Synthesis of spacer-equipped di-, tri-, and the tetrasaccharide fragments of the deacetylated O-PS1 of Citrobacter gillenii O9a,9b, and a related pentasaccharide

Abstract

The title rhamnooligosaccharides [α-d-Rhap4NAc-(1→3)–α-d-Rhap4NAc-1-O-(CH₂)₅COOMe, α-d-Rhap4NAc-(1→3)-α-d-Rhap4NAc-(1→3)–α-d-Rhap4NAc-1-O-(CH₂)₅COOMe, α-d-Rhap4NAc-(1→2)-α-d-Rhap4NAc-(1→3)-α-d-Rhap4NAc-(1→3)–α-d-Rhap4NAc-1-O-(CH₂)₅COOMe, and α-d-Rhap4NAc-(1→3)-α-d-Rhap4NAc-(1→2)-α-d-Rhap4NAc-(1→3)-α-d-Rhap4NAc-(1→3)–α-d-Rhap4NAc-1-O-(CH₂)₅COOMe] were synthesized in a stepwise fashion from 5-methoxycarbonylpentyl 4-azido-4,6-dideoxy-2-O-benzyl-α-d-mannopyranoside and orthogonally protected 1-thioglycoside glycosyl donors. The amorphous, final products were fully characterized as corresponding per-O-acetyl derivatives.

1. Introduction

Citrobacter gillenii O9a,9b is one of Citrobacter strains that are found in the intestinal tract of some vertebrates, and may be associated with meningitis, brain abscesses, and neonatal sepsis in humans.¹ A characteristic feature² of C. gillenii O9a,9b is the presence of two different O-polysaccharides (PS) as parts of the lipopolysaccharide (LPS). One of them (PS2) is a homopolymer composed of α-(1→2)-linked 4-acetamido-4,6-dideoxy-α-d-mannopyranose (N-acetyl-d-perosamine, d-Rhap4NAc) residues. The other one (PS1) is also built up of N-acetyl-d-perosamine, and its tetrasaccharide repeating unit has the structure: →3)-α-d-Rhap4NAc-(1→2)-α-d-Rhap4NAc-(1→2)-α-d-Rhap4NAc-(1→3)–α-d-Rhap4NAc-(1→. The NMR data suggest² that the O-PS1 is partially 2-O-acetylated, and that the degree of acetylation of the →3-linked N-acetyl-d-perosamine residues is ~70%. Gel-permeation chromatography of products of mild acid hydrolysis of the C. gillenii O9a,9b LPS yielded PS1 and PS2 in 11.4 and 18% yield, respectively.

Rabbit antiserum raised against C. gillenii O9a,9b bacteria reacted with PS1 in Ouchterlony double immunodiffusion test and immunoblotting. PS2 and LPS from Vibrio cholerae O:1, which has a structurally related O-PS, were not reactive with the same sera. It was reasoned² that the unreactivity was due to the small molecular size of PS2, and to the different N-acyl group present in the O-PS from V. cholerae O:1. The structure of the tetrasaccharide repeating unit of PS1 lacking the O-acetyl group is the same as the tetrasaccharide part of a pentasaccharide repeating unit from the O-chain PS of Escherichia hermanii,³ the bacteria often isolated from infected wounds. It is noteworthy, in this context, that the serological reactivity of the LPS of C. gillenii O9a,9b was not affected by O-deacetylation.²

2. Results and Discussion

This laboratory has been involved in developing a conjugate vaccine for cholera using synthetic fragments of the O-PS of V. cholerae O:1 as the antigenic component.^4–8,9 The considerable structural similarity between the O-PS of V. cholerae O:1 and C. gillenii O9a,9b lies in the presence of the (1→2)-α-linked d-perosamine in both O-PSs. The two polysaccharides differ in the N-acyl groups present, i.e., acetyl in C. gillenii O9a,9b as compared to 3-deoxy-l-glycerotetronyl group in V. cholerae O:1, and in absence of the (1→3)-interglycosidic linkages and O-acetyl groups in V. cholerae O:1. The usefulness of pure, well defined fragments of the O-PS of C. gillenii O9a,9b as probes for inhibition studies and studies of cross-reactivity as well as our experience in the chemical synthesis of perosamine-containing oligosaccharides^10–15 prompted us to synthesize fragments that mimic partial structures of the O-PS of C. gillenii O9a,9b.

Because O-acetyl groups are not present in the O-PS of V. cholerae O:1, and their presence in the O-PS of C. gillenii O9a,9b was found non-essential for serology,² we designed the synthetic schemes to yield non-acetylated oligosaccharides (Scheme 1–Scheme 4). To obtain the tetrasaccharide repeating unit and the two oligosaccharide fragments from which it is formed, we chose a stepwise synthetic strategy starting with the linker (spacer)-equipped glycosyl acceptor 12. In this way, as the linker chosen can be readily transformed into derivatives suitable for conjugation, the final oligosaccharides were obtained in form amenable to both inhibition/binding studies and preparation of neoglycoconjugates by a variety of protocols.^16–18

Scheme 1.

Scheme 4.

The hitherto unknown compound 12 was obtained from mesylate 1¹⁹ following a series of known conversions where a number of minor modifications led to improved yields of desired intermediates and/or made the individual steps experimentally less demanding. Consequently, the known^20,21 azido compound 2 was prepared by combining advantages of each of the previous two protocols leading to it.^20,21 We used mesylate 1, recommended by Eis and Ganem²⁰ but prepared by an improved procedure,¹⁹ and treated it with NaN₃²⁰ in the presence of a crown ether, as used by Bundle et al.²¹ with methyl 6-deoxy-1,2-O-isopropylidene-4-O-trifluoromethanesulfonyl-α-d-mannopyranoside. In this way, we were able to readily introduce the azido function while bypassing the need to use of the better, trifluoromethanesulfonyloxy leaving group,²¹ which would be cost prohibitive when working on a large scale. Unlike with the conversion in the absence of the crown ether, only slight discoloration of the reaction mixture occurred during the displacement reaction, and the crystalline azide 2 was obtained consistently in 80–90% yield.

Precursors to glycosyl donors bearing selectively removable protecting groups at position O-2 or O-3, benzyl ethers 3 and 4, which were required for the synthesis of oligosaccharides containing (1→2)- and (1→3)-linkages, were obtained by selective benzylation of 2 by phase transfer catalysis and benzylation via stannylation, respectively. The reaction time of the phase-transfer mediated alkylation could be shortened, and the yield of the conversion²² 2→3 + 4 could be improved by using a larger relative amount of the phase-transfer catalyst. The mixture was resolved by column chromatography, and the 2-benzyl ether was now obtained crystalline. Some amount of the 3-benzyl ether 4 was obtained from this reaction but the bulk of this compound was obtained by benzylation of the 2,3-stannylene acetal of 2. However, instead of applying the original procedure²⁰ leading to 4, which required removal of large amount of unchanged BnBr, we employed the benzylation protocol by Nashed²³ (c.f. Experimental). Acetolysis of 3 gave the hitherto unknown 1,3-di-O-acetyl derivatives 5 (α) and 6 (β). The 1,3-di-O-acetyl derivative 5 and its 1,2-O-acetylated analog 7, prepared as described,^14,21 were subsequently transformed to the corresponding 1-thioglycosides 8 and 10,^14,21 respectively. The β anomer 9, formed when 5 was treated with ethanethiol and isolated by chromatography along with 8, was also fully characterized.

	R	R1	R2	R3	R4
1	H	OMe	H	H	OMs
2	H	OMe	H	H	N3
3	H	OMe	Bn	H	N3
4	H	OMe	H	Bn	N3
5	H	OAc	Bn	Ac	N3
6	OAc	H	Bn	Ac	N3
7	H	OAc	Ac	Bn	N3
8	H	SEt	Bn	Ac	N3
9	SEt	H	Bn	Ac	N3
10	H	SEt	Ac	Bn	N3
11	H		Bn	Ac	N3
12	H		Bn	H	N3

5-Methoxycarbonylpentyl glycoside 12 was prepared from acetate 11 by Zemplén deacetylation. The latter was obtained from 6 and 5-methoxycarbonylpentanol²⁴ by SnCl₄-mediated²⁵ glycosylation, essentially as described for preparation of the similar 3-O-benzyl derivative.⁷ It is worth noting that the conversion 6→11 was fast and afforded a high yield (~80%) of the 1,2-trans glycoside, even though the reaction could not involve the acetoxonium ion intermediate, which was the case in previous situations involving a participating group at O-2.^7,25

Oligosaccharides 16, 21 and 26 were synthesized (Scheme 1–Scheme 3) by NIS and silver triflate-mediated condensations of thioglycoside donors 8 or 10^14,21 with acceptors 12, 14 and 19. The products thus obtained (13, 18 and 23) were deacetylated (Zemplén), to afford glycosyl acceptors (14, 19 and 24) for further extension of the oligosaccharide chain. It is noteworthy that the fast-atom-bombardment (FAB) mass-spectrum of compound 13 showed a peak at m/z 685.4, instead of the expected peak at m/z 710 ([M + H]⁺). It reflected reduction of one azido group to an amino group during the course of mass-spectrometric analysis.²⁶ The same phenomenon was observed with all compounds containing more then one azido group.

Scheme 3.

While the one-pot conversion of a single azide group to the N-acetamido group by catalytic hydrogenolysis in the presence of Ac₂O and MeOH works well,²⁷ similar conversions described here with compounds containing multiple azido groups were achieved more cleanly using a two step process. The azido groups in 14, 19, and 24 were first reduced with H₂S in aqueous pyridine,^21,28 and subsequent N-acetylation with acetic anhydride in presence of methanol gave the acetamido derivatives 15, 20 and 25. Catalytic hydrogenolysis of the foregoing substances then gave the target oligosaccharide fragments 16, 21 and 26.

The purpose of making the related pentasaccharide 31 was its use in inhibition and other immunochemical studies with antibodies specific for the O-PS of C. gillenii O9a,9b. It was obtained by coupling of donor 8 with tetrasaccharide acceptor 24, to give fully protected pentasaccharide 28, followed by conversions (Scheme 4) similar to those described above.

The molecular mass of the deprotected oligosaccharides was verified by mass spectrometry and their structures follows from the mode of synthesis. The compounds were fully characterized as per-O-acetates 17, 22, 27 and 32 whose NMR data were fully consistent with their structures.

3. Experimental

3.1 General methods

Unless stated otherwise, optical rotations were measured at ambient temperature for solutions in chloroform (c ~1), with a Perkin-Elmer automatic polarimeter, Model 341 or with a Jasco automatic polarimeter, Model P-2000. All reactions were monitored by thin-layer chromatography (TLC) on Silica gel 60 coated glass slides (Whatman or Analtech). Column chromatography was performed by gradient elution from columns of silica gel. Solvent mixtures less polar than those used for TLC were used at the onset of development. NMR spectra of monosaccharide derivatives were measured at 22 °C, at 300 MHz (¹H) and 75 MHz (¹³C), with a Varian Mercury spectrometer. NMR spectra of oligosaccharides were measured at 300 or 600 MHz (¹H) and 75 or 150 MHz (¹³C), with a Varian Mercury or Bruker Avance spectrometers. All assignments were supported by homonuclear and heteronuclear 2-dimensional correlation spectroscopy, run with the software supplied with the spectrometers. When reporting assignments of NMR signals of oligosaccharides, sugar residues in oligosaccharides are serially numbered, beginning with the one bearing the aglycon, and are identified by a Roman numeral superscript in listings of signal assignments. When reporting NMR data, the nuclei belonging to the spacer aglycon are denoted with a prime (′). ¹H NMR spectra of some compounds containing NHAc groups showed presence of isomers and/or considerable broadening of signals, and coupling constants in such cases are not reported. When presence of isomers was evident, only resonances characteristic of the more abundant isomer are reported. Liquid Chromatography-Electron Spray-Ionization Mass Spectrometry (ESI-MS) was performed with a Hewlett–Packard 1100 MSD spectrometer. Attempts have been made to obtain correct analytical data for all new compounds. However, some compounds tenaciously retained traces of solvents, despite exhaustive drying, and analytical figures for carbon could not be obtained within ±0.4%. The [α]_D values reported for such materials may not be quite accurate. Structures of these compounds follow unequivocally from the mode of synthesis and m/z values found in their mass spectra, and TLC and NMR spectroscopy verified their purity. Palladium-on-charcoal catalyst (5%, ESCAT 103) was a product of Engelhard Industries. 1,2-Di-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (7) was prepared as described.²¹ Before final freeze-drying of the deprotected oligosaccharides 16, 21, 26, and 31, their solutions in water (HPLC quality) were passed through the Anotop syringe filter (Whatman, 0.2 µm porosity). Solutions in organic solvents other than alcohols were dried with anhydrous Na₂SO₄, and concentrated at <40 °C/2 kPa.

3.2 General procedure for the NIS/AgOTf-mediated glycosylations

A mixture of glycosyl donor (1.3 mmol), acceptor (1 mmol) and finely powdered 4 Å molecular sieves (0.5 g) in CH₂Cl₂ (10–15 mL) was stirred under argon for 15 min. The mixture was cooled to ~10 °C, and solid NIS (1.4 mmol) was added, followed by a solution of AgOTf (0.4 mmol) in toluene (4 mL). The stirring was continued for 3 min at the same temperature, cooling was terminated, and when TLC showed that the reaction was complete (~15 min) the mixture was neutralized with Et₃N, partitioned between CH₂Cl₂ and aqueous sodium hydrogen carbonate, concentrated, and the residue was purified by chromatography.

3.4 Methyl 4-azido-4,6-dideoxy-α-d-mannopyranoside (2)

A mixture of methyl 4-O-methanesulfonyl-6-deoxy-α-d-mannopyranoside¹⁹ (1, 142.2 g, 0.555 mol), NaN₃ (87 g, 1.338 mol), and dicyclohexano-15-crown-5 (3.7 g, 16.8 mmol) in DMF (1 L) was heated with vigorous stirring at 100 °C until TLC (3:1 hexane–acetone) showed that all starting material was consumed. DMF was evaporated from the yellow reaction mixture, the residue was treated with CH₂Cl₂, and the mixture was filtered through a Celite pad. The filtrate was concentrated and the residue was purified by chromatography to give, after crystallization from isopropyl ether–hexane, 95.5 g (85%) of pure 2, mp. 84–85 °C, ref.20, mp 83–84 °C, 72% yield. CIMS m/z 221 [M + NH₄]⁺.

3.5 Methyl 4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (3) and methyl 4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (4)

3.5.1

A mixture of 2 (20 g, 98.4 mmol) and Bu₄NBr (2.7 g, 8.375 mmol), CH₂Cl₂ (300 mL), aqueous 20% NaOH (300 mL) and BnBr (12 mL, 98.4 mmol) was vigorously stirred overnight, when TLC (3:1 hexane–EtOAc) showed almost complete conversion of the starting material. The phases were separated, the aqueous phase was extracted with CH₂Cl₂, and the combined organic phase was washed with water and concentrated. Chromatography gave first methyl 4-azido-2,3-di-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (2.95 g, 7.8%), [α]_D +60.4 (c 0.9), ref. 22 [α]_D +65.9 (c 2.5). CIMS: m/z 401 [M + NH₄]⁺.

Eluted next was the 2-O-benzyl derivative 3 (18.97 g, 65%), mp 35–36 °C (from hexane), [α]_D +6.4 (c 0.7), ref.,²² [α]_D +8.54 (c 5.2), for amorphous 3 obtained in 56% yield. CIMS: m/z 311 [M + NH₄]⁺. Anal. Calcd for C₁₄H₁₉N₃O₄: C, 57.33; H, 6.53; N 14.33. Found: C, 57.08; H, 6.46; N, 14.36.

Eluted last was the 3-O-benzyl derivative 4, (4.75 g, 16.5%), CIMS: m/z 311 [M + NH₄]⁺. ¹H NMR data recorded for 3 and 4 agreed with those reported.^20–22

3.5.2

Bu₂SnO (40.0 g, 164.7 mmol) was added to a solution of 2 (33 g, 162.4 mmol) in MeOH (~1 L) and the mixture was heated under reflux until a clear solution was obtained (~1–2 h). Toluene (~100 mL) was added and solvents were evaporated. After the residue had been dried in a vacuum oven at 40 °C for 2 h, it was dissolved in DMF (200 mL), benzyl bromide (38 mL, 317 mmol) and Bu₄NI (1 g) was added, and the mixture was heated at ~100 °C until TLC showed complete disappearance of the starting material (4–6 h). After concentration, excess of BnBr was evaporated at 60 °C/133 Pa, and the residue was purified by chromatography to give the desired compound 4 as a light-yellow thick oil (40 g, 84%), which was identical with the material described above.

3.6 1,3-Di-O-acetyl-4-azido-2-O-benzyl-4,6-dideoxy-α- (5) and β-d-mannopyranose (6)

A solution of 3 (19.86 g, 67.7 mmol) in Ac₂O–AcOH–H₂SO₄ (50:20:0.01, 480 mL) was kept at room temperature for 45 min, when TLC (40:1 toluene–acetone) showed that the reaction was complete. NaOAc trihydrate was added, to neutralize H₂SO₄, and the pH was adjusted to ~7 with aqueous Na₂CO₃ and NaHCO₃. The mixture was extracted with dichloromethane to give, after concentration, crystalline residue 5 (23 g, 93%), mp 60–62 °C (from ethanol), [α]_D +49.5 (c 0.7). ¹H NMR (300 MHz, CDCl₃): δ 6.12 (d, 1 H, J_1,2 = 2.0 Hz, H-1), 5.05 (dd, 1 H, J_2,3 = 3.2, J_3,4 = 10.4 Hz, H-3), 4.73, 4.55 (2 d, 1 H each, ²J = 12.5 Hz, CH₂Ph), 3.81 (dd, 1 H, H-2), 3.74–3.63 (m, 2 H, H-4,5), 2.11, 2.06 (2 s , 3 H each, 2 COCH₃), 1.36 (d, 3 H, J_5,6 = 6.0 Hz, H-6). ¹³C NMR (75 MHz, CDCl₃): δ 90.93 (C-1), 73.41 (C-2), 72.90 (CH₂Ph), 71.85 (C-3), 69.44 (C-5), 62.04 (C-4), 20.95, 20.77 (2 COCH₃), 18.39 (C-6). CIMS: m/z 381 [M + NH₄]⁺. Anal. Calcd for C₁₇H₂₁N₃O₆: C, 56.19; H, 5.83; N, 11.56. Found: C, 56.43; H, 5.82; N, 11.62.

The mother liquor was purified by chromatography, to give first 400 mg of the α-anomer 5, total yield, 95%.

Eluted next was a small amount of material that was identified by NMR spectroscopy as the β-anomer 6, [α]_D -12.7 (c, 5.1). ¹H NMR (300 MHz, CDCl₃): δ 5.65 (d, 1 H, J_1,2 = 0.9 Hz, H-1), 4.83, 4.65 (2 d, partially overlapped, ²J = 12.2 Hz, CH₂Ph), 4.78 (dd, partially overlapped, J_2,3 = 3.2, J_3,4 = 10.5 Hz, H-3), 4.01 (bd, 1-H, H-2), 3.64 (t, J = 10.0 Hz, H-4), 3.44 – 3.34 (m, 1H, H-5), 2.11, 2.00 (2 s, 3 H each, 2 COCH₃), 1.40 (d, 3 H, J_5,6 = 6.0 Hz, H-6). ¹³C NMR (75 MHz, CDCl₃): δ 92.14 (C-1), 74.94 (CH₂Ph), 74.32 (C-3), 73.91 (C-2), 71.84 (C-5), 61.81 (C-4), 20.79, 20.63 (2 COCH₃), 18.20 (C-6).

3.7 Ethyl 3-O-acetyl-4-azido-2-O-benzyl-4,6-dideoxy-1-thio-α- (8) and β-d-mannopyranoside (9)

Ethanethiol (7.8 mL, 105 mmol) followed by BF₃·Et₂O (10.3 mL, 83.7 mmol) was added to a solution of 5^14,21 (30 g, 82.6 mmol) in dry CH₂Cl₂ (660 mL), which had been stirred with molecular sieves (4Å, 3 g) for 15 min. The stirring was continued for 20 min at room temperature, when TLC (10:1 hexane–EtOAc) showed that the reaction was complete. After neutralization with aqueous sodium hydrogen carbonate, the product was extracted with CH₂Cl₂, the organic layer was washed with water and concentrated. The residue was purified by chromatography to give first the α-anomer 8 (25 g), [α]_D +123 (c 1.6). ¹H NMR (300 MHz, CDCl₃): δ 5.30 (d, 1 H, J_1,2 = 1.2 Hz, H-1), 5.04 (dd, 1 H, J_2,3 = 3.3, J_3,4 = 10.3 Hz, H-3), 4.71, 4.54 (2 d, 1 H each, ²J = 12.3 Hz, CH₂Ph), 4.04–3.96 (m, 2 H, H-2,5), 3.68 (t, 1 H, J = 10.2 Hz, H-4), 2.70–2.51 (m, 2 H, CH₂CH₃), 2.07 (s, 3 H, COCH₃), 1.37 (d, 3 H, J_5,6 = 6.2 Hz, H-6), 1.27 (t, 3 H, J = 7.3 Hz, CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 81.53 (J_C-1,H-1 = 164.2 Hz, C-1), 76.34 (C-2), 72.71 (C-3), 72.49 (CH₂Ph), 67.22 (C-5), 62.84 (C-4), 25.22 (CH₂CH₃), 20.78 (COCH₃), 18.25 (C-6), 14.74 (CH₂CH₃). CIMS: m/z 383 [M + NH₄]⁺. Anal. Calcd for C₁₇H₂₃N₃O₄S: C, 55.87; H, 6.34; N, 11.50; S, 8.77. Found: C, 55.99; H, 6.23; N, 11.54; S, 8.75.

Eluted later was the β-anomer 9 (2.2 g), [α]_D -95 (c 1.6). ¹H NMR (300 MHz, CDCl₃): δ 4.79 (dd, partially overlapped, J_2,3 = 3.2, J_3,4 = 10.4 Hz, H-3), 4.80, 4.62 (2 d, partially overlapped, ²J ~12.0 Hz, CH₂Ph), 4.60 (d, partially overlapped, J_1,2 ~1.0 Hz, H-1), 4.03 (dd, 1 H, H-2), 3.65 (t, 1 H, H-4), 3.31–3.22 (m, 1 H, H-5), 2.71 (q, 2 H, J = 7.4 Hz, CH₂CH₃), 1.98 (s, 3 H, COCH₃), 1.40 (d, 3 H, J_5,6 = 6.0 Hz, H-6), 1.29 (t, 3 H, J = 7.5 Hz, CH₂CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 84.31 (J_C-1,H-1 = 150.6 Hz, C-1), 77.41 (C-2), 76.18 (C-3), 76.08 (CH₂Ph), 75.65 (C-5), 62.60 (C-4), 27.00 (CH₂CH₃), 21.03 (COCH₃), 18.95 (C-6), 15.34 (CH₂CH₃). CIMS: m/z 383 [M + NH₄]⁺. Anal. Calcd for C₁₇H₂₃N₃O₄S: C, 55.87; H, 6.34; N, 11.50; S, 8.77. Found: C, 56.17; H, 6.26; N, 11.52; S, 8.76.

The intermediate, mixed fraction (1.85 g) was purified by chromatography, to give more 8 (665 mg, total yield, 85%) and 9 (625 mg, total yield, 9.4%).

3.8 5-Methoxycarbonylpentyl 4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (12)

SnCl₄ (3.5 mL, 29.5 mmol) was added to a solution of 5 (8.25 g, 22.7 mmol) in dry CH₂Cl₂ (200 mL) and the mixture was stirred for 20 min with exclusion of atmospheric moisture. A solution of 5-(methoxycarbonyl)pentanol²⁴ (4.3 g, 29.5 mmol) in dry CH₂Cl₂ (30 mL) was added to a slightly yellow reaction mixture and, when the reaction was complete (~40 min, TLC in 40:1 toluene–acetone), the mixture was neutralized with aqueous, saturated sodium hydrogen carbonate. The product was extracted with CH₂Cl₂ and, after concentration, chromatography gave 5-methoxycarbonylpentyl 3-O-acetyl-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (11, 8.28 g, 80%). ¹H NMR (300 MHz, CDCl₃): δ 5.09 (dd, 1 H, J_2,3 = 3.3, J_3,4 = 10.3 Hz, H-3), 4.73 (d, 1 H, J_1,2 = 1.8 Hz, H-1), 4.66, 4.57 (2 d, 1 H each, ²J = 12.1 Hz, CH₂Ph), 3.80 (dd, 1 H, H-2), 3.67 (s, partially overlapped, COOCH₃), 3.67–3.43 (m, partially overlapped, H-4,5,1′a), 3.37–3.34 (2 t, 1 H, J = 6.5 Hz, H-1′b), 2.31 (t, 2 H, J = 7.3 Hz, H-5′a,b), 2.07 (s, 3 H, COCH₃), 1.71–1.51 (m, 4 H, H-4′a,b,2′a,b), 1.39–1.31 (m, 5 H, H-3′a,b, incl. d, 1.35, J_5,6 ~5.8 Hz, H-6). ¹³C NMR (75 MHz, CDCl₃): δ 97.57 (J_C-1,H-1 = 169.1 Hz, C-1), 75.00 (C-2), 73.14 (CH₂Ph), 72.53 (C-3), 67.53 (C-1′), 66.82 (C-5), 62.74 (C-4), 51.43 (COOCH₃), 33.84 (C-5′), 28.92 (C-2′), 25.58 (C-3′), 24.57 (C-4′), 20.89 (COCH₃), 18.38 (C-6). FABMS: m/z 450 [M + H]⁺, 472 [M + Na]⁺.

A solution of the foregoing acetylated compound 11 (5.66 g, 15.58 mmol) in MeOH (150 mL) was treated with 1 M NaOMe (~5 mL). After 18 h, TLC (5:1 hexane–EtOAc) showed that the deacetylation of 12 was complete. The solution was neutralized with Amberlite IR-120 (H⁺) cation-exchange resin, concentrated, and chromatography of the residue gave 12 (4.38 g, 85%), [α]_D +19.4 (c 4). ¹H NMR (300 MHz, CDCl₃): δ 4.80 (d, 1 H, J_1,2 = 1.5 Hz, H-1), 4.73, 4.57 (2 d, 1 H each, ²J = 11.7 Hz, CH₂Ph), 3.84 (ddd, 1 H, J_2,3 = 3.7, J_3,4 = 10.1 Hz, H-3), 3.67 (dd, partially overlapped, 1 H, H-2), 3.66 (s, partially overlapped, COOCH₃), 3.63, 3.60 (2 t, partially overlapped, J = 6.8 Hz, H-1′a), 3.53–3.44 (m, 1 H, H-5), 3.36, 3.33 (2 t, 1 H, H-1′b), 3.26 (t, 1 H, J = 9.9 Hz, H-4), 2.60 (d, 1 H, J_3,OH = 10.3 Hz, OH), 2.30 (t, 2 H, J = 7.4 Hz, H-5′a,b), 1.68–1.50 (m, 4 H, H-4′a,b,2′a,b), 1.39–1.26 (m, 5 H, H-3′a,b, incl. d, 1.31, J_5,6 = 6.2 Hz, H-6). ¹³C NMR (75 MHz, CDCl₃): δ 96.53 (C-1), 77.36 (C-2), 72.84 (CH₂Ph), 70.22 (C-3), 67.28 (C-1′), 66.39 (2 C, C-4,5), 51.36 (COOCH₃), 33.73 (C-5′), 28.85 (C-2′), 25.51 (C-3′), 24.47 (C-4′), 18.24 (C-6). FABMS: m/z 408 [M + H]⁺, 430 [M + Na]⁺. Anal. Calcd for C₂₀H₂₉N₃O₆: C, 58.95; H, 7.17; N, 10.31. Found: C, 59.10; H, 7.19; N, 10.23.

3.9 5-Methoxycarbonylpentyl (3-O-acetyl-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (13)

The glycosyl donor 8 (7.18 g, 19.65 mmol) and the acceptor 12 (6.16 g, 15.12 mmol) were coupled as described in General procedure, to give after chromatography (10:1→5:1 hexane–EtOAc) 13 (9.385 g, 89%), [α]_D +65 (c 0.8). ¹H NMR (300 MHz, C₆D₆): δ 5.63 (dd, 1 H, J_2,3 = 3.2, J_3,4 = 10.0 Hz, H-3^II), 5.28 (d, 1 H, J_1,2 = 1.7 Hz, H-1^II), 4.81 (d, 1 H, J_1,2 = 1.7 Hz, H-1^I), 4.61, 4.48 (2 dd, 1 H each, ²J = 11.8 and 12.1 Hz, 2 CH₂Ph), 4.22 (dd, 1 H, H-2^II), 4.14 (dd, 1 H, J_2,3 = 3.2, J_3,4 = 9.8 Hz, H-3^I), ~3.87 (m, partially overlapped, H-5^II), 3.81 (t, partially overlapped, H-4^II), 3.76 (dd, 1 H, H-2^I), 3.71–3.55 (m, 2 H, H-4^I,5^I), 3.47, 3.44 (2 t, 1 H, J = 6.4 Hz, H-1′a), 3.38 (s, 3 H, COOCH₃), 3.13, 3.09 (2 t, 1 H, H-1′b), 2.06 (t, 2 H, J = 7.4 Hz, H-5′), 1.74 (s, 3 H, COCH₃), 1.52–1.42 (m, 2 H, H-4'a,b), 1.35–1.09 (m, 10 H, H-2′a,b,3'a,b, incl. 2 d at 1.24, 1.22, J_5,6 = 6.1 Hz, H-6^I,II). ¹³C NMR (75 MHz, C₆D₆): δ 100.34 (C-1^II), 97.88 (C-1^I), 78.39 (C-3^I), 78.15 (C-2^I), 76.25 (C-2^II), 73.72, 73.43 (2 CH₂Ph), 72.85 (C-3^II), 68.64 (C-5^II), 68.20 (C-5^I), 68.05 (C-1′), 65.53 (C-4^I), 63.55 (C-4^II), 51.38 (COOCH₃), 34.20 (C-5′), 29.60 (C-2′), 26.25 (C-3′), 25.19 (C-4′), 20.79 (COCH₃), 18.97 (C-6^I), 18.84 (C-6^II). FABMS: m/z 685.4 [M + H – 2 N + 2 H]⁺, 843.3 [M + Cs]⁺, 717.1 [M + Li]⁺. Anal. Calcd for C₃₅H₄₆N₆O₁₀: C, 59.14; H, 6.52; N, 11.82. Found: C, 58.95; H, 6.52; N, 11.72.

3.10 5-Methoxycarbonylpentyl (4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (14)

De-O-acetylation (Zemplén) of 13 and chromatography (15:1 toluene–EtOAc) gave 14 (4.23 g, 94%), [α]_D +54 (c 1.2). ¹H NMR (C₆D₆, 300 MHz): δ 5.22 (d, 1 H, J_1,2 = 1.8 Hz, H-1^II), 4.81 (d, 1 H, J_1,2 = 1.8 Hz, H-1^I), 4.50, 4.40 (2 dd, 2 H each, ²J = 11.9 Hz, 2 CH₂Ph), 4.12 (dd, partially overlapped, 1 H, J_3,4 = 3.1, J_2,3 = 9.7 Hz, H-3^I), ~4.11 (ddd, partially overlapped, H-3^II), 3.90 (dd, 1 H, H-2^II), 3.81–3.75 (m, partially overlapped, H-2^I,5^II), 3.72 (t, partially overlapped, J = 9.7 Hz, H-4^I), 3.66–3.56 (m, 1 H, H-5^I), 3.34 (t, partially overlapped, J = 9.9 Hz, H-4^II), 3.48, 3.45 (2 t, 1 H, J = 6.2 Hz, H-1′a), 3.37 (s, 3 H, COOCH₃), 3.12, 3.09 (2 t, 1 H, H-1′b), 2.17 (d, 1 H, J_3,OH = 10.1 Hz, OH), 2.06 (t, 2 H, J = 7.4 Hz, H-5′a,b), 1.52–1.42 (m, 2 H, H-4'a,b), 1.37–1.09 (m, 10 H, H-2′a,b, 3'a,b, incl. 2 d at 1.28, 1.23, J_5,6 = 6.2 Hz, H-6^I,II, in that order). ¹³C NMR (75 MHz, C₆D₆): δ 100.05 (C-1^II), 97.69 (C-1^I), 78.96 (C-3^I), 78.37 (C-2^II), 78.13 (C-2^I), 73.56, 73.16 (2 CH₂Ph), 71.25 (C-3^II), 68.17 (C-5^I), 68.13 (C-1′), 68.09 (C-5^II), 67.03 (C-4^II), 65.69 (C-4^I), 51.37 (COOCH₃), 34.20 (C-5′), 29.59 (C-2′), 26.25 (C-3′), 25.19 (C-4′), 18.04 (C-6^I), 18.88 (C-6^II). FABMS: m/z 643.4 [M + H - 2N + 2H]⁺, 691.4 [M + Na]⁺. Anal. Calcd for C₃₃H₄₄N₆O₉: C, 59.57; H, 6.63; N, 12.57. Found: C, 59.45; H, 6.76; N, 12.76.

3.11 5-Methoxycarbonylpentyl (4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (15)

A gentle stream of H₂S was passed through a solution of 14 (500 mg, 0.75 mmol) in 2:1 pyridine–water (5 mL) for 30 min at 40 °C. The mixture was stirred at this temperature for 16 h, when TLC (10:1:0.1 CH₂Cl₂–MeOH–conc. NH₄OH) showed that the reaction was complete. A stream of nitrogen was passed through the reaction mixture for 15 min, and the solution was co-concentrated with several co-evaporations of water to remove pyridine. After co-evaporation with toluene, to remove water, the residue was dissolved in MeOH (20 mL) and Ac₂O (1 mL) was added. After 16 h, TLC (3:2 CH₂Cl₂–acetone) showed that the reaction was complete. The mixture was concentrated and chromatography of the residue (2:1→1:1 CH₂Cl₂–acetone) gave 15 (390 mg, 75%), mp. 176.5–177 °C (from acetone), [α]_D +35.4 (c 0.8). ¹H NMR (600 MHz, ~1:1 CDCl₃–C₆D₆): δ 5.87 (d, 1 H, J_4,NH = 9.8 Hz, NH^I), 5.18 (d, 1 H, J_4,NH = 9.2 Hz, NH^II), 5.00 (d, 1 H, J_1,2 = 1.3 Hz, H-1^II), 4.80 (d, 1 H, J_1,2 = 2.0 Hz, H-1^I), 4.78–4.50 (4 d partially overlapped, 2 CH₂Ph), 4.25 (q, 1 H, J = 9.8 Hz, H-4^I), 4.00 (q, 1 H, J = 9.8 Hz, H-4^II), 3.94 (dd, J_2,3 = 3.1, J_3,4 = 10.4 Hz, H-3^I), 3.70 (dd, , J_2,3 = 3.4 Hz, H-2^II), 3.69 (bt, H-2^I), 3.67 (m, 2 H, H-3^II,5^I), 3.60 (m, 1 H, 1′a), 3.45 (m, 1 H, H-5^II), 3.42 (s, 3 H, COOCH₃), 3.27 (m, 1 H, H-1′b), 2.66 (bd, 1 H, OH), 2.21–2.12 (m, 2 H, H-5′a,b), 1.80, 1.79 (2 s, 6 H, 2 COCH₃), 1.59–1.52 (m, 3 H, H-4'a,b,2′a), 1.48–1.42 (m, 1 H, H-2′b), 1.40–1.29 (m, 1 H, H-3′a), 1.27 (d, 3 H, J_5,6 = 6.0 Hz, H-6^I), 1.25–1.19 (m, 1 H, H-3′b), 1.10 (d, 3 H, J_5,6 = 6.2 Hz, H-6^II). ¹³C NMR (150 MHz, ~1:1 CDCl₃–C₆D₆): δ 99.26 (C-1^II), 97.12 (C-1^I), 77.52 (C-2^II), 77.33 (C-2^I), 76.66 (C-3^I), 72.70, 72.01 (2 CH₂Ph), 69.41 (C-3^II), 67.99 (C-5^II), 67.84 (C-5^I), 66.85 (C-1′), 53.99 (C-4^II), 52.68 (C-4^I), 51.22 (COOCH₃), 33.82 (C-5′), 28.62 (C-2′), 26.00 (C-3′), 24.44 (C-4′), 23.13, 23.08 (2 COCH₃), 18.09 (C-6^I), 17.94 (C-6^II). FABMS: m/z 701 [M + H]⁺, 723 [M + Na]⁺. Anal. Calcd for C₃₇H₅₂N₂O₁₁: C, 63.41; H, 7.49; N, 4.00. Found: C, 63.44; H, 7.39; N, 3.97.

3.12 5-Methoxycarbonylpentyl (4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-4,6-dideoxy-α-d-mannopyranoside (16) and 5-methoxycarbonylpentyl (4-acetamido-2,3-di-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-2-O-acetyl-4,6-dideoxy-α-d-mannopyranoside (17)

A mixture of compound 15 (140 mg) and 5% palladium-on-charcoal catalyst (140 mg) in methanol (40 mL), was stirred under hydrogen overnight at 45 °C. TLC (5:1 CHCl₃–MeOH) showed that the reaction was complete. After filtration and evaporation of the solvent, the residue was eluted from a small column of silica gel using 5:1 CHCl₃–MeOH as eluent to give, after freeze-drying and additional drying at 40 °C/133 Pa, 16 (99.5 mg, 96%), [α]_D +8.2 (c 1.7, H₂O). Definite signals in the ¹H NMR spectrum (600 MHz, D₂O, 50 °C) were at δ 5.18 (d, 1 H, J_1,2 = 1.7 Hz, H-1^II), 5.08 (d, 1 H, J_1,2 = 1.8 Hz, H-1^I), 4.23 (dd, 1 H, J_2,3 = 3.0 Hz, H-2^I), 4.19 (t, J = 10.3 Hz, H-4^I), ~4.12 (dd, overlapped, H-3^I), ~4.11 (m, overlapped, H-3^II,5^II), ~4.09 (overlapped,H- 4^II), ~4.08 (overlapped, H-2^II), ~4.07 (m, H-5^I), 3.98–3.94 (m, 4 H, H-1′a, incl. s at 3.96 for COOCH₃), 3.80, 3.78 (2 t, 1 H, J = 6.0 Hz, H-1′b), 2.67 (t, 2 H, J = 7.5 Hz, H-5′a,b), 2.30, 2.29 (2 s, 6 H, 2 NHCOCH₃), 1.92–1.85 (m, 4 H, H-4′a,b,2′a,b), 1.69–1.62 (m, 2 H, H-3′a,b), 1.46 (d, 3 H, J_5,6 = 6.2 Hz, H-6^I), 1.44 (d, 3 H, J_5,6 = 5.8 Hz, H-6^II). ¹³C NMR (D₂O, 50°C): δ 180.41 (COOCH₃), 177.67, 177.41 (2 NHCOCH₃), 105.26 (C-1^II), 102.75 (C-1^I), 79.99 (C-3^I), 72.68 (C-2^II), 72.33 (C-2^I) 71.34, 71.27 (C-3^II,5^II), 70.96 (C-1′), 70.60 (C-5^I), 56.04 (C-4^II), 55.20 (COOCH₃), 55.07 (C-4^I,II), 36.72 (C-5′), 31.26 (C-2′), 28.07 (C-3′), 27.13 (C-4′), 25.33, 25.25 (2 COCH₃), 19.96, 19.90 (C-6^I,II). FABMS: m/z 543 [M + Na]⁺. The corresponding per-O-acetyl derivative 17 showed [α]_D +58 (c 0.9, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 6.09 (d, 1 H, J_4,NH = 9.5 Hz, NH^I), 5.68 (d, 1 H, J_4,NH = 10.0 Hz, NH^II), 5.18 (dd, 1 H, J_2,3 = 3.1 Hz, H-2^I), 5.09 (dd, 1 H, J_2,3 = 3.3, J_3,4 = 10.9 Hz, H-3^II), 4.99 (d, 1 H, J_1,2 = 2.0 Hz, H-1^II), 4.90 (dd, 1 H, H-2^II), 4.70 (d, 1 H, H-1^I), 4.18 (q, partially overlapped, J = 10.1 Hz, H-4^II), 4.13 (q, partially overlapped, J = 10.9 Hz, H-4^I), 4.07 (dd, 1 H, H-3^I), 3.88 (m, 1 H, H-5^II), 3.77 (m, 1 H, H-5^I), 3.69–3.66 (m, 4 H, H-1′a, incl. 3.67, s, COOCH₃), 3.42, 3.40 (2 t, 1 H, J = 5.1 Hz, H-1′b), 2.36 (m, 2 H, H-5′a,b), 2.23, 2.12, 2.01, 2.06, 1.96 (5 s, 15 H, 5 COCH₃), 1.67 (m, 3 H, H-2′a,4′a,b), 1.58 (m, 1 H, H-2′b), 1.49 (m, 1 H, H-3′a), 1.35 (m, 1 H, H-3′b), 1.26 (d, 3 H, J_5,6 = 6.2 Hz, H-6^I), 1.23 (d, 3 H, J_5,6 = 6.3 Hz, H-6^II). ¹³C NMR (150 MHz, CDCl₃): δ 97.97 (C-1^II), 97.25 (C-1^I), 73.49 (C-3^I), 70.45 (C-2^I), 69.95 (C-2^II), 68.84 (C-5^II), 67.95 (C-3^II), 67.63 (C-5^I), 66.86 (C-1′), 52.61 (C-4^I), 51.58 (COOCH₃), 51.34 (C-4^II), 33.86 (C-5′), 28.55 (C-2′), 25.88 (C-3′), 24.29 (C-4′), 23.40, 23.33 (2 NHCOCH₃), 21.20, 20.96, 20.81 (3 COCH₃), 17.92 (C-6^II), 17.75 (C-6^I). ESI-MS (m/z): 647.3023; calcd for [M + H]⁺ 6747.3027. Anal. Calcd for C₂₉H₄₆N₂O₁₄: C, 53.86; H, 7.17; N, 4.33. Found: C, 54.04; H, 7.26; N, 4.29.

3.13 5-Methoxycarbonylpentyl (2-O-acetyl-4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (18)

Reaction of 10^14,21 (2.2 g, 6.02 mmol) and 14 (3.125 g, 4.67 mmol) as described in the General procedure gave, after chromatography (20:1→5:1 hexane–EtOAc), the fully protected trisaccharide 18 (4.275 g, 95%), [α]_D +79.6 (c 1.2). ¹H NMR (600 MHz, C₆D₆): δ 5.87 (dd, 1 H, J_1,2 = 1.8, J_2,3 = 3.1 Hz, H-2^III), ~5.25 (d, partially overlapped, H-1^III), ~5.24 (d, partially overlapped, H-1^II), 4.81 (d, 1 H, J_1,2 = 1.7 Hz, H-1^I), 4.74–4.39 (6 d, partially overlapped, 6 H, 3 CH₂Ph), 4.22 (dd, 1 H, J_2,3 = 3.1, J_3,4 = 9.7 Hz, H-3^II), 4.13 (dd, partially overlapped, J_2,3 = 3.3, J_3,4 = 9.2 Hz, H-3^I), 4.12 (dd, partially overlapped, H-2^II), 4.10 (dd, 1 H, J_3,4 = 10.0 Hz, H-3^III), 3.94 (m, 1 H, H-5^III), 3.85–3.77 (m, 3 H, H-5^II,2^I,4^II in that order), 3.69 (t, 1 H, J = 9.9 Hz, H-4^I), 3.63 (t, 1 H, J = 9.9 Hz, H-4^III), 3.59 (m, partially overlapped, H-5^I), 3.47, 3.46 (2 t, J = 6.7 Hz, 1 H, H-1′a), 3.36 (s, 3 H, COOCH₃), 3.12, 3.11 (2 t, 1 H, H-1′b), 2.05 (t, 2 H, J = 7.3 Hz, H-5′a,b), 1.65 (s, 3 H, COCH₃), 1.49–1.44 (m, 2 H, H-4′a,b), 1.36–1.29 (m, partially overlapped, H-2′a,b), 1.31 (d, partially overlapped, J_5,6 = 5.9 Hz, H-6^III), 1.25 (d, J_5,6 = 6.1 Hz, H-6^I), 1.20 (d, J_5,6 = 6.0 Hz, H-6^II), 1.18–1.11 (m, 2 H, H-3′a,b). ¹³C NMR (150 MHz, C₆D₆): δ 100.93 (C-1^III), 100.04 (C-1^II), 97.32 (C-1^I), 80.17 (C-3^I), 79.71 (C-3^II), 78.34 (C-2^I), 77.80 (C-2^II), 76.93 (C-3^III), 73.02, 72.98, 72.02 (3 CH₂Ph), 68.81 (C-5^II) 68.54 (C-5^III), 68.22 (C-1′), 67.99 (C-5^I), 67.95 (C-2^III), 65.26 (C-4^I), 65.19 (C-4^II), 64.96 (C-4^III), 51.39 (COOCH₃), 34.18 (C-5′), 29.61 (C-2′), 26.28 (C-3′), 25.18 (C-4′), 20.73 (COCH₃), 19.09, 19.06 (2C) (C-6^I–III). FABMS: m/z 946 [M + H - 2N + 2 H]⁺. Anal. Calcd for C₄₈H₆₁N₉O₁₃: C, 59.31; H, 6.33; N, 12.97. Found: C, 59.47; H, 6.34; N, 12.89.

3.14 5-Methoxycarbonylpentyl (4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (19)

Deacetylation of 18 (5.45 g, 5.6 mmol) as described for 13 gave, after chromatography and crystallization, 19 (3.75 g, 98%), mp. 37–40 °C (from isopropyl ether), [α]_D +76 (c 1.9). ¹H NMR (600 MHz, C₆D₆): δ 5.31 (d, 1 H, J_1,2 = 1.5 Hz, H-1^III), 5.26 (d, 1 H, J_1,2 = 1.5 Hz, H-1^II), 4.82 (d, 1 H, J_1,2 = 1.6 Hz, H-1^I), 4.59–4.34 (6 d, partially overlapped, 6 H, 3 CH₂Ph), 4.24 (dd, 1 H, J_2,3 = 3.1 Hz, H-2^III), 4.21 (dd J_2,3 = 3.2, J_3,4 = 9.8 Hz, H-3^II), ~4.14 (dd, partially overlapped, H-2^II), 4.13 (dd, partially overlapped, J_2,3 = 3.1, J_3,4 = 9.9 Hz, H-3^I), 3.90 (m, partially overlapped, H-5^III), 3.88 (dd, partially overlapped, J_3,4 = 9.8 Hz, H-3^III), 3.84–3.80 (m, 2 H, H-2^I,5^II), 3.77 (t, 1 H, J = 9.9 Hz, H-4^II), 3.70 (t, J = 9.9 Hz, H-4^I), 3.60 (m, 1 H, H-5^I), 3.51 (t, 1 H, J = 9.9 Hz, H-4^III), 3.47, 3.45 (2 t, 1 H, J = 6.6 Hz, H-1′a), 3.36 (s, 3 H, COOCH₃), 3.12, 3.10 (2 t, 1 H, H-1′b), 2.05 (t, 2 H, J = 7.4 Hz, H-5′a,b), 1.50–1.43 (m, 2 H, H-4′a,b), 1.36–1.29 (m, 5 H, H-2′a,b,6^III), 1.26 (d, 3 H, J_5,6 = 6.1 Hz, H-6^I), 1.20 (d, 3 H, J_5,6 = 6.0 Hz, H-6^II), 1.17–1.12 (m, 2 H, H-3′a,b). ¹³C NMR (150 MHz, C₆D₆): δ 102.61 (C-1^III), 100.01 (C-1^II), 97.44 (C-1^I), 80.01 (C-3^I), 79.35 (C-3^II), 79.01 (C-3^III), 78.36 (C-2^I), 77.96 (C-2^II), 73.08, 73.00, 71.93 (3 CH₂Ph), 68.71 (C-5^II), 68.34 (C-5^III), 68.19 (C-1′), 68.05 (C-5^I), 67.83 (C-2^III), 65.32 (C-4^I), 65.27 (C-4^II), 64.59 (C-4^III), 51.38 (COOCH₃), 34.19 (C-5′), 29.61 (C-2′), 26.27 (C-3′), 25.19 (C-4′), 19.08, 19.05, 19.04 (C-6^I–III). FABMS: m/z 904 [M + H - 2N + 2 H]⁺, 952 [M + Na]⁺. Anal. Calcd for C₄₆H₅₉N₉O₁₂: C, 59.41; H, 6.39; N, 13.55. Found: C, 59.49; H, 6.55; N, 13.55.

3.15 5-Methoxycarbonylpentyl (4-acetamido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (20)

Reduction of 19 (1 g, 1.075 mmol) with H₂S followed by N-acetylation, as described for 14, gave 20 (0.9 g, 86%). ¹H NMR (600 MHz, CD₃OD at 45 °C): δ 5.06 (d, 1 H, J_1,2 = 1.8 Hz, H-1^II), 4.93 (d, 1 H, J_1,2 = 1.8 Hz, H-1^III), 4.80 (d, 1 H, J_1,2 = 1.8 Hz, H-1^I), 4.79–4.41 (6 d, partially overlapped, 6 H, 3 CH₂Ph), 4.18–4.13 (m, 2 H, H-4^I,II), 4.01–3.97 (m, 4 H, H-2^III,4^III,3^I,II in that order), 3.88 (m, 1 H, H-5^II), 3.82 (m, partially overlapped, H-5^III), 3.79 (bt, partially overlapped, H-2^I), 3.78 (bt, partially overlapped, H-2^II), 3.72 (m, 1 H, H-5^I ), 3.69–3.63 (m, 5 H, H-1′a, 3^III, incl. s, 3.64, COOCH₃), 3.42, 3.41 (2 t, 1 H, J = 6.1 Hz, H-1′b), 2.33 (t, 2 H, J = 7.3 Hz, H-5′a,b), 1.97, 1.96, 1.88 (3 s, 3 H each, 3 COCH₃), 1.66–1.61 (m, partially overlapped, H-4′a,b), 1.61–1.56 (m, partially overlapped, H-2′a,b), 1.43–1.38 (m, 2 H, H-3′a,b), 1.18, 1.15, 1.13 (3 d, 3 H each, J_5,6 6.2 Hz, H-6^I–III, in that order). ¹³C NMR (150 MHz, CD₃OD, 45°C): δ 103.45 (C-1^III), 100.58 (C-1^II), 98.93 (C-1^I), 79.38 (C-2^II), 78.85 (C-2^I), 78.11, 77.97, 77.91 (C-3^I–III), 74.05, 74.01, 72.07 (3 CH₂Ph), 69.74 (C-5^II), 69.54 (C-5^III), 68.96 (C-5^I), 68.59 (C-1′), 68.40 (C-2^III), 53.90, 53.83 (C-4^I,II), 53.12 (C-4^III), 51.99 (COOCH₃), 34.68 (C-5′), 30.09 (C-2′), 26.81 (C-3′), 25.69 (C-4′), 23.18, 22.99, 22.94 (3 NHCOCH₃), 18.54, 18.46, 18.28 (C-6^I–III). FABMS: m/z 984.5 [M + Li]⁺, 1000.50 [M + Na]⁺. FAB HRMS: m/z 1000.4783 (100%); calcd for C₅₂H₇₁N₃NaO₁₅, 1000.4783.

3.16 5-Methoxycarbonylpentyl (4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-4,6-dideoxy-α-d-mannopyranoside (21) and 5-methoxycarbonylpentyl (4-acetamido-2,3-di-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-2-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido--2-O-acetyl-4,6-dideoxy-α-d-mannopyranoside (22)

Compound 20 (0.75 g) was treated with hydrogen, as described for 15. Elution of the crude product from a small silica gel column (5:1→3:2 CH₂Cl₂-MeOH) gave compound 21 (345 mg, 64%) [α]_D +78.9 (c 3.7, H₂O). Structurally significant signals in the 1H NMR spectrum (D₂O at 50 °C, 600 MHz) were at: δ 5.13 (bd, 2 H, H-1^II,III), 5.02 (bd, 1 H, H-1^I), 4.21 (bdd, 1 H, H-2^I), 3.93, 3.92, 3.79, 3.75 (4 t, 2 H, J = 6.5 Hz, H-1′a.b), 2.62 (t, 2 H, J = 7.4 Hz, H-5′a,b), 2.27. 2.26, 2.25 (3 bs, 12 H, 3 NHCOCH₃), 1.43–1.41 (m, H-6^I–III). Structurally significant signals in the ¹³C NMR spectrum (D₂O at 50 °C, 600 MHz) were at: δ 105.25, 105.13 (C-1^II,III), 102.75 (C-1^I), 52.11 (COOCH₃), 36.86 (C-5′), 31.31 (C-2′), 28.09 (C-3′), 27.16 (C-4′), 25.35, 25.32, 25.25 (3 NHCOCH₃), 20.03, 19.98, 19.95 (C-6^I,III). FABMS: m/z 708.5 [M + H]⁺, 730.5 [M + Na]⁺. The corresponding per-O-acetyl derivative 22 showed [α]_D +50 (c 1.2, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 6.38 (d, 1 H, J_4,NH = 9.5 Hz, NH^I), 5.83 (d, 1 H, J_4,NH = 9.9 Hz, NH^III), 5.80 (d, 1 H, J_4,NH = 10.0 Hz, NH^II), 5.14 (dd, 1 H, J_1,2 = 1.9 Hz, H-2^I), 5.05 (dd, 1 H, J_2,3 = 2.6, J_3,4 = 10.8 Hz, H-3^III), 5.02 (dd, 1 H, J_1,2 = 2.0, J_2,3 = 3.3 Hz, H-2^II), 4.92–4.90 (m, 3 H, H-2^III,1^III,II in that order), 4.70 (d, 1 H, J_1,2 = 1.9 Hz, H-1^I), 4.16 (t, partially overlapped, J = 10.0 Hz, H-4^III), 4.15–4.04 (m, partially overlapped, H-4^I,II,H-3^I in that order), 3.87 (dd, partially overlapped, H-3^II), 3.83 (m, partially overlapped, H-5^I ), ~3.77 (m, partially overlapped, H-5^III), ~3.75 (m, partially overlapped, H-5^I), 3.69–3.65 (m, 4 H, H-1′a, incl. 3.68, s, COOCH₃), 3.42, 3.40 (2 t, 1 H, J = 5.4 Hz, H-1′b), 2.36 (m, 2 H, H-5′a,b), 2.19, 2.17, 2.14 (3 s, 9 H, 3 NHCOCH₃), 2.05, 2.02, 2.00, 1.97 (4 s, 12 H, 4 COCH₃), 1.74–1.25 (4 m, 6 H, H-4′a,b,2′b,3′a,3′b in that order), 1.25, 1.23, 1.21 (3 d, 9 H, J_5,6 = 6.2 Hz, H-6^I,II,III in that order). ¹³C NMR (150 MHz, CDCl₃): δ 98.44 (C-1^II), 97.85 (C-1^III), 97.05 (C-1^I), 73.63 (C-3^I), 72.76 (C-3^II), 70.53 (C-2^I), 70.37 (C-2^II), 69.49 (C-2^III), 68.82 (C-5 ^III), 68.44 (C-5^II), 68.05 (C-3^III), 67.55 (C-5^I), 68.84 (C-1′), 52.37 (C-4^I), 52.14 (C-4^II), 51.49 (COOCH₃), 51.02 (C-4^III), 33.78 (C-5′), 28.44 (C-2′), 25.76 (C-3′), 24.24 (C-4′), 23.26, 23.15, 21.12, 20.95, 20.88, 20.67 (7 COCH₃), 17.75, 17.66 (3 C, C-6^I–III). ESI-MS (m/z): 914.3589; calcd for C₃₉H₆₁N₃O₁₉K: 914.3536. Anal. Calcd for C₃₉H₆₁N₃O₁₉: C, 53.48; H, 7.02; N, 4.80. Found: C, 53.76; H, 6.98; N, 4.56.

3.17 5-Methoxycarbonylpentyl (3-O-acetyl-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (23)

Reaction of 8 (1.8 g, 5 mmol) and 19 (3.75 g, 3.86 mmol) as described in General procedure gave, after chromatography (15:1→5:1 hexane–EtOAc), the protected tetrasaccharide 23 (4.45 g, 94%), mp. 112.5–113.5 °C (EtOH–EtOAc), [α]_D +37.2 (c 1.1). ¹H NMR (600 MHz, C₆D₆): δ 5.53 (dd, 1 H, J_2,3 = 3.1, J_3,4 = 10.1 Hz, H-3^IV), 5.36 (d, 1 H, J_1,2 = 1.6 Hz, H-1^IV), 5.32 (d, 1 H, J_1,2 = 1.8 Hz, H-1^III), 5.26 (d, 1 H, J_1,2 = 1.4 Hz, H-1^II), 4.82 (d, 1 H, J_1,2 = 1.8 Hz, H-1^I), 4.61–4.05 (8 d, partially overlapped, 4 CH₂Ph), 4.49 (bt, H-2^III), 4.27 (dd, partially overlapped, H-3^II), 4.15 (dd, 1 H, J_2,3 = 3.0, J_3,4 = 9.6 Hz, H-3^I), ~ 4.13 (m, 2 H, H-2^II,IV), 4.00 (dd, 1 H, J_2,3 = 2.8, J_3,4 = 9.8 Hz, H-3^III), 3.92–3.81 (m, partially overlapped, H-5^IV,III,II in that order), 3.82 (m, H-2^I,4^IV), 4.81 (t, partially overlapped, H-4^II), 4.76 (t, partially overlapped, H-4^III), 3.74 (t, partially overlapped, H -4^I), 3.60 (m, 1 H, H-5^I), 3.48, 3.46 (2 t, 1 H, J = 6.4 Hz, H-1′a), 3.36 (s, 3 H, COOCH₃), 3.13, 3.11 (2 t, 1 H, H-1′b), 2.05 (t, 2 H, J = 7.3 Hz, H-5′a,b), 1.70 (s, 3 H, COCH₃), 1.50–1.45 (m, 2 H, H-4′a,b), 1.36–1.30 (m, 2 H, H-2′), 1.30 (d, partially overlapped, H-6^IV), 1.29 (d, partially overlapped, H-6^III), 1.25 (d, 3 H, J_5,6 = 6.2 Hz, H-6^I), 1.22 (d, 3 H, J_5,6 = 6.0 Hz, H-6^II), 1.17–1.14 (m, 2 H, H-3'a,b). ¹³C NMR (150 MHz, C₆D₆): δ 102.19 (C-1^III), 100.06 (C-1^II), 99.59 (C-1^IV), 97.36 (C-1^I), 80.11 (C-3^I), 79.43 (C-3^III), 79.01 (C-3^II), 78.53 (C-2^I), 77.90 (C-2^II), 75.52 (C-2^IV), 73.29 (C-2^III), 73.12, 73.09 (2 CH₂Ph), 73.01 (C-3^IV), 72.97, 72.78 (2 CH₂Ph), 68.98 (C-5^III), 68.77 (C-5^II), 68.71 (C-5^IV), 68.20 (C-1′), 68.01 (C-5^I), 65.51 (C-4^II), 65.34 (C-4^I), 65.10 (C-4^III), 63.52 (C-4^IV), 51.37 (COOCH₃), 34.18 (C-5′), 29.62 (C-2′), 26.28 (C-3′), 25.19 (C-4′), 20.79 (COCH₃), 19.05, 19.03 18.89 (4 C, C-6^I–IV). FABMS: m/z 1207.6 [M + H – 2 N + 2 H]⁺, 1255 [M + Na]⁺. Anal. Calcd for C₆₁H₇₆N₁₂O1₆: C, 59.40; H, 6.21; N, 13.63. Found: C, 59.43; H, 6.11, N, 13.54.

3.18 Methoxycarbonylpentyl (4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (24)

Deacetylation of 23 (4.27 g, 3.46 mmol) as for 13, except that the reaction was started at 50 °C, to solubilize the starting material, gave pure 24 (4.03 g, 97%), which crystallized on standing but could not be crystallized from common solvents, mp. 53–57 °C, [α]_D +10.0 (c 0.4). ¹H NMR (600 MHz, C₆D₆): δ 5.30 (bd, partially overlapped, H-1^IV), 5.29 (d, partially overlapped, H-1^III), 5.26 (d, 1 H, J_1,2 = 1.7 Hz, H-1^II), 4.83 (d, 1 H, J_1,2 = 1.8 Hz, H-1^I), 4.63–4.01 (8 d, partially overlapped, 4 CH₂Ph), 4.45 (bdd, 1 H, H-2^III), 4.25 (dd, 1 H, J_2,3 = 3.0, J_3,4 = 9.8 Hz, H-3^II), 4.14 (dd, 1 H, J_2,3 = 3.1, J_3,4 = 10.0 Hz, H-3^I), 4.11 (dd, 1 H, J_2,3 = 3.0 Hz, H-2^II), 4.05 (dd, 1 H, J_2,3 = 3.6, J_3,4 = 9.9 Hz, H-3^IV), 3.97 (dd, 1 H, J_2,3 = 3.5, J_3,4 = 9.7 Hz, H-3^III), 3.86–3.81 (m, partially overlapped, H-5^II,III), 3.82 (dd, partially overlapped, H-2^I), 3.80 (t, partially overlapped, J = 9.8 Hz, H-4^II), ~3.74 (m, partially overlapped, H-5^IV), 3.72 (m. partially overlapped, H-4^I,2^IV), 3.62–3.56 (m, 2 H, H-5^I,4^III), 3.49, 3.47 (2 t, 1 H, J = 6.5 Hz, H-1′a), 3.37 (s, partially overlapped, COOCH₃), 3.35 (t, partially overlapped, J = 10.0 Hz, H-4^IV), 3.15, 3.13 (2 t, 1 H, H-1′b), 2.06 (t, 2 H, J = 7.4 Hz, H-5′a,b), 1.51–1.46 (m, 2 H, H-4′a,b), 1.38–1.32 (m, partially overlapped, H-2′a,b), 1.31–1.21 (4 d, partially overlapped, H-6^I–IV), 1.20–1.13 (m, 2 H, H-3′a,b). ¹³C NMR (150 MHz, C₆D₆): δ 102.22 (C-1^III), 100.10 (C-1^II), 98.96 (C-1^IV), 97.33 (C-1^I), 80.16 (C-3^I), 79.20 (C-3^III), 78.86 (C-3^II), 78.49 (C-2^I), 77.88 (C-2^II), 77.41 (C-2^IV), 73.11, 73.06, 72.81, 72.66 (4 CH₂Ph), 72.97 (C-2^III), 71.10 (C-3^IV), 68.79. 68.77 (C-5^II,III), 68.23 (C-1′), 68.21 (C-5^IV), 68.00 (C-5^I), 67.03 (C-4^IV), 65.67 (C-4^II), 65.33 (C-4^I), 65.10 (C-4^III), 51.43 (COOCH₃), 34.20 (C-5′), 29.61 (C-2′), 26.28 (C-3′), 25.19 (C-2′), 19.01, 18.95, 18.75 ( 4C, C-6^I–IV). FABMS: m/z 1165.6 [M + H – 2 N + 2 H]⁺. Anal. Calcd for C₅₉H₇₄N₁₂O₁₅: C, 59.48; H, 6.26; N, 14.11. Found: C, 59.50; H, 6.27; N, 13.83.

3.19 5-Methoxycarbonylpentyl (4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-acetamido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (25)

Treatment of 24 (1.42 g, 1.19 mmol) as for compound 19 but in pyridine and water (3:1), to assist solubility of the starting material, gave the corresponding, intermediate tetrakis-amine (1 g). FABMS: m/z 1087.62 [M + H]⁺, 1109.61 [M + Na]⁺.

A solution of the foregoing amine was N-acetylated, as described for preparation of 15, to give after chromatography 25 (1.05 g, 70% from 24), [α]_D +30.0 (c 0.2). Structurally significant ¹H NMR (600 MHz, C₆D₆) signals were at: δ 7.62, 6.73, 5.86, 5.48 (4 bs, NH^I–IV), 5.21, 5.10, 5.07 (3 bs, H-1^II–IV), 4.80 (bs, H-1^I), 4.37–4.28 (m, 4 H, H-4^I–IV), 3.60–3.58, 3.24–3.22 (2 m, 2 H, H-1′a,b), 3.38 (s, 3 H, COOCH₃), 2.12–2.09 (m, 2 H, H-5′a,b), 2.00, 1.99, 1.87, 1.43 (4 s, 4 NHCOCH₃), 1.46, 1.31, 1.24, 1.23 (4 d, H-6^I–IV). The spectrum taken in CD₃OD showed peaks at: δ 5.08, 5.01, 4.96 (3 d, 1 H each, H-1^II–IV), 4.80 (d, 1 H, J_1,2 = 1.9 Hz, H-1^I), 4.82–4.36 (8 d, 1 H, each, 4 CH₂Ph), 4.15 (bt, partially overlapped, H-4^I), 3.78 (bdd, 1 H, H-2^I), 3.64 (s, partially overlapped, COOCH₃), 3.42, 3.40 (2 t, 1 H, J = 6.2 Hz, H-1′b), 2.43 (t, 2 H, J = 7.3 Hz, H-5′a,b), 1.99, 1.98, 1.96, 1.93 (4 s, 3 H each, 4 NHCOCH₃), 1.18, 1.17, 1.12, 1.10 (4 d, 12 H, J_5,6 ~6.3 Hz, H-6^I–IV). Structurally significant ¹³C NMR (150 MHz, C₆D₆) signals were at: δ 101.20, 99.40, 97.84 (br, C-1^II–IV), 96.89 (br, C-1^I), 51.03 (COOCH₃), 23.27, 23.16. 23.09, 22.96 (NHCOCH₃). The spectrum taken in CD₃OD showed peaks at: δ 102.19, 100.66 (3 C) (C-1^II–IV), 98.94 (C-1^I), 74.25, 74.00, 73.92, 72.75 (4 CH₂Ph), 68.57 (C-1′), 52.06 (COOCH₃), 23.30, 23.25, 22.91, 22.88 (4 NHCOCH₃), 18.68, 18.67, 18.57, 18.26 (C-6^I–IV). FABMS: m/z 1278 [M + Na]⁺.

3.20 5-Methoxycarbonylpentyl (4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-4,6-dideoxy-α-d-mannopyranoside (26) and 5-methoxycarbonylpentyl (4-acetamido-2,3-di-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-acetamido-3-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-2-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-2-O-acetyl-4,6-dideoxy-α-d-mannopyranoside (27)

Hydrogenolysis of 25 (960 mg) and processing as described for 16, gave the deprotected, amorphous tetrasaccharide 26 in virtually theoretical yield, [α]_D +62.4 (c 6.6, H₂O). ¹H NMR (600 MHz, D₂O at 50 °C): δ 5.27 (d, 1 H, J_1,2 = 1.6 Hz, H-1^III), 5.20, d, 1 H, J_1,2 = 1.7 Hz, H-1^II), 5.15 (d, 1 H, J_1,2 = 1.7 Hz, H-1^IV), 5.05 (d, 1 H, J_1,2 = 1.7 Hz, H-1^I), 4.33 (bdd, 1 H, H-2^II), 4.25 (bt, 1 H, H-2^I), ~4.23 (m, partially overlapped, H-3^III), ~4.19 (m, partially overlapped, H-3^II), 4.12 (m, partially overlapped, H-3^I), ~4.09 (m, partially overlapped, H-2^IV), 4.03 (bdd, 1 H, H-2^III), 3.96 (m, 4 H, H-1′a, incl s, 3.96 COOCH₃), 3.80, 3.78 (2 t, 1 H, J = 6.1 Hz, H-1′b), 2.66 (t, 2 H, J = 7.4 Hz, H-5′a,b), 2.31, 2.30, 2.29, 2.28 (4 s, 3 H each, 4 NHCOCH₃), 1.92–1.85 (m, 4 H, H-2′a,b4′a,b), 1.69–1.62 (m, 2 H, H-3′a,b), 1.46–1.45 (m, 12 H, H-6^I–IV). ¹³C NMR (150 MHz, D₂O at 50°C): δ 105.47 (C-1^II), 105.21 (C-1^1V), 103.90 (C-1^III), 102.72 (C-1^I), 81.60 (C-2^III), 80.52 (C-3^I), 80.02 (C-3^II), 72.58 (C-2^IV), 72.39 (C-2^I), 72.20 (C-2^II), 70.98 (C-1′), 52.08 (COOCH₃), 36.70 (C-5′), 31.23 (C-2′), 28.04 (C-3′), 27.10 (C-4′), 25.43, 25.34, 25.30, 25.27 (4 NHCOCH₃), 20.20, 20.05, 19.90, 19.86 (C-6^I–IV). FABMS: m/z 917.5 [M + Na]⁺. The corresponding per-O-acetyl derivative 27 showed [α]_D +67, c 1.1, CHCl₃). ¹H NMR (600 MHz, CDCl₃): δ 6.98 (d, 1 H, J_4,NH = 9.3 Hz, NH^IV), 6.38 (d, 1 H, J_4,NH = 9.8 Hz, NH^I), 5.82 (bd, 2 H, NH^II,III), 5.16–5.13 (m, 3 H, H-2^I,2^IV,3^IV, in that order), 5.06 (dd, 1 H, J_2,3 = 3.2 Hz, J_3,4 = 8.7 Hz, H-3^III), 4.95 (bdd, 1 H, H-2^II), 4.90 (d, 1 H, J_1,2 = 2.0 Hz, H-1^II), 4.88 (d, 1 H, J_1,2 = 2.8 Hz, H-1^III), 4.86 (bd, 1 H, J_1,2 ~1 Hz, H-1^IV), 4.71 (d, 1 H, J_1,2 = 1.7 Hz, H-1^I), 4.20 (bq, 1 H, H-4^IV), 4.13 (bq, 1 H, H-4^I), 4.04–3.99 (m, 3 H, H-3^II,4^III,3^I, in that order), 3.94–3.81 (m, 4 H, H-4^II,5^II,2^III,5^IV, in that order), 3.75 (m, partially overlapped, H-5^I), 3.73–3.65 (m, 5 H, H-5^III,1′a, incl. 3.68, s, COOCH₃), 3.42, 3.40 (2 t, 1 H, J = 5.2 Hz, H-1′b), 2.36 (t, 2 H, J = 7.0 Hz, H-5′), 2.19, 2.14, 2.13, 2.08, 2.07, 2.03, 2.01, 1.98, 1.97 (9 s, 3 H each, 9 COCH₃), 1.71–1.61 (m, 3 H, H-4′a,b,2′a), 1.60–1.55 (m, H-2′b), 1.45, 1.38 (2 m, 2 H, H-3′a,b), 1.29, 1.25, 1.23, 1.20 (4 d, J_5,6 = 1.2 Hz, H-6^IV,I,III,II in that order). ¹³C NMR (CDCl₃, 150 MHz): δ 99.55 (C-1^III), 98.55 (C-1^II), 97.85 (C-1^IV), 97.06 (C-1^I), 74.59 (C-3^I), 74.21 (C-2^III), 72.04 (C-3^II), 70.95 (C-2^II), 69.71 (C-3^III), 70.55, 68.88 (C-2^I,IV), 68.81 (C-3^IV), 68.56 (C-5^III), 68.50 (C-5^IV), 68.19 (C-5^II), 67.46 (C-5^I), 66.81 (C-1′), 52.74 (C-4^II), 52.30 (2 C, C-4^I,III), 51.58 (COOCH₃), 50.88 (C-4^IV), 33.80 (C-5′), 28.48 (C-2′), 25.77 (C-3′), 24.21 (C-4′), 23.49, 23.36, 23.26, 23.16 (4 NHCOCH₃), 21.14, 21.01, 20.91, 20.79, 20.78 (5 COCH₃), 18.07, 17.78, 17.74, 17.73 (C-6^I–IV). ESI-MS (m/z): 1105.4944 [M + H]⁺; calcd for C₄₉H₇₇N₄O₂₄: 1105.4928. Anal. Calcd for C₄₉H₇₆N₄O₂₄: C, 53.25; H, 6.93; N, 5.07. Found: C, 53.07; H, 6.97; N, 5.00.

3.21 5-Methoxycarbonylpentyl (3-O-acetyl-4-azido-2-O-benzyl-4,6-dideoxy-α-_D-mannopyranosyl)-(1→3)-(4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (28)

Condensation of the glycosyl donor 8 (1 g, 2.73 mmol) and acceptor 24 (2.61 g, 2.19 mmol), performed as described in General procedure, afforded pure (TLC, NMR) 28 (2.37 g, 73%), [α]_D +49.8. ¹H NMR (600 MHz, C₆D₆): δ 5.55 (dd, 1 H, J_2,3 = 3.3, J_3,4 = 10.1 Hz, H-3^V), 5.39 (d, 1 H, J_1,2 = 1.6 Hz, H-1^III), 5.36 (d, 1 H, J_1,2 = 1.6 Hz, H-1^IV), 5.28 (d, 1 H, J_1,2 = 1.6 Hz, H-1^V), 5.24 (d, 1 H, J_1,2 = 1.6 Hz, H-1^II), 4.81 (d, 1 H, J_1,2 = 1.6 Hz, H-1^I), 4.61–4.36 (m, 11 H, 5 CH₂Ph, incl. bdd, 4.46, H-2^III), 4.27 (dd, 1 H, J_2,3 = 3.1, J_3,4 = 9.9 Hz, H-3^II), 4.22 (dd, J_2,3 = 3.0, J_3,4 = 10.1 Hz, H-3^IV), 4.19 (dd, H-2^V), 4.13 (dd, partially overlapped, J_2,3 = 3.2, J_3,4 = 9.9 Hz, H-3^I), ~4.11 (dd, partially overlapped, H-2^II), 4.00 (dd, 1 H, J_2,3 = 2.9, J_3,4 = 9.9 Hz, H-3^III), 3.89 (dd, partially overlapped, H-2^IV), 3.87 (m, partially overlapped, H-5^III,II,IV, in that order), ~3.82 (m, partially overlapped, H-2^I), 3.80 (t, partially overlapped, H-4^II), 3.79 (t, partially overlapped, H-4^V), ~3.73 (m, partially overlapped, H-5^V), 3.71 (t, partially overlapped, H-4^IV), 3.69 (t, partially overlapped, H-4^I), 3.66 (t, partially overlapped, H-4^III), 3.58 (m, partially overlapped, H-5^I), 3.48, 3.46 (2 t, 1 H, J = 6.5 Hz, H-1′a), 3.37 (s, 3 H, COOCH₃), 3.14, 3.12 (2 t, 1 H, H-1′b), 2.05 (t, 2 H, J = 7.3 Hz, H-5′a,b), 1.72 (s, 3 H, COCH₃), 1.50–1.45 (m, 2 H, H-4′a,b), 1.37–1.31 (m, partially overlapped, H-2′a,b, incl. 1.33 ,d, J_5,6 = 6.2 Hz, H-6^III), 1.28 (d, J_5,6 = 6.1 Hz, H-6^IV), 1.24 (d, partially overlapped, J_5,6 = 6.2 Hz, H-6^I), 1.23, (d, partially overlapped, H-6^V), 1.22 (d, partially overlapped, H-6^II). ¹³C NMR (150 MHz, C₆D₆): δ 102.21 (C-1^III), 100.26 (C-1^V), 100.08 (C-1^II), 99.60 (C-1^IV), 97.38 (C-1^I), 80.18 (C-3^I), 79.04 (C-3^III), 78.96 (C-3^II), 78.47 (C-2^I), 77.87 (C-2^II), 77.45 (C-2^IV), 77.43 (C-3^IV), 76.21 (C-2^V), 73.85 (C-2^III), 73.73, 73.10, 73.07 (3 CH₂Ph), 72.94 (2 C, CH₂Ph, C-3^V), 72.67 (CH₂Ph), 69.27 (C-5^IV), 68.82, 68.78 (C-5^II,III), 68.70 (C-5^V), 68.24 (C-1′), 68.02 (C-5^I), 65.62 (C-4^II), 65.56 (C-4^III), 65.37, 65.34 (C-4^I,IV), 63.46 (C-4^V), 51.38 (COOCH₃), 34.19 (C-5′), 29.62 (C-2′), 26.30 (C-3′), 25.20 (C-4′), 20.79 (COCH₃), 19.09 (C-6^III), 19.05 (C-6^IV), 19.03, 18.97 (C-6^I,IV), 18.75 (C-6^II). FABMS: m/z 1468.7 [M + H – 2 N + 2 H]⁺, 1516.6 [M + Na]⁺. Anal. Calcd for C₇₄H₉₁N₁₅O₁₉: C, 59.47; H, 6.14; N, 14.06. Found: C, 59.30; H, 6.21; N, 13.93.

3.22 5-Methoxycarbonylpentyl (4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-azido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-azido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (29)

Deacetylation of 28 (2.25 g, 1.505 mmol) as described for 12, except that 1:2 MeOH–toluene mixture was used as solvent, gave 29 (2.05 g, 94%), [α]_D +69.2 (c 2.4). ¹H NMR (600 MHz , C₆D₆): δ 5.37 (d, 1 H, J_1,2 = 1.7 Hz, H-1^III), 5.36 (d, 1 H, J_1,2 = 1.7 Hz, H-1^IV), 5.24 (d, 1 H, J_1,2 = 1.7 Hz, H-1^II), 5.20 (d, 1 H, J_1,2 = 1.3 Hz, H-1^V), 4.81 (d, 1 H, J_1,2 = 1.7 Hz, H-1^I), 4.60–4.21 (10 d, partially overlapped, 5 CH₂Ph), 4.46 (bt, 1 H, H-2^III), 4.27 (dd, 1 H, J_2,3 = 3.1, J_3,4 = 9.9 Hz, H-3^II), 4.18 (dd, 1 H, J_2,3 = 3.0, J_3,4 = 10.2 Hz, H-3^IV), 4.12 (dd, 1 H, J_2,3 = 3.2, J_3,4 = 9.9 Hz, H-3^I), 4.10 (dd, 1 H, H-2^II), 4.02 (dd, 1 H, J_2,3 = 3.6, J_3,4 = 9.8 Hz, H-3^V), 3.99 (dd, 1 H, J_3,4 = 9.9 Hz, H-3^III), 3.88–3.82 (m, 5 H, H-2^IV,2^V,5^III,5^IV,5^II, in that order), 3.81 (bt, 1 H, H-2^I), 3.80 (t, 1 H, J = 10.0 Hz, H-4^II), 3.74 (t, 1 H, J = 10.0 Hz, H-4^IV), 3.68 (t, partially overlapped, J = 10.0 Hz, H-4^I), 3.66 (t, partially overlapped, J = 10.0 Hz, H-4^III), 3.59 (m, 2 H, H-5^V,5^I, in that order), 3.48, 3.46 (2 t, 1H, J = 6.6 Hz, H-1′a), 3.36 (s, 3 H, COOCH₃), 3.30 (t, 1 H, J = 9.9 Hz, H-4^V), 3.14, 3.12 (2 t, 1 H, H-1′b), 2.05 (t, 2 H, J = 7.2 Hz, H-5′a,b), 1.50–1.45 (m, 2 H, H-4′a,b), 1.37–1.31 (m, partially overlapped, H-2′a,b), 1.31 (d, partially overlapped, J_5,6 = 6.2 Hz, H-6^III), 1.30 (d, 3 H, J_5,6 = 6.2 Hz, H-6^IV), 1.23 (d, 3 H, J_5,6 = 6.2 Hz, H-6^I), 1.22 (d, 3 H, J_5,6 = 6.2 Hz, H-6^V), 1.21 (d, 3 H, J_5,6 = 6.1 Hz, H-6^II), 1.18–1.13 (m, 2 H, H-3′a,b). ¹³C NMR (C₆D₆, 150 MHz): δ 102.19 (C-1^III), 100.04 (C-1^II), 99.86 (C-1^V), 99.34 (C-1^IV), 97.34 (C-1^I), 80.34 (C-3^I), 79.05 (C-3^III), 78.97 (C-3^II), 78.44 (C-2¹), 78.30 (C-2^V), 77.92 (C-3^IV), 77.83 (C-2^II), 77.38 (C-2^IV), 73.83 (C-2^III), 73.54, 73.07, 73.02, 72.68, 72.62 (5 CH₂Ph), 71.28 (C-3^V), 69.17 (C-5^IV), 68.83, 68.76 (C-5^II,III), 68.25 (C-1′), 68.10 (C-5^V), 67.99 (C-5^I), 66.86 (C-4^V), 65.58 (C-4^II), 65.52 (C-4^IV), 65.50 (C-4^III), 65.30 (C-4^I), 51.41 (COOCH₃), 34.19 (C-5′), 29.62 (C-2′), 26.29 (C-3′), 25.19 (C-4′), 19.07, 19.04, 19.02 (2 C), 18.78 (C-6^I–V). FABMS: m/z 1426.6 [M + H – 2 N + 2 H]⁺, 1474.5 [M + Na]⁺. Anal.Calcd for C₇₂H₈₉N₁₅O₁₈: C, 59.53; H, 6.18; N 14.46. Found: C, 59.56; H, 6.25; N, 14.57.

3.23 5-Methoxycarbonylpentyl (4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-acetamido-3-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-2-O-benzyl-4,6-dideoxy-α-d-mannopyranoside (30)

Reduction of 29 (1.935 g, 1.33 mmol) in 3:1 pyridine-H₂O was performed as described for 24. After work-up and chromatography (EtOAc–methanol 5:1 to 3:1), the intermediate pentakis-amine {(: m/z 1322.67 [M+H]⁺) was N-acetylated, as described above for similar conversions, and chromatography (CH₂Cl₂–acetone 1:1→2:3) gave 30 (1.5 g, 74%), [α]_D +31.2 (c 1.8). FABMS: m/z 1532.8 [M + H]⁺, 1554.8 [M + Na]⁺. Structurally significant resonances in the ¹H NMR (600 MHz, CD₃OD at 45 °C) were at: δ 5.15, 5.08, 5.07, 5.02 (4 d, 1 H each, J_1,2 ~1.6 Hz, H-1^II–V), 4.79 (d, 1 H, J_1,2 = 1.6 Hz, H-1^I), 4.80–4.41 (10 d, partially overlapped, 5 CH₂Ph), 3.64 (s, 3 H, COOCH₃), 2.33 (t, 2 H, J = 7.5 Hz, H-5′), 1.98, 1.97, 1.95, 1.92, 1.89 (5 s, 3 H each, 5 NHCOCH₃), 1.19, 1.18, 1.14, 1.30, 1.12 (5 d, partially overlapped, H-6^I–V). Structurally significant resonances in the ¹³C NMR (150 MHz, CD₃OD at 45 °C) were at: δ 102.20, 100.92, 100.28, 100.16 (C-1^II–V), 99.12 (C-1^I), 74.28, 74.15, 74.02, 73.96, 73.19 (5 CH₂Ph), 68.78 (C-1′), 52.14 (COOCH₃), 23.50, 23.44, 23.21, 23.06, 23.03 (5 NHCOCH₃), 18.90, 18.77, 18.73, 18.72, 18.46 (C-6^I–V).

3.24 5-Methoxycarbonylpentyl (4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-4,6-dideoxy-α-d-mannopyranoside (31) and 5-methoxycarbonylpentyl (4-acetamido-2,3-di-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-2-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→2)-(4-acetamido-3-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-(4-acetamido-2-O-acetyl-4,6-dideoxy-α-d-mannopyranosyl)-(1→3)-4-acetamido-2-O-acetyl-4,6-dideoxy-α-d-mannopyranoside (32)

Hydrogenolysis of 30 (1.4 g, 0.91 mmol), performed as described for 25 gave, after freeze-drying, the deprotected pentasaccharide 31 in virtually theoretical yield, [α]d +79.2 (c 5.1, H₂O). Structurally significant resonances in the ¹H NMR spectrum (600 MHz, D₂O at 50 °C) were at 5.26 (d, 1 H, J_1,2 = 1.4 Hz, H-1^III), 5.22, 5.19, 5.15 (3 d, J_1,2 ~1.6 Hz, H-1^II,IV,V), 5.05 (d, 1 H, J_1,2 = 1.5 Hz, H-1^I), 4.24 (dd, 1 H, J_2,3 = 3.1 Hz, H-2^I), 4.04 (d, 1 H, J_2,3 = 3.0 Hz, H-2^III), 3.95 (m, 4 H, H-1′a, incl s, COOCH₃), 3.80, 3.78 (2 t, 1 H, H-1′b), 2.66 (t, 2 H, J = 7.6 Hz, H-5′a,b), 2.31, 2.30, 2.28 (3 s, 15 H, 5 NHCOCH₃), 1.91–1.84 (m, 4 H, H-2′a,b,4′a,b), 1.68–1.60 (m, 2 H, H-3′a,b), 1.48–1.44 (5 d, partially overlapped, H-6^I–V). ¹³C NMR (150 MHz, D₂O at 50 °C): δ 105.26, 105.22, 105.07 (C-1^II,IV,V), 103.84 (C-1^III), 102.71 (C-1^I), 81.48 (C-2^III), 80.56, 80.25, 79.67 (C-3^I,II,IV), 70.99 (C-1′), 52.06 (COOCH₃), 36.69 (C-5′), 31.21 (C-2′), 28.01 (C-3′), 25.08 (C-4′), 25.41, 25.29, 25.28, 25.34, 25.22 (5 NHCOCH₃), 20.13, 20.04, 19.94, 19.87, 19.83 (C-6^I–V). FABMS: m/z 1082.6 [M + H]⁺, 1104.6 [M + Na]⁺. The corresponding per-O-acetyl derivative 32 showed [α]_D +57 (c 1.1, CHCl3). ESI-MS (m/z): 1351.6146 [M + NH₄]⁺, calcd for C₅₉H₉₅N₆O₂₉: 1351.6143. ¹H NMR (CDCl₃, 600 MHz): δ 7.33 (bd, 1H, NH^IV), 6.39 (d, 1 H, J_4,NH = 10.1 Hz, NH^I), 5.98 (bs, 1 H, NH^II), 5.86 (bd, 1 H, NH^III), 5.78 (d, 1 H, J_4,NH = 9.9 Hz NH^V), 5.18 (bdd, 1 H, H-2^IV), 5.15 (dd, 1 H, J_1,2 = 1.9, J_2,3 = 3.2 Hz, H-2^I), 5.07 (dd, partially overlapped, H-3^V), ~5.07 (d, partially overlapped, H-1^V), ~5.06 (dd, partially overlapped, H-3^III), 4.95 (dd, 1 H, J_1,2 = 2.0, J_2,3 = 3.1 Hz, H-2^V), 4.92 (bt, 1 H, H-2^II), 4.90 (d, 1 H, J_1,2 = 2.0 Hz, H-1^II), 4.84 (d, 1 H, J_1,2 = 1.7 Hz, H-1^IV), 4.83 (bd, 1 H, J_1,2 ~3 Hz, H-1^III), 4.71 (d, 1 H, J_1,2 = 1.9 Hz, H-1^I), 4.16, t, partially overlapped, J ~10.0 Hz, H-4^V), 4.14 (t, partially overlapped, H-4^IV), 4.12 (t, partially overlapped, J = 10.4 Hz, H-4^I), 4.04–3.97 (m, 4 H, H-3^IV,3^II,4^III,3^I, in that order), 3.96–3.89 (m, 3 H, H-5^V,4^II,5^II), 3.86 (bt, 1 H, H-2^III), 3.80 (m, 1 H, H-5^IV), 3.74 (m, 1 H, H-5^I), 3.70–3.65 (m, 5 H, H-5^III,1′a, incl 3.68, s COOCH₃), 3.41, 3.40 (2 t, 1 H, J = 4.9 Hz, H-1′b), 2.37 (t, 2 H, J = 6.9 Hz, H-5′a,b), 2.22–1.97 (10 s, 33 H, 11COCH₃), 1.71–1.63 (m, 3 H, H-2′a,4′a,b), 1.61–1.54 (m, 1 H, H-2′b), 1.53–1.46 (m, 1 H, H-3′a), 1.37–1.30 (m, 1 H, H-3′b), 1.28–1.20 (6 d, 15 H, partially overlapped, J_5,6 ~6.2 Hz, H-6^I–V). ¹³C NMR (CDCl₃, 150 MHz): δ 99.60 (C-1^III), 98.58 (C-1^II), 97.81 (b, C-1^IV), 97.31 (C-1^V), 97.02 (C-1^I), 74.77 (b, C-3^I), 73.64 (b, C-2^III), 72.05 (b, C-3^II), 71.47 (b, C-3^IV), 71.03 (C-2^II), 70.59 (C-2^I), 69.96 (C-3^III), 69.87 (C-2^IV), 69.57 (C-2^V), 68.57 (C-5^V), 68.48 (C-5^III), 68.39 (C-5^IV), 68.09 (C-5^II), 68.00 (C-3^V), 67.45 (C-5^I), 66.72 (C-1′), 52.79 (C-4^II), 52.40 (C-4^III), 52.26 (2 C, C-4^I,IV), 51.56 (COCH₃), 51.19 (C-4^V), 33.79 (C-5′), 28.47 (C-2′), 25.72 (C-3′), 24.16 (C-4′), 23.46, 23.37, 23.33 (2 C), 23.13 (5 NHCOCH₃), 21.07, 21.04, 20.99, 20.93, 20.73 (5 COCH₃), 18.08, 17.83, 17.76, 17.70, 17.64 (C-6^I–V). Anal. Calcd for C₅₉H₉₁N₅O₂₉: C, 53.11; H, 6.87; N, 5.25. Found: C, 52.99; H, 6.99; N, 5.01.

Scheme 2.