Synthetic oligosaccharides as tools to demonstrate cross-reactivity between polysaccharide antigens

Abstract

Escherichia coli O148 is a non-encapsulated enterotoxigenic (ETEC) Gram negative bacterium that can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. The surface-exposed O-specific polysaccharide (O-SP) of the lipopolysaccharide of this bacterium is considered both a virulence factor and a protective antigen. It is built up of the linear tetrasaccharide repeating unit 3)-α-d-Rhap-(1→2)-α-d-Glcp-(1→3)-α-d-GlcNAcp-(1→3)-α-d-Rhap-(1→ differing from that of the O-SP of Shigella dysenteriae type 1 (SD) only in that the latter contains a d-Galp residue in place of the glucose moiety of the former. The close similarity of the O-SP's of these bacteria indicated a possible cross-reactivity. To answer this question we synthesized several oligosaccharide fragments of E. coli O148 O-SP, up to a dodecasaccharide, as well as their bovine serum albumin or recombinant diphtheria toxin conjugates. Immunization of mice with these conjugates induced anti O-SP-specific serum IgG antibody responses. The antisera reacted equally well with the LPS's of both bacteria, indicating cross-reactivity between the SD and E. coli O148 O-PS's that was further supported by Western-blot and dot-blot analyses, as well as by inhibition of binding between the antisera and the O-SP's of both bacteria.

INTRODUCTION

Escherichia coli O148 is an enterotoxigenic (ETEC) bacterium that has been identified as a cause of enteric infections in children and adults, including dysentery, hemorrhagic colitis, and hemolytic uremic syndrome. This disease is endemic in developing countries and affects travelers to those areas.^1–5 The main carriers of ETEC bacteria are food and water⁵ and their spreading is facilitated by poor sanitary conditions. As has been reported recently regarding the spread of the bacterium E. coli O104 in several Western-European countries, even the best sanitary conditions cannot always prevent an epidemic. It is likely that infections caused by various ETEC serotypes are underreported because of insufficient surveillance and difficulties in serotyping. In early 2009, the World Health Organization declared the development of a vaccine against ETEC an urgency.^6,7

E. coli O148 has been proposed to be a precursor to Shigella dysenteriae type 1 (SD), the most virulent of all Shigellae.⁸ Virulence of these two bacteria is related to their O-specific polysaccharide (O-SP): those that lack a fully developed O-SP are not considered virulent.⁹ The repeating unit of the O-SP of E. coli O148 (A) is similar to the O-SP of SD

• [3)-α-l-Rhap-(1→2)-α-d-Glcp-(1→3)-α-d-GlcpNAc-(1→3)-α-l-Rhap-(1→]_n A

• [3)-α-l-Rhap-(1→2)-α-d-Galp-(1→3)-α-d-GlcpNAc-(1→3)-α-l-Rhap-(1→]_n B

(B), the only difference being in the chirality of a single carbon atom in the tetrasaccharide repeating unit: the galactose residue in SD is replaced by a d-glucose moiety in the E. coli repeating unit, while the anomeric configurations and the locations of all of the interglycosidic linkages are preserved. The two bacteria have the same genes for O antigen synthesis, except that in SD, a glucosyltransferase gene is interrupted by a deletion, and a galactosyltransferase gene located on a plasmid is responsible for the transfer of galactose to synthesize the O-antigen.⁸ The two bacteria also have chemically identical LPS cores and O-chain–core linkage regions.¹⁰

Based on the close similarity of the O-SP's of E. coli O148 and SD we hypothesized that they may cross-react, i.e. antibodies raised against one of the O-SP's or their fragments will react (bind) not only with the saccharides of the homologous organism but also with those of the cross-reacting ones. We note, however, that cross-reactivity between similar surface polysaccharides of different bacteria is not obvious. We approached this question by using synthetic oligosaccharides.

We have reported the synthesis of a panel of oligosaccharides related to the O-SP of SD up to a tetracosasaccharide^11–13 and demonstrated that the immunogenicity of their BSA conjugates in mice is influenced by the size of the saccharides, their loading on the protein, as well as by the identity of the non-reducing terminus.^12,14

In this paper, we report our initial studies that may lead to the development of a single component neoglycoconjugate vaccine against two enteric bacteria, namely SD and E. coli O148, consisting of a covalent conjugate of an oligosaccharide portion of the O-specific oligosaccharide portion of only one of them, covalently attached to an immunogenic protein. The idea behind using a protein conjugate of synthetic or natural oligosaccharides to induce anti-carbohydrate serum is not new: it dates back to the early part of the twentieth century when Avery and Goebel showed that a covalent conjugate of the capsular polysaccharide of Type 3 pneumococcus with horse serum globulin elicited anti-polysaccharide-specific antibodies in rabbits.¹⁵ The antisera conferred both active and passive protection against the homologous organism. Goebel also showed that a covalent conjugate of cellobiuronic acid with horse serum globulin elicited cellobiuronic acid-specific antibodies in rabbits that precipitated the Type 3 pneumococcal polysaccharide.^16,17 Goebel's neoglycoprotein protected rabbits against challenge by the homologous organism. Based on this idea, several commercial vaccines have been developed for human use, consisting of protein conjugates of bacterial polysaccharides. These include vaccines against infections by Haemophilus influenzae type b, Neisseria meningitidis serotypes A, C, Y, W-135, pneumococci, and Salmonella typhi.¹⁸ Recently, it has been shown that a tetanus toxoid conjugate of synthetic fragments of the capsular material of H. influenzae type b consisting of an average of eight ribosyl-ribitol-phosphate repeating units is an efficacious vaccine for both children and adults.¹⁹ The potential of synthetic oligosaccharide fragments of bacterial cell-surface glycans as antibacterial vaccines has generated increasing interest in the field that led to the synthesis of numerous bacterial oligosaccharides^20–24 and improved conjugation methods.²⁵

Here, we first report the chemical synthesis of oligosaccharide components 1–7 of the O-SP of E. coli O148, ranging from tetra- to dodecasaccharides (Figure 1), then test their immunogenicity and cross-reactivity with the O-SP of SD. Oligosaccharides 1–7 are equipped with a heterobifunctional spacer to allow their one-point conjugation to the proteins bovine serum albumin (BSA) and recombinant diphtheria toxin. These constructs, injected without an adjuvant at a schedule and dosage compatible with use for humans, were used to evaluate the antibody response to the native O-SP's in mice. We also describe the isolation of purified LPS's of E. coli O148 and SD and report on their binding to mice sera elicited by various protein conjugates of the synthetic oligosaccharides, and to sera generated in rabbits by heat-killed E. coli O148 and SD. The selection of this panel of oligosaccharides was based on our observations on the immunogenicity of BSA conjugates of SD-related oligosaccharides in mice.^12,14 In those studies, we found that the conjugates containing an N-acetyl-D- glucosamine or a D-galactose residue at the non-reducing end of the oligosaccharide portion induced statistically significantly higher O-SP-specific IgG antibody levels in mice than those having an L-rhamnose moiety at that position. It is to be expected that mapping the immunogenicity of the protein conjugates of the synthesized oligosaccharides will contribute to the development of oligosaccharide-based semisynthetic vaccines against enteric diseases.

Figure 1.

Spacer-equipped oligosaccharide fragments of the O-SP of E. coli O148 synthesized in this study

RESULTS

Synthetic studies

We envisioned a blockwise approach to the target oligosaccharides 1–7 that required mono-, di-, tri-, and tetrasaccharide building units (Figure 2). For example, the retrosynthetic analysis of the dodecasaccharide 7 called for the tetrasaccharide donor 8 as the crucial intermediate that was prepared from the rhamnosyl donor 9¹¹ and the trisaccharide acceptor 10 that in turn was constructed by condensation of the the glucosyl donor 11 and the disaccharide alcohol 12.²⁶ The spacer-linked tetrasaccharide 13 was obtained from the imidate 8. The synthesis of the dodecasaccharide 14 was completed by two iterative sequential chain extension steps using the tetrasaccharide donor 8. This was followed by global deprotection and installation of the linker to afford 7, in which the spacer features a keto functionality to be used for the conjugation to proteins, using the oxime methodology.²⁷ The syntheses of the oligosaccharides 1–6 were carried out in a similar fashion, as described in detail below.

Figure 2.

Retrosynthetic strategy toward the targeted oligosaccharides

The tetrasaccharide block 8 was prepared from the trisaccharide 10, assembly of which required the D-glucose derivative 11 that was prepared as shown in Scheme 1. The starting material was orthoester 15 that was deacetylated (→ 16) followed by O-benzylation to afford compound 17.²⁸ Next, we attempted to convert orthoester 17 into the diacetate 18, using acetic acid.²⁹ It has been noted that in such conversions the acetic acid must be meticulously dry, because even traces of water would lead to complete hydrolysis.^28,29 A safer alternative is the use of AcOSi(CH₃)₃ instead of acetic acid.³⁰ Thus, boiling a solution of orthoester 17 in AcOSi(CH₃)₃ afforded the diacetate 18 in 93% yield after chromatographic purification. Next, 18 was converted to thioglucoside 19 with trimethylsilylthiophenol³¹ in the presence of BF₃.Et₂O in 84% yield. Subsequently, the acetyl group in 19 was removed by NaOMe in MeOH to afford the alcohol 20. The β anomeric configuration was ascertained from the J_1,2 coupling constant whose value was 9.5 Hz. Next, compound 20 was reacted with 4-methoxybenzyl chloride in the presence of NaH to afford 21 in 91% yield. Subsequent hydrolysis of the thioglucosyl linkage by the action of (CF₃CO₂)₂Hg¹¹ in moist CH₂Cl₂ afforded the hemiacetal 22 in 97% yield, from which the Schmidt-type donor 11 was prepared in nearly quantitative yield by treatment with CCl₃CN/Cs₂CO₃.

Scheme 1a.

Synthesis of glucosyl trichloroacetimidate 11

^aReagents and conditions: (a) NaOMe, MeOH, quant.; (b) BnBr, NaH, DMF, 95%; (c) AcOSi(CH₃)₃ (excess), reflux, 2 h, 93%; (d) PhSSi(CH₃)₃, BF₃, Et₂O, CH₂Cl₂, 0 °C, 90 min, 84%; (e) MeONa, MeOH, 4 h, 97%; (f) MBnCl, NaH, DMF, 91%; (g) (CF₃CO₂)₂Hg, CH₂Cl₂, H₂O, 97%; (h) CCl₃CN, Cs₂CO₃, CH₂Cl₂, quantitative.

With the glucosyl imidate 11 in hand, synthesis of the trisaccharide donor 28 was undertaken. (Scheme 2) First, the disaccharide alcohol 12²⁶ was glucosylated with compound 11 using TMSOTf activation. Under these conditions, an inseparable 4:1 mixture of the α and β-linked trisaccharides 23 was obtained in a combined yield of 90 %. Attempted activation of 11 by other Lewis acids, e. g. BF₃-etherate failed to improve the anomeric stereoselectivity. Next, the azido group was converted to acetamido by catalytic hydrogenation over palladium-on-charcoal in the presence of Et₃N, followed by reaction with Ac₂O (→ 24). Subsequent treatment with ammonium ceric nitrate in a mixture of CH₃CN and water removed the methoxybenzyl group. To our delight, the required trisaccharide alcohol 10 could be isolated in a pure form in 57% yield after column chromatographic purification without the β glucosyl-linked minor product 25 that was also recovered in 11% yield. The synthesis of the trisaccharide 28 was completed by (i) acetylation of HO-2 of the glucose residue in 10 (→ 26, quantitative), (ii) removal of the trimethylsilylethyl group by treatment of compound 26 with trifluoroacetic acid³² (→ 27, 80%) and (iii) exposure of the hemiacetal 27 to CCl₃CN/DBU to afford the trisaccharide trichloroacetimidate 28 in 95%yield.

Scheme 2a.

Synthesis of the trisaccharide trichloroacetimidate 28.

^aReagents and conditions: (a) 1.6 equivalents of 11, TMSOTf, −40 °C → 0 °C, 2h, 90%; (b) H₂ (200 psi), Pd/C, Et₃N, EtOAc, EtOH, Ac₂O; (c) Ce(NH₄)₂(NO₃)₆, CH₃CN, H₂O, yields: 57% for 10, 11% for 25; (d) Ac₂O, C 5H₅N, DMAP, CH₂Cl₂, quantitative; (e) TFA, CH₂Cl₂, 80%; (f) CCl₃CN, DBU, CH₂Cl₂, 95%.

The tetrasaccharide building block 8 was synthesized by coupling the trisaccharide acceptor 10 with the rhamnosyl donor 9¹¹ under TMSOTf activation, to afford tetrasaccharide 29 in 91% yield (Scheme 3). Routine removal of the trimethylsilylethyl group with trifluoroacetic acid in CH₂Cl₂³² (→ 30, 65%) followed by reaction with CCl₃CN/DBU yielded the tetrasaccharide trichloroacetimidate 8 in 78% yield.

Scheme 3a.

Synthesis of the tetrasaccharide trichloroacetimidate 8.

^aReagents and conditions: (a) 4.5 equivalents of 9, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 3 h, 91%, (b) TFA, CH₂Cl₂, 23 °C, 3 h, 65%; (c) CCl₃CN, DBU, CH₂Cl₂, 0 °C → 23 °C, 1 h, 78%.

As a prelude to the assembly of higher-membered oligosaccharides, the tetrasaccharide donor 8 was condensed with methoxycarbonylpentanol 31³³ under TMSOTf promotion, to afford compound 13 in 81% yield (Scheme 4). We favor aglycon 31 as the linker because it is stable under a variety of conditions and can easily be converted to several reactive species for incorporation into proteins. Routine removal of the monochloroacetyl group by thiourea³⁴ afforded the tetrasaccharide alcohol 32 that was subjected to further chain extension steps with glycosyl donors as presented in Scheme 5. Condensation of the alcohol 32 with the di- 33²⁶, tri- (28), and tetrasaccharide (8) donors using TMSOTf as the activator in CH₂Cl₂ afforded the fully protected hexa- (34), hepta- (35), and octasaccharides (36) in the range of 60 to 91% yields. For further chain extension, the temporary monochloroacetyl group was removed from the fully protected octasaccharide 36 by treatment with thiourea to afford the alcohol 37 (77%). Glycosylation of the octamer 37 with the di- (33), tri-(28), and tetrasaccharides (8) using TMSOTf as the activator yielded the deca- (38), undeca- (39), and dodecasaccharides (40) (Scheme 5). In these condensations, the donors were used in 2- to 3-fold molar excesses, and the isolated yields were in the 60–91% range. Although the isolated yields of the protected oligosaccharides were, in most cases, acceptable, the procedure was not without the need for repeated column chromatographic purification, steps that undoubtedly contribute to the often moderate yields.

Scheme 4a.

Synthesis of the spacer-linked tetrasaccharide acceptor 32

^aReagents and conditions: (a) 1.4 equivalents of 31, TMSOTf, CH₂Cl₂, 0 °C, 1 h, 81%; (b) CS(NH₄)₂, C₅H₅N, DMF, 23 °C, 12 h, 83%.

Scheme 5a.

Synthesis of the higher-membered oligosaccharides 34–40

Reagents and conditions: (a) 2.3 equivalents of 33, TMSOTf, CH₂Cl₂, 0 °C, 45 min, 91%; (b) 2.3 equivalents of 28, TMSOTf, CH₂Cl₂, 0 °C, 40 min, 60%; (c) 2.1 equivalents of 8, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 88%; (d) CS(NH₄)₂, C₅H₅N, DMF, 23 °C, 16 h, 77%; (e) 5.2 equivalents of 33, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 88%; (f) 3.5 equivalents of 28, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 86%; (g) 1.9 equivalents of 8, TMSOTf, CH₂Cl₂, 0 °C → 23 °C, 1 h, 64%.

Preparation of the targets 1–7 from the fully protected tetra- to dodecasaccharides 13, 34, 35, 36, and 38–40 (denoted C in Scheme 6) proceeded in four stages: (i) base-promoted removal of the O-acyl protecting groups, (ii) hydrogenolytic removal of the O-benzyl groups (→ D), (iii) aminolysis with ethylenediamine (→ E), and (iv) N-acylation with ketohexanoic anhydride (→ F). Removal of all the protecting groups was ascertained by the ¹H and ¹³C NMR spectra of the oligosaccharides so obtained that were consistent with the proposed structures.

Scheme 6.

Removal of the protecting groups and attachment of the linker moiety

Pg = protecting group

In addition to further transformations as described below, three representative intermediates 14, 41, and 42 (Figure 3) were used in detailed NMR studies.

Figure 3.

Tetra- 41, octa- (42), and dodecasaccharide (14) methoxycarbonylpentyl glycosides

Nuclear magnetic resonance studies

The three methoxycarbonylpentyl glycosides 14, 41, and 42 were examined in detail by ¹H and ¹³C NMR spectroscopy, using 2D COSY-30 and TOCSY to assign the ¹H spectra, and 2D HSQC and HMBC to confirm assignments for the ¹³C spectra. ¹H chemical shifts for the glycosides 41 and 42 are shown in Table 1, and for 14 in Table 2. Vicinal and geminal ¹H-¹H coupling constants are listed for 41 and 42 in Table 3, and for 14 in Table 4. ¹³C chemical shifts are shown for 41 and 42 in Table 5, and for 14 in Table 6. Finally, values of the ¹J_C-1,H-1 coupling constants measured by ¹H-coupled 2D HSQC for 41, 42, and 14 may be compared in Table 7. The observation of exclusively large ¹J_C-1,H-1 values (168.9–175.8 Hz) by ¹H-coupled 2D HSQC indicates the α anomeric configuration for all linkages in the three oligosaccharide glycosides, as synthetically designed. Similar repeating structural environments in glycosides 42 and 14 cause equivalence or near-equivalence of the chemical shifts of residues in the same immediate environment. These residues are Glc C and G in 42, and residues Glc C, G and K; GlcNAc F and J; Rha D and H; and Rha E and I, in 14. For example, the multiplet patterns in the ¹H-coupled 2D HSQC spectra of 42 (Figure 4a) and 14 (Figure 4b) are remarkably similar. However, close inspection of these spectra reveals intensity differences due to the coincidence of resonances of residues in equivalent environments. According to the ¹H chemical shifts of 14 (Table 2), the structural environment of GlcNAc B is observably different to those of GlcNAc F and J, apparently because of the proximity of GlcNAc B to the unique Rha A residue at the glycoside terminus. Characteristic ¹³C-1 chemical shifts were observed for the different types of residues: GlcNAc at ~95 ppm, Glc at 98.5 ppm, Rha A at ~100 ppm, Rha glycosidically attached to Glc at 102.1–102.3 ppm, and Rha glycosidically bound to Rha at 102.7–102.8 ppm. All of the linkage positions in the three oligosaccharide glycosides 41, 42, and 14 were confirmed by the observation of specific connectivities in the 2D HMBC spectra, for one or two ¹³C,H pairs per linkage. Interpretation of the ¹H–¹H coupling constants (Tables 3 and 4) for the glycosides 41, 42, and 14 according to Karplus considerations confirmed the types of sugar residues present, and their chair conformations.

Table 1.

¹H NMR chemical shifts (ppm) of the E. coli O148 tetra- (41) and octasaccharide (42) 5-methoxycarbonylpentyl glycosides

	Tetrasaccharide 41					Octasaccharide 42
	A	B	C	D	MeOCO	A	B	C	D	E	F	G	H	MeOCO
	Rha	GlcNAc	Glc	Rha	Pentyl	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha	Pentyl
H-1	4.806	4.981	5.534	5.106	3.702	4.801	4.980	5.564	5.103	5.123	5.038	5.564	5.127	3.695
H-1'	---	---	---	---	3.527	---	---	---	---	---	---	---	---	3.512
H-2	4.067	4.128	3.660	4.069	1.627	4.066	4.136	3.662	4.182	4.247	4.140	3.662	4.077	1.612
H-3	3.810	4.032	3.730	3.788	1.377	3.825	4.067	3.754	3.886	3.968	4.081	3.754	3.802	1.377
H-4	3.525	3.785	3.493	3.471	1.627	3.547	3.803	3.488	3.572	3.576	3.794	3.486	3.490	1.612
H-5	3.722	~4.012	~3.639	3.840	2.404	3.717	~4.041	3.658	3.901	3.915	~4.053	3.658	3.857	2.391
H-6	1.312	3.808	3.852	1.293	---	1.314	3.841	3.876	1.303	1.349	3.845	3.868	1.303	---
H-6'	---	3.808	3.802	---	---	---	.3.841	3.796	---	---	3.804	3.796	---	---
MeO	---	---	---	---	3.690	---	---	---	---	---	---	---	---	3.685
NAc	---	2.041	---	---		---	2.053a	---	---	---	2.061a	---	---	---

^aInterchangeable.

Table 2.

¹H NMR chemical shifts (ppm) of the E. coli dodecasaccharide 5-methoxycarbonylpentyl glycoside 14

	Dodecasaccharide residues
	A	B	C	D	E	F	G	H	I	J	K	L	MeOCO
	Rha	GlcNAcGlc		Rha	Rha	GlcNAcGlc		Rha	Rha	GlcNAcGlc		Rha	Pentyl
H-1	4.801	4.980	5.581	5.121	5.136	5.044	5.580	5.118	5.136	5.044	5.578	5.139	3.692
H-1'	---	---	---	---	---	---	---	---	---	---	---	---	3.506
H-2	4.065	4.145	3.662	4.191	4.258a	4.150	3.662	4.189	4.261a	4.150	3.662	4.083	1.612
H-3	3.833	4.088	3.767	3.896	3.987	4.102	3.767	3.895	3.987	4.102	3.767	3.810	1.380
H-4	3.560	3.811	3.483	3.586	3.590	3.797	3.483	3.585	3.590	3.797	3.481	3.501	1.612
H-5	3.713	4.052	3.661	~3.905	3.928	4.064	3.661	~3.904	3.590	4.064	3.661	3.865	2.386
H-6	1.315	3.838	3.890	1.311	1.356	3.860	3.890	1.308	1.356	3.860	3.881	1.308	---
H-6'	---	3.814	3.793	---	---	3.804	3.793	---	---	3.804	3.793	---	---
MeO	---	---	---	---	---	---	---	---	---	---	---	---	3.684
NAc	---	2.061b	---	---	---	2.072b	---	---	---	2.070b	---	---	---

^a,bInterchangeable within each letter group.

Table 3.

¹H—¹H coupling constants (Hz) of the E. coli O148 tetra- (41) and octasaccharide (42) 5-methoxycarbonylpentyl glycosides

	Tetrasaccharide residues				Octasaccharide residues
	A	B	C	D	A	B	C	D	E	F	G	H
	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha
J 1,2	2.1	3.6	3.6	1.8	1.7	3.6	3.6	1.7	~2.1	3.6	3.6	1.8
J 2,3	3.3	10.6	10.0	3.6	3.6	10.7	9.8	3.2	3.3	10.7	9.8	3.6
J 3,4	10.1	8.8	8.9	10.0	10.2	8.7	9.1	9.6	10.6	8.7	8.8	10.1
J 4,5	9.8	10.2	10.1	9.8	9.8	10.0	10.2	9.4	9.7	9.8	10.0	9.8
J 5,6	6.3	nra	2.3	6.3	6.2	nr	2.3	6.2	6.2	2.0	2.1	6.4
J 5,6'	---	nr	4.2	---	---	nr	4.6	---	---	3.9	4.6	---
J 6,6'	---	nr	12.3	---	---	nr	12.2	---	---	12.4	12.0	---

^anr, not resolved.

Table 4.

¹H—¹H coupling constants (Hz) of the E. coli O148 dodecasaccharide 5-methoxycarbonylpentyl glycoside 14

	Dodecasaccharide residues
	A	B	C	D	E	F	G	H	I	J	K	L
	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha
J 1,2	1.8	3.6	3.8	1.8	1.5	3.6	3.7	1.7	2.0	3.6	3.7	1.8
J 2,3	3.1	10.6	10.0	3.1	3.4	10.5	10.0	3.1	3.3	10.5	10.0	3.3
J 3,4	9.9	8.5	9.1	9.7	9.9	8.6	9.1	9.8	9.9	8.6	9.1	10.1
J 4,5	9.6	10.2	10.0	9.6	9.7	9.9	10.0	9.7	9.7	9.9	10.1	9.7
J 5,6	6.2	3.0	2.2	6.2	6.2	2.5	2.2	6.3	6.2	2.5	2.1	6.2
J 5,6'	---	4.3	5.1	---	---	4.9	5.1	---	---	4.9	5.1	---
J 6,6'	---	12.8	12.3	---	---	~11.7	12.3	---	---	~11.7	12.2	---

Table 5.

¹³C NMR chemical shifts (ppm) of the E. coli O148 tetra- (41) and octasaccharide (42) 5-methoxycarbonylpentyl glycosides

	Tetrasaccharide residues					Octasaccharide residues
	A	B	C	D	MeOCO	A	B	C	D	E	F	G	H	MeOCO
	Rha	GlcNAc	Glc	Rha	Pentyl	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha	Pentyl
C-1	100.23	94.94	98.49	102.25	68.52	100.32	94.98	98.47	102.14	102.73	94.88	98.47	102.26	68.39
C-2	67.48	52.65	77.15	70.58	24.74a	67.54	52.66	77.30	70.37	67.49	52.67	77.12	70.65	24.84b
C-3	75.91	75.98	73.14	70.78	25.66	76.01	76.22	73.33c	78.90	75.67	75.82	73.18c	70.87	25.76
C-4	71.00	71.69	69.81	72.62	28.86a	71.03	71.87	69.99	72.01	71.08	71.87	70.07	72.68	28.99b
C-5	69.41	72.49	72.95	69.99	34.33	69.39	72.55	72.99	69.99	69.99	72.63	72.99	69.95	34.31
C-6	17.42	60.71	60.88	17.34	---	17.53	60.86	61.08	17.47d	17.68	60.95	61.08	17.58d	---
MeO	---	---	---	---	52.85	---	---	---	---	---	---	---	---	52.74
NAc	---	22.89	---	---	---	---	22.93e	---	---	---	22.95e	---	---	---
NC=O	---	174.81	---	---	---	---	174.58	---	---	---	174.49	---	---	---
OC=O	---	---	---	---	178.33	---	---	---	---	---	---	---	---	177.77

^a,b,c,d,eInterchangeable within each letter group.

Table 6.

¹³C NMR chemical shifts (ppm) of the E. coli O148 dodecasaccharide 5-methoxycarbonylpentyl glycoside 14

	Dodecasaccharide residues
	A	B	C	D	E	F	G	H	I	J	K	L	MeOCO
	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha	Pentyl
C-1	100.38	95.00	98.49	102.15	102.77a	94.87	98.51b	102.15	102.73a	94.87	98.46b	102.25	68.31
C-2	67.58	52.74	77.29	70.41c	67.50d	52.66	77.29	70.40c	67.49d	52.66	77.11	70.69	24.91e
C-3	76.08	76.19f	73.27g	78.98h	75.70	75.78e	73.23g	78.96h	75.70	76.19f	73.23g	70.94	25.82
C-4	71.05	71.97	70.12	72.02i	71.11	71.97	70.12	72.03i	71.11	71.97	70.12	72.72	29.09e
C-5	69.37	72.62	73.02	70.05	69.92	72.66j	73.02	69.92	69.92	72.70j	73.02	69.92	34.31
C-6	17.68	60.97	61.23k	17.60	17.75	61.09l	61.21k	17.60	17.75	61.05l	61.21k	17.55	---
MeO	---	---	---	---	---	---	---	---	---	---	---	---	52.66
NAc	---	22.98m	---	---	---	22.98m	---	---	---	22.96m	---	---	---
NC=O	---	174.46	---	---	---	174.41n	---	---	---	174.33n	---	---	---
OC=O	---	---	---	---	---	---	---	---	---	---	---	---	177.38

^aInterchangeable within each letter group.

Table 7.

¹J_C-1,H-1 coupling constants (Hz) of the E. coli O148 tetra- (41), octa- (42) and dodecasaccharide 14 5-methoxycarbonylpentyl glycosides

	Tetrasaccharide residues				Octasaccharide residues
	A	B	C	D	A	B	C	D	E	F	G	H
	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha
1 J C-1,H-1	170.0	171.6	175.8	172.6	170.0	171.0	174.4	173.0	170.6	172.0	174.4	172.4

	Dodecasaccharide residues
	A	B	C	D	E	F	G	H	I	J	K	L
	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha	Rha	GlcNAc	Glc	Rha
1 J C-1,H-1	169.9	168.9	175.3	171.8	171.1	168.9	175.3	171.8	171.1	168.9	175.3	172.8

Figure 4a.

¹H-coupled 2D HSQC spectrum of octasaccharide 42.

Figure 4b.

¹H-coupled 2D HSQC spectrum of dodecasaccharide 14.

Conjugation of the oligosaccharides to proteins

Oligosaccharides do not elicit an immune response when injected into mammals. However, they can be converted to immunogens when covalently linked to immunogenic proteins. The conjugation should use reactions that are compatible with the acid and base-sensitivities of the proteins. The protocol should allow the introduction of required copies of the oligosaccharide haptens and the unreacted saccharides should be recoverable in their original, reactive form. Of the numerous possible approaches, selective oxime formation between a keto-derivatized carbohydrate and an aminooxy-equipped protein counterpart stands out with its simple operation, mild conditions, and applicability to a variety of saccharide haptens, ranging from mono- to polysaccharides.^14,27 In our approach (Scheme 7), the spacer-equipped oligosaccharides 1–7 (F) were combined with the aminooxy-derivatized proteins I to afford the conjugates J, at pH 7.2 or below.

Scheme 7.

Synthesis of neoglycoproteins by oxime-conjugation between carbohydrates and proteins.

(Scheme 7.) The aminooxy moiety was introduced into the protein carriers in a two-step process. First, the proteins BSA or recombinant diphtheria toxoid (G) were reacted with succinimidyl (3-bromoacetamido)propionate to afford structure H featuring a labile bromine moiety that, upon reacting with 2-aminooxypropanethiol,³⁵ allowed the formation of a stable thioether linkage connecting the derivatized protein and the aminooxy moiety (→ I). The conjugates were purified through a BioGel P10 column to afford neoglycoproteins having an average of up to 15 oligosaccharide chains, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Serum antibody responses

The immunogenicity of the conjugates containing the E. coli O148 oligosaccharides and those made from oligosaccharide portions related to the LPS of SD¹⁴ (Table 8) was estimated in groups of ten mice (Table 9). Three subcutaneous injections containing 2.5 μg of saccharides in the form of conjugates in phosphate buffered saline (PBS) were given without adjuvant at 2 week intervals. We note that almost all of the synthetic oligosaccharide-based carbohydrate-protein conjugates reported in the literature have been administered in Freund's complete adjuvant followed by two injections in Freund's incomplete adjuvant. Since Freund's adjuvant cannot be given to humans, the extrapolation of the experimental data for human use is at least questionable. All conjugates induced significantly higher IgG antibody levels against both LPS's than the control group that received PBS at the same schedule (not shown, p<0.0001). There were no statistical differences between the anti-SD IgG levels induced either by the SD or by the E. coli oligosaccharide conjugates (data in column 5, Table 9) with one exception: the conjugate of the E. coli 10mer 5 (item no 5) induced a statistically lower anti-SD level than did the conjugate of the SD 10mer (44) conjugate (item no 11, 4.6 EU vs 11.8 EU, P=0.03). Similarly, there were no statistical differences between the anti-E. coli O148 levels induced by any of the conjugates (Column 6, Table 9). The highest antibody levels against both LPS's were induced by the BSA conjugate of the E. coli octasaccharide 4 (item no 4). Some of the differences, e.g. the higher anti-SD response by the conjugate of the E. coli 8-mer 4 versus that of the SD octasaccharide 43 (item 10), may be a consequence of different loadings on the protein. In general, the conjugates that induced high anti SD IgG levels also induced high anti-E. coli O148 IgG levels. These data support the assumption that immunity to either of these bacteria would protect the host against both of them.

Table 8.

Oligosaccharides related to S. dysentariae type 1 lipopolysaccharide that were used in this study

Table 9.

Composition of protein conjugates of synthetic oligosaccharides related to Shigella dysenteriae type 1 and E. coli O148, and the geometric means of their anti-LPS serum IgG

Item no	Conjugate/OS size	Non-reducing terminus	Average number of OS chains per protein	Anti SD LPS IgG GM[EU]	Anti Ec148 LPS IgG GM [EU]
1	1-EC-BSA /4-mer	Rha(1)	ND	ND	ND
2	2-EC-BSA /6mer	GlcNAc	ND	ND	ND
3	3-EC-BSA /7mer	Glc	ND	ND	ND
4	4-EC-BSA/8-mer	Rha(2)	14	33.3	155.6
5	5-EC-BSA/10-mer	GlcNAc	15	4.6	14.7
6	6-EC-BSA/11mer	Glc	ND	ND	ND
7	6-EC-DT/11mer	Gal	11	12.9	39.8
8	7-EC-BSA/12mer	Rha(2)	13	11.2	56.4
9	7-EC-DT/12mer	Rha(2)	12	14.5	45.7
10	43-SD-DT/8mer	Rha(2)	7	14.4	ND
11	44-SD-DT/10mer	GlcNAc	12	11.8	33.7
12	45-SD-DT/11mer	Gal	11	9.5	48.0
13	46-SD-DT/12mer	Rha(2)	7	17.9	ND
14	47-SD-DT/13mer	Rha(1)	7	13.5	ND

Abbreviations: BSA, bovine serum albumin; DT, diphtheria toxoid; EC, Escherichia coli; EU, elisa unit; GM, geometric mean; IgG, immunoglobulin G; LPS, lipopolysaccharide; Rha(1), the rhamnose unit to which GlcNAc is attached; Rha(2), the rhamnose unit which is linked to either Gal or Glc; OS, oligosaccharide; SD, Shigella dysenteriae type 1.

All groups vs control: p<0.000

Immunoblotting

Western blot assays were performed to evaluate if sera of immunized animals cross-react with LPS's of both bacteria. Sera used in these experiments were induced either in rabbits by heat-killed bacteria or in mice by the conjugates of synthetic oligosaccharides corresponding to their O-SP subunits: compound 7 (12mer) in the case of E. coli O148 and compounds 44, 45, 46, 47 (10, 11, 12, and 13mer) (Table 9) in the case of SD.¹⁴ The results presented in Figure 5 show that all sera reacted with both LPS's with similar intensity, and in both directions.

Figure 5.

Western immunoblot of S. dysenteriae type 1 and E. coli O148 LPS with sera induced by whole bacteria or synthetic oligosaccharides bound to the recombinant Diphteria Toxin (DT). The terminal sugars are as follow: 10mer: GlcNAc; 11mer: Gal; 12 and 13mers: Rha. EC: E. coli O148, SD: S. dysenteriae type 1

1. S. dysenteriae type 1 LPS,

2. E. coli O148 LPS

In a dot-blot experiment, BSA conjugates of the monosaccharides galactose and glucose (not described in the experimental) did not bind to sera raised to either bacterium. The BSA conjugate of compound 1 (4mer), representing one repeating unit of E. coli O148 O-SP, bound to sera raised by either bacterium less strongly than did conjugates of compounds 2–7, which underwent binding with approximately the same intensity.

Competitive inhibition of binding

In order to corroborate the results of the immunoblotting experiments and demonstrate further that the cross-reactivity between SD and E. coli O148 is due to the O-SP portion of their respective lipopolysaccharides, we analyzed the inhibition of binding between sera induced by DT conjugates of SD 12mer (compound 46) and E. coli O148 12mer (compound 7) and their respective LPS's (Tables 10 and 11), using the O-SP of the unrelated bacterium Bordetella bronchiseptica as the control. The data in Tables 10 and 11 demonstrate dose-related inhibition of binding within similar ranges, thus confirming that the cross reactivity between SD and E. coli O148 is, indeed, due to their respective O-SP's.

Table 10.

Competitive inhibition of anti-S. dysenteriae type 1 serum (induced by rDT/Sd1-12mer) binding to the homologous LPS by different dosages of S. dysenteriae type 1, E. coli O148 or B. bronchiseptica RB50 O-SPs.

Inhibitor	Inhibition (%)
	Inhibitor per well (μg)
	0.04	0.2	5	80
S. dysenteriae type 1 O-SP	23	39	69	75
E. coli O148 O-SP	21	37	71	79
B. bronchiseptica O-SP	5	9	14	25

Table 11.

Competitive inhibition of anti-E. coli O148 serum (induced by rDT/EcO148-12mer) binding to the homologous LPS by different dosages of S. dysenteriae type 1, E. coli O148 or B. bronchiseptica RB50 O-SPs.

Inhibitor	Inhibition (%)
	Inhibitor per well (μg)
	0.04	0.2	5	80
S. dysenteriae type 1 O-SP	23	54	58	80
E. coli O148 O-SP	30	42	72	83
B. bronchiseptica O-SP	8	9	8	24

DISCUSSION

As part of our program directed towards the development of carbohydrate based conjugate vaccines against human pathogenic bacteria, we were intrigued as to whether there exists cross-reactivity between the O-PS's of SD and E. coli O148, based on their close structural similarity. Both bacteria are infectious Gram-negative human pathogens that can cause severe enteric infections in children and adults, including dysentery, hemorrhagic colitis, and hemolytic uremic syndrome. Of the two, SD is the more common pathogen. Both are water and food borne organisms, and their transmission is facilitated by unhygienic conditions. The severity of these diseases calls for the development of vaccines against enteropathogenic Gram-negative bacteria. We hypothesized that cross reactivity between the O-SP's may serve as a basis for protection against both bacteria by a single monovalent vaccine. We decided to explore cross-reactivity by using oligosaccharide fragments of the O-SP's of both bacteria, in the form of covalent conjugates with immunogenic proteins. Such oligosaccharides are difficult to isolate in homogeneous form from the native O-SP because of the difficulty of its site-specific fragmentation by either chemical or enzymatic processes. Therefore, we decided to prepare the required oligosaccharides by chemical syntheses.

Starting from appropriately protected/functionalized monosaccharide intermediates, di-, tri-, and tetra-saccharide building blocks were prepared that were combined using a blockwise approach to yield fully protected tetra- to dodecasaccharides, from which removal of the protecting groups afforded the targeted oligosaccharides. The identity and purity of the synthetic oligosaccharides were ascertained by detailed ¹H and ¹³C NMR spectroscopy, in addition to mass spectrometric analyses. The synthetic oligosaccharides were equipped with a linking arm featuring an oxo function for covalent attachment to immunogenic proteins, including bovine serum albumin and recombinant diphtheria toxin, by using the oxime method.²⁷ The level of incorporation was determined by MALDI-TOF mass spectrometry and reached up to an average of fifteen oligosaccharide chains per protein molecule. The neoglycoconjugates, subcutaneously injected in groups of ten mice, induced IgG antibodies with O-SP specificities shown by immunochemical experiments.

Western-blot analysis demonstrated that sera raised against heat-killed SD or E. coli O148 bacteria in rabbits or against the synthetic oligosaccharide-protein conjugates in mice reacted with the lipopolysaccharides of either SD or of E. coli O148, in both directions. The dot blot assays indicate the importance of the length of the oligosaccharide chains. BSA conjugates of the monosaccharides galactose and glucose failed to react with either of the antisera, and the conjugate of the tetrasaccharide 1 reacted weakly as compared with the conjugates of the hexa- to dodecasaccharides 2–7, which reacted with comparable intensities. These qualitative observations support the view that a chain length exceeding a complete repeating unit is necessary for specific antigenic recognition by either SD or E.coli O148 antibodies. Further proof for the cross-reactivity was provided by competitive inhibition of binding between sera raised by the neoglycoconjugates and their respective homologous LPS.

In summary, we have synthesized a number of oligosaccharide fragments of the O-SP of E. coli O148, which were bound covalently to two proteins. Using these synthetic experimental vaccines we have demonstrated cross-reactivity between the O-SP's of SD and E. coli O148, in both directions. The observed cross-reactivity suggests that anti O-SP-based immunity against one of these organisms would provide protection to both, an observation that should be conducive to a single monovalent vaccine against the two organisms.

EXPERIMENTAL SECTION

General chemical synthesis

All chemicals were commercial grade and used without purification. Solvents for chromatography were distilled prior to use. Anhydrous solvents were obtained from Aldrich. All glycosylation reactions were carried out after drying the reacting partners at 10 microns or less for 12 h except for the reaction with imidate 11 that was used immediately after its preparation. Glycosylation reactions as well as preparation of trichloroacetimidates were carried out under argon. Column chromatography was performed on silica gel 60 (0.040–0.063 mm) and thin layer chromatography was performed on glass-supported silica gel layers obtained from Analtech (Uniplate, Silica gel GHLF) or on HPTLC plates (Kieselgel 60 F₂₅₄, Merck). Visualization was carried out by inspection under UV light (254 nm), by iodine adsorption, and by charring using a solution of ammonium cerium(IV) sulfate and ammonium molybdate in sulfuric acid. Column chromatography was performed on silica gel 60 (230–400 mesh) obtained from EMD Chemicals Inc. ¹H and ¹³C NMR spectra were recorded on a Bruker DRX spectrometer at 500 and 125 MHz, respectively. Chemical shifts are recorded in ppm relative to internal references. For ¹H: 0.00 for (CH₃)₄Si, or 3.30 for CD₂ HOD. For ¹³C: 48.90 for CD₃OD or 77.00 for CDCl₃. Atmospheric pressure electrospray ionization mass spectrometry (API-ES-MS) was carried out on an Agilent LC/MSD SL spectrometer.

General procedure for the deprotection of oligosaccharides 13, 34–36, and 38–40

The protected oligosaccharide C (Scheme 6) was dissolved in anh. CHCl₃ to which anh. MeOH was added. To the resulting solution was added a solution of NaOMe in MeOH (25 wt %) at room temperature until the pH of the solution reached approx. 12 as estimated with a moistened indicator paper. After 24 h, the solution was neutralized with Dowex (H⁺) resin. Next, the resin was removed by filtration and the volatiles were removed under reduced pressure. To a solution of the residue so obtained in EtOH was added Pd/C and the resulting mixture was stirred under hydrogen (200 psi) for 24 h. Removal of the catalyst by filtration through a layer of Celite followed by concentration afforded the deprotected oligosaccharides as 5-methoxycarbonyl glycosides D. In addition to further transformations as described below, compounds 14, 41, and 42 (Figure 3) were used in NMR studies.

General procedure for the preparation of the spacer-linked oligosaccharides 1–7

The deprotected oligosaccharide was dissolved in 1,2-diaminoethane. After 24 h at room temperature, the solution was diluted with water followed by evaporation of the volatiles by freeze-drying. The residue was purified by chromatography through a Bio-Gel P-2 column, using 0.02 M pyridine acetate in water as the eluant. The fractions containing carbohydrates as determined by the phenol-sulfuric acid assay were combined and the solution was freeze-dried. This cycle was repeated two more times to afford intermediate E (Scheme 7) as a white amorphous solid. To a stirred solution of derivative E in MeOH were added triethylamine and 5-ketohexanoic anhydride.²⁷ After 10 min, the solution was concentrated under reduced pressure. To the residue was added water and the mixture was stirred with a magnetic stirring bar. The flask containing the solution was immersed in ice-water. The clear solution was siphoned off with a Pasteur pipette the tip of which was covered with tissue paper. The solution so obtained was freeze-dried. To the residue was added 0.02 M pyridine-acetate buffer. The mixture was filtered through a pad of tissue paper to remove residual solid particles. The clear solution was passed through a Biogel P-4 column using 0.02 M pyridine-acetate as the eluant. The fractions containing carbohydrates were pooled and freeze-dried. The residue was dissolved in water and the solution was freeze-dried. This cycle was repeated two more times to afford compound F as a white amorphous solid.

Synthesis and/or characterization of compounds the new compounds described in this paper

5-{2-[(5'-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (1)

¹H NMR (D₂O): δ 5.53 (d, 1H, J = 3.6 Hz), 5.11 (br s, 1H), 4.98 (d, 1H, J = 3.6 Hz), 4.80 (br s, 1H), 4.13 (dd, 1H, J = 3.6 Hz, J = 10.4 Hz), 3.30 (br s, 4H), 2.58 (t, 2H, J = 7.4 Hz), 2.25–2.24 (m, 4H), 2.20, 2.04 (2 s, 2 × 3H), 1.83–1.77 (m, 2H), 1.64–1.54 (m, 4H), 1.40–1.37 (m, 2H), 1.31, 1.29 (2 d, 2 × 3H); ¹³C NMR (D₂O): δ 216.2, 177.9, 177.0, 174.8, 102.2, 100.2, 98.5, 94.9, 77.1, 75.89, 75.85, 73.1, 72.9, 72.6, 72.5, 71.7, 71.0, 70.7, 70.5, 70.0, 69.8, 69.4, 68.4, 67.4, 60.8, 60.7, 52.6, 42.8, 39.3, 39.2, 36.5, 35.6, 30.0, 28.9, 25.8, 25.7, 22.9, 20.33, 20.28, 17.4, 17.3. HRMS: m/z calcd for [C₄₀H₆₉N₃O₂₂]H⁺: 944.4451 Found 944.4449.

5-{2-[(5'-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl 2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (2)

¹H NMR (D₂O, partial): δ 5.5 (d, 1H, J = 3.6 Hz), 5.09, 5.07 (2 d, 2 × 1H, J = 1.6 Hz), 5.05, 4.97 (2 d, 2 × 1H, J = 3.8 Hz), 4.79 (d, 1H, J = 1.6 Hz), 3.30 (br, 4H), 2.57 (t, 2H, J = 7.3 Hz), 2.23, 2.21 (2 t, 2 × 2H, J ~ 7 Hz), 2.19, 2.04, 2.03 (3 s, 3 × 3H), 1.80 (m, 2H), 1.63–1.54 (m, 4H), 1.38–1.27 (m, 11H); ¹³C NMR (D₂O): δ 216.2, 177.9, 177.0, 175.2, 174.8, 102.8, 102.2, 100.2, 98.5, 95.01, 94.95, 78.8, 77.3, 76.3, 76.0, 75.9, 73.1, 73.0, 72.6, 72.5, 72.0, 71.8, 71.7, 71.1, 71.0, 70.5, 70.3, 70.1, 70.0, 69.8, 69.4, 68.5, 67.6, 67.5, 61.0, 60.9, 60.8, 54.4, 52.7, 42.9, 39.4, 39.3, 36.5, 35.6, 30.1, 29.0, 25.9, 25.7, 22.9, 22.7, 20.4, 17.6, 17.5, 17.4. API-ES-MS: m/z calcd for [C₅₄H₉₂N₄O₃₁]H⁺: 1293.6 Found 1293.5.

5-{2-[(5'-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (3)

¹H NMR (D₂O, partial): δ 5.53, 5.38 (2 d, 2 × 1H, J ~ 3.6 Hz for each), 5.10, 5.07 (2 d, 2 × 1H, J ~ 1.6 Hz each), 5.03, 4.97 (2 d, 2 × 1H, J ~ 3.6 Hz each), 4.79 (d, 1H, J = 1.6 Hz), 4.22, 4.16 (2 br, 2 × 1H), 3.30 (br, 4H), 2.57 (t, 1H, J = 7.3 Hz), 2.24–2.20 (m, 4H), 2.19 (s, 3H), 2.03 (s, 6H), 1.80 (m, 2H), 1.65–1.54 (m, 4H), 1.39–1.26 (m, 11H); ¹³C NMR (D₂O): δ 216.3, 178.0, 177.1, 174.9, 174.8, 102.8, 102.2, 100.33, 100.29, 98.6, 95.2, 95.0, 78.9, 78.6, 77.4, 76.3, 76.0, 75.9, 73.6, 73.2, 73.02, 72.98, 72.5, 72.4, 72.1, 71.8, 71.2, 71.14, 71.08, 70.4, 70.2, 70.2, 69.9, 69.8, 69.5, 68.6, 67.6, 67.5, 60.93, 60.90, 60.83, 52.7, 43.0, 39.4, 39.3, 36.6, 35.7, 30.1, 29.0, 25.9, 25.8, 23.0, 20.4, 17.7, 17.54, 17.50.

API-ES-MS: m/z calcd for [C₆₀H₁₀₂N₄O₃₆]H⁺: 1455.6. Found 1455.6

5-{2-[(5'-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-L-rhamnopyranosyl-(1→2)- α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (4)

¹H NMR (D₂O, partial): δ 5.53 (br, 2H), 5.10 (br, 2H), 5.08 (br, 1H), 5.02 and 4.97 (2 d, 2 × 1H, J ~ 3.6 Hz), 4.79 (br, 1H), 4.22, 4.16, 4.13, 4.11 (4 br, 4 × 1H), 4.07 (br, 2H), 3.93 (dd, 1H, J = 3.3 Hz, J = 10.0 Hz), 3.30 (m, 4H); ¹³C NMR (D₂O): δ 216.3, 178.0, 177.1, 174.9, 174.8, 102.7, 102.3, 102.2, 100.2, 98.5, 95.0, 94.9, 78.8, 77.3, 77.2, 76.3, 75.93, 75.91, 75.7, 73.2, 73.1, 73.0, 72.6, 72.5, 74.5, 72.0, 71.8, 71.7, 71.1, 71.0, 70.8, 70.6, 70.3, 70.1, 70.0, 69.8, 69.4, 68.5, 67.5, 60.9, 60.7, 42.9, 39.4, 39.3, 36.5, 35.6, 30.0, 29.9, 25.9, 25.7, 22.9, 20.4, 17.6, 17.5, 17.4, 17.3. HRMS: m/z calcd for [C₆₆H₁₁₂N₄O₄₀]H⁺: 1601.6931. Found: 1601.7007.

5-{2-[(5'-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl 2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)- α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (5)

¹H NMR (D₂O, partial): δ 5.54 (d, 2H, J ~ 3.5 Hz), 5.10 (br s, 2H), 5.08 (br s, 2H), 5.06, 5.03, 4.98 (3 d, 3 × 1H, J = 3.6 Hz), 4.80 (d, 1H, J ~ 1.5 Hz), 4.23, 4.17 (2 br s, 2 × 1H), 4.17 (br s, 2H), 3.30 (s, 4H), 2.58 (t, 2H, J ~ 7.1 Hz), 2.38 (t, ~ 7.0 Hz), 2.24, 2.22 (2 t, 2 × 1H. J ~ 7.0 Hz), 2.20, 2.05, 2.04, 2.03 (4 s, 4 × 3H), 1.83–1.77 (m, 2H), 1.65–1.55 (m, 4H), 1.34 (d, 6H, J ~ 6.3 Hz), 1.31 (d, 3H, J = 6.3 Hz), 1.30 (d, 6H, J ~ 6.3 Hz); ¹³C NMR (D₂O): δ 216.2, 177.9, 177.0, 175.2, 174.80, 174.78, 102.76, 102.72, 102.1, 100.2, 98.5, 95.02, 94.95, 94.9, 78.8, 77.3, 76.26, 76.0, 75.9, 75.6, 73.1, 73.0, 72.6, 72.5, 72.0, 71.8, 71.7, 71.13, 71.08, 71.0, 70.5, 70.3, 70.1, 70.0, 69.96, 69.8, 69.4, 68.5, 67.6, 67.5, 61.0, 60.9, 54.4, 52.7, 42.9, 39.4, 39.3, 36.5, 35.6, 30.1, 29.0, 25.9, 25.7, 22.92, 22.91, 22.7, 20.4, 17.6, 17.5, 17.4. HRMS: m/z calcd for [C₈₀H₁₃₅N₅O₄₉]H⁺: 1950.8304. Found 1950.8314.

5-{2-[(5'-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (6)

¹H NMR (D₂O, partial): δ 5.54 (br s, 2H), 5.39 (d, 1H, J = 3.8 Hz), 5.11, 5.08 (2 br s, 2 × 2H), 5.04, 5.03 (2 d, 2 × 1H, J ~ 3.6 Hz), 4.98 (d, 1H, J = 3.6 Hz, 4.80 (br d, 1H, J ~ 1.5 Hz), 4.23, 4.17 (2 br s, 2 × 2H), 3.30 (s, 4H), 2.23, 2.22 (2 t, 2 × 1H, J ~ 7 Hz), 2.19, 2.04, 2.03 (3 s, 3 × 3H), 1.83–1.77 (m, 2H), 1.64–1.53 (m, 4H), 1.33 (d, 6H, J ~ 6.3 Hz), 1.30 (d, 3H, J = 6.3 Hz), 1.29 (d, 6H, J = 6.3 Hz); ¹³C NMR (D₂O): δ 216.3, 178.0, 177.1, 174.9, 174.83, 174.81, 102.7, 102.2, 100.28, 100.25, 98.5, 95.1, 95.0, 78.8, 78.5, 77.3, 76.3, 75.9, 73.6, 73.2, 73.0, 72.9, 72.5, 72.4, 72.0, 71.8, 71.2, 71.1, 71.0, 70.3, 70.1, 70.0, 69.8, 69.7, 69.4, 68.5, 67.6, 67.5, 60.87, 60.86, 60.8, 52.7, 42.9, 39.4, 39.3, 36.5, 35.6, 30.1, 29.0, 25.9, 25.7, 22.9, 20.4, 17.6, 17.5, 17.4. HRMS: m/z calcd for [C₈₆H₁₄₅N₅O₅₄]H⁺: 2112.8832. Found 2112.8816.

5-{2-[(5'-Oxohexanoyl)amino]ethyl-amino}carbonylpentyl α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (7)

¹H NMR (D₂O, partial): δ 5.57 (br s, 3H), 5.13 (br s, 2H), 5.06 (br d, 2H, J = 3.5 Hz), 5.00 (br s, 1H, J = 3.5 Hz), 4.83 (br s, 1H), 4.26, 4.19 (2 br s, 2 × 2H), 3.33 (br s, 4H), 2.16 (t, 2H, J = 7.3 Hz), 2.27, 2.26 (2 t, 2 × 2H, J ~ 7 Hz), 2.10–2.04 (m, 9H), 1.86–1.80 (m, 2H), 1.67–1.57 (m, 4H), 1.42–1.30 (m, 18H); ¹³C NMR (D₂O): δ 216.2, 177.9, 177.1, 174.84, 174.81, 174.8, 102.72, 102.69, 102.3, 102.2, 102.1, 100.2, 98.5, 94.95, 94.92, 78.8, 77.3, 77.1, 76.3, 75.92, 75.90, 75.6, 73.20, 73.16, 73.02, 73.01, 72.96, 72.93, 72.92, 72.91, 72.6, 72.52, 72.48, 72.04, 72.02, 71.99, 71.8, 71.7, 71.10, 71.07, 71.06, 71.02, 70.99, 70.8, 70.6, 70.3, 70.2, 70.12, 70.08, 70.07, 70.06, 70.05, 70.0, 69.9, 69.85, 69.80, 69.4, 68.5, 67.5, 67.4, 60.93, 60.92, 60.91, 60.87, 60.86, 60.83, 60.76, 60.75, 60.73, 60.72, 60.70, 52.7, 42.9, 39.39, 39.37, 39.3, 36.5, 35.6, 30.1, 29.0, 25.9, 25.7, 22.9, 20.4, 17.62, 17.59, 17.51, 17.47, 17.43, 17.40, 17.39, 17.37. API-ES-MS: m/z calcd for [C₉₂H₁₅₅N₅O₅₈]H⁺: 2260.2. Found 2259.8.

(2-O-Benzoyl-4-O-benzyl-3-O-chloroacetyl-α-l-rhamnopyranosyl-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranose trichloroacetimidate (8)

To a solution of 30 (13.0 g, 8.7mmol) in CH₂Cl₂ (50 mL) were added CCl₃CN (15 mL) and DBU (0.3 mL) under cooling in ice-water. The mixture was allowed to reach room temperature. After 1 h the solution was concentrated and the residue purified by column chromatography using hexanes-EtOAc 3:2 containing 0.1% Et₃N to afford 8 (11.1 g, 78%) as an amorphous material. ¹H NMR (CDCl₃, partial): δ 8.75 (s, 1H), 8.15, 8.13, 8.00, 7.96 (4 s, 4 × 1H), 7.67–7.59 (m, 2H), 7.55–7.49 (m 2H), 7.37–7.02 (m, 24H), 6.29 (d, 1H, J = 11.7 Hz), 5.75 (d, 1H, J = 9.5 Hz), 5.63–5.60 (2 br s, 2 × 1 H), 5.40 (dd, 1H, J =3.5 Hz, J = 9.2 Hz), 5.19 (t, 1H, J = 9.6 Hz), 5.16 (br s, 1H), 4.93 (d, 1H, J = 3.4 Hz each), 4.79 (d, 1H, J = 3.2 Hz), 4.74, 4.69, 4.66, 4.62, 5.53 (5 d, 5 × 1H, J ~ 11 Hz), 4.52 (d, 2H, J ~ 11 Hz), 4.45 (dt, 1H, J = 3.4 Hz, J = 10.0 Hz), 4.35, 4.34, 4.30 (3 d, 3 × 1H, J ~ 11 Hz), 4.24 (dd, 1H, J = 3.3 Hz, J = 10.0 Hz), 3.49 (t, 1H, J = 9.6 Hz), 2.13, 2.00, 1.79 (3 s, 3 × 3H), 1.40, 1.09 (2 d, 2 × 3H, J = 6.3 each); ¹³C NMR (CDCl₃): δ 170.6, 170.4, 169.4, 165.9, 165.5, 165.2, 160.1, 138.2, 137.9, 137.7, 137.6, 137.1, 134.2, 133.4, 129.84, 129.80, 129.77, 129.2, 128.9, 128.7, 128.44, 128.39, 128.3, 128.35, 128.3, 128.25, 128.18, 128.1, 128.05, 128.02, 127.98, 127.94, 127.8, 127.7, 127.54, 127.48, 127.35, 127.32, 99.6, 99.4, 96.8, 94.8, 90.6, 80.9, 78.8, 78.42, 78.38, 78.0, 77.6, 75.7, 75.44, 75.37, 74.8, 74.4, 73.9, 73.5, 71.5, 71.4, 70.1, 68.7, 68.6, 68.3, 68.0, 67.8, 60.9, 51.9, 40.4, 23.1, 21.0, 20.7, 18.2, 17.4.

2-(Trimethylsilyl)ethyl (3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (10)

A mixture of 24 (approx 35 g, 27.0 mmol) in CH₃CN (350 mL) and water (60 mL) was treated with ammonium cerium(IV)nitrate (34 g, 62 mmol) in portions. The mixture was cooled to 0 °C then was decolorized with 10 % aq NaHSO₃. The solution was then extracted with saturated aq NaHCO₃. The mixture was filtered through a Celite layer and the solids were washed with CHCl₃. The combined organic layers were washed with brine several times. Concentration of the organic layer yielded a syrupy residue which was chromatograped through silica gel using hexanes–EtOAc 5:1 → 2:1 as the eluant to afford 10 (17.8 g, 15 mmol) as a syrupy material. ¹H NMR (CDCl₃, partial): δ 8.12–8.05 (m, 2H), 7.58–7.53 (m, 1H), 7.43–7.41 (m, 2H), 7.38–7.19 (m, 16H), 7.19–7.15 (m, 2H), 7.10–7.07 (m, 2H), 5.46 (m, 1H), 5.14 (t, 1H, J = 9.9 Hz), 5.11 (d, 1H, J = 3.6 Hz), 4.87 (d, 1H, J = 3.6 Hz), 4.86 (d, 1H, J = 11.0 Hz), 4.84 (d, 1H, J = 12 Hz), 4.81 (d, 1H, J = 11.8 Hz), 4.76 (d, 1H, J = 11.5 Hz), 4.73 (d, 1H, J = 11.6 Hz), 4.68 (d, 1H, J = 11.0 Hz), 4.30 (d, 1H, J = 12.0 Hz), 4.20 (d, 1H, J = 12.0 Hz), 3.36 (dd, 1H, J = 6.2 Hz, J = 10.0 Hz), 3.29 (dd, 1H, J = 8.6 Hz, J = 10.0 Hz), 2.04, 1.90, 1.61 (3 s, 3 × 3H), 1.44 (d, 3H, J = 6.3 hz), 1.00–0.88 (m, 2H), 0.03 (s, 9H); ¹³C NMR (CDCl₃): δ 170.6, 170.1, 169.8, 165.2, 138.4, 138.0, 137.7, 137.3, 133.5, 129.7–126.9, 99.7, 96.9, 93.9, 82.8, 79.7, 77.1, 75.41, 75.39, 75.1, 74.3, 73.2, 72.9, 72.7, 70.6, 70.4, 68.8, 68.7, 68.0, 67.8, 65.4, 61.1, 51.4, 22.5, 20.7, 20.6, 18.0, 17.8, −1.5. API-ES-MS: m/z calcd for [C₆₄H₇₉NO₁₈Si]H⁺: 1179.4. Found 1179.2.

3,4,6-Tri-O-benzyl-2-O-(4-methoxybenzyl)-α,β-d-glucopyranosyl trichloroacetimidate (11)

To a stirred mixture of compound 22 (12 g, 21 mmol) and Cs₂CO₃ (1.2 g, 3.7 mmol) in anh. CH₂Cl₂ (100 mL) was added Cl₃CCN (25 mL, 243 mmol) under ice-cooling. After 30 min at room temperature the mixture was extracted with brine 3 times followed by drying (Na₂SO₄). Removal of the volatiles under reduced pressure afforded a syrup which was used in the next step without further purification.

5-(Methoxycarbonyl)pentyl (2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-α-l-rhamnopyranosyl-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (13)

To a stirred solution of compounds 8 (3.0 g, 1.83 mmol) and 31 (375 mg, 2.5 mmol) in CH₂Cl₂ (35 mL) was added TMSOTf (15 μL) under external ice-water cooling. After 1 h the solution was treated with Et₃N (~0.1 mL) followed by removal of the volatiles under vacuum. Column chromatography of the residue using hexanes–EtOAc 2:1 → 3:2 as the eluant gave 13 (2.4 g, 81%) as an amorphous substance. ¹H NMR (CDCl₃, partial): δ 8.13–8.11 (m, 2H), 7.98–7.97 (m, 2H), 7.66–7.59 (m, 2H), 7.52–7.45 (m, 4H), 7.35–7.15 (m, 19H), 7.09–7.02 (m, 6H), 5.72 (d, 1H, J = 5.7 Hz), 5.60 (dd, 1H, J = 1.7 Hz, J = 3.5 Hz), 5.40 (dd, 1H, J = 3.6 Hz, J = 10.3 Hz), 5.39 (br s, 1H), 5.19 (t, 1H, J = 9.9 Hz), 5.14 (br d, 1H, J ~ 1.8 Hz), 4.90 (d, 1H, J = 3.5 Hz), 4.80 (d, 1H J = 1.9 Hz), 4.78 (d, 1H, J = 3.3 Hz), 4.74 (d, 1H, J = 10.5 Hz), 4.43 (dt, 1H, J = 3.5 Hz, J = 10.2 Hz), 4.17 (dd, 1H, J = 2.9 Hz, J = 9.7 Hz), 3.67 (s, 3H), 3.37 (t, 1H, J = 9.6 Hz), 2.34 (t, 1H, J = 7.6 Hz), 2.14, 2.01,1.75 (3 s, 3 × 3H), 1.69–1.55 (m, 4H), 1.42–1.37 (m, 2H), 1.36, 1.08 (2 d, 2 × 3H, J = 6.3 Hz each); ¹³C NMR (CDCl₃): δ 174.0, 170.7, 170.5, 169.4, 166.0, 165.7, 165.3, 138.3, 138.0, 137.8, 137.5, 137.4, 133.9, 133.4, 129.8–127.3, 99.8, 99.5, 97.4, 96.5, 80.9, 79.5, 78.8, 78.4, 78.0, 77.9, 75.7, 75.4, 75.2, 75.1, 74.4, 73.9, 73.5, 71.4, 70.4, 70.1, 68.58, 68.55, 68.1, 98.0, 67.9, 67.7, 60.8, 51.9, 51.4, 40.4, 33.8, 29.0, 25.6, 24.6, 23.0, 21.1, 20.7, 18.2, 17.4. API-ES-MS: m/z calcd for [C₈₈H₁₀₀ClNO₂₆]H⁺: 1622.6. Found 1622.8.

Methoxycarbonylpentyl α-L-rhamnopyranosyl-(1→2)- α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)-α-L-rhamnopyranoside (14)

For ¹H and ¹³C NMR data, see Tables 2, 4, 6, and 7.

1,2-Di-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranose (18)

A stirred solution of compound 17 (5.0 g) in Me₃SiOAc (120 mL) was refluxed for 2 h. The solution was concentrated and the residue purified by column chromatography using hexanes–EtOAc (2:1) to afford 18 (4.6 g, 93%) as a syrup. ¹H NMR (CDCl₃): δ 7.41–7.12 (m, 15H), 5.61 (d, 1H, J = 7.8 Hz), 5.11 (dd,1 H, J = 8.3 Hz, J = 9,4 Hz), 4.80, 4.77, 4.67, 4.62, 4.54, 4.49 (6 d, J ~ 11 Hz each), 3.82 (t, 1H, J = 9.2 Hz), 3.77–3.69 (m, 3H), 3.61–3.57 (m, 1H), 2.07, 1.92 (2 s, 2 × 3H); ¹³C NMR (CDCl₃): δ 169.4, 138.0–127.7, 92.2, 82.7, 75.7, 75.1, 75.0, 73.5, 72.1, 68.0, 20.9, 20.7. HRMS: m/z calcd for [C₃₁H₃₈O₈]NH₄⁺: 552.2597. Found 552.2593.

Phenyl 2-O-acetyl-3,4,6-tri-O-benzyl-1-thio-β-d-glucopyranoside (19)

To a stirred solution of di-acetate 18 (2.4 g, 4.5 mmol), PhSSiMe₃ (2.0 mL) in CH₂Cl₂ (15 mL) was added BF₃. Et₂O (0.8 mL) under cooling with ice-water. The cooling bath was then removed and the solution was allowed to reach room temperature. After 90 min total reaction time to the solution was added Et₃N (2 mL) followed by removal of the volatiles under reduced pressure to yield a solid which was stirred in MeOH for 14 h. Filtration followed by drying afforded crystalline 19 (2.2 g, 84 %): mp 114–116 °C; ¹H NMR (CDCl₃): δ 7.52–7.48 (m, 2H), 7.33–7.19 (m, 18H), 5.02 (m, 2H), 4.80–4.77 (m, 2H), 4.67 (d, 1H, J = 11.0 Hz), 4.61 (d, 1H, J = 10.0 Hz), 4.59 (d, 1H, J = 12.0 Hz), 4.57 (d, 1H, J = 11.0 Hz), 4.53 (d, 1H, J = 12.0 Hz), 3.79 (dd, 1H, J = 1.8 Hz, J = 11.0 Hz), 3.72 (dd, 1H, J = 4.8 Hz, J = 11.0 Hz), 3.70–3.65 (m, 2H), 3.54 (m, 1H), 1.99 (s, 3H); ¹³C NMR (CDCl₃): δ 169.4, 138.1, 138.0, 137.8, 132.9, 132.2, 128.7–127.5, 85.9, 84.3, 79.3, 77.7, 75.2, 75.0, 73.3, 71.7, 68.8, 20.9. HRMS: m/z calcd for [C₃₅H₃₆O₆S]NH₄⁺: 602.2576. Found 602.2556.

Phenyl 3,4,6-tri-O-benzyl-1-thio-β-d-glucopyranoside (20)

To a solution of 19 (38 g) in CH₂Cl₂ (150 mL) were added sequentially MeOH (50 mL) and NaOMe (10 mL of a 25 % solution of NaOMe in MeOH) at room temperature. After 4 h the solution was treated with Dowex50 (H⁺) until its pH dropped to approx 3. The solids were removed by filtration and the solution so obtained was extracted with 5% aq. NaHCO₃ then washed with H₂O twice. The organic layers were combined and concentrated. Hexanes were added to and evaporated from the residue. The solids so obtained were triturated with hexanes followed by filtration to afford 20 (34 g, 97 %) as a crystalline solid: mp 74–76 °C; ¹H NMR (CDCl₃): δ 7.61–7.51 (m, 2H), 7.39–7.11 (m, 18H), 4.90, 4.83, 4.81, 4.60, 4.56, 4.53 (6 d, 6 × 1H, J ~ 11Hz), 4.49 (d, 1H, J = 9.5 Hz), 3.78 (dd, 1H, J = 10.4 Hz), 3.72 (dd, 1H, J = 4.2 Hz, J = 10.4 Hz), 3.62–3.57 (m, 2H), 3.55–3.47 (m, 2H); ¹³C NMR (CDCl₃): δ 138.4, 138.2, 138.0, 132.8, 131.8, 128.9–127.5, 88.0, 85.9, 79.3, 77.3, 75.2, 75.0, 73.3, 72.5, 68.9. API-ES-MS: m/z calcd for [C₃₃H₃₄O₅S]NH₄⁺: 560.2. Found 560.2.

Phenyl 3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-1-thio-β-d-glucopyranoside (21)

To a stirred solution of 20 (33.5 g, 62 mmol) in DMF (150 mL) was added NaH (5.0 g, ~ 125 mmol of a 60% suspension in mineral oil) in portions under ice-water cooling. After 15 min, MBnCl (11 mL, 81 mmol) was added dropwise. The mixture was allowed to reach room temperature in approx. 30 min. To the stirred mixture was added MeOH (excess) under cooling with ice-water. The volatiles were removed by distillation under reduced pressure and the residue was equilibrated between CHCl₃ and water. The organic layer was concentrated. Trituration of the residue in hexanes followed by filtration afforded 21 (38.4 g, 91%) as a colorless crystalline solid: mp 86–87 °C; ¹H NMR (CDCl₃): δ 7.76–7.73 (m, 2H), 7.47–7.26 (m, 20H), 6.99–6.92 (m, 2H), 5.02, 4.95 (2 d, 2 × 1 H, J = 11.3 Hz each), 4.94, 4.91 (2 br s, 2 × 1H), 4.77, 4.76 (2 d, 2 × 1H, J = 10.2 Hz), 4.70 (d, 1H, J = 12.0 Hz), 4.69 (d, 1H, J = 10.7 Hz), 4.63 (d, 1H, J = 12.0 Hz), 3.86 (s, 3H), 3.89–3.73 (m, 4H), 3.67–3.54 (m, 2H); ¹³C NMR (CDCl₃): δ 159.3, 138.4, 138.2, 138.0, 133.8, 131.8, 130.1–127.3, 113.8, 87.4, 86.7, 80.5, 79.0, 77.7, 75.7, 74.97, 74.95, 73.3, 68.9, 55.2. HRMS: m/z calcd for [C₄₁H₄₂O₆S]NH₄⁺: 680.3046. Found 680.3057.

3,4,6-Tri-O-benzyl-2-O-(4-methoxybenzyl)-α,β-d-glucopyranose (22)

To a stirred mixture of 21 (33.0 g, 48 mmol), CH₂Cl₂ (500 mL), and water (2 mL) was added (CF₃CO₂)₂Hg (33.0 g, 77 mmol) under cooling in ice-water. Afte 5 min the mixture was treated with saturated aqueous KI (~ 50 mL). The organic layer was separated and washed with water. The residue obtained after removal of the volatiles was purified by column chromatography using hexanes–EtOAc 2:1 → 3:2 as the eluant to afford 16 (27.7 g, 97%) as a colorless syrup. ¹H NMR (CDCl₃, partial): δ 7.36–7.22 (m, 15H), 7.16–7.09 (m, 2H), 6.85–6.80 (m, 2H), 5.15 (d, J = 3.6 Hz), 4.93, 4.82, 4.81, 4.69, 4.61, 4.58 (6 d, 6 × 1H, J ~ 11 Hz), 3.94 (t, J = 9.0 Hz), 3.78 (s, 3H); ¹³C NMR (CDCl₃, partial): δ 159.4, 138.7, 138.2, 137.8, 129.9–127.6, 113.9, 91.3, 81.7, 79.6, 77.6, 75.6, 75.0, 73.4, 72.8, 70.2, 68.6, 55.2. API-ES-MS: m/z calcd for [C₃₅H₃₈O₇]NH₄⁺: 588.3. Found 588.2.

2-(Trimethylsilyl)ethyl [3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-α,β-d-glucopyranosyl]-(1→3)-(4,6-di-O-acetyl-2azido-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (23)

To a stirred solution of 11 prepared from hemiacetal 22 (15.0 g, 26.3 mmol) and 12 (12.0 g, 16.4 mmol) in CH₂Cl₂ (100 mL) was added TMSOTf (50 μL) at approx. −40 °C then the solution was allowed to reach 0 °C in 2 h. The solution was extracted with aq 5% NaHCO₃, dried and concentrated. Column chromatographic purification of the residue using hexanes–EtOAc 10:1 → 2:1 as the eluant afforded 23 (19.0 g, 90%) as a colorless syrup. ¹H NMR (CDCl₃, partial): δ 8.17–8.12 (m, 4H), 7.60–7.07 (m, 25H), 6.80–6.70 (m, 4H), 5.56 (m), 5.35 (d, J = 3.6 Hz), 5.15 (t, J = 10.0 Hz), 5.00 (d, J = 3.6 Hz), 3.70 (s, 3H), 2.06, 1.76 (2 s), 1.40 (d, J = 6.3 Hz), 1.02–0.88 (m), 0.03 (s, 9H); ¹³C NMR (CDCl₃): δ 170.7, 169.3, 165.9, 159.4, 138.8–127.6, 98.6, 97.0, 93.0, 81.4, 79.5, 79.0, 77.3, 75.7, 75.4, 74.6, 73.3, 72.9, 72.7, 71.3, 69.3, 67.7, 67.4, 67.3, 65.2, 62.4, 55.0, 20.6, 17.9, 17.8, −1.5. API-ES-MS: m/z calcd for [C₇₀H₈₃N₃O₁₈Si]H⁺: 1282.6. Found 1282.8.

2-(Trimethylsilyl)ethyl [3,4,6-tri-O-benzyl-2-O-(4-methoxybenzyl)-α,β-d-glucopyranosyl]-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (24)

A mixture consisting of 23 (33 g, 10.9 mmol), Et₃N (7.5 mL), EtOAc (30 mL), EtOH (140 mL), and 10% palladium-on-charcoal (5.5 g) was stirred under hydrogen at 200 psi at room temperature for 1 h. The mixture was treated with Ac₂O (11 mL) then was filtered through a Celite layer followed by concentration. A solution of the residue in CHCl₃ was washed with water, was dried (Na₂SO₄) and was concentrated. The material so obtained was used in the next step without further purification. ¹H NMR (CDCl₃, partial): δ 8.11–8.06 (m, 2H), 7.62–7.55 (m, 1H), 7.49–7.43 (m, 2H), 7.41–7.36 (m, 2H), 7.35–7.28 (m, 4H), 7.27–7.17 (m, 12H), 7.16–7.10 (m, 2H), 7.08–7.02 (m, 2H), 6.76–6.69 (m, 2H), 5.57 (d, 1H, J = 9.8 Hz), 5.42 (m, 1H), 5.19 (t, 1H, J = 9.8 Hz), 5.03 (d, 1H, J = 3.5 Hz), 3.71 (s, 3H), 2.02, 1.71, 1.52 (3 s, 3 × 3H), 1.41 (d, 1H, J = 6.3 Hz), 0.99-0–86 (m, 2H), 0.01 (s, 9H); ¹³C NMR (CDCl₃): δ 170.8, 170.0, 169.2, 165.6, 159.3, 138.8, 138.5, 137.8, 137.7, 130.4–127.4, 113.8, 113.6, 99.2, 97.0, 95.2, 81.5, 80.1, 79.9, 77.5, 75.6, 75.4, 74.7, 73.9, 73.5, 72.9, 71.3, 69.5, 69.4, 68.1, 65.6, 55.3, 52.0, 22.9, 20.9, 20.8, 18.2, 18.0, −1.3. API-ES-MS: m/z calcd for [C₇₂H₈₇NO₁₉Si]H⁺: 1299.5. Found 1299.4.

2-(Trimethylsilyl)ethyl (3,4,6-tri-O-benzyl-β-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (25)

Compound 25 (approx. 3.5 g, 3 mmol) was obtained after the isomer 10 was eluted as described above. ¹H NMR (CDCl₃, partial): δ 8.16–8.09 (m, 2H), 7.69–7.63 (m, 1H), 7.58–7.50 (m, 1H), 7.39–7.17 (m, 27H), 7.16–7.13 (m, 2H), 7.12–7.06 (m, 2H), 5.81 (d, 1H, J = 8.9 Hz), 5.44 (br, 1H), 2.01, 1.91, 1.66 (3 s, 3 × 3H), 1.47 (d, 3H, J = 6.3 Hz), 1.00–0.88 (m, 3H), 0.03 (s, 9H); ¹³C NMR (CDCl₃): δ 172.0, 170.7, 169.6, 166.8, 165.8, 138.8, 138.3, 138.2, 137.9, 137.6, 137.5, 137.4, 134.0, 129.8–127.4, 104.1, 96.9, 94.7, 85.3, 84.3, 79.9, 79.7, 78.6, 76.7, 76.6, 75.5, 75.3, 74.9, 74.8, 74.7, 74.5, 73.7, 73.6, 73.3, 73.2, 69.4, 69.0, 68.2, 68.09, 68.06, 67.6, 65.5, 61.4, 52.2, 42.5, 22.8, 20.7, 20.5, 18.2, 17.9, −1.4. API-ES-MS: m/z calcd for [C₆₄H₇₉NO₁₈Si]H⁺: 1179.4. Found 1179.2.

2-(Trimethylsilyl)ethyl (2-O-acetyl-3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (26)

To a solution of compound 10 (4.3 g, 3.6 mmol) in CH₂Cl₂ (20 mL) were added C₅H₅N (5 mL), Ac₂O (5 mL), and a catalytic amount of 4-dimethylaminopyridine. The solution was kept at ~ 45 °C for 1 h followed by removal of the volatiles under reduced pressure. Toluene was added to and removed from the residue several times to afford 26 (quant.) that was used in the next step without further purification. ¹H NMR (CDCl₃, partial): δ 8.11–8.08 (m, 2H), 7.59–7.56 (m, 1H), 7.45–7.42 (m, 2H), 7.36–7.18 (m, 16H), 7.15–7.14 (m. 2H), 7.11–7.07 (m, 2H), 5.89 (d, 1H, J = 9.5 Hz), 5.47 (m, 1H), 5.14 (t, 1H, J = 9.7 Hz), 5.12, 5.08 (2d, 2 × 1H, J ~ 3.6 Hz), 4.82 (d, 1H, J = 1.8 Hz), 4.76 (dd, 1H, J = 7.4 Hz, J = 11.0 Hz), 4.61 (d, 1H, J = 11.5 Hz), 4.45 (dd, 1H, J = 3.5 Hz, J = 10.3 Hz), 4.28–4.25 (m 2H), 4.02 (dd, 1H, J = 9.3 Hz, J = 10.8 Hz), 3.54–3.46 (m, 3H), 3.34 (dd, 1H, J = 6.8 Hz, J = 9.5 Hz), 3.30 (dd, 1H, J = 9.5 Hz, J = 10.0 Hz), 2.04, 1.97, 1.81, 1.60 (4 s, 4 × 3H), 1.42 (d, 3H, J = 6.3 Hz), 1.00–0.88 (m, 2H), 0.02 (s, 9H); ¹³C NMR (CDCl₃): δ 171.0, 170.6, 169.9, 169.2, 165.2, 138.3, 138.0, 137.7, 137.4, 133.6, 129.7–126.9, 97.0, 95.5, 93.6, 79.9, 79.8, 77.5, 75.5, 76.2, 74.4, 73.3, 73.0, 72.8, 72.7, 70.8, 70.3, 68.8, 68.7, 68.0, 67.7, 65.4, 61,2, 51.1, 22.6, 20.75, 20.68, 20.64, 18.0, 17.8, −1.5. API-ES-MS: m/z calcd for [C₆₆H₈₁NO₁₉Si]H⁺: 1221.4. Found 1221.0.

(2-O-Acetyl-3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-l-rhamnopyranose (27)

A solution of 26 (4.5 g, 4.1 mmol) in a mixture of CH₂Cl₂ (20 mL) and CF₃CO₂H (100 mL) was allowed to stand at rt for 4 h. Removal of the volatiles under reduced pressure followed by column chromatography (hexanes-EtoAc 2:1 → 1:1) of the resulting syrup afforded 27 (3.3 g, 80%) as an amorphous material. ¹H NMR (D₂O): δ 8.05–8.01 (m, 2H), 7.54–7.50 (m, 1H), 7.39–7.17 (m, 18H), 7.13–7.06 (m, 4H), 4.01 (dd, 1H, J = 9.0 Hz, J =10.6 Hz), 3.83 (t, 1H, J ~ 10.3 Hz), 3.80 (d, 1H, J = 3.3 Hz), 3.49, 3.45 (2 d, 2 × 1H, J ~ 9.6 Hz), 2.02, 1.95, 11.78, 1.65 (4 s, 4 × 3H), 1.41 (d, 3H, J = 6.3 Hz); ¹³C NMR (CDCl₃): δ 171.1, 170.7, 170.6, 169.3, 165.2, 138.2, 137.9, 137.7, 137.2, 133.5–126.9, 95.5, 93.4, 92.1, 80.0, 79.8, 77.5, 75.6, 75.1, 74.4, 73.2, 72.9, 72.7, 72.3, 70.9, 70.3, 69.3, 68.7, 68.0, 67.7, 61.3, 51.3, 22.6, 20.72, 20.67, 18.1. API-ES-MS: m/z calcd for [C₆₁H₆₉NO₁₉]H⁺: 1121.2. Found 1121.2.

(2-O-Acetyl-3,4,6-tri-O-benzyl-〈-D-glucopyranosyl)-(1→3)-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl trichloroacetimidate (28)

To a stirred solution of compound 27 (3.0 g, 2.7 mmol) in anh. CH₂Cl₂ (50 mL) were added CCl₃CN (3 mL) and DBU 0.9 mL) cooled at 0 °C. The solution was allowed to reach room temperature in approx. 1 h. the solution was concentrated and the residue chromatographed through silica gel using hexanes-EtOAc 3:2 as the eluant, containing 0.1 % Et₃N to afford 28 (3.2 g, 95%) as an amorphous material. ¹H NMR (D₂O): δ 8.70 (s, 1H), 8.10 (d, 2H, J = 7.8 Hz), 7.60 (t, 1H, J = 7.3 Hz), 7.50 (t, 2H, J = 7.9 Hz), 7.36–7.18 (m, 16H), 7.13, 7.09 (m, 2 × 2H), 6.27 (d, 1H, J = 1.7 Hz), 5.95 (d, 1H, J = 10.0 Hz), 5.71 (m, 1H), 5.16–5.10 (m, 3H), 4.78, 4.25 (2 × 1H, J = 11.5 Hz), 4.71–4.66 (m, 3H), 4.59 (d, 1H, J = 11.5 Hz), 4.45 (dt, 1H, J = 3.8 Hz, J = 10.6 Hz), 4.39 (d, 1H, J = 11.5 Hz), 4.32 (dd, 1H, J = 3.2 Hz, J = 9.8 Hz), 4.32 (d, 1H, J = 12.5 Hz), 4.22 (d, 1H, J = 12.5 Hz), 4.08–3.98 (m, 3H), 3.91 (m, 1H), 3.84 (dd, 1H, J = 9.0 Hz, J = 10.0 Hz), 3.81 (d, 2H, J = 3.1 Hz), 3.57 (t, 1H, J = 9.6 Hz), 3.48 (dd, 1H, J = 1.5 Hz, J = 10.2 Hz), 3.28 (t, 1H, J = 9.5 Hz), 3.21 (dd, 1H, J = 7.0 Hz, J = 10.1 Hz), 2.03, 1.97, 1.82, 1.64, (4 s, 4 × 3H), 1.46 (d, 3H, J = 6.3 Hz); ¹³C NMR (CDCl₃): δ 171.1, 170.6, 170.0, 169.3, 165.0, 164.9, 160.0, 138.3, 138.0, 137.4, 137.3, 133.9, 138.3, 138.0, 137.4, 137.3, 133.9, 129.9, 128.9–127.4, 95.6, 95.0, 94.0, 90.6, 80,0, 79.1, 77.6, 75.6, 75.5, 74.5, 73.3, 73.0, 72.7, 72.5, 71.1, 70.8, 70.3, 68.9, 68.1, 66.9, 61.3, 51.1, 22.7, 20.8, 20.7, 18.1, 14.2.

2-(Trimethylsilyl)ethyl (2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-α-l-rhamnopyranosyl-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (29)

To a stirred solution of compounds 9 (36 g, 62.1 mmol) and 10 (16.4 g, 13.9 mmol) in CH₂Cl₂ (180 mL) was added TMSOTf (60 μL) under cooling in ice-water. The solution was allowed to reach room temperature. After 3 h the solution was treated with Et₃N (0.5 mL). The solution was washed sequentially washed with 2 % aq NaHCO₃ and water. The solution was concentrated and the residue purified by column chromatography using hexanes-EtOAc 5:1 → 2:1 as the eluant to afford 29 (20.0 g, 91%) as an amorphous solid. ¹H NMR (CDCl₃, partial): δ 8.09 (m, 2H), 7.94 (m, 2H), 7.62–7.56 (m, 2H), 7.48–7.41 (m, 4H), 7.32–6.99 (m, 25H), 5.69 (d, 1H, J = 9.2 Hz), 5.57 (dd, 1H, J = 1.7 Hz, J = 3.4 Hz), 5.37 (dd, 1H, J = 3.5 Hz, J = 9.2 Hz), 5.35 (m, 1H), 5.16 (dd, 1H, J = 9.2 Hz, J = 10.3 Hz), 5.12 (d, 1H, J = 1.6 Hz), 4.86 (d, 1H, J = 3.4 Hz), 4.81 (d, 1H, J = 1.9 Hz), 4.76 (d, 1H, J = 3.5 Hz), 4.71 (d, 1H, J = 10.8 Hz), 4.64 (d, 1H, J = 11.3 Hz), 4.63 (d, 1H, J = 11.0 Hz), 4.59 (d, 1H, J = 11.6 Hz), 4.51 (d, 1H, J = 11.3 Hz), 4.47 (d, 1H, J = 11.3 Hz), 4.39 (dt, 1H, J = 3.3 Hz, J = 10.5 Hz), 4.18 (dd, 1H, J = 3.3 Hz, J = 10.5 Hz), 4.00 (m, 1H), 3.35 (t, 1H, J = 9.8 Hz), 2.12, 2.01, 1.74 (3 s, 3 × 3H), 1.37 (d, 3H, J = 6.3 Hz), 1.09 (d, 1H, J = 6.3 Hz), 1.03–0.89 (m, 2H), 0.03 (s, 9H); ¹³C NMR (CDCl₃): δ 170.7, 170.4, 169.4, 165.9, 165.7, 165.3, 139.8–137.3, 133.9, 133.4, 99.8, 99.4, 96.8, 96.3, 80.9, 79.6, 78.7, 78.0, 77.8, 77.3, 75.7, 75.3, 75.2, 75.0, 74.4, 73.9, 73.5, 71.4, 70.5, 70.2, 68.7, 68.5, 68.1, 68.0, 67.8, 65.4, 60.9, 51.9, 40.4, 23.0, 21.1, 20.7, 18.2, 17.9, 17.4, −1.4. API-ES-MS: m/z calcd for [C₈₆H₁₀₀ClNO₂₄Si]H⁺: 1596.2. Found 1596.4.

(2-O-Benzoyl-4-O-benzyl-3-O-chloroacetyl-α-l-rhamnopyranosyl-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranose (30)

To a solution of compound 29 (21.5 g, 13.4 mmol) in CH₂Cl₂ (100 mL) was added trifluoroacetic acid (400 mL) at room temperature. After 3 h the volatiles were removed under reduced pressure. Toluene was added to and evaporated from the residue four times followed by column chromatographic purification using hexanes–EtOAc 1:1 → 2:3 as the eluant to afford 30 (13.0 g, 65%) as a solid material. ¹H NMR (CDCl₃, partial): δ 8.11–7.92 (m, 4H), 7.61 (t, 2H, J = 7.2 Hz), 7.46 (t, 4H, J = 7.7 Hz), 7.35–7.01 (m, 25H), 5.81 (d, 1H, J = 9.8 Hz), 5.59, 5.46 (2 br, 2 × 1H), 5.39 (dd, 1H, J = 3.6 Hz, 9.5 Hz), 5.24 (br, 1H), 5.15 (m, 2H), 2.11, 2.01, 1.76 (3 s, 3 × 3H), 1.35, 1.09 (2 d, 2 × 3H, J = 6.3 Hz); ¹³C NMR (CDCl₃): δ 170.9, 170.7, 169.4, 166.0, 165.7, 165.3, 138.3, 137.9, 137.8, 137.6, 133.8, 133.4, 129.9–127.4, 99.7, 99.2, 95.9, 92.1, 80.9, 79.5, 78.5, 78.4, 78.0, 75.7, 75.3, 75.1, 74.6, 74.5, 73.9, 73.5, 71.4, 70.7, 70.1, 68.6, 68.5, 68.0, 67.8, 60.9, 51.8, 40.4, 23.0, 21.0, 20.7, 18.30, 18.27, 17.4. HRMS: m/z calcd for [C₈₁H₈₈ClNO₂₄]H⁺: 1494.5463. Found 1494.5516.

5-(Methoxycarbonyl)pentyl (2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (32)

A mixture consisting of compound 13 (3.3 g, 2.0 mmol), thiourea (3 g, 39.5 mmol), C₅H₅N (4 mL), and DMF (35 mL) was stirred at room temperature for 12 h. The volatiles were removed by distillation under reduced pressure. The residue was stirred in CHCl₃ (50 mL) followed by removal of the solids by filtration. Concentration afforded a syrup that was purified by column chromatography using hexanes–EtOAc 2:1 → 3:2 as the eluant to afford 32 (2.6 g, 83%) as an amorphous substance. ¹H NMR (CDCl₃, partial): δ 8.16, 8.00 (2 m, 2 × 2H), 7.68–7.59 (m, 2H), 7.55–7.45 (m, 4H), 7.41–7.18 (m, 1pH), 7.13–7.03 (m, 6H), 5.69 (d, 1H, J = 9.9 Hz), 5.42 (br s, 2H), 5.24 (br s, 1H), 5.19 (t, 1H, J = 9.8 Hz), 4.96 (d, 1H, J = 3.6 Hz), 4.77, 4.73, 4.69, 4.64, 4.56, 4.55 (6 d, 6 × 1H, J ~ 11Hz each), 4.44 (dt, 1H, J = 3.4 Hz, J = 10.0 Hz), 3.69 (s, 3H), 2.36 (t, 2H, J = 7.6) 2.05, 2.03, 1.70 (3 s, 3 × 3H), 1.69–1.59 (m, 4H), 1.46–1.39 (m, 2H), 1.38, 1.28 (2 d, 2 × 3 H, J = 6.3 Hz each); ¹³C NMR (CDCl₃): δ 174.1, 170.8, 170.5, 169.0, 166.2, 165.7, 138.333, 138.327, 138.2, 137.74, 137.67, 134.0, 133.4, 129.9–127.4, 99.4, 98.5, 97.5, 95.7, 81.5, 81.4, 79.6, 78.0, 75.9, 75.7, 75.4, 75.3, 74.5, 74.4, 73.6, 73.3, 71.5, 70.6, 70.0, 69.3, 68.5, 68.4, 68.1, 68.0, 61.0, 51.9, 51.5, 33.9, 29.1, 25.7, 24.6, 23.0, 21.1, 20.8, 18.1, 17.9. API-ES-MS: m/z calcd for [C₈₆H₉₉NO₂₅]H⁺: 1546.6. Found 1546.7.

5-(Methoxycarbonyl)pentyl (2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)- (1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (34)

To a stirred solution of imidate 33²⁶ (820 mg, 0.98 mmol) and compound 32 (650 mg, 0.42 mmol) in CH₂Cl₂ (10 mL) was added TMSOTf (8 μL) under external cooling with ice-water. After 45 min the cooling bath was removed and the solution was treated with Et₃N (~ 0.1 mL). Concentration under reduced pressure followed by column chromatographic purification of the residue using hexanes–EtOAc 3:2 → 2:3 as the eluant afforded 34 (0.85 g, 91%) as an amorphous substance. ¹H NMR (CDCl₃, partial): δ 8.12–8.07 (m, 2H), 8.03–7.99 (m, 4H), 7.67–7.57 (m, 2H), 7.53–7.44 (m, 6H), 7.37–7.10 (m, 29H), 7.04–6.97 (m, 2H), 5.75 (d, 1H, J = 9.7 Hz), 5.57 (dd, 1H, J = 1.5 Hz, J = 3.4 Hz), 5.41, 5.38 (2 br m, 2 × 1H), 5.36 (d, 1H, J = 9.7 Hz), 5.21, 5.20 (2 d, 2 × 1H, J = 1.5 Hz each), 5.06, 4.98 (2 d, 2 × 1H, J = 9.7 Hz each), 5.02 (d, 1H, J = 7 Hz), 4.91 (d, 1H, J = 3.4 Hz), 4.15, 4.10 (2 dd, 2 × 1 H, J = 3.0, J = 9.9 Hz each), 3.66 (s, 3H), 2.33 (t, 2H, J = 7.4 Hz), 1.98, 1.96, 1.91, 1.87, 1.77, 1.69, 1.45 (7 s, 7 × 3H), 1.66–1.57 (m, 4H), 1.43–1.40 (m, 2H), 1.37, 1.24, 1.11 (3 d, 3 – 3H, J = 6.3 Hz each); ¹³C NMR (CDCl₃): δ 174.0, 171.1, 170.6, 170.5, 170.3, 169.9, 169.0, 168.9, 165.6, 165.3, 165.0, 138.3, 138.1, 137.7, 137.6, 137.3, 133.9, 133.8, 133.3, 129.8–126.8, 99.3, 99.2, 98.5, 97.4, 95.8, 93.7, 81.1, 80.1, 79.5, 78.8, 77.9, 75.70, 75.6, 75.2, 74.5, 74.4, 73.4, 72.9, 72.4, 71.5, 71.2, 70.0, 69.4, 69.1, 68.5, 68.4, 68.1, 67.94, 67.85, 67.3, 67.1, 60.9, 60.7, 51.5, 51.4, 50.9, 33.8, 29.0, 25.6, 24.6, 22.9, 22.4, 21.2, 20.61, 20.59, 20.5, 20.4, 18.18, 18.16, 17.9, 17.5. TOF-MS-ES⁺: m/z calcd for [C₁₂₀H₁₃₈N₂O₃₈]NH₄⁺: 2232.9 Found 2232.9.

5-(Methoxycarbonyl)pentyl (2-O-acetyl-3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-3,4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (35)

To a solution of compounds 28 (1.19 g, 0.9 mmol) and 32 (650 mg, 0.4 mmol) in CH₂Cl₂ (10 mL) was added TMSOTf (2 μL) under ice-cooling. After 40 min the solution was allowed to reach room temperature in approx. 40 min. The solution was treated with Et₃N (0.1 mL). The syrupy residue obtained after removal of the volatiles under reduced pressure was purified by column chromatography using hexanes–EtOAc 2:1 → 4:3 as the eluant afforded unreacted 32 (344 mg) followed by 35 (320 mg, 60% based on recovered 32. ¹H NMR (CDCl₃, partial): δ 8.15–7.95 (m, 7H), 7.65–7.45 (m, 10H), 7.30–6.95 (m, 43H), 5.77, 5.70 (2 d, 2 × 1H, J ~ 9.7 Hz), 5.62 (br s, 2H), 5.42 (br m, 2H), 5.30 (dd, 1H, J = 3.6 Hz, J = 9.5 Hz), 5.24 (t, 1H, J = 9.6 Hz), 4.86 (d, 1H, J = 3.5 Hz), 3.65 (s, 3H), 2.35 (t, 2H, J = 7.5 Hz), 2.05, 1.98, 1.96, 1.93, 1.88, 1.77 (6 s, 6 × 3H); ¹³C NMR (CDCl₃): δ 174.0,173.9, 171.2, 171.0, 170.6, 170.1, 169.9, 169.2, 168.8, 165.6, 165.4, 165.2, 138.3, 138.2, 138.0, 137.9, 137.8, 137.6, 137.5, 137.3, 137.1, 133.9, 133.7, 133.2, 129.8–126.5, 100.0, 99.4, 98.7, 98.5, 97.6, 96.7, 94.9, 81.3, 80.1, 80.0, 79.6, 79.5, 78.8, 77.9, 75.7, 75.6, 75.2, 75.0, 74.7, 74.5, 74.4, 74.1, 73.9, 73.5, 72.8, 72.6, 72.4, 71.5, 71.2, 70.1, 69.4, 69.1, 68.5, 68.4, 68.1, 67.94, 67.85, 67.3, 67.1, 60.8, 51.6, 51.4, 50.9, 33.8, 29.0, 25.6, 24.6, 22.9, 22.4, 21.2, 20.61, 20.59, 20.5, 20.4, 18.2, 17.9, 17.5. TOF-MS-ES⁺: m/z calcd for [C₁₄₇H₁₆₆N₂O₄₃]NH₄⁺: 2665.1 Found 2665.1.

5-(Methoxycarbonyl)pentyl) (2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-3,4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (36)

TMSOTf (7.5 μL) was added to a stirred solution of compounds 8 (2.6 g, 1.6 mmol) and 32 (1.3 g, 0.84 mmol) in CH₂Cl₂ (15 mL) under ice-water cooling. After 5 min the cooling bath was removed and the solution was allowed to reach room temperature. After 1 h Et₃N (0.2 mL) was added followed by removal of the volatiles. The residue was purified by column chromatography (hexanes–EtOAc 2:1 → 3:2) to give 36 (2.2 g, 88%) as an amorphous solid. ¹H NMR (CDCl₃, partial): δ 8.11, 8.09, 8.05, 8.03, 8.01, 7.99, 7.97, 7.95 (8 s, 8 × 1H), 7.66–7.57 (m, 4H), 7.52–7.43 (m, 8H), 7.32–6.94 (m, 50H), 5.79, 5.75 (2 d, 2 × 1H, J = 9.9 Hz each), 5.60–5.54 (br m, 2H), 5.40 (br s, 1H), 5.37 (dd, 1H, J = 3.5 Hz, J = 9.2 Hz), 5.3, 5.28 (1 br s, 2 × 1H), 5.22 (t, 1H, J = 9.7 Hz), 5.20 (br s, 1H), 5.11–5.07 (m, 2H), 4.95, 4.89 (2 d, 2 × 1H, J ~ 3 Hz), 4.92 (d, 1H, J = 11.0 Hz), 4.82 (d, 1H, J = 11.0 Hz), 4.81 (br s, 1H), 4.17 (dd, 1H, J = 2.9 Hz, J = 9.6 Hz), 4.09 (d, 1H, J = 11.0 Hz), 3.66 (s, 3H), 2.33 (t, 2H, J = 9.5 Hz), 2.06, 2.00, 1.96, 1.84, 1.83, 1.67 (6 s, 6 × 3H), 1.66–1.56 (m, 4H), 1.42–1.34 (m, 2H), 1.39, 1.17, 1.12, 0.93 (4 d, 4 × 3H, J = 6.3 each); ¹³C NMR (CDCl₃): δ 173.9, 170.6, 170.5, 170.3, 169.4, 168.9, 165.9, 165.6, 165.6, 165.5, 165.4, 165.2, 138.3, 138.24, 138.15, 137.9, 137.7, 137.6, 137.38, 137.35, 133.9, 133.4, 133.3, 129.7–126.8, 100.0, 99.5, 99.2, 98.8, 98.2, 97.3, 96.7, 95.6, 81.3, 80.8, 80.0, 79.5, 79.3, 78.8, 78.4, 78.1, 77.92, 77.89, 75.7, 75.6, 75.3, 75.2, 74.9, 74.5, 74.4, 74.3, 74.1, 73.9, 73.42, 73.39, 72.5, 71.3, 71.1, 70.3, 70.1, 69.8, 69.6, 69.5, 68.4, 68.3, 68.2, 68.0, 67.9, 67.83, 67.79, 67.7, 60.7, 51.7, 51.4, 40.4, 33.8, 28.9, 25.5, 24.5, 23.1, 22.9, 21.1, 21.0, 20.7, 20.4, 18.12, 18.06, 17.6, 17.1. TOF-MS-ES⁺: m/z calcd for [C₁₆₇H₁₈₅ClN₂O₄₈]Na⁺: 3044.2 Found 3044.2.

5-(Methoxycarbonyl)pentyl) (2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-3,4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (37)

A mixture of compound 36 (2.14 g, 0.7 mmol), thiourea (13.2 mmol), C₅H₅N (1 mL), and DMF (25 mL) was stirred at room temperature for 16 h. The solution was concentrated under reduced pressure. To the residue CHCl₃ (50 mL) was added. The solids were removed by filtration then were washed with CHCl₃ thrice. The combined solutions were washed with brine thrice. Concentration of the organic layer afforded a syrup that was purified by column chromatography using hexanes–EtOAc (3:2 → 5:4) as the eluant to afford 37 (1.6 g, 77%) as a colorless syrup.¹H NMR (CDCl₃, partial): δ 8.14–8.97 (4 m, 4 × 2H), 7.67–7.55 (m, 4H), 7.54 (m, 8H), 7.38–7.13 (m, 40H), 7.10–703 (m, 10H), 5.75 (dd, 2H, J = 9.9 Hz, J = 12.0 Hz), 5.57 (dd, 1H, J = 1.4 Hz), J = 3.5 Hz), 5.40 (m, 1H), 5.37 (dd, 1H, J = 1.5 Hz, J = 3.5 Hz), 5.32 (m, 1H), 5.29 (br s, 1H), 5.23–5.18 (m, 3H), 5.07 (dd, 1H, J = 9.1 Hz, J = 10.0 Hz), 4.95 (d, 1H, J = 3.5 Hz), 2.33 (t, 2H, J = 7.4 Hz), 1.98, 1.96, 1.90, 1.85, 1.76, 1.68 (6 s, 6 × 3H), 1.67–1.57 (m, 4H), 1.42–1.34 (M, 2H), 1.39, 1.16, 1.13, 1.11 (4 d, 4 × 3H, J ~ 6.3 Hz each); ¹³C NMR (CDCl₃): δ 173.9, 170.6, 170.5, 170.3, 168.9, 166.1, 165.6, 165.4, 165.3, 138.21, 138.20, 138.1, 138.0, 137.7,137.59, 137.57, 137.3, 133.9, 133.2, 129.77–126.70, 99.2, 99.1, 98.8, 98.4, 98.2, 97.3, 95.8, 95.5, 81.31, 81.28, 81.2, 80.0, 79.5, 78.7, 77.9, 77.8, 76.6, 75.8, 75.59, 75.56, 75.5, 75.22, 75.18, 74.6, 74.5, 74.4, 74.3, 74.1, 74.0, 73.38, 73.35, 73.2, 72.4, 71.2, 71.1, 70.4, 69.9, 69.8, 69.6, 69.4, 69.1, 68.3, 68.2, 68.0, 67.9, 67.79, 67.75, 67.7, 60.8, 60.7, 51.5, 51.4, 33.8, 28.9, 25.5, 24.5, 23.0, 22.8, 21.1, 20.8, 20.6, 20.4, 18.1, 17.9, 17.62, 17.60. TOF-MS-ES⁺: m/z calcd for [C₁₆₅H₁₈₄N₂O₄₇]Na⁺: 2968.2 Found 2968.2.

5-(Methoxycarbonyl)pentyl) (2-acetamido-3,4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-3,4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (38)

A stirred solution of compounds 33 (1.0 g, 1.2 mmol) and 37 (0.66 g, 0.23 mmol) in CH₂Cl₂ (10 mL) was treated with TMSOTf (5 μL) under external cooling with ice-water. After 5 min the cooling bath was removed, then the solution was allowed to reach room temperature over a period of 1 h. To the solution was added Et₃N (approx 0.1 mL) followed by concentration under reduced pressure and column chromatography of the resulting syrup to afford 38 (0.73 g, 88%). ¹H NMR (CDCl₃, partial): δ 8.12–8.06 (m, 2H), 8.03–7.94 (m, 8H), 7.67–7.32 (m, 16H), 7.31–7.12 (m, 34H), 7.17–7.00 (m, 16H), 6.98–6.92 (m, 4H), 5.74 (t, 1H, J = 9.1 Hz), 5.54 (m, 1H), 5.39 (br s, 1H), 3.66 (s, 3H), 2.33 (t, 2H, J = 7.5 Hz), 1.98, 1.95, 1.91, 1.88, 1.85, 1.82, 1.77, 1.75, 1.67, 1.45 (10 s, 10 × 3H), 1.68–1.56 (m, 4H), 1.44–1.40 (m, 2H), 1.38, 1.22, 1.00 (3 d, 3 × 3H, J = 6.3 each), 1.25 (d, 6H, J = 6.3 Hz). ¹³C NMR (CDCl₃): δ 174.1, 171.2, 170.7, 170.63, 170.57, 170.5, 170.4, 170.0, 169.1, 169.0, 168.9, 165.7, 165.5, 165.45, 165.41, 165.1, 138.45, 138.36, 138.24, 138.22, 137.82, 137.79, 137.74, 137.73, 137.68, 137.49, 137.45, 134.0, 133.9, 133.3, 129.9–126.8, 99.5, 99.27, 99.25, 99.0, 98.7, 98.4, 97.5, 96.2, 95.8, 93.8, 93.6, 93.2, 81.3, 81.2, 80.24, 80.20, 79.7, 79.6, 78.9, 78.0, 77.9, 75.7, 75.5, 75.3, 74.63, 74.56, 74.5, 74.2, 73.5, 73.4, 73.0, 72.7, 72.52, 72.45, 71.6, 71.3, 71.2, 70.1, 70.0, 69.7, 69.5, 69.3, 69.1, 68.9, 68.7, 68.5, 68.3, 68.1, 68.0, 67.9, 67.8, 67.7, 67.6, 67.4, 67.2, 60.9, 60.8, 60.7, 51.5, 51.4, 51.2, 51.0, 33.9, 29.0, 25.7, 24.6, 23.1, 23.0, 22.5, 22.4, 21.27, 21.26, 20.73, 20.70, 20.68, 20.59, 20.56, 20.5, 20.4, 18.3, 18.2, 18.1, 18.0, 17.7, 17.5. TOF-MS-ES⁺: m/z calcd for [C₁₉₉H₂₂₃N₃O₆₀]Na⁺: 3637.2. Found 3637.4.

5-(Methoxycarbonyl)pentyl) (2-O-acetyl-3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)- (2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-3,4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (39)

To a stirred solution of 28 (1.00 g, 0.82 mmol), 37 (0.66 g, 0.23 mmol) in CH₂Cl₂ (15 mL) was added TMSOTf (5 μL) under cooling in ice-water. After 15 min the cooling bath was removed then the solution was allowed to reach room temperature in 1 h. The solution was treated with Et₃N (~ 0.1 mL) followed by removal of the volatiles. Column chromatographic purification of the residue using hexanes-EtOAc (3:2 → 5:4) as the eluant afforded 39 (0.95 g, 91%) as an amorphous substance purity of which was about 95% (NMR).¹H NMR (CDCl₃, partial): δ 8.11–8.08 (m), 8.02–7.95 (m, 7.64–7.54 (m), 7.50–7.39 (m), 7.32–6.89 (m), 5.83 (d, 1H, J = 10 Hz), 5.73 (t, 2 × 1H, J = 9.3 Hz), 5.55, 5.54 (2 d, 2 × 1H, J = 3.5 Hz each), 5.42–5.38 (2 m, 2 × 1H), 3.66 (s, 3H), 2.33 (t, 2H, J = 7.5 Hz), 1.96, 1.95, 1.93, 1.88, 1.85, 1.82, 1.75, 1.71, 1.67, 1.63 (10 s, 10 × 3H), 1.38, 1.19, 0.99 (3 d, 3 × 3 H, J = 6.3 Hz), 1.14 (d, 6H, J = 6.3 Hz); ¹³C NMR (CDCl₃): δ 174.0, 171.1, 170.60, 170.57, 170.5, 170.3, 170.1, 169.2, 168.9, 165.7, 165.5, 165.4, 165.0, 138.4, 138.3, 138.20, 138.16, 137.8, 137.7, 137.6, 137.44, 137.40, 134.0, 133.7, 133.29, 133.26, 129.8–125.3, 99.4, 99.3, 99.20, 98.9,98.7, 98.4, 97.4, 96.20, 95.7, 95.5, 94.2, 81.3, 81.1, 80.20, 80.16, 79.9, 79.6, 79.0, 78.0, 77.9, 77.5, 76.6, 75.73, 75.70, 75.6, 75.3, 74.6, 74.5, 74.4, 74.3, 7.35, 73.4, 73.2, 73.0, 72.8, 72.5, 72.4, 71.20, 71.16, 70.5, 70.3, 70.1, 70.0, 69.5, 69.2, 68.9, 68.6, 68.4, 68.1, 68.04, 67.99, 67.9, 67.7, 60.78, 60.76, 53.4, 51.5, 51.4, 50.9, 33.9, 31.6, 29.0, 25.6, 24.6, 23.0, 22.9, 22.7, 22.6, 21.4, 21.24, 21.21, 20.8, 20.70, 20.68, 20.53, 20.51, 20.2, 18.2, 18.1, 17.9, 17.7, 17.4, 14.2, 14.1, 11.4. TOF-MS-ES⁺: m/z calcd for [C₂₂₆H₂₅₁N₃O₆₅]Na⁺: 4069.5. Found 4069.6.

5-(Methoxycarbonyl)pentyl) (2-O-benzoyl-4-O-benzyl-3-O-chloroacetyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)- (2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-3,4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranosyl)-(1→2)-(3,4,6-tri-O-benzyl-α-d-glucopyranosyl)-(1→3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy-α-d-glucopyranosyl)-(1→3)-(2-O-benzoyl-4-O-benzyl-α-l-rhamnopyranoside (40)

To a stirred solution of imidate 8 (1.6 g, 0.98 mmol) and octasaccharide 37 (1.5 g, 0.52 mmol) in CH₂Cl₂ (15 mL) was added TMSOTf (7.5 μL) under cooling in ice-water. The solution was allowed to reach room temperature. After 1 h the solution was treated with Et₃N (0.1 mL) followed by concentration. Repeated column chromatography of the residue using hexanes-EtOAc (3:2 → 6:5) as the eluant afforded 40 (1.45 g, 64%) as an amorphous material. ¹H NMR (CDCl₃, partial): δ 8.15–7.97 (m, 12H), 7.7–7.57 (m, 7H), 7.55–7.43 (m, 14H), 7.35–7.21 (m, 46H), 7.21–6.96 (m, 26H), 3.69 (s, 3H), 5.83–5.74 (m, 3H), 5.63–5.55 (m, 3H), 5.43 (br s, 1H), 5.39 (dd, 1H, J = 3.5 Hz, J = 9.2 Hz), 5.34 (br s, 1H), 5.29 (br s, 3H), 5.25–5.19 (m, 3H), 5.16–5.09 (m, 3H), 4.97 (d, 1H, J = 3.2 Hz), 2.36 (t, 4H, J = 7.5 Hz), 2.09, 2.01, 1.98, 1.90, 1.88, 1.86, 1.82, 1.77, 1.70 (9 s, 9 × 3H), 1.70–1.60 (m, 4H), 1.43–1.37 (m, 2H), 1.41, 1.19, 1.18, 1.12, 1.09, 0.95 (6 d, 6 × 3H, J = 6.3 each); ¹³C NMR (CDCl₃): δ 174.0, 170.64, 170.62, 170.58, 170.5, 170.4, 170.3, 169.4, 168.9, 166.0, 165.6, 165.52, 165.46, 165.44, 165.42, 165.3, 165.2, 138.36, 138.35, 138.3, 138.2, 138.23, 138.16, 138.0, 137.80, 137.75, 137.70, 137.69, 137.67, 137.5, 137.43, 137.41, 137.39, 137.38, 133.93, 133.87, 133.4, 133.3, 133.2, 129.8 – 126.8, 100.1, 99.6, 99.2, 98.9, 98.3, 97.4, 96.8, 95.9, 95.7, 81.28, 81.25, 80.8, 80.2, 80.1, 79.6, 79.40, 78.9, 78.8, 78.4, 78.1, 77.9, 75.72,75.67, 75.65, 75.4, 75.2, 74.9, 74.6, 74.5, 74.4, 74.2, 74.1, 73.9, 73.45, 73.43, 73.38, 72.5, 72.4, 71.3, 71.2, 71.1, 70.4, 70.2, 69.9, 69.6, 69.5, 69.4, 68.44, 68.39, 68.2, 68.1, 68.0, 67.9, 67.8, 67.72, 67.68, 60.8, 60.65, 51.7, 51.5, 51.3, 25.6, 24.6, 23.2, 23.0, 22.9, 21.2, 21.1, 21.0, 20.7, 20.5, 18.2, 18.1, 18.0, 17.6, 17.5, 17.1. TOF-MS-ES⁺: m/z calcd for [C₂₄₆H₂₇₀ClN₃O₇₀]Na⁺: 4443.7. Found 4444.0.

5-Methoxycarbonylpentyl α-L-rhamnopyranosyl-(1→2)- α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)- α-L-rhamnopyranoside (41)

For ¹H and ¹³C NMR data, see Tables 1, 3, 5, and 7.

5-Methoxycarbonylpentyl α-L-rhamnopyranosyl-(1→2)- α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)- α-L-rhamnopyranosyl-(1→2)- α-D-glucopyranosyl-(1→3)-2-acetamido-2-deoxy-α-D-glucopyranosyl)-(1→3)- α-L-rhamnopyranoside (42)

For ¹H and ¹³C NMR data, see Tables 1, 3, 5, and 7.

NMR Spectroscopy

NMR spectra were measured at 300 K, by use of a Bruker DRX-500 spectrometer equipped with a 5 mm HCN cryoprobe. Glycoside 41 was examined as its solution in D₂O, but for 42 and 14, the D₂O lock signal was found to be insensitive to shimming. Therefore, spectra for 41 and 42 were recorded for solutions in D₂O with 20% v/v of acetone-d₆ present. Due to the instability of the acetone-d₆ lock signal in the presence of a strong D₂O resonance, the best procedure found was to shim on acetone-d₆, then lock on D₂O for data acquisition. Acetone was used as an internal chemical shift reference at δ = 2.225 for ¹H NMR and 31.0 for ¹³C NMR spectra. The methods used for 1D and 2D NMR data acquisition were similar to those reported by us recently for a set of glycolipids.³⁶

Growth of bacteria, isolation of LPS and O-SP

E. coli O148 strain 201 and rabbit typing antiserum were obtained from the Centers for Disease Control and Prevention (Atlanta, GA) and cultured in Tryptic Soy Broth (Difco Laboratories) for 20 h at 37 °C with stirring and aeration. The pH was maintained at ~7.5 by the addition of ammonium hydroxide. The identity of the bacteria was confirmed by the Clinical Microbiology Laboratory at the NIH, Bethesda, MD. LPS was extracted by the hot phenol method.³⁷ LPS was purified by ultracentrifugation twice at 35,000 rpm for 5 h, at 4 °C (Sorvall Discovery 100SE, Kendro, Asheville, NC). The contents of proteins and nucleic acids in the final LPS preparation were less than 1.5%. To isolate O-SP, LPS (100 mg) was treated with 10 mL of 1% acetic acid at 100 °C for 1.5 h. Lipid A was removed by ultracentrifugation as above, and the soluble products were separated by gel chromatography on a BioGel P-10 (1 × 100 cm) column equilibrated with 0.05 M pyridinium acetate buffer at pH 5.5.The chromatography was monitored with a differential refractometer (Knauer, Germany). O-SP was eluted in the void volume fraction with a yield of 28%.

Conjugation of oligosaccharides

To 15 mg of BSA (Sigma, St. Louis, MO) or rDT (H21G)³⁸ in 2.2 mL of Buffer A (PBS, 0.1% glycerol, 5 mM EDTA, pH 7.2), 4 mg of N-succinimidyl 3-(bromoacetamido) propionate (SBAP, Pierce, Rockford, IL) in 40 μL DMSO was added at pH 7.2, at rt with mixing. After 1.5 h, the solution was applied to a Sephadex G-50 column (1 × 50 cm) in PBS. The void volume fraction containing bromoacetylated protein (Pr-Br) was concentrated using an Amicon Ultra-15 centrifuge filter device (Millipore, Billerica, MA) to 2.6 mL. 13 mg of the protein was recovered), and 0.1 mL of the solution was removed for analysis. To 12 mg of Pr-Br in 2.4 mL Buffer A, 10 mg O-(3-thiopropyl)hydroxylamine were added in 300 μL of 1M KCl, reacted at pH 7.2, r.t. with mixing for 3 h. Next, the solution was passed through Sephadex G-50 (1×50 cm) and the void volume fraction containing aminooxylated protein (Pr-ONH₂) was concentrated to 2.6 mL as above, and 0.2 mL removed for analysis. Pr-ONH₂ (10 mg) was treated with the oligosaccharide (10 mg) in 3 mL of Buffer A at pH 7.2 at rt with mixing for 12 h. The solution was then passed through Sepharose G-50 in PBS and the void volume fraction was collected and analyzed for sugar and protein contents and molecular mass.

Analytical methods

Protein concentration was measured by the BCA Protein Assay (Thermo Scientific, Rockford, Il) according to the manufacturer's protocol. Sugar was quantitated by the anthrone assay.³⁹ SDS-PAGE and Western Blot were performed using standard protocols. LPS, (2 μg) was loaded into 14% Tris-Glycine gels and transferred into polyvinylidene (PDVF) membranes according to the manufacturer's instructions (Bio-Rad, Hercules, CA). After the transfer, the membranes were blocked with 1% BSA in PBS and incubated for 3 h with anti-SD or anti-E. coli O148 sera raised by whole bacteria or by the synthetic oligosaccharide/protein conjugates diluted 1:100 in blocking buffer. After washing 3 × 15 min with PBS + 0.05% Tween, the membranes were incubated for 1 h with phosphatase labeled goat anti-mouse IgG (KPL, Gaithersburg, MD) diluted 1:500 in blocking buffer, washed again as above, and visualized using BCIP/NBT phosphate substrate (KPL, Gaithersburg, MD). For dot-blot analyses, 2 μg of conjugates were pipetted onto stripes of a nitrocellulose membrane (Invitrogen, Carlsbad, CA) and developed the same way as the Western Blot. MALDI-TOF mass spectra of the derivatized proteins and of the conjugates were obtained with an OmniFlex MALDI-TOF instrument (Bruker Daltonics, Billerica, MA) operated in the linear mode. Samples for analysis were desalted, and a 1 μL aliquot was mixed with 20 μL of a saturated sinapinic acid matrix solution made in 30% aq. CH₃CN containing 0.1% trifluoroacetic acid. 1 μL of the solution so obtained was applied to and dried on the sample stage.

Immunization

All animal experiments were approved by the National Institute of Child Health and Human Development Animal Care and Use Committee. Five to six-week-old female NIH general purpose mice were immunized subcutaneously 3 times at 2 week intervals, with 2.5 μg of oligosaccharide as a conjugate in 0.1 mL phosphate buffered saline (PBS). Groups of 10 mice were exsanguinated 7 days after the third injection.⁴⁰ Controls received PBS.

Serology

IgG antibodies were measured by ELISA using BSA or human serum albumin as appropriate for blocking. Antibody levels were calculated relative to a pool of highest antibody level sera obtained from mice immunized 3 times with S. dysenteriae type 1 conjugates¹⁴ and assigned a value of 100 ELISA units (EU). Results were computed with an ELISA immunoassay data processing program provided by the Biostatistics and Information Management Branch, CDC.⁴¹ Competitive inhibition ELISA was done by incubating sera from mice injected with either S. dysenteriae type 1 or E. coli O148 synthetic 12-mers conjugated to rDT, diluted in PBS to give an A₄₀₅ of 1.0, with 0.04, 0.2, 5, or 80 μg/well of either O-SP, incubated for 1 h at 37 °C, followed by incubation at 4 °C for 12 h. The assay was then continued as described above. Sera with inhibitor were compared to the same serum dilution without an inhibitor. Percent inhibition was defined as (1-A₄₀₅ adsorbed serum/A₄₀₅ non-adsorbed serum) × 100%.

Statistics

ELISA values are expressed as the geometric mean (GM). Unpaired t tests were used to compare GMs of different groups.