Towards the complete synthetic O-antigen of Vibrio cholerae O1, serotype inaba: improved synthesis of the conjugation-ready upstream terminal hexasaccharide determinant

Abstract

Synthesis of the upstream terminal hexasaccharide part of the lipopolysaccharides (LPS) of Vibrio cholerae O1, serotype Inaba has been improved. The key improvements include but are not limited to optimized conditions for the stereoselectivity of glycosylation reactions involved and fewer number of synthetic steps, compared to previous approaches. Particularly noteworthy is conducting the glycosylation of the very reactive glycosyl acceptor 8-azido-3,6-dioxaoctanol with the fully assembled hexasaccharide trichloroacetimidate under thermodynamic control. It produced the desired α glycoside with an α : β ratio of 7 : 1, compared with the ratio of 1.1 : 1, observed when the coupling was conducted conventionally. Several substances, which were previously obtained in purity acceptable only for synthetic intermediates, were now obtained in the analytically pure state and were fully characterized. The structure of the key trisaccharide glycosyl acceptor was confirmed by single-crystal X-ray structure determination.

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Introduction

The upstream, terminal part of the lipopolysaccharides (LPS) of most Gram-negative bacteria consist of several copies of oligosaccharide, sometimes monosaccharide repeating units. The said macromolecules are termed O-specific polysaccharides (O-SP) and are recognized as protective antigens of these pathogens. O-SPs confer specificity to their homologous antibodies and they, or fragments thereof, are paramount in the development of synthetic/conjugate vaccines. We have been involved in work towards vaccines from synthetic fragments of O-SPs for a number of years. One of our major targets has been a conjugate vaccine for cholera from synthetic fragments of O-SPs of Vibrio cholerae.^1–3

The O-SP of Vibrio cholerae O1, serotype Inaba consists of 12–18 repeats⁴ of (1→2)-α-linked perosamine (4-amino-4,6-dideoxy-D-mannose) whose amino group is acylated with 3-deoxy-L-glycero-tetronic acid. We have previously synthesized hexasaccharide fragments of the O-SP and have determined essential immune responses of conjugates therefrom in mice.⁵ Chemical structures of O-SPs of the two strains (Inaba and Ogawa) of Vibrio cholerae O1 are the same, except that the terminal, upstream perosamine residue in the Ogawa strain carries a methyl group at O-2. To be able to compare immune responses of the conjugates from the hexasaccharide fragments of the O-SP of both serotypes of Vibrio cholerae O1 with those of similar conjugates from synthetic polymers representing the complete O-SPs, we intend to synthesize glycoconjugates from the analogous octadecasaccharides. Syntheses of such structures are much more involved undertakings. Previous syntheses of the hexasaccharide antigens comprised up to more than 40 linear steps, depending on the individual approach.^1,2 The key intermediates within our strategy towards the octadecasaccharides will be synthons derived from the related hexasaccharides. With the aim to increase the feasibility of a large-scale synthesis required by future immunization studies and decrease the number of synthetic steps involved in making such substances, the objective of this work was to test the practicability and scalability of the current, new synthetic scheme. Thus, we synthesized on large scale the trisaccharide glycosyl donor and acceptor 5 and 4, respectively, and used these to prepare, also on large scale, the related hexasaccharide 3 (Fig. 1). These substances are versatile intermediates, which we intend to use to make considerably larger fragments of the O-SP, up to the complete bacterial O-specific antigen, octadecasaccharide. Using hexasaccharide 3, we proceeded to complete the synthesis of the title hexasaccharide 1. The present pathway is the shortest published to date (33 linear steps). An additional advantage of the approach described here is that the stereoselectivity of the critical glycosylation reaction, which converted the terminal determinant to a conjugation-ready form, was substantially increased by controlling it thermodynamically.^2,6

Fig. 1 Retrosynthetic analysis of the hexasaccharide 1.

Results and discussion

Synthesis of the title hexasaccharide started with the known⁷ methyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (10). It was treated with Ac₂O in presence of H₂SO₄, and the formed (Scheme 1) 1-O-acetyl-4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α,β-D-mannopyranose (11)⁷ where the α-anomer largely predominated was resolved by chromatography. Unlike previously, the α-acetate 11a was now obtained crystalline for the first time. We planned to use the latter as a glycosyl donor under BF₃·Et₂O or TMSOTf catalysis^8,9 to make the corresponding 2-trimethylsilylethyl (SE) glycoside 13a, and perhaps also higher oligosaccharides in this series, thereby improving the economy of the overall synthesis. Unfortunately, this approach was unsuccessful: only the corresponding 1-OH compound was formed. Similarly unsuccessful was the reaction of the corresponding 2-O-acetyl derivative 12 (→13b), prepared conventionally¹⁰ (these reactions are not described in the Experimental). Explanation for these failures was not sought, but in their extensive synthetic study towards this class of substances, Saksena et al.¹¹ observed that formation of 2-trimethylsilylethyl mannopyranosides is a complex process whose outcome is largely unpredictable. In contrast with the unsuccessful Lewis acid mediated reaction of 11 or 12 with 2-trimethylsilylethanol, the BF₃·Et₂O-catalyzed reaction of 11 with ethanethiol readily produced ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-1-thio-α,β-D-mannopyranoside (7a and hitherto unknown 7b),¹¹ which was fully characterized. The latter mixture of anomers was converted to its corresponding SE glycoside 13a as described,¹¹ and the pure α-anomer 7a was used as glycosyl donor towards oligosaccharide synthesis (Schemes 2 and 3). The initial glycosyl acceptor 8⁷ (Scheme 1) was prepared by conventional debenzoylation (Zemplén¹²) from the foregoing 2-O-benzoate 13a.

Scheme 1 Synthesis of the monosaccharide building blocks.

Scheme 2 Synthesis of the disaccharide acceptor 6.

Scheme 3 Synthesis of trisaccharide donor 5 and acceptor 4.

With suitably equipped glycosyl donor and acceptor at hand, we set out for oligosaccharide synthesis on the multigram scale (see Experimental). Accordingly, glycosyl acceptor 8 and thioglycoside donor 7a were coupled using NIS/TMSOTf as promoter, affording disaccharide 14 (94%, Scheme 2). The NMR spectra of the disaccharide showed signals characteristic of the presence of both donor and acceptor moieties (165.3 ppm for the benzoyl carbonyl carbon from the donor and −1.32 ppm for SiMe₃ carbon from the acceptor), while the α-configuration of the interglycosidic linkage was confirmed from the corresponding NMR spectra, mainly J_C,H at the newly formed glycosidic linkage (173.3 Hz).¹³ Zemplén debenzoylation of 14 furnished disaccharide acceptor 6,⁷ which was subsequently used for chain elongation (Scheme 3). Coupling of disaccharide acceptor 6 and thioglycoside donor 7a in presence of NIS-TMSOTf at 0 °C produced trisaccharide 15 (93%, Scheme 3). Formation of the desired trisaccharide 15 was confirmed by HRMS and the stereochemistry of the newly formed interglycosidic linkage followed from the ¹³C–¹H coupling constant for the anomeric carbon center at 99.2 ppm (C-1^III, J_C–H = 173.3 Hz). Zemplén debenzoylation of 15 furnished trisaccharide acceptor 4, which was obtained crystalline (CCDC no. 1939745†). Compound 4 crystallizes with 2 independent molecules in the asymmetric unit of the unit cell. Only one free –OH group is available on each molecule for hydrogen bonding, and in each case, a hydrogen bond is formed to an O5 acceptor on a mannopyranoside ring in an adjacent molecule. A modest number of weaker C–H⋯O and C–H⋯N interactions are also observed. Given the limited number of free hydroxyl groups available for hydrogen bonding, the majority of the intermolecular interactions will be weaker non-polar van der Waals type interactions. In this case, the conformations of the polysaccharide chains of the two independent molecules are more likely to reflect a conformational energy minimum for the chain, then would be the case if extensive hydrogen bonding were present. Significantly, the relative conformations of the trisaccharide chains of both independent molecules are very similar, while the orientations of the substituent groups (especially the benzyl and trimethylsilylethyl groups) show greater variation (Fig. 2). Further analysis of the conformation of X-ray data for compound 4 will be reported in a subsequent communication.

Fig. 2 Stereo projections of the two independent molecules of compound 4 at 120 K. Thermal ellipsoids are plotted at the 50% level, and hydrogen atoms have been omitted for clarity. The projection directions have been chosen to highlight the similarity of the core conformations of the two trisaccharide chains.

Compared to the NMR spectra of parent compound 15, disappearance of the carbonyl carbon signal at 165.3 ppm in the ¹³C NMR spectrum of 4 and upfield shift of H-2^III (from δ 5.59 ppm, dd, J_2–3 = 2.9 Hz, J_2–1 = 2.0 Hz to δ 3.98 ppm, ddd, J_2–3 = 2.8 Hz, J_2-OH-2 = 1.7 Hz, J_2–1 = 1.3 Hz) in the ¹H NMR spectrum confirmed the removal of the benzoyl group and formation of the corresponding 2-hydroxy product 4. Treatment of the foregoing 2-trimethylsilyl ethyl glycoside 15 with trifluoroacetic acid (TFA) produced the corresponding trisaccharide hemiacetal 16 (89%). Absence of signals for the 2-(trimethylsilylethyl) group in the ¹H NMR spectrum of 16, together with presence of two anomeric C-1^I signals in the ¹³C NMR spectrum (δ 93.5 and 93.2 ppm) confirmed the successful hydrolysis and formation of the desired hemiacetal 16. Subsequent base-catalyzed reaction of 16 with trichloroacetonitrile and DBU produced the corresponding trichloroacetimidate donor 5 (Scheme 3).

Large-scale glycosylation (24.3 g of trichloroacetimidate donor 5 with 19 g of trisaccharide acceptor 4) produced hexasaccharide 3 (92%, Scheme 4) with excellent stereoselectivity (α : β ∼ 34 : 1, as shown from integration of signals at δ 5.32 ppm and 5.59 ppm for H-1^V of 3a and H-2^VI of 3b, respectively, in the NMR spectrum of the crude reaction mixture). Strong contours for anomeric carbons at 101.2 ppm (C-1^II), 101.06 ppm (C-1^III), 101.04 ppm (C-1^IV), 101.03 ppm (C-1^V), 100.3 ppm (C-1^VI), 99.16 ppm (C-1^I) in the HSQC spectrum confirmed the formation of the hexasaccharide. That the predominant isomer contained the desired α-configuration followed from the ¹³C–¹H coupling constant for the ¹³C carbon involved in the newly formed interglycosidic linkage. For the major isomer, ¹³C signal of C-1^IV at δ 101.04 ppm showed coupling constant J_C-1–H-1 = 174.4 Hz whereas the same for the minor isomer was at δ 96.3 with J_C-1–H-1 = 155.5 Hz (Fig. 3).

Scheme 4 Synthesis of the hexasaccharide derivative 3.

Fig. 3 (a) Comparison of ¹³C-NMR spectra of compounds 3a (150 MHz, C₆D₆) and 3b (150 MHz, CDCl₃); (b) ¹³C–¹H coupled 2D-NMR spectrum of compound 3a (C₆D₆); (c) ¹³C–¹H coupled 2D-NMR spectrum of compound 3b (CDCl₃).

Debenzoylation of hexasaccharide 3a under Zemplén conditions produced the 2-hydroxyl group-free intermediate 17.¹⁴ The six azido groups in the foregoing hexasaccharide were transformed, into amines by H₂S reduction (→18,¹⁴ 92%, Scheme 5). The next task was to introduce the N-3-deoxy-L-glycero-tetronoyl groups into the foregoing hexaamine 18. This was performed conventionally^2,6 with 2,4-di-O-acetyl-3-deoxy-L-glycero-tetronic acid¹⁵ and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDAC). For easier isolation and next reaction, the crude product was directly acetylated, to give the fully protected hexasaccharide amide 19 (84% over two steps, Scheme 5).

Scheme 5 Synthesis of the title hexasaccharide.

To convert compound 19 into a conjugation-ready form, aglycone in the silyl ethyl glycoside had to be replaced with a suitably equipped linker molecule. Cleavage of 19 with trifluoroacetic acid (TFA), and subsequent conversion of the reducing sugar 20, thus obtained (Scheme 5), to the corresponding trichloroacetimidate donor 21 brought the synthesis of the title antigen to a synthetic step that proved difficult in the past. In most situations across carbohydrate chemistry, synthesis of the 1,2-trans-glycosidic linkage is not problematic because a participating group can be introduced into 2-positions of the glycosyl donors. However, in our case, the 2-O-position of the donor is glycosylated and, therefore, no participating group could be introduced. Furthermore, as we found during our previous syntheses of oligosaccharides within the Vibrio cholerae O1 series, the selectivity of α-mannosylation is impaired when the glycosylation is conducted with donors having the 3-deoxy-L-glycero-tetronic acid side chain already in place.^2,6,16 It is especially noteworthy that when we^7,14 previously glycosylated a fully assembled hexasaccharide Vibrio cholerae O1 donor with a primary hydroxyl group-containing linker molecule, the reaction showed almost no stereoselectivity. This was the reason why more recent syntheses of similar oligosaccharides utilized glycosyl donors containing 4-azido groups,^1,17 unlike in the earlier works where oligosaccharides related to the O-SP of Vibrio cholerae O1 were synthesized using glycosyl donors where the tetronamido side chain was already in place.^18,19 Similarly, in the initial reaction of 21 with 8-azido-3,6-dioxaoctanol (22) under Ogawa's¹⁴ conditions, the α and β glycosides were formed in a ratio of ∼1.1 : 1. When we took advantage of the glycosylation under thermodynamic control developed in this laboratory,^2,6 the stereoselectivity of the same glycosylation increased by many folds (α : β ∼ 7 : 1). Conversion of oligosaccharides into conjugation-ready forms often involves α-mannosylation of very reactive aglycons. Such reactions are characterized by poor stereo selectivity. Performing such reactions under thermodynamic control remarkably increases the stereo selectivity of such conversions, and constitutes a considerable improvement of the synthesis of this and similar conjugation-ready oligosaccharides over existing protocols. Conventional deacetylation (Zemplén¹²) of 2a, followed by hydrogenation/hydrogenolysis (Pd/C) yielded the final glycoside 1 (71%, over two steps). The structure of compound 1 was confirmed by HRMS and NMR spectra.

Conclusions

We have modified existing approaches leading to antigenic determinants of Vibrio cholerae O1 and verified scalability of reactions involved. Intermediates to oligosaccharides in this series, up to and including the hexasaccharide have been successfully prepared on multigram scales. Using such hexasaccharide, the synthesis of the terminal hexasaccharide fragment of the O-specific polysaccharide of Vibrio cholerae O1, serotype Inaba was improved. The number of linear synthetic steps toward the compound was reduced, and the stereoselectivity of the critical 1,2-trans-glycosylation of the very reactive 8-azido-3,6-dioxaoctanol with the fully assembled hexasaccharide trichloroacetimidate was markedly increased, from α : β = 1.1 : 1 to 7 : 1 thereby increasing considerably the yield of the conjugation-ready title compound manifold.

Experimental

Materials and methods, crystallography

Unless specified otherwise, all reagents and solvents were purchased from Sigma-Aldrich Chemical Company and used as supplied. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 glass slides. Spots were visualized by charring with H₂SO₄ in EtOH (5% v/v) and/or UV light. Melting points were determined with a Kofler hot stage. Optical rotations were measured at ambient temperature with a Jasco P-2000 digital polarimeter. NMR spectra were measured at 25 °C for solutions in benzene-d₆, methanol-d₄ or CDCl₃, at 400 MHz, 500 MHz or 600 MHz for ¹H, and at 100 MHz, 125 MHz or 150 MHz for ¹³C with Bruker Avance Spectrometers. Assignments of NMR signals were aided by 1D and 2D experiments (1H–1H homonuclear decoupling, APT, COSY, HSQC, TOCSY and HMBC) run with the software supplied with the spectrometer. Chemical shifts were referenced to that of tetramethylsilane (0 ppm) or signals of residual non-deuterated solvents. Crystals of 4 suitable for X-ray data collection were obtained by slow evaporation of ethanol from ethanolic solution. X-ray intensity measurements were collected at low temperature from a colorless needle-shaped crystal using a Bruker Kappa APEX II 4K CCD diffractometer with MoKα radiation. An Oxford Cryosystems 700 low temperature system was used to generate a stream of cold N₂ gas that cooled the sample crystal to 120(2) K during data collection. Data were collected using both ω and ϕ scans with a scan width of 0.50° per frame and a rate of 30 s per frame, with the detector center located 40.0 mm from the crystal at 2θ = 30.00° or 60.00°. The data were processed using the Bruker APEX III software package. The crystal structure was solved and refined using the SHELXL²⁰ software package. The absolute configuration (Flack) parameter, determined from the X-ray data during the refinement, correctly identified the absolute configuration of the structure, which was also established by the known configuration of the α-D-mannopyranoside rings. The positions of H atoms attached to C and O atoms were calculated using idealized sp² or sp³ geometry and included as riding atoms in the least-squares refinement. For methyl and –OH hydrogens, the torsion angles about the X-Me or C–OH bond were also optimized during the refinement. Details of the crystal data and structure refinement are given in ESI.† The 7 N solution of NH₃ in MeOH was purchased from Sigma-Aldrich. Solutions in organic solvents were dried with anhydrous MgSO₄ and concentrated at reduced pressure at <40 °C.

1-O-Acetyl-4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranose (11a) and 1-O-acetyl-4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-β-D-mannopyranose (11b)

This compound was prepared from 115 g (289.4 mmol) of 10 as described,⁷ yielding 115.7 g (94%) of anomeric mixture of 11a and 11b (α/β ∼ 9 : 1) as white solid. R_f = 0.45 (hexane/ethyl acetate, 4/1). Chromatography gave first 11a (104.4 g, 85%). m.p. 107–108 °C (EtOAc–hexane). [α]_D +1.5 (c 1.0, CHCl₃), lit¹⁷ [α]_D +1.9 (c 1.9, CHCl₃) for amorphous material. ¹H NMR (400 MHz, CDCl₃): δ 8.07–8.05 (d, 2H, J = 7.9 Hz, Ar–H), 7.59 (t, 1H, J = 7.3 Hz, Ar–H), 7.49–7.43 (t, 2H, J = 7.4 Hz, Ar–H), 7.36–7.24 (m, 5H, Ar–H), 6.15 (d, 1H, J_1–2 = 1.4 Hz, H-1), 5.56 (2d, 1H, J_2–3 = 2.8, J_2–1 = 1.9 Hz, H-2), 4.80 (d, 1H, J = 11.2 Hz, CH₂Ph), 4.59 (d, 1H, J = 11.2 Hz, CH₂Ph), 3.91 (dd, 1H, J_3–4 = 9.7 Hz, J_3–2 = 3.2 Hz, H-3), 3.67 (dq, 1H, J_5–4 = 10.1 Hz, J_5–6 = 6.0 Hz, H-5), 3.57 (2d, 1H, J_4–3 = 9.8 Hz, J_4–5 = 10.0 Hz, H-4), 2.12 (s, 3H, COCCH₃), 1.39 (d, 3H, J_6–5 = 6.0 Hz, CH₃). ¹³C{1H} NMR (100 MHz, CDCl₃): δ 168.4 (C=O), 165.4 (C=O), 136.9, 133.4, 129.9 (2C), 129.3, 128.5 (2C), 128.4 (2C), 128.2 (2C), 127.9, 91.1 (C-1), 75.8 (C-3), 71.6 (CH₂Ph), 69.3 (C-2), 66.7 (C-5), 63.8 (C-4), 20.9 (COCH₃), 18.7 (C-6). HRMS (ESI-TOF): m/z [M + Na]⁺ calcd for C₂₂H₂₃O₆N₃Na 448.1485, found 448.1488. Anal. calcd for C₂₂H₂₃O₆N₃: C, 62.11; H, 5.45; N, 9.88. Found C, 62.20; H, 5.40; N, 10.06.

Continued elution gave the β-linked derivative 11b as colorless syrup 11.3 g, 9.2%. R_f = 0.43 (hexane/ethyl acetate, 4/1). [α]_D −51.1 (c 1.0, CHCl₃), lit¹⁷ [α]_D −50.2 (c 1.2, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 8.11–8.09 (d, 2H, J = 7.9 Hz, Ar–H), 7.59 (t, 1H, J = 7.3 Hz, Ar–H), 7.49–7.43 (t, 2H, J = 7.7 Hz, Ar–H), 7.36–7.24 (m, 5H, Ar–H), 5.82 (d, 1H, J_1–2 = 3.1 Hz, H-2), 5.77 (s, 1H, H-1), 4.80 (d, 1H, J = 11.2 Hz, CH₂Ph), 4.56 (d, 1H, J = 11.2 Hz, CH₂Ph), 3.68 (dd, 1H, J_3–4 = 9.6 Hz, J_3–2 = 3.0 Hz, H-3), 3.53 (t, 1H, J = 9.7 Hz, H-4), 3.39 (m, 1H, H-5), 2.04 (s, 3H, COCCH₃), 1.45 (d, 3H, J_6–5 = 6.1 Hz, CH₃). ¹³C{1H} NMR (100 MHz, CDCl₃): δ 168.8 (C=O), 165.8 (C=O), 136.6, 133.4, 129.9 (2C), 129.5, 128.5 (2C), 128.4 (2C), 128.3 (2C), 128.1, 91.1 (C-1), 78.0 (C-3), 72.1 (C-2), 71.4 (CH₂Ph), 66.8 (C-5), 63.6 (C-4), 20.7 (COCH₃), 18.6 (C-6). HRMS (ESI-TOF): m/z [M + Na]⁺ calcd for C₂₂H₂₃O₆N₃Na 448.1485, found 448.1481. Anal. calcd for C₂₂H₂₃O₆N₃: C, 62.11; H, 5.45; N, 9.88. Found C, 62.21; H, 5.47; N, 10.01.

Ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-1-thio-α-D-mannopyranoside (7a) and ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-1-thio-β-D-mannopyranoside (7b)

To a stirred suspension of anomeric mixture of compounds 11a and 11b (115 g, 270.3 mmol) and powdered 4 Å molecular sieves (10.0 g) in dichloromethane (800 mL), ethanethiol (39 mL, 540.6 mmol) followed by boron trifluoride etherate (100 mL, 811 mmol) were added dropwise at 0 °C and the mixture was stirred at room temperature overnight, when TLC (4 : 1 hexane–EtOAc) showed that the reaction was complete. The mixture was neutralized with NEt₃ (113 mL, 811 mmol), filtered through a Celite pad and the filtrate was concentrated with Chlorox in the receiving flask, to give crude product. A solution of the residue in DCM (300 mL) was washed with aq NaHCO₃, and the aqueous layer was backwashed with DCM (3× 100 mL). Concentration of the organic phase and chromatography (10 : 1 hexane–EtOAc) gave first the α-linked glycoside (7a, 97.3 g, 84%) as colorless syrup. R_f = 0.70 (hexane/ethyl acetate, 4/1). [α]_D +63.7 (c 1.3, CHCl₃), lit¹¹ [α]_D +65 (c 1.45, CHCl₃), ¹H NMR (400 MHz, CDCl₃): δ 8.07–8.05 (d, 2H, J = 7.8 Hz, Ar–H), 7.59 (app t, 1H, J = 7.5, 7.3 Hz, Ar–H), 7.49–7.43 (t, 2H, J = 7.7 Hz, Ar–H), 7.36–7.24 (m, 5H, Ar–H), 5.62 (t, 1H, J = 1.5 Hz, H-2), 5.34 (s, 1H, H-1), 4.74 (d, 1H, J = 11.3 Hz, CH₂Ph), 4.56 (d, 1H, J = 11.2 Hz, CH₂Ph), 3.96 (dq, 1H, J_5–4 = 10.0 Hz, J_5–6 = 6.2 Hz, H-5), 3.84 (dd, 1H, J_3–4 = 10.1 Hz, J_3–2 = 3.2 Hz, H-3), 3.55 (t, 1H, J = 9.9 Hz, H-4), 2.71–2.56 (m, 2H, SCH₂CH₃), 1.38 (d, 3H, J_6–5 = 6.3 Hz, CH₃), 1.29 (t, 3H, J = 7.4 Hz, SCH₂CH₃). ¹³C{1H} NMR (100 MHz, CDCl₃): δ 165.6 (C=O), 137.0, 133.3, 129.9 (2C), 129.7, 128.43 (2C), 128.4 (2C), 128.2 (2C), 127.9, 82.4 (C-1), 76.4 (C-3), 71.5 (CH₂Ph), 69.8 (C-2), 67.5 (C-5), 64.6 (C-4), 25.7 (SCH₂CH₃), 18.6 (C-6), 14.9 (SCH₂CH₃). HRMS (ESI-TOF): m/z [M + Na]⁺ calcd for C₂₂H₂₅O₄N₃SNa 450.1463, found 450.1459.

Continued elution gave the β-linked glycoside 7b as colorless syrup (2.1 g, 1.8%, Total 99.4 g, 86% overall, α/β ∼ 46 : 1). R_f = 0.69 (hexane/ethyl acetate, 4/1). Data for 7b, [α]_D −97.2 (c 1.0, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ 8.13–8.09 (d, 2H, J = 7.8 Hz, Ar–H), 7.57 (app t, 1H, J = 7.5, 7.4 Hz, Ar–H), 7.47–7.42 (t, 2H, J = 7.7 Hz, Ar–H), 7.38–7.25 (m, 5H, Ar–H), 5.86 (d, 1H, J_2–3 = 3.2 Hz, H-2), 4.86 (d, 1H, J = 11.2 Hz, CH₂Ph), 4.70 (s, 1H, H-1), 4.56 (d, 1H, J = 11.8 Hz, CH₂Ph), 3.61 (dd, 1H, J_3–4 = 9.9 Hz, J_3–2 = 3.3 Hz, H-3), 3.49 (t, 1H, J = 9.5 Hz, H-4), 3.61 (dq, 1H, J_5–4 = 9.7 Hz, J_5–6 = 6.2 Hz, H-5), 2.77–2.68 (m, 2H, SCH₂CH₃), 1.45 (d, 3H, J_6–5 = 6.3 Hz, CH₃), 1.30–1.24 (t, 3H, J = 7.3 Hz, SCH₂CH₃). ¹³C{1H} NMR (125 MHz, CDCl₃): δ 165.8 (C=O), 136.9, 133.3 (2C), 130.1, 129.4 (2C), 128.42 (2C), 128.39 (2C), 128.37, 127.9, 82.2 (C-1), 79.3 (C-3), 75.4 (C-5), 71.4 (CH₂Ph), 69.2 (C-2), 63.9 (C-4), 25.6 (SCH₂CH₃), 18.9 (C-6), 14.8 (SCH₂CH₃). HRMS (ESI-TOF): m/z [M + Na]⁺ calcd for C₂₂H₂₅O₄N₃SNa 450.1463, found 450.1461. Anal. calcd for C₂₂H₂₅O₄N₃S: C, 61.81; H, 5.89; N, 9.83. Found C, 61.93; H, 5.89; N, 9.58.

2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (13a)¹¹

Method 1. Attempted use of anomeric acetates 11a and 11b (α/β ∼ 9 : 1), prepared as described above, as glycosyl donors.

When compounds 11a and 11b (0.25 g, 0.6 mmol) were treated with 2-(trimethylsilyl) ethanol (0.25 mL, 1.8 mmol) in presence of either boron trifluoride etherate (0.12 mL, 0.9 mmol) or TMSOTf (0.1 mL, 0.6 mmol) as described above for the synthesis of thioglycosides 7a and 7b, TLC showed that a complex mixture was formed where the product of hydrolysis of the anomeric OAc group largely predominated. Optimization of reaction conditions for this approach was not attempted.

Method 2. From anomeric mixture of compounds 7a and 7b.

This compound was prepared as described¹¹ from 36 g (84.2 mmol) of anomeric mixture of 7a and 7b resulting in 38.3 g (94%) of pure compound 13a as white solid. R_f = 0.45 (hexane/ethyl acetate, 9/1). Mp. 75–76 °C (hexane), lit¹¹ mp. 73–75 °C. [α]_D −0.6 (c 1.0, CHCl₃), lit¹¹ [α]_D −1 (c 1, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 8.06–8.05 (d, 2H, J = 7.8 Hz, Ar–H), 7.57 (app t, 1H, J = 7.5, 7.2 Hz, Ar–H), 7.47–7.43 (m, 2H, Ar–H), 7.34–7.24 (m, 5H, Ar–H), 5.52 (s, 1H, H-2), 4.89 (s, 1H, H-1), 4.76 (d, 1H, J = 11.7 Hz, CH₂Ph), 4.56 (d, 1H, J = 11.3 Hz, CH₂Ph), 3.93 (dd, 1H, J_3–4 = 9.8 Hz, J_3–2 = 3.0 Hz, H-3), 3.78 (ddd, 1H, J = 6.8, 6.6, 6.4 Hz, OCH₂CH₂Si), 3.63 (ddd, 1H, J = 6.0, 6.2, 4.1 Hz, OCH₂CH₂Si), 3.56–3.47 (m, 2H, H-4,5), 1.37 (d, 3H, J_6–5 = 6.5 Hz, CH₃), 1.01–0.87 (m, 2H, OCH₂CH₂Si), 0.28 (s, 9H, (CH₃)₃Si). ¹³C{1H} NMR (100 MHz, CDCl₃): δ 165.9 (C=O), 137.5, 133.3, 129.9 (2C), 129.7, 128.4 (2C), 128.3 (2C), 128.2 (2C), 127.8, 97.3 (C-1), 76.2 (C-3), 71.4 (CH₂Ph), 68.1 (C-2), 66.9 (C-5), 65.5 (OCH₂Si), 64.4 (C-4), 18.7 (CH₃), 17.9 (CH₂Si), −1.33 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + Na]⁺ calcd for C₂₅H₃₃O₅N₃SiNa 506.2087, found 506.2089.

2-(Trimethylsilyl)ethyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (8)⁷

This compound was prepared as described⁷ from 38 g (78.6 mmol) of 13a giving pure compound 8 as colorless syrup (28 g, 94%). R_f = 0.47 (hexane/ethyl acetate, 4/1). [α]_D +122.1 (c 1.6, CHCl₃), lit⁷ [α]_D +121 (c 1.0, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ 7.41–7.30 (m, 5H, Ar–H), 4.82 (d, 1H, J_1–2 = 1.5 Hz, H-1), 4.71, 4.66 (2d, 1H each, ²J = 11.3 Hz, CH₂Ph), 3.95 (t, 1H, J = 1.5 Hz, H-2), 3.76 (dd, 1H, J = 9.9, 6.2 Hz, OCH₂CH₂Si), 3.73 (dd, 1H, J_3–4 = 9.7 Hz, J_3–2 = 3.5 Hz, H-3), 3.58–3.37 (m, 3H, OCH₂CH₂Si, H-4,5), 2.39 (d, 1H, J = 1.6 Hz, OH), 1.32 (d, 3H, J_6–5 = 6.2 Hz, CH₃), 0.98–0.82 (m, 2H, OCH₂CH₂Si), 0.01 [s, 9H, (CH₃)₃Si]. ¹³C{1H} NMR (100 MHz, CDCl₃): δ 137.3, 128.6, 128.2 (2C), 128.1 (2C), 98.5 (C-1), 78.4 (C-3), 72.0 (CH₂Ph), 67.4 (C-2), 66.5 (C-5), 65.2 (OCH₂Si), 64.1 (C-4), 18.4 (CH₃), 17.9 (CH₂Si), −1.34 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₁₈H₃₃O₄N₄Si 397.2271, found 397.2275.

2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (14)

To a solution of the glycosyl acceptor 8 (26 g, 68.5 mmol) and thioglycoside donor 7a (32.2 g, 75.3 mmol) in dry DCM (500 mL) was added 10 g of 4 Å powdered molecular sieves, and the mixture was stirred for 15 min. N-Iodosuccinimide (22.1 g, 90.4 mmol) was added, which resulted in slight pink color development. The stirring was continued for another 5 min, the reaction mixture was cooled at 0 °C, and TMSOTf (5.3 mL, 29.4 mmol) was added dropwise. Red color developed immediately and, after 20 min, when TLC (solvent 1, R_f = 0.72 at 4 : 1 hexane–EtOAc. Solvent 2, R_f = 0.26 at neat toluene) in both solvents showed complet conversion, the reaction was quenched by addition of triethylamine (4 mL, 29.4 mmol). The precipitate formed was filtered off (a pad of Celite) directly into a separating funnel containing excess of 2 : 1 (v/v) sodium thiosulfate (10%)-sodium bicarbonate (saturated) solution. The organic layer was extracted with DCM (3× 50 mL), dried and concentrated. The residue was chromatographed (10 : 1 hexane–EtOAc) and title compound 14 was obtained as colorless syrup (47.9 g, 94%), [α]_D +23.8 (c 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 8.05–8.03 (d, 2H, J = 7.5 Hz, Ar–H), 7.58 (t, 1H, J = 7.5 Hz, Ar–H), 7.48–7.45 (t, 2H, J = 7.5 Hz, Ar–H), 7.38–7.33 (m, 4H, ArH), 7.31–7.24 (m, 5H, ArH), 7.17 (t, 1H, J = 7.5 Hz, Ar–H), 5.62 (t, 1H, J = 2.5 Hz, H-2^II), 4.94 (d, 1H, J_1–2 = 1.6 Hz, H-1^II), 4.78 (d, 1H, J = 11.2 Hz, CH₂Ph), 4.71 (d, 1H, J_1–2 = 1.4 Hz, H-1^I), 4.69 (d, 1H, J = 11.5 Hz, CH₂Ph), 4.61 (d, 1H, J = 11.5 Hz, CH₂Ph), 4.57 (d, 1H, J = 11.5 Hz, CH₂Ph), 3.90 (dd, 1H, J_3–4 = 9.8 Hz, J_3–2 = 3.1 Hz, H-3^II), 3.83 (t, 1H, J = 2.2 Hz, H-2^I), 3.75 (dd, 1H, J_3–4 = 9.8 Hz, J_3–2 = 2.9 Hz, H-3^I), 3.71 (m, 1H, H-5^I), 3.66 (m, 1H, OCH_aCH₂Si), 3.52–3.47 (m, 2H, H-4^II, H-5^II), 3.43 (m, 1H, OCH_bCH₂Si), 3.33 (t, 1H, J = 9.9 Hz, H-4^I), 1.34 (d, 3H, J_6–5 = 6.2 Hz, CH₃, H-6^I), 1.29 (d, 3H, J_6–5 = 6.3 Hz, CH₃, H-6^II), 0.94–0.82 (m, 2H, CH₂CH₂Si), 0.01 [s, 9H, (CH₃)₃Si]. ¹³C{1H} NMR (150 MHz, CDCl₃): δ 165.3 (C=O), 137.6, 137.1, 133.2, 129.9 (2C), 129.7, 128.5 (2C), 128.4 (2C), 128.3 (2C), 127.94 (2C), 127.90 (2C), 127.8 (2C), 99.5 (C-1^II, J_C-1,H-1 = 173.3 Hz), 98.2 (C-1^I, J_C-1,H-1 = 167.7 Hz), 77.8 (C-3^I), 75.3 (C-3^II), 74.4 (C-2^I), 72.1 (CH₂Ph), 71.4 (CH₂Ph), 67.7 (C-2^II), 67.6 (C-5^I), 66.9 (C-5^II), 65.2 (OCH₂CH₂Si), 64.2 (C-4^I), 64.1 (C-4^II), 18.7 (CH₃, C-6^I), 18.6 (CH₃, C-6^II), 17.5 (CH₂Si), −1.32 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₃₈H₅₂O₈N₇Si 762.3647, found 762.3637. Anal. calcd for C₃₈H₄₈O₈N₆Si: C, 61.27; H, 6.5; N, 11.28. Found C, 61.54; H, 6.36; N, 11.09.

2-(Trimethylsilyl)ethyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (6)⁷

This compound was prepared as described⁷ from 47.5 g (63.8 mmol) of 14, to give 38.4 g (94%) of pure compound 6 as colorless syrup. R_f = 0.41 at hexane/ethyl acetate, 4/1. [α]_D +101.7 (c 1.1, CHCl₃), lit⁷ [α]_D +102 (c 1.0, CHCl₃). ¹H NMR (500 MHz, CDCl₃): δ 7.41–7.29 (m, 10H, Ar–H), 4.94 (d, 1H, J_1–2 = 1.4 Hz, H-1^II), 4.72, 4.68, 4.64, 4.61 (partially overlapped 4d, 4H, ²J ∼ 11.5 Hz, 2 CH₂Ph), 4.66 (partially overlapped d, 1H, J_1–2 = 4.4 Hz, H-1^I), 3.99 (m, 1H, H-2^II), 3.88 (t, 1H, J = 2.5 Hz, H-2^I), 3.75–3.68 (m, 3H, H-3^I,II, OCH_aCH₂Si), 3.61 (m, 1H, H-5^I), 3.51–3.40 (m, 3H, H-4^I, H-5^II, OCH_bCH₂Si), 3.29 (t, 1H, J = 10.0 Hz, H-4^II), 2.29 (d, 1H, J = 1.7 Hz, OH), 1.30, 1.29 (overlapped 2d, 6H, J_6–5 = 6.2 Hz, CH₃, H-6^I,II), 0.94–0.82 (m, 2H, CH₂CH₂Si), 0.01 [s, 9H, (CH₃)₃Si]. ¹³C{1H} NMR (125 MHz, CDCl₃): δ 137.4, 137.1, 128.6 (2C), 128.5 (2C), 128.3 (2C), 128.2 (2C), 127.9 (2C), 100.7 (C-1^II), 98.3 (C-1^I), 77.9 (C-3^I), 77.6 (C-3^II), 73.8 (C-2^I), 72.1 (2C, CH₂Ph), 67.2 (C-2^II), 67.1 (C-5^I), 66.9 (C-5^II), 65.2 (OCH₂Si), 64.3 (C-4^II), 63.8 (C-4^I), 18.6 (CH₃), 18.4 (CH₃), 17.7 (CH₂Si), −1.32 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₃₁H₄₈O₇N₇Si 658.3384, found 658.3389.

2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (15)

To a solution of disaccharide acceptor 6 (38 g, 59.3 mmol) and thioglycoside donor 7a (28 g, 65.2 mmol) in dry DCM (500 mL) was added 10 g of 4 Å powdered molecular sieves, and the mixture was stirred for 15 min. N-Iodosuccinimide (19.2 g, 78.2 mmol) was added, which resulted in slight pink color development. The stirring was continued for another 5 min, and the reaction mixture was cooled at 0 °C. TMSOTf (4.7 mL, 26.1 mmol) was added, whereupon red color developed immediately. After 20 min at 0 °C, when TLC in 2 solvents (1. R_f = 0.72 at 4 : 1 hexane–EtOAc; 2. R_f = 0.32 at neat toluene) showed that the reaction was complete, the reaction was terminated by addition of triethylamine (3.7 mL). The precipitate formed was filtered through a pad of Celite directly into a separating funnel containing excess of 2 : 1 (v/v) sodium thiosulfate (10%)–sodium bicarbonate (saturated) solution. The mixture was extracted with DCM (3× 50 mL), the combined organic layers were dried, concentrated, and chromatography (12 : 1 hexane : EtOAc) gave product 15 as colorless syrup (55.8 g, 93%). [α]_D +34.0 (c 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 8.07–8.05 (d, 2H, J = 7.3 Hz, Ar–H), 7.61 (t, 1H, J = 7.4 Hz, Ar–H), 7.50–7.47 (t, 2H, J = 7.7 Hz, Ar–H), 7.38–7.37 (m, 2H, ArH), 7.34–7.25 (m, 11H, ArH), 7.20–7.15 (m, 2H, Ar–H), 5.59 (dd, 1H, J_2–3 = 2.9 Hz, J_2–1 = 2.0 Hz, H-2^III), 4.97 (d, 1H, J_1–2 = 1.6 Hz, H-1^II), 4.89 (d, 1H, J_1–2 = 1.6 Hz, H-1^III), 4.76 (d, 1H, J = 11.0 Hz, CH₂Ph), 4.71 (d, 1H, J = 11.7 Hz, CH₂Ph), 4.65 (d, 1H, J_1–2 = 1.6 Hz, H-1^I), 4.63 (d, 1H, J = 11.6 Hz, CH₂Ph), 4.60–4.55 (3d, 3H, J = 11.5 Hz, 11.4 Hz, 11.3 Hz, 3CH₂Ph), 3.88–3.85 (m, 2H, H-2^II, H-3^III), 3.82 (t, 1H, J = 2.3 Hz, H-2^I), 3.73 (dd, 1H, J_3–4 = 9.9 Hz, J_3–2 = 2.9 Hz, H-3^II), 3.73–3.68 (m, 2H, H-3^I, OCH_aCH₂Si), 3.60–3.51 (m, 2H, H-5^III, H-5^II), 3.49–3.40 (m, 3H, H-4^III, H-5^I, OCH_bCH₂Si), 3.35 (t, 1H, J = 10.0 Hz, H-4^II), 3.22 (t, 1H, J = 9.9 Hz, H-4^I), 1.28, 1.27 (overlapped 2d, 3H each, J_6–5 = 6.1 Hz, CH₃, H-6^II, H-6^III), 1.23 (d, 3H, J_6–5 = 6.1 Hz, CH₃, H-6^I), 0.93–0.82 (m, 2H, CH₂CH₂Si), 0.01 [s, 9H, (CH₃)₃Si]. ¹³C{1H} NMR (150 MHz, CDCl₃): δ 165.3 (C=O), 137.4, 137.3, 137.1, 133.3, 129.9, 129.8 (2C), 128.6, 128.5 (2C), 128.46 (2C), 128.45 (2C), 128.4 (2C), 128.3 (2C), 128.1 (2C), 128.0 (2C), 127.9 (2C), 100.4 (C-1^II, J_C–H = 173.7 Hz), 99.2 (C-1^III, J_C–H = 173.3 Hz), 98.3 (C-1^I, J_C–H = 168.6 Hz), 77.7 (C-3^I), 76.7 (C-3^II), 75.3 (C-3^III), 74.1 (C-2^II), 74.0 (C-2^I), 72.1 (2C, 2 CH₂Ph), 71.4 (CH₂Ph), 67.7 (C-5^II), 67.64 (C-5^III), 67.6 (C-2^III), 67.0 (C-5^I), 65.2 (OCH₂CH₂Si), 64.5 (C-4^I), 64.1 (C-4^III), 64.0 (C-4^II), 18.7 (CH₃, C-6^I), 18.6 (CH₃, C-6^II), 18.58 (CH₃, C-6^III), 17.8 (CH₂Si), −1.32 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₅₁H₆₇O₁₁N₁₀Si 1023.4760, found 1023.4742. Anal. calcd for C₅₁H₆₃O₁₁N₉Si: C, 60.88; H, 6.31; N, 12.53. Found C, 61.17; H, 6.05; N, 12.32.

2-(Trimethylsilyl)ethyl 4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (4)

To the solution of trisaccharide 15 (23 g, 22.8 mmol) in a mixture of dry DCM (50 mL) and dry MeOH (50 mL), methanolic NaOMe (1 M, 10 mL) was added under Ar, and the mixture was stirred at room temperature overnight, when TLC (R_f = 0.54 at 4 : 1 hexane : EtOAc) showed that the reaction was complete and that a much slower moving product was formed. The mixture was neutralized with Dowex 50W resin, filtered, and the solvent was removed. The crude product was passed through a short pad of silica and elution with 5 : 1 hexane–EA afforded pure 4 as white solid. Crystallization from hot hexane gave needles (19.4 g, 94%), mp. 72–73 °C, [α]_D +102.86 (c 1.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 7.40–7.29 (m, 15H, Ar–H), 4.96 (d, 1H, J_1–2 = 1.3 Hz, H-1^III), 4.95 (d, 1H, J_1–2 = 1.6 Hz, H-1^II), 4.72 (d, 1H, J = 11.4 Hz, CH₂Ph), 4.69–4.64 (m, 3H, 2 CH₂Ph, H-1^I), 4.62 (d, 1H, J = 11.5 Hz, CH₂Ph), 4.61 (d, 1H, J = 11.6 Hz, CH₂Ph), 4.57 (d, 1H, J = 11.6 Hz, CH₂Ph), 3.98 (ddd, 1H, J_2–3 = 2.8 Hz, J_2-OH-2 = 1.7 Hz, J_2–1 = 1.3 Hz, H-2^III), 3.93 (t, 1H, J = 2.2 Hz, H-2^II), 3.81 (t, 1H, J = 2.3 Hz, H-2^I), 3.74–3.71 (m, 1H, H-3^II), 3.71–3.67 (m, 3H, H-3^III, H-3^I, OCH_aCH₂Si), 3.56–3.51 (m, 1H, H-5^II), 3.51–3.47 (m, 1H, H-5^III), 3.47–3.38 (m, 3H, H-5^I, OCH_bCH₂Si, H-4^III), 3.32 (t, 1H, J = 10.0 Hz, H-4^II), 3.22 (t, 1H, J = 9.9 Hz, H-4^I), 2.27 (d, 1H, J_2-OH-2 = 1.8 Hz, OH), 1.28 (2d, 3H each, J_6–5 = 6.1 Hz, CH₃, H-6^II, H-6^I), 1.18 (d, 3H, J_6–5 = 6.1 Hz, CH₃, H-6^III), 0.92–0.83 (m, 2H, CH₂CH₂Si), 0.01 [s, 9H, (CH₃)₃Si]. ¹³C{1H} NMR (150 MHz, CDCl₃): δ 137.31, 137.26, 137.1, 128.6 (2C), 128.57, 128.3 (2C), 128.26 (2C), 128.21 (2C), 128.17 (2C), 128.1 (2C), 128.0 (2C), 100.42 (C-1^II), 100.4 (C-1^III), 98.3 (C-1^I), 77.6 (C-3^II), 77.5 (C-3^I), 76.9 (C-3^III), 73.9 (C-2^II), 73.2 (C-2^I), 72.2 (2C, 2 CH₂Ph), 72.1 (CH₂Ph), 67.7 (C-5^II), 67.3 (C-5^III), 67.1 (C-2^III), 67.0 (C-5^I), 65.2 (OCH₂CH₂Si), 64.4 (C-4^I), 64.2 (C-4^II), 63.8 (C-4^III), 18.64 (CH₃, C-6^I), 18.56 (CH₃, C-6^III), 18.3 (CH₃, C-6^II), 17.7 (CH₂Si), −1.32 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₄₄H₆₃O₁₀N₁₀Si 919.4498; found 919.4483. Anal. calcd for C₄₄H₅₉O₁₀N₉Si: C, 58.58; H, 6.59; N, 13.97. Found C, 58.77; H, 6.44; N, 13.77.

4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-D-mannopyranose (16)

A solution of glycoside 16 (30 g, 30 mmol) in trifluoroacetic acid (TFA, 120 mL) was stirred at room temperature for 2 hours, when TLC (4 : 1 hexane–EtOAc) showed that the reaction was complete and that a much slower moving product was formed. The mixture was concentrated and a solution of the residue in DCM was washed with saturated Na₂CO₃ solution. The aqueous layer was extracted with DCM (3× 50 mL), and the combined organic phase was dried and concentrated. The residue was chromatographed (R_f = 0.27 at 4 : 1 hexane–EtOAc) to give pure product 16 as white foam (23.9 g, 89%). ¹H NMR for the major α anomer (600 MHz, CDCl₃): δ 8.07–8.06 (d, 2H, J = 7.3 Hz, Ar–H), 7.61 (t, 1H, J = 7.5 Hz, Ar–H), 7.50–7.47 (t, 2H, J = 7.7 Hz, Ar–H), 7.39–7.27 (m, 13H, Ar–H), 7.20–7.16 (m, 2H, Ar–H), 5.59 (dd, 1H, J_2–3 = 2.9 Hz, J_2–1 = 2.0 Hz, H-2^III), 5.08 (dd, 1H, J_1-1-OH = 3.2 Hz, J_1–2 = 1.8 Hz, H-1^I), 4.98 (d, 1H, J_1–2 = 1.6 Hz, H-1^II), 4.89 (d, 1H, J_1–2 = 1.6 Hz, H-1^III), 4.77 (d, 1H, J = 11.6 Hz, CH₂Ph), 4.71 (d, 1H, J = 11.7 Hz, CH₂Ph), 4.65–4.55 (m, 4H, 4CH₂Ph), 3.89–3.84 (m, 3H, H-2^I, H-2^II, H-3^III), 3.75 (dd, 1H, J_3–4 = 10.0 Hz, J_3–2 = 2.8 Hz, H-3^I), 3.73 (dd, 1H, J_3–4 = 10.0 Hz, J_3–2 = 2.9 Hz, H-3^II), 3.68 (m, 1H, H-5^I), 3.58 (m, 1H, H-5^III), 3.52 (m, 1H, H-5^II), 3.47 (t, 1H, J = 10.0 Hz, H-4^III), 3.35 (t, 1H, J = 10.0 Hz, H-4^II), 3.22 (t, 1H, J = 10.0 Hz, H-4^I), 2.54 (d, 1H, J_1-OH-1 = 3.5 Hz, OH), 1.28, 1.27 (overlapped 2d, 3H each, J_6–5 = 6.1 Hz each, CH₃, H-6^II, H-6^I), 1.23 (d, 3H, J_6–5 = 6.1 Hz, CH₃, H-6^III). ¹³C{1H} NMR (150 MHz, CDCl₃): δ 165.3 (C=O), 137.4, 137.3, 137.1, 133.3, 129.9, 129.7, 128.52, 128.5 (2C), 128.44 (2C), 128.43, 128.4 (2C), 128.3 (2C), 128.2 (2C), 128.1 (2C), 128.04 (2C), 128.0, 127.9, 100.3 (C-1^II), 99.2 (C-1^III), 93.5 (C-1^I), 77.0 (C-3^I), 76.6 (C-3^II), 75.3 (C-3^III), 74.0 (C-2^II), 73.9 (C-2^I), 72.2 (CH₂Ph), 72.0 (CH₂Ph), 71.3 (CH₂Ph), 67.7 (C-5^II), 67.63 (C-5^III), 67.6 (C-2^III), 67.2 (C-5^I), 64.4 (C-4^I), 64.1 (C-4^III), 64.0 (C-4^II), 18.7 (CH₃, C-6^I), 18.6 (CH₃, C-6^II), 18.5 (CH₃, C-6^III). HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₄₆H₅₅O₁₁N₁₀ 923.4052; found 923.4054.

4-Azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-D-mannopyranosyl trichloroacetimidate (5)

1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU, 1.95 mL, 13 mmol) was added at 0 °C to a solution of hemiacetal 16 (23.5 g, 26 mmol) and trichloroacetonitrile (3.9 mL, 39 mmol) in dry DCM (100 mL). The mixture was stirred for 2 h, when TLC (R_f = 0.57 at 4 : 1 hexane–EtOAc) showed complete consumption of starting material and formation of a faster moving product. The mixture was concentrated to a small volume and applied onto a short column of silica gel. Elution with 10 : 1 hexane–EtOAc gave trichloroacetimidate donor 5 as colorless syrup (24.8 g, 91%). ¹H NMR for the major isomer (600 MHz, CDCl₃): δ 8.59 (s, 1H, NH), 8.07–8.06 (d, 2H, J = 7.8 Hz, Ar–H), 7.61 (t, 1H, J = 7.5 Hz, Ar–H), 7.50–7.47 (t, 2H, J = 7.6 Hz, Ar–H), 7.41–7.39 (m, 2H, Ar–H), 7.36–7.21 (m, 13H, Ar–H), 6.07 (s, 1H, H-1^I), 5.61 (s, 1H, H-2^III), 4.99 (s, 1H, H-1^II), 4.98 (s, 1H, H-1^III), 4.78 (d, 1H, J = 11.5 Hz, CH₂Ph), 4.75 (d, 1H, J = 11.5 Hz, CH₂Ph), 4.67–4.56 (m, 4H, 4CH₂Ph), 3.93–3.86 (m, 3H, H-2^I, H-2^II, H-3^III), 3.75–3.71 (m, 2H, H-3^I, H-3^II), 3.66–3.55 (m, 3H, H-5^I, H-5^II, H-5^III), 3.49 (t, 1H, J = 9.9 Hz, H-4^III), 3.38 (t, 1H, J = 10.0 Hz, H-4^II), 3.33 (t, 1H, J = 10.0 Hz, H-4^I), 1.32 (d, 3H, J_6–5 = 6.3 Hz, CH₃, H-6^II), 1.30 (d, 3H, J_6–5 = 6.1 Hz, CH₃, H-6^I), 1.28 (d, 3H, J_6–5 = 6.2 Hz, CH₃, H-6^III). ¹³C{1H} NMR (150 MHz, CDCl₃): δ 165.3 (C=O), 159.9 (C=O), 137.3, 137.1, 136.8, 133.3, 129.9 (2C), 129.7, 128.6 (2C), 128.5 (2C), 128.44 (2C), 128.42 (2C), 128.4 (2C), 128.3 (2C), 128.2 (2C), 128.1 (2C), 127.9, 100.4 (C-1^II), 99.2 (C-1^III), 96.2 (C-1^I), 76.7 (C-3^I), 76.5 (C-3^II), 75.3 (C-3^III), 73.8 (C-2^I), 72.5 (CH₂Ph), 72.2 (CH₂Ph), 71.9 (C-2^II), 71.4 (CH₂Ph), 70.1 (C-5^II), 68.1 (C-5^III), 67.7 (C-2^III), 67.6 (C-5^I), 64.0 (C-4^I), 63.9 (C-4^III), 63.7 (C-4^II), 18.64 (CH₃, C-6^I), 18.62 (CH₃, C-6^II), 18.5 (CH₃, C-6^III). HRMS (ESI-TOF): m/z [M + 18]⁺ calcd for C₄₈H₅₁O₁₁N₁₀Cl₃ 1048.2804; found 1048.2810.

2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-[4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)]₄-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (3a) and 2-(Trimethylsilyl)ethyl 4-azido-2-O-benzoyl-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-β-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (3b)

To a solution of the trisaccharide acceptor 4 (19 g, 21 mmol) and trisaccharide trichloroacetimidate donor 5 (24.3 g, 23.2 mmol) in dry DCM (300 mL) was added 5 g of 4 Å powdered molecular sieves, and the mixture was stirred for 15 min. The mixture was cooled to −40 °C, TMSOTf (1.7 mL, 9.3 mmol) was added dropwise at the rate of 0.1 mL min⁻¹ over a period of 17 min using a syringe pump. After additional 30 min at −40 °C TLC in two solvents (1. R_f = 0.74 at 4 : 1 hexane–EtOAc and 2. R_f = 0.40 at neat toluene) showed that the glycosyl acceptor was completely consumed. Two faster moving spots were formed, one of which strongly predominated, indicating formation of anomeric hexasaccharides. The mixture was neutralized with NEt₃ (1.3 mL, 9.3 mmol), filtered through Celite pad and filtrate combined with the washings were concentrated. The residue was chromatographed (8 : 1 hexane–EtOAc) to give first the α-linked hexasaccharide (3a, 33.72 g, 90%) as colorless syrup, [α]_D +73.0 (c 2.0, CHCl₃). ¹H NMR (600 MHz, C₆D₆): δ 8.20–8.19 (d, 2H, J = 7.3 Hz, Ar–H), 7.43–7.38 (d, 2H, J = 7.4 Hz, Ar–H), 7.38–7.31 (m, 10H, Ar–H), 7.31–7.19 (m, 11H, Ar–H), 7.19–7.08 (m, 5H, Ar–H), 7.06–7.01 (m, 5H, Ar–H), 5.93 (dd, 1H, J_2–3 = 2.9 Hz, J_2–1 = 1.9 Hz, H-2^VI), 5.32 (d, 1H, J = 1.3 Hz, H-1^V), 5.25 (d, 1H, J = 1.1 Hz, H-1^IV), 2.23 (d, 1H, J = 1.2 Hz, H-1^III), 5.21 (d, 1H, J = 1.2 Hz, H-1^II), 5.16 (d, 1H, J = 1.3 Hz, H-1^VI), 4.89 (d, 1H, J = 1.3 Hz, H-1^I), 4.59 (d, 1H, J = 11.5 Hz, CH₂Ph), 4.49–4.43 (dd, 2H, I = 16.6, 11.5 Hz, CH₂Ph), 4.42–4.33 (m, 7H, CH₂Ph), 4.31 (d, 1H, J = 11.8 Hz, CH₂Ph), 4.30 (d, 1H, J = 11.3 Hz, CH₂Ph), 4.15 (t, 1H, J = 2.4 Hz, H-2^IV), 4.12 (dd, 1H, J_3–4 = 10.0 Hz, J_3–2 = 3.2 Hz, H-3^VI), 4.10 (t, 1H, J = 2.2 Hz, H-2^III), 4.09 (t, 1H, J = 2.4 Hz, H-2^II), 4.05 (t, 1H, J = 2.3 Hz, H-2^V), 3.98 (t, 1H, J = 2.4 Hz, H-2^I), 3.96–3.88 (m, 6H, H-5^VI, H-3^V, H-3^IV, H-3^III H-3^II, H-3^I), 3.80 (m, 1H, H-5^V), 3.77–3.68 (m, 5H, OCH_bCH₂Si, H-4^VI, H-5^IV, H-5^III, H-5^I), 3.67–3.61 (m, 2H, H-5^II, H-4^I), 3.60–3.51 (m, 4H, H₄^V, H-4^IV, H-4^III, H-4^II), 3.34 (m, 1H, OCH_bCH₂Si), 1.33 (d, 3H, J_6–5 = 6.4 Hz, CH₃, H-6^II), 1.32 (d, 3H, J_6–5 = 6.5 Hz, CH₃, H-6^V), 1.30 (2d, 6H, J = 6.1 Hz, H-6^VI, H-6^I), 1.28 (d, 3H, J_6–5 = 6.5 Hz, CH₃, H-6^IV), 1.26 (d, 3H, J_6–5 = 6.0 Hz, CH₃, H-6^III), 0.85–0.76 (m, 2H, CH₂CH₂Si), 0.06 (s, 9H, (CH₃)₃Si). ¹³C{1H} NMR (150 MHz, C₆D₆): δ 165.9 (C=O), 138.3, 138.1, 137.9, 137.89, 137.88 (2C), 133.7, 130.7, 130.5 (2C), 129.3 (2C), 129.28 (2C), 129.24 (2C), 129.22 (2C), 129.2 (2C), 129.17 (2C), 129.15 (2C), 129.1 (2C), 129.07 (2C), 129.05 (3C), 129.0 (2C), 128.9 (3C), 128.8 (3C), 128.7 (2C), 128.6, 101.2 (C-1^II, J_C-1,H-1 = 173.2 Hz), 101.06 (C-1^III, J_C-1,H-1 = 173.8 Hz), 101.04 (C-1^IV, J_C-1,H-1 = 174.4 Hz), 101.03 (C-1^V, J_C-1,H-1 = 174.2 Hz), 100.3 (C-1^VI, J_C-1,H-1 = 172.8 Hz), 99.16 (C-1^I, J_C-1,H-1 = 170.9 Hz), 78.7 (C-3^I), 77.9 (C-3^IV), 77.8 (2C, C-3^II, C-3^III), 77.6 (C-3^V), 76.1 (C-3^VI), 75.0 (C-2^V), 74.6 (C-2^I), 73.99 (C-2^II), 73.97 (C-2^IV), 73.7 (C-2^III), 72.8 (CH₂Ph), 72.7 (3C, 3× CH₂Ph), 72.3 (CH₂Ph), 71.6 (CH₂Ph), 68.8 (2C, C-5^I, C-5^IV), 68.7 (C-5^V), 68.6 (C-5^III), 68.5 (2C, C-5^VI, C-2^VI), 67.9 (C-5^II), 65.7 (OCH₂CH₂Si), 65.5 (C-4^V), 65.2 (C-4^IV), 65.1 (C-4^III), 65.05 (C-4^VI), 65.02 (C-4^II), 64.9 (C-4^I), 19.14 (3× CH₃, C-6^VI, C-6^III, C-6^II), 19.11 (CH₃, C-6^V), 19.09 (CH₃, C-6^IV), 19.08 (CH₃, C-6^I), 18.2 (CH₂Si), −0.98 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₉₀H₁₁₂O₂₀N₁₉Si 1806.8100; found 1806.8083. Anal. calcd for C₉₀H₁₀₈O₂₀N₁₈Si: C, 60.39; H, 6.08; N, 14.08. Found C, 60.22; H, 5.92; N, 13.89.

Continued elution gave the β-linked hexasaccharide 3b as colorless syrup (0.98 g, 2.5%, Total yield of the glycosylation, 34.7 g, 92%, α/β ∼ 34 : 1). Data for 3b, [α]_D +17.6 (c 2.0, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 8.02–7.98 (d, 2H, J = 7.1 Hz, Ar–H), 7.56 (t, 1H, J = 7.5 Hz, Ar–H), 7.50–7.47 (d, 2H, J = 7.5 Hz, Ar–H), 7.46–7.43 (t, 2H, J = 7.8 Hz, Ar–H), 7.43–7.36 (m, 8H, Ar–H), 7.36–7.31 (m, 6H, Ar–H), 7.31–7.22 (m, 5H, Ar–H), 7.22–7.17 (m, 3H, Ar–H), 7.17–7.09 (m, 5H, Ar–H), 7.04 (m, 1H, Ar–H), 5.59 (dd, 1H, J_2–3 = 2.8 Hz, J_2–1 = 2.0 Hz, H-2^VI), 5.31 (d, 1H, J = 0.9 Hz, H-1^V), 4.99 (d, 1H, J = 1.0 Hz, H-1^II), 4.87 (s, 1H, H-1^VI), 4.87 (s, 1H, H-1^III), 4.79 (d, 1H, J = 10.8 Hz, CH₂Ph), 4.76 (d, 1H, J = 11.0 Hz, CH₂Ph), 4.71 (d, 1H, J = 11.8 Hz, CH₂Ph), 4.70 (d, 1H, J = 10.8 Hz, CH₂Ph), 4.68 (d, 1H, J = 1.5 Hz, H-1^I), 4.66 (d, 1H, J = 10.8 Hz, CH₂Ph), 4.60 (d, 1H, J = 11.6 Hz, CH₂Ph), 4.57 (d, 1H, J = 11.0 Hz, CH₂Ph), 4.54 (d, 1H, J = 11.6 Hz, CH₂Ph), 4.52 (d, 1H, J = 11.60 Hz, CH₂Ph), 4.46 (d, 1H, J = 10.8 Hz, CH₂Ph), 4.24 (d, 1H, J = 11.3 Hz, CH₂Ph), 4.15 (dd, 1H, J = 3.3, 1.5 Hz, H-2^III), 4.10 (t, 1H, J = 2.2 Hz, H-2^II), 4.03 (d, 1H, J = 1.9 Hz, H-2^IV), 3.99 (d, 1H, J = 11.5 Hz, CH₂Ph), 3.92 (m, 1H, H-5^V), 3.89 (t, 1H, J = 2.2 Hz, H-2^I), 3.86 (t, 1H, J = 2.0 Hz, H-2^V), 3.83 (dd, 1H, J_3–4 = 9.5 Hz, J_3–2 = 3.0 Hz, H-3^VI), 3.81 (dd, 1H, J_3–4 = 11.3 Hz, J_3–2 = 3.0 Hz, H-3^V), 3.77–3.69 (m, 4H, OCH_aCH₂Si, H-1^IV, H-3^II, H-3^I), 3.68 (dd, 1H, J_3–4 = 10.1 Hz, J_3–2 = 3.4 Hz, H-3^III), 3.63 (t, 1H, J = 10.0 Hz, H-4^III), 3.54 (m, 1H, H-5^II), 3.51–3.36 (m, 6H, OCH_bCH₂Si, H-4^VI, H-4^V, H-5^VI, H-5^III, H-5^I), 3.34 (t, 1H, J = 10.0 Hz, H-4^II), 3.33 (t, 1H, J = 10.0 Hz, H-4^I), 3.29 (t, 1H, J = 9.9 Hz, H-4^IV), 3.00 (dd, 1H, J_3–4 = 9.8 Hz, J_3–2 = 2.3 Hz, H-3^IV), 2.46 (dq, 1H, J_5–4 = 3.8 Hz, J_5–6 = 6.1 Hz, H-5^IV), 1.34 (d, 3H, J_6–5 = 6.1 Hz, CH₃, H-6^V), 1.31 (d, 3H, J_6–5 = 6.5 Hz, CH₃, H-6^I), 1.29 (d, 3H, J_6–5 = 6.5 Hz, CH₃, H-6^II), 1.22 (d, 3H, J_6–5 = 6.1 Hz, CH₃, H-6^IV), 1.17 (d, 3H, J_6–5 = 6.2 Hz, CH₃, H-6^III), 1.12 (d, 3H, J_6–5 = 6.0 Hz, CH₃, H-6^VI), 0.95–0.83 (m, 2H, CH₂CH₂Si), 0.01 (s, 9H, (CH₃)₃Si). ¹³C{1H} NMR (150 MHz, CDCl₃): δ 165.4 (C=O), 138.2, 137.9, 137.4, 137.2, 137.1, 136.9, 133.1, 129.9 (2C), 129.8, 128.7 (2C), 128.6 (2C), 128.5 (2C), 128.45 (2C), 124.43 (3C), 128.1 (3C), 128.06 (3C), 128.03 (3C), 128.0 (3C), 127.8 (3C), 127.7 (3C), 127.6 (2C), 127.3, 100.1 (C-1^II, J_C-1,H-1 = 170.5 Hz), 99.4 (C-1^VI, J_C-1,H-1 = 173.7 Hz), 98.33 (C-1^I, J_C-1,H-1 = 169.3 Hz), 98.26 (C-1^III, J_C-1,H-1 = 170.8 Hz), 97.4 (C-1^V, J_C-1,H-1 = 177.3 Hz), 96.3 (C-1^IV, J_C-1,H-1 = 155.5 Hz), 80.8 (C-3^IV), 78.3 (C-3^I), 77.8 (C-3^II), 77.7 (C-3^V), 75.5 (C-3^VI), 75.3 (C-3^III), 74.1 (C-2^V), 72.9 (C-2^I), 72.7 (CH₂Ph), 72.5 (C-2^II), 72.2 (CH₂Ph), 71.9 (CH₂Ph), 71.5 (CH₂Ph), 71.3 (CH₂Ph), 70.6 (C-5^IV), 69.9 (CH₂Ph), 69.2 (C-2^III), 67.7 (C-5^II), 67.6 (C-5^VI), 67.43 (C-5^III), 67.40 (C-2^VI), 67.10 (C-5^I), 66.9 (C-5^V), 66.6 (C-2^IV), 65.2 (OCH₂CH₂Si), 64.5 (C-4^I), 64.4 (C-4^II), 64.3 (C-4^V), 64.05 (C-4^VI), 64.01 (C-4^IV), 62.9 (C-4^III), 18.9 (CH₃, C-6^V), 18.6 (CH₃, C-6^I), 18.5 (CH₃, C-6^II), 18.3 (CH₃, C-6^IV), 18.23 (CH₃, C-6^III), 18.21 (CH₃, C-6^VI), 17.7 (CH₂Si), −1.33 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₉₀H₁₁₂O₂₀N₁₉Si 1806.8100; found 1806.8089. Anal. calcd for C₉₀H₁₀₈O₂₀N₁₈Si: C, 60.39; H, 6.08; N, 14.08. Found C, 60.46; H, 6.15; N, 14.06.

2-(Trimethylsilyl)ethyl [4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl-(1→2)]₅-4-azido-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (17)¹⁴

This compound was prepared as described¹⁴ from 2 g (1.1 mmol) of 3a giving 1.8 g (94%) of pure compound 17 as colorless syrup. R_f = 0.55 at 4 : 1 hexane–EtOAc. [α]_D +115.3 (c 1.0, CHCl₃), lit¹⁴ [α]_D +112. ¹H NMR (600 MHz, C₆D₆): δ 7.38–7.34 (m, 4H, Ar–H), 7.34–7.29 (m, 6H, Ar–H), 7.29–7.24 (m, 7H, Ar–H), 7.24–7.18 (m, 7H, Ar–H), 7.18–7.12 (m, 4H, Ar–H), 7.12–7.08 (m, 2H, Ar–H), 5.29 (d, 1H, J = 1.3 Hz, H-1^VI), 5.25 (brs, 1H, H-1^V), 5.21 (d, 1H, J = 1.3 Hz, H-1^II), 5.19 (brs, 2H, H-1^III, H-1^IV), 4.88 (d, 1H, J = 1.4 Hz, H-1^I), 4.47–4.31 (m, 8H, CH₂Ph), 4.31–4.27 (m, 2H, CH₂Ph), 4.19–4.13 (m, 3H, 2 CH₂Ph, H-2^V), 4.01 (t, 1H, J = 2.2 Hz, H-2^II), 4.09–4.06 (m, 2H, H-2^III, H-2^IV), 4.01 (brs, 1H, H-2^VI), 3.97 (t, 1H, J = 2.3 Hz, H-2^I), 3.96–3.86 (m, 5H, H-3^I–V), 3.80–3.60 (m, 9H, H-5^I–VI, H-4^II, H-3^VI, OCH_aCH₂Si), 3.59–3.49 (m, 4H, H-4^I–V), 3.46 (t, 1H, J = 10.0 Hz, H-4^VI), 3.33 (m, 1H, OCH_bCH₂Si), 2.08 (d, 1H, J = 1.8 Hz, H-2^VI-OH), 1.32 (d, 3H, J_6–5 = 6.3 Hz, CH₃, H-6), 1.31–1.23 (m, 15H, 5 CH₃, H-6), 0.83–0.76 (m, 2H, CH₂CH₂Si), 0.06 (s, 9H, (CH₃)₃Si). ¹³C{1H} NMR (150 MHz, C₆D₆): δ 138.1 (2C), 138.0 (2C), 137.9 (2C), 129.33 (3C), 129.3, 129.28 (3C), 129.23 (3C), 129.2 (3C), 129.13 (3C), 129.1 (2C), 128.98 (2C), 128.96 (2C), 128.8 (3C), 128.75 (2C), 128.7 (2C), 128.6 (2C), 101.5 (C-1^VI), 101.2 (C-1^IV), 101.1 (C-1^V), 100.06 (C-1^II), 100.04 (C-1^III), 99.2 (C-1^I), 78.7 (C-3^I), 78.5 (C-3^VI), 77.82 (C-3^IV), 77.8 (2C, C-3^II,II), 77.7 (C-3^V), 74.6 (C-2^I), 74.0 (C-2^IV), 73.8 (C-2^III), 73.7 (C-2^II), 73.3 (C-2^V), 72.8 (CH₂Ph), 72.7 (CH₂Ph), 72.69 (CH₂Ph), 72.6 (CH₂Ph), 72.3 (CH₂Ph), 71.8 (CH₂Ph), 68.83 (C-5^V), 68.78 (C-5^VI), 68.72 (2C, C-5^I,III), 68.3 (C-5^II), 67.9 (C-5^IV), 67.6 (C-2^VI), 65.7 (OCH₂CH₂Si), 65.5 (C-4^II), 65.14, 65.12, 65.1 and 65.07, (4C, C-4^I, C-4^III, C-4^IV and C-4^V), 64.5 (C-4^VI), 19.14 (2× CH₃, 2× C-6), 19.11 (CH₃, C-6), 19.1 (CH₃, C-6), 19.08 (CH₃, C-6), 18.9 (CH₃, C-6), 18.2 (CH₂Si), −0.98 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₈₃H₁₀₈O₁₉N₁₉Si 1702.7838; found 1702.7826.

2-(Trimethylsilyl)ethyl [4-amino-3-O-benzyl-4,6-dideoxy-α-D-mannopyranosyl-(1→2)]₅-4-amino-3-O-benzyl-4,6-dideoxy-α-D-mannopyranoside (18)¹⁴

Compound 17 (0.75 g, 0.45 mmol) was dissolved in pyridine/triethylamine 7 : 3 (20 mL). Hydrogen sulfide (H₂S) was passed through the above solution for 2 h and the resulting dark solution was stirred at room temperature, in the same flask equipped with an empty balloon, to ensure exclusion of atmospheric oxygen, overnight. TLC (R_f = 0.51 at DCM–MeOH 10 : 1) showed that the reaction was complete and that a slower moving product was formed. The reaction mixture was concentrated to dryness and coevaporated with toluene. The crude product was chromatographed and eluted with 1–3% ammonia (7 N solution in MeOH) in DCM (v/v). Compound 18 was obtained as a white foam (0.62 g, 92%), [α]_D −5.0 (c 2.1, CHCl₃), ¹H NMR (600 MHz, CDCl₃): δ 7.37–7.27 (m, 30H, Ar–H), 5.09 (s, 1H, H-1), 5.08–5.06 (s, 3H, 3× H-1), 5.01 (s, 1H, H-1^VI), 4.78 (s, 1H, H-1^I), 4.70–4.65 (m, 6H, CH₂Ph), 4.51 (d, 1H, J = 11.5 Hz, CH₂Ph), 4.47 (d, 1H, J = 12.0 Hz, CH₂Ph), 4.44–4.38 (m, 4H, CH₂Ph), 4.08–4.06 (m, 2H, 2× H-2), 4.03 (brs, 3H, 3× H-2), 3.92 (t, 1H, J = 2.3 Hz, H-2^I), 3.77–3.72 (m, 1H, OCH_aCH₂Si), 3.65–3.46 (m, 12H, 6× H-3, 6× H-5), 3.46–3.42 (m, 1H, OCH_bCH₂Si), 2.88–2.79 (m, 6H, 6× H-4), 1.38–1.21 (m, 18H, 12× NH₂, 2× CH₃, 2× H-6), 1.18–1.14 (m, 12H, 4× CH₃, 4× H-6), 0.96–0.90 (m, 1H, CH₂CH_aSi), 0.89–0.84 (m, 1H, CH₂CH_bSi), 0.02 (s, 9H, (CH₃)₃Si). ¹³C{1H} NMR (150 MHz, CDCl₃): δ 137.9 (2C), 137.8 (2C), 137.7, 137.6, 128.6 (2C), 128.56 (3C), 128.53 (2C), 128.5 (3C), 128.4 (2C), 128.36 (3C), 128.32 (3C), 128.2 (2C), 128.1 (2C), 128.0 (3C), 127.98 (3C), 127.9 (2C), 101.1 (C-1), 100.98 (C-1), 100.96 (C-1), 100.94 (2C, 2× C-1), 98.8 (C-1), 79.7 (C-3), 79.2 (3C, 3× C-3), 78.8 (C-3), 78.7 (C-3), 73.2 (C-2), 73.1 (C-2), 73.0 (C-2), 72.9 (C-2), 72.8 (C-2), 71.4 (CH₂Ph), 71.2 (CH₂Ph), 71.15 (CH₂Ph), 71.1 (CH₂Ph), 71.0 (2C, 2× CH₂Ph), 70.3 (4C, 4 C-5), 69.6 (C-5), 69.5 (C-5), 66.5 (C-2), 64.7 (OCH₂CH₂Si), 53.7 (2C, 2× C-4), 53.6 (3C, 3× C-4), 53.3 (C-4), 18.3 (CH₃, C-6), 18.2 (3C, CH₃, 3× C-6), 18.1 (CH₃, C-6), 17.9 (CH₃, C-6), 17.8 (CH₂Si), −1.3 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + H⁺] calcd for C₈₃H₁₁₇O₁₉N₆Si 1529.8143; found 1529.8136.

2-(Trimethylsilyl)ethyl 2-O-acetyl-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-[3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranosyl-(1→2)]₄-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranoside (19)

EDAC (0.45 g, 2.4 mmol) was added portion wise at room temperature to a stirred solution of amino sugar 18 (0.5 g, 0.33 mmol) and 2,4-di-O-acetyl-3-deoxy-L-glycero-tetronic acid¹⁵ (0.5 g, 2.5 mmol) in dry dichloromethane (20 mL). Stirring was continued overnight, when TLC showed complete consumption of starting material and formation of a much faster moving product (15 : 1 DCM–MeOH). The mixture was diluted with DCM (25 mL), washed with aq NaHCO₃ solution (3× 10 mL), brine (3× 10 mL), dried and concentrated. HRMS analysis of the crude product confirmed formation of the desired coupling product only (HRMS (ESI-TOF): m/z [M + H⁺] calcd for C₁₃₁H₁₇₇O₄₉N₆Si 2646.1312; found 2646.1313). The crude product was dissolved in dry pyridine (2 mL) and treated with acetic anhydride (0.1 mL) overnight at room temperature. TLC showed complete conversion of the starting material and formation of a faster moving product (R_f = 0.54 at 15 : 1 DCM–MeOH). After concentration, a solution of the residue in DCM (10 mL) was washed with cold 4 N HCl (3× 10 mL), aq NaHCO₃ solution (3× 10 mL), the phases were separated, and the aqueous phase was backwashed with DCM. The organic phase was dried, concentrated, and chromatography (DCM–MeOH–Py 10 : 1 : 0.1) gave pure 19 as colorless syrup (738 mg, 84% over 2 steps), [α]_D −11.6 (c 1.0, CHCl₃), ¹H NMR (600 MHz, CD₃OD): δ 7.45–7.41 (m, 4H, Ar–H), 7.39–7.37 (m, 6H, Ar–H), 7.36–7.32 (m, 6H, Ar–H), 7.31–7.25 (m, 10H, Ar–H), 7.25–7.18 (m, 4H, Ar–H), 5.40 (t, 1H, J = 2.0 Hz, H-2^VI), 5.10–5.00 (m, 10H, 6× H-2′, H-1^II, H-1^III, H-1^IV, H-1^V), 4.77 (s, 1H, H-1^I), 4.65–4.55 (m, 12H, 11× CH₂Ph and H-1^VI at 4.56), 4.41 (d, 1H, J = 11.3 Hz, CH₂Ph), 4.21–4.03 (m, 22H, 12× H-4′, 6× H-4^I–VI, 4× H-2^II–V), 3.98–3.91 (m, 6H, 6× H-3^I–VI), 3.88–3.76 (m, 7H, H-2^I, 5× H-5, OCH_aCH₂Si), 3.71 (m, 1H, H-5), 4.48 (m, 1H, OCH_bCH₂Si), 2.15–1.98 (m, 51H, 6× H_a,b-3′ incl. 12 s at 2.14, 2.11, 2.08, 2.07, 2.06, 2.05, 2.04, 2.03, 2.02, 2.00, 1.998, 1.99 for 13× COCH₃), 1.14–1.11 (m, 6H, 2× H-6), 1.06–1.02 (m, 12H, 4× H-6), 0.96–0.92 (m, 1H, CH₂CH_aSi), 0.90–0.86 (m, 1H, CH₂CH_bSi), 0.02 (s, 9H, (CH₃)₃Si). ¹³C{1H} NMR (150 MHz, CD₃OD): δ 172.9 (CH₃C=O), 172.87 (CH₃C=O), 172.8 (CH₃C=O), 172.79 (CH₃C=O), 172.77 (CH₃C=O), 172.7 (CH₃C=O), 172.61 (3C, 3× CH₃C=O), 172.60 (CH₃C=O), 172.5 (2C, 2× CH₃C=O), 171.84 (NHC=O), 171.8 (NHC=O), 171.78 (NHC=O), 171.76 (NHC=O), 171.73 (NHC=O), 171.69 (NHC=O), 171.5 (CH₃C=O), 139.8, 139.7, 139.68, 139.63 (2C), 139.6, 129.7, 129.67 (2C), 129.6 (2C), 129.5 (2C), 129.4 (3C), 129.23 (3C), 129.2 (3C), 129.1 (2C), 129.0 (2C), 128.9 (2C), 128.8 (2C), 128.7 (2C), 128.6 (2C), 128.5, 102.5 and 102.3 (4C, 4× C-1^II–V), 100.9 (C-1^VI), 99.8 (C-1^I), 77.0, 76.9, 76.6, 76.5, 76.4 (10C, 5× C-3, 5× C-2), 75.6 (C-3^VI), 73.0 (CH₂Ph), 72.8 (3C, 3× CH₂Ph), 72.6 (CH₂Ph), 72.56 (CH₂Ph), 72.5 (C-2′), 72.43 (2C, 2× C-2′), 72.4 (3C, 3× C-2′), 69.8 (C-5), 69.7 (C-5), 69.6 (C-5), 69.5 (C-5), 68.9 (2C, C-5, C-2^VI), 68.8 (C-5), 66.1 (OCH₂CH₂Si), 61.3 (4C, 4× C-4′), 61.25 (C-4′), 61.2 (C-4′), 53.6 (C-4), 53.4 (C-4), 53.3 (C-4), 53.2 (3C, 3× C-4), 32.4 (C-3′), 32.3 (5C, 5× C-3′), 20.9 (COCH₃), 20.83 (5C, 5× COCH₃), 20.81 (COCH₃), 20.78 (COCH₃), 20.73 (COCH₃), 20.71 (3C, 3× COCH₃), 20.70 (COCH₃), 18.7 (CH₃, C-6), 18.57 (CH₃, C-6), 18.54 (CH₂Si), 18.48 (2C, CH₃, 2× C-6), 18.4 (CH₃, C-6), 18.3 (CH₃, C-6), −1.2 [3C, (CH₃)₃Si]. HRMS (ESI-TOF): m/z [M + H⁺] calcd for C₁₃₃H₁₇₉O₅₀N₆Si 2688.1418; found 2688.1423. Anal. calcd for C₁₃₃H₁₇₈O₅₀N₆Si: C, 59.41; H, 6.67; N, 3.13. Found C, 59.51; H, 6.66; N, 3.06.

2-O-Acetyl-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranosyl-(1→2)-[3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranosyl-(1→2)]₄-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranose (20)

A solution of 19 (700 mg, 0.26 mmol) in TFA (12 mL) was kept at room temperature for 2 hours, when TLC (R_f = 0.31 at 15 : 1 DCM–MeOH) showed that the reaction was complete and that a much slower moving product was formed. The mixture was processed as described above for a similar reaction, and chromatography (19 : 1 DCM–MeOH) gave 20 as foam (565 mg, 84%). ¹H NMR (600 MHz, CD₃OD) for the major anomer: δ 8.14–7.92 (m, 6H, NH), 7.43–7.17 (m, 30H, Ar–H), 5.39 (brs, 1H, H-2^VI), 5.09–5.00 (11H, 6× H-2′, 5× H-1^I–V at 5.083, 5.076, 5.056, 5.056, 5.012), 4.69–4.55 (m, 12H, 11× CH₂Ph and H-1^VI at 4.56), 4.41 (d, 1H, J = 11.4 Hz, CH₂Ph), 4.20–4.03 (m, 21H, 12× H-4′, 4× H-2, 4× H-4 and H-5^VI), 3.97–3.78 (m, 14H, H-2^I, 6× H-3, 2× H-4, 5× H-5), 2.17–1.95 (m, 51H, 6× H_a,b-3′ incl. 12 s at 2.136, 2.13, 2.10, 2.09, 2.08, 2.07, 2.06, 2.05, 2.03, 2.02, 2.01, 2.00, 1.99 for 13× COCH₃), 1.16–0.99 (m, 18H, 6× H-6). ¹³C{1H} NMR (150 MHz, CD₃OD): δ 172.98 (CH₃C=O), 172.95 (CH₃C=O), 172.9 (CH₃C=O), 172.87 (CH₃C=O), 172.84 (CH₃C=O), 172.80 (CH₃C=O), 172.78 (CH₃C=O), 172.76 (CH₃C=O), 172.7 (CH₃C=O), 172.6 (2C, 2× CH₃C=O), 172.5 (CH₃C=O), 171.85 (NHC=O), 171.8 (NHC=O), 171.78 (NHC=O), 171.76 (NHC=O), 171.73 (NHC=O), 171.7 (NHC=O), 171.6 (CH₃C=O), 139.8, 139.76, 139.72, 139.64, 139.64, 139.6, 129.7, 129.6 (2C), 129.55 (2C), 129.5 (2C), 129.4 (2C), 129.2 (2C), 129.19 (2C), 129.1 (2C), 129.0 (3C), 128.86 (3C), 128.84 (2C), 128.80 (2C), 128.7 (2C), 128.6 (2C), 128.5, 102.4 and 102.2 (4C, 4× C-1^II–V), 100.9 (C-1^VI), 94.5 (C-1^I), 76.9, 76.8, 76.6, 76.56, 76.5, 75.6, 75.5 (11C, 6× C-3, 5 C-2), 72.9 (2C, 2× CH₂Ph), 72.8 (3C, 3× CH₂Ph), 72.6 (CH₂Ph), 72.5 (C-2′), 72.47 (C-2′), 72.45 (2C, 2× C-2′), 72.4 (2C, 2× C-2′), 69.76 (C-5), 69.7 (C-5), 69.5 (2 C, C-5), 68.9 (2C, C-5, C-2^VI), 68.3 (C-5), 61.3 (4C, 4× C-4′), 61.25 (C-4′), 61.2 (C-4′), 53.9 (C-4), 53.8 (C-4), 53.5 (C-4), 53.4 (C-4), 53.3 (C-4), 53.2 (C-4), 32.4 (6C, 6× C-3′), 20.9 (COCH₃), 20.8 (6C, 6× COCH₃), 20.78 (COCH₃), 20.74 (COCH₃), 20.72 (2C, 2× COCH₃), 20.70 (2C, 2× COCH₃), 18.7 (CH₃, C-6), 18.6 (CH₃, C-6), 18.5 (3C, CH₃, 3× C-6), 18.3 (CH₃, C-6). HRMS (ESI-TOF): m/z [M + H⁺] calcd for C₁₂₈H₁₆₇O₅₀N₆ 2588.0710; found 2588.0725.

8-Azido-3,6-dioxaoctyl 2-O-acetyl-3-O-benzyl-[4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranosyl]-(1→2)-[3-O-benzyl-[4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranosyl-(1→2)]₄-3-O-benzyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranoside (2)

1,8-Diazabicyclo[5,4,0]undec-7-ene (DBU, 15 μL, 0.1 mmol) was added at 0 °C to a stirred solution of hemiacetal 21 (500 mg, 0.19 mmol) and trichloroacetonitrile (30 μL, 0.29 mmol) in dry DCM (10 mL). The stirring was continued for 2 hours when TLC (15 : 1 DCM–MeOH) showed complete formation of a faster moving product. The mixture was concentrated, and passed through a small pad of silica gel which was eluted with (DCM : MeOH = 19 : 1) containing a few drops of triethylamine, to obtain pure compound 21 as light brown syrup (448 mg, 85%). HRMS: m/z [M + NH₄⁺] calcd for C₁₃₀H₁₇₀O₅₀N₈Cl₃ 2748.0071; found 2748.0081. The material was sufficiently pure for the next step.

Method 1. To a solution of the hexasaccharide trichloroacetimidate donor 21 (100 mg, 0.04 mmol) and glycosyl acceptor 22 (35 mg, 0.2 mmol) in dry DCM (7 mL) was added 0.1 g of 4 Å powdered molecular sieves, and the mixture was stirred for 15 min. The mixture was cooled to 0 °C, and TMSOTf (3 μL, 0.016 mmol) was added. After 30 min at 0 °C, when TLC (3 : 2 toluene–acetone) showed complete consumption of the glycosyl donor, the reaction was terminated by addition of triethylamine (1 drop). The mixture was filtered through a pad of Celite directly into a separating funnel containing excess of sodium bicarbonate (saturated) solution. The mixture was extracted with DCM (3× 5 mL), the combined organic layers were dried and concentrated, to give crude product (65.3 mg, 65%). NMR showed that the reaction produced the two anomers with poor selectivity (α : β = 1.1 : 1).

Method 2. To a solution of the hexasaccharide trichloroacetimidate donor 21 (200 mg, 0.08 mmol) and acceptor 22 (70 mg, 0.4 mmol) in dry toluene (10 mL with drops of dry DCM to aid solubility of synthons) was added 0.1 g of 4 Å powdered molecular sieves, and the mixture was stirred for 15 min. The reaction mixture was warmed to 100 °C (bath), and TMSOTf (6 μL, 0.03 mmol) was added. After 1.5 hours at that temperature, when TLC (R_f = 0.43 at 3 : 2 toluene–acetone) showed complete consumption of the glycosyl donor, the mixture was processed as described above. ¹H NMR of the crude mixture showed the ratio of the α and β anomers formed was 7 : 1. Chromatography (3 : 2 toluene–acetone) gave first the α-linked hexasaccharide (2a, 132 mg, 66%) as colorless syrup. [α]_D −10.8 (c 2.0, CHCl₃). ¹H NMR (600 MHz, CD₃OD): δ 7.74–7.71 (m, 2H, Ar–H), 7.40–7.38 (m, 5H, Ar–H), 7.36–7.33 (m, 5H, Ar–H), 7.30–7.26 (m, 6H, Ar–H), 7.23–7.19 (m, 6H, Ar–H), 7.16–7.14 (m, 4H, Ar–H), 7.13–7.10 (m, 2H, Ar–H), 5.40 (brs, 1H, H-2^VI), 5.09–5.00 (m, 10H, 6× H-2′, 4× H-1^II–V at 5.09, 5.065, 5.06 and 5.02), 4.83 (d, 1H, J_1–2 = 1.5 Hz, H-1^I), 4.66–4.55 (m, 12H, 11× CH₂Ph and H-1^VI at 4.57), 4.41 (d, 1H, J = 11.3 Hz, CH₂Ph), 4.20–4.05 (m, 22H, 12× H-4′, 4× H-2^II–^V, 6× H-4^I–VI), 3.99–3.92 (m, 6H, 5× H-3^II–V, H-2^I), 3.91–3.88 (m, 2H, H-3, H-5), 3.86–3.79 (m, 4H, 4× H-5), 3.78–3.73 (m, 2H, OCH_a, H-5), 3.69–3.63 (m, 8H, 8× OCH₂), 3.59 (m, 1H, OCH_b), 3.37 (t, 2H, J = 5.0 Hz, CH₂N₃), 2.15–1.98 (m, 51H, 6× H_a,b-3′ incl. 12 s at 2.145, 2.143, 2.12, 2.08, 2.07, 2.06, 2.04, 2.03, 2.02, 2.00, 1.999 and 1.99 for 13× COCH₃), 1.15–1.11 (m, 6H, 2× CH₃, 2× H-6), 1.09–1.02 (m, 12H, 4× CH₃, 4× H-6). ¹³C{1H} NMR (150 MHz, CD₃OD): δ 172.88 (CH₃C=O), 172.84 (CH₃C=O), 172.82 (CH₃C=O), 172.77 (CH₃C=O), 172.75 (CH₃C=O), 172.68 (CH₃C=O), 172.59 (2C, 2× CH₃C=O), 172.58 (2C, 2× CH₃C=O), 172.54 (2C, 2× CH₃C=O), 171.81 (2C, 2× NHC=O), 171.76 (NHC=O), 171.74 (NHC=O), 171.71 (NHC=O), 171.67 (NHC=O), 171.5 (CH₃C=O), 139.74, 139.72, 139.70 (2C), 139.6 (2C), 129.9, 129.7 (2C), 129.6 (2C), 129.56 (2C), 129.5 (2C), 129.4 (2C), 129.22 (2C), 129.21 (2C), 129.18 (2C), 129.1 (2C), 129.0 (2C), 128.9 (2C), 128.8 (2C), 128.7 (2C), 128.6, 128.5, 126.3, 102.4 and 102.3 (4C, 4× C-1^II–V), 100.9 (C-1^VI), 100.5 (C-1^I), 76.98, 76.9, 76.6, 76.5, 76.4, 75.6 (11C, 6× C-3, 5 C-2), 72.86 (2C, 2× CH₂Ph), 72.8 (2C, 2× CH₂Ph), 72.6 (CH₂Ph), 72.54 (CH₂Ph), 72.5 (C-2′), 72.4 (3C, 3× C-2′), 72.3 (2C, 2× C-2′), 71.7 (OCH₂), 71.5 (OCH₂), 71.4 (OCH₂), 71.2 (OCH₂), 69.8 (C-5), 69.7 (2C, 2× C-5), 69.6 (C-5), 69.5 (C-5), 68.9 (C-2^VI), 68.8 (C-5), 68.1 (OCH₂), 61.3 (4C, 4× C-4′), 61.24 (C-4′), 61.2 (C-4′), 53.5 (C-4), 53.4 (C-4), 53.3 (2C, 2× C-4), 53.2 (2C, 2× C-4), 51.7 (CH₂N₃), 32.42 (C-3′), 32.4 (2C, 2× C-3′), 32.36 (3C, 3× C-3′), 21.5 (COCH₃), 20.9 (COCH₃), 20.84 (5C, 5× COCH₃), 20.82 (COCH₃), 20.8 (COCH₃), 20.73 (3C, 3× COCH₃), 20.7 (COCH₃), 18.7 (CH₃, C-6), 18.6 (CH₃, C-6), 18.5 (CH₃, C-6), 18.47 (CH₃, C-6), 18.4 (CH₃, C-6), 18.3 (CH₃, C-6). HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₁₃₄H₁₈₁O₅₂N₁₀ 2762.1826; found 2762.1836. Anal. calcd for C₁₃₄H₁₇₇O₅₂N₉: C, 59.61; H, 6.50; N, 4.59. Found C, 59.48; H, 6.60; N, 4.47.

Continued elution gave the β-linked hexasaccharide 2b as colorless syrup (19 mg, 9%, total yield of glycosylation, 151 mg, 75%, α/β ∼ 6.9 : 1). Data for 2b: [α]_D −18.3 (c 0.9, CHCl₃). ¹H NMR (600 MHz, CD₃OD): δ 7.52–7.17 (m, 30H, Ar–H), 5.41 (t, 1H, J = 2.1 Hz, H-2^VI), 5.21 (d, 1H, J_1–2 = 1.9 Hz, H-1^I), 5.15–4.98 (m, 9H, 6× H-2′, 3× H-1^II–V at 5.10, 5.08 and 5.04), 4.73–4.55 (m, 12H, 11× CH₂Ph and H-1^VI at 4.58), 4.49 (s, 1H, H-1^I), 4.43 (d, 1H, J = 11.6 Hz, CH₂Ph), 4.24–4.03 (m, 22H, 12× H-4′, 4× H-2^II–V, 6× H-4^I–VI), 3.99–3.75 (m, 13H, H-2^I, 6× H-3^II–V, 6× H-5), 3.69–3.63 (m, 8H, 8× OCH₂), 3.59 (m, 2H, OCH₂), 3.38 (2d, 2H, J = 4.5 Hz, CH₂N₃), 2.15–1.98 (m, 51H, 6× H_a,b-3′ incl. 11 s at 2.11, 2.09, 2.08, 2.06, 2.057, 2.05, 2.04, 2.03, 2.02, 2.01 and 2.00 for 13× COCH₃), 1.15–1.11 (m, 6H, 2× CH₃, 2× H-6), 1.09–1.02 (m, 12H, 4× CH₃, 4× H-6). ¹³C{1H} NMR (150 MHz, CD₃OD): δ 172.9 (CH₃C=O), 172.88 (CH₃C=O), 172.84 (CH₃C=O), 172.78 (4C, 4× CH₃C=O), 172.6 (2C, 2× CH₃C=O), 172.59 (3C, 3× CH₃C=O), 171.82 (NHC=O), 171.81 (NHC=O), 171.77 (NHC=O), 171.75 (NHC=O), 171.72 (NHC=O), 171.7 (NHC=O), 171.6 (CH₃C=O), 139.8, 139.73, 139.71, 139.65, 139.6, 139.5, 129.7 (2C), 129.6 (2C), 129.5 (2C), 129.4 (2C), 129.3 (2C), 129.24 (2C), 129.2 (2C), 129.1 (2C), 129.0 (2C), 128.96 (2C), 128.9 (2C), 128.8 (2C), 128.7 (2C), 128.6 (2C), 128.5 (2C), 101.5 (3C, 3× C-1^II–V), 100.8 (3C, 3× C-1^II–V), 76.9, 76.6, 76.2, 75.6 (11C, 6× C-3, 5 C-2), 73.2 (2C, 2× CH₂Ph), 72.8 (3C, 3× CH₂Ph), 72.6 (CH₂Ph), 72.54, 72.5, 72.4, 72.40, 72.39 (6C, 6× C-2′), 71.6 (OCH₂), 71.5 (OCH₂), 71.4 (OCH₂), 71.1 (OCH₂), 69.8 (OCH₂), 69.8 (C-5), 69.7 (3C, 3× C-5), 69.6 (C-5), 69.5 (C-5), 68.9 (C-2^VI), 61.3 (C-4′), 61.28 (2C, 2× C-4′), 61.26 (2C, 2× C-4′), 61.23 (C-4′), 53.7 (C-4), 53.4 (2C, 2× C-4), 53.3 (3C, 3× C-4), 51.7 (CH₂N₃), 32.5 (C-3′), 32.4 (2C, 2× C-3′), 32.38 (3C, 3× C-3′), 20.98 (COCH₃), 20.85 (2C, 2× COCH₃), 20.83 (3C, 3× COCH₃), 20.8 (COCH₃), 20.78 (COCH₃), 20.75 (COCH₃), 20.7 (4C, 4× COCH₃), 18.7 (CH₃, C-6), 18.6 (CH₃, C-6), 18.5 (3CH₃, 3× C-6), 18.4 (CH₃, C-6). HRMS (ESI-TOF): m/z [M + NH₄⁺] calcd for C₁₃₄H₁₈₁O₅₂N₁₀ 2762.1826; found 2762.1838. Anal. calcd for C₁₃₄H₁₇₇O₅₂N₉: C, 59.61; H, 6.50; N, 4.59. Found C, 58.99; H, 6.50; N, 4.49.

8-Amino-3,6-dioxaoctyl [4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranosyl-(1→2)]₅-4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-α-D-mannopyranopyranoside (1)

To a solution of the linker-equipped hexasaccharide 2a (100 mg, 0.04 mmol) in dry MeOH (10 mL), methanolic NaOMe (1 M, 1 mL) was added with exclusion of moisture and atmospheric CO₂, and the mixture was kept at room temperature for 6 hours, when TLC (3 : 2 toluene–acetone) showed that the starting material was consumed and that a much slower moving product was formed (R_f = 0.34 at 4 : 1 DCM–methanol with 1 drop of acetic acid). After neutralization (Dowex 50W H⁺ resin) and filtration, the solvent was removed, and the residue, showing correct HRMS (ESI-TOF: m/z [M + H⁺] calcd for C₁₀₈H₁₅₂O₃₉N₉ 2199.0187; found 2199.0188), was used for the next step without further purification.

To a solution of the above product in MeOH (2 mL), 10 mg of Pd–C was added, and the mixture was stirred at room temperature under 100 Psi pressure of hydrogen gas for 4 hours. TLC showed complete consumption of starting material and presence of a much more polar product. HRMS analysis confirmed the completion of global reduction. The mixture was filtered over a Celite pad, the solids were washed several times with methanol and the solvent was removed. A solution of the product in MeOH : H₂O (2 : 1) was filtered through a 0.2 μm porosity syringe filter and lyophilized to collect pure product as white foam (42 mg, 71% over 2 steps), [α]_D +1.8 (c 1.0, CH₃OH). ¹H NMR (600 MHz, CD₃OD): δ 5.16–5.11 (m, 4H, 4× H-1^II–V), 4.98 (s, 1H, H-1^VI), 4.87 (s, 1H, H-1^I), 4.22–4.14 (m, 6H, 6× H-2′), 4.13–4.08 (m, 4H, 4× H-2^II–V), 4.05–3.98 (m, 5H, 4× H-3^II–V, H-2^VI at 4.02), 3.98–3.83 (m, 13H, 2× H-3^I,VI, 6× H-4^I–VI, 5× H-5^II–VI), 3.83–3.78 (m, 3H, H-2^I, H-5^I, OCH_a), 3.77–3.71 (m, 12H, 12× H-4′), 3.71–3.52 (m, 10H, 10× OCH₂), 3.13 (t, 1H, J = 4.8 Hz, OCH₂CH_aNH₂), 2.06–1.97 (m, 6H, 6× H-3′_a), 1.87–1.78 (m, 6H, 6× H-3′_b), 1.22–1.10 (m, 18H, 6× H-6). ¹³C{1H} NMR (150 MHz, CD₃OD): δ 178.0 (5C, 5× NHC=O), 177.9 (NHC=O), 103.8 (C-1^VI), 102.7, 102.5, 105.46, 102.4 (4C, 4× C-1^II–V), 100.4 (C-1^I), 79.6, 79.23, 79.2 (5C, 5× C-2), 71.6, 71.4, 71.30 (3C, 3× OCH₂), 70.9 (C-2), 70.7 (6C, 6× C-2′), 70.0, 69.5, 69.4, 69.3 (11C, 6× C-3, 5× C-5), 68.7 (C-5), 68.1 (OCH₂), 67.8 (OCH₂), 59.4 (6C, 6× C-4′), 54.8–54.2 (6C, C-4^I–VI), 40.7 (CH₂NH₂), 38.3 (6C, 6× C-3′), 18.4 (2C, CH₃, 2× C-6), 18.3 (3C, CH₃, 3× C-6), 18.2 (CH₃, C-6). HRMS (ESI-TOF): m/z [M + H⁺] calcd for C₆₆H₁₁₈O₃₉N₇ 1632.7465; found 1632.7467.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was supported by the Intramural Research Program of the National Institutes of Health, NIDDK.

Notes and references

1. P. Kováč, Studies toward a rationally designed conjugate vaccine for cholera using synthetic carbohydrate antigens, in Protein-carbohydrate interactions in infectious diseases. Studies toward a rationally designed conjugate vaccine for cholera using synthetic carbohydrate antigens, ed. C. Bewley, Royal Society of Chemistry London, 2006, vol. 10, pp. 175–220.
2. S. J. Hou and P. Kovac, Carbohydr. Res., 2010, 345, 999–1007.
3. X. Lu, H. B. Pfister, S. E. Soliman and P. Kováč, Eur. J. Org. Chem., 2018, 2944–2957.
4. S. N. Chatterjee and K. Chaudhuri, Biochim. Biophys. Acta, Mol. Basis Dis., 2003, 1639, 65–79.
5. M. D. Meeks, R. Saksena, X. Ma, T. K. Wade, R. K. Taylor, P. Kováč and W. F. Wade, Infect. Immun., 2004, 72, 4090.
6. R. Adamo and P. Kováč, Eur. J. Org. Chem., 2007, 988–1000.
7. Y. Ogawa, P.-s. Lei and P. Kováč, Carbohydr. Res., 1996, 288, 85–98.
8. H. Paulsen and M. Brenken, Liebigs Ann. Chem., 1988, 649–654.
9. H. Paulsen and M. Paal, Carbohydr. Res., 1984, 135, 53–69.
10. D. R. Bundle, M. Gerken and T. Peters, Carbohydr. Res., 1988, 174, 239–251.
11. R. Saksena, J. Zhang and P. Kováč, J. Carbohydr. Chem., 2002, 21, 453–470.
12. G. Zemplén, Berichte der deutschen chemischen Gesellschaft (A and B Series), 1926, 59, 1254–1266.
13. K. Bock and C. Pedersen, J. Chem. Soc., Perkin Trans. 2, 1974, 293–297.
14. Y. Ogawa, P.-s. Lei and P. Kováč, Carbohydr. Res., 1996, 293, 173–194.
15. A. Arencibia-Mohar, A. Ariosa-Alvarez, O. Madrazo-Alonso, G. E. Abreu, L. García -Imia, G. Sierra-González and V. Verez-Bencomo, Carbohydr. Res., 1998, 306, 163–170.
16. R. Adamo and P. Kováč, Eur. J. Org. Chem., 2006, 2803–2809.
17. N. V. Ganesh, J. M. Sadowska, S. Sarkar, L. Howells, J. McGiven and D. R. Bundle, J. Am. Chem. Soc., 2014, 136, 16260–16269.
18. M. Gotoh and P. Kovác, J. Carbohydr. Chem., 1993, 12, 981–983.
19. X. Ma, R. Saksena, A. Chernyak and P. Kováč, Org. Biomol. Chem., 2003, 1, 775–784.
20. G. M. Sheldrick, Acta Crystallogr., Sect. C: Struct. Chem., 2015, 71, 3–8.

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Footnote

† Electronic supplementary information (ESI) available: Copy of ¹H, ¹³C NMR of all compounds, ¹H, ¹³C, COSY, and HSQC NMR spectra of new compounds and table for crystallographic and structural refinement parameters (pdf). CCDC 1939745. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c9ra08232h

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