.09 – 3.95 (m, 3H), 3.85 (dd, J = 9.5, 2.7 Hz, 1H), 3.78 (dd, J = 13.7, 7.7 Hz, 4H), 3.72 – 3.57 (m, 7H), 3.55 – 3.48 (m, 2H), 3.46 – 3.39 (m, 1H), 2.82 (t, J = 2.4 Hz, 1H), 1.29 (d, J = 6.1 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 101.3, 97.5, 82.8, 81.5, 78.8, 78.1, 75.4, 74.1, 73.4, 73.4, 72.9, 72.1, 71.0, 70.5, 68.9, 65.7, 64.7, 62.2, 60.9, 59.4, 18.1. HRMS (ESI) (m/z) calculated for C₂₁H₃₇O₁₇P, [M+H]⁺ 593.1841, found 593.1842.

4.2.43. 2-Propyn-1-yl 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→3)-4-O-acetyl-2-O-benzoyl-α-L-rhamnopyranoside (72)

To a solution of 38 (0.077 g, 0.129 mmol) in DCM (3 mL), 2,4,6-tri-tert-butylpyrimidine (TTBP) (0.1120 g, 0.451 mmol), diphenyl sulfoxide (Ph₂SO) (0.0643 g, 0.318 mmol) and ground 4Å molecular sieves (0.1285 g) were added and stirred at room temperature for 16 h. After cooling down to −78 °C, trifluoromethanesulfonic anhydride (Tf₂O) (30 μL, 0.178 mmol) was added to the mixture and stirred for 10 min. Then, compound 27 (0.069 g, 0.155 mmol) in DCM (0.5 mL) was added dropwise. The mixture was allowed to warm to room temperature while stirring for 4 h before the molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10%, 20%, 30% EtOAc/Hexanes) to afford compound 71 (70 mg, 58%) as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₅₃H₅₆O₁₅, [M+NH₄]⁺ 950.3957, found 950.3938. To a solution of 71 (0.070 g, 0.075 mmol) in DCM (3 mL) and MeOH (1 mL), DDQ (0.1023 g, 0.451 mmol) was added and stirred at room temperature for 16 h. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. Na₂SO₃, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. HRMS (ESI) (m/z) calculated for C₂₆H₃₂O₁₃, [M+NH₄]⁺ 570.2181, found 570.2112. The crude compound was dissolved in pyridine (2 mL), Ac₂O (1 mL) was added and stirred at room temperature for 16 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (40% EtOAc/Hexanes) to afford compound 72 (39.2 mg, 77%, 2 steps) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 8.11 – 8.04 (m, 2H), 7.59 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.7 Hz, 2H), 5.51 (dd, J = 3.1, 2.0 Hz, 1H), 5.33 – 5.22 (m, 3H), 5.06 – 4.98 (m, 2H), 4.90 (dd, J = 10.3, 3.6 Hz, 1H), 4.26 (d, J = 2.4 Hz, 2H), 4.21 (dd, J = 10.0, 3.3 Hz, 1H), 4.18 (d, J = 3.5 Hz, 2H), 4.06 (dt, J = 10.3, 3.5 Hz, 1H), 3.89 (dq, J = 9.8, 6.1 Hz, 1H), 2.46 (t, J = 2.4 Hz, 1H), 2.16 (s, 3H), 2.11 (s, 3H), 1.97 (s, 3H), 1.91 (s, 3H), 1.52 (s, 3H), 1.28 (d, J = 6.3 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.7, 170.1, 169.9, 169.7, 169.6, 165.6, 133.7, 130.2, 129.3, 128.7, 96.6, 92.5, 78.4, 75.4, 71.9, 71.0, 70.1, 69.8, 68.7, 68.0, 67.7, 67.5, 61.9, 55.1, 20.9, 20.7, 20.7, 20.1, 17.7. HRMS (ESI) (m/z) calculated for C₃₂H₃₈O₁₆, [M+NH₄]⁺ 696.2498, found 696.2486.

4.2.44. 2-Propyn-1-yl α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside (5)

To a solution of 72 (61.4 mg, 0.0905 mmol) in MeOH (1 mL) and THF (1 mL), NaOMe (0.1 mL of a 2.41 M solution, 0.241 mmol) was added. The mixture was stirred at room temperature for 3 h before the reaction was quenched with AcOH. The reaction mixture was concentrated and absorbed onto celite. The crude product was purified by column chromatography on silica gel (15% MeOH/EtOAc) to afford compound 5 (30 mg, 91%) as a white solid. ¹H NMR (500 MHz, CD₃OD) δ 4.96 – 4.93 (m, 2H), 4.29 – 4.20 (m, 2H), 4.00 – 3.94 (m, 2H), 3.82 – 3.75 (m, 2H), 3.73 – 3.67 (m, 2H), 3.64 (dq, J = 9.7, 6.1 Hz, 1H), 3.50 (t, J = 9.5 Hz, 1H), 3.43 (dd, J = 9.8, 3.8 Hz, 1H), 3.38 – 3.33 (m, 1H), 2.88 (t, J = 2.4 Hz, 1H), 1.29 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, CD₃OD) δ 99.6, 97.9, 80.0, 78.5, 76.0, 75.0, 73.5, 72.0, 71.5, 70.4, 68.9, 62.3, 55.0, 18.0. HRMS (ESI) (m/z) calculated for C₁₅H₂₄O₁₀, [M+Na]⁺ 387.1262, found 387.1276.

4.2.45. Phenyl 2-O-[(4-Methoxyphenyl)methyl]-3,4-O-(1-methylethylidene)-6-O-(2-naphthalenylmethyl)-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-O-(2-naphthalenylmethyl)-1-thio-β-D-glucopyranoside (73)

To a solution of 42 (0.27 g, 0.562 mmol) in DCM (10 mL), TTBP (0.4907 g, 1.98 mmol), Ph₂SO (0.2850 g, 1.41 mmol) and ground 4Å molecular sieves (0.63 g) were added and stirred at room temperature for 2 h. After cooling down to −60 °C, Tf₂O (0.130 mL, 0.773 mmol) was added to the mixture. Then, compound 35 (0.2709 g, 0.541 mmol) in DCM (1.5 mL) was added dropwise when the temperature reached −40 °C. The mixture was allowed to warm to room temperature while stirring for 1.5 h before the reaction was quenched with TEA. The molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10%, 15% EtOAc/Hexanes) to afford compound 73 (0.17 g, 33%) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.81 (s, 1H), 7.76 (d, J = 7.6 Hz, 1H), 7.74 – 7.66 (m, 4H), 7.65 – 7.59 (m, 2H), 7.57 – 7.51 (m, 3H), 7.48 – 7.30 (m, 12H), 7.27 (d, J = 8.6 Hz, 1H), 6.90 (d, J = 8.5 Hz, 2H), 6.60 (d, J = 8.6 Hz, 2H), 5.60 (d, J = 3.4 Hz, 1H), 5.52 (s, 1H), 5.24 (d, J = 10.9 Hz, 1H), 4.76 (d, J = 10.9 Hz, 1H), 4.70 (d, J = 9.7 Hz, 1H), 4.60 (d, J = 12.5 Hz, 1H), 4.49 – 4.44 (m, 1H), 4.42 – 4.30 (m, 4H), 4.18 (dd, J = 17.1, 7.7 Hz, 2H), 3.86 – 3.76 (m, 2H), 3.71 (s, 3H), 3.64 (t, J = 9.3 Hz, 1H), 3.53 (dd, J = 10.6, 8.1 Hz, 1H), 3.43 – 3.36 (m, 2H), 3.28 (dd, J = 8.1, 3.5 Hz, 1H), 3.19 (dd, J = 10.7, 4.3 Hz, 1H), 1.14 (s, 3H), 1.06 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 159.2, 137.1, 136.4, 135.5, 133.3, 133.3, 133.3, 133.1, 132.9, 132.2, 129.7, 129.5, 129.3, 129.3, 128.6, 128.2, 128.0, 128.0, 127.9, 127.9, 127.8, 127.8, 126.6, 126.5, 126.4, 126.2, 126.1, 126.0, 125.8, 125.7, 125.5, 113.7, 109.0, 102.0, 95.7, 88.5, 82.3, 80.1, 76.5, 75.8, 75.3, 74.5, 73.5, 72.5, 70.5, 69.9, 69.5, 68.9, 65.7, 55.4, 28.1, 26.3. HRMS (ESI) (m/z) calculated for C₅₈H₅₈O₁₁S, [M+NH₄]⁺ 980.4038, found 980.4061.

4.2.46. Phenyl 3,4-di-O-acetyl-2-O-[(4-Methoxyphenyl)methyl]-6-O-(2-naphthalenylmethyl)-α-D-galactopyranosyl-(1→3)-4,6-di-O-acetyl-2-O-(2-naphthalenylmethyl)-1-thio-β-D-glucopyranoside (74)

To a solution of 73 (0.281 g, 0.292 mmol) in MeOH (4 mL) and DCM (1 mL), CSA (0.0555, 0.239 mmol) was added, and the mixture was stirred at room temperature for 3 d before the reaction was quenched with TEA. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (1%, 2%, 3% MeOH/DCM) to afford a light yellow syrup. HRMS (ESI) (m/z) calculated for C₄₈H₅₀O₁₁S, [M+NH₄]⁺ 852.3412, found 852.3405. The syrup was dissolved in pyridine (1.5 mL), Ac₂O (0.8 mL) was added and stirred at room temperature for 16 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (40% EtOAc/Hexanes) to afford compound 74 (0.118 g, 40%, 2 steps) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 8.02 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.74 (dd, J = 14.5, 7.6 Hz, 2H), 7.69 – 7.64 (m, 2H), 7.59 – 7.54 (m, 3H), 7.48 – 7.37 (m, 5H), 7.32 (dd, J = 5.0, 1.8 Hz, 3H), 7.23 (d, J = 8.6 Hz, 2H), 7.11 (dd, J = 8.4, 1.4 Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 5.42 (dd, J = 10.6, 3.2 Hz, 1H), 5.32 (d, J = 2.5 Hz, 1H), 5.26 (d, J = 3.4 Hz, 1H), 5.21 (dd, J = 19.4, 9.7 Hz, 2H), 4.88 (d, J = 9.7 Hz, 1H), 4.57 – 4.48 (m, 4H), 4.15 (dd, J = 12.2, 5.5 Hz, 1H), 4.09 – 4.02 (m, 2H), 4.01 – 3.93 (m, 2H), 3.83 (dd, J = 10.6, 3.5 Hz, 1H), 3.78 (s, 3H), 3.62 (t, J = 9.3 Hz, 1H), 3.40 (ddd, J = 9.8, 5.4, 2.4 Hz, 1H), 3.19 (dd, J = 10.4, 7.0 Hz, 1H), 2.90 (dd, J = 10.4, 5.0 Hz, 1H), 2.09 (s, 3H), 2.05 (s, 3H), 1.95 (s, 3H), 1.93 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.8, 170.1, 170.1, 169.5, 159.6, 135.6, 135.0, 133.5, 133.5, 133.3, 133.2, 132.9, 132.0, 129.9, 129.9, 129.2, 128.3, 128.3, 128.0, 127.9, 127.8, 127.7, 126.7, 126.2, 126.2, 126.1, 126.1, 125.8, 125.6, 113.9, 96.9, 87.8, 79.2, 77.6, 76.0, 75.8, 72.9, 72.4, 70.7, 69.9, 69.3, 68.3, 67.1, 62.5, 55.4, 21.1, 21.0, 21.0, 20.8. HRMS (ESI) (m/z) calculated for C₅₆H₅₈O₁₅S, [M+NH₄]⁺ 1020.3835, found 1020.3796.

4.2.47. 2-Propyn-1-yl 3,4-di-O-acetyl-2-O-[(4-Methoxyphenyl)methyl]-6-O-(2-naphthalenylmethyl)-α-D-galactopyranosyl-(1→3)-4,6-di-O-acetyl-2-O-(2-naphthalenylmethyl)-α-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranoside (76)

To a solution of 74 (0.1993 g, 0.199 mmol) in acetone/water 99:1 (7 mL), NBS (0.0418 g, 0.235 mmol) was added. The mixture was stirred at 0 °C for 30 min before the reaction mixture was concentrated and re-dissolved in DCM. After washing with sat. Na₂SO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (40% EtOAc/Hexanes) to afford compound 75 (0.1548 mg, 85%) as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₅₀H₅₄O₁₆, [M+NH₄]⁺ 928.3750, found 928.3765. To a solution of 75 (52.6 mg, 0.0577 mmol) in DCM (1 mL), TTBP (50.6 mg, 0.204 mmol), Ph₂SO (29.7 mg, 0.147 mmol) and ground 4Å molecular sieves (0.1177 g) were added and stirred at room temperature for 3 h. After cooling down to −60 °C, Tf₂O (0.0135 mL, 0.0802 mmol) was added to the mixture. Then, compound 27 (22 mg, 0.0493 mmol) in DCM (0.5 mL) was added dropwise when the temperature reached −40 °C. The mixture was allowed to warm to room temperature while stirring for 1 h before the reaction was quenched with TEA. The molecular sieves were filtered through a celite pad. The reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (10%, 20%, 30%, 40% EtOAc/Hexanes) to afford compound 76 (25.8 mg, 33%) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 8.04 (d, J = 7.3 Hz, 2H), 7.82 – 7.74 (m, 5H), 7.73 – 7.69 (m, 1H), 7.65 (dd, J = 18.2, 8.4 Hz, 2H), 7.56 (dd, J = 12.2, 6.5 Hz, 2H), 7.46 – 7.35 (m, 9H), 7.30 (dd, J = 8.4, 1.1 Hz, 1H), 7.24 – 7.19 (m, 3H), 7.14 (d, J = 8.5 Hz, 2H), 7.04 (dd, J = 8.4, 1.2 Hz, 1H), 6.79 (d, J = 8.6 Hz, 2H), 5.75 – 5.70 (m, 1H), 5.49 (d, J = 3.4 Hz, 1H), 5.38 (dd, J = 10.6, 3.2 Hz, 1H), 5.18 (d, J = 2.2 Hz, 1H), 5.10 (d, J = 9.6 Hz, 1H), 5.07 (d, J = 3.9 Hz, 2H), 4.95 (d, J = 11.7 Hz, 1H), 4.72 (d, J = 10.5 Hz, 1H), 4.59 – 4.49 (m, 3H), 4.46 (d, J = 11.7 Hz, 1H), 4.39 – 4.31 (m, 2H), 4.30 – 4.21 (m, 3H), 4.07 – 3.94 (m, 3H), 3.92 – 3.85 (m, 2H), 3.80 – 3.72 (m, 6H), 3.65 (t, J = 9.6 Hz, 1H), 3.08 (dd, J = 10.3, 6.4 Hz, 1H), 2.86 (dd, J = 10.3, 5.5 Hz, 1H), 2.45 (t, J = 2.3 Hz, 1H), 2.07 (s, 3H), 1.97 (s, 2H), 1.84 (s, 3H), 1.32 (d, J = 6.2 Hz, 3H), 1.27 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.9, 170.2, 169.9, 169.4, 166.0, 159.4, 135.7, 135.6, 134.6, 133.6, 133.4, 133.2, 133.0, 132.9, 130.1, 129.9, 129.8, 129.5, 128.6, 128.5, 128.1, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.3, 126.5, 126.3, 126.2, 126.2, 126.1, 126.0, 126.0, 125.9, 125.8, 125.7, 113.8, 96.9, 96.7, 91.1, 78.7, 78.5, 78.3, 77.4, 75.3, 75.1, 73.3, 73.1, 72.8, 72.6, 72.6, 71.7, 70.9, 69.8, 69.5, 68.8, 68.2, 68.0, 67.4, 66.8, 62.1, 55.4, 54.9, 29.8, 21.0, 21.0, 20.8, 20.2, 18.0. HRMS (ESI) (m/z) calculated for C₇₇H₇₈O₂₁, [M+NH₄]⁺ 1356.5374, found 1356.5398.

4.2.48. 2-Propyn-1-yl 3,4-di-O-acetyl-6-O-(2-naphthalenylmethyl)-α-D-galactopyranosyl-(1→3)-4,6-di-O-acetyl-2-O-(2-naphthalenylmethyl)-α-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranoside (77)

To a solution of 76 (82.3 mg, 0.0614 mmol) in DCM (2 mL), TFA (10% in DCM, v/v, 2 mL) was added, and the mixture was stirred at 0 °C for 1.5 h. After quenching with TEA, the reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (40% EtOAc/Hexanes) to afford compound 77 (67.7 mg, 90%) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 7.94 (d, J = 7.7 Hz, 2H), 7.84 – 7.78 (m, 4H), 7.77 (dd, J = 5.7, 3.4 Hz, 1H), 7.73 – 7.69 (m, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 7.8 Hz, 2H), 7.48 – 7.38 (m, 8H), 7.35 (t, J = 7.4 Hz, 1H), 7.25 – 7.21 (m, 2H), 7.17 (t, J = 7.7 Hz, 2H), 7.01 (d, J = 8.3 Hz, 1H), 5.73 (s, 1H), 5.54 (d, J = 3.0 Hz, 1H), 5.20 (d, J = 1.9 Hz, 1H), 5.11 – 5.04 (m, 2H), 5.01 (dd, J = 10.6, 2.9 Hz, 1H), 4.92 – 4.85 (m, 2H), 4.80 (d, J = 3.6 Hz, 1H), 4.71 (d, J = 11.2 Hz, 1H), 4.50 (d, J = 11.2 Hz, 1H), 4.39 (dd, J = 9.7, 2.6 Hz, 1H), 4.31 (t, J = 6.3 Hz, 1H), 4.28 (s, 2H), 4.09 – 4.02 (m, 2H), 3.99 – 3.94 (m, 2H), 3.91 (d, J = 11.8 Hz, 1H), 3.81 – 3.61 (m, 6H), 2.90 (d, J = 6.3 Hz, 2H), 2.47 (s, 1H), 2.07 (s, 3H), 2.01 (s, 3H), 1.79 (s, 3H), 1.43 (d, J = 6.1 Hz, 3H), 1.28 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 170.7, 170.7, 170.3, 170.0, 165.9, 135.7, 135.4, 134.5, 133.4, 133.4, 133.2, 133.1, 133.0, 132.9, 129.8, 129.2, 128.4, 128.1, 128.0, 127.9, 127.9, 127.9, 127.8, 127.8, 127.6, 126.9, 126.3, 126.3, 126.1, 126.0, 126.0, 125.9, 125.9, 125.8, 125.6, 125.2, 99.6, 96.7, 90.9, 78.9, 78.5, 77.2, 75.8, 75.4, 75.1, 72.6, 71.8, 71.6, 70.6, 70.3, 68.8, 68.7, 67.8, 67.4, 67.3, 67.1, 61.9, 54.9, 29.8, 20.9, 20.9, 20.7, 19.9, 18.1. HRMS (ESI) (m/z) calculated for C₆₉H₇₀O₂₀, [M+NH₄]⁺ 1237.4799, found 1237.4772.

4.2.49. 2-Propyn-1-yl 3,4-di-O-acetyl-6-O-(2-naphthalenylmethyl)-2-O-[2-propyn-1-yloxy-(2-cyanoethoxy)-phosphono]-α-D-galactopyranosyl-(1→3)-4,6-di-O-acetyl-2-O-(2-naphthalenylmethyl)-α-D-glucopyranosyl-(1→3)-2-O-benzoyl-4-O-(2-naphthalenylmethyl)-α-L-rhamnopyranoside (78)

To a solution of 77 (0.0762 g, 0.0625 mmol) in DCM (3 mL), 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (50 μL, 0.157 mmol) and 5-phenyl-1H-tetrazole (0.0187 g, 0.128 mmol) were added at 0 °C. The mixture was allowed to warm to room temperature while stirring for 16 h to afford the phosphoramidite product. HRMS (ESI) (m/z) calculated for C₇₈H₈₇N₂O₂₁P, [M+H]⁺ 1419.5612, found 1419.5613. Dry MeOH (0.3 mL,7.42 mmol) and 5-phenyl-1H-tetrazole (0.0278 g, 0.190 mmol) were added to the reaction mixture and stirred at room temperature for 5 h to afford the phosphite. HRMS (ESI) (m/z) calculated for C₇₃H₇₆NO₂₂P, [M+NH₄]⁺ 1367.4935, found 1367.4923. THF (1 mL) and H₂O₂ (30% in H₂O, 20 μL, 0.272 mmol) were added to the mixture and stirred at room temperature for 1 h. The reaction mixture was diluted with DCM and washed with sat. NaHCO₃ and brine. The organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (1%, 2% MeOH/DCM) to afford compound 78 (0.0484 g, 57%, 3 steps) as a light yellow syrup. ¹H NMR (500 MHz, CDCl₃) δ 8.07 (dd, J = 9.2, 8.1 Hz, 2H), 7.81 – 7.76 (m, 5H), 7.73 (d, J = 6.6 Hz, 1H), 7.71 – 7.63 (m, 2H), 7.56 (d, J = 6.8 Hz, 2H), 7.47 – 7.38 (m, 9H), 7.31 (app t, J = 8.2 Hz, 1H), 7.25 – 7.18 (m, 3H), 7.04 (d, J = 8.4 Hz, 1H), 5.76 (s, 1H), 5.54 (d, J = 3.5 Hz, 1H), 5.40 (dd, J = 10.5, 3.2 Hz, 1H), 5.25 (d, J = 3.4 Hz, 1H), 5.15 – 5.09 (m, 1H), 5.07 (s, 1H), 4.97 – 4.89 (m, 1H), 4.87 (t, J = 9.7 Hz, 1H), 4.75 (d, J = 10.4 Hz, 1H), 4.65 – 4.60 (m, 1H), 4.60 – 4.52 (m, 2H), 4.48 (d, J = 11.7 Hz, 1H), 4.37 (dd, J = 9.7, 2.8 Hz, 1H), 4.35 – 4.29 (m, 1H), 4.29 – 4.24 (m, 2H), 4.14 – 4.08 (m, 3H), 4.02 – 3.96 (m, 1H), 3.95 – 3.88 (m, 2H), 3.85 – 3.76 (m, 2H), 3.76 – 3.68 (m, 4H), 3.67 – 3.61 (m, 1H), 3.16 (dd, J = 10.2, 6.7 Hz, 1H), 2.85 (dd, J = 10.3, 5.3 Hz, 1H), 2.72 – 2.66 (m, 2H), 2.46 (t, J = 2.1 Hz, 1H), 2.06 (s, 3H), 2.04 (s, 3H), 1.88 (s, 3H), 1.32 (m, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 170.7, 170.3, 170.2, 169.9, 166.0, 135.6, 135.5, 134.4, 133.7, 133.4, 133.2, 133.2, 133.0, 132.9, 130.1, 129.4, 128.7, 128.7, 128.6, 128.1, 128.0, 128.0, 127.9, 127.8, 127.8, 127.7, 127.4, 126.5, 126.3, 126.3, 126.1, 126.0, 126.0, 125.8, 125.6, 116.8, 96.8, 95.8, 90.6, 78.6, 78.5, 78.0, 77.4, 75.4, 75.1, 72.9, 72.6, 72.0, 71.5, 71.4, 69.1, 68.8, 68.0, 67.8, 67.3, 66.7, 62.4, 62.4, 62.2, 55.0, 55.0, 54.9, 29.8, 20.9, 20.9, 20.7, 20.7, 20.2, 19.5, 19.5, 18.0. HRMS (ESI) (m/z) calculated for C₇₃H₇₆NO₂₃P, [M+Na]⁺ 1388.4438, found 1388.4411.

4.2.50. 2-Propyn-1-yl 2-O-(methyl-hydrogen phosphate)-α-D-galactopyranosyl-(1→3)-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside (6)

To a solution of 78 (0.0484 g, 0.0354 mmol) in DCM (3 mL) and MeOH (1 mL), DDQ (0.0648 g, 0.285 mmol) was added and stirred at room temperature for 5 d. The reaction mixture was concentrated and then re-dissolved in EtOAc. After washing with sat. Na₂SO₃, sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. HRMS (ESI) (m/z) calculated for C₄₀H₅₂NO₂₃P, [M+NH₄]⁺ 963.3006, found 963.3051. The crude compound was dissolved in pyridine (2 mL), Ac₂O (0.5 mL) was added and stirred at room temperature for 16 h before the reaction was quenched with MeOH. The reaction mixture was concentrated and then re-dissolved in DCM. After washing with sat. NaHCO₃ and brine, the organic layer was dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography on silica gel (1%, 2%, 2.5% MeOH/DCM) to afford compound 79 as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₄₆H₅₈NO₂₆P, [M+NH₄]⁺ 1089.3323, found 1089.3357. To a solution of 79 in dry DCM (1 mL), dry TEA (1 mL) was added and stirred at room temperature for 16 h. The reaction mixture was concentrated and then the crude product was purified by column chromatography on silica gel (10%, 15% MeOH/DCM) to afford compound 80 as a light yellow syrup. HRMS (ESI) (m/z) calculated for C₄₃H₅₅O₂₆P, [M+NH₄]⁺ 1036.3057, found 1036.3154. To a solution of 80 in EtOH (1 mL), NH₄OH (2 mL) was added, and the mixture was stirred at room temperature for 4 d. The solvent was blown off, and then the crude product was purified by column chromatography on silica gel (60/20/1, 60/20/2.5, 60/20/5, 60/30/5 EtOAc/MeOH/H₂O) to afford compound 6 (4.2 mg, 19%, 4 steps) as a white solid. ¹H NMR (500 MHz, D₂O) δ 5.62 (d, J = 3.8 Hz, 1H), 5.08 (d, J = 3.7 Hz, 1H), 5.02 (d, J = 1.4 Hz, 1H), 4.34 – 4.24 (m, 4H), 4.18 – 4.14 (m, 1H), 4.06 (d, J = 2.9 Hz, 1H), 4.02 – 3.95 (m, 3H), 3.83 (dd, J = 9.7, 3.1 Hz, 1H), 3.81 – 3.71 (m, 6H), 3.68 (dd, J = 9.9, 3.7 Hz, 1H), 3.65 (d, J = 10.9 Hz, 3H), 3.58 (t, J = 9.7 Hz, 1H), 2.92 (t, J = 2.3 Hz, 1H), 1.32 (d, J = 6.2 Hz, 3H). ¹³C NMR (126 MHz, D₂O) δ 98.5, 97.4, 95.5, 79.1, 78.7, 76.0, 75.3, 73.0, 71.2, 70.3, 70.1, 69.7, 69.6, 69.2, 69.1, 68.2, 66.6, 60.5, 60.0, 54.7, 53.2, 16.6. HRMS (ESI) (m/z) calculated for C₂₂H₃₇O₁₈P, [M−H]⁻ 619.1645, found 619.1632.

Highlights.

• The capsular polysaccharides of Streptococcus pneumoniae serotypes 6A and 6B are attractive targets for antibody generation.

• Optimal epitopes for the capsular polysaccharides are unknow.

• Synthesis of glycans that encompass varied “frames” along the polysaccharide will allow identification of optimal epitopes.

• Six glycan “frames” from the capsular polysaccharide were synthesized.

• Glycans included propargyl groups to allow short-tethered conjugation to virus-like particles.