A Rational Synthesis of a Branched Decaarabinofuranoside Related to the Fragments of Mycobacterial Polysaccharides

Abstract

A rational synthesis of the branched decaarabinofuranoside with 4-(2-azidoethoxy)phenyl aglycone (a Janus aglycone) related to the non-reducing terminal fragments of the arabinogalactan and lipoarabinomannan from Mycobacterium tuberculosis was proposed. Since the most challenging step is the formation of a 1,2-cis glycosidic linkage, we have significantly simplified access to a library of oligoarabinofuranosides derived from Mycobacterium tuberculosis polysaccharides using a silylated Ara-β-(1→2)-Ara disaccharide as the glycosyl donor. The application of a Janus aglycone also allowed us to reduce the number of reaction steps in glycoside synthesis. The obtained arabinans can be useful to further prepare conjugates as antigens for creating tuberculosis screening assays.

1. Introduction

The main causative agent of tuberculosis is Mycobacterium tuberculosis [1,2], and mycobacterial infections have received significant attention due to the increasing number of cases worldwide [3]. The components of the mycobacterial cell wall include arabinogalactan (AG) and lipoarabinomannan (LAM), which have a common motif consisting of α-(1→5), α-(1→3), and β-(1→2) glycosidic bonds. Due to the critical roles of AG and LAM in infectivity and pathogenicity of Mycobacterium tuberculosis, their structural fragments are currently attracting unprecedented attention for the study of pathways of biosynthesis of these glycopolymers [4], the development of diagnostics [5,6] and vaccines [7,8,9], as well as new effective antimicrobial drugs. It was found that neoglycoconjugates derived from synthetic fragments of LAM with bovine serum albumin (BSA), as well as neoglycoconjugates based on the non-reducing terminal branched hexasaccharide fragment (Ara₆) with mycobacterial recombinant proteins, are important for the diagnosis of tuberculosis [6,10].

It should be noted that, despite many papers describing the preparation of oligoarabinofuranosides [11,12,13,14,15,16,17,18,19,20,21,22,23,24], the synthesis of the fragments of the mycobacterial cell wall remains a significant challenge. This is especially true for the preparation of oligoarabinofuranosides containing a large number of monosaccharide units (≥10), and the number of such studies remains limited.

It is known that 1,2-trans-α-arabinofuranosides can be easily synthesized using glycosyl donors with a participating acyl group at O-2. In contrast, the preparation of 1,2-cis-β-arabinofuranosides is more challenging, and 2-O-benzyl-containing glycosyl donors are often used for this purpose [13,16,18,20,21,22,24,25,26]. It should be noted that the removal of O-benzyl protective groups is incompatible with the azido group in the aglycone, which makes the synthesis less straightforward and requires additional steps [13].

We have recently showcased the benefits of glycosyl donors with O-2 protected by a triisopropylsilyl (TIPS) nonparticipating group (Figure 1, Block A, R = (t-Bu)₂Si<) in the synthesis of the terminal oligoarabinoside fragments of M. tuberculosis such as penta- and hexaarabinofuranoside with α-(1→3)-, α-(1→5)- and β-(1→2)-linkages, containing 2-azidoethyl aglycone (Figure 2). Block A with a different type of remote protection was used by us in the synthesis of diarabinofuranosides Ara-β-(1→2)-Ara with 4-(2-azidoethoxy)phenyl (AEP) and 4-(3-azidopropoxy)phenyl (APP) aglycones. Moreover, the linear α-(1→5)-, β-(1→2)-linked tetrasaccharide AEP glycoside, as well as branched α-(1→3)-, α-(1→5)-, β-(1→2)-linked tetrasaccharide as an APP glycoside, were synthesized from Block A, (R = (t-Bu)₂Si<) (Figure 2).

On the other hand, oligoarabinofuranosides with β-(1→2)-linked residues can be assembled using an Ara-β-(1→2)-Ara diarabinofuranoside glycosyl donor (Figure 1, Block B). In this case, the main difficulty is caused by the necessity to create a 1,2-trans-glycosidic linkage in the absence of a 2-O-acyl participating group. We have already demonstrated the successful stereoselective introduction of the silylated Ara-β-(1→2)-Ara disaccharide moiety (Figure 1, Block B), leading to more complex arabinans, including the formation of tetra [28] and branched hexaarabinofuranoside [30] 3 bearing APP aglycone and linear hexaarabinofuranoside with AEP aglycone [29] (Figure 2, Scheme 1).

4-(ω-Chloroalkoxy)phenyl aglycones, due to their dual function, may be called Janus aglycones in analogy to the known nanoparticles [31]. 4-(ω-Chloroalkoxy)phenyl aglycones can be converted to 4-(ω-azidoalkoxy)phenyl arabinofuranosides [27]. The azido group in aglycone can be used for further preparation of corresponding glycosides with an amino group in aglycone [10] or it can be modified by using click-chemistry methods [32]. Moreover, both 4-(ω-chloroalkoxy)phenyl and 4-(ω-azidoalkoxy)phenyl glycosides are precursors of various glycosyl donors useful for the synthesis of more complex oligoarabinofuranosides [33].

It should be noted that the synthesis of oligosaccharides demanded selective multi-step protection and deprotection of numerous hydroxy groups and control of regio- and stereoselectivity. In this regard, developing strategies aimed at reducing the number of reaction steps still remains an important task. Since the most problematic glycosylation step is the creation of a 1,2-cis-glycosidic linkage, we significantly simplified the access to the library of oligoarabinofuranosides using Ara-β-(1→2)-Ara disaccharide as a glycosyl donor. Moreover, the application of 4-(ω-chloroalkoxy)phenyl aglycones also allows us to significantly reduce the number of reaction steps in glycoside synthesis.

Earlier, we synthesized branched hexaarabinofuranoside 1 with α-(1→3)-, α-(1→5)-, β-(1→2)-linkages and successfully converted it into deprotected AEP glycoside 3 for further conjugation [30] (Figure 1, Scheme 1). However, the synthesis of hexaarabinofuranoside 1 by glycosylation of benzoylated diol 7 with diarabinofuranoside glycosyl donor 6 under NIS/Et₃SiOTf promotion was complicated by the formation of Et₃Si-substituted products of monoglycosylation of the glycosyl acceptor at both the primary and the secondary position.

In the current work, we aimed to test an alternative promotion system in the synthesis of branched hexaarabinofuranoside 2 containing the homologous 4-(2-chloroethoxy)phenyl (CEP) aglycone. Additionally, we focused on converting hexaarabinofuranoside 2 to a glycosyl donor (2→4) with subsequent preparation of deprotected decaarabinofuranoside 5 bearing AEP aglycone (Scheme 1).

We hope that the application of our rational strategy based on the use of silylated Ara-β-(1→2)-Ara disaccharide 6 (Block B) will allow us easy access to the library of oligoarabinofuranosides. These oligosaccharides can then be used to create conjugates that act as antigens for tuberculosis screening assays.

2. Results

2.1. Synthesis of Branched α-(1→5)-, α-(1→3)-, β-(1→2)-Linked Hexaarabinofuranoside 2 with CEP Aglycone

Alternative Synthesis of the Known Silylated Ara-β-(1→2)-Ara p-Tolyl Thioglycoside 6 [28]

For the synthesis of hexaarabinofuranoside 2 with a 4-(2-chloroethoxy)phenyl (CEP) aglycone, similar to the preparation of hexaarabinofuranoside [30] 1 with a homologous 4-(3-chloropropoxy)phenyl (CPP) aglycone, we planned to use silylated Ara-β-(1→2)-Ara p-tolyl thioglycoside 6 with five TIPS groups [28]. In this study, we aim to investigate the conversion of silylated disaccharides 10 and 13, which contain acetyl and CPP groups at the anomeric position, to Ara-β-(1→2)-Ara p-tolyl thioglycoside 6 (Scheme 2). For this purpose, the previously obtained hemiacetal 9 [28] with five TIPS groups was acetylated with Ac₂O/pyridine (Py), resulting in the single α-anomer of acetate 10. Next, thiolysis of 10 was carried out with p-TolSH/BF₃·Et₂O at 40 °C for 40 min, followed by silylation of the resulting desilylation products. After silica gel chromatography, disaccharide 6 was isolated in 63% yield.

Alternatively, known disaccharide 11 with CPP aglycone was desilylated with TBAF in THF. The α-linked CPP-disaccharide 12, isolated in 81% yield, contained 3% of substitution product with 4-(3-fluoropropoxy)phenyl aglycone. Moreover, silica gel chromatography failed to remove Bu₄N⁺ (15% according to NMR). The resulting crude pentaol 12 was silylated by TIPSOTf in 2,4,6-collidine to give α-linked CPP-disaccharide 13, also containing the product of substitution of a chlorine atom in the CPP aglycone with fluorine (3%). At the next stage, thiolysis of 13 with TolSH/BF₃·Et₂O was performed at −5 °C→0 °C for 2.5 h to give the target α-linked thioglycoside 6 isolated in a 50% yield. To the best of our knowledge, the products of the cleavage of the inter-saccharide glycosidic bond were not observed during thiolysis of 10 and 13. In contrast, the use of the acetylated disaccharide Ara-β-(1→2)-Ara with a 4-(ω-chloroalkoxy)phenyl aglycone [28] resulted in significant amounts of products arising from cleavage of this bond.

2.2. Synthesis of Branched Hexaarabinofuranoside 2 with the Use of Silylated Ara-β-(1→2)-Ara p-Tolyl Thioglycoside 6

At the next step, we performed the glycosylation of the diol of disaccharide with CEP aglycone 8 [28] with the Ara-β-(1→2)-Ara glycosyl donor 6 under AgOTf/NIS promotion. However, instead of the desired hexaarabinofuranoside 2, the formation of α-(1→5)-linked tetraarabinofuranoside 14 was observed.

The anomeric signals of tetraarabinofuranoside 14 were found at δ_H 5.06 (s, 1H, H-1^III), 5.20 (d, 1H, J 2.6 Hz, H-1^IV), 5.37 (s, 1H, H-1^II), and 5.83 (s, 1H, H-1^I) in the ¹H NMR spectrum, which correlated with signals at δ_C 104.8 (C-1^I), 105.0 (C-1^IV), 105.4 (C-1^II), and 106.3 (C-1^III) in the ¹³C NMR spectrum. An additional confirmation of the structure of tetraarabinofuranoside 14 and the formation of a new glycosidic linkage at the primary position followed from the fact that the ¹H–¹³C HMBC spectrum showed a correlation between the signals of H-1^II at δ_H 5.37 ppm (s, 1H) and C-5^I at δ_C 66.0 ppm, a correlation between the signals of H-1^III at δ_H 5.06 ppm (s, 1H) and C-5^II at δ_C 64.4 ppm, and a correlation between the signals of H^IV at δ_H 5.20 (d, 1H, J 2.6 Hz) and C-2^III at δ_C 90.8 ppm.

Then, we introduced the second disaccharide Ara-β-(1→2)-Ara fragment at C-3^II of disaccharide 8 under TfOH/NIS promotion to form the key hexaarabinofuranoside 2 in 90% yield.

Moreover, we successfully obtained hexaarabinofuranoside 2 in a single step by bis-glycosylation of the diol 8 with p-tolyl thioglycoside Ara-β-(1→2)-Ara 6 under TfOH/NIS promotion in very high yield (97%) (Scheme 3).

Comparison of the ¹H and ¹³C NMR spectra for hexaarabinofuranosides with CPP and CEP aglycones (1 and 2, respectively), as expected, revealed many common features.

The signals of ten TIPS groups in the ²⁹Si NMR spectrum for hexaarabinofuranosides 1, 2 were observed in a similar range from δ_Si 12.3 to 13.9, and the signals of the corresponding carbon atoms of the isopropyl groups ((CH₃)₂CH)₃Si) in the ¹³C NMR spectrum were observed in the range from δ_C 11.9 to 18.2. The signals of the isopropyl carbon atoms correlated with the ¹H NMR proton signals observed at δ_H 0.86–1.15 (m, 210H, 10 × ((CH₃)₂CH)₃Si).

The signals of the α-anomeric protons of the monosaccharide residues of hexaarabinofuranosides 2, 1 are found at δ_H 5.10 (s, 1H, H-1^III), 5.23 (s, 1H, H-1^V), 5.27 (s, 1H, H-1^II), 5.79 (s, 1H, H-1^I), and 5.11 (s, 1H, H-1^III), 5.23 (s, 1H, H-1^V), 5.27 (s, 1H, H-1^II), 5.80 (s, 1H, H-1^I), respectively. While the following signals for the two β-anomeric protons of the monosaccharide residues of hexaarabinofuranosides 2, 1 are present at δ_H 5.17 (d, 1H, J 2.5 Hz, H-1^VI), 5.25 (d, 1H, J 2.8 Hz, H-1^IV), and 5.17 (d, 1H, J 2.7 Hz, H-1^VI), 5.25 (d, 1H, J 2.8 Hz, H-1^IV), respectively.

The signals of the anomeric carbon atoms for all monosaccharide residues for hexaarabinofuranosides 2, 1 in the ¹³C NMR spectra appeared at δ_C 104.7 (C-1^IV), 104.8 (C-1^I), 105.4 (C-1^VI), 106.0 (C-1^II), 106.3 (C-1^V), 106.9 (C-1^III), and 104.69 (C-1^IV), 104.71 (C-1^I), 105.4 (C-1^VI), 105.0 (C-1^II), 106.3 (C-1^V), 106.8 (C-1^III), respectively. It is noteworthy that the low-field positions of the signals of C-1^IV and C-1^VI are untypical for β-arabinofuranosides, presumably due to the presence of bulky TIPS groups.

Moreover, similar correlations, confirming the formation of the glycosidic bonds between the corresponding monosaccharide residues in hexaarabinofuranosides 2, 1 were observed in a ¹H–¹³C HMBC spectrum: the correlation of the H-1^II proton signal at δ_H 5.27 (s, 1H) with the C-5^I carbon atom signal at δ_C 65.3; the correlation of the H-1^III proton signal at δ_H 5.1 (s, 1H) with the C-5^II carbon atom signal at δ_C 65.14; the correlation of the H-1^IV proton signal at δ_H 5.25 (d, 1H, J 2.8 Hz) with the C-2^III carbon atom signal at δ_C 90.5; and the correlation of the H-1^VI proton signal at δ_H 5.17 (d, 1H, J 2.5 Hz) with the C-2^V carbon atom signal at δ_C 91.3.

The H-2^III signal at δ_H 4.14 (d, 1H, J 0.8 Hz) correlates with the C-1^IV signal at δ_C 104.7; the H-2^III signal at 4.14 (d, 1H, J 1.1 Hz, H-2^III) correlates with the C-1^IV signal at δ_C 104.7 for hexaarabinofuranosides 2 and 1, respectively. Correlation of the H-2^V signal at 4.08 (d, 1H, J 1.2 Hz) with the C-1^VI signal at δ_C 105.4 and correlation of the H-2^V signal at 4.09 (d, 1H, J 1.3 Hz, H-2^V) with the C-1^VI signal at δ_C 105.4 were also found for hexaarabinofuranosides 2 and 1, respectively.

2.3. Synthesis of Branched α-(1→5)-, α-(1→3)-, β-(1→2)-Linked Decaarabinofuranoside 5 with 4-(2-Azidoethoxy)phenyl Aglycone

2.3.1. Conversion of the Branched α-(1→5)-, α-(1→3)-, β-(1→2)-Linked Hexaarabinofuranoside 2 to Glycosyl Donor 4

In the obtained hexaarabinofuranoside 2, CEP aglycone was cleaved under oxidative conditions ((NH₄)₂Ce(NO₃)₆ in aqueous MeCN—CH₂Cl₂). After purification by silica gel column chromatography, hemiacetal 15 (70%) as a mixture of anomers (α:β = 1:0.45 according to NMR data), contaminated with the product 16 of migration of the benzoyl group from O-2 to O-1, was obtained (Scheme 4). The ratio of the compounds in the mixture (15α:15β:16 = 1:0.45:0.28 according to NMR data) was determined by integration of the signals of corresponding anomeric protons of the residues I in the ¹H NMR spectrum: 5.62 (s, 1H, H-1^Iα) for 15α, 5.72 (t, 1H, J 5.1 Hz, H-1^Iβ) for 15β, and 6.60 (d, 1H, J 4.5 Hz, H-1^I) for 16. The formation of the β-linked product 16 of the migration of the benzoyl group followed from the low-field position of the anomeric proton δ_H 6.60 (d, 1H, J 4.5 Hz, H-1^I) in the ¹H NMR spectrum, which correlated with the signal of anomeric carbon atom of monosaccharide residue δ_C 97.04 (C-1^I) in the ¹³C NMR spectrum.

The mixture of hemiacetal 15 and β-linked benzoate 16 was treated with CF₃C(NPh)Cl in the presence of Cs₂CO₃ (Scheme 5), then subjected to gel chromatography on Bio-Beads S-X1 in toluene. N-Phenyltrifluoroacetimidate 4 was obtained as a mixture of anomers (α:β ~ 1:1.3 according to NMR data) in high yield (96%). It should be noted that the characteristic signal of β-linked benzoate 16 in the low-field position δ_H 6.60 (d, 1H, J 4.5 Hz, H-1^I minor) was absent in the ¹H NMR spectrum. On the contrary, characteristic signals related to the N-phenyltrifluoroacetimidoyl group were present. The ratio of α and β isomers of N-phenyltrifluoroacetimidate 4 was determined by integrating the following signals in the ¹H NMR spectrum: 6.51–6.57 (m, 2H, PhN (H-2, H-6)β) and 6.85–6.91 (m, 2H, PhN (H-2, H-6)α). Probably, a reverse migration of the benzoyl group from O-1 to O-2 for benzoate 16 took place under the basic conditions used for the imidate formation.

2.3.2. Synthesis of the Protected Decaarabinofuranoside 18 with CEP Aglycone

The glycosylating ability of the obtained N-phenyltrifluoroacetimidate of hexaarabinofuranoside 4 was tested in the synthesis of branched α-(1→5)-, α-(1→3)-, β-(1→2)-linked decaarabinofuranoside 18 with cleavable CEP aglycone. To this end, benzoylated tetraarabinofuranoside α-(1→5)-linked glycosyl acceptor 17 [33] was glycosylated by N-phenyltrifluoroacetimidate 4 promoted by TfOH (Scheme 5). After gel chromatography on Bio-Beads S-X1 in toluene, decaarabinofuranoside 18 was isolated in 72% yield (51% over 3 steps starting from 2). It should be noted that the use of glycosyl donor 4 generated from 2 and having a 2-O-acyl participating group ensures the creation of 1,2-trans-(α) glycosidic linkage. The signals of ten TIPS groups were observed for decaarabinofuranoside in the ²⁹Si NMR spectrum. It is important to note that no desilylation products were found despite the fact that TfOH was used as the promotor.

The anomeric configuration of decaarabinofuranoside 18 was confirmed by NMR spectroscopy. For α-linked Ara residues, the following signals of the anomeric protons were observed: δ_H 5.10 (s, 1H, H-1^VII), 5.23 (s, 1H, H-1^IX), 5.29 (s, 1H, H-1^VI), 5.37 (s, 2H, 2 × H-1), 5.38–5.41 (m, 3H, H-2^VI, 2 × H-1), 5.82 (s, 1H, H-1^I). The following signals for two β-anomeric protons of the monosaccharide residues of decaarabinofuranoside 18 are present: δ_H 5.15 (d, 1H, J 2.6 Hz, H-1^X) and δ_H 5.24 (d, 1H, J 2.8 Hz, H-1^VIII). The signals of the anomeric carbon atoms for all monosaccharide residues for decaarabinofuranoside 18 as in case of branched hexaarabinofuranosides 1 [30], 2 and α-(1→5)-, β-(1→2)-linked tetraarabinofuranoside [28] resonated in a low-field region at δ_C 104.8 (C-1^VIII), 104.9 (C-1^I), 105.5 (C-1^X), 105.85 (C-1^VI), 105.89 (C-1), 106.0 (3C, C-1^II, C-1^III, C-1^IV), 106.2 (C-1^IX), and 106.9 (C-1^VII) in the ¹³C NMR spectra. Moreover, correlations confirming the presence of glycosidic bonds between the corresponding monosaccharide residues of decaarabinofuranoside 18 were found in its ¹H–¹³C HMBC spectrum: the H-1^VI signal at δ_H 5.29 (s, 1H) correlates with the C-5^V signal at δ_C 65.3; the H-1^VII signal at δ_H 5.10 (s, 1H) correlates with the C-5^VI signal at δ_C 64.8; the H-1^VIII signal at δ_H 5.24 (d, 1H, J 2.8 Hz, H-1^VIII) correlates with the C-2^VII signal at δ_C 90.6; the H-1^IX signal at δ_H 5.23 (s, 1H, H-1^IX) correlates with the C-3^VI signal at δ_C 81.0 (C-3^VI); and the H-1^X signal at δ_H 5.15 (d, 1H, J 2.6 Hz, H-1^X) correlates with the C-2^IX signal at δ_C 91.4 (C-2^IX).

2.3.3. Synthesis of the Branched α-(1→5)-, α-(1→3)-, β-(1→2)-Linked Decaarabinofuranoside 5 with AEP Aglycone

The chlorine atom in the aglycone of the resulting decaarabinofuranoside 18 was replaced with an azido group (NaN₃, DMF, 18-crown-6) to give the corresponding AEP glycoside 19 in 84% yield (Scheme 5). Then all TIPS groups in azide 19 were removed by treatment with TBAF in THF in the presence of AcOH at 40 °C to give partially protected decaarabinofuranoside 20 isolated in a mixture with n-Bu₄N⁺ salts after silica gel chromatography, which was treated with MeONa in MeOH with subsequent acetylation (Scheme 6). After purification by silica gel chromatography, acetylated decasaccharide 21 (14% over 3 steps) was obtained. The overall low deprotection yield can be attributed to the difficulty of the removal of the numerous (ten) triisopropylsilyl groups, followed by purification from by-products.

The anomeric configuration of acetylated decasaccharide 21 was verified by NMR spectroscopy. As expected, the signals of the two β-anomeric protons were found at δ_H 5.39 (d, 1H, J 4.7 Hz, H-1^X) and δ_H 5.44 (d, 1H, J 4.7 Hz, H-1^VIII), which correlated with the signals of the anomeric carbon atoms in the characteristic regions at δ_C 99.29 (C-1^X) and 99.50 (C-1^VIII), respectively. The obtained decasaccharide 21 was deacetylated by treatment with MeONa in MeOH to give the target deprotected decasaccharide 5 in 85% yield.

The presence of two β-anomeric Ara^VIII and Ara^X residues of the target deprotected decasaccharide 5 was confirmed by the signals of the C-1^VIII and C-1^X carbon atoms at δ_C 102.5, 102.6 (C-1^VIII, C-1^X). In the HSQC spectrum, the C-1^VIII and C-1^X carbon atom signals correlated with the proton signals at δ_H 5.02–5.06 (m, 2H, H-1^VIII, H-1^X). The signals of the eight α-linked arabinofuranose moieties were observed in the characteristic low-field regions at δ_C 107.1 (C-1^IX), 107.4 (C-1^VII), 108.7 (C-1^I), 109.58, 109.63, 109.70 (2C), and 109.74 (C-1^II-VI). The upfield position of C-2^VIII and C-2^X at δ_C 79.19 additionally confirms the β-configuration of the residues VIII and X. On the contrary, the low-field position of C-2^VII, C-2^IX at δ_C 89.3 (C-2^VII), 89.6 (C-2^IX), and C-3^VI at δ_C 84.6, respectively, indicated that those residues are glycosylated.

3. Discussion

We suggested an alternative synthesis of diarabinofuranoside 6 and found that during the thiolysis of silylated disaccharides Ara-β-(1→2)-Ara (10 and 13) containing acetyl or CPP groups at the anomeric position, no products of cleavage of the inter-saccharide glycosidic bond were observed.

It is important to note that the previously proposed synthesis of 6 included the reaction of acetylated Ara-β-(1→2)-Ara bearing 4-(ω-chloroalkoxy)phenyl aglycone with TolSH/BF₃·Et₂O, which required a higher temperature and a longer time (20 °C for 16 h, then 60 °C for 2 h). Moreover, thiolysis of acetylated Ara-β-(1→2)-Ara was complicated by the formation of monosaccharides [28]. We can conclude that the protective groups in Ara-β-(1→2)-Ara have a significant effect on the course of the thiolysis. Thus, the presence of electron-donating TIPS groups is favorable for the reaction.

We tested various promotor systems, such as NIS/TfOH, NIS/TESOTf [30], and NIS/AgOTf to activate silylated Ara-β-(1→2)-Ara p-tolyl thioglycoside 6 in the synthesis of branched hexasaccharide 2. No glycosylation of the secondary 3′-OH of diarabinofuranoside glycosyl acceptor 14 was observed under mild activation conditions (NIS/AgOTf), and only α-(1→5)-linked tetraarabinofuranoside 14 was obtained. On the contrary, the use of a stronger activation system (NIS/TfOH) made possible stereospecific glycosylation of both primary (5′-OH) and secondary (3′-OH) hydroxy groups and afforded the desired hexaarabinofuranoside 2 in exceptionally high yield. We can conclude that the use of (NIS/TfOH) is more preferable for bis-glycosylation.

It should be noted that, like in the case of hexaarabinofuranoside 1 with the CPP aglycone, an exclusively α-configuration of the glycosidic linkage was observed in hexaarabinofuranoside 2 bearing the CEP aglycone. On the contrary, as we showed earlier, in the case of the use of Ara-β-(1→2)-Ara disaccharide glycosyl donor, which contains only O-benzoyl substituents, the absence of stereocontrol (α:β = 1:2) was observed [28].

TfOH also effectively facilitated the reaction between N-phenyltrifluoroacetimidate of hexaarabinofuranoside 4 and α-(1→5)-linked benzoylated tetrasaccharide glycosyl acceptor 17, resulting in the formation of decaarabinofuranoside 18. As we mentioned above, the nature of the protective group in the glycosyl donor is very essential in the outcome of glycosylation. Recently, we observed an unusual oligomerization of arabinofuranosides promoted by TfOH during the glycosylation of the primary hydroxyl group of the same α-(1→5)-linked tetraarabinofuranoside 17, which bears a 4-(2-chloroethoxy)phenyl aglycone by benzoylated N-phenyltrifluoroacetimidate [33]. To explain these unusual results, we suggested that silylated disaccharide Ara-β-(1→2)-Ara glycosyl donor 6 has superior reactivity compared to “disarmed” benzoylated glycosyl donors. Due to the higher reactivity of the former, side reactions are suppressed, and we did not observe the formation of the oligomerization products.

As the most challenging step in glycosylation is creating a 1,2-cis glycosidic linkage, we have significantly simplified access to a library of oligoarabinofuranosides from Mycobacterium tuberculosis using silylated Ara-β-(1→2)-Ara disaccharide. In addition, the use of a 4-(2-chloroethoxy)phenyl aglycone allowed us to reduce the number of reaction steps in glycosidic synthesis.

4. Materials and Methods

General methods. All reactions sensitive to air and/or moisture were carried out under an argon atmosphere. The reactions were performed with the use of commercial reagents (Aldrich (St. Louis, MO, USA), Fluka (Waltham, MA, USA), Acros Organics (Geel, Belgium)). Anhydrous solvents were purified and dried (where appropriate) according to standard procedures [34]. Dichloromethane was distilled over P₂O₅ and then over CaH₂ and stored over 4 Å molecular sieves (MS 4 Å). Powdered MS 4 Å and 3 Å molecular sieves (MS 4 Å and MS 3 Å, respectively) (Fluka, Seelze, Germany) were activated before glycosylation reactions by heating at 220 °C in high vacuum (0.2 mbar) for 6 h. Column chromatography was performed on silica gel 60 (40–63 μm, Merck, Darmstadt, Germany) using a Büchi C-815 Flash chromatograph (Büchi Labotechnic AG, Flawil, Switzerland). Thin-layer chromatography was carried out on plates with silica gel 60 on aluminum foil (Merck). Spots of compounds were visualized under UV light (254 nm) and by heating the plates (at ca. 150 °C) after immersion in a 1:10 (v/v) mixture of 85% aqueous H₃PO₄ and 95% EtOH. Gel permeation chromatography was performed on a 400 × 20 mm column packed with Bio-Beads S-X3 (200–400 mesh, Bio-Rad, Hercules, CA, USA) or on a 450 × 30 mm column packed with Bio-Beads S-X1 (200–400 mesh). A procedure for “co-evaporation” with water (or toluene) involved (multiple) addition of water (or toluene) and evaporation of volatiles on a rotary evaporator. Amberlite MB-3 mixed-bed ion-exchange resin (Fluka) (1 mL) was washed with H₂O (10 mL), 50% EtOH (5 mL), EtOH (5 mL), 50% EtOH (5 mL), and H₂O (10 mL) before use. ¹H, ¹³C, ¹⁹F, and ²⁹Si NMR spectra were recorded on a Bruker AVANCE NEO 300 spectrometer (300.23, 75.50, 282.47, and 59.65 MHz for ¹H, ¹³C, ¹⁹F, and ²⁹Si, respectively) or on a Bruker AVANCE 600 spectrometer (Billerica, MA, USA, 600.13 and 150.92 MHz for ¹H and ¹³C, respectively). The ¹H NMR chemical shifts are referred to the residual signal of CHCl₃ (δ_H 7.27 ppm), CHD₂OD (δ_H 3.31 ppm) for solutions in CD₃OD, and HDO (δ_H 4.79 ppm) for solutions in D₂O. The ¹³C NMR shifts to the central line of the CDCl₃ signal (δ_C 77.00 ppm), CD₃OD signal (δ_C 49.00 ppm), or the signal of external 1,4-dioxane in D₂O (δ_C 67.40 ppm). The ¹⁹F chemical shifts are given relative to the signal of external CFCl₃ (δ_F 0.00 ppm). The ²⁹Si chemical shifts are given relative to the signal of external Me₄Si (δ_Si 0.00 ppm). Assignments of the signals in the NMR spectra were performed using ¹H–¹H and ¹H–¹³C 2D-spectroscopy (COSY, HSQC, HMBC) and DEPT-135 experiments. Position of silyl groups was determined from ¹H–²⁹Si HMBC experiments. High-resolution mass spectra (HRMS, electrospray ionization (ESI)) were recorded in a positive ion mode on Bruker micrOTOF II or maXis mass spectrometers for 2 × 10⁻⁵ M solutions in MeCN. Optical rotations were measured using a JASCO P-2000 automatic digital polarimeter (Hachioji, Japan).

4-(2-Chloroethoxy)phenyl 2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→3)-[2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→5)]-2-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranoside (2)

(1) A mixture of disaccharide thioglycoside 6 (37 mg, 0.31 mmol) and tetrasaccharide alcohol 14 (19 mg, 0.25 mmol) was dried in vacuo for 2 h, then anhydrous CH₂Cl₂ (2 mL) was added under argon. Freshly activated powdered MS 4 Å (200 mg) (100 mg per 1 mL of solvent) was added under argon to the resulting solution. The suspension was stirred under argon at ~22 °C for 1 h, cooled to −60 °C, then NIS (7 mg, 0.31 mmol) and TfOH (1 μL) were added. Then, the temperature was allowed to rise slowly until −30 °C during 0.5 h and was kept at −30 °C for 10 min. Then the reaction was quenched by the addition of Py (50 μL), diluted with CH₂Cl₂ (15 mL), and filtered through a Celite pad. The solids were washed with CH₂Cl₂ (5 × 10 mL), and the filtrate was washed with a mixture of satd aq Na₂S₂O₃ (50 mL) and satd aq NaHCO₃ (50 mL). The aqueous layer was extracted with CH₂Cl₂ (2 × 5 mL). The combined organic extracts were filtered through a cotton wool plug, concentrated and dried in vacuo, then dissolved in toluene (2 mL) and subjected to chromatography on Bio-Beads S-X1 in toluene to give α-linked hexasaccharide 2 (52 mg, 90%); R_f = 0.50 (light petroleum–EtOAc 10:1); ${\left[\mathrm{\alpha }\right]}_D^{28}$ +31.0 (c 1.0 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 0.95–1.15 (m, 210H, 10 × ((CH₃)₂CH)₃Si), 3.63 (dd, 1H, J 9.5 Hz, J 4.5 Hz, H-5^IVa or H-5^VIa), 3.66 (dd, 1H, J 10.5 Hz, J 5.4 Hz, H-5^Va), 3.71 (dd, 1H, J 9.5 Hz, J 4.5 Hz, H-5^IVa or H-5^VIa), 3.78 (t, 2H, J 6.0 Hz, CH₂Cl), 3.73–3.99 (m, 11H, 8 × H-5, H-4^IV, H-4^VI, H-4^V), 4.03–4.05 (m, 2H, H-2^IV, H-2^VI), 4.09 (d, 1H, J 1.3 Hz, H-2^V), 4.11 (td, 1H, J 5.9 Hz, J 3.9 Hz, H-4^III), 4.14 (d, 1H, J 1.1 Hz, H-2^III), 4.15–4.19 (m, 1H, H-5^Ib), 4.17 (t, 2H, J 6.0 Hz, CH₂O), 4.22 (dd, 1H, J 7.0 Hz, J 2.1 Hz, H-3^II), 4.30 (d, 1H, J 0.9 Hz, H-3^VI), 4.29–4.32 (m, 1H, H-4^II), 4.33 (d, 1H, J 0.8 Hz, H-3^IV), 4.34 (d, 1H, J 4.0 Hz, H-3^III), 4.45 (dd, 1H, J 3.7 Hz, J 1.4 Hz, H-3^V), 4.60 (q, 1H, J 4.2 Hz, H-4^I), 5.11 (s, 1H, H-1^III), 5.17 (d, 1H, J 2.7 Hz, H-1^VI), 5.23 (s, 1H, H-1^V), 5.25 (d, 1H, J 2.8 Hz, H-1^IV), 5.27 (s, 1H, H-1^II), 5.41 (d, 1H, J 2.1 Hz, H-2^II), 5.65 (dd, 1H, J 4.5 Hz, J 2.0 Hz, H-3^I), 5.71 (d, 1H, J 1.6 Hz, H-2^I), 5.80 (s, 1H, H-1^I), 6.82–6.87 (m, 2H, OC₆H₄O (H-3, H-5)), 7.03–7.09 (m, 2H, OC₆H₄O (H-2, H-6)), 7.38–7.44 (m, 2H, 2^II-O-PhCO (H-3, H-5)), 7.44–7.62 (m, 7H, PhCO), 8.01–8.05 (m, 2H, 2^II-O-PhCO (H-2, H-6)), 8.10–8.13 (m, 2H, PhCO (H-2, H-6)), 8.13–8.16 (m, 2H, PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 11.93 (((CH₃)₂CH)₃Si), 11.96 (((CH₃)₂CH)₃Si), 11.97 (((CH₃)₂CH)₃Si), 12.01 (((CH₃)₂CH)₃Si), 12.18 (((CH₃)₂CH)₃Si), 12.23 (((CH₃)₂CH)₃Si), 12.24 (2 × ((CH₃)₂CH)₃Si), 12.3 (((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 17.9 (2 × ((CH₃)₂CH)₃Si), 17.95 (2 × ((CH₃)₂CH)₃Si), 17.96 (2 × ((CH₃)₂CH)₃Si), 18.02 (2 × ((CH₃)₂CH)₃Si), 18.03 (((CH₃)₂CH)₃Si), 18.05 (((CH₃)₂CH)₃Si), 18.08 (((CH₃)₂CH)₃Si), 18.09 (2 × ((CH₃)₂CH)₃Si), 18.11 (((CH₃)₂CH)₃Si), 18.12 (4 × ((CH₃)₂CH)₃Si), 18.17 (((CH₃)₂CH)₃Si), 18.19 (((CH₃)₂CH)₃Si), 41.9 (CH₂Cl), 63.7 (C-5^IV, C-5^VI), 63.8 (C-5^IV, C-5^VI), 64.1 (C-5^V), 64.7 (C-5^III), 65.1 (C-5^II), 65.3 (C-5^I), 68.7 (CH₂O), 77.5 (C-3^I), 77.6 (C-3^V), 78.1 (C-2^IV or C-2^VI), 78.2 (C-3^VI), 78.3 (C-2^IV or C-2^VI), 78.48 (C-3^III, C-3^IV), 78.51 (C-3^III, C-3^IV), 81.0 (C-3^II), 81.9 (C-2^I), 82.2 (C-4^II), 82.9 (C-4^I), 84.0 (C-2^II), 85.68 (C-4^IV, C-4^VI), 85.74 (C-4^IV, C-4^VI), 87.6 (C-4^III), 88.2 (C-4^V), 90.5 (C-2^III), 91.3 (C-2^V), 104.69 (C-1^IV), 104.71 (C-1^I), 105.7 (C-1^VI), 106.0 (C-1^II), 106.3 (C-1^V), 106.8 (C-1^III), 115.8 (OC₆H₄O (C-3, C-5)), 118.3 (OC₆H₄O (C-2, C-6)), 128.2 (2^II-O-PhCO (C-3, C-5)), 128.4 (PhCO (C-3, C-5)), 128.6 (PhCO (C-3, C-5)), 129.1 (PhCO (C-1)), 129.5 (PhCO (C-1)), 129.81 (PhCO (C-1)), 129.84 (2^II-O-PhCO (C-2, C-6)), 129.95 (PhCO (C-2, C-6)), 130.03 (PhCO (C-2, C-6)), 132.8 (2^II-O-PhCO (C-4)), 133.2 (PhCO (C-4)), 133.4 (PhCO (C-4)), 151.0 (OC₆H₄O (C-1)), 153.5 (OC₆H₄O (C-4)), 165.1 (2^II-O-PhCO), 165.5 (PhCO), 165.6 (PhCO); ²⁹Si INEPT NMR (60 MHz, CDCl₃): δ 12.36, 12.93, 13.26, 13.48, 13.51, 13.53, 13.56, 13.79, 13.83, 13.87; HRMS (ESI): m/z [M + 2NH₄]²⁺ Calcd for C₁₄₉H₂₇₇ClN₂O₂₉Si₁₀²⁺ 2855.7294; Found: 2855.7251;

(2) A mixture of disaccharide thioglycoside 6 (127 mg, 0.11 mmol) and disaccharide 8 (22 mg, 0.03 mmol) was dried in vacuo for 2 h, then anhydrous CH₂Cl₂ (3 mL) was added under argon. Freshly activated powdered MS 4 Å (300 mg) (100 mg per 1 mL of solvent) was added under argon to the resulting solution. The suspension was stirred under argon at ~22 °C for 1 h, cooled to −60 °C, then NIS (7 mg, 0.31 mmol) and TfOH (1 μL) were added. Then, the temperature was allowed to rise slowly to −30 °C during 0.5 h and was kept at −30 °C for 10 min. Then the reaction was quenched by the addition of Py (50 μL), diluted with CH₂Cl₂ (15 mL), and filtered through a Celite pad. The solids were washed with CH₂Cl₂ (5 × 10 mL), and the filtrate was washed with a mixture of satd aq Na₂S₂O₃ (50 mL) and satd aq NaHCO₃ (50 mL). The aqueous layer was extracted with CH₂Cl₂ (2×5 mL). The combined organic extracts were filtered through a cotton wool plug, concentrated and dried in vacuo, then dissolved in toluene (2 mL) and subjected to chromatography on Bio-Beads S-X1 in toluene to give hexasaccharide 2 (80 mg, 97%).

2,3,5-Tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→3)-[2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→5)]-2-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-d-arabinofuranosyl N-phenyltrifluoroacetimidate (4)

To a solution of hemiacetal of hexasaccharide 15 (31 mg, 0.012 mmol) in CH₂Cl₂ (1 mL), Cs₂CO₃ (30 mg, 0.093 mmol) and CF₃C(NPh)Cl [35] (4 μL, 0.023 mmol) were added at 0 °C. The reaction mixture was stirred at 20 °C for 3h. The reaction mixture was diluted with CH₂Cl₂ (20 mL) and then filtered through the cotton wool plug. The filtrate concentrated under reduced pressure, and the residue was dried in vacuo and subjected to gel chromatography on Bio-Beads S-X1 in toluene to give imidate 4 as a mixture of anomers (α:β = ~1:1.3 according to NMR), (32 mg, 96%). R_f = 0.76 (light petroleum–EtOAc, 8.5:1.5); selected signals: ¹H NMR (300 MHz, CDCl₃): δ 0.78–1.35 (m, 483H), 3.61–4.01 (m, 38H), 4.01–4.19 (m, 16H), 4.19–4.39 (m, 13H), 4.43–4.48 (m, 2H), 4.51–4.59 (m, 1H), 4.61–4.71 (m, 1H), 5.08 (s, 1H), 5.10 (s, 1H), 5.16–5.28 (m, 11H), 5.36 (d, 1H, J 1.9 Hz), 5.62 (d, 1H, J 3.7 Hz), 5.73 (s, 1H), 5.84–5.97 (m, 2H), 6.51–6.57 (m, 2H, PhN (H-2, H-6)β), 6.79 (s, 1H, H-1), 6.85–6.91 (m, 2H, PhN (H-2, H-6)α), 6.98–7.10 (m, 2H, PhN (H-4)), 7.12–7.20 (m, 2H, PhN (H-3, H-5)), 7.22–7.31 (m, 5H, PhCO (H-3, H-5)), 7.36–7.63 (m, 25H, PhCO (H-3, H-5); PhCO (H-4), 7.97–8.17 (m, 16H, PhCO (H-2, H-6)); ¹³C NMR (76 MHz, CDCl₃): δ 12.0 (((CH₃)₂CH)₃Si), 12.2 (((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 18.0 (((CH₃)₂CH)₃Si), 18.1 (((CH₃)₂CH)₃Si), 63.7, 63.8, 64.1, 64.6, 64.7, 67.8, 78.3, 78.4, 83.7, 85.7, 87.6, 88.2, 90.8, 91.2, 97.3 (C-1), 105.4 (C-1), 105.89 (C-1), 106.91 (C-1), 119.3 (PhN (C-2, C-6)), 119.7 (PhN (C-2, C-6)), 124.0, 128.2, 128.4, 128.49, 128.53, 128.6, 129.85 (PhCO (C-2, C-6)), 129.94 (PhCO (C-2, C-6)), 130.0 (PhCO (C-2, C-6)), 132.9 (PhCO (C-4)), 133.4 (PhCO (C-4)), 133.6 (PhCO (C-4)), 165.1 (CO), 165.4 (CO); ²⁹Si INEPT NMR (60 MHz, CDCl₃): δ 12.39, 12.45, 12.84, 12.94, 13.26, 13.38, 13.47, 13.50, 13.53, 13.55, 13.74, 13.79, 13.83, 13.88, 13.91; ¹⁹F NMR (282 MHz, CDCl₃): δ −65.76 (CF₃); HRMS (ESI): m/z [M + NH₄]⁺ Calcd for C₁₄₉H₂₇₀F₃N₂O₂₈Si₁₀⁺ 2872.7404; found: 2872.7378.

4-(2-Azidoethoxy)phenyl β-d-arabinofuranosyl-(1→2)-α-d-arabinofuranosyl-(1→3)-[β-d-arabinofuranosyl-(1→2)-α-d-arabinofuranosyl-(1→5)]-α-d-arabinofuranosyl-(1→5)-α-d-arabinofuranosyl-(1→5)-α-d-arabinofuranosyl-(1→5)-α-d-arabinofuranosyl-(1→5)-α-d-arabinofuranosyl-(1→5)-α-d-arabinofuranoside (5)

Acetylated decasaccharide 21 (1.5 mg, 0.0006 mmol) was dissolved in anhydrous CH₂Cl₂ (0.2 mL) and anhydrous MeOH (0.8 mL), followed by the addition of 1 M methanolic MeONa (20 μL). The reaction mixture was kept at ~20 °C for 24 h. Then, the reaction mixture was neutralized with Dowex 50W X8 (H⁺) ion-exchange resin (the resin was washed with MeOH before addition) and then filtered. The resin was washed with MeOH (3 × 20 mL). The combined filtrate was concentrated under reduced pressure. The residue was co-evaporated with toluene (2 × 2 mL), dried in vacuo, dissolved in water (1 mL), and applied on a small column (ID 5 mm) packed with Amberlite MB-3 mixed-bed ion-exchange resin (1 mL), which was eluted with H₂O (15 mL). The eluate was lyophilized and additionally purified by reversed phase chromatography on a Sep-Pak C18 cartridge (particle size: 55–105 μm, pore size: 125 Å, sorbent substrate: silica, sorbent weight: 360 mg), gradient: 0→100% MeCN in H₂O) to give deprotected decasaccharide 5 (0.8 mg, 85%). R_f = 0.50 (MeCN–H₂O 7.5:2.5); ${\left[\mathrm{\alpha }\right]}_D^{28}$ +15.1 (c 0.2 in MeOH); ¹H NMR (600 MHz, CD₃OD): δ 3.54–3.58 (m, 2H, CH₂N₃), 3.61–3.88 (m, 21H, H-5^I-Xa, H-4^{VIII, X}, H-5^{I-V, VII-X}b), 3.89–3.95 (m, 5H, H-5^VIb, H-3^II-V), 3.99–4.06 (m, 12H, H-2^{II-V, VIII, X}, H-3^{I, VI-X}), 4.06–4.11 (m, 4H, H-4^II-V), 4.12 (dd, 2H, J 5.4 Hz, J 4.4 Hz, CH₂O), 4.13–4.15 (m, 3H, H-2^VII, H-2^IX, H-4^I), 4.16 (dd, 1H, J 2.7 Hz, J 1.5 Hz, H-2^VI), 4.17–4.21 (m, 1H, H-4^VI), 4.21 (dd, 1H, J 4.0 Hz, J 1.8 Hz, H-2^I), 4.94–4.98 (m, 5H, H-1^II-VI), 5.02–5.06 (m, 2H, H-1^VIII, H-1^X), 5.09 (d, 1H, J 2.2 Hz, H-1^VII), 5.17 (d, 1H, J 2.4 Hz, H-1^IX), 5.42 (d, 1H, J 2.0 Hz, H-1^I), 6.86–6.91 (m, 2H, OC₆H₄O (H-3, H-5)), 6.97–7.03 (m, 2H, OC₆H₄O (H-2, H-6)); ¹³C NMR (151 MHz, CD₃OD): δ 51.4 (CH₂N₃), 62.45 (C-5^VII, C-5^IX), 62.53 (C-5^VII, C-5^IX), 64.38 (C-5^VIII, C-5^X), 64.41 (C-5^VIII, C-5^X), 67.9 (2C, C-5^I-VI), 68.0 (C-5^I-VI), 68.3 (3C, C-5^I-VI), 69.0 (CH₂O), 75.8 (C-3^VIII, C-3^X), 75.9 (C-3^VIII, C-3^X), 76.3 (C-3^VII, C-3^IX), 76.5 (C-3^VII, C-3^IX), 78.8 (3C, C-2^VIII, C-2^X, C-3^I-V), 78.9 (C-2^VIII, C-2^X, C-3^I-V), 79.2 (3C, C-2^VIII, C-2^X, C-3^I-V), 81.6 (C-2^VI), 83.03 (C-4^VI), 83.3 (C-2^II-V), 83.8 (C-2^I), 83.95 (C-4^I-V, C-4^VII-X), 84.03 (C-4^I-V, C-4^VII-X), 84.2 (4C, C-4^I-V, C-4^VII-X), 84.3 (C-4^I-V, C-4^VII-X), 84.4 (2C, C-4^I-V, C-4^VII-X), 84.6 (C-3^VI), 89.3 (C-2^VII), 89.6 (C-2^IX), 102.5 (C-1^VIII, C-1^X), 102.6 (C-1^VIII, C-1^X), 107.1 (C-1^IX), 107.4 (C-1^VII), 108.7 (C-1^I), 109.58 (C-1^II-VI), 109.63 (C-1^II-VI), 109.70 (2C, C-1^II-VI), 109.74 (C-1^II-VI), 116.6 (OC₆H₄O (C-3, C-5)), 119.3 (OC₆H₄O (C-2, C-6)), 152.7 (OC₆H₄O (C-1)), 155.2 (OC₆H₄O (C-4)); HRMS (ESI): m/z [M + NH₄]⁺ Calcd for C₅₈H₉₃N₄O₄₂⁺ 1517.5259; found: 1517.5244.

p-Tolyl 2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-1-thio-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranoside (6)

(1) To a solution of acetate 10 (41 mg, 0.037 mmol) in ClCH₂CH₂Cl (1 mL), p-TolSH (9 mg, 0.074 mmol) and BF₃·Et₂O (2 μL, 0.02 mmol) were added at 20 °C. The reaction mixture was stirred at 40 °C for 1 h. Then, the reaction mixture was diluted with CH₂Cl₂ (50 mL) and washed with H₂O (50 mL). The organic phase was filtered through a cotton wool plug, concentrated, and dried in vacuo. The residue was dissolved in 2,4,6-collidine (1 mL), then i-Pr₃SiOTf (450 mL, mmol) was added. The reaction mixture was stirred at 70 °C for 1 h and then diluted with CH₂Cl₂ (50 mL), washed with 1 M KHSO₄ (50 mL), H₂O (50 mL), and NaHCO₃ (50 mL). Combined organic extracts were filtered through a cotton wool plug, concentrated, and dried in vacuo. The residue was purified by silica gel chromatography (gradient: 0%→15% CH₂Cl₂ in petroleum ether) to give known [28] silylated disaccharide 6 (27 mg, 63%).

(2) To a solution of CPP-glycoside 13 (91 mg, 0.075 mmol) in ClCH₂CH₂Cl (2 mL), p-TolSH (19 mg, 0.15 mmol) and BF₃·Et₂O (4 μL, 0.02 mmol) at −5 °C were added. Then, the temperature was raised to 0 °C and the reaction mixture was stirred at 0 °C for 2.5 h. Then, the reaction mixture was diluted with CH₂Cl₂ (50 mL) and washed with H₂O (50 mL). The organic extract was filtered through a cotton wool plug, concentrated, and dried in vacuo. The residue was purified by silica gel chromatography (gradient: 0%→15% CH₂Cl₂ in petroleum ether) to give known [28] silylated disaccharide 6 (44 mg, 50%; 54% with respect to the reacted starting material).

2,3,5-Tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-1-O-acetyl-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranose (10)

To a solution of known arabinofuranose 9 [28] (48 mg, 0.045 mmol) in anhydrous Py (1 mL), Ac₂O (1 mL) was added at 0 °C (ice–water bath). The reaction mixture was stirred at 20 °C for 24 h. The reaction was quenched by the addition of MeOH (1 μL) at 0 °C (ice–water bath), then concentrated under reduced pressure, co-evaporated with toluene (5×5 mL), and dried in vacuo to give acetate 10 (42 mg, 84%); R_f = 0.50 (light petroleum–CH₂Cl₂ 1:1); ${\left[\mathrm{\alpha }\right]}_D^{28}$ −1.7 (c 2.1 in CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 0.99–1.20 (m, 105H, 5 × ((CH₃)₂CH)₃Si), 2.05 (s, 3H, CH₃CO), 3.68 (dd, 1H, J 8.4 Hz, J 3.6 Hz, H-5^IIa), 3.71–3.82 (m, 2H, H-5^Ia, H-5^Ib), 3.82–3.99 (m, 2H, H-4^II, H-5^IIb), 4.04 (dd, 1H, J 2.7 Hz, J 1.0 Hz, H-2^II), 4.21 (d, 1H, J 0.8 Hz, H-2^I), 4.25 (td, 1H, J 6.4 Hz, J 2.4 Hz, H-4^I), 4.32 (d, 1H, J 1.1 Hz, H-3^II), 4.56 (d, 1H, J 2.5 Hz, H-3^I), 5.25 (d, 1H, J 2.7 Hz, H-1^II), 6.16 (s, 1H, H-1^I); ¹³C NMR (76 MHz, CDCl₃): δ 12.0 (2 × ((CH₃)₂CH)₃Si), 12.2 (2 × ((CH₃)₂CH)₃Si), 12.3 ((CH₃)₂CH)₃Si), 17.9 (2 × ((CH₃)₂CH)₃Si), 17.97 (2 × ((CH₃)₂CH)₃Si), 18.01 ((CH₃)₂CH)₃Si), 18.0 ((CH₃)₂CH)₃Si), 18.07 ((CH₃)₂CH)₃Si), 18.11 (3 × ((CH₃)₂CH)₃Si), 21.2 (CH₃CO), 63.6 (C-5^II), 64.2 (C-5^I), 77.1 (C-3^I), 78.0 (C-2^II), 78.1 (C-3^II), 86.0 (C-4^II), 90.4 (C-2^I), 90.8 (C-4^I), 101.0 (C-1^I), 105.3 (C-1^II), 170.1 (CO); ²⁹Si INEPT NMR (60 MHz, CDCl₃) δ 13.67 (5^I-O-TIPS, 5^II-O-TIPS), 13.80 (5^I-O-TIPS, 5^II-O-TIPS), 13.96 (3^II-O-TIPS), 14.02 (3^I-O-TIPS), 14.29 (2^II-O-TIPS); HRMS (ESI): m/z [M + NH₄]⁺ Calcd for C₅₇H₁₂₄NO₁₀Si₅⁺ 1122.8066; Found: 1122.8063; [M + K]⁺ Calcd for C₅₇H₁₂₀KO₁₀Si₅⁺: 1143.7359; found: 1143.7357.

4-(3-Chloropropoxy)phenyl 2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranoside (13)

Known 4-(3-chloropropoxy)phenyl 3,5-O-(di-tert-butylsilylene)-2-O-[2-O-(triisopropylsilyl)-β-d-arabinofuranosyl]-α-d-arabinofuranoside 11 [28] (147 mg, 0.19 mmol) was dissolved in THF (2 mL), then 1 M TBAF in THF (0.77 mL, 0.77 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was kept at 0 °C for 3 h. After that, the reaction mixture was concentrated under reduced pressure, co-evaporated with toluene (2 × 2 mL), and dried in vacuo. The residue was purified by silica gel chromatography (gradient: 5%→10% MeOH in CH₂Cl₂) to give 4-(3-chloropropoxy)phenyl 2-O-(β-d-arabinofuranosyl)-α-d-arabinofuranoside 12, containing 3 mol. % of 4-(3-fluoropropoxy)phenyl 2-O-(β-d-arabinofuranosyl)-α-d-arabinofuranoside (72 mg, 81%) and 15 mol. % of n-Bu₄N⁺ salts. Selected signals for 12: ¹H NMR (300 MHz, CD₃OD): δ 2.18 (p, 2H, J 6.2 Hz, CH₂), 3.62–3.86 (m, 7H), 3.94–4.09 (m, 5H), 4.13–4.20 (m, 1H, H-3^I), 4.33–4.38 (m, 1H, H-2^I), 5.03 (d, 1H, J 4.2 Hz, H-1^II), 5.56 (d, 1H, J 2.5 Hz, H-1^I), 6.79–6.92 (m, 2H, OC₆H₄O), 6.93–7.05 (m, 2H, OC₆H₄O); selected signals for minor 4-(3-fluoropropoxy)phenyl 2-O-(β-d-arabinofuranosyl)-α-d-arabinofuranoside: ¹H NMR (300 MHz, CD₃OD): 4.60 (dt, 2H, J 47.3 Hz, J 5.9 Hz, CH₂F), ¹⁹F NMR (282 MHz, CD₃OD): δ −223.86 (tt, ²J_H–F 47.3 Hz, ³J_H–F 25.4 Hz, CH₂F). Then, i-Pr₃SiOTf (0.42 mL, 1.57 mmol) was added to the solution of crude pentaol 12 (72 mg, 0.16 mmol) in 2,4,6-collidine (1.5 mL) at 20 °C. The reaction mixture was stirred at 80 °C for 20 h and then diluted with CH₂Cl₂ (30 mL), washed with 1 M KHSO₄ (3 × 30 mL), and satd aq NaHCO₃ (3 × 30 mL). Organic extracts were filtered, concentrated under reduced pressure, and purified by silica gel chromatography (gradient: petroleum ether–EtOAc, 0%→6%) to give silylated disaccharide 13, containing 3% 4-(3-fluoropropoxy)phenyl derivative (155 mg, 80%). R_f = 0.80 (light petroleum–EtOAc 10:1); ${\left[\mathrm{\alpha }\right]}_D^{22}$ +21.1 (c 0.25 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 1.03–1.22 (m, 105H, ((CH₃)₂CH)₃Si), 2.15 (dt, 1H, J 25.8 Hz, J 6.0 Hz, CH₂CH₂F), 2.22 (p, 2H, J 6.1 Hz, CH₂), 3.71 (dd, 1H, J 9.3 Hz, J 4.5 Hz, H-5^IIa), 3.75 (t, 2H, J 6.4 Hz, CH₂Cl), 3.78 (dd, 1H, J 10.7 Hz, J 5.6 Hz, H-5^Ia), 3.87 (dd, 1H, J 10.7 Hz, J 5.3 Hz, H-5^Ib), 3.88 (dd, 1H, J 10.4 Hz, J 4.5 Hz, H-4^II), 3.94 (dd, 1H, J 10.4 Hz, J 9.3 Hz, H-5^IIb), 4.01 (d, 1H, J 2.9 Hz, H-2^II), 4.07 (t, 2H, J 5.8 Hz, CH₂O), 4.20 (td, 1H, J 5.5 Hz, J 4.4 Hz, H-4^I), 4.33 (d, 1H, J 1.1 Hz, H-3^II), 4.41 (dd, 1H, J 2.2 Hz, J 1.0 Hz, H-2^I), 4.52 (dd, 1H, J 4.5 Hz, J 2.2 Hz, H-3^I), 4.65 (dt, 1H, J 47.1 Hz, J 5.8 Hz, CH₂F), 5.29 (d, 1H, J 2.8 Hz, H-1^II), 5.53 (d, 1H, J 0.8 Hz, H-1^I), 6.78–6.84 (m, 2H, OC₆H₄O (H-3, H-5)), 6.94–7.00 (m, 2H, OC₆H₄O (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 11.98 (((CH₃)₂CH)₃Si), 12.03 (((CH₃)₂CH)₃Si), 12.30 (((CH₃)₂CH)₃Si), 12.32 (((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 17.96 (((CH₃)₂CH)₃Si), 17.98 (((CH₃)₂CH)₃Si), 18.08 (((CH₃)₂CH)₃Si), 18.11 (((CH₃)₂CH)₃Si), 18.14 (((CH₃)₂CH)₃Si), 18.15 (((CH₃)₂CH)₃Si), 32.4 (CH₂), 41.6 (CH₂Cl), 63.7 (C-5^II), 63.8 (C-5^I), 64.9 (CH₂O), 77.1 (C-3^I), 78.1 (C-2^II), 78.3 (C-3^II), 85.9 (C-4^II), 87.9 (C-4^I), 91.2 (C-2^I), 104.8 (C-1^II), 105.6 (C-1^I), 115.3 (OC₆H₄O (C-3, C-5)), 118.1 (OC₆H₄O (C-2, C-6)), 151.6 (OC₆H₄O (C-1)), 153.7 (OC₆H₄O (C-4)); ²⁹Si INEPT NMR (60 MHz, CDCl₃): δ 13.36 (3^I-O-TIPS), 13.65 (5^I-O-TIPS, 5^II-O-TIPS), 13.70 (3^II-O-TIPS), 14.05 (2^II-O-TIPS); HRMS (ESI): m/z [M + NH₄]⁺ Calcd for C₆₄H₁₃₁ClNO₁₀Si₅⁺ 1248.8302; found: 1248.8296.

4-(2-Chloroethoxy)phenyl 2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→5)-2-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranoside (14)

A mixture of disaccharide thioglycoside 6 (82 mg, 0.07 mmol) and disaccharide diol 8 [28] (19 mg, 0.25 mmol) was dried in vacuo for 2 h, then anhydrous CH₂Cl₂ (2 mL) was added under argon. Freshly activated powdered MS 4 Å (200 mg) (100 mg per 1 mL of solvent) was added under argon to the resulting solution. The suspension was stirred under argon at ~22 °C for 1 h, cooled to −60 °C, then NIS (16 mg, 0.7 mmol) and, after 10 min, AgOTf (1 mg) was added. Then, the temperature was allowed to rise slowly to −20 °C and was kept at −20 °C for 1 h. Then, the reaction was quenched by the addition of satd aq NaHCO₃ (50 μL), diluted with CH₂Cl₂ (15 mL), and filtered through a Celite pad. The solids were washed with CH₂Cl₂ (5 × 10 mL), and the filtrate was washed with a mixture of satd aq Na₂S₂O₃ (50 mL) and satd aq NaHCO₃ (50 mL). The aqueous layer was extracted with CH₂Cl₂ (2 × 5 mL). The combined organic extracts were filtered through a cotton wool plug, concentrated and dried in vacuo, then dissolved in toluene (2 mL) and subjected to chromatography on Bio-Beads S-X1 in toluene. The fractions eluted just after the void volume were collected, concentrated under reduced pressure, and the residue was purified by silica gel chromatography (gradient: EtOAc in petroleum ether, 0%→30%) to give α-linked tetrasaccharide 14 (36 mg, 80%). R_f = 0.20 (light petroleum–EtOAc 10:1); ¹H NMR (600 MHz, CDCl₃): δ 0.89 –1.14 (m, 105H, 5 × ((CH₃)₂CH)₃Si), 3.43 (d, 1H, J 4.1 Hz, HO-3^II), 3.66 (dd, 1H, J 9.5 Hz, J 4.6 Hz, H-5^IVa), 3.68 (dd, 1H, J 11.1 Hz, J 3.9 Hz, H-5^IIa), 3.73 (dd, 1H, J 10.4 Hz, J 6.4 Hz, H-5^IIIa), 3.77–3.80 (m, 1H, H-5^IIIb), 3.79 (t, 2H, J 5.9 Hz, CH₂Cl), 3.82 (dd, 1H, J 10.3 Hz, J 4.6 Hz, H-4^IV), 3.90 (dd, 1H, J 10.2 Hz, J 9.4 Hz, H-5^IVb), 3.92 (dd, 1H, J 11.1 Hz, J 3.7 Hz, H-5^Ia), 3.98 (d, 1H, J 3.0 Hz, H-2^IV), 4.00 (dd, 1H, J 11.2 Hz, J 3.5 Hz, H-5^IIb), 4.07 (td, 1H, J 6.0 Hz, J 3.6 Hz, H-4^III), 4.17 (d, 1H, J 1.2 Hz, H-2^III), 4.19 (t, 2H, J 6.0 Hz, CH₂O), 4.21 (dd, 1H, J 11.0 Hz, J 4.5 Hz, H-5^Ib), 4.29 (d, 1H, J 1.1 Hz, H-3^IV), 4.31 (dt, 1H, J 7.1 Hz, J 3.5 Hz, H-3^II), 4.34–4.38 (m, 2H, H-4^II, H-3^III), 4.58–4.63 (m, 1H, H-4^I), 5.06 (s, 1H, H-1^III), 5.17 (dd, 1H, J 3.7 Hz, J 1.4 Hz, H-2^II), 5.20 (d, 1H, J 2.6 Hz, H-1^IV), 5.37 (d, 1H, J 1.4 Hz, H-1^II), 5.68 (dd, 1H, J 5.0 Hz, J 1.8 Hz, H-3^I), 5.75 (d, 1H, J 1.7 Hz, H-2^I), 5.83 (s, 1H, H-1^I), 6.84–6.91 (m, 2H, OC₆H₄O (H-3, H-5)), 7.04–7.11 (m, 2H, OC₆H₄O (H-2, H-6)), 7.41–7.51 (m, 6H, 3 × PhCO (H-3, H-5)), 7.55–7.64 (m, 3H, 3 × PhCO (H-4)), 7.99–8.04 (m, 2H, 2^II-O-PhCO (H-2, H-6)), 8.07–8.11 (m, 2H, 2^I-O-PhCO (H-2, H-6)), 8.09–8.14 (m, 2H, 3^I-O-PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 11.9 (((CH₃)₂CH)₃Si), 12.0 (((CH₃)₂CH)₃Si), 12.1 (((CH₃)₂CH)₃Si), 12.2 (((CH₃)₂CH)₃Si), 12.3 (((CH₃)₂CH)₃Si), 17.9 (2 × ((CH₃)₂CH)₃Si), 17.95 (3 × ((CH₃)₂CH)₃Si), 17.97 (((CH₃)₂CH)₃Si), 18.06 (((CH₃)₂CH)₃Si), 18.09 (((CH₃)₂CH)₃Si), 18.13 (((CH₃)₂CH)₃Si), 18.14 (((CH₃)₂CH)₃Si), 41.9 (CH₂Cl), 63.7 (C-5^IV), 64.4 (C-5^II), 64.6 (C-5^III), 66.0 (C-5^I), 68.7 (CH₂O), 76.1 (C-3^II), 77.3 (C-3^I), 77.9 (C-3^III), 78.0 (C-2^IV), 78.1 (C-3^IV), 82.1 (C-2^I, C-4^II), 82.5 (C-4^I), 85.8 (C-4^IV), 86.9 (C-2^II), 88.2 (C-4^III), 90.8 (C-2^III), 104.8 (C-1^I), 105.0 (C-1^IV), 105.4 (C-1^II), 106.3 (C-1^III), 115.8 (OC₆H₄O (C-3, C-5)), 118.3 (OC₆H₄O (C-2, C-6)), 128.4 (PhCO (C-3, C-5)), 128.5 (PhCO (C-3, C-5)), 128.6 (PhCO (C-3, C-5)), 128.9 (PhCO (C-1)), 129.1 (PhCO (C-1)), 129.3 (PhCO (C-1)), 129.87 (PhCO (C-2, C-6)), 129.92 (PhCO (C-2, C-6)), 129.94 (PhCO (C-2, C-6)), 133.4 (PhCO (C-4)), 133.5 (PhCO (C-4)), 133.6 (PhCO (C-4)), 150.7 (OC₆H₄O (C-1)), 153.7 (OC₆H₄O (C-4)), 165.5 (2^I-O-PhCO), 165.7 (3^I-O-PhCO), 166.8 (2^II-O-PhCO); ²⁹Si INEPT NMR (60 MHz, CDCl₃): δ 13.54, 13.59, 13.70, 13.80, 13.89; HRMS (ESI): m/z [M + NH₄]⁺ Calcd for C₉₄H₁₉₃ClNO₂₁Si₅⁺ 1810.9777; found: 1810.9764; [M + K]⁺ Calcd for C₉₄H₁₅₃ClKO₂₁Si₅⁺: 1831.9071; found: 1831.9063.

2,3,5-Tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→3)-[2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→5)]-2-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-d-arabinofuranose (15) and 2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→3)-[2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→5)]-2-O-benzoyl-α-d-arabinofuranosyl-(1→5)-1,3-di-O-benzoyl-β-d-arabinofuranose (16)

CEP glycoside 2 (44 mg, 0.016 mmol) was dissolved in CH₂Cl₂ (2 mL) and MeCN (1 mL), then H₂O (0.1 mL) was added at 0 °C, followed by (NH₄)₂Ce(NO₃)₆ (52.6 mg, 0.096 mmol). The reaction mixture was stirred at 0 °C for 4 h. Then, Na₂SO₃ (110 mg) and H₂O (1 mL) were added to the reaction mixture at 0 °C, and the reaction mixture was kept at 0 °C for 16 h. Then, the reaction mixture was diluted with CH₂Cl₂ (50 mL), washed with H₂O (50 mL), concentrated under reduced pressure and purified by silica gel column chromatography (gradient: petroleum ether–EtOAc, 3%→8%) to give hemiacetal 15 (29 mg, 70%) as a mixture of anomers 15, and contaminated with the product 16 of migration of benzoyl group from O-2 to O-1. The ratio of 15α:15β:16 = 1:0.45:0.28 according to NMR; R_f = 0.50 (light petroleum–EtOAc, 8.5:1.5); selected signals: ¹H NMR (300 MHz, CDCl₃): δ 0.75–1.18 (m, 363H, ((CH₃)₂CH)₃Si), 3.40 (s, 1H, HO-1^Iα), 4.65 (ddd, 1H, J 6.3 Hz, J 5.2 Hz, J 3.3 Hz, H-4^Iα), 5.05 (s, 1H, H-1^III of 16), 5.10 (s, 1H, H-1^IIIβ), 5.10 (s, 1H, H-1^IIIα), 5.16 (s, 1H, H-1^Vβ), 5.17–5.18 (m, 2H, H-1^IIβ, H-1^II or H-1^V of 16), 5.31 (d, 1H, J 3.2 Hz, H-2^IIβ), 5.35 (d, 1H, J 2.0 Hz, H-2^IIα), 5.40–5.48 (m, 2H, H-3^Iα, H-3^I of 16), 5.51 (d, 1H, J 2.4 Hz, H-2^Iα), 5.56 (dd, 1H, J 6.2 Hz, J 4.7 Hz, H-2^Iβ), 5.63 (s, 1H, H-1^Iα), 5.72 (dd, 1H, J 5.5 Hz, J 5.5 Hz, H-1^Iβ), 5.86 (dd, 1H, J 6.2 Hz, J 5.0 Hz, H-3^Iβ), 6.60 (d, 1H, J 4.5 Hz, H-1^I of 16), 7.33–7.66 (m, 16H, PhCO (H-3, H-4, H-5)), 7.92–8.19 (m, 11H, PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 11.97 (((CH₃)₂CH)₃Si), 11.99 (((CH₃)₂CH)₃Si), 12.02 (((CH₃)₂CH)₃Si), 12.03 (((CH₃)₂CH)₃Si), 12.2 (((CH₃)₂CH)₃Si), 12.28 (((CH₃)₂CH)₃Si), 12.31 (((CH₃)₂CH)₃Si), 12.36 (((CH₃)₂CH)₃Si), 12.39 (((CH₃)₂CH)₃Si), 17.95 (((CH₃)₂CH)₃Si), 17.97 (((CH₃)₂CH)₃Si), 17.99 (((CH₃)₂CH)₃Si), 18.01 (((CH₃)₂CH)₃Si), 18.03 (((CH₃)₂CH)₃Si), 18.05 (((CH₃)₂CH)₃Si), 18.08 (((CH₃)₂CH)₃Si), 18.09 (((CH₃)₂CH)₃Si), 18.11 (((CH₃)₂CH)₃Si), 18.13 (((CH₃)₂CH)₃Si), 18.14 (((CH₃)₂CH)₃Si), 18.17 (((CH₃)₂CH)₃Si), 18.18 (((CH₃)₂CH)₃Si), 18.20 (((CH₃)₂CH)₃Si), 63.7 (C-5), 63.76 (2 × C-5), 63.82 (C-5), 64.16 (C-5), 64.18 (C-5), 64.5 (C-5), 64.7 (C-5), 65.3 (C-5^II minor), 65.4 (C-5^IIα), 66.0 (C-5^IIβ), 66.6 (C-5^Iα), 67.4 (C-5^I of 16), 67.6 (C-5^Iβ), 76.5 (C-3^Iβ), 76.7 (C-2^I of 16), 77.4, 77.5, 77.7, 78.08, 78.14, 78.19, 78.21, 78.23, 78.25, 78.28, 78.32, 78.37, 78.43, 78.47, 78.52, 79.7, 80.06 (C-3^IIβ), 80.12, 80.6, 80.7 (C-3^IIα), 81.0 (C-3^I of 16), 81.1, 81.8 (C-4^Iα), 82.1, 82.2, 82.6 (C-2^Iα), 83.6 (C-2^IIα), 83.7 (C-2^II of 16), 84.1 (C-2^IIβ), 85.7 (C-4^IV, C-4^VI), 85.7 (C-4^IV, C-4^VI), 85.6 (C-4^IV, C-4^VI), 85.7 (C-4^IV, C-4^VI), 85.8 (C-4^IV, C-4^VI), 87.5 (C-4^III), 87.54 (C-4^III), 87.59 (C-4^III of 16), 87.8 (C-4^V), 88.3 (C-4^V), 88.4 (C-4^V of 16), 90.5 (C-2^III), 90.70 (C-2^III), 90.74 (C-2^III of 16), 91.2 (C-2^V), 91.4 (C-2^V of 16), 91.6 (C-2^V), 95.4 (C-1^Iβ), 97.0 (C-1^I of 16), 100.9 (C-1^Iα), 104.7 (C-1), 104.75 (C-1), 104.78 (C-1 minor), 105.2 (C-1), 105.5 (C-1), 105.48 (C-1 of 16), 105.93 (C-1), 105.94 (C-1 of 16), 105.98 (C-1), 106.04 (C-1), 106.2 (C-1), 106.4 (C-1 of 16), 106.69 (C-1^III of 16), 106.73 (C-1^IIIβ), 106.8 (C-1^IIIα), 128.20 (PhCO (C-3, C-5) of 16), 128.23 (PhCO (C-3, C-5)), 128.27 (PhCO (C-3, C-5)), 128.30 (PhCO (C-3, C-5)), 128.37 (2 × PhCO (C-3, C-5)), 128.44 (PhCO (C-3, C-5) of 16), 128.5 (PhCO (C-3, C-5)), 129.3 (PhCO (C-1)), 129.4 (PhCO (C-1)), 129.48 (PhCO (C-1)), 129.49 (PhCO (C-1)), 129.54 (PhCO (C-1)), 129.8 (PhCO (C-1)), 129.85 (PhCO (C-2, C-6)), 129.86 (PhCO (C-2, C-6)), 129.92 (PhCO (C-2, C-6) of 16), 129.94 (PhCO (C-2, C-6)), 130.0 (2 × PhCO (C-2, C-6)), 130.1 (PhCO (C-2, C-6)), 132.9 (PhCO (C-4)), 133.07 (PhCO (C-4)), 133.12 (PhCO (C-4)), 133.2 (PhCO (C-4)), 133.27 (PhCO (C-4)), 133.29 (PhCO (C-4)), 133.5 (PhCO (C-4) of 16), 165.16 (PhCO of 16), 165.20 (PhCO), 165.58 (PhCO), 165.59 (PhCO), 165.7 (PhCO), 165.8 (2 × PhCO); HRMS (ESI): m/z [M + NH₄]⁺ Calcd for C₁₄₁H₂₆₆NO₂₈Si₁₀⁺ 2701.7109; found: 2701.7093.

4-(2-Chloroethoxy)phenyl 2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→3)-[2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→5)]-2-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranoside (18)

A mixture of hexasaccharide imidate 5 (32 mg, 0.011 mmol) and known tetrasaccharide glycosyl acceptor 17 [33] (21 mg, 0.013 mmol) was dried in vacuo for 2 h, then anhydrous CH₂Cl₂ (2 mL) was added under argon. Freshly activated powdered MS 4 Å (200 g) (100 mg per 1 mL of solvent) was added under argon to the resulting solution. The suspension was stirred under argon at ~22 °C for 1 h, then cooled to −60 °C, followed by the addition of TfOH (1 μL). Then, the temperature was allowed to rise during 1 h to −40 °C and this temperature was kept for 1 h. After an additional 20 min, the reaction was quenched by the addition of Py (50 µL). The reaction mixture was diluted with CH₂Cl₂ (15 mL) and filtered through a Celite pad. The solids were washed with CH₂Cl₂ (5 × 10 mL), and the filtrate was washed with satd aq NaHCO₃ (20 mL). The aqueous layer was extracted with CH₂Cl₂ (2 × 5 mL). The combined organic layer was filtered through a cotton wool plug, concentrated under reduced pressure, the residue was dried in vacuo, then dissolved in toluene (2 mL) and subjected to chromatography on Bio-Beads S-X1 in toluene. The fractions eluted just after the void volume were collected and concentrated under reduced pressure to give decasaccharide 18 (34 mg, 72%; 51% over 3 steps starting from 2). R_f = 0.28 (light petroleum–EtOAc 6:1); ${\left[\mathrm{\alpha }\right]}_D^{23}$ +31.9 (c 1.13 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 0.91–1.16 (m, 210H, 10 × ((CH₃)₂CH)₃Si), 3.63 (dd, 1H, J 9.3 Hz, J 4.4 Hz, H-5^VIIIa or H-5^Xa), 3.65 (dd, 1H, J 10.7 Hz, J 5.5 Hz, H-5^IXa), 3.78 (t, 2H, J 5.9 Hz, CH₂Cl), 3.69–3.98 (m, 16H, H-5^Xa or H-5^VIIIa, H-5^I-VIIa, H-5^VI-Xb, H-4^VIII-X), 4.02–4.04 (m, 2H, H-2^VIII, H-2^X), 4.06 (d, 1H, J 1.2 Hz, H-2^IX), 4.08–4.12 (m, 2H, H-4^VII, H-5^Vb), 4.13 (s, 1H, H-2^VII), 4.16 (t, 2H, J 5.9 Hz, CH₂O), 4.14–4.24 (m, 5H, H-5^I-IV, H-3^VI), 4.26–4.28 (m, 1H, H-4^VI), 4.29 (s, 1H, H-3^X), 4.32 (s, 1H, H-3^VIII), 4.34 (d, 1H, J 3.9 Hz, H-3^VII), 4.45 (d, 1H, J 3.4 Hz, H-3^IX), 4.55–4.64 (m, 5H, H-4^I-V), 5.10 (s, 1H, H-1^VII), 5.15 (d, 1H, J 2.6 Hz, H-1^X), 5.23 (s, 1H, H-1^IX), 5.24 (d, 1H, J 2.8 Hz, H-1^VIII), 5.29 (s, 1H, H-1^VI), 5.37 (s, 2H, 2 × H-1), 5.38–5.41 (m, 3H, H-2^VI, 2 × H-1), 5.50 (d, 1H, J 4.4 Hz, H-3^V), 5.58 (d, 1H, J 1.1 Hz, H-2^V), 5.62–5.67 (m, 6H, H-3^II-IV, H-2^II-IV), 5.75–5.79 (m, 2H, H-3^I, H-2^I), 5.82 (s, 1H, H-1^I), 6.80–6.86 (m, 2H, OC₆H₄O (H-3, H-5)), 7.02–7.08 (m, 2H, OC₆H₄O (H-2, H-6)), 7.18–7.27 (m, 8H, 4 × PhCO (H-3, H-5)), 7.34–7.55 (m, 24H, 7 × PhCO (H-3, H-5), 10 × PhCO (H-4)), 7.55–7.61 (m, 1H, PhCO (H-4)), 7.84–7.92 (m, 8H, 4 × PhCO (H-2, H-6)), 7.97–8.06 (m, 10H, 5 × PhCO (H-2, H-6)), 8.06–8.11 (m, 2H, PhCO (H-2, H-6)), 8.09–8.14 (m, 2H, PhCO (H-2, H-6));¹³C NMR (151 MHz, CDCl₃): δ 11.97 (((CH₃)₂CH)₃Si), 11.99 (((CH₃)₂CH)₃Si), 12.01 (((CH₃)₂CH)₃Si), 12.1 (((CH₃)₂CH)₃Si), 12.2 (((CH₃)₂CH)₃Si), 12.27 (2 × ((CH₃)₂CH)₃Si), 12.29 (2 × ((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 17.94 (((CH₃)₂CH)₃Si), 17.96 (((CH₃)₂CH)₃Si), 17.97 (((CH₃)₂CH)₃Si), 18.02 (((CH₃)₂CH)₃Si), 18.04 (((CH₃)₂CH)₃Si), 18.06 (((CH₃)₂CH)₃Si), 18.09 (((CH₃)₂CH)₃Si), 18.13 (((CH₃)₂CH)₃Si), 18.19 (((CH₃)₂CH)₃Si), 18.20 (((CH₃)₂CH)₃Si), 41.9 (CH₂Cl), 63.8 (C-5^VIII, C-5^X), 63.9 (C-5^VIII, C-5^X), 64.3 (C-5^IX), 64.9 (C-5^VII), 65.2 (C-5^VI), 65.3 (C-5^V), 65.8 (2C, C-5^I, C-5^II, C-5^III, C-5^IV), 65.86 (C-5^I, C-5^II, C-5^III, C-5^IV), 65.91 (C-5^I, C-5^II, C-5^III, C-5^IV), 68.8 (CH₂O), 77.2 (C-3^I), 77.2 (2C, C-3^II, C-3^III, C-3^IV, C-3^V), 77.3 (C-3^II, C-3^III, C-3^IV, C-3^V), 77.6 (C-3^II, C-3^III, C-3^IV, C-3^V), 77.7 (C-3^IX), 78.2 (C-2^VIII or C-2^X), 78.3 (C-3^X), 78.4(C-2^VIII or C-2^X), 78.5 (2C, C-3^VII, C-3^VIII), 81.0 (C-3^VI), 81.5 (2C, C-2^II, C-2^III, C-2^IV, C-2^V), 81.6 (2C, C-2^II, C-2^III, C-2^IV, C-2^V), 81.9 (C-2^I), 82.2 (3C, C-4^II, C-4^III, C-4^IV, C-4^V, C-4^VI), 82.26 (C-4^II, C-4^III, C-4^IV, C-4^V, C-4^VI), 82.30 (C-4^II, C-4^III, C-4^IV, C-4^V, C-4^VI), 82.8 (C-4^I), 84.0 (C-2^VI), 85.7 (C-4^VIII, C-4^X), 85.6 (C-4^VIII, C-4^X), 87.7 (C-4^VII), 88.6 (C-4^IX), 90.6 (C-2^VII), 91.4 (C-2^IX), 104.8 (C-1^VIII), 104.9 (C-1^I), 105.5 (C-1^X), 105.85 (C-1^VI), 105.89 (C-1), 106.0 (3C, C-1^II, C-1^III, C-1^IV), 106.2 (C-1^IX), 106.9 (C-1^VII), 115.9 (OC₆H₄O (C-3, C-5)), 118.3 (OC₆H₄O (C-2, C-6)), 128.1 (PhCO (C-3, C-5)), 128.15 (PhCO (C-3, C-5)), 128.17 (PhCO (C-3, C-5)), 128.20 (PhCO (C-3, C-5)), 128.3 (PhCO (C-3, C-5)), 128.4 (PhCO (C-3, C-5)), 128.6 (PhCO (C-3, C-5)), 128.49 (3 × PhCO (C-3, C-5)), 128.54 (PhCO (C-3, C-5)), 129.1–129.29 (PhCO (C-1)), 129.29 (PhCO (C-1)), 129.36 (PhCO (C-1)), 129.41 (PhCO (C-1)), 129.5 (PhCO (C-1)), 129.78 (3 × PhCO (C-2, C-6)), 129.81 (2 × PhCO (C-2, C-6)), 129.83 (3 × PhCO (C-2, C-6)), 129.86 (PhCO (C-2, C-6)), 129.92 (PhCO (C-2, C-6)), 130.0 (PhCO (C-2, C-6)), 132.75 (PhCO (C-4)), 132.82 (PhCO (C-4)), 132.9 (PhCO (C-4)), 133.0 (PhCO (C-4)), 133.1 (2 × PhCO (C-4)), 133.2 (PhCO (C-4)), 133.3 (PhCO (C-4)), 133.35 (PhCO (C-4)), 133.44 (PhCO (C-4)), 133.5 (PhCO (C-4)), 150.8 (OC₆H₄O (C-1)), 153.7 (OC₆H₄O (C-4)), 165.0 (PhCO), 165.2 (PhCO), 165.2 (PhCO), 165.2 (PhCO), 165.2 (PhCO), 165.3 (PhCO), 165.37 (PhCO), 165.44 (PhCO), 165.5 (PhCO), 165.57 (PhCO), 165.64 (PhCO); ²⁹Si INEPT NMR (60 MHz, CDCl₃): δ 12.30 (3^VII-O-TIPS), 12.84 (3^IX-O-TIPS), 13.21 (5-O-TIPS), 13.44 (5-O-TIPS), 13.49 (3^X-O-TIPS, 5-O-TIPS), 13.50 (3^X-O-TIPS, 5-O-TIPS), 13.54 (5-O-TIPS), 13.76 (3^VIII-O-TIPS), 13.82 (2^VIII-O-TIPS, 2^X-O-TIPS), 13.86 (2^VIII-O-TIPS, 2^X-O-TIPS); HRMS (ESI): m/z [M + 2NH₄]²⁺ Calcd for C₂₂₅H₃₄₁ClN₂O₅₃Si₁₀²⁺ 2117.0710; found: 2117.0702.

4-(2-Azidoethoxy)phenyl 2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→3)-[2,3,5-tris-O-(triisopropylsilyl)-β-d-arabinofuranosyl-(1→2)-3,5-bis-O-(triisopropylsilyl)-α-d-arabinofuranosyl-(1→5)]-2-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranoside (19)

A mixture of decasaccharide CEP glycoside 18 (30 mg, 0.007 mmol), NaN₃ (3 mg, 0.042 mmol), and 18-crown-6 (2 mg, 0.006 mmol) in DMF (1 mL) was stirred at 80 °C for 16 h. The reaction mixture was concentrated under reduced pressure, co-evaporated with toluene (2×2 mL), and dried in vacuo. The residue was dissolved in EtOAc (50 mL), washed with water (50 mL), and the aqueous layer was extracted with EtOAc (5 mL). The combined organic extracts were filtered through a cotton wool plug, concentrated under reduced pressure, and purified by silica gel column chromatography (gradient: EtOAc in petroleum ether, 2%→20%) to give azide 19 (25 mg, 84%); ${\left[\mathrm{\alpha }\right]}_D^{22}$ +29.7 (c 2.55 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 0.91–1.13 (m, 210H, 10 × ((CH₃)₂CH)₃Si), 3.56 (t, 2H, J 5.0 Hz, CH₂N₃), 3.63 (dd, 1H, J 9.3 Hz, J 4.4 Hz, H-5^VIIIa or H-5^Xa), 3.66 (dd, 1H, J 10.5 Hz, J 5.5 Hz, H-5^IXa), 3.69–3.98 (m, 16H, H-5^Xa or H-5^VIIIa, H-5^I-VIIa, H-5^VI-Xb, H-4^VIII-X), 4.03 (d, 1H, J 2.8 Hz, H-2^VIII), 4.04 (d, 1H, J 2.7 Hz, H-2^X), 4.07 (d, 1H, J 1.4 Hz, H-2^IX), 4.08 (t, 2H, J 5.1 Hz, CH₂O), 4.07–4.13 (m, 2H, H-4^VII, H-5^Vb), 4.13 (d, 1H, J 1.2 Hz, H-2^VII), 4.14–4.24 (m, 5H, H-5^I-IVb, H-3^VI), 4.27 (ddd, 1H, J 7.2 Hz, J 5.0 Hz, J 1.9 Hz, H-4^VI), 4.29 (d, 1H, J 1.0 Hz, H-3^X), 4.32 (d, 1H, J 0.9 Hz, H-3^VIII), 4.34 (dt, 1H, J 4.0 Hz, J 0.8 Hz, H-3^VII), 4.45 (dd, 1H, J 3.3 Hz, J 1.6 Hz, H-3^IX), 4.54–4.64 (m, 5H, H-4^I-V), 5.09 (s, 1H, H-1^VII), 5.16 (d, 1H, J 2.7 Hz, H-1^X), 5.23 (s, 1H, H-1^IX), 5.24 (d, 1H, J 2.8 Hz, H-1^VIII), 5.29 (s, 1H, H-1^VI), 5.37 (s, 1H, H-1), 5.37 (s, 1H, H-1), 5.37–5.41 (m, 3H, H-2^VI, 2 × H-1), 5.50 (d, 1H, J 4.4 Hz, H-3^V), 5.58 (d, 1H, J 1.2 Hz, H-2^V), 5.61–5.67 (m, 6H, H-3^II-IV, H-2^II-IV), 5.74–5.79 (m, 1H, H-3^I), 5.77 (s, 1H, H-2^I), 5.82 (s, 1H, H-1^I), 6.80–6.86 (m, 2H, OC₆H₄O (H-3, H-5)), 7.02–7.08 (m, 2H, OC₆H₄O (H-2, H-6)), 7.19–7.27 (m, 8H, 4 × PhCO (H-3, H-5)), 7.34–7.55 (m, 24H, 7 × PhCO (H-3, H-5), 10 × PhCO (H-4)), 7.55–7.61 (m, 1H, PhCO (H-4)), 7.84–7.92 (m, 8H, 4 × PhCO (H-2, H-6)), 7.97–8.06 (m, 10H, 5 × PhCO (H-2, H-6)), 8.06–8.11 (m, 2H, PhCO (H-2, H-6)), 8.09–8.14 (m, 2H, PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 11.97 (((CH₃)₂CH)₃Si), 11.99 (((CH₃)₂CH)₃Si), 12.01 (((CH₃)₂CH)₃Si), 12.04 (((CH₃)₂CH)₃Si), 12.2 (((CH₃)₂CH)₃Si), 12.26 (2 × ((CH₃)₂CH)₃Si), 12.28 (((CH₃)₂CH)₃Si), 12.29 (((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 17.9 (((CH₃)₂CH)₃Si), 17.96 (((CH₃)₂CH)₃Si), 17.97 (((CH₃)₂CH)₃Si), 18.02 (((CH₃)₂CH)₃Si), 18.04 (((CH₃)₂CH)₃Si), 18.06 (((CH₃)₂CH)₃Si), 18.09 (((CH₃)₂CH)₃Si), 18.13 (((CH₃)₂CH)₃Si), 18.19 (((CH₃)₂CH)₃Si), 18.20 (((CH₃)₂CH)₃Si), 50.2 (CH₂N₃), 63.8 (C-5^VIII, C-5^X), 63.9 (C-5^VIII, C-5^X), 64.3 (C-5^IX), 64.8 (C-5^VII), 65.2 (C-5^V, C-5^VI), 65.3 (C-5^V, C-5^VI), 65.76 (2C, C-5^I, C-5^II, C-5^III, C-5^IV), 65.83 (C-5^I, C-5^II, C-5^III, C-5^IV), 65.9 (C-5^I, C-5^II, C-5^III, C-5^IV), 67.6 (CH₂O), 77.10 (C-3^I), 77.2 (2C, C-3^II, C-3^III, C-3^IV, C-3^V), 77.3 (C-3^II, C-3^III, C-3^IV, C-3^V), 77.6 (C-3^II, C-3^III, C-3^IV, C-3^V), 77.7 (C-3^IX), 78.15 (C-2^VIII or C-2^X), 78.24 (C-3^X), 78.4 (C-2^VIII or C-2^X), 78.51 (C-3^VII, C-3^VIII), 78.53 (C-3^VII, C-3^VIII), 81.0 (C-3^VI), 81.5 (2C, C-2^II, C-2^III, C-2^IV, C-2^V), 81.59 (C-2^II, C-2^III, C-2^IV, C-2^V), 81.61 (C-2^II, C-2^III, C-2^IV, C-2^V), 81.9 (C-2^I), 82.15 (C-4^II, C-4^III, C-4^IV, C-4^V, C-4^VI), 82.17 (C-4^II, C-4^III, C-4^IV, C-4^V, C-4^VI), 82.19 (C-4^II, C-4^III, C-4^IV, C-4^V, C-4^VI), 82.26 (C-4^II, C-4^III, C-4^IV, C-4^V, C-4^VI), 82.30 (C-4^II, C-4^III, C-4^IV, C-4^V, C-4^VI), 82.8 (C-4^I), 84.0 (C-2^VI), 85.7 (C-4^VIII, C-4^X), 85.8 (C-4^VIII, C-4^X), 87.6 (C-4^VII), 88.3 (C-4^IX), 90.6 (C-2^VII), 91.4 (C-2^IX), 104.8 (C-1^VIII), 104.9 (C-1^I), 105.6 (C-1^X), 105.8 (C-1^VI), 105.87 (C-1^II, C-1^III, C-1^IV, C-1^V), 105.94 (C-1^II, C-1^III, C-1^IV, C-1^V), 105.97 (2C, C-1^II, C-1^III, C-1^IV, C-1^V), 106.2 (C-1^IX), 106.9 (C-1^VII), 115.6 (OC₆H₄O (C-3, C-5)), 118.3 (OC₆H₄O (C-2, C-6)), 128.10 (PhCO (C-3, C-5)), 128.14 (PhCO (C-3, C-5)), 128.16 (PhCO (C-3, C-5)), 128.20 (PhCO (C-3, C-5)), 128.3 (PhCO (C-3, C-5)), 128.4 (PhCO (C-3, C-5)), 128.46 (PhCO (C-3, C-5)), 128.49 (2 × PhCO (C-3, C-5)), 128.50 (PhCO (C-3, C-5)), 128.54 (PhCO (C-3, C-5)), 129.0 (PhCO (C-1)), 129.1 (PhCO (C-1)), 129.15 (PhCO (C-1)), 129.17 (3 × PhCO (C-1)), 129.22 (PhCO (C-1)), 129.28 (PhCO (C-1)), 129.34 (PhCO (C-1)), 129.4 (PhCO (C-1)), 129.76 (PhCO (C-2, C-6)), 129.78 (2 × PhCO (C-2, C-6)), 129.80 (PhCO (C-2, C-6)), 129.81 (PhCO (C-2, C-6)), 129.83 (3 × PhCO (C-2, C-6)), 129.86 (PhCO (C-2, C-6)), 129.92 (PhCO (C-2, C-6)), 130.0 (PhCO (C-2, C-6)), 132.7 (PhCO (C-4)), 132.8 (PhCO (C-4)), 132.9 (PhCO (C-4)), 133.0 (PhCO (C-4)), 133.1 (PhCO (C-4)), 133.2 (PhCO (C-4)), 133.2 (PhCO (C-4)), 133.3 (PhCO (C-4)), 133.4 (PhCO (C-4)), 133.44 (PhCO (C-4)), 133.49 (PhCO (C-4)), 150.7 (OC₆H₄O (C-1)), 153.6 (OC₆H₄O (C-4)), 165.01 (PhCO), 165.11 (PhCO), 165.14 (PhCO), 165.18 (PhCO), 165.23 (PhCO), 165.3 (PhCO), 165.37 (PhCO), 165.44 (PhCO), 165.5 (PhCO), 165.57 (PhCO), 165.64 (PhCO); ²⁹Si INEPT NMR (60 MHz, CDCl₃): δ 12.27, 12.83, 13.20, 13.44, 13.48, 13.50, 13.53, 13.76, 13.81, 13.85; HRMS (ESI): m/z [M + 2NH₄]²⁺ Calcd for C₂₂₅H₃₄₁N₅O₅₃Si₁₀²⁺ 2120.5912; Found: 2120.5897.

4-(2-Azidoethoxy)phenyl 2,3,5-tri-O-acetyl-β-d-arabinofuranosyl-(1→2)-3,5-di-O-acetyl-α-d-arabinofuranosyl-(1→3)-[2,3,5-tri-O-acetyl-β-d-arabinofuranosyl-(1→2)-3,5-di-O-acetyl-α-d-arabinofuranosyl-(1→5)]-2-O-acetyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-acetyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-benzoyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-acetyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-acetyl-α-d-arabinofuranosyl-(1→5)-2,3-di-O-acetyl-α-d-arabinofuranoside (21)

Protected AEP decasaccharide 19 (25 mg, 0.059 mmol) was dissolved in THF (1 mL), then AcOH (4 μL, 0.059 mmol) and 1 M TBAF in THF (178 μL, 0.18 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was stirred at 40 °C for 3 h. Then, the reaction mixture was concentrated under reduced pressure, co-evaporated with toluene (2 × 2 mL), dried in vacuo, and purified by silica gel column chromatography (gradient: MeOH in CH₂Cl₂, 2%→20%) to give benzoylated polyol of decasaccharide 20 (12 mg) isolated as a mixture with n-Bu₄N⁺ salts according to MS and NMR data. R_f = 0.42 (CH₂Cl₂–MeOH 4:1); HRMS (ESI): m/z [M + Na]⁺ Calcd for C₁₃₅H₁₃₃N₃NaO₅₃ 2666.7696; found: 2666.7635. Then, the obtained reaction mixture was dissolved in MeOH (1 mL), followed by the addition of 1 M methanolic MeONa (50 µL, 0.05 mmol). The reaction mixture was kept at ~20 °C for 16 h. Then, the reaction mixture was neutralized with Dowex 50W×8 (H⁺) ion-exchange resin (the resin was washed with MeOH before addition) and then filtered. The resin was washed with MeOH (50 mL). The filtrate was concentrated under reduced pressure, and the residue was dried in vacuo. The obtained crude product was dissolved in anhydrous Py (1 mL), then Ac₂O (1 mL) was added at 0 °C (ice–water bath). The reaction mixture was stirred at 20 °C for 24 h. The reaction was quenched by the addition of MeOH (1 μL) at 0 °C (ice–water bath), then concentrated under reduced pressure, co-evaporated with toluene (5 × 5 mL), and dried in vacuo. The residue was purified by silica gel chromatography (gradient: 5%→70% acetone in petroleum ether) to give acetylated decasaccharide 21 (2 mg, 14% over 3 steps). R_f = 0.20 (light petroleum–acetone 1:1); ${\left[\mathrm{\alpha }\right]}_D^{23}$ +53.9 (c 0.15 in CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 2.08 (s, 9H, 3 × CH₃CO), 2.08 (s, 9H, 3 × CH₃CO), 2.09 (s, 6H, 2 × CH₃CO), 2.09 (s, 3H, CH₃CO), 2.10 (s, 3H, CH₃CO), 2.11 (s, 3H, CH₃CO), 2.11 (s, 15H, 5 × CH₃CO), 2.11 (s, 3H, CH₃CO), 2.12 (s, 6H, 2 × CH₃CO), 2.14 (s, 3H, CH₃CO), 2.14 (s, 3H, CH₃CO), 3.58 (t, 2H, J 5.0 Hz, CH₂N₃), 3.70–3.78 (m, 6H, H-5^I-VIa), 3.86–3.90 (m, 1H, H-5^VIb), 3.92–3.98 (m, 5H, H-5^I-Vb), 4.11–4.13 (m, 3H, CH₂O), 4.10–4.26 (m, 15H, H-4^II-X, H-3^VI, H-5^VII-Xa, H-5^VIIb or H-5^IXb), 4.25 (d, 1H, J 2.0 Hz, H-2^VII), 4.29 (d, 1H, J 1.7 Hz, H-2^IX), 4.32 (dd, 1H, J 11.5 Hz, J 3.9 Hz, H-5^VIIb or H-5^IXb), 4.34–4.39 (m, 3H, H-4^I, H-5^VIIIb, H-5^Xb), 4.95–5.01 (m, 4H, H-2^VIII, H-2^X, H-3^VII, H-3^IX), 5.02 (s, 1H, H-1^VII), 5.04–5.07 (m, 3H, H-3^V, H-2^VI, H-1^IX), 5.12–5.18 (m, 12H, H-1^II-VI, H-2^II-V, H-3^II-IV), 5.29 (dd, 1H, J 5.3 Hz, J 1.9 Hz, H-3^I), 5.32–5.37 (m, 3H, H-3^VIII, H-3^X, H-2^I), 5.39 (d, 1H, J 4.7 Hz, H-1^X), 5.44 (d, 1H, J 4.7 Hz, H-1^VIII), 5.61 (s, 1H, H-1^I), 6.84–6.88 (m, 2H, OC₆H₄O (H-3, H-5)), 6.98–7.03 (m, 2H, OC₆H₄O (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 20.3 (CH₃CO), 20.4 (CH₃CO), 20.6 (CH₃CO), 20.67 (CH₃CO), 20.69 (CH₃CO), 20.73 (CH₃CO), 20.75 (CH₃CO), 20.80 (CH₃CO), 50.2 (CH₂N₃), 63.4 (C-5^VII, C-5^IX), 63.7 (C-5^VII, C-5^IX), 64.2 (C-5^VI), 65.0 (C-5^I-V, C-5^VIII, C-5^X), 65.1 (C-5^I-V, C-5^VIII, C-5^X), 65.18 (C-5^I-V, C-5^VIII, C-5^X), 65.24 (3C, C-5^I-V, C-5^VIII, C-5^X), 65.7 (C-5^I-V, C-5^VIII, C-5^X), 67.6 (CH₂O), 75.5 (C-3^VIII, C-3^X), 75.7 (C-3^VIII, C-3^X), 76.4 (C-3^I), 76.6 (C-3^II-V, C-3^VII, C-3^IX, C-2^VIII, C-2^X), 76.7 (C-3^II-V, C-3^VII, C-3^IX, C-2^VIII, C-2^X), 76.8 (C-3^II-V, C-3^VII, C-3^IX, C-2^VIII, C-2^X), 76.9 (C-3^II-V, C-3^VII, C-3^IX, C-2^VIII, C-2^X), 77.1 (C-3^II-V, C-3^VII, C-3^IX, C-2^VIII, C-2^X), 77.4 (C-3^II-V, C-3^VII, C-3^IX, C-2^VIII, C-2^X), 77.7 (C-3^II-V, C-3^VII, C-3^IX, C-2^VIII, C-2^X), 78.9 (C-4^VIII, C-4^X), 78.6 (C-4^VIII, C-4^X), 80.3 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 80.7 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 80.8 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 81.1 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 81.2 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 81.47 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 81.51 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 81.53 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 81.68 (2C, C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 81.73 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 81.9 (C-4^I), 82.0 (C-2^I), 82.2 (C-4^II-VII, C-4^IX, C-3^VI, C-2^II-V), 83.4 (C-2^VI), 83.9 (C-2^IX), 84.2 (C-2^VII), 99.3 (C-1^X), 99.5 (C-1^VIII), 104.8 (C-1^I), 105.2 (C-1^II-VII, C-1^IX), 105.35 (3C, C-1^II-VII, C-1^IX), 105.39 (C-1^II-VII, C-1^IX), 105.43 (C-1^II-VII, C-1^IX), 105.7 (C-1^II-VII, C-1^IX), 115.6 (OC₆H₄O (C-3, C-5)), 118.3 (OC₆H₄O (C-2, C-6)), 150.4 (OC₆H₄O (C-1)), 153.8 (OC₆H₄O (C-4)), 169.7 (CO), 169.7 (CO), 169.9 (CO), 170.00 (CO), 170.02 (CO), 170.1 (CO), 170.2 (CO), 170.18 (CO), 170.20 (CO), 170.3 (CO), 170.46 (CO), 170.51 (CO), 170.6 (CO); HRMS (ESI): m/z [M + 2NH₄]²⁺ Calcd for C₁₀₀H₁₃₉N₅O₆₃²⁺ 1208.8908; found: 1208.8948.

5. Conclusions

In summary, a synthesis of the branched α-(1→3)-, α-(1→5), β-(1→2)-linked hexaarabinofuranoside with CEP aglycone using silylated Ara-β-(1→2)-Ara disaccharide was accomplished. The CEP aglycone in hexaarabinofuranoside was cleaved, and the obtained hemiacetal was converted to the new glycosyl donor with N-phenyltrifluoroacetimidoyl leaving group. The coupling of N-phenyltrifluoroacetimidate of hexaarabinofuranoside and benzoylated tetraarabinofuranoside α-(1→5)-linked glycosyl acceptor promoted by TfOH successfully led to the branched α-(1→5)-, α-(1→3)-, β-(1→2)-linked decaarabinofuranoside with CEP aglycone. After the replacement of the chlorine atom with an azido group in the aglycone of the resulting decaarabinofuranoside and following removal of silyl and acyl groups, the deprotected decaarabinofuranoside bearing 4-(2-azidoethoxy)phenyl aglycone (AEP) was obtained.

The synthesized decaarabinofuranoside and neoglycoconjugates derived from them expand the library of antigens based on oligoarabinofuranosides related to LAM and AG fragments of mycobacteria developed by us. It is important to note that such libraries hold promise for the creation of new multi-antigen/multi-epitope assays for the serodiagnosis of tuberculosis and leprosy, as it has been established that tests based on a single antigen may not be equally sensitive in all regions endemic for tuberculosis and leprosy [10].