Super-Armed Thiomannopyranosides in the Synthesis of a Mannose-Capped Trisaccharide of Mycobacterium tuberculosis Lipoarabinomannan

Abstract

Silylation of phenyl 1-thio-α-d-mannopyranoside, ethyl 1-thio-α- and β-d-mannopyranosides under different conditions was studied. Low-temperature NMR analysis revealed that the silylated products typically exist as an equilibrium of two chair conformations with a predominance of the axially rich ¹C₄ conformation. The dependence of the ratio of conformers on the anomeric configuration, the type of silyl groups and the nature of the aglycone was established. The fully silylated phenyl 1-thio-α-d-mannopyranoside, ethyl 1-thio-α- and β-d-mannopyranosides were tested as glycosyl donors in the synthesis of di- and trisaccharides, including one-pot synthesis. In all cases, only α-linked oligosaccharides as mixtures of conformers were formed. The obtained deprotected Manp-(1→2)-α-d-Manp-(1→5)-α-d-Araf trisaccharide 2-azidoethyl glycoside, related to the non-reducing terminal fragment of the mannose-capped lipoarabinomannan (ManLAM) of Mycobacterium tuberculosis, can be used for further preparation of conjugates with proteins to provide antigens, which are important for development of new tuberculosis screening assays.

1. Introduction

Mycobacterial infections have received significant attention due to the rising number of cases globally, while tuberculosis (TB), which is caused by Mycobacterium tuberculosis, continues to be one of the most dangerous infectious diseases [1].

A major mycobacterial cell wall glycan is lipoarabinomannan (LAM), which plays a critical role in mycobacteria–host interactions [2]. A significant number of immune processes related to mycobacterial infection are associated with LAM, which serves as the main non-protein antigen in mycobacteria [3]. The structure of LAM consists of a phosphatidyl-myo-inositol moiety, a core d-mannopyranose region and d-arabinofuranose domain with α-(1→5)-, α-(1→3)-, and β-(1→2)-glycosidic bonds. The terminal β-(1→2)-diarabinofuranoside fragments of the LAM of M. tuberculosis and others pathogenic strains, such as M. bovis and M. leprae, are often glycosylated with one to three α-1,2-linked mannopyranose residues (resulting in ManLAM) [4]. Endocytic receptors, including the macrophage mannose receptor and DC-SIGN, present on the surface of dendritic cells, thus playing an important role in host–cell recognition, interact predominantly with mannose-containing caps found on the ManLAM [5].

The LAM fragments are attractive targets for the development of TB diagnostic and therapeutic strategies [3,6,7,8,9]. It should be noted that despite the fact that many papers describe preparation of oligoarabinofuranosides [10,11,12,13,14,15,16,17,18,19,20,21,22], the synthesis of LAM fragments containing d-mannopyranose and d-arabinofuranose residues and, most importantly, neoglycoconjugates, thereof remains a challenge [23,24,25,26,27,28].

Generally, during the synthesis of oligoarabinofuranosides, capped with monosaccharide d-mannopyranose residues, the stereoselective formation of an α-glycosidic linkage is enabled by the presence of a participating acyl group at O-2 of the glycosyl donor [27]. Oligosaccharides with di- and trimannose caps can be synthesized by stepwise introduction of monosaccharide blocks. However, such a strategy increases the number of synthetic steps due to protective groups manipulations. Direct use of di- and trimannose building blocks is more challenging, since the effect of a neighboring participating group at O-2 cannot be applied to control the stereoselectivity. For example, when partially benzylated α-(1→2)-linked dimannopyranose thioglycosides were used for the glycosylation of the primary position of β-(1→2)-linked diarabinofuranosides, the formation of anomeric mixtures was observed [29].

A 1,2-trans glycosidic linkage can also be created by the use of polysilylated “super-armed” glycosyl donors. The conformational features of a number of silylated glycosyl donors were investigated previously (for a review, see [30]). In particular, it was shown that 6-O-benzyl-2,3,4-tris-O-(tert-butyldimethylsilyl (TBS))-1-thio-α-d-mannopyranoside exists as a mixture of ⁴C₁ and ¹C₄ conformers in an 1:1 ratio (¹H NMR, −35 °C) [31]. It was found that its reactions with “armed” phenyl tri-O-benzyl-β-d-thioglucosides led predominantly to α-linked glycosides (as was established after desilylation) [31]. Except the above data, no other information is currently available, to the best of our knowledge, regarding the synthesis and reactivity of variously protected polysilylated thiomannopyranosides, which could be valuable for synthesizing biologically important oligosaccharides.

In this work, we aimed at studying the silylation of phenyl 1-thio-α-d-mannopyranoside, ethyl 1-thio-α- and β-d-mannopyranosides under different conditions. Additionally, we focused on the investigation of conformational behavior and glycosylation ability of the obtained polysilylated thiomannopyranosides. We planned to perform one-pot synthesis of mannose-capped trisaccharide of the M. tuberculosis ManLAM and to compare our results with the use of α-(1→2)-linked dimannopyranoside (Block (A), Figure 1).

Recently, we successfully applied [32,33,34,35,36,37] silylated Ara-β-(1→2)-Ara diarabinofuranosides as glycosyl donors lacking 2-O-acyl participating groups for stereoselective 1,2-trans-glycosylation leading to the non-reducing terminal fragments of the arabinogalactan and LAM of M. tuberculosis (Block (B), Figure 1).

The current research may be regarded as an extension of super–armed concept based on the difference of the reactivity between glycosyl donors having silyl and benzyl-protecting groups [30]. We focused on the investigation of the reactivity of polysilylated mannopyranosyl donors with disarmed glycosyl acceptors, having benzoyl protection. It allows us to realize a benzyl-free strategy [34] and to expand the library of terminal oligoarabinofuranosides (with 2-azidoethyl, 4-(2-azidoethoxy)phenyl (AEP) and 4-(3-azidopropoxy)phenyl (APP) aglycones) related to the fragments of M. tuberculosis LAM [34]. These oligosaccharides can be used for further preparation of conjugates with proteins to provide antigens, which are important for development of new tuberculosis screening assays [38].

2. Results and Discussion

2.1. Silylation of Phenyl 1-thio-α-d-Mannopyranoside (1): The Analysis of Conformational Features of Super-Armed Silylated Thiomannopyranosides 2, 5, 7 According to NMR Data

First, silylation of phenyl 1-thio-α-d-mannopyranoside (1) was investigated under various conditions (Scheme 1). According to Method A (TIPSOTf, 2,4,6-collidine, 90 °C, 45 h), fully silylated mannopyranoside (2) as the main product (63%) and a series of regioisomers (3–5) with one hydroxy group were obtained. As expected, the presence of several conformations is typical for compounds 2 and 5 due to the ring flip after the introduction of bulky silyl substituents at O-3 and O-4.

Silylation of phenyl 1-thio-α-d-mannopyranoside (1) under conditions of Method B (TIPSCl (6 equiv.), imidazole, DMF, 90 °C, 52 h) was incomplete and less selective. We obtained the following compounds: mannopyranoside 6 with two TIPS groups at O-3 and O-6, as well as mannopyranosides 3–5 with OH groups at O-4, O-3 and O-2, respectively. It should be noted that we did not observe the formation of fully silylated mannopyranoside (2) at all.

Besides, we directly obtained mannopyranoside 6 (82%) with two TIPS groups at O-3 and O-6 by silylation of phenyl 1-thio-α-d-mannopyranoside (1) under milder conditions of Method C (TIPSCl (2 equiv.), imidazole, DMF, 70 °C, 72 h). Subsequent introduction of TES groups under the conditions of Method D (TESOTf, 2,4,6-collidine, 20 °C, 2 h) resulted in formation of a fully silylated thioglycoside 7.

We analyzed the ¹C₄ (A) to ⁴C₁ (B) ratio for super-armed silylated thiomannopyranosides 2, 5, 7 according to NMR data (

Table 1. The ¹C₄ (A) to ⁴C₁ (B) ratio for 1-thio-d-mannopyranosides 2, 5, 7, 10, 13 and for di- and trisaccharides 15, 18, 22, 24 containing TIPS groups at the mannopyranosyl residues according to ¹H NMR (600 MHz, CDCl₃).

	1C4 (A):4C1(B), T (K)
	For 2:10:1.6; 243; For 5:1:0; 298; For 7:10:1.4; 303; 1:1, 240
	For 10:10:3.4; 240; For 13:1:0; 298; 1:0, 240
	For 15:10:3, 298; 10:7.6; 240; For 18:1:0; 303; 10:3.4; 240
	For 22:10:3, 303; 10:8.6, 236; For 24:10:3, 303; 10:8.6, 244

,

Table 2. ¹H NMR data for super-armed thiomannopyranosides 2, 5, 7, 10, 13 (600 MHz, CDCl₃, δ_H (ppm)/J (Hz)).

Donor (T (K))	H-1 δH/J1,2	H-2 δH/J2,3	H-3 δH/J3,4	H-4 δH/J4,5	H-5 δH/J5,6 or J5,4	H-6a δH/J6a,6b or J6a,5	H-6b δH/J6b,6a or J6b,5
2 (323)	5.40, 7.7	4.20–4.24	4.15–4.19	3.90/<1	3.96–4.01	3.86/10.7, 7.6	3.92–3.97
2 (298)	5.38/7.6	4.17–4.24	4.11–4.16	3.84–3.90	3.94–4.01	3.85/10.7, 7.6	3.90–3.94
2A (243)	5.35/8.5	4.14/2.0	4.09/2.2	3.82–3.87	3.94/4.6, 3.2	3.82–3.87	3.88/10.8, 7.4
2B (243)	5.34–5.35	4.33/1.8 a	3.94–3.97	4.06–4.09	4.11–4.14	3.77/10.0, 8.5	4.04
5 (298)	5.06/9.3	3.83/2.9, 4.9 (OH)	4.23/3.3 a	4.08/1.6	3.97–4.01	3.99/12.4, 5.8	4.08/8.8
7A (240)	5.31/8.8	3.98/2.2	3.91–3.94	3.77–3.86	3.77–3.86	3.77–3.86	3.77–3.86
7B (240)	5.32/1.4	4.21/1.9 a	3.91–3.94	4.05/9.3, 8.6	3.99–4.03	3.77–3.86	3.94–3.96
10A (240)	5.12/8.5	4.08/1.9	4.07/2.3	3.79–3.85	3.79–3.85	3.79–3.85	3.87/11.2, 1.9
10B (240)	5.19/1.4	4.14/1.5	4.01–4.05	4.01–4.05	3.93/10.6, 5.8	3.70/10.1, 8.7	4.00/1.8
13 (298)	5.14/5.7	4.59/2.6	4.16/3.7	4.26/<1	3.83	4.18/10.4, 4.8	4.61/8.6
13 (240)	5.11/5.7	4.54/2.4	4.10/3.8	4.22/<1	3.79	4.05/10.3, 4.6	4.63/9.2

^a An apparent coupling constant (J_app) is shown.

and

Table 3. ¹³C NMR data for super-armed thiomannopyranosides 2, 5, 7, 10, 13 (151 MHz, CDCl₃, δ_C (ppm).

Donor (T (K))	C-1	C-2	C-3	C-4	C-5	C-6
2 (323)	88.6	72.4	77.3	73.1	77.7	65.1
2 (298)	88.3	71.8	77.1	73.0	77.6	64.7
2A (243)	87.4	71.1	76.5	72.3	77.9	63.7
2B (243)	88.9	75.6	74.9	69.9	76.6	63.8
5 (298)	82.2	67.0	73.5	71.1	82.0	61.9
7A (240)	86.0	70.2	76.2	71.2	78.3	62.9 or 63.0
7B (240)	88.9	75.0	74.4	68.7	75.9	62.9 or 63.0
10A (240)	80.8	71.2	76.5	72.4	78.5	63.1
10B (240)	82.9	75.3	75.0	70.0	76.0	63.7
13 (298)	84.7	68.0	74.3	72.1	81.1	65.0
13 (240)	84.3	67.4	73.8	71.3	80.7	64.4

). The A:B ratio for the mannosyl donor 2 could not be measured at 298 K and 323 K since the peaks in the NMR spectra were too broad for characterization. At the same time, we observed 10/1.6 ratio for two conformers of 2, when the ¹H NMR spectrum was recorded at 243 K.

The values J_H1–H2 = 8.5 Hz (243 K), J_H1–H2 = 7.6 Hz (298 K), J_H1–H2 = 7.7 Hz (323 K) for silylated phenyl 1-thio-α-d-mannopyranoside (2) suggest a trans-diaxial relationship between H-1 and H-2, corresponding to ¹C₄ (A) conformer. Conversely, the value of J_H2–H1 = 1.8 Hz (243 K) indicated trans-diequatorial relationship between H-1 and H-2 and the existence of ⁴C₁ (B) conformer. At the same time, the compound 5 with OH group at O-2 exists exclusively as ¹C₄ conformer even at 298 K. We found the correlation of the signal of the anomeric proton of 5 at δ_H 5.06 (d, 1H, J 9.3 Hz, H-1) ppm in a ¹H NMR spectrum with the carbon signal at δ_C 82.2 (C-1). For phenyl 1-thio-α-d-mannopyranoside (7), containing less bulky TES-groups at O-2 and O-4, the abundance of the ⁴C₁ conformer increased (¹C₄ (A) to ⁴C₁ (B) ratio = 1/1 (240 K)). On the contrary, ⁴C₁ conformation is typical for compounds 3 (J_H1–H2 = 1.8 Hz) and 4 (J_H1–H2 = 1.6 Hz).

2.2. The Comparison of Silylation of Ethyl 1-thio-α- and β-d-Mannopyranosides (8, 9)

The data obtained above were compared with the results of the silylation of ethyl 1-thio-α- and β-d-mannopyranosides (8 and 9), obtained from corresponding ethyl tetra-O-acetyl-1-thio-α- and β-d-mannopyranosides [39,40]. We used the same conditions as for phenyl 1-thio-α-d-mannopyranoside 1 (TIPSOTf (6 equiv.), 2,4,6-collidine, 90 °C, 45 h) (Scheme 2).

During the silylation of ethyl 1-thio-α-d- mannopyranoside (8), fully silylated mannopyranoside 10 in ¹C₄ conformation and partially silylated derivatives 11 and 12 in ⁴C₁ conformation were obtained. On the contrary, silylation of ethyl 1-thio-β-d-mannopyranoside (9) led exclusively to the product of complete silylation 13 in 83% yield. We compared ¹C₄ (A) to ⁴C₁ (B) ratio for ethyl 1-thio-α- and β-d-mannopyranosides (10 and 13) (Table 1, Table 2 and Table 3). We observed that silylated ethyl 1-thio-β-d-mannopyranoside 13 exists exclusively as a slightly distorted ¹C₄ conformer at 298 K and 240 K (J_H1–H2 = 5.7 Hz), while for silylated ethyl 1-thio-α-d-mannopyranoside 10 the formation of the mixture of conformers is typical (¹C₄ (A) to ⁴C₁ (B) ratio = 10/3.4 (240 K) with predominance of axially rich conformation ¹C₄.

2.3. A Model Reaction: Synthesis of 1,2-α-Linked Methyl Mannopyranoside 16

The reactivity of fully silylated phenyl 1-thio-α-d-mannopyranoside 2 was tested in a model reaction with methyl 3,4,6-tri-O-benzoyl-α-d-mannopyranoside (14) [41] under NIS/TfOH promotion (Scheme 3). After gel chromatography in toluene, dimannopyranoside 15 was obtained as a mixture of conformers. The ¹C₄ (A) to ⁴C₁ (B) ratio of conformers for the monosaccharide residue at the non-reducing end of methyl dimannopyranoside 15 was 10/3 at 298 K and 10/7.6 at 240 K (Table 1). The anomeric protons for ¹C₄ (A) and ⁴C₁ (B) conformers of the silylated residue of 15 (240 K) resonate at δ_H 4.98 (d, 1H, J 6.6 Hz, H-1^II, A) and 4.68 (d, 1H, J 1.2 Hz, H-1^II, B) ppm in ¹H NMR spectra, correlating to the carbon signals at δ_C 99.7 (C-1^II, A) and 102.2 (C-1^II, B) ppm in ¹³C NMR spectra (

Table 4. ¹H NMR data for di- and trisaccharides 15, 18, 22, 24 containing mannopyranosyl residues with TIPS groups at the non-reducing end (600 MHz, CDCl₃, δ_H (ppm).

Residue (T (K))	H-1 δH/J1,2	H-2 δH/J2,3	H-3 δH/J3,4	H-4 δH/J4,5	H-5 δH/J5,6 or J5,4	H-6a δH/J6a,6b or J6a,5	H-6b δH/J6b,6a or J6b,5
15AII (298)	5.05/6.5	4.16/6.5, 2.0	4.06/2.2 a	3.76/4.6, 2.3	3.70/7.8, 4.6, 3.2	3.80/10.7, 3.3	3.85/10.7, 8.0
15BII (298)	4.76/1.8	4.21/1.9 a	4.27–4.34	4.09/9.3 a	3.67–3.70	3.72–3.76	4.06/10.1
15AII (240)	4.98/6.6	4.12/1.8	3.95–4.05	3.71/4.8, 2.2	3.65–3.70	3.74/10.9, 3.6	3.81/10.7, 8.1
15BII (240)	4.68/1.2	4.14/2.3	4.23/9.3, 1.8	3.95–4.05	3.65–3.70	3.65–3.70	3.95–4.05
18AII (303)	5.05/6.8	4.21/6.8, 1.9	4.11/<1	3.94/3.1	3.77/6.9, 3.6	3.92/10.6, 6.8	3.80/10.7, 4.0
18AII (240)	4.96/7.0	4.15/7.1, 1.9	4.04/2.2 a	3.93/3.0 a	3.69–3.74	3.76/10.6, 4.6	3.86/10.8, 6.5
18BII (240)	4.72/<1	4.12–4.13	4.23/9.4, 1.7	4.07/9.3 a	3.63/8.3 a	3.69–3.74	3.81–3.85
22AIII (303)	5.06/6.6	4.20/6.6, 2.0	4.08/2.2	3.82–3.89	3.70–3.77	3.80/10.8, 3.5	3.82–3.89
22BIII (303)	4.79/<1	4.22/1.9 a	4.32/9.1, 1.9	4.10/9.2 a	n.d	n.d	n.d
22AIII (236)	5.00/6.9	4.12–4.21	3.98–4.04	3.77–3.98	3.63–3.71	3.74/10.7, 3.7	3.77–3.98
22BIII (236)	4.71/<1	4.12–4.21	4.23/9.2	3.98–4.04	3.63–3.71	3.63–3.71	3.77–3.98
24AIII (303)	5.06/6.6	4.20/6.6, 2.0	4.08/2.2	3.83/4.3, 2.4	3.69–3.77	3.80/10.8, 3.5	3.86/10.8, 7.6
24BIII (303)	4.79/1.1	4.22/1.5	4.31/9.3, 1.5	4.10/9.2	n.d	n.d	4.04/9.2
24AIII (244)	5.01/6.7	4.13–4.17	4.00–4.09	3.79–3.84	3.65–3.78	3.65–3.78	3.79–3.84
24BIII (244)	4.72/1.4	4.13–4.17	4.24/9.3, 2.1	4.00–4.09	3.65–3.78	3.65–3.78	3.97/7.7

^a An apparent coupling constant (J_app) is shown.

and

Table 5. ¹³C NMR data for di- and trisaccharides 15, 18, 22, 24 containing mannopyranosyl residues with TIPS groups at the non-reducing end (151 MHz, CDCl₃, δ_C (ppm).

Residue (T (K))	C-1	C-2	C-3	C-4	C-5	C-6
15AII (298)	99.9	72.6	77.5	73.2	78.4	64.0
15BII (298)	102.4	74.4	73.8	70.1	77.7	64.7
15AII (240)	99.7	72.0	77.2	72.6	78.0	63.2
15BII (240)	102.2	73.7	73.5	69.5	77.1	64.2
18AII (303)	99.69	72.3	77.3	72.8	79.5	63.2
18AII (240)	99.67	71.4	77.0	71.8	79.1	62.2
18II (240)	102.9	73.7	73.5	69.1	77.0	63.5
22AIII (303)	99.9	72.5	77.5	73.1	77.5	63.7
22BIII (303)	102.5	74.5	73.8	70.2	77.7	64.8
22AIII (236)	99.6	71.7	77.1	72.3	78.5	62.8
22BIII (236)	102.3	73.8	73.4	69.4	77.0	64.1
24AIII (303)	99.9	72.5	77.5	73.1	78.7	63.7
24BIII (303)	102.6	74.5	73.8	70.2	77.7	64.8
24AIII (244)	99.7	71.8	77.2	72.3	78.5	62.9
24BIII (244)	102.4	73.9	73.4	69.5	77.0	64.2

). In addition to the characteristic high field position of C-1 (¹C₄ (A)) signals compared with low field position of C-1 (⁴C₁ (B)) signals, the characteristic positions of C-3 for¹C₄ (A) and ⁴C₁ (B) conformers in ¹³C NMR spectra could be noted. For example, we observed the carbon signals at δ_C 77.2 (C-3^II, A) and 73.5 (C-3^II, B) for 15 (240 K). The formation of an α-glycoside linkage in the dimannopyranoside 15 followed from ¹H–¹³C HMBC NMR spectrum. We observed ¹J_C1–H1 = 171.2 Hz (240 K) for the major axially rich conformation ¹C₄ and ¹J_C1–H1 = 168.4 Hz for the minor ⁴C₁ conformation. The TIPS groups in the resulting dimannopyranoside were removed (Bu₄NF, AcOH, THF, 40 °C, 2 h) leading to the formation of disaccharide 16, as expected, in the usual ⁴C₁ conformation.

2.4. Synthesis of Thioglycoside of α-(1→2)-Linked Dimannopyranoside 18 with TIPS Protection at the Non-Reducing End

At the next step, glycosylation of phenyl 3,4,6-tri-O-benzoyl-1-thio-α-d-mannopyranoside (17) [42] with fully silylated α-configured thiomannopyranosides (2, 10) and β-configured thiomannopyranoside 13, was studied under various conditions (Scheme 4). The coupling of 17 with 2 with NIS/TfOH promotion resulted in the formation of a chromatographically inseparable mixture of the target α-(1→2)-linked dimannopyranoside 18 with silylated N-glycosylsuccinimide 19 in a ratio of 18:19 = 2.5:1 (NMR data). Glycosylation of thioglycoside 17 with silylated ethyl 1-thio-α-d-mannopyranoside 10 under preactivation conditions (benzenesulfenylpiperidine (BSP), 2,4,6-tri-tert-butylpyrimidine (TTBP), Tf₂O) also gave a chromatographically inseparable mixture of α-(1→2)-linked dimannopyranoside 18 and piperidine-1-yl glycoside 20 (16 mol% according to ¹H NMR data) (Scheme 4).

Glycosylation of phenyl 3,4,6-tri-O-benzoyl-1-thio-α-d-mannopyranoside (17) with silylated ethyl 1-thio-β-d-mannopyranoside 13 with NIS/TfOH promotion proved to be the best way to synthesize 1,2-α-linked dimannopyranoside 18, obtained in 86% yield.

Based on these results, we can conclude that the optimal conditions for the preparation of the key 1,2-α-linked dimannopyranoside 18 were as follows: (1) an excess of the glycosyl acceptor; (2) temperature range from −78 to −50 °C; (3) NIS/TfOH promoting system. It should be noted that in all cases we observed the formation of only α-glycosydic linkage in 18. Besides, the use of the silylated ethyl 1-thio-β-d-mannopyranoside 13 led to less side reactions and enabled simpler purification.

The dependence of the ¹C₄ (A) to ⁴C₁ (B) ratio of conformers on nature of the aglycone in α-(1→2)-linked dimannopyranosides with O-methyl and S-phenyl aglycones (15 and 18) was also established. For methyl mannopyranoside 15 the portion of ¹C₄ (A) conformer is lower (10/3 (298 K); 10/7.6 (240 K)) than in the case of phenyl 1-thiomannopyranoside 18 (1/0 (303 K); 10/3.4 (240 K)) (Table 1). The anomeric protons for ¹C₄ (A) and ⁴C₁ (B) conformers of silylated residues of 18 (240 K) resonated in the same region as for 15 at δ_H 4.96 (d, 1H, J 7.0 Hz, H-1^II A) and 4.72 (s, 1H, H-1^II B) ppm in ¹H NMR spectra, correlating to the carbon signals at δ_C 99.7 (C-1^II, A) and 102.9 (C-1^II, B) ppm in ¹³C NMR spectra (Table 4 and Table 5). Besides, we observed the carbon signals δ_C 77.0 (2 C, C-3^II, A; C-5^II B) and δ_C 73.5 (C-3^II, B) for 18 (240 K) in the same region as for 15.

2.5. Synthesis of Mannose-Capped Trisaccharide of the M. tuberculosis ManLAM

At the next step, we investigated the applicability of α-(1→2)-linked dimannopyranoside 18 with TIPS groups at the non-reducing end for the preparation of mannose-capped trisaccharide of the M. tuberculosis ManLAM. We studied two strategies to access protected trisaccharide 22 bearing 2-chloroethyl aglycone according to [2+1] convergent way and performing one-pot synthesis (Scheme 5 and Scheme 6).

To this end, the 2-chloroethyl arabinofuranoside 21 [43], synthesized by us earlier, was glycosylated with dimannothiopyranoside 18 under NIS/TfOH promotion to give trisaccharide 22 in 78% yield as a mixture of conformers according to NMR data (Scheme 6). The formation of α-glycoside linkage in the trisaccharide 22 followed from ¹H–¹³C HMBC NMR spectrum. We observed ¹J_C1–H1 = 176.8 Hz (236 K) for the conformer A in the axially rich ¹C₄ conformation and ¹J_C1–H1 = 169.2 Hz for the minor conformer B in the ⁴C₁ conformation.

Alternatively, based on the difference in reactivity of 10, 17 and 21, we performed one-pot synthesis of trisaccharide 22. At the first step, we glycosylated phenyl 3,4,6-tri-O-benzoyl-1-thio-α-d-mannopyranoside 17 (1.2 equiv.) with fully silylated ethyl 1-thio-α-d-mannopyranoside 10 (1 equiv.) under NIS/TfOH promotion at −78 → −40 °C. Then 2-chloroethyl arabinofuranoside 21 was added followed by an additional portion of NIS/TfOH and the temperature was raised to 0 °C. Since the reaction was not complete after 20 h at 0 °C, an additional portion of arabinofuranoside 21 (2 equiv.) was added. The desired trisaccharide 22 was isolated in 49% yield (over two steps) along with partially protected trisaccharide with three TIPS groups (5%) and α-(1→5)-linked mannoarabinofuranoside 23 (27%). It is interesting to note that we observed the formation of only α-glycosidic linkages in the obtained mannoarabinofuranoside 23 despite the absence of a participating group at O-2 both in the monosaccharide mannosyl donor 17.

Then we replaced the chlorine atom in the aglycone of the resulting trisaccharide 22 with azido group (NaN₃, DMF, 18-crown-6) to give 2-azidoethyl glycoside 24 in 84% yield (Scheme 7). According to NMR data, the ¹C₄ (A) to ⁴C₁ (B) ratios for trisaccharides 22 and 24 were 10/3 and 10/8.6 at 303 K and 236 (244) K, respectively (Table 1). We found the signals of the anomeric protons of 22 for conformer A at δ_H 5.06 (d, 1H, J 6.6 Hz, H-1^III), 5.09 (d, 1H, J 1.8 Hz, H-1^II), 5.14 (s, 1H, H-1^I) in the ¹H NMR spectrum, correlating with the carbon signals at δ_C 99.9 (C-1^III), 100.11 (C-1^II), 105.87 (C-1^I) at 303 K. The signals of the anomeric protons of 22 for conformer B were found at δ_H 4.79 (s, 1H, H-1^III), 5.18 (s, 1H, H-1^I), 5.27 (s, 1H, H-1^II), correlating with the carbon signals at δ_C 98.5 (C-1^II), 102.5 (C-1^III), 105.9 (C-1^I) at 303 K (Table 4 and Table 5).

Then all TIPS groups in azide 24 were removed by treatment with TBAF in THF in the presence of AcOH at 40 °C to give partially protected trisaccharide 25 as expected, in the usual ⁴C₁ conformation. Debenzoylation with MeONa in MeOH led the target deprotected 2-azidoethyl 5-O-[α-d-mannopyranosyl-2-O-(α-d-mannopyranosyl)-α-d-arabinofuranoside (26) in 78% yield (Scheme 7).

3. Materials and Methods

3.1. General Methods

All reactions sensitive to air and/or moisture were carried out under 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 [44]. Dichloromethane was distilled over P₂O₅ and then over CaH₂ and stored over 4 Å molecular sieves (MS 4 Å). Powdered MS 4 Å (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). 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 toluene involved addition of toluene and evaporation of volatiles on a rotary evaporator. ¹H, ¹³C and ²⁹Si NMR spectra were recorded on a Bruker AVANCE NEO 300 spectrometer (Billerica, MA, USA, 300.23, 75.50 and 59.65 MHz for ¹H, ¹³C and ²⁹Si, respectively) or on a Bruker AVANCE 600 spectrometer (Billerica, MA, USA, 600.13, 150.92 and 119 MHz for ¹H, ¹³C and ²⁹Si respectively).The ¹H NMR chemical shifts are referred to the residual signal of CHCl₃ (δ_H 7.27 ppm) for solutions in CDCl₃, C₆HD₅ (δ_H 7.16 ppm) for solutions in C₆D₆ or CHD₂OD (δ_H 3.31 ppm) for solutions in CD₃OD, the ¹³C NMR shifts–to the central line of CDCl₃ signal (δ_C 77.00 ppm), C₆D₆ (δ_C 128.06 ppm) or CD₃OD signal (δ_C 49.00 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). 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).

3.2. Silylation of Phenyl 1-thio-α-d-Mannopyranoside (1): Phenyl 2,3,4,6-Tetrakis-O-(Triisopropylsilyl)-1-thio-α-d-mannopyranoside (2), Phenyl 2, 3,6-tris-O-(Triisopropylsilyl)-1-thio-α-d-mannopyranoside (3), Phenyl 2,4,6-tris-O-(Triisopropylsilyl)-1-thio-α-d-mannopyranoside (4), Phenyl 3,4,6-tris-O-(Triisopropylsilyl)-1-thio-α-d-mannopyranoside (5), Phenyl 3,6-bis-O-(Triisopropylsilyl)-1-thio-α-d-mannopyranoside (6)

(1) i-Pr₃SiOTf (0.630 mL, 2.34 mmol) was added to the solution of tetraol 1 (106 mg, 0.389 mmol) in 2,4,6-collidine (1.5 mL) at 20 °C. The reaction mixture was stirred at 90 °C for 45 h and then allowed to reach 20 °C, diluted with CH₂Cl₂ (40 mL), washed with H₂O (40 mL), 1 M KHSO₄ (3 × 40 mL), H₂O (40 mL), and satd aq NaHCO₃ (3 × 40 mL). Organic extracts were dried with Na₂SO₄, filtered, concentrated under reduced pressure, and purified by silica gel chromatography (gradient: 0 → 24% EtOAc in petroleum ether) to give silylated derivatives 2–5.

Data for phenyl 2,3,4,6-tetrakis-O-(triisopropylsilyl)-1-thio-α-d-mannopyranoside (2):

219 mg (0.244 mmol, 63%), R_f = 0.73 (petroleum ether–CH₂Cl₂, 4:1), [α]_D²¹ + 99.7 (c 0.90, CHCl₃); ¹H NMR (600 MHz, CDCl₃, 323 K): δ 1.01–1.34 (m, 84H, 4 × ((CH₃)₂CH)₃Si), 3.86 (dd, 1H, J 10.7 Hz, J 7.6 Hz, H-6a), 3.90 (s, 1H, H-4), 3.92–3.97 (m, 1H, H-6b), 3.96–4.01 (m, 1H, H-5), 4.15–4.19 (m, 1H, H-3), 4.20–4.24 (m, 1H, H-2), 5.40 (d, 1H, J 7.7 Hz, H-1), 7.16–7.21 (m, 1H, PhS (H-4)), 7.21–7.25 (m, 2H, PhS (H-3, H-5)), 7.55–7.59 (m, 2H, PhS (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 12.1 (((CH₃)₂CH)₃Si), 13.0 (((CH₃)₂CH)₃Si), 13.2 (((CH₃)₂CH)₃Si), 13.6 (((CH₃)₂CH)₃Si), 17.9 (((CH₃)₂CH)₃Si), 18.0 (((CH₃)₂CH)₃Si), 18.2 (((CH₃)₂CH)₃Si), 18.3 (((CH₃)₂CH)₃Si), 18.4 (((CH₃)₂CH)₃Si), 18.51 (((CH₃)₂CH)₃Si), 18.53 (((CH₃)₂CH)₃Si), 65.1 (C-6), 72.3 (C-2), 73.1 (C-4), 77.3 (C-3), 77.7 (C-5), 88.6 (C-1), 126.3 (PhS (C-4)), 128.4 (PhS (C-3, C-5)), 131.1 (PhS (C-2, C-6)).

¹H NMR (600 MHz, CDCl₃, 298 K): δ 0.89–1.38 (m, 84H, 4 × ((CH₃)₂CH)₃Si), 3.85 (dd, 1H, J 10.7 Hz, J 7.6 Hz, H-6a), 3.84–3.90 (m, 1H, H-4), 3.90–3.94 (m, 1H, H-6b), 3.94–4.01 (m, 1H, H-5), 4.11–4.16 (m, 1H, H-3), 4.17–4.24 (m, 1H, H-2), 5.38 (d, 1H, J 7.6 Hz, H-1), 7.17–7.21 (m, 1H, PhS (H-4)), 7.21–7.26 (m, 2H, PhS (H-3, H-5)), 7.55–7.61 (m, 2H, PhS (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 11.9 (((CH₃)₂CH)₃Si), 12.7 (((CH₃)₂CH)₃Si), 13.0 (((CH₃)₂CH)₃Si), 13.3 (((CH₃)₂CH)₃Si), 17.89 (((CH₃)₂CH)₃Si), 17.94 (((CH₃)₂CH)₃Si), 18.20 (((CH₃)₂CH)₃Si), 18.24 (((CH₃)₂CH)₃Si), 18.4 (((CH₃)₂CH)₃Si), 18.45 (((CH₃)₂CH)₃Si), 18.49 (((CH₃)₂CH)₃Si), 64.7 (C-6), 71.8 (C-2), 72.9 (C-4), 77.1 (C-3), 77.6 (C-5), 88.3 (C-1), 126.3 (PhS (C-4)), 128.4 (PhS (C-3, C-5)), 131.1 (PhS (C-2, C-6));

¹H NMR (600 MHz, CDCl₃, 243 K, A:B = 10:1.6): δ 0.97–1.16 (m, 84H, ((CH₃)₂CH)₃Si), 1.26 (p, 3H, J 7.5 Hz, ((CH₃)₂CH)₃Si), 3.77 (dd, 1H, J 10.0 Hz, J 8.5 Hz, H-6a, B), 3.82–3.87 (m, 2H, H-4, A, H-6a, A), 3.88 (dd, 1H, J 10.8 Hz, J 7.4 Hz, H-6b, A), 3.94 (ddd, 1H, J 7.8 Hz, J 4.6 Hz, J 3.2 Hz, H-5, A), 3.94–3.97 (m, 1H, H-3, B), 4.04 (d, 1H, J 9.7 Hz, H-6b, B), 4.06–4.09 (m, 1H, H-4, B), 4.09 (d, 1H, J 2.2 Hz, H-3, A), 4.11–4.14 (m, 1H, H-5, B), 4.14 (dd, 1H, J 8.4 Hz, J 2.0 Hz, H-2, A), 4.33 (t, 1H, J 1.8 Hz, H-2, B), 5.34–5.35 (m, 1H, H-1, B), 5.35 (d, 1H, J 8.5 Hz, H-1, A), 7.18–7.24 (m, 1H, Ph (H-4)), 7.22–7.28 (m, 3H, Ph (H-3, H-5)), 7.54–7.59 (m, 1H, Ph (H-2, H-6), B), 7.57–7.62 (m, 2H, Ph (H-2, H-6), A); ¹³C NMR (151 MHz, CDCl₃): δ 11.3 (((CH₃)₂CH)₃Si, B), 11.5 (((CH₃)₂CH)₃Si, A), 12.2 (((CH₃)₂CH)₃Si, A), 12.6 (((CH₃)₂CH)₃Si, A), 12.9 (((CH₃)₂CH)₃Si, B), 13.1 (((CH₃)₂CH)₃Si, A), 13.9 (((CH₃)₂CH)₃Si, B), 14.2 (((CH₃)₂CH)₃Si, B), 17.7 (((CH₃)₂CH)₃Si, B), 17.79 (((CH₃)₂CH)₃Si, A), 17.81 (((CH₃)₂CH)₃Si, B), 17.9 (((CH₃)₂CH)₃Si, A), 18.0 (((CH₃)₂CH)₃Si, A), 18.08 (((CH₃)₂CH)₃Si, A), 18.14 (((CH₃)₂CH)₃Si, B), 18.2 (((CH₃)₂CH)₃Si, A), 18.27 (((CH₃)₂CH)₃Si, A), 18.31 (((CH₃)₂CH)₃Si, A), 18.4 (((CH₃)₂CH)₃Si, A), 18.6 (((CH₃)₂CH)₃Si, B), 18.7 (((CH₃)₂CH)₃Si, B), 63.7 (C-6, A), 63.8 (C-6, B), 69.9 (C-4, B), 71.1 (C-2, A), 72.3 (C-4, A), 74.9 (C-3, B), 75.6 (C-2, B), 76.5 (C-3, A), 76.6 (C-5, B), 77.9 (C-5, A), 87.4 (C-1, A), 88.9 (C-1, B), 126.2 (PhS (C-4), A), 126.7 (PhS (C-4), B), 128.3 (PhS (C-3, C-5), A), 128.6 (PhS (C-3, C-5), B), 130.7 (PhS (C-2, C-6), A), 131.7 (PhS (C-2, C-6), B), 135.2 (PhS (C-1), B), 136.4 (PhS (C-1), A);

¹H NMR (300 MHz, C₆D₆): δ 0.93–1.54 (m, 84H, ((CH₃)₂CH)₃Si), 4.03–4.25 (m, 4H, H-4, H-6), 4.28–4.39 (m, 1H, H-5), 4.41 (d, 1H, J 2.4 Hz, H-3), 4.49–4.57 (m, 1H, H-2), 5.77 (d, 1H, J 7.3 Hz, H-1), 6.91–7.04 (m, 1H, Ph (H-4)), 7.06–7.12 (m, 2H, Ph (H-3, H-5)), 7.61–8.09 (m, 2H, Ph (H-2, H-6));selected signals ¹³C NMR (76 MHz, C₆D₆): δ 12.4 (((CH₃)₂CH)₃Si), 13.3 (((CH₃)₂CH)₃Si), 13.6 (((CH₃)₂CH)₃Si), 13.8 ((CH₃)₂CH)₃Si)), 18.25 (((CH₃)₂CH)₃Si), 18.29 (((CH₃)₂CH)₃Si), 18.5 (((CH₃)₂CH)₃Si), 18.6 (((CH₃)₂CH)₃Si), 18.8 (((CH₃)₂CH)₃Si), 18.8 (((CH₃)₂CH)₃Si), 18.9 (((CH₃)₂CH)₃Si), 65.6 (C-6), 77.70, 89.5 (C-1), 126.9 (PhS (C-4)), 129.0 (PhS (C-3, C-5)), 131.3 (PhS (C-2, C-6)), 136.6 (PhS (C-1));

Data for phenyl 2,3,6-tris-O-(triisopropylsilyl)-1-thio-α-d-mannopyranoside (3): 45.4 mg (0.061 mmol, 16%), R_f = 0.37 (petroleum ether–CH₂Cl₂, 4:1). [α]_D²² +58.7 (c 1.13, CHCl₃); ¹H NMR (300 MHz, C₆D₆): δ 1.01–1.39 (m, 63H, ((CH₃)₂CH)₃Si), 2.77 (d, 1H, J 2.2 Hz, OH), 4.11 (dd, 1H, J 10.2 Hz, J 5.7 Hz, H-6a), 4.17 (dd, 1H, J 10.2 Hz, J 5.1 Hz, H-6b), 4.29 (td, 1H, J 9.0 Hz, J 2.2 Hz, H-4), 4.37 (dd, 1H, J 9.0 Hz, J 2.5 Hz, H-3), 4.43–4.52 (m, 1H, H-5), 4.66 (dd, 1H, J 2.5 Hz, J 1.8 Hz, H-2), 5.75 (dd, 1H, J 1.8 Hz, J 0.7 Hz, H-1), 6.89–6.97 (m, 1H, Ph (H-4)), 7.00–7.09 (m, 2H, Ph (H-3, H-5)), 7.59–7.68 (m, 2H, Ph (H-2, H-6)); ¹³C NMR (76 MHz, C₆D₆): δ 12.2 (((CH₃)₂CH)₃Si), 13.3 (((CH₃)₂CH)₃Si), 13.4 (((CH₃)₂CH)₃Si), 18.2 (((CH₃)₂CH)₃Si), 18.45 (((CH₃)₂CH)₃Si), 18.50 (((CH₃)₂CH)₃Si), 18.6 (((CH₃)₂CH)₃Si), 18.7 (((CH₃)₂CH)₃Si), 66.0 (C-6), 70.6 (C-4), 74.5 (C-5), 74.9 (C-3), 75.7 (C-2), 90.6 (C-1), 127.6 (PhS (C-4)), 129.4 (PhS (C-3, C-5)), 131.7 (PhS (C-2, C-6)), 135.7 (PhS (C-1)); ²⁹Si INEPT NMR (60 MHz, C₆D₆): δ 12.6 (2-O-TIPS), 14.1 (3-O-TIPS), 15.0 (6-O-TIPS); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₄₈H₁₀₀NO₅SSi₄ 914.6394; Found: 914.6393.

Data for phenyl 2,4,6-tris-O-(triisopropylsilyl)-1-thio-α-d-mannopyranoside (4): 28.1 mg (0.038 mmol, 10%) R_f = 0.26 (petroleum ether–CH₂Cl₂, 4:1). [α]_D²² +78.4 (c 1.32, CHCl₃); ¹H NMR (300 MHz, C₆D₆): δ 0.89–1.47 (m, 63H, 3 × ((CH₃)₂CH)₃Si), 2.25 (d, 1H, J 9.8 Hz, 3-HO), 4.00 (ddd, 1H, J 9.8 Hz, J 8.2 Hz, J 3.4 Hz, H-3), 4.11 (dd, 1H, J 10.7 Hz, J 2.5 Hz, H-6a), 4.16 (dd, 1H, J 10.8 Hz, J 4.2 Hz, H-6b), 4.27 (dd, 1H, J 9.3 Hz, J 8.1 Hz, H-4), 4.34 (ddd, 1H, J 9.1 Hz, J 4.3 Hz, J 2.5 Hz, H-5), 4.47 (dd, 1H, J 3.4 Hz, J 1.6 Hz, H-2), 5.74 (d, 1H, J 1.6 Hz, H-1), 6.92–7.02 (m, 1H, PhS (H-4)), 7.05–7.14 (m, 2H, PhS (H-3, H-5)), 7.59–7.67 (m, 2H, PhS (H-2, H-6)); ¹³C NMR (76 MHz, C₆D₆): δ 12.4 (((CH₃)₂CH)₃Si), 12.8 (((CH₃)₂CH)₃Si), 13.6 (((CH₃)₂CH)₃Si), 18.16 (((CH₃)₂CH)₃Si), 18.18 (((CH₃)₂CH)₃Si), 18.31 (((CH₃)₂CH)₃Si), 18.34 (((CH₃)₂CH)₃Si), 18.82 (((CH₃)₂CH)₃Si), 18.85 (((CH₃)₂CH)₃Si), 63.6 (C-6), 71.0 (C-4), 73.9 (C-3), 74.8 (C-2), 75.5 (C-5), 88.7 (C-1), 127.4 (PhS (C-4)), 129.3 (PhS (C-3, C-5)), 131.4 (PhS (C-2, C-6)), 135.7 (PhS (C-1)); ²⁹Si INEPT NMR (60 MHz, C₆D₆): δ 12.2 (4-O-TIPS), 13.3 (6-O-TIPS), 15.5 (2-O-TIPS); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₃₉H₈₀NO₅SSi₃ 758.5060; Found: 758.5052.

Data for phenyl 3,4,6-tris-O-(triisopropylsilyl)-1-thio-α-d-mannopyranoside (5): 25 mg (0.034 mmol, 9%), R_f = 0.17 (petroleum ether–CH₂Cl₂, 4:1). [α]_D²² +30.3 (c 1.22, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 0.99–1.21 (m, 63H, 3 × ((CH₃)₂CH)₃Si), 2.16 (d, 1H, J 4.9 Hz, OH), 3.83 (ddd, 1H, J 9.3 Hz, J 4.9 Hz, J 2.9 Hz, H-2), 3.99 (dd, 1H, J 12.4 Hz, J 5.8 Hz, H-6a), 3.97–4.01 (m, 1H, H-5), 4.08 (dd, 1H, J 3.8 Hz, J 1.6 Hz, H-4), 4.08 (ddd, 1H, J 12.4 Hz, J 8.8 Hz, H-6b), 4.23 (t, 1H, J 3.3 Hz, H-3), 5.06 (d, 1H, J 9.3 Hz, H-1), 7.20–7.28 (m, 3H, PhS (H-3, H-4, H-5)), 7.58–7.63 (m, 2H, PhS (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 12.0 (((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 12.6 (((CH₃)₂CH)₃Si), 18.0 (2 × ((CH₃)₂CH)₃Si), 18.09 (((CH₃)₂CH)₃Si), 18.11 (((CH₃)₂CH)₃Si), 18.16 (((CH₃)₂CH)₃Si), 18.24 (((CH₃)₂CH)₃Si), 61.9 (C-6), 67.0 (C-2), 71.1 (C-4), 73.5 (C-3), 82.0 (C-5), 82.2 (C-1), 127.4 (PhS (C-4)), 128.7 (PhS (C-3, C-5)), 132.6 (PhS (C-1)), 132.5 (PhS (C-2, C-6)); ²⁹Si INEPT NMR (119 MHz, CDCl₃): δ 13.7 (4-O-TIPS), 14.0 (6-O-TIPS), 15.2 (3-O-TIPS); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₃₉H₈₀NO₅SSi₃ 758.5060; Found: 758.5058.

(2) To the solution of tetraol 1 (100 mg, 0.367 mmol) in DMF (2 mL) imidazole (300 mg, 4.41 mmol) and TIPSCl (0.471 mL, 2.2 mmol) were added. The reaction mixture was stirred at 90 °C for 52 h, then co-evaporated with toluene (3 × 10 mL). The residue was diluted with CH₂Cl₂ (50 mL), washed with H₂O (50 mL), 1 M KHSO₄ (3 × 30 mL), H₂O (30 mL), satd aq NaHCO₃ (3 × 30 mL), concentrated and dried in vacuo. After purification by silica gel chromatography (gradient: 0 → 24% CH₂Cl₂ in petroleum ether) silylated derivatives 3 (60.0 mg, 0.081 mmol, 21%), 4 (70.0 mg, 0.094 mmol, 22%), 5 (56 mg, 0.076 mmol, 21%), 6 (55 mg, 0.094 mmol, 26%) were obtained.

(3) To the solution of tetraol 1 (136 mg, 0.5 mmol) in DMF (2 mL) imidazole (170 mg, 2.5 mmol) and TIPSCl (0.234 mL, 1.1 mmol) were added. The reaction mixture was stirred at 20 °C for 96 h, then co-evaporated with toluene (3 × 10 mL). The residue was diluted with CH₂Cl₂ (50 mL), washed with H₂O (50 mL), 1 M KHSO₄ (3 × 30 mL), H₂O (30 mL), satd aq NaHCO₃ (3 × 30 mL), concentrated and dried in vacuo. After purification by silica gel chromatography (gradient: 2 → 10% EtOAc in petroleum ether) silylated derivative 6 (250 mg, 0.427 mmol, 85%) was obtained; R_f = 0.75 (petroleum ether–EtOAc, 4:1). [α]_D²⁶ +77.3 (c 1.15, CHCl₃); ¹H NMR (300 MHz, CDCl₃): δ 1.01–1.28 (m, 42H, 2 × ((CH₃)₂CH)₃Si), 2.81 (d, 1H, J 1.4 Hz, OH-2), 2.99 (d, 1H, J 1.9 Hz, OH-4), 3.87 (td, 1H, J 9.0 Hz, J 1.8 Hz, H-4), 3.93–3.97 (m, 2H, H-6a, H-6b), 4.05 (dd, 1H, J 8.6 Hz, J 3.4 Hz, H-3), 4.10–4.19 (m, 2H, H-2, H-5), 5.59 (d, 1H, J 1.4 Hz, H-1), 7.24–7.33 (m, 3H, SPh (H-3, H-4, H-5)), 7.46–7.51 (m, 3H, SPh (H-2, H-6)); ¹³C NMR (75 MHz, CDCl₃): δ 11.8 (((CH₃)₂CH)₃Si), 12.5 (((CH₃)₂CH)₃Si), 17.88 (4 C, ((CH₃)₂CH)₃Si), 17.93–18.18 (4 C, ((CH₃)₂CH)₃Si), 65.6 (C-6), 71.5 (C-4), 71.7 (C-5), 72.6 (C-2), 73.4 (C-3), 87.1 (C-1), 127.3 (SPh (C-4)), 129.0 (SPh (C-3, C-5)), 131.5 (SPh (C-2, C-6)); HRMS (ESI): m/z [M+Na]⁺Calcd for C₃₀H₅₆NaO₅SSi₂⁺ 607.3279; Found: 607.3282;

3.3. Phenyl 3,6-bis-O-(Triisopropylsilyl)-2,4-bis-O-(Triethylsilyl)-1-thio-α-d-mannopyranoside (7)

TESOTf (0.135 μL, 0.6 mmol) was added to the solution of diol 6 (116.6 mg, 0.2 mmol) in 2,4,6-collidine (2 mL) at 20 °C. The reaction mixture was stirred at 20 °C for 2 h, then diluted with CH₂Cl₂ (50 mL), washed with H₂O (50 mL), satd aq NaHCO₃ (50 mL), concentrated and dried in vacuo. After purification by silica gel chromatography (gradient: 1 → 6% EtOAc in petroleum ether) silylated derivative 7 (126 mg, 0.155 mmol, 78%) was obtained. R_f = 0.50 (petroleum ether–CH₂Cl₂, 2:1); [α]_D²⁰ +9.4 (c 0.99, CHCl₃); ¹H NMR (600 MHz, CDCl₃, 303 K, A:B = 10:0.14) Signals were too broad for full assignment. Selected signals: δ 5.23 (br. s, 1H, H-1, B), 5.26 (d, 1H, J 6.3 Hz, H-1, A);¹H NMR (600 MHz, CDCl₃, 240 K, A:B = 1:1): δ 0.52–0.62 (m, 12H, 2 × (CH₃CH₂)₃Si), 0.62–0.76 (m, 12H, 2 × (CH₃CH₂)₃Si), 0.88–0.98 (m, 36H, 2 × (CH₃CH₂)₃Si A, 2 × (CH₃CH₂)₃Si B), 0.99–1.17 (m, 84H, 2 × ((CH₃)₂CH)₃Si A, 2 × ((CH₃)₂CH)₃Si B), 3.77–3.86 (m, 5H, H-6a B, H-4 A, H-5 A, H-6a A, H-6b A), 3.91–3.94 (m, 2H, H-3 B, H-3 A), 3.94–3.96 (m, 1H, H-6b B), 3.98 (dd, 1H, J 8.7 Hz, J 2.2 Hz, H-2 A), 3.99–4.03 (m, 1H, H-5 B), 4.05 (dd, 1H, J 9.3 Hz, J 8.6 Hz, H-4 B), 4.21 (t, 1H, J 1.9 Hz, H-2 B), 5.31 (d, 1H, J 8.8 Hz, H-1 A), 5.32 (d, 1H, J 1.4 Hz, H-1 B), 7.18–7.33 (m, 6H, PhS (H-4) A, PhS (H-4) B, PhS (H-3, H-5) A, PhS (H-3, H-5) B), 7.51–7.55 (m, 2H, PhS (H-2, H-6) B), 7.55–7.60 (m, 2H, PhS (H-2, H-6) A); ¹³C NMR (151 MHz, CDCl₃): δ 4.4 ((CH₃CH₂)₃Si), 4.6 ((CH₃CH₂)₃Si), 4.8 ((CH₃CH₂)₃Si), 5.1 ((CH₃CH₂)₃Si), 6.90 ((CH₃CH₂)₃Si), 6.92 ((CH₃CH₂)₃Si), 6.7 ((CH₃CH₂)₃Si), 7.1 ((CH₃CH₂)₃Si), 11.49 (((CH₃)₂CH)₃Si), 11.51 (((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 13.4 (((CH₃)₂CH)₃Si), 17.8 (2 × ((CH₃)₂CH)₃Si), 17.7 (2 × ((CH₃)₂CH)₃Si), 18.18 (((CH₃)₂CH)₃Si), 18.23 (((CH₃)₂CH)₃Si), 18.33 (((CH₃)₂CH)₃Si), 18.34 (((CH₃)₂CH)₃Si), 62.9 (C-6 A, C-6 B), 63.0 (C-6 A, C-6 B), 68.7 (C-4 B), 70.2 (C-2 A), 71.2 (C-4 A), 74.6 (C-3 B), 75.0 (C-2 B), 75.9 (C-5 B), 76.2 (C-3 A), 78.3 (C-5 A), 86.0 (C-1 A), 88.8 (C-1 B), 126.2 (PhS (C-4) A), 126.6 (PhS (C-4) B), 128.5 (PhS (C-3, C-5) A, PhS (C-3, C-5) B), 128.6 (PhS (C-3, C-5) A, PhS (C-3, C-5) B), 130.4 (PhS (C-2, C-6) A), 130.7 (PhS (C-2, C-6) B), 135.6 (PhS (C-1) B), 136.1 (PhS (C-1) A); HRMS (ESI): m/z [M+Na]⁺Calcd for C₄₂H₈₄NaO₅SSi₄⁺ 835.5009; Found: 835.5009.

3.4. Silylation of Ethyl 1-thio-α-and β-d-Mannopyranosides (8 and 9): Ethyl 2,3,4,6-Tetrakis-O-(Triisopropylsilyl)-1-thio-α-d-mannopyranoside (10), Ethyl 2,3,6-tris-O-(Triisopropylsilyl)-1-thio-α-d-mannopyranoside (11) and Ethyl 2,4,6-tris-O-(Triisopropylsilyl)-1-thio-α-d-mannopyranoside (12)

i-Pr₃SiOTf (0.720 mL, 2.68 mmol) was added to the solution of ethyl 1-thio-α-d-mannopyranoside 8 (108 mg, 0.483 mmol) in 2,4,6-collidine (1.5 mL) at 20 °C. The reaction mixture was stirred at 90 °C for 45 h and then allowed to reach 20 °C, diluted with CH₂Cl₂ (40 mL), washed with H₂O (40 mL), 1 M KHSO₄ (3 × 40 mL), H₂O (40 mL) and satd aq NaHCO₃ (3 × 40 mL). Organic extracts were dried with Na₂SO₄, filtered, concentrated under reduced pressure and purified by silica gel chromatography (gradient: 10 → 22% CH₂Cl₂ in petroleum ether) to give silylated derivatives 10–12.

Data for ethyl 2,3,4,6-tetrakis-O-(triisopropylsilyl)-1-thio-α-d-mannopyranoside (10): 208 mg (0.254 mmol, 51%) R_f = 0.69 (petroleum ether–CH₂Cl₂, 2:1). [α]_D²⁰ +58.7 (c 1.02, CHCl₃); ¹H NMR (600 MHz, CDCl₃, 240 K, A:B = 10:3.4): δ 0.98–1.13 (m, 168H, 4 × ((CH₃)₂CH)₃Si, A&, B), 1.19 (q, 3H, J 7.3 Hz, ((CH₃)₂CH)₃Si, A), 1.23 (t, 3H, J 7.4 Hz, CH₃CH₂S, B), 1.25 (t, 3H, J 7.4 Hz, CH₃CH₂S, A), 2.51 (dq, 1H, J 13.3 Hz, J 7.6 Hz, CH₃CHHS, B), 2.57 (dq, 1H, J 13.0 Hz, J 7.5 Hz, CH₃CHHS, A), 2.71 (dq, 1H, J 13.0 Hz, J 7.2 Hz, CH₃CHHS, B), 2.76 (dq, 1H, J 13.0 Hz, J 7.3 Hz, CH₃CHHS, A), 3.70 (dd, 1H, J 10.1 Hz, J 8.7 Hz, H-6a, B), 3.79–3.85 (m, 3H, H-4, A, H-5, A, H-6a, A), 3.87 (dd, 1H, J 11.2 Hz, J 1.9 Hz, H-6b, A), 3.93 (dp, 1H, J 10.6 Hz, J 5.8 Hz, H-5, B), 4.00 (dd, 1H, J 10.1 Hz, J 1.8 Hz, H-6b, B), 4.01–4.05 (m, 2H, H-3, B, H-4, B), 4.07 (t, 1H, J 2.3 Hz, H-3, A), 4.08 (dd, 1H, J 8.5 Hz, J 1.9 Hz, H-2, A), 4.14 (t, 1H, J 1.5 Hz, H-2, B), 5.12 (d, 1H, J 8.5 Hz, H-1, A), 5.19 (d, 1H, J 1.4 Hz, H-1, B); ¹³C NMR (151 MHz, CDCl₃): δ 11.4 (((CH₃)₂CH)₃Si, B), 11.5 (((CH₃)₂CH)₃Si, A), 12.1 (((CH₃)₂CH)₃Si, A), 12.6 (((CH₃)₂CH)₃Si, A), 13.0 (((CH₃)₂CH)₃Si, B), 13.1 (((CH₃)₂CH)₃Si, A), 13.7 (((CH₃)₂CH)₃Si, B), 14.1 (((CH₃)₂CH)₃Si, B), 14.4 (CH₃CH₂S, B), 14.8 (CH₃CH₂S, A), 17.66 (((CH₃)₂CH)₃Si, B), 17.74 (((CH₃)₂CH)₃Si, B), 17.78 (((CH₃)₂CH)₃Si, A), 17.83 (((CH₃)₂CH)₃Si, A), 18.01 (((CH₃)₂CH)₃Si, A), 18.05 (((CH₃)₂CH)₃Si, A), 18.17 (((CH₃)₂CH)₃Si, B), 18.23 (((CH₃)₂CH)₃Si, A), 18.25 (((CH₃)₂CH)₃Si, B), 18.30 (((CH₃)₂CH)₃Si, A), 18.32 (((CH₃)₂CH)₃Si, A&, B), 18.4 (((CH₃)₂CH)₃Si, A&, B), 18.5 (((CH₃)₂CH)₃Si, B), 18.6 (((CH₃)₂CH)₃Si, B), 23.8 (CH₃CH₂S, B), 24.3 (CH₃CH₂S, A), 63.1 (C-6, A), 63.7 (C-6, B), 70.0 (C-4, B), 71.2 (C-2, A), 72.4 (C-4, A), 75.0 (C-3, B), 75.3 (C-2, B), 76.0 (C-5, B), 76.50 (C-3, A), 78.54 (C-5, A), 80.8 (C-1, A), 82.9 (C-1, B); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₄₄H₁₀₀NO₅SSi₄⁺ 866.6394; Found: 866.6389.

Data for ethyl 2,3,6-tris-O-(triisopropylsilyl)-1-thio-α-d-mannopyranoside (11): 87 mg (0.125 mmol, 26%), R_f = 0.44 (petroleum ether–CH₂Cl₂, 2:1) [α]_D²⁰ +48.6 (c 1.00, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 1.05–1.18 (m, 63H, 3 × ((CH₃)₂CH)₃Si), 1.28 (t, 4H, J 7.4 Hz, SCH₂CH₃), 2.49 (d, 1H, J 2.0 Hz, OH-4), 2.57 (dq, 1H, J 13.0 Hz, J 7.5 Hz, SCH₂CH₃), 2.63–2.71 (m, 1H, SCH₂CH₃), 3.90–3.99 (m, 5H, H-3, H-4, H-5, H-6a, H-6b), 4.17 (dd, 1H, J 2.6 Hz, J 1.7 Hz, H-2), 5.17 (d, 1H, J 1.7 Hz, H-1); ¹³C NMR (151 MHz, CDCl₃): δ 11.7 (((CH₃)₂CH)₃Si), 12.9 ((CH₃)₂CH)₃Si), 13.0 ((CH₃)₂CH)₃Si), 15.0 (SCH₂CH₃), 17.90 ((CH₃)₂CH)₃Si), 17.92 ((CH₃)₂CH)₃Si), 18.17 ((CH₃)₂CH)₃Si), 18.21 ((CH₃)₂CH)₃Si), 18.3 ((CH₃)₂CH)₃Si), 25.0 (SCH₂CH₃), 65.1 (C-6), 67.0 (C-4), 73.2 (C-5), 74.3 (C-3), 74.6 (C-2), 85.4 (C-1); HRMS (ESI): m/z [M+Na]⁺ Calcd for C₃₅H₇₆NaO₅SSi₃⁺ 715.4613; Found: 715.4611.

Data for ethyl 2,4,6-tris-O-(triisopropylsilyl)-1-thio-α-d-mannopyranoside (12): 18 mg (0.026 mmol, 6%), R_f = 0.33 (petroleum ether–CH₂Cl₂, 2:1). [α]_D²⁰ + 80.73 (c 1.01, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 1.03–1.22 (m, 63H, 3 × ((CH₃)₂CH)₃Si), 1.27 (t, 4H, J 7.4 Hz, SCH₂CH₃), 2.21 (d, 1H, J 10.5 Hz, OH-3), 2.54 (dq, 1H, J 12.9 Hz, J 7.5 Hz, SCH₂CH₃), 2.69 (dq, 1H, J 12.9 Hz, J 7.3 Hz, -SCH₂CH₃), 3.68 (ddd, 1H, J 10.5 Hz, J 8.5 Hz, J 3.5 Hz, H-3), 3.81–3.86 (m, 2H, H-4, H-6b), 3.91 (ddd, 1H, J 8.7 Hz, J 6.4 Hz, J 1.9 Hz, H-5), 4.05 (dd, 1H, J 10.5 Hz, J 1.9 Hz, H-6a), 4.15 (dd, 1H, J 3.5 Hz, J 1.5 Hz, H-2), 5.27 (d, 1H, J 1.5 Hz, H-1); ¹³C NMR (151 MHz, CDCl₃): δ 11.9 ((CH₃)₂CH)₃Si), 12.6 ((CH₃)₂CH)₃Si), 13.1 (((CH₃)₂CH)₃Si), 14.5 (-SCH₂CH₃), 17.92 ((CH₃)₂CH)₃Si), 17.97 ((CH₃)₂CH)₃Si), 18.04 ((CH₃)₂CH)₃Si), 18.4 ((CH₃)₂CH)₃Si), 24.0 (-SCH₂CH₃), 63.4 (C-6), 70.9 (C-4), 73.4 (C-3), 74.1 (C-2), 74.5 (C-5), 83.1 (C-1); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₃₅H₈₁NO₅SSi₃⁺ 710.5060; Found: 710.5060.

3.5. Ethyl 2,3,4,6-Tetrakis-O-(Triisopropylsilyl)-1-thio-β-d-mannopyranoside (13)

i-Pr₃SiOTf (0.720 mL, 2.68 mmol) was added to the solution of ethyl 1-thio-β-d-mannopyranoside (9) (100 mg, 0.445 mmol) in 2,4,6-collidine (1.5 mL) at 20 °C. The reaction mixture was stirred at 90 °C for 45 h and then allowed to reach 20 °C, diluted with CH₂Cl₂ (40 mL), washed with H₂O (40 mL), 1 M KHSO₄ (3 × 40 mL), H₂O (40 mL), and satd aq NaHCO₃ (3 × 40 mL). Organic extracts were dried with Na₂SO₄, filtered, concentrated under reduced pressure and purified by silica gel chromatography (gradient: 0 → 5% CH₂Cl₂ in petroleum ether) to give silylated derivative 13 (317.5 mg, 0.374 mmol, 84%), R_f = 0.49 (petroleum ether–CH₂Cl₂, 4:1). [α]_D²⁰ −55.2 (c 1.01, CHCl₃); ¹H NMR (600 MHz, CDCl₃, 298 K): δ 1.03–1.21 (m, 84H, 4 × ((CH₃)₂CH)₃Si), 1.26 (t, 3H, J 7.4 Hz, CH₃CH₂S), 2.59 (dq, 1H, J 12.5 Hz, J 7.4 Hz, CH₃CHHS), 2.63 (dq, 1H, J 12.6 Hz, J 7.5 Hz, CH₃CHHS), 3.83 (dd, 1H, J 8.6 Hz, J 4.8 Hz, H-5), 4.16 (t, 1H, J 3.2 Hz, H-3), 4.18 (dd, 1H, J 10.4 Hz, J 4.8 Hz, H-6a), 4.26 (d, 1H, J 3.7 Hz, H-4), 4.59 (dd, 1H, J 5.7 Hz, J 2.6 Hz, H-2), 4.61 (dd, 1H, J 10.4 Hz, J 8.6 Hz, H-6b), 5.14 (d, 1H, J 5.7 Hz, H-1); ¹³C NMR (151 MHz, CDCl₃): δ 12.1 (((CH₃)₂CH)₃Si), 12.5 (((CH₃)₂CH)₃Si), 12.80 (((CH₃)₂CH)₃Si), 12.81 (((CH₃)₂CH)₃Si), 15.1 (CH₃CH₂S), 18.01 (((CH₃)₂CH)₃Si), 18.02 (((CH₃)₂CH)₃Si), 18.13 (((CH₃)₂CH)₃Si), 18.15 (((CH₃)₂CH)₃Si), 18.23 (((CH₃)₂CH)₃Si), 18.3 (((CH₃)₂CH)₃Si), 18.5 (((CH₃)₂CH)₃Si), 18.6 (((CH₃)₂CH)₃Si), 26.7 (CH₃CH₂S), 65.0 (C-6), 68.0 (C-2), 72.1 (C-4), 74.3 (C-3), 81.1 (C-5), 84.7 (C-1);

¹H NMR (600 MHz, CDCl₃, 240 K): δ 0.99–1.13 (m, 84H, 4 × ((CH₃)₂CH)₃Si), 1.24 (t, 3H, J 7.4 Hz, CH₃CH₂S), 2.56 (dq, 1H, J 12.7 Hz, J 7.4 Hz, CH₃CHHS), 2.59 (dq, 1H, J 12.7 Hz, J 7.5 Hz, CH₃CHHS), 3.79 (dd, 1H, J 9.1 Hz, J 4.6 Hz, H-5), 4.05 (dd, 1H, J 10.3 Hz, J 4.6 Hz, H-6a), 4.10 (t, 1H, J 3.0 Hz, H-3), 4.22 (d, 1H, J 3.8 Hz, H-4), 4.54 (dd, 1H, J 5.7 Hz, J 2.4 Hz, H-2), 4.63 (dd, 1H, J 10.3 Hz, J 9.2 Hz, H-6b), 5.11 (d, 1H, J 5.7 Hz, H-1); ¹³C NMR (151 MHz, CDCl₃): δ 11.6 (((CH₃)₂CH)₃Si), 12.0 (((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 12.5 (((CH₃)₂CH)₃Si), 15.3 (CH₃CH₂S), 17.87 (2 × ((CH₃)₂CH)₃Si), 17.94 (((CH₃)₂CH)₃Si), 18.00 (((CH₃)₂CH)₃Si), 18.1 (((CH₃)₂CH)₃Si), 18.2 (((CH₃)₂CH)₃Si), 18.3 (((CH₃)₂CH)₃Si), 18.4 (((CH₃)₂CH)₃Si), 26.7 (CH₃CH₂S), 64.4 (C-6), 67.4 (C-2), 71.3 (C-4), 73.8 (C-3), 80.7 (C-5), 84.3 (C-1); HRMS (ESI): m/z [M+Na]⁺ Calcd for C₄₄H₉₆NaO₅SSi₄⁺ 871.5948; Found: 871.5949.

3.6. Synthesis of 1,2-α-Linked Methyl and Phenyl Mannopyranosides

3.6.1. General Procedure for the Synthesis of 1,2-α-Linked Methyl and Phenyl Mannopyranosides 15, 18 with NIS/TfOH

A mixture of silylated glycosyl donor (2 or 13) and glycosyl acceptor (14 or 17) was dried in vacuo for 2 h, then anhydrous CH₂Cl₂ (2 mL) was added under argon. Freshly activated powdered MS 4 Å (200 mg) were added under argon to the resulting solution. The suspension was stirred under argon at ~22 °C for 1 h, then cooled to −60 °C. Solid NIS (17–22 mg, 1 eq. of glycosyl donor’s moles) was added followed by TfOH (1 μL, 0.012 mmol). Then the temperature was allowed to rise slowly until appearance of persistent characteristic iodine color at −50 °C (for 2 + 14 or 13 + 14) or −30 °C (for 13 + 17) and was kept at that temperature for 2 h. Then the reaction was quenched by addition of Py (20 μL). The reaction mixture was diluted with CH₂Cl₂ (20 mL) and filtered through a Celite pad. The solids were washed with CH₂Cl₂ (20 mL) and the filtrate was successively washed with a mixture of satd aq Na₂S₂O₃ (40 mL) and satd aq NaHCO₃ (40 mL). The aqueous layer was extracted with CH₂Cl₂ (2×5 mL). Combined organic extracts were filtered through a cotton wool plug, concentrated and dried in vacuo. The residue was then dissolved in toluene (2 mL) and subjected to gel chromatography on Bio-Beads S-X3 in toluene. The fractions eluted just after the void volume were collected, concentrated under reduced pressure.

3.6.2. Methyl 2-O-[2,3,4,6-Tetrakis-O-(Triisopropylsilyl)-α-d-mannopyranosyl]-3,4,6-tri-O-Benzoyl-α-d-mannopyranoside (15)

A mixture of silylated α-d-mannopyranoside 2 (88 mg, 0.1 mmol) and methyl mannopyranoside 14 (40 mg, 0.08 mmol) was treated according to General procedure. Concentrated fractions were additionally purified by silica gel chromatography in petroleum ether–CH₂Cl₂ (gradient: 0 → 35% CH₂Cl₂ in petroleum ether) to give α-linked methyl dimannopyranoside 15 as a mixture of conformers (59 mg, 0.046 mmol, 58%); R_f = 0.66 (petroleum ether–EtOAc, 6:1). [α]_D²⁰ +7.3 (c 1.14, CHCl₃);¹H NMR (600 MHz, CDCl_3, 298 K, A:B = 10:3): δ 0.80–1.34 (m, 84H, 4 × ((CH₃)₂CH)₃Si), 3.45 (s, 3H, OCH₃ A), 3.48 (s, 3H, OCH₃ B), 3.67–3.70 (m, 1H, H-5^II B), 3.70 (ddd, 1H, J 7.8 Hz, J 4.6 Hz, J 3.2 Hz, H-5^II A), 3.72–3.76 (m, 1H, H-6^IIa B), 3.76 (dd, 1H, J 4.6 Hz, J 2.3 Hz, H-4^II A), 3.80 (dd, 1H, J 10.7 Hz, J 3.3 Hz, H-6^IIa A), 3.85 (dd, 1H, J 10.7 Hz, J 8.0 Hz, H-6^IIb A), 4.06 (d, 1H, J 10.1 Hz, H-6^IIb B), 4.06 (t, 1H, J 2.2 Hz, H-3^II A), 4.09 (t, 1H, J 9.3 Hz, H-4^II B), 4.16 (dd, 1H, J 6.5 Hz, J 2.0 Hz, H-2^II A), 4.21 (t, 1H, J 1.9 Hz, H-2^II B), 4.30 (dt, 1H, J 9.8 Hz, J 5.6 Hz, H-5^I A), 4.27–4.34 (m, 2H, H-5^I B, H-3^II B), 4.35 (dd, 1H, J 3.2 Hz, J 1.9 Hz, H-2^I B), 4.43 (dd, 1H, J 11.6 Hz, J 7.5 Hz, H-6^Ia B), 4.51 (d, 2H, J 5.6 Hz, H-6^Ib A, H-6^Ib A), 4.55 (dd, 1H, J 11.7 Hz, J 3.4 Hz, H-6^Ib B), 4.66 (dd, 1H, J 3.3 Hz, J 1.9 Hz, H-2^I A), 4.76 (d, 1H, J 1.8 Hz, H-1^II B), 4.88 (d, 1H, J 1.8 Hz, H-1^I A), 5.05 (d, 1H, J 6.5 Hz, H-1^II A), 5.07 (d, 1H, J 2.0 Hz, H-1^I B), 5.63 (dd, 1H, J 10.0 Hz, J 3.3 Hz, H-3^I B), 5.71 (t, 1H, J 9.9 Hz, H-4^I B), 5.75 (t, 1H, J 9.9 Hz, H-4^I A), 5.81 (dd, 1H, J 10.1 Hz, J 3.4 Hz, H-3^I A), 7.24–7.38 (m, 6H, C3 × PhCO (H-3, H-5)), 7.41–7.54 (m, 3H, 3 × PhCO (H-4)), 7.86–7.99 (m, 6H, 3 × PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 12.0 (((CH₃)₂CH)₃Si A&,B), 12.6 (((CH₃)₂CH)₃Si A), 12.81 (((CH₃)₂CH)₃Si A), 12.84 (((CH₃)₂CH)₃Si B), 12.9 (((CH₃)₂CH)₃Si A), 14.1 (((CH₃)₂CH)₃Si B), 14.3 (((CH₃)₂CH)₃Si, B), 17.90 (((CH₃)₂CH)₃Si B), 17.92 (((CH₃)₂CH)₃Si A&,B), 17.94 (((CH₃)₂CH)₃Si A), 18.0 (((CH₃)₂CH)₃Si B), 18.10 (((CH₃)₂CH)₃Si, A), 18.13 (((CH₃)₂CH)₃Si, B), 18.16 (((CH₃)₂CH)₃Si A), 18.19 (((CH₃)₂CH)₃Si A), 18.22 (((CH₃)₂CH)₃Si A), 18.4 (2 × ((CH₃)₂CH)₃Si A), 18.48 (((CH₃)₂CH)₃Si, B), 18.50 (((CH₃)₂CH)₃Si B), 18.7 (((CH₃)₂CH)₃Si B), 18.9 (((CH₃)₂CH)₃Si B), 54.75 (OCH₃ B), 54.81 (OCH₃, A), 64.0 (C-6^II A), 64.7 (C-6^II B), 65.0 (C-6^I B), 65.2 (C-6^I A), 68.1 (C-4^I B), 68.5 (C-5^I B), 68.8 (C-4^I A), 68.9 (C-5^I A), 70.1 (C-4^II B), 71.1 (C-2^I A), 72.0 (C-3^I A), 72.3 (C-3^I B), 72.6 (C-2^II A), 73.2 (C-4^II A), 73.8 (C-3^II B), 74.4 (C-2^II B), 75.4 (C-2^I B), 77.5 (C-3^II A), 77.7 (C-5^II B), 78.4 (C-5^II A), 99.6 (C-1^I B), 99.9 (C-1^II A), 101.4 (C-1^I A), 102.4 (C-1^II B), 128.1 (PhCO (C-3, C-5), A), 128.18 (PhCO (C-3, C-5), A), 128.22 (PhCO (C-3, C-5) A&,B), 128.3 (2 × PhCO (C-3, C-5) B), 129.4 (PhCO (C-1)), 129.5 (PhCO), 129.6 (PhCO (C-2, C-6) A&B), 129.7 (PhCO (C-2, C-6) B), 129.77 (PhCO (C-2, C-6)A&B), 129.84 (PhCO (C-2, C-6) A), 132.7 (PhCO (C-4) A&B), 132.8 (PhCO (C-4) A), 132.9 (PhCO (C-4) B), 133.0 (PhCO (C-4) A), 133.1 (PhCO (C-4) B), 165.4 (4^I-O-PhCO A), 166.0 (3^I-O-PhCO A), 166.1 (6^I-O-PhCO A); ²⁹Si INEPT NMR (60 MHz, CDCl₃): δ 11.3 (TIPS A), 12.8 (TIPS A), 13.6 (TIPS A), 15.5 (TIPS A);

¹H NMR (600 MHz, CDCl₃, 240 K, A:B = 10:7.6): δ 0.62–1.35 (m, 168H, 3 × ((CH₃)₂CH)₃Si, A, 3 × ((CH₃)₂CH)₃Si, B), 3.46 (s, 3H, OCH₃, A), 3.49 (s, 3H, OCH₃, B), 3.65–3.70 (m, 3H, H-6^IIa, B, H-5^II, A, H-5^II, B), 3.71 (dd, 1H, J 4.8 Hz, J 2.2 Hz, H-4^II, A), 3.74 (dd, 1H, J 10.9 Hz, J 3.6 Hz, H-6^IIa, A), 3.81 (dd, 1H, J 10.7 Hz, J 8.1 Hz, H-6^IIb, A), 3.95–4.05 (m, 3H, H-6^IIb,B, H-3^II, A, H-4^II, B), 4.12 (dd, 1H, J 6.8 Hz, J 1.8 Hz, H-2^II, A), 4.14 (d, 1H, J 2.3 Hz, H-2^II, B), 4.23 (dd, 1H, J 9.3 Hz, J 1.8 Hz, H-3^II, B), 4.27–4.40 (m, 4H, H-5^I, A, H-5^I, B, H-2^I, B, H-6^Ia,B), 4.46 (dd, 2H, J 11.7 Hz, J 7.8 Hz, H-6^Ia, A), 4.48 (dd, 1H, J 11.9 Hz, J 3.7 Hz, H-6^Ib, A), 4.54 (dd, 1H, J 11.2 Hz, J 2.3 Hz, H-6^Ib, B), 4.58 (dd, 1H, J 3.2 Hz, J 1.9 Hz, H-2^I, A), 4.68 (d, 1H, J 1.2 Hz, H-1^II, B), 4.92 (s, 1H, H-1^I, A), 4.98 (d, 1H, J 6.6 Hz, H-1^II, A), 5.08 (s, 1H, H-1^I, B), 5.59 (dd, 1H, J 10.1 Hz, J 3.4 Hz, H-3^I, B), 5.69 (t, 1H, J 9.7 Hz, H-4^I, B), 5.72 (t, 1H, J 10.1 Hz, H-4^I, A), 5.83 (dd, 1H, J 10.1 Hz, J 3.5 Hz, H-3^I, A), 7.28–7.42 (m, 14H, 3 × PhCO (H-3, H-5), A, 3 × PhCO (H-3, H-5) B), 7.44–7.58 (m, 6H, 3 × PhCO (H-4) A, 3 × PhCO (H-4) B), 7.88–8.02 (m, 12H, 3 × PhCO (H-2, H-6) A, 3 × PhCO (H-2, H-6) B); ¹³C NMR (151 MHz, CDCl₃): δ 11.48 (((CH₃)₂CH)₃Si, B), 11.52 (((CH₃)₂CH)₃Si, A), 12.2 (((CH₃)₂CH)₃Si, A), 12.37 (((CH₃)₂CH)₃Si, B), 12.41 (((CH₃)₂CH)₃Si, A), 12.6 (((CH₃)₂CH)₃Si, A), 13.95 (((CH₃)₂CH)₃Si, B), 14.04 (((CH₃)₂CH)₃Si, B), 17.7 (((CH₃)₂CH)₃Si, B), 17.6 (((CH₃)₂CH)₃Si,B), 17.78 (((CH₃)₂CH)₃Si, A, ((CH₃)₂CH)₃Si, B), 17.81 (((CH₃)₂CH)₃Si, A), 17.91 (((CH₃)₂CH)₃Si, B), 17.98 (((CH₃)₂CH)₃Si, A), 18.02 (2 × ((CH₃)₂CH)₃Si, A), 18.1 (((CH₃)₂CH)₃Si, A), 18.3 (2 × ((CH₃)₂CH)₃Si, A), 18.36 (2 × ((CH₃)₂CH)₃Si, B), 18.44 (((CH₃)₂CH)₃Si, B), 18.7 (((CH₃)₂CH)₃Si,B), 54.7 (OCH₃, B), 54.8 (OCH₃, A), 63.2 (C-6^II, A), 64.2 (C-6^II, B), 64.8 (C-6^I, B), 64.9 (C-6^I, A), 67.1 (C-4^I, B), 68.0 (2C, C-4^I, A, C-5^I, B), 68.5 (C-5^I, A), 69.5 (C-4^II, B), 71.2 (C-2^I, A), 71.3 (C-3^I, A), 72.0 (C-2^II, A), 72.1 (C-3^I, B), 72.6 (C-4^II, A), 73.5 (C-3^II, B), 73.7 (C-2^II, B), 74.8 (C-2^I, B), 77.1 (C-5^II, B), 77.2 (C-3^II, A), 78.0 (C-5^II, A), 99.2 (C-1^I, B), 99.7 (C-1^II, A), 100.9 (C-1^I, A), 102.2 (C-1^II, B), 128.1 (PhCO (C-3, C-5)), 128.2 (PhCO (C-3, C-5)), 128.25 (2 × PhCO (C-3, C-5)), 128.30 (PhCO (C-3, C-5)), 128.33 (PhCO (C-3, C-5)), 128.7 (PhCO (C-1)), 128.8 (2 × PhCO (C-1)), 128.9 (PhCO (C-1)), 129.07 (PhCO (C-1)), 129.14 (PhCO (C-1)), 129.5 (2 × PhCO (C-2, C-6)), 129.56 (PhCO (C-2, C-6)), 129.58 (PhCO (C-2, C-6)), 129.65 (PhCO (C-2, C-6)), 129.73 (PhCO (C-2, C-6)), 132.9 (PhCO (C-4)), 133.0 (PhCO (C-4)), 133.1 (2 × PhCO (C-4)), 133.3 (2 × PhCO (C-4)), 165.2 (PhCO), 165.3 (PhCO), 165.9 (2 × PhCO), 166.18 (PhCO), 166.24 (PhCO); ²⁹Si INEPT NMR (119 MHz, CDCl₃): δ 11.6 (TIPS A), 11.7 (TIPS B), 12.3 (TIPS B), 12.8 (TIPS B), 13.38 (TIPS A), 13.44 (TIPS B), 14.0 (TIPS A), 15.6 (TIPS A); HRMS (ESI): m/z [M+Na]⁺ Calcd for C₇₀H₁₁₆NaO₁₄Si₄⁺ 1315.7334; Found: 1315.7330.

3.6.3. Methyl 2-O-(α-d-Mannopyranosyl)-3,4,6-tri-O-benzoyl-α-d-mannopyranoside (16)

Protected methyl dimannopyranoside 15 (57 mg, 0.04 mmol) was dissolved in THF (2 mL), then AcOH (20 µL, 0.35 mmol) and 1 M TBAF in THF (700 µL, 0.7 mmol) were added. The reaction mixture was stirred at 40 °C for 2.5 h, then concentrated under reduced pressure, co-evaporated with toluene (5 × 5 mL) and dried in vacuo. The residue was purified by silica gel chromatography CH₂Cl₂–MeOH (gradient: 0 → 10% MeOH in CH₂Cl₂) to give tetraol isolated in a mixture with Bu₄N. The obtained mixture was subjected to the gel chromatography on Bio-Beads S-X3 in toluene. The fractions eluted just after the void volume were collected, concentrated under reduced pressure to give α-linked methyl dimannopyranoside 16 (21 mg, 0.031 mmol, 71%). R_f = 0.55 (CH₂Cl₂–MeOH, 10:1). [α]_D²⁵ +18.9 (c 1.00, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 3.45 (s, 3H, CH₃O), 3.65–3.74 (m, 2H, H-6^IIa, H-5^II), 3.87 (d, 1H, J 11.3 Hz, H-6^IIb), 3.92–3.97 (m, 2H, H-3^II, H-4^II), 3.97–4.02 (m, 1H, HO), 4.04 (s, 1H, H-2^II), 4.24 (dd, 1H, J 3.2 Hz, J 1.8 Hz, H-2^I), 4.29 (ddd, 1H, J 9.8 Hz, J 5.1 Hz, J 3.5 Hz, H-5^I), 4.35 (s, 1H, HO), 4.42 (s, 1H, HO), 4.50 (dd, 1H, J 12.0 Hz, J 5.2 Hz, H-6^Ia), 4.52 (s, 1H, 2^II-HO), 4.53 (dd, 1H, J 12.1 Hz, J 3.4 Hz, H-6^Ib), 4.91 (d, 1H, J 1.9 Hz, H-1^I), 4.97 (d, 1H, J 1.5 Hz, H-1^II), 5.72 (dd, 1H, J 10.0 Hz, J 3.1 Hz, H-3^I), 5.81 (t, 1H, J 10.0 Hz, H-4^I), 7.28–7.35 (m, 4H, 3^I-O-PhCO (H-3, H-5), 4^I-O-PhCO (H-3, H-5)), 7.35–7.40 (m, 2H, 6^I-O-PhCO (H-3, H-5)), 7.40–7.45 (m, 1H, 3^I- or 4^I-O-PhCO (H-4)), 7.45–7.52 (m, 2H, 4^I- or 3^I-O-PhCO (H-4), 6^I-O-PhCO (H-4)), 7.90 (s, 4H, 3^I-O-PhCO (H-2, H-6), 4^I-O-PhCO (H-2, H-6)), 7.96–8.03 (m, 2H, 6^I-O-PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 55.3 (CH₃O), 61.0 (C-6^II), 63.5 (C-6^I), 66.4 (C-4^II), 67.5 (C-4^I), 68.7 (C-5^I), 70.9 (C-2^II), 71.4 (C-3^I), 71.5 (C-3^II), 73.0 (C-5^II), 76.5 (C-2^I), 99.7 (C-1^I), 102.1 (C-1^II), 128.35 (PhCO (C-3, C-5)), 128.43 (PhCO (C-3, C-5)), 128.5 (PhCO (C-3, C-5)), 129.0 (PhCO (C-1)), 129.1 (PhCO (C-1)), 129.6 (PhCO (C-2, C-6)), 129.67 (PhCO (C-2, C-6)), 129.69 (PhCO (C-1)), 129.8 (PhCO (C-2, C-6)), 133.1 (6^I-O-PhCO (C-4))), 133.29 (3^I-O-PhCO (C-4)), 4^I-O-PhCO (C-4))), 133.34 (3^I-O-PhCO (C-4)), 4^I-O-PhCO (C-4))), 165.55 (3^I-O-PhCO, 4^I-O-PhCO), 165.61 (3^I-O-PhCO, 4^I-O-PhCO), 166.2 (6^I-O-PhCO); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₃₄H₄₀NO₁₄⁺ 686.2443; Found: 686.2438.

3.6.4. Phenyl 2-O-[2,3,4,6-Tetrakis-O-(Triisopropylsilyl)-α-d-mannopyranosyl]-3,4,6-tri-O-benzoyl-1-thio-α-d-mannopyranoside (18)

(a) A mixture of silylated phenyl 1-thio-α-d-mannopyranoside 2 (77 mg, 0.09 mmol) and alcohol 17 [42] (39 mg, 0.07 mmol) was treated according to General procedure to give unseparable mixture (98 mg) of disaccharide 18 (85% purity according to NMR data) with N-glycoside 19 in 2.5:1 ratio according to NMR data (HRMS (ESI): m/z [M+Na]⁺ Calcd for C₄₆H₉₅NNaO₇Si₄⁺ 908.6078; Found: 908.6068)).

(b) A mixture of ethyl 1-thio-α-d-mannopyranoside 10 (49 mg, 0.06 mmol), TTBP (31 mg, 0.13 mmol), BSP (14 mg, 0.07 mmol) was dried in vacuo for 2 h, then anhydrous CH₂Cl₂ (1 mL) was added under argon and freshly activated powdered MS 4 Å (200 mg) were added under argon to the resulting solution. The suspension was stirred under argon at ~22 °C for 1 h, then cooled to −78 °C. After 10 min Tf₂O (15 μL) was added. Then the temperature was allowed to rise slowly until −60 °C and the solution of phenyl mannopyranoside 17 [42] (40 mg, 0.07 mmol) (previously dried in vacuo for 2 h) in anhydrous CH₂Cl₂ (1 mL) was added. Then the temperature was allowed to rise slowly to −50 °C during 0.5 h and was kept at −50 °C for 1 h. Then the reaction was quenched by addition of satd aq NaHCO₃ (20 µL). The reaction mixture was diluted with CH₂Cl₂ (15 mL) and filtered through a Celite pad. The solids were washed with CH₂Cl₂ (20 mL) and the filtrate was successively washed with satd aq NaHCO₃ (50 mL). The aqueous layer was extracted with CH₂Cl₂ (2 × 5 mL). Combined organic extracts were filtered through a cotton wool plug, concentrated and dried in vacuo. The residue was dried in vacuo, then dissolved in toluene (2 mL) and subjected to gel chromatography on Bio-Beads S-X3 in toluene. The fractions eluted just after the void volume were collected, concentrated under reduced pressure and additionally purified by silica gel chromatography in petroleum ether–CH₂Cl₂ (gradient: 0 → 11% CH₂Cl₂ in petroleum ether) to give of α-linked phenyl dimannopyranoside 18 (41 mg, 52% according to NMR) isolated as an inseparable mixture with piperidin-1-yl glycoside 20 (16 mol% according to NMR); HRMS (ESI): m/z [M+H]⁺ Calcd for C₄₇H₁₁₁NO₅Si₄⁺ 872.6830; Found: 872.6827)).

(c) A mixture of phenyl 1-thio-β-d-mannopyranoside 13 (49 mg, 0.08 mmol) and alcohol 17 [42] (63 mg, 0.07mmol) was treated according to General procedure. Residue obtained after gel chromatography was additionally purified by silica gel chromatography in petroleum ether–EtOAc (gradient: 0 → 8% EtOAc in petroleum ether) to give α-linked phenyl dimannopyranoside 18 (79.3 mg, 0.058 mmol, 86%); R_f = 0.66 (petroleum ether–EtOAc, 10:1); [α]_D²⁰ +56.3 (c 1.01, CHCl₃); ¹H NMR (600 MHz, CDCl₃, 303 K): δ 0.88–1.35 (m, 84H, 3 × ((CH₃)₂CH)₃Si), 3.77 (dt, 1H, J 6.9 Hz, J 3.6 Hz, H-5^II), 3.80 (dd, 1H, J 10.7 Hz, J 4.0 Hz, H-6^IIb), 3.92 (dd, 1H, J 10.6 Hz, J 6.8 Hz, H-6^IIa), 3.94 (t, 1H, J 3.1 Hz, H-4^II), 4.11 (s, 1H, H-3^II), 4.21 (dd, 1H, J 6.8 Hz, J 1.9 Hz, H-2^II), 4.48 (dd, 1H, J 11.8 Hz, J 8.2 Hz, H-6^Ia), 4.54 (dd, 1H, J 11.8 Hz, J 3.1 Hz, H-6^Ib), 4.86 (d, 1H, J 3.4 Hz, H-2^I), 4.86–4.93 (m, 1H, H-5^I), 5.05 (d, 1H, J 6.8 Hz, H-1^II), 5.73 (dd, 1H, J 10.1 Hz, J 3.4 Hz, H-3^I), 5.76 (s, 1H, H-1^I), 5.80 (t, 1H, J 9.9 Hz, H-4^I), 7.18 (dd, 3H, J 12.6 Hz, J 7.1 Hz, PhS (H-3, H-4, H-5)), 7.28–7.38 (m, 6H, 3 × PhCO (H-3, H-5)), 7.44–7.57 (m, 5H, 3 × PhCO (H-4), PhS (H-2, H-6)), 7.86–7.90 (m, 2H, PhCO (H-2, H-6)), 7.90–7.94 (m, 3H, PhCO (H-2, H-6)), 7.96–8.01 (m, 2H, PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 12.0 (((CH₃)₂CH)₃Si), 12.6 (((CH₃)₂CH)₃Si), 12.9 (((CH₃)₂CH)₃Si), 13.0 (((CH₃)₂CH)₃Si), 18.02 (((CH₃)₂CH)₃Si), 18.03 (((CH₃)₂CH)₃Si), 18.1 (((CH₃)₂CH)₃Si), 18.15 (((CH₃)₂CH)₃Si), 18.23 (((CH₃)₂CH)₃Si), 18.3 (((CH₃)₂CH)₃Si), 18.5 (2 × ((CH₃)₂CH)₃Si), 63.2 (C-6^II), 64.7 (C-6^I), 68.6 (C-4^I), 70.0 (C-5^I), 72.1 (C-3^I), 72.3 (C-2^II), 72.8 (C-4^II), 73.6 (C-2^I), 77.3 (C-3^II), 79.5 (C-5^II), 88.5 (C-1^I), 99.7 (C-1^II), 127.0 (PhS (C-4)), 128.1 (PhCO (C-3, C-5)), 128.2 (PhCO (C-3, C-5)), 128.3 (PhCO (C-3, C-5)), 128.9 (PhS (C-3, C-5)), 129.3 (PhCO (C-1)), 129.4 (PhCO (C-1)), 129.6 (PhCO (C-2, C-6)), 129.8 (PhCO (C-2, C-6)), 129.9 (PhCO (C-2, C-6)), 131.3 (PhS (C-2, C-6)), 132.8 (PhCO (C-4)), 132.9 (PhCO (C-4)), 133.1 (PhCO (C-4)), 134.3 (PhS (C-1)), 165.4 (PhCO), 166.0 (PhCO), 166.1 (PhCO); ²⁹Si INEPT NMR (119 MHz, CDCl₃): δ 12.0 (TIPS), 12.9 (TIPS), 13.9 (TIPS), 15.7 (2^II-O-TIPS); ¹H NMR (600 MHz, CDCl₃, 240 K, A:B = 10:3.4): δ 0.78–1.28 (m, 126H, 3 × ((CH₃)₂CH)₃Si), 3.63 (t, 1H, J 8.3 Hz, H-5^II B), 3.69–3.74 (m, 2H, H-6^IIa B, H-5^II A), 3.76 (dd, 1H, J 10.6 Hz, J 4.6 Hz, H-6^IIa A), 3.81–3.85 (m, 1H, H-6^IIb B), 3.86 (dd, 1H, J 10.8 Hz, J 6.5 Hz, H-6^IIb A), 3.93 (t, 1H, J 3.0 Hz, H-4^II A), 4.04 (t, 1H, J 2.2 Hz, H-3^II A), 4.07 (t, 1H, J 9.3 Hz, H-4^II B), 4.12–4.13 (m, 1H, H-2^II B), 4.15 (dd, 1H, J 7.1 Hz, J 1.9 Hz, H-2^II A), 4.23 (dd, 1H, J 9.4 Hz, J 1.7 Hz, H-3^II B), 4.33 (dd, 1H, J 11.7 Hz, J 8.8 Hz, H-6^Ia B), 4.43 (dd, 1H, J 11.8 Hz, J 8.9 Hz, H-6^Ia A), 4.51 (dd, 1H, J 11.9 Hz, J 2.5 Hz, H-6^Ib A), 4.55 (dd, 1H, J 11.9 Hz, J 2.3 Hz, H-6^Ib B), 4.61 (d, 1H, J 3.6 Hz, H-2^I B), 4.72 (s, 1H, H-1^II B), 4.73 (d, 1H, J 3.5 Hz, H-2^I A), 4.89–4.99 (m, 2H, H-5^I A, H-5^I B), 4.96 (d, 1H, J 7.0 Hz, H-1^II A), 5.55 (dd, 1H, J 10.1 Hz, J 3.3 Hz, H-3^I B), 5.70 (dd, 1H, J 10.3 Hz, J 3.5 Hz, 3^I A), 5.72–5.80 (m, 2H, 4^I B, 4^I A), 5.80 (s, 1H, H-1^I A), 5.91 (s, 1H, H-1^I B), 7.16 (t, 2H, J 7.6 Hz, PhS (H-3, H-5) A), 7.19–7.26 (m, 4H, PhS (H-3, H-5) B, PhS (H-4) A, PhS (H-4) B), 7.30–7.42 (m, 12H, 3 × PhCO (H-3, H-5) A, 3 × PhCO (H-3, H-5) B), 7.47–7.60 (m, 10H, 3 × PhCO (H-4) A, 3 × PhCO (H-4) B, PhS (H-2, H-6) A, PhS (H-2, H-6) B), 7.86–7.90 (m, 4H, PhCO (H-2, H-6) A, PhCO (H-2, H-6) B), 7.92 (d, 4H, J 7.8 Hz, PhCO (H-2, H-6) A, PhCO (H-2, H-6) B), 7.97–8.02 (m, 4H, PhCO (H-2, H-6) A, PhCO (H-2, H-6) B); ¹³C NMR (151 MHz, CDCl₃): δ 11.4 (((CH₃)₂CH)₃Si B), 11.5 (((CH₃)₂CH)₃Si A), 12.1 (((CH₃)₂CH)₃Si A), 12.4 (((CH₃)₂CH)₃Si B), 12.5 (((CH₃)₂CH)₃Si A), 12.7 (((CH₃)₂CH)₃Si A), 13.9 (((CH₃)₂CH)₃Si B), 14.1 (((CH₃)₂CH)₃Si B), 17.7 (((CH₃)₂CH)₃Si), 17.79 (((CH₃)₂CH)₃Si), 17.84 (((CH₃)₂CH)₃Si), 17.88 (((CH₃)₂CH)₃Si), 17.92 (((CH₃)₂CH)₃Si), 18.01 (((CH₃)₂CH)₃Si), 18.03 (((CH₃)₂CH)₃Si), 18.2 (((CH₃)₂CH)₃Si), 18.3 (((CH₃)₂CH)₃Si), 18.40 (((CH₃)₂CH)₃Si), 18.45 (((CH₃)₂CH)₃Si), 18.48 (((CH₃)₂CH)₃Si), 18.7 (((CH₃)₂CH)₃Si), 62.2 (C-6^II A), 63.5 (C-6^II B), 64.3 (C-6^I B), 64.4 (C-6^I B), 66.8 (C-4^I A), 67.7 (C-4^I A), 69.1 (2 C, C-4^II B, C-5^I B), 69.4 (C-5^I A), 71.4 (C-2^II A), 71.5 (C-3^I A), 71.8 (C-4^II A), 72.6 (C-3^I B), 73.5 (C-3^II B), 73.7 (C-2^II B), 74.2 (C-2^I A), 76.5 (C-2^I B), 77.0 (2 C, C-3^II A, C-5^II B), 79.1 (C-5^II A), 85.7 (C-1^I A), 88.2 (C-1^I A), 99.7 (C-1^II A), 102.9 (C-1^II B), 127.0 (PhS (C-4) B), 127.1 (PhS (C-4) A), 128.17 (PhCO (C-3, C-5)), 128.21 (PhCO (C-3, C-5)), 128.3 (PhCO), 128.36 (PhCO), 128.41 (PhCO), 128.5 (PhCO), 128.6 (PhCO), 128.9 (PhS (C-3, C-5) A), 129.0 (PhS (C-3, C-5) B), 129.1 (PhCO (C-1)), 129.58 (PhCO (C-2, C-6)), 129.66 (PhCO (C-2, C-6)), 129.71 (PhCO (C-2, C-6)), 129.8 (PhCO (C-2, C-6)), 130.3 (PhS (C-2, C-6) B), 131.2 (PhS (C-2, C-6) A), 133.06 (PhCO (C-4)), 133.11 (PhCO (C-4)), 133.3 (PhCO (C-4)), 133.38 (PhCO (C-4)), 133.43 (PhCO (C-4)), 133.5 (PhCO (C-4)), 133.7 (PhS (C-1) A), 165.1 (PhCO), 165.2 (PhCO), 165.9 (PhCO), 166.0 (PhCO), 166.2 (PhCO); ²⁹Si INEPT NMR (119 MHz, CDCl₃): δ 12.0, 12.2, 12.6, 12.9, 13.5, 14.0, 14.3, 15.8; HRMS (ESI): m/z [M+Na]⁺ Calcd for C₇₅H₁₁₈NaO₁₃SSi₄⁺ 1393.7262; Found: 1393.7262.

3.7. Synthesis of Mannose-Capped Trisaccharide of M. tuberculosis: 2-Chloroethyl 2,3-di-O-benzoyl-5-O-[3,4,6-tri-O-benzoyl-α-d-mannopyranosyl-2-O-{2,3,4,6-tetrakis-O-(Triisopropylsilyl)-α-d-mannopyranosyl}]-α-d-arabinofuranoside (22) and 2-Chloroethyl 2,3-di-O-benzoyl-5-O-(3,4,6-tri-O-benzoyl-α-d-mannopyranosyl)-α-d-arabinofuranoside (23)

(a) A mixture of 2-chloroethyl α-d-arabinofuranoside 21 [43] (14 mg, 0.033 mmol) and dimannothiopyranoside 18 (36 mg, 0.026 mmol) was dried in vacuo for 2 h, then dissolved in anhydrous CH₂Cl₂ (1.5 mL) under argon. Freshly activated powdered MS 4 Å (150 mg) were added under argon the resulting solution. The suspension was stirred under argon at ~22 °C for 1 h, then cooled to −10 °C. Solid NIS (6 mg, 0.03 mmol) was added followed by TfOH (1.4 µL, 0.017 mmol). Then the temperature was allowed to rise to 0 °C during 1 h and was kept at 0 °C for 16 h. Then the reaction was quenched by addition of Et₃N (15 μL). The reaction mixture was diluted with CH₂Cl₂ (20 mL) and filtered through a Celite pad. The solids were washed with CH₂Cl₂ (20 mL) and the filtrate was successively washed with a mixture of satd aq Na₂S₂O₃ (40 mL) and satd aq NaHCO₃ (40 mL). The aqueous layer was extracted with CH₂Cl₂ (2 × 5 mL). Combined organic extracts were filtered through a cotton wool plug, concentrated and dried in vacuo. The residue was dried in vacuo, then purified by silica gel chromatography in petroleum ether–EtOAc (gradient: 0 → 12% EtOAc in petroleum ether) to give α-linked trisaccharide 22 as a mixture of conformers (34 mg, 0.020 mmol, 78%). R_f = 0.45 (petroleum ether–EtOAc, 5:1), [α]_D²⁴ −1.3 (c 1.07, CHCl₃); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₉₀H₁₃₇NClO₂₀Si₄⁺ 1698.8494; Found: 1698.8512.

(b) A mixture of ethyl 1-thio-α-d-mannopyranoside 10 (47 mg, 0.055 mmol) and phenyl mannothiopyranoside 17 (39 mg, 0.067 mmol) was dried in vacuo for 2 h, then anhydrous CH₂Cl₂ (2 mL) was added under argon. Freshly activated powdered MS 4 Å (100 mg) were added under argon to the resulting solution and the suspension was stirred under argon at ~22 °C for 1 h, then cooled to −78 °C. Solid NIS (15 mg, 0.067 mmol) was added followed by TfOH (0.75 µL, 0.009 mmol). Then the temperature was allowed to rise to −40 °C during 1 h. After that, 2-chloroethyl α-d-arabinofuranoside 21 [43] (31 mg, 0.073 mmol) in anhydrous CH₂Cl₂ (0.6 mL), solid NIS (15.5 mg, 0.07 mmol) and TfOH (0.9 μL, 0.011 mmol) were added in this order to the reaction mixture. Then the temperature was allowed to rise to 0 °C during 1 h and was kept at 0 °C for 20 h. Since the reaction was not complete (according to TLC), an additional portion of arabinofuranoside 21 (16 mg, 0.038 mmol) in anhydrous CH₂Cl₂ (0.2 mL), solid NIS (9 mg, 0.04 mmol) and TfOH (0.75 µL, 0.009 mmol). were added. After additional 3 h at 0 °C the reaction was quenched by addition of Py (15 μL). The reaction mixture was diluted with CH₂Cl₂ (15 mL) and filtered through a Celite pad. The solids were washed with CH₂Cl₂ (20 mL) and the filtrate was successively washed with a mixture of satd aq Na₂S₂O₃ (20 mL) and satd aq NaHCO₃ (20 mL). The aqueous layer was extracted with CH₂Cl₂ (2 × 5 mL). Combined organic extracts were filtered through a cotton wool plug, concentrated and dried in vacuo. The residue was dried in vacuo, then dissolved in toluene (2 mL) and subjected to gel chromatography on Bio-Beads S-X3 in toluene. The fractions eluted just after the void volume were collected, concentrated under reduced pressure. The fist fraction was additionally purified by silica gel chromatography in petroleum ether–EtOAc (gradient: 5 → 15% EtOAc in petroleum ether) to give an α-linked trisaccharide 22 as a mixture of conformers (46 mg, 0.027 mmol, 49%) and a partially protected trisaccharide with three TIPS groups (4 mg, 0.0026 mmol, 5%, HRMS (ESI): m/z [M+Na]⁺ Calcd for C₈₁H₁₁₃ClNaO₂₀Si₃⁺ 1547.6714; Found: 1547.6706)). The second fraction (after gel chromatography on Bio-Beads S-X3) was also subjected to the silica gel chromatography in petroleum ether–EtOAc (gradient: 8 → 32% EtOAc in petroleum ether) to give α-linked benzoylated disacharide 23 (16 mg, 0.018 mmol, 27%).

• Data for conformer A:

¹H NMR (600 MHz, CDCl₃, 303 K, A:B = 10:3): δ 0.80–1.35 (m, 84H, 4 × ((CH₃)₂CH)₃Si), 3.70–3.77 (m, 3H, H-5^III, CH₂Cl), 3.80 (dd, 1H, J 10.8 Hz, J 3.5 Hz, H-6^IIIa), 3.82–3.89 (m, 3H, H-6^IIIb, H-4^III, CHHO), 3.93 (dd, 1H, J 10.6 Hz, J 2.4 Hz, H-5^Ia), 4.03–4.08 (m, 1H, CHHO), 4.08 (t, 1H, J 2.2 Hz, H-3^III), 4.16 (dd, 1H, J 10.6 Hz, J 7.0 Hz, H-5^Ib), 4.20 (dd, 1H, J 6.6 Hz, J 2.0 Hz, H-2^III), 4.47 (dd, 1H, J 12.4 Hz, J 8.3 Hz, H-6^IIa), 4.52–4.61 (m, 3H, H-5^II, H-6^IIb, H-4^I), 4.70 (dd, 1H, J 3.5 Hz, J 1.8 Hz, H-2^II), 5.06 (d, 1H, J 6.6 Hz, H-1^III), 5.09 (d, 1H, J 1.8 Hz, H-1^II), 5.14 (s, 1H, H-1^I), 5.51 (dd, 1H, J 6.4 Hz, J 2.4 Hz, H-3^I), 5.54 (d, 1H, J 2.5 Hz, H-2^I), 5.78 (t, 1H, J 9.8 Hz, H-4^II), 5.87 (dd, 1H, J 10.1 Hz, J 3.4 Hz, H-3^II), 7.16–7.23 (m, 2H, PhCO (H-3, H-5)), 7.27–7.33 (m, 5H, 2 × PhCO (H-3, H-5), PhCO (H-4)), 7.36–7.41 (m, 4H, 2 × PhCO (H-3, H-5)), 7.42–7.56 (m, 4H, 4 × PhCO (H-4)), 7.86–7.92 (m, 4H, 2 × PhCO (H-2, H-6)), 7.93–7.98 (m, 2H, PhCO (H-2, H-6)), 8.01–8.06 (m, 4H, 2 × PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 11.9 (((CH₃)₂CH)₃Si), 12.7 (((CH₃)₂CH)₃Si), 12.9 (((CH₃)₂CH)₃Si), 13.0 (((CH₃)₂CH)₃Si), 17.91 (((CH₃)₂CH)₃Si), 17.93 (((CH₃)₂CH)₃Si), 18.1 (((CH₃)₂CH)₃Si), 18.17 (((CH₃)₂CH)₃Si), 18.23 (((CH₃)₂CH)₃Si), 18.3 (((CH₃)₂CH)₃Si), 18.45 (((CH₃)₂CH)₃Si), 18.46 (((CH₃)₂CH)₃Si), 42.7 (CH₂Cl), 63.7 (C-6^III), 65.1 (C-6^II), 67.2 (C-5^I), 67.8 (CH₂O), 68.8 (C-4^II), 69.1 (C-5^II), 71.8 (C-2^II), 72.1 (C-3^II), 72.5 (C-2^III), 73.1 (C-4^III), 77.2 (C-3^I), 77.5 (C-3^III), 78.7 (C-5^III), 81.0 (C-4^I), 82.5 (C-2^I), 99.9 (C-1^III), 100.1 (C-1^II), 105.9 (C-1^I), 128.09 (PhCO (C-3, C-5)), 128.11 (PhCO (C-3, C-5)), 128.2 (PhCO (C-3, C-5)), 128.36 (PhCO (C-3, C-5)), 128.41 (PhCO (C-3, C-5)), 129.1 (PhCO (C-1)), 129.2 (PhCO (C-1)), 129.5 (PhCO (C-2, C-6)), 129.8 (PhCO (C-2, C-6)), 129.87 (PhCO (C-2, C-6)), 129.94 (PhCO (C-2, C-6)), 130.0 (PhCO (C-2, C-6)), 132.66 (PhCO (C-4)), 132.69 (PhCO (C-4)), 132.9 (PhCO (C-4)), 133.4 (2 × PhCO (C-4)), 165.47 (CO), 165.55 (CO), 165.58 (CO), 165.7 (CO), 166.0 (CO); ²⁹Si INEPT NMR (119 MHz, CDCl₃): δ 11.5, 12.8, 13.7, 15.6;

• Data for conformer B (selected signals):

¹H NMR (600 MHz, CDCl₃, 303 K, A:B = 10:3): δ 3.96 (dd, 1H, J 10.5 Hz, J 2.3 Hz, H-5^Ia), 4.10 (t, 1H, J 9.2 Hz, H-4^III), 4.22 (t, 1H, J 1.9 Hz, H-2^III), 4.32 (dd, 1H, J 9.1 Hz, J 1.9 Hz, H-3^III), 4.42 (dd, 1H, J 12.3 Hz, J 8.2 Hz, H-6^IIa), 4.79 (s, 1H, H-1^III), 5.18 (s, 1H, H-1^I), 5.27 (s, 1H, H-1^II), 5.56 (dd, 1H, J 6.2 Hz, J 2.5 Hz, H-3^I), 5.71 (dd, 1H, J 9.9 Hz, J 3.0 Hz, H-3^II), 5.75 (t, 1H, J 9.7 Hz, H-4^II); ¹³C NMR (151 MHz, CDCl₃): δ 11.9 (((CH₃)₂CH)₃Si), 12.9 (((CH₃)₂CH)₃Si), 14.1 (((CH₃)₂CH)₃Si), 14.3 (((CH₃)₂CH)₃Si), 18.1 (((CH₃)₂CH)₃Si), 18.2 (((CH₃)₂CH)₃Si), 18.50 (((CH₃)₂CH)₃Si), 18.52 (((CH₃)₂CH)₃Si), 18.7 (((CH₃)₂CH)₃Si), 18.9 (((CH₃)₂CH)₃Si), 42.7 (CH₂Cl), 64.7 (C-6^III), 65.0 (C-6^II), 66.9 (C-5^I), 67.8 (CH₂O), 68.1 (C-4^II), 68.8 (C-5^II), 70.2 (C-4^III), 72.4 (C-3^II), 73.8 (C-3^III), 74.5 (C-2^III), 75.6 (C-2^II), 77.0 (C-3^I), 77.7 (C-5^III), 80.8 (C-4^I), 82.5 (C-2^I), 98.5 (C-1^II), 102.5 (C-1^III), 105.9 (C-1^I);

¹H NMR (600 MHz, CDCl₃, 236 K, A:B = 10:8.6): δ 0.62–1.37 (m, 168H, 4 × ((CH₃)₂CH)₃Si A, 4 × ((CH₃)₂CH)₃Si B), 3.63–3.71 (m, 3H, H-6^IIIa B, H-5^III B, H-5^III A), 3.74 (dd, 1H, J 10.7 Hz, J 3.7 Hz, H-6^IIIa A), 3.77–3.98 (m, 11H, CH₂Cl A, CH₂Cl B, H-4^III A, H-6^IIIb A, CHHO A, CHHO B, H-5^Ia A, H-5^Ia B, H-6^IIIb B), 3.98–4.04 (m, 2H, H-4^III B, H-3^III A), 4.08 (dt, 2H, J 11.1 Hz, J 5.5 Hz, CHHO A, CHHO B), 4.12–4.21 (m, 4H, H-2^III B, H-2^III A, H-5^Ib A, H-5^Ib B), 4.23 (d, 1H, J 9.2 Hz, H-3^III B), 4.31–4.37 (m, 1H, H-6^IIa A or B), 4.38–4.46 (m, 2H, H-6^IIa A or B, H-2^II B), 4.51–4.62 (m, 7H, H-6^IIb A, H-6^IIb B, H-5^II A, H-5^II B, H-4^I A, H-4^I B, H-2^II A), 4.71 (s, 1H, H-1^III B), 5.00 (d, 1H, J 6.9 Hz, H-1^III A), 5.08 (s, 1H, H-1^I A), 5.12 (s, 1H, H-1^II A), 5.13 (s, 1H, H-1^I B), 5.25 (s, 1H, H-1^II B), 5.48–5.52 (m, 2H, H-3^I A, H-2^I B), 5.52 (d, 1H, J 2.2 Hz, H-2^I A), 5.54 (dd, 1H, J 6.6 Hz, J 2.5 Hz, H-3^I B), 5.66–5.77 (m, 3H, H-3^II B, H-4^II B, H-4^II A), 5.91 (dd, 1H, J 10.1 Hz, J 3.4 Hz, H-3^II A), 7.16–7.65 (m, 30H, 5 × PhCO (H-3, H-4, H-5) A, 5 × PhCO (H-3, H-4, H-5) B), 7.86–8.07 (m, 20H, 5 × PhCO (H-2, H-6) A, 5 × PhCO (H-2, H-6) B); ¹³C NMR (151 MHz, CDCl₃): δ 11.4 (((CH₃)₂CH)₃Si), 11.4 (((CH₃)₂CH)₃Si), 12.2 (((CH₃)₂CH)₃Si), 12.4 (((CH₃)₂CH)₃Si), 12.5 (((CH₃)₂CH)₃Si), 12.7 (((CH₃)₂CH)₃Si), 14.0 (((CH₃)₂CH)₃Si), 14.1 (((CH₃)₂CH)₃Si), 17.7 (2 × ((CH₃)₂CH)₃Si), 17.76 (2 × ((CH₃)₂CH)₃Si), 17.81 (((CH₃)₂CH)₃Si), 17.9 (((CH₃)₂CH)₃Si), 17.99 (((CH₃)₂CH)₃Si), 18.03 (((CH₃)₂CH)₃Si), 18.06 (((CH₃)₂CH)₃Si), 18.13 (((CH₃)₂CH)₃Si), 18.26 (((CH₃)₂CH)₃Si), 18.27 (((CH₃)₂CH)₃Si), 18.36 (((CH₃)₂CH)₃Si), 18.38 (((CH₃)₂CH)₃Si), 18.5 (((CH₃)₂CH)₃Si), 18.7 (((CH₃)₂CH)₃Si), 43.0 (CH₂Cl), 62.8 (C-6^III A), 64.1 (C-6^III B), 64.6 (C-6^II A, C-6^II B), 64.7 (C-6^II A, C-6^II B), 66.4 (C-5^I B), 66.7 (C-5^I A), 67.1 (C-4^II B), 67.35 (CH₂O A or B), 67.40 (CH₂O A or B), 68.0 (C-4^II A), 68.2 (C-5^II B), 68.8 (C-5^II A), 69.4 (C-4^III B), 71.1 (C-3^II A), 71.7 (C-2^III A), 71.96 (C-2^II A, C-3^II B), 71.99 (C-2^II A, C-3^II B), 72.29 (C-4^III A), 73.38 (C-3^III B), 73.8 (C-2^III B), 75.1 (C-2^II B), 76.5 (C-3^I B), 76.7 (C-3^I A), 77.0 (C-5^III B), 77.1 (C-3^III A), 78.5 (C-5^III A), 80.0 (C-4^I B), 80.5 (C-4^I A), 82.1 (C-2^I A), 82.3 (C-2^I B), 98.2 (C-1^II B), 99.6 (C-1^III A), 99.7 (C-1^II A), 102.3 (C-1^III B), 105.4 (2 C, C-1^I A, C-1^I B), 128.05 (PhCO (C-3, C-5)), 128.10 (PhCO (C-3, C-5)), 128.11 (PhCO (C-3, C-5)), 128.2 (PhCO (C-3, C-5)), 128.26 (PhCO (C-3, C-5)), 128.28 (PhCO (C-3, C-5)), 128.33 (4 × PhCO (C-3, C-5)), 128.40 (PhCO (C-1)), 128.44 (3 × PhCO (C-1)), 128.8 (PhCO (C-1)), 128.86 (PhCO (C-1)), 128.88 (PhCO (C-1)), 128.92 (PhCO (C-1)), 129.2 (PhCO (C-1)), 129.3 (PhCO (C-1)), 129.4 (2 × PhCO (C-2, C-6)), 129.5 (PhCO (C-2, C-6)), 129.66 (PhCO (C-2, C-6)), 129.70 (PhCO (C-2, C-6)), 129.72 (PhCO (C-2, C-6)), 129.77 (PhCO (C-2, C-6)), 129.79 (PhCO (C-2, C-6)), 129.80 (PhCO (C-2, C-6)), 129.9 (PhCO (C-2, C-6)), 132.8 (PhCO (C-4)), 132.87 (PhCO (C-4)), 132.92 (PhCO (C-4)), 133.1 (PhCO (C-4)), 133.2 (PhCO (C-4)), 133.3 (PhCO (C-4)), 133.5 (4 × PhCO (C-4)), 165.3 (CO), 165.4 (CO), 165.46 (CO), 165.52 (CO), 165.53 (CO), 165.58 (CO), 165.62 (CO), 165.7 (CO), 166.06 (CO), 166.09 (CO); ²⁹Si INEPT NMR (119 MHz, CDCl₃): δ 11.9 (2 × TIPS), 12.3 (TIPS), 12.9 (TIPS), 13.4 (TIPS), 13.5 (TIPS), 14.0 (TIPS), 15.7 (TIPS).

• Data for benzoylated disaccharide (23):

R_f = 0.33 (petroleum ether–EtOAc, 2:1); [α]_D²⁵ +5.0 (c 1.00, CHCl₃); ¹H NMR (600 MHz, CDCl₃): δ 2.30 (d, 1H, J 4.5 Hz, OH-2^II), 3.72–3.78 (m, 1H, CH₂Cl), 3.85–3.92 (m, 1H, CH₂aO), 4.01–4.09 (m, 2H, CH₂bCl, H-5a^I), 4.22 (dd, 1H, J 11.3 Hz, J 5.1 Hz, H-5b^I), 4.37–4.43 (m, 1H, H-2^II), 4.50 (dd, 1H, J 11.9 Hz, J 5.0 Hz, H-6a^II), 4.52–4.58 (m, 2H, H-4^I, H-5^II), 4.63 (dd, 1H, J 11.9 Hz, J 2.9 Hz, H-6b^II), 5.16 (d, 1H, J 1.8 Hz, H-1^II), 5.30 (s, 1H, H-1^I), 5.57 (d, 1H, J 1.6 Hz, H-2^I), 5.61 (dd, 1H, J 5.5 Hz, J 1.6 Hz, H-3^I), 5.74 (dd, 1H, J 9.9 Hz, J 3.2 Hz, H-3^II), 5.97 (t, 1H, J 10.0 Hz, H-4^II), 7.30–7.54 (m, 7H, Ph(H-3, H-4, H-5)), 7.57–7.62 (m, 1H, Ph(H-4)), 7.91–7.99 (m, 2H, Ph(H-2, H-6)), 7.99–8.04 (m, 1H, Ph(H-2, H-6)), 8.06–8.13 (m, 2H, Ph(H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 42.7 (CH₂Cl), 63.4 (C-6^II), 66.8 (C-5^I), 67.0 (C-4^II), 67.7 (CH₂O), 69.0 (C-5^II), 69.3 (C-2^II), 72.6 (C-3^II), 77.2 (C-3^I), 81.9 (C-4^I), 82.3 (C-2^I), 99.9 (C-1^II), 105.8 (C-1^I), 128.30 (Ph(C-3, C-5)), 128.34 (Ph(C-3, C-5)), 128.4 (Ph(C-3, C-5)), 128.48 (Ph(C-3, C-5)), 128.53 (Ph(C-3, C-5)), 129.0 (Ph(C-1)), 129.1 (Ph(C-1)), 129.2 (Ph(C-1)), 129.3 (Ph(C-1)), 129.7 (Ph(C-2, C-6)), 129.78 (Ph(C-2, C-6)), 129.82 (Ph(C-2, C-6)), 129.9 (Ph(C-2, C-6)), 130.0 (Ph(C-2, C-6)), 133.0 (Ph(C-4)), 133.2 (Ph(C-4)), 133.3 (Ph(C-4)), 133.5 (2 × Ph(C-4)), 165.3 (PhCO), 165.5 (PhCO), 165.6 (PhCO), 165.8 (PhCO), 166.2 (PhCO); HRMS (ESI): m/z [M+H]⁺ Calcd for C₄₈H₄₃ClNaO₁₅⁺ 917.2183; Found: 872.6827.

3.8. 2-Azidoethyl 2,3-di-O-Benzoyl-5-O-[3,4,6-tri-O-benzoyl-α-d-mannopyranosyl-2-O-{2,3,4,6-tetrakis-O-(Triisopropylsilyl)-α-d-mannopyranosyl}]-α-d-arabinofuranoside (24)

Trisaccharide 23 (32 mg, 0.019 mmol), NaN₃ (7.4 mg, 0.11 mmol) and 18-crown-6 (3 mg, 0.016 mmol) in DMF (0.6 mL), was stirred at 80 °C for 22 h. Then the reaction mixture was concentrated, the residue was co-evaporated with toluene (4 × 5 mL), concentrated under reduced pressure (bath temperature ~35 °C) and purified by silica gel column chromatography (gradient: 0 → 15% EtOAc in petroleum ether) to give azide 24 (29 mg, 0.017 mmol, 90%). R_f = 0.45 (petroleum ether–EtOAc, 5:1); [α]_D²² −8.9 (c 0.96; CHCl₃);

• Data for conformer A:

¹H NMR (600 MHz, CDCl₃, 303 K, A:B =10:3): δ 0.82–1.33 (m, 84H, 4 × ((CH₃)₂CH)₃Si), 3.49 (dd, 3H, J 5.8 Hz, J 4.6 Hz, CH₂N₃), 3.69–3.77 (m, 2H, H-5^III, CHHO), 3.80 (dd, 1H, J 10.8 Hz, J 3.5 Hz, H-6^IIIa), 3.83 (dd, 1H, J 4.3 Hz, J 2.4 Hz, H-4^III), 3.86 (dd, 1H, J 10.8 Hz, J 7.6 Hz, H-6^IIIb), 3.93 (dd, 1H, J 10.6 Hz, J 2.3 Hz, H-5^Ia), 4.02 (dt, 1H, J 10.3 Hz, J 5.0 Hz, CHHO), 4.08 (t, 1H, J 2.2 Hz, H-3^III), 4.16 (dd, 1H, J 10.6 Hz, J 6.9 Hz, H-5^Ib), 4.20 (dd, 1H, J 6.6 Hz, J 2.0 Hz, H-2^III), 4.46 (dd, 1H, J 12.4 Hz, J 8.3 Hz, H-6^IIa), 4.53–4.60 (m, 3H, H-6^IIb, H-5^II, H-4^I), 4.70 (dd, 1H, J 3.4 Hz, J 1.9 Hz, H-2^II), 5.06 (d, 1H, J 6.6 Hz, H-1^III), 5.09 (d, 1H, J 2.0 Hz, H-1^II), 5.14 (s, 1H, H-1^I), 5.53 (dd, 1H, J 6.3 Hz, J 2.4 Hz, H-3^I), 5.54 (d, 1H, J 2.6 Hz, H-2^I), 5.78 (t, 1H, J 9.8 Hz, H-4^II), 5.88 (dd, 1H, J 10.1 Hz, J 3.4 Hz, H-3^II), 7.16–7.21 (m, 2H, PhCO (H-3, H-5)), 7.26–7.33 (m, 5H, 2 × PhCO (H-3, H-5), PhCO (H-4)), 7.36–7.40 (m, 4H, 2 × PhCO (H-3, H-5)), 7.41–7.48 (m, 2H, 2 × PhCO (H-4)), 7.46–7.52 (m, 1H, PhCO (H-4)), 7.50–7.56 (m, 2H, PhCO (H-4)), 7.87–7.93 (m, 4H, 2 × PhCO (H-2, H-6)), 7.92–7.97 (m, 3H, PhCO (H-2, H-6)), 8.00–8.06 (m, 4H, 2 × PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CDCl₃): δ 11.9 (((CH₃)₂CH)₃Si), 12.7 (((CH₃)₂CH)₃Si), 12.9 (((CH₃)₂CH)₃Si), 13.0 (((CH₃)₂CH)₃Si), 17.91 (((CH₃)₂CH)₃Si), 17.92 (((CH₃)₂CH)₃Si), 18.1 (((CH₃)₂CH)₃Si), 18.5 (d, J 2.3 Hz, ((CH₃)₂CH)₃Si), 18.2 (((CH₃)₂CH)₃Si), 18.3 (((CH₃)₂CH)₃Si), 18.45 (((CH₃)₂CH)₃Si), 18.46 (((CH₃)₂CH)₃Si), 50.7 (CH₂N₃), 63.7 (C-6^III), 65.1 (C-6^II), 66.6 (CH₂O), 67.1 (C-5^I), 68.8 (C-4^II), 69.2 (C-5^II), 71.8 (C-2^II), 72.0 (C-3^II), 72.5 (C-2^III), 73.1 (C-4^III), 77.2 (C-3^I), 77.5 (C-3^III), 78.7 (C-5^III), 80.9 (C-4^I), 82.6 (C-2^I), 99.9 (C-1^III), 100.1 (C-1^II), 105.9 (C-1^I), 128.10 (PhCO (C-3, C-5)), 128.12 (PhCO (C-3, C-5)), 128.2 (PhCO (C-3, C-5)), 128.6 (PhCO (C-3, C-5)), 128.4 (PhCO (C-3, C-5)), 129.10 (PhCO (C-1)), 129.13 (PhCO (C-1)), 129.5 (PhCO (C-2, C-6)), 129.8 (PhCO (C-2, C-6)), 129.9 (PhCO (C-2, C-6)), 129.95 (PhCO (C-2, C-6)), 130.00 (PhCO (C-2, C-6)), 132.67 (PhCO (C-4)), 132.71 (PhCO (C-4)), 132.9 (PhCO (C-4)), 133.3 (2 × PhCO (C-4)), 165.5 (CO), 165.58 (CO), 165.60 (CO), 165.9 (CO), 166.0 (CO); ²⁹Si INEPT NMR (119 MHz, CDCl₃): δ 11.5 (TIPS), 12.8 (TIPS), 13.7 (TIPS), 15.6 (TIPS);

• Data for conformer B (selected signals):

¹H NMR (600 MHz, CDCl₃, 303 K, A:B = 10:3): δ 3.96 (dd, 1H, J 10.6 Hz, J 2.4 Hz, H-5^Ia), 4.04 (t, 1H, J 9.2 Hz, H-6^IIIb), 4.10 (d, 1H, J 9.2 Hz, H-4^III), 4.22 (t, 1H, J 1.5 Hz, H-2^III), 4.31 (dd, 1H, J 9.3 Hz, J 1.5 Hz, H-3^III), 4.41 (dd, 1H, J 12.1 Hz, J 8.0 Hz, H-6^IIa), 4.44 (d, 1H, J 2.4 Hz, H-2^II), 4.79 (d, 1H, J 1.1 Hz, H-1^III), 5.17 (s, 1H, H-1^I), 5.27 (s, 1H, H-1^II), 5.58 (dd, 1H, J 6.5 Hz, J 2.5 Hz, H-3^I), 5.69–5.76 (m, 2H, H-3^II, H-4^II); ¹³C NMR (151 MHz, CDCl₃): δ 11.91 (((CH₃)₂CH)₃Si), 12.90 (((CH₃)₂CH)₃Si), 14.08 (((CH₃)₂CH)₃Si), 14.33 (((CH₃)₂CH)₃Si), 18.05 (((CH₃)₂CH)₃Si), 18.16 (((CH₃)₂CH)₃Si), 18.49 (((CH₃)₂CH)₃Si), 18.52 (((CH₃)₂CH)₃Si), 18.68 (((CH₃)₂CH)₃Si), 18.88 (((CH₃)₂CH)₃Si), 50.69 (CH₂N₃), 64.75 (C-6^III), 64.96 (C-6^II), 66.62 (CH₂O), 66.77 (C-5^I), 68.05 (C-4^II), 68.79 (C-5^II), 70.16 (C-4^III), 72.36 (C-3^II), 73.78 (C-3^III), 74.47 (C-2^III), 75.68 (C-2^II), 77.03 (C-3^I), 77.66 (C-5^III), 80.76 (C-4^I), 82.70 (C-2^I), 98.51 (C-1^II), 102.55 (C-1^III), 105.93 (C-1^I);

¹H NMR (600 MHz, CDCl₃, 244 K, A:B =10:8.6): δ 0.72–1.32 (m, 168H, 4 × ((CH₃)₂CH)₃Si A, 4 × ((CH₃)₂CH)₃Si B), 3.43–3.57 (m, 3H, CH₂N₃ A, CH₂N₃ B), 3.65–3.78 (m, 6H, H-6^IIIa B, H-5^III B, H-5^III A, CHHO A, CHHO B, H-6^IIIa A), 3.79–3.84 (m, 2H, H-4^III A, H-6^IIIb A), 3.91 (dd, 1H, J 10.9 Hz, J 1.8 Hz, H-5^Ia A), 3.93 (dd, 1H, J 10.5 Hz, J 1.9 Hz, H-5^Ia B), 3.97 (d, 1H, J 7.7 Hz, H-6^IIIb B), 4.00–4.09 (m, 4H, H-4^III B, H-3^III A, CHHO A, CHHO B), 4.13–4.17 (m, 2H, H-2^III B, H-2^III A), 4.17–4.22 (m, 2H, H-5^Ib A, H-5^Ib B), 4.24 (dd, 1H, J 9.3 Hz, J 2.1 Hz, H-3^III B), 4.35 (dd, 1H, J 12.1 Hz, J 8.6 Hz, H-6^IIa B), 4.42 (dd, 1H, J 11.5 Hz, J 8.3 Hz, H-6^IIa A), 4.43–4.45 (m, 1H, H-2^II B), 4.51–4.61 (m, 6H, H-6^IIb A, H-4^I A, H-4^I B, H-5^II A, H-5^II B, H-6^IIb B), 4.61 (dd, 1H, J 3.3 Hz, J 1.8 Hz, H-2^II A), 4.72 (d, 1H, J 1.4 Hz, H-1^III B), 5.01 (d, 1H, J 6.7 Hz, H-1^III A), 5.10 (s, 1H, H-1^I A), 5.12 (d, 1H, J 1.7 Hz, H-1^II A), 5.14 (s, 1H, H-1^I B), 5.26 (d, 1H, J 1.7 Hz, H-1^II B), 5.52 (d, 1H, J 2.3 Hz, H-2^I B), 5.53 (d, 1H, J 2.1 Hz, H-2^I A), 5.54 (dd, 1H, J 6.5 Hz, J 2.2 Hz, H-3^I A), 5.58 (dd, 1H, J 6.6 Hz, J 2.0 Hz, H-3^I B), 5.69–5.77 (m, 3H, H-3^II B, H-4^II B, H-4^II A), 5.92 (dd, 1H, J 10.3 Hz, J 3.3 Hz, H-3^II A), 7.16–7.59 (m, 30H, 5 × PhCO (H-3, H-4, H-5) A, 5 × PhCO (H-3, H-4, H-5) B), 7.85–8.07 (m, 20H, 5 × PhCO (H-2, H-6) A, 5 × PhCO (H-2, H-6) B); ¹³C NMR (151 MHz, CDCl₃): δ 11.41 (((CH₃)₂CH)₃Si B), 11.43 (((CH₃)₂CH)₃Si A), 12.2 (((CH₃)₂CH)₃Si A), 12.46 (((CH₃)₂CH)₃Si B), 12.51 (((CH₃)₂CH)₃Si A), 12.7 (((CH₃)₂CH)₃Si A), 14.0 (((CH₃)₂CH)₃Si B), 14.1 (((CH₃)₂CH)₃Si B), 17.7 (((CH₃)₂CH)₃Si B), 17.75 (((CH₃)₂CH)₃Si B), 17.77 (2 × ((CH₃)₂CH)₃Si A), 17.83 (((CH₃)₂CH)₃Si Bi), 17.96 (((CH₃)₂CH)₃Si B), 18.00 (((CH₃)₂CH)₃Si A), 18.04 (((CH₃)₂CH)₃Si A), 18.1 (((CH₃)₂CH)₃Si A), 18.2 (((CH₃)₂CH)₃Si A), 18.27 (((CH₃)₂CH)₃Si A), 18.28 (((CH₃)₂CH)₃Si A), 18.37 (((CH₃)₂CH)₃Si B), 18.40 (((CH₃)₂CH)₃Si B), 18.5 (((CH₃)₂CH)₃Si B), 18.7 (((CH₃)₂CH)₃Si B), 50.28 (CH₂N₃ B), 50.32 (CH₂N₃ A), 62.9 (C-6^III A), 64.2 (C-6^III B), 64.63 (C-6^II B), 64.7 (C-6^II A), 66.3 (C-5^I B), 66.6 (C-5^I A, CH₂O A, CH₂O B), 67.2 (C-4^II B), 68.1 (C-4^II A), 68.3 (C-5^II B), 68.8 (C-5^II A), 69.5 (C-4^III B), 71.2 (C-3^II A), 71.8 (C-2^III A), 71.97 (C-2^II A), 72.02 (C-3^II B), 72.3 (C-4^III A), 73.4 (C-3^III B), 73.9 (C-2^III B), 75.3 (C-2^II B), 76.5 (C-3^I B), 76.7 (C-3^I A), 77.0 (C-5^III B), 77.2 (C-3^III A), 78.5 (C-5^III A), 80.1 (C-4^I B), 80.6 (C-4^I A), 82.4 (C-2^I A), 82.6 (C-2^I B), 98.2 (C-1^II B), 99.66 (C-1^III A), 99.72 (C-1^II A), 102.4 (C-1^III B), 105.47 (C-1^I A), 105.49 (C-1^I B), 128.07 (PhCO (C-3, C-5)), 128.12 (2 × PhCO (C-3, C-5)), 128.2 (PhCO (C-3, C-5)), 128.25 (PhCO (C-3, C-5)), 128.30 (3 × PhCO (C-3, C-5)), 128.34 (2 × PhCO (C-3, C-5)), 128.4 (PhCO (C-1)), 128.46 (PhCO (C-1)), 128.48 (PhCO (C-1)), 128.87 (PhCO (C-1)), 128.91 (PhCO (C-1)), 128.93 (PhCO (C-1)), 129.1 (PhCO (C-1)), 129.2 (PhCO (C-1)), 129.3 (PhCO (C-1)), 129.4 (2 × PhCO (C-2, C-6)), 129.5 (PhCO (C-2, C-6)), 129.66 (PhCO (C-2, C-6)), 129.71 (PhCO (C-2, C-6)), 129.73 (PhCO (C-2, C-6)), 129.80 (PhCO (C-2, C-6)), 129.81 (PhCO (C-2, C-6)), 129.84 (PhCO (C-2, C-6)), 129.9 (PhCO (C-2, C-6)), 132.8 (PhCO (C-4)), 132.87 (PhCO (C-4)), 132.91 (PhCO (C-4)), 133.1 (PhCO (C-4)), 133.2 (PhCO (C-4)), 133.3 (PhCO (C-4)), 133.5 × PhCO (C-4)), 165.3 (CO), 165.4 (CO), 165.48 (CO), 165.54 (CO), 165.58 (CO), 165.64 (2 × CO), 165.7 (CO), 166.07 (CO), 166.10 (CO); ²⁹Si INEPT NMR (119 MHz, CDCl₃): δ 11.8 (TIPS A), 11.9 (TIPS B), 12.3 (TIPS B), 12.9 (TIPS B), 13.4 (TIPS A), 13.5 (TIPS B), 14.0 (TIPS A), 15.7 (TIPS A); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₉₀H₁₃₇N₄O₂₀Si₄⁺ 1705.8898; Found: 1705.8909.

3.9. 2-Azidoethyl 2,3-di-O-Benzoyl-5-O-[3,4,6-tri-O-benzoyl-α-d-mannopyranosyl-2-O-[α-d-mannopyranosyl]-α-d-arabinofuranoside (25)

Trisaccharide 24 (30 mg, 0.019 mmol) was dissolved in THF (1 mL) followed by the addition of AcOH (7.5 μL, 0.13 mmol) and 1 M TBAF in THF (275 µL, 0.275 mmol). After stirring at 40 °C for 3.5 h the reaction mixture was concentrated under reduced pressure, co-evaporated with toluene (5 × 5 mL) and dried in vacuo. The residue was purified by silica gel chromatography CH₂Cl₂–MeOH (gradient: 0 → 8% MeOH in CH₂Cl₂) to give trisaccharide 25 (11 mg, 0.010 mmol, 60%). R_f = 0.21 (CH₂Cl₂–MeOH, 20:1); [α]_D²⁶ +1.7 (c 0.96; CHCl₃); ¹H NMR (600 MHz, CD3OD): δ 3.46–3.55 (m, 2H, CH₂N₃), 3.65 (t, 1H, J 9.6 Hz, H-4^III), 3.71 (dd, 1H, J 11.7 Hz, J 6.2 Hz, H-6^IIIa), 3.77–3.83 (m, 2H, H-5^III, CHHO), 3.86 (dd, 1H, J 9.5 Hz, J 3.4 Hz, H-3^III), 3.88 (dd, 1H, J 11.8 Hz, J 2.3 Hz, H-6^IIIb), 3.99 (dd, 1H, J 3.4 Hz, J 1.7 Hz, H-2^III), 3.98–4.05 (m, 1H, CHHO), 4.05 (dd, 1H, J 11.3 Hz, J 2.8 Hz, H-5^Ia), 4.24 (dd, 1H, J 11.3 Hz, J 4.1 Hz, H-5^Ib), 4.39 (dd, 1H, J 3.3 Hz, J 1.8 Hz, H-2^II), 4.46 (dd, 1H, J 12.0 Hz, J 3.5 Hz, H-6^IIa), 4.49 (dd, 1H, J 12.0 Hz, J 4.5 Hz, H-6^IIb), 4.53–4.59 (m, 2H, H-4^I, H-5^II), 4.90 (d, 1H, J 1.8 Hz, H-1^III), 5.37 (d, 2H, J 1.9 Hz, H-1^II), 5.39 (s, 1H, H-1^I), 5.51 (d, 1H, J 1.8 Hz, H-2^I), 5.72 (dd, 1H, J 5.6 Hz, J 1.8 Hz, H-3^I), 5.74 (dd, 1H, J 10.1 Hz, J 3.2 Hz, H-3^II), 5.84 (t, 1H, J 10.0 Hz, H-4^II), 7.28–7.46 (m, 11H, 5 × PhCO (H-3, H-5), PhCO (H-4)), 7.46–7.62 (m, 4H, 4 × PhCO (H-4)), 7.82–7.87 (m, 4H, 2 × PhCO (H-2, H-6)), 7.92–7.97 (m, 2H, PhCO (H-2, H-6)), 8.03–8.10 (m, 4H, 2 × PhCO (H-2, H-6)); ¹³C NMR (151 MHz, CD3OD): δ 51.9 (CH₂N₃), 63.0 (C-6^III), 64.6 (C-6^II), 67.5 (C-5^I), 67.8 (CH₂O), 68.6 (C-4^III), 68.9 (C-4^II), 70.2 (C-5^II), 72.1 (C-2^III), 72.6 (C-3^III), 73.0 (C-3^II), 75.6 (C-5^III), 77.7 (C-2^II), 78.7 (C-3^I), 83.1 (C-4^I), 84.1 (C-2^I), 100.4 (C-1^II), 104.2 (C-1^III), 107.1 (C-1^I), 129.5 (PhCO (C-3, C-5)), 129.6 (3 × PhCO (C-3, C-5)), 129.7 (PhCO (C-3, C-5)), 130.4 (PhCO (C-1)), 130.48 (PhCO (C-1)), 130.50 (2 × PhCO (C-1)), 130.6 (PhCO (C-2, C-6)), 130.6 (PhCO (C-2, C-6)), 130.7 (PhCO (C-2, C-6)), 130.9 (2 × PhCO (C-2, C-6)), 134.3 (PhCO (C-4)), 134.55 (PhCO (C-4)), 134.60 (2 × PhCO (C-4)), 134.7 (PhCO (C-4)), 166.7 (PhCO), 167.1 (2 × PhCO), 167.6 (2 × PhCO); HRMS (ESI): m/z [M+NH₄]⁺ Calcd for C₅₄H₅₇N₄O₂₀ 1081.3561; Found: 1081.3576.

3.10. 2-Azidoethyl 5-O-[α-d-Mannopyranosyl-2-O-(α-d-mannopyranosyl)]-α-d-arabinofuranoside (26)

Partially protected trisaccharide (25) (11 mg, 0.011 mmol) was dissolved in anhydrous CH₂Cl₂ (0.5 mL) and MeOH (1 mL) followed by an addition of 1 M methanolic MeONa (45 μL). The reaction mixture was kept at ~22 °C for 18 h, then neutralized with Dowex 50W × 8 (H+) ion-exchange resin (the resin was washed with MeOH before addition) and then filtered. The filtrate was concentrated under reduced pressure, and the residue was dried in vacuo and 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). The collected fraction was lyophilized to give deprotected trisaccharide 26 (4.3 mg, 0.0082 mmol, 78%). R_f = 0.49 (CH₂Cl₂–MeOH–H₂O 40:10:3); [α]_D²⁵ +72.68 (c 1.0, CHCl₃); ¹H NMR (600 MHz, CD₃OD): δ 3.38 (ddd, 2H, J 13.3 Hz, J 6.0 Hz, J 3.6 Hz, CH₂N₃a), 3.46 (ddd, 3H, J 13.3 Hz, J 7.1 Hz, J 3.5 Hz, CH₂N₃b), 3.56–3.66 (m, 4H, OCH₂a, H-5^III, H-4^II, H-4^III), 3.67–3.73 (m, 5H, H-5a^I, H-6a^II, H-6a^III, H-3^III, H-5^II), 3.81–3.89 (m, 5H, OCH₂b, H-5b^I, H-6b^II, H-6b^III, H-3^II), 3.89–3.92 (m, 2H, H-2^II, H-3^I), 3.98 (dd, 1H, J 3.3 Hz, J 1.8 Hz, H-2^III), 4.00 (dd, 1H, J 4.1 Hz, J 1.9 Hz, H-2^I), 4.04 (ddd, 1H, J 6.9 Hz, J 5.1 Hz, J 3.2 Hz, H-4^I), 4.91 (d, 1H, J 1.9 Hz, H-1^I), 4.98 (d, 1H, J 1.8 Hz, H-1^III), 5.14 (d, 1H, J 1.8 Hz, H-1^II); ¹³C NMR (151 MHz, CD₃OD): δ 51.9 (CH₂N₃), 63.0 (C-6^II), 63.1 (C-6^III), 67.7 (C-5^I), 67.9 (OCH₂), 68.8 (C-4^II), 69.0 (C-4^III), 71.9 (C-2^III), 72.1 (C-3^II), 72.4 (C-3^III), 74.7 (C-5^III), 75.0 (C-5^II), 78.8 (C-3^I), 80.3 (C-2^II), 83.5 (C-4^I), 83.8 (C-2^I), 100.3 (C-1^II), 104.2 (C-1^III), 109.7 (C-1^I); HRMS (ESI): m/z [M+Na]⁺ Calcd for C₁₉H₃₃N₃NaO₁₅ 566.1804; Found: 566.1806.

4. Conclusions

In summary, silylation of phenyl 1-thio-α-d-mannopyranoside, ethyl 1-thio-α- and β-d-mannopyranosides under different conditions was studied. Low-temperature NMR analysis revealed that the silylated products 2, 5, 7, 10, 13 typically exist as an equilibrium of two chair conformations with a predominance of the axially rich ¹C₄ conformation. The ring flip was induced by simultaneous introduction of bulky silyl substituents at O-3 and O-4 in mannopyranosides. The dependence of the ratio of conformers on the anomeric configuration, the type of silyl groups and the nature of the aglycone for silylated mannopyranosides was established. The fully silylated phenyl 1-thio-α-d-mannopyranoside (2), ethyl 1-thio-α- and β-d-mannopyranosides (10, 13) were involved in the synthesis of α-1,2-linked methyl- and phenyl mannopyranosides (15, 18) and trisaccharide (22) with 2-chloroethyl aglycone. In particular, based on the difference in reactivity of (10), (17) and (21), we performed one-pot synthesis of trisaccharide (22). In all cases, regardless of conformational preference of silylated thiomannopyranosides (¹C₄ or ⁴C₁), the formation of only α-linked oligosaccharides also existing as mixtures of conformers was observed. Thus, the use of polysilylated thiomannopyranosides led to the same α-stereoselectivity as in case of the use of mannopyranosyl donors with 2-O-acyl participating group. The exploring of the one-pot methodology could significantly reduce the number of stages.

The obtained deprotected Manp-(1→2)-α-d-Manp-(1→5)-α-d-Araf trisaccharide 2-azidoethyl glycoside 26, related to the non-reducing terminal fragment of the M. tuberculosis ManLAM, can be used for further preparation of conjugates with proteins to provide antigens, which are important for the development of new tuberculosis screening assays.