Structural Studies of the O-Acetyl-Containing O-Antigen from a Shigella flexneri Serotype 6 Strain and Synthesis of Oligosaccharide Fragments Thereof

FULL PAPER

DOI: 10.1002/ejoc.201300180

Structural Studies of the O-Acetyl-Containing O-Antigen from a Shigella

flexneri Serotype 6 Strain and Synthesis of Oligosaccharide Fragments Thereof

Pierre Chassagne,^[a,b,c][‡]Carolina Fontana,^[d]Catherine Guerreiro,^[a,b]

Charles Gauthier,^{[a,b][‡‡]}Armelle Phalipon,^[e,f]Göran Widmalm,^[d]and

Laurence A. Mulard*^[a,b]

Keywords: Carbohydrates / Glycosylation / Oligosaccharides / NMR spectroscopy / Structure elucidation / Polysaccharides

Extensive analysis by NMR spectroscopy of the delipidated

lipopolysaccharide of Shigella flexneri serotype 6 strain

MDC 2924-71 confirmed the most recently reported structure

multistep linear strategy relying on late-stage acetylation at

O-3_C. Thus, the 3_C-O-acetylated and non-O-acetylated tar-

gets were synthesized from common protected intermedi-

ates. Rhamnosylation was most efficiently achieved by using

imidate donors, including at O-4 of a benzyl galacturonate

acceptor. In contrast, a thiophenyl 2-deoxy-2-trichloroacet-

of the O-antigen repeating unit as {Ǟ4)-β-

D-GalpA-(1Ǟ3)-β-

D

-GalpNAc-(1Ǟ2)-α- -Rhap_3Ac/4Ac-(1Ǟ2)-α-L

L

-Rhap-(1Ǟ},

and revealed the non-stoichiometric acetylation at O-3_C/4_C.

Input from the CASPER program helped to ascertain the fine

distribution of the three possible patterns of O-acetylation.

The non-O-acetylated repeating unit (ABCD) corresponded

to about 2/3 of the population, while 1/4 was acetylated at

amido-D-galactopyranoside precursor was preferred for

chain elongation involving residue B. Final Pd/C-mediated

deprotection ensured O-acetyl stability. All of the target mo-

lecules represent parts of the O-antigen of S. flexneri 6, a

O-3_C(_3AcCDAB), and 1/10 at O-4_C(_4AcCDAB). Di- to tetra- prevalent serotype. Non-O-acetylated oligosaccharides are

saccharides with a GalpA residue (A) at their reducing end

also fragments of the Escherichia coli O147 O-antigen.

were synthesized as their propyl glycosides following a

resistance.^[3]It is endemic worldwide, and it remains a

major health concern, especially in the child population of

the most impoverished areas.^[3,4]S. flexneri – one of the

four species of Shigella – is prevalent in developing coun-

tries, where it accounts for the endemic disease.^[3,5]Numer-

ous S. flexneri serotypes – varying in their geographic and

temporal distributions – have been isolated from patients.

In recent years, S. flexneri serotype 6 (SF6) has been iden-

tified as an increasingly prevalent serotype in several set-

tings worldwide,^[3,6]and evidence strongly supports the in-

clusion of SF6 as one of the key valences in a broad-cover-

age Shigella vaccine.^[4,7]

S. flexneri serotypes are defined on the basis of the

carbohydrate repeating unit of the surface O-antigen (O-

Ag), the polysaccharide part of the bacterial lipopolysac-

charide (LPS).^[8]Protection against reinfection by the ho-

mologous serotype, suggesting serotype-specific natural im-

munity, has been established following Shigella infec-

tion.^[6a,9]These observations provided strong evidence that

S. flexneri O-Ags are major targets of the host’s adaptive

immune system. Accordingly, several LPS-based vaccine

candidates against shigellosis have been developed and even

evaluated in clinical trials.^[4,10]Along these lines, we have

investigated a promising alternative to the use of material

of biological origin. It involves the design of synthetic oligo-

saccharide haptens to serve as functional mimics of the nat-

ural O-Ag of interest.^[11]The strategy relies on the availabil-

Introduction

Shigellosis, an invasive infection of the human colon, has

been identified as one of the major diarrhoeal diseases

worldwide.^[1]In its most classical expression, it is charac-

terized by a triad of fever, intestinal cramps, and bloody

diarrhoea.^[2]Also known as bacillary dysentery, this highly

contagious infection is associated with increased antibiotic

[a] Institut Pasteur, Unité de Chimie des Biomolécules,

28 rue du Dr Roux, 75015 Paris, France

Fax: +33-1-45688404

E-mail: laurence.mulard@pasteur.fr

[b] CNRS UMR3523, Institut Pasteur,

28 rue du Dr Roux, 75015 Paris, France

[c] Université Paris Descartes Sorbonne Paris Cité, Institut

Pasteur,

rue du Dr Roux, 75015 Paris, France

[d] Department of Organic Chemistry, Arrhenius Laboratory,

Stockholm University,

10691 Stockholm, Sweden

[e] Institut Pasteur, Pathogénie Microbienne Moléculaire,

28 rue du Dr Roux, 75015 Paris, France

[f] INSERM U786, Institut Pasteur,

28 rue du Dr Roux, 75015 Paris, France

[‡] Present address: Glycom A/S, DTU,

Bld 201, 2800 Kgs Lyngby, Denmark

[‡‡]Present address: Université de Poitiers, Institut de Chimie

IC2MP UMR-CNRS 7285,

86022 Poitiers, France

Supporting information for this article is available on the

WWW under http://dx.doi.org/10.1002/ejoc.201300180.

Eur. J. Org. Chem. 2013, 4085–4106

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L. A. Mulard et al.

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ity of well-defined synthetic frame-shifted fragments of the

O-Ag.^[12]In this paper, this is addressed for the first time

for SF6.

Knowledge of the exact repeating unit (RU) of the O-Ag

of interest is a major prerequisite to launching such a strat-

egy. Considering the numerous revised structures of S.

flexneri O-Ags published recently,^[13a–13c]in addition to the

various structures reported for the SF6 O-Ag,^[14]our first Figure 1. Comparison of the structures of the repeating units of

the O-Ag from E. coli O147 (top) and SF6 (bottom). The sugar

residues are denoted A–D.

concern was to ascertain the exact molecular composition

of the O-Ag of SF6.

This paper describes the elucidation by NMR spec-

troscopy of the RU and acetylation pattern of the O-Ag

from SF6 strain MDC 2924-71, and also the first synthesis

of di- to tetrasaccharide fragments of the O-Ag.

by integration of the N-acetyl signals of residue B in the

region δ = 2.04–2.09 ppm (Figure 2a) relative to the anom-

eric signals in the core region for α-Galp (δ = 5.86 ppm)

and α-Glcp (δ = 5.63 ppm), as described by Kubler-Kielb

et al.^[14c]A direct inspection of the ¹H NMR spectra in

the region of δ = 2.14–2.21 ppm suggested that different

O-acetylation patterns were present in the lower molecular

weight (mw) fraction, which is consistent with reported re-

sults.^[14c]A fraction of intermediate mw showed resonances

characteristic of both regions of the PS. As the present

study was undertaken to establish the location of O-acetyl

groups in the O-Ag, a fraction of higher mw, corresponding

to about a dozen RUs, was used in the NMR studies. Pre-

cautions were taken to minimize acetyl migration, such as

keeping the pH at 6 or below, and keeping the sample tem-

perature as low as possible, between 0 and 17 °C.

Results and Discussion

Structural Investigation of the O-Ag from SF6 Strain

MDC 2924-71

The most recent structural investigations of the full-

length SF6 O-Ag^[14d,15]reported a structure similar to that

of the O-Ag of Escherichia coli O147.^[16]The basic RU is a

linear tetrasaccharide made of one d-galacturonic acid (A),

one N-acetyl-d-galactosamine (B) and two l-rhamnose resi-

dues (C, D). The only difference between these two polysac-

charides (PS) is that the O-Ag of SF6 is O-acetylated (Fig-

ure 1). The occurrence and position of O-acetyl (OAc)

groups in a PS may influence its antigenicity,^[17]and more

importantly, its immunogenicity,^[18]and thus it is particu-

larly important to establish the exact location(s) of such

OAc groups. Acetylation in the SF6 O-Ag was identified at

O-3 of rhamnose C.^[14d]However, the main problem when

determining the O-acetylation pattern in a native PS is

whether acetyl migration or deacetylation has occurred;

these processes can easily take place given appropriate spa-

tial arrangements, either in the course of PS extraction or

purification, or even during the acid-mediated delipidation

procedure of the LPS.^[19]Consequently, when deacetylation

occurs during the delipidation procedure, one does not

know whether OAc groups were present in the native LPS.

It has been possible to determine the locations of OAc

groups by NMR spectroscopy directly on the intact LPS,^[20]

but this is highly dependent on the preparation, and is not

always feasible. Nevertheless, one can often obtain infor-

mation at least about the presence or absence of OAc

1

Figure 2. (a) Spectral region of the H NMR spectrum of the SF6

groups from a one-dimensional H NMR spectrum of the

PS containing the N- and O-acetyl resonances. (b) Selected region

LPS in D₂O as solvent. In the first part of this study, we

confirm the acetylation at O-3_C, as was previously report-

1

of the H,¹³C-BS-CT-HMBC NMR spectrum of the PS from SF6

showing correlations from the carbonyl carbon atoms to the methyl

ed,^[14d,15]and describe in detail the population distribution protons of the O-acetyl and N-acetyl groups (residues C and B,

respectively). The residues of the non-O-acetylated population are

denoted by the respective non-primed capital letters, whereas the

O-acetylated populations are denoted by primed (major) and

double-primed capital letters (minor).

of OAc groups on rhamnose C in the SF6 O-Ag.

The LPS was isolated from SF6 strain MDC 2924-71 ac-

cording to a known protocol.^[21]It was delipidated under

mild acidic conditions to yield a PS corresponding to the

O-Ag covalently linked to the core through residue B. The

PS was purified by gel-permeation chromatography. Dif- plexity. Two H signals for OAc at δ = 2.158 (minor form)

The ¹H NMR spectrum revealed a material of high com-

1

ferent fractions were collected, and the average number of and 2.207 ppm (major form) could be easily identified (Fig-

1

RU per O-Ag was estimated from their H NMR spectra ure 2a). As a consequence, several sets of signals were found

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O-Antigen from a Shigella flexneri Serotype 6

for all the residues due to partial O-acetylation. For in- large downfield shift of the 3-H signal (δ = 5.071 ppm) in

stance, the N-acetyl (NAc) signals at δ = 2.037, 2.042, and the spin system originating from the proton signal at δ =

2.086 ppm suggest three different populations of residue B. 1.289 ppm (6-H, major O-acetylated population, denoted

The ratio of the different populations was estimated by inte- CЈ in Figure 3), indicates acetylation at O-3_C. Intra-residual

gration of the OAc signals with respect to the NAc signals. NOEs from the OAc signal at δ = 2.207 ppm to the reso-

The major and minor O-acetylated populations corre- nances at δ = 4.282 (2-H) and 5.071 ppm (3-H) supported

sponded to about 1/4 and 1/10 of the total, respectively, and this substitution pattern (Figure 4c). In addition to the sig-

the population without OAc corresponded to the remaining nals due to residue B in the non-O-acetylated population,

1

2/3. A band-selective constant-time H,¹³C-HMBC experi- two new spin systems corresponding to minor populations

ment^[22]confirmed the presence of two OAc signals at δ = were identified. Both spin systems were assigned from 1-H

1

174.43 and 174.49 ppm, as well as three NAc signals at δ = to 4-H by using H,¹H-TOCSY experiments with different

175.36, 175.82, and 175.88 ppm (Figure 2b).

mixing times, and the respective 5-H protons were identified

To facilitate the identification of the resonances corre- by intra-residual NOE correlations from the anomeric pro-

sponding to the population of the non-O-acetylated O-Ag, tons. Based on their relative intensities, the spin systems

1

the H and ¹³C chemical shifts of the E. coli O147 O-Ag^[16]originating from the 1-H signals at δ = 4.512 and 4.716 ppm

were reassigned at pD = 5 by using 1D and 2D NMR ex- were assigned to the major and minor O-acetylated popula-

periments. Due to the pD change, the signals for C-5_A, C- tions, respectively. The chemical shift displacements, in par-

6_A, and 4_A-H were shifted downfield to δ_C= 74.91 and ticular upfield by 0.2 ppm in the former case, were attrib-

174.42 ppm, and δ_H= 4.10 ppm, while the signals for C-1_Duted to the perturbation by the OAc group in the neigh-

1

and 5_D-H were found at δ_C= 100.24 ppm and δ_H

=

boring residue. The H chemical shifts of residues B and C

3.72 ppm, respectively. The remaining chemical shift differ- of the O-acetylated populations are compiled in Table 1.

ences were less than 0.04 ppm and 0.25 ppm for ¹H and ¹³

resonances, respectively.

C

By comparison with the E. coli O147 O-Ag, the reso-

nances at δ = 1.23–1.27 ppm in the SF6 O-Ag were assigned

to 6-H in the non-O-acetylated rhamnose C and rham-

nose D. Two conspicuous signals of lower intensity at δ =

1

1.151 and 1.289 ppm, which are absent from the H NMR

spectrum of the E. coli O147 PS, corresponded to the minor

and major O-acetylated forms, respectively. At this point we

1

used the CASPER program,^[23]which is able to predict H

and ¹³C NMR chemical shifts of oligo- and polysaccha-

1

rides. The H chemical shifts of rhamnosyl residues C and

D in the RUs, mono-acetylated either at O-3_C, O-4_C, O-3_D,

or O-4_D, were predicted. The signals from the 6_C-H were

calculated to resonate at δ = 1.28 ppm in the 3_C-OAc RU

and at δ = 1.16 ppm in the 4_C-OAc RU, whereas the signals

from the 6_D-H were calculated to resonate at δ = 1.33 ppm

in the 3_D-OAc RU and at δ = 1.20 ppm in the 4_D-OAc RU.

These predictions suggest that the major and minor O-acet- Figure 3. Selected regions of the ¹H,¹H-TOCSY NMR spectrum

(t_mix= 120 ms) of the SF6 PS showing the spin system of residues

_3AcC (denoted CЈ), C (denoted C), and _4AcC (denoted CЈЈ).

ylated populations corresponded to RUs of the O-Ags acet-

ylated at O-3_Cor O-4_C, respectively.

The ¹H chemical shifts of the two variants of the O-acet-

ylated residue C were assigned by using H,¹H-TOCSY ex-

Inter-residue correlations observed in the ¹H,¹H-NOESY

1

periments with increasing mixing times. In both cases, the spectrum were consistent with acetylation at O-3_Cand

spin systems could be fully characterized by starting from O-4_C. In both cases, long-range NOE correlations were ob-

6-H. Subsequent correlations were observed to 5-H, 4-H, served from 2_C-H in the O-acetylated residue C to the re-

and 3-H (Figure 3a and b) as well to 2-H (Figure 3c). The spective 1_B-H (Figure 4d) and from 4_C-H in the O-acet-

resonances from the anomeric protons were then readily as- ylated residue C to the respective NAc in residue B (Fig-

sociated with their respective 2-H signals (Figure 3d). In the ure 4e and f), indicating that residue B is linked to the O-

spin system originating from the proton signal at δ = acetylated rhamnose C. NOE correlations were also ob-

1.151 ppm (minor O-acetylated population, denoted CЈЈ in served between 6_C-H and the proton signals at δ = 5.421

Figure 3), the large downfield shift of proton 4-H signal (δ and 5.384 ppm in the major and minor populations, respec-

= 4.823 ppm) suggests acetylation at O-4_C. This was con- tively (Figure 4g). By comparison with the chemical shifts

firmed by intra-residual NOE correlations in the ¹H,¹H- in the PS from E. coli O147, those resonances can be attrib-

NOESY spectrum from the OAc signal at δ = 2.158 ppm to uted to 1_D-H, suggesting that the O-acetylated residue C is

the 6-H resonance at δ = 1.151 ppm (Figure 4a) and to the linked to rhamnose D. The NOE correlations observed in

1

4-H resonance at δ = 4.823 ppm (Figure 4c). Likewise, the the H,¹H-NOESY spectrum from the NAc signal at δ =

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pattern is in agreement with that reported.^[14d]Interestingly,

the dominance of the population of non-O-acetylated RU

is, to the best of our knowledge, a new finding, and suggests

that the S. flexneri strain MDC 2924-71, used in this study,

belongs to the newly introduced type 6.^[15]The observed

NOEs are in good agreement with a 3D model generated

by CarbBuilder.^[25]

Chemical Synthesis of Di- to Tetrasaccharides Representing

SF6 O-Ag Fragments Bearing Residue A at Their Reducing

End

Having confirmed the structure of the RU of the SF6 O-

Ag, we turned to the synthesis of di- to tetrasaccharides

having a galactopyranosiduronic acid residue A at the re-

ducing end. These oligosaccharides were synthesized both

in their non-O-acetylated form 1 (DA-Pr), 2 (CDA-Pr), 4

(BCDA-Pr), and as their mono-O-acetylated counterparts

3 (_3AcCDA-Pr) and 5 (B_3AcCDA-Pr). All were isolated as

β-propyl glycosides in order to lock their reducing end in a

form mimicking the natural linkages found in the O-Ag.

The choice of propyl glycosides derived from our assump-

tions that an allyl aglycon would (i) be easily introduced

into commercially available d-galactose, (ii) be fully orthog-

onal to most other conventional protecting groups used in

glycochemistry, in particular to acetate, (iii) be smoothly

converted into a propyl group upon concomitant Pd/C-me-

diated hydrogenolysis of benzyl ether or benzyl esters, and

(iv) in due course, have the potential to serve as an anchor

for chemoselective modification,^[26]opening the way to a

variety of SF6-related glycoconjugates. To reduce the

number of synthetic intermediates, the 3_C-O-acetyl moiety

was introduced at a late stage of the synthesis. Masking of

the corresponding hydroxy group before functionalization

was achieved by using a p-methoxybenzyl (PMB) ether.

Stepwise chain extension starting from a galactopyranosid-

uronate acceptor allowed the investigation and optimization

of each glycosylation step by using appropriate monosac-

charide precursors selected from trichloroacetimidate

(TCA),^[27]thioglycoside,^[28]or N-phenyltrifluoroacetimid-

ate^[29](PTFA) donors.

Figure 4. Selected regions of the ¹H,¹H-NOESY NMR spectrum

(t_mix= 150 ms) of the SF6 PS showing intra- and inter-residue

NOE correlations in residues _3AcC (denoted CЈ), C (denoted C),

and _4AcC (denoted CЈЈ).

1

Table 1. H NMR chemical shifts (ppm) of selected resonances of

the O-acetylated populations from the SF6 O-Ag.

Atom

Major

Minor

B

C

B

C

1-H

2-H

3-H

4-H

5-H

6-H

NAc

OAc

4.512

4.014

3.899

4.300

3.633

n.d.^[a]

2.086

5.164

4.282

5.071

3.540

3.800

1.289

4.716

4.055

3.868

4.301

3.685

n.d.

5.159

4.238

4.095

4.823

3.857

1.151

2.037

2.207

2.158

[a] n.d. = not determined.

2.086 and 2.042 ppm, and from the OAc signal at δ =

2.207 ppm, were also confirmed by using DPFGSE CSSF-

NOESY experiments.^[24]From the data in Table 1, it can be

Synthesis of Disaccharide DA-Pr (1)

Glycosylation at O-4 of a galacturonide acceptor is

seen that acetylation at O-3_Cstrongly affects the chemical thought to be disfavored compared to the corresponding

shifts of the NAc group and 1_B-H. This can be explained galactopyranoside.^[30]In addition, the reactivity of galacto-

in terms of the close spatial proximity of these groups, as pyranosiduronic acid esters possessing a free axial hydroxy

shown by the NOE correlations in Figure 4b and c (denoted group has been shown to depend significantly on the anom-

by primed capital letters). On the other hand, acetylation eric configuration. Interestingly, experimental data and

at O-4_Cdoes not significantly influence the chemical shifts theoretical calculations agree that OH-4 is more nucleo-

of residue B.

philic in the β than in the α anomers.^[31]Thus, taking ad-

All these results are consistent with the structure Ǟ2)-α- vantage of the successful use of 2,3-di-O-benzyl-d-galacto-

l-Rhap3Ac/4Ac-(1Ǟ2)-α-l-Rhap-(1Ǟ4)-β-d-GalpA-(1Ǟ3)- pyranosiduronic acid esters as acceptors in α-(1Ǟ4)- or β-

β-d-GalpNAc-(1Ǟ, in which about 2/3 corresponds to the (1Ǟ4)-glycosylation reactions involved in the synthesis of

non-O-acetylated form (CDAB), 1/4 is acetylated at O-3_Chomogalacturonans^[31,32]or rhamnogalacturonans,^[32b,33]

(

_3AcCDAB), and 1/10 at O-4_C(_4AcCDAB). This substitution we selected benzyl (allyl 2,3-di-O-benzyl-β-d-galactopyr-

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O-Antigen from a Shigella flexneri Serotype 6

anosid)uronate^[34](9) as the precursor to residue A. This silyl) as catalyst (Scheme 1). The α-l-(1Ǟ4)-linked disac-

precursor was prepared from commercially available β-d- charide (i.e., 11; NMR spectroscopic data for C-1_D: δ =

1

galactose pentaacetate via allyl glycoside 6, obtained as a 99.7 ppm, J_CH= 170.4 Hz) was isolated in a pleasing 91%

crystalline material in a non-optimized 54% yield over three yield, which confirmed the good nucleophilic properties of

steps (Scheme 1).^[35]Benzylation of acetal 6 and subsequent OH-4 of β-d-galactopyranosiduronic acid esters. A two-

acidic hydrolysis of the benzylidene protecting group gave step deprotection sequence was designed so as to prevent

4,6-diol 7 (89%) on a multigram scale.^[36]Of the numerous any risk of β-elimination upon treatment of uronate inter-

well-established oxidation protocols envisioned for the con- mediates with base. Thus, Pd/C-mediated hydrogenolysis of

version of diol 7 into known uronic acid 8,^[37]we favored the benzyl ether protecting groups was run first. Ethyl acet-

the 2,2,6,6-tetramethyl-1-piperidinyloxy/[bis(acetoxy)iodo]- ate was used in this reaction to solubilize uronic acid 12

benzene (TEMPO/BAIB) system,^[38]which we thought resulting from concomitant cleavage of the benzyl ester in

would be compatible with the allyl aglycon. To our satisfac- fully protected 11. Saponification of the acetyl protecting

tion, treatment of diol 7 with a catalytic amount of groups of crude acid 12 provided DA-Pr glycoside 1 in 51%

TEMPO and excess BAIB in CH₂Cl₂/water (2:1) gave the isolated yield over two steps, following RP-HPLC purifica-

expected carboxylic acid (i.e., 8), which was smoothly con- tion. Satisfactorily, no by-products related to β-elimination

verted into benzyl ester 9 upon reaction with benzyl brom- were identified.

ide in the presence of potassium hydrogen carbonate. When

running the oxidation/protection sequence on a multigram

scale, acceptor 9 was isolated in a good 74% yield over the

two steps. Next, allyl glycoside 9 was condensed with the

Synthesis of Trisaccharides CDA-Pr (2) and _3AcCDA-Pr (3)

readily accessible 2,3,4-tri-O-acetyl-l-rhamnosyl trichloro-

Having ascertained that galacturonate 9 was an efficient

acetimidate^[39](10) by using TMSOTf (TMS = trimethyl- acceptor even when using disarmed donors, the next step

consisted of identifying a suitable rhamnosyl donor, a pre-

cursor to residue D, that would be compatible with chain

elongation at O-2. As concomitant CO₂Bn Ǟ CO₂Me

transesterification has been observed during acetyl removal

under methanolysis conditions,^[33a]we turned to donors

bearing a levulinoyl (Lev) ester at C-2 in view of its stereo-

directing potency^[40]and convenient selective removal in the

presence of other esters, including benzyl uronates.^[40,41]

Thus, acceptor 9 was treated with known rhamnosyl tri-

chloroacetimidate^[42]14 in CH₂Cl₂containing a catalytic

amount of TMSOTf (Scheme 2). To our surprise, the reac-

tion did not go to completion, even when 1.3 or up to

1.5 equiv. of donor were used (Table 2, Entries 1 and 2).

This unexpected outcome was, in part, explained by re-

arrangement of the donor into β-glycosylamide 19 (¹³C

1

NMR spectroscopic data for C-1: δ = 78.4 ppm, J_CH

=

154.3 Hz), which was isolated as a major by-product. The β

configuration of the glycosidic linkage in 19 was ascertained

from NOESY 1D NMR spectroscopic data, which indi-

cated spatial proximity between 1-H (δ = 5.32 ppm) and

both 3-H (δ = 3.74 ppm) and 5-H (δ = 3.55 ppm). Although

formation of the β-glycosylamide could not be avoided,

changing CH₂Cl₂for toluene resulted in completion of the

reaction in the presence of only 1.2 equiv. of TCA 14. Re-

ducing the amount of donor resulted in the formation of

less of the by-product, thus facilitating the purification to

Scheme 1. Synthesis of A acceptor 9 and of target DA-Pr disac- give disaccharide 17 in 94% yield (Table 2, Entry 3). As

charide 1. Reagents and conditions: (a) AllOH, BF₃·OEt₂, CH₂Cl₂,

with donor 10, the α stereochemistry of the newly formed

0 °C to room temperature, 24 h; (b) NaOMe, MeOH, room tem-

glycosidic linkage was obvious from the ¹J_CHcoupling con-

perature, 18 h; (c) benzaldehyde dimethyl acetal, CSA (camphorsul-

stant at C-1_D(¹J_CH= 172.7 Hz). In our search for an im-

fonic acid), MeCN, room temperature, 2 h, 54 % over 3 steps;

proved method that would avoid glycosylamide formation,

we referred to previous work by C. Vogel, which suggested

that β-d-galactopyranosiduronate acceptors could react at

O-4 with numerous types of donors.^[32b]In one approach,

acetylation of known hemiacetal^[42]13 gave donor 15 as a

4.5:1 mixture of α/β anomers (96%). In a second approach,

(d) NaH, BnBr, DMF, 0 °C to room temperature, 2 h; (e) AcOH

(80 % aq.), 80 °C, 2 h, 89 % over 2 steps; (f) TEMPO, BAIB,

CH₂Cl₂/H₂O, room temperature, 1 h; (g) KHCO₃, BnBr, DMF,

room temperature, 16 h, 74% over 2 steps; (h) TMSOTf, CH₂Cl₂,

–40 °C to room temperature, 1 h, 91%; (i) H₂, Pd/C 10%, EtOAc,

room temperature, 16 h; (j) NaOH, THF/H₂O, room temperature,

24 h, 51% over 2 steps.

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the same hemiacetal was treated with N-phenyltrifluoro- [4-(dimethylamino)pyridine] to give ester 21, which was

acetimidoyl chloride in acetone containing excess cesium converted into hemiacetal 22 following a two-step anomeric

carbonate^[43]to give the corresponding PTFA donor (i.e., deallylation procedure involving isomerisation of the allyl

16) as a 4:1 α/β mixture (95%). Both the acetate and PTFA ether into the corresponding prop-1-enyl ether with a cat-

donors – 15 and 16 – were treated with acceptor 9 in the ionic iridium complex^[46]and its subsequent iodine-medi-

presence of catalytic TMSOTf (Scheme 2). With the acetate ated hydrolysis (90%).^[47]The hemiacetal was transformed

donor, the glycosylation resulted in an 89% isolated yield of either into TCA 23 (97%) by reaction with trichloroaceto-

disaccharide 17, which compared favorably with published nitrile in the presence of catalytic DBU (1,8-diazabi-

data,^[32b]by using 1.5 equiv. of donor 15 and 0.15 equiv. of cyclo[5.4.0]undec-7-ene), or into PTFA donor 24 (98%) un-

TMSOTf in CH₂Cl₂(Table 2, Entry 4). Under conditions der conditions similar to those used to prepare the corre-

optimized for TCA donor 14 (Table 2, Entry 3), a re- sponding 3,4-di-O-benzyl derivative (i.e., 16). The outcome

warding 96% glycosylation yield was obtained with PTFA of the TMSOTf-mediated [C + DA] assembly was similar

donor 16 (Table 2, Entry 5). As already observed,^[44]the use to that of the [D + A] glycosylation. Briefly, trisaccharide

1

of donor 16 (1.2 equiv.) avoided the formation of any un- 26 (NMR spectroscopic data for C-1_C: δ = 98.9 ppm, J_CH

wanted glycosylamide or other by-products that hampered = 172.5 Hz) was isolated in higher yield – 91 vs. 84% –

the purification. Removal of the levulinoyl group by using when rhamnosyl C donor 23 (1.5 equiv.) and DA acceptor

hydrazine hydrate in buffered medium gave target acceptor 18 were set to react in toluene rather than in a chlorinated

18 (90%).

solvent (Table 3, Entries 1 and 2). Fortunately, while a lower

proportion of rearranged β-glycosylamide 25 formed as a

by-product, which facilitated the purification, reducing the

amount of donor 23 to 1.2 equiv. had no influence on the

glycosylation yield (92%, Table 3, Entry 3). To our satisfac-

tion, the formation of by-products was minimal, and

glycosylation at O-2_Dof acceptor 18 remained high-yielding

(89%) when TCA donor 23 was substituted by its PTFA

equivalent 24 (Scheme 3, Table 3, Entry 4). The high-yield-

ing removal of the 2_C-levulinoyl ester of glycosylation prod-

uct 26 by reaction with hydrazine hydrate in pyridine/AcOH

gave alcohol 27 (93%), which could serve either as an inter-

mediate for the synthesis of CDA-Pr target 2, or as an ac-

ceptor in the synthesis of tetrasaccharides 4 and 5; Pd/C-

mediated benzyl ether hydrogenolysis, benzyl ester cleavage,

and concomitant allyl reduction gave propyl glycoside 2 in

a good 69% yield after RP-HPLC purification. This final

deprotection step was run in methanol, and it is noteworthy

that despite the neutral conditions used, methyl ester 32 was

also isolated, albeit in low yield (5%). Taken together with

an independent report with a similar outcome,^[48]this en-

couraged us to use a THF/H₂O solvent for subsequent hy-

drogenolysis reactions. Alternatively, acetylation at O-3_C

was a prerequisite for obtaining _3AcCDA-Pr target 3. Oxi-

dative removal of the PMB ether was attempted by using

DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) or

CAN (ceric ammonium nitrate). When run in CH₂Cl₂/H₂O

in the presence of DDQ (3.0 equiv.), the reaction was slow,

and degradation increased with time. The best isolated yield

of alcohol 28 was 45%. In contrast, treatment of fully pro-

tected 26 with CAN (3.0 equiv.) in MeCN/H₂O resulted in

a faster and cleaner conversion, giving alcohol 28 in a good

71% yield. When the amount of CAN was increased to

Scheme 2. Synthesis of DA disaccharide acceptor 18. Reagents and

conditions: (a) see ref.^[42], 94%; (b) Ac₂O, pyridine, 0 °C to room

temperature, 2 h, 96%; (c) PTFACl, Cs₂CO₃, acetone, room tem-

perature, 2 h, 95%; (d) 9, TMSOTf, see Table 2; (e) H₂NNH₂·H₂O,

AcOH/pyridine, 0 °C to room temperature, 1.5 h, 90%.

Table 2. Conditions for the synthesis of disaccharide 17 from ac-

ceptor 9.

Entry Donor (equiv.) Solvent

T [°C]

Yield of 17 [%]

1

2

3

4

5

14 (1.3)

14 (1.5)

14 (1.2)

15 (1.5)

16 (1.2)

CH₂Cl₂–40 Ǟ room

temperature

CH₂Cl₂–40 Ǟ room

temperature

toluene –10 Ǟ room

temperature

CH₂Cl₂–10 Ǟ room

temperature

70

84

94

89

96

toluene –10 Ǟ room

temperature

The formation of the C–D glycosidic linkage was in- 4.0 equiv., the oxidative unmasking of OH-3_Cwas ac-

spired by the above results. Hence, the 3-O-PMB analogs celerated, and only alcohol 28 was formed. Under the best

of donors 14 and 16 – rhamnosyl TCA 23 and PTFA 24, conditions, CAN-mediated removal of the 3_C-PMB ether,

respectively – were examined as precursors to residue C. and subsequent acetylation of the crude intermediate gave

They were prepared in three steps from allyl 4-O-benzyl-3- 3_C-O-acetyl trisaccharide 29 in a rewarding 88% yield. This

O-p-methoxybenzyl-α-l-rhamnoside^[45](20) (Scheme 3). trisaccharide was subjected to conventional hydrazinolysis

Thus, alcohol 20 was treated with levulinic acid in the pres- of the levulinoyl ester at O-2_C. Acetyl migration to the vici-

ence of DCC (N,NЈ-dicyclohexylcarbodiimide) and DMAP nal hydroxy group could not be avoided, and a 10:1 mixture

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O-Antigen from a Shigella flexneri Serotype 6

of the OH-2_Cand OH-3_Cregiosiomers – 30 (NMR spectro- chain terminator, thus limiting the number of protecting

scopic data for 3_C-H: δ = 4.96 ppm), and 31 (NMR spectro- group manipulations, was preferred. In view of our work

scopic data for 2_C-H: δ = 5.04 ppm), respectively – was iso- involving β-linked N-acetylglucosamine residues,^[49]a tri-

lated (93%); Pd/C/hydrogen-mediated final deprotection of chloroacetamide moiety was chosen to act as a masked

the mixture of the two alcohols in THF/H₂O gave the corre- acetamido functionality and ensure the required 1,2-trans

sponding mono-O-acetylated trisaccharides, _3AcCDA-Pr (3, stereoselectivity in the glycosylation reactions. It was also

78%) and _2AcCDA-Pr (33, 2%; O-acetylated at 2_C). The 4_C- hypothesized that Pd/C-mediated hydrodechlorination of

O-acetyl isomer was not detected. This outcome suggests the trichloroacetamide functionality at the final stage of the

that acetyl migration did not occur under the neutral condi- synthesis would permit full recovery of the acetamido moi-

tions used for hydrogenolysis.

ety without perturbation of the 3_C-acetate.^[42]Since the

known 3,4,6-tri-O-acetyl-2-deoxy-2-trichloroacetamido-d-

galactopyranosyl trichloroacetimidate^[50]did not meet or-

thogonality criteria, we turned to perbenzylated analogs in

order to (i) facilitate glycosylation by using an armed do-

nor, (ii) avoid the remote α-stereodirecting effect attributed

to esterification at O-4 of galactose and galactosamine do-

nors,^[51]and (iii) minimize the number of final deprotection

steps. As for the construction of the C–D and D–A link-

ages, donors activated in the form of TCA^[52](40) or PTFA

(41) were evaluated as precursors to residue B. Towards this

aim, β-pyranose tetraacetate 35 was prepared in four steps

as described^[50]in 43% overall yield on a multigram scale

from galactosamine hydrochloride 34. It was adequately

transformed into β-allyl glycoside 36 (92%) when treated

with allyl alcohol and stoichiometric TMSOTf in CH₂Cl₂

(Scheme 4). Transesterification gave triol 37, and subse-

quent selective O-benzylation gave protected intermediate

38 (86%). Anomeric allyl cleavage proceeded smoothly to

give key hemiacetal 39 in high yield (93%). This hemiacetal

was thus readily accessible (74%) in four steps from pure β-

tetraacetate 35. Treatment of hemiacetal 39 under standard

conditions gave either known TCA donor 40 (94%) or its

PTFA equivalent 41 (88%). The identification of oxazoline

42 (5%) by NMR spectroscopy could explain the lower iso-

lated yield in the latter case.

Scheme 3. Synthesis of C donors 23 and 24, and of CDA trisac-

charides 2 and 3. Reagents and conditions: (a) LevOH, DCC,

DMAP, CH₂Cl₂, room temperature, 2 h, quantitative;

–

(b) (i) [Ir(COD){PCH₃(C₆H₅)₂}₂]⁺PF₆(COD = 1,5-cyclooc-

tadiene), H₂, THF, room temperature, 2 h; (ii) I₂, THF/H₂O, room

temperature, 1 h, 90 %; (c) CCl₃CN, DBU, DCE (1,2-dichloro-

ethane), room temperature, 20 min, 97 %; (d) PTFACl, Cs₂CO₃,

acetone, room temperature, 4 h, 98%; (e) 18, TMSOTf, –10 °C, see

Table 3; (f) H₂NNH₂·H₂O, AcOH/pyridine, 0 °C to room tempera-

ture, 30 min, 93%; (g) DDQ, CH₂Cl₂/H₂O, room temperature, 3 h,

45 %; or CAN, MeCN/H₂O, room temperature, 1.5 h, 71 %;

(h) CAN, MeCN/H₂O, room temperature, 30 min; (i) Ac₂O,

DMAP, pyridine, room temperature, 2 h, 88 % over 2 steps;

(j) H₂NNH₂·H₂O, AcOH/pyridine, 0 °C to room temperature,

1.5 h, 93%; (k) Pd/C (10%), H₂, MeOH, room temperature, 24 h,

69% for 2, 5% for 32; (l) Pd/C (10%), H₂, THF/H₂O, room tem-

perature, 20 h, 78% for 3, 2% for 33.

With the two galactosaminyl donors in hand, we set out

to explore the best conditions to form tetrasaccharide 50

from the above-mentioned trisaccharide alcohol (i.e., 27;

Scheme 5). Glycosylation of acceptor 27 with trichloroacet-

imidate 40 was found to be problematic. When the reaction

was run at –10 °C in toluene containing catalytic TMSOTf

(Table 4, Entry 1), the product of α-glycosylation (i.e., 51),

identified by mass spectrometry analysis and NMR spectro-

1

scopic data for C-1_B(δ = 96.0 ppm, J_CH= 175.2 Hz), was

isolated in a significant 15% yield, in addition to the re-

quired β-linked tetrasaccharide (i.e., 50; NMR spectro-

scopic data for C-1_B: δ = 100.7 ppm, J_CH= 162.4 Hz). Al-

Table 3. Conditions for the synthesis of trisaccharide 26.

1

Entry

Donor (equiv.)

Solvent

Yield of 26 [%]

though the β-glycoside was the major product according to

TLC analysis, it co-eluted with the glycosylamide by-prod-

uct 53, as suggested by mass spectrometry data, and could

not be isolated as a pure material. Mass spectrometry

analysis also indicated the formation of the silylated ac-

ceptor (i.e., 52) under these conditions. Lowering the tem-

perature to –78 °C improved the selectivity remarkably,

since only traces of the unwanted α-linked glycosylation

product remained according to TLC observation. However,

1

2

3

4

23 (1.5)

23 (1.2)

24 (1.2)

DCE

84

91

92

89

toluene

Synthesis of Tetrasaccharides BCDA-Pr (4) and B_3AcCDA-

Pr (5)

Since chain elongation at residue B was not planned, an the reactivity of the acceptor dropped tremendously.

N-acetyl-d-galactosamine precursor that would act as a Changing toluene for CH₂Cl₂and TMSOTf for

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L. A. Mulard et al.

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Scheme 4. Synthesis of B donors 40 and 41. Reagents and conditions: (a) see ref.^[50], 43% over 4 steps; (b) AllOH, TMSOTf, CH₂Cl₂,

room temperature, 14 h, 92%; (c) NaOMe, MeOH, room temperature, 2 h; (d) NaH, BnBr, DMF, –10 °C to 0 °C, 1.5 h, 86% for 38, 77%

for 48 over 2 steps; (e) (i) [Ir(COD){PCH₃(C₆H₅)₂}₂]⁺PF₆, H₂, THF, room temperature, 2 h; (ii) I₂, THF/H₂O, room temperature, 1 h,

–

93%; (f) CCl₃CN, DBU, DCE, room temperature, 1 h, 94%; (g) PTFACl, Cs₂CO₃, acetone, room temperature, 4 h, 88%; (h) PhSH,

BF₃·OEt₂, CH₂Cl₂, room temperature, 2.5 h, 89%; (i) (CCl₃CO)₂O, NaOMe, MeOH, 0 °C, 2 h; (j) Ac₂O, pyridine, 0 °C to room tempera-

ture, 16 h; (k) PhSH, BF₃·OEt₂, CH₂Cl₂, room temperature, 16 h, 64% over 3 steps; (l) NIS/TfOH (NIS = N-iodosuccinimide), CH₂Cl₂/

H₂O, 0 °C, 30 min, 77%.

Scheme 5. Synthesis of tetrasaccharides 4 and 5. Reagents and conditions: (a) see Table 4; (b) Pd/C (10%), H₂, THF/H₂O, room tempera-

ture, 48 h, 59% for 4, 53% for 5; (c) CAN, MeCN/H₂O, room temperature, 30 min; (d) Ac₂O, DMAP, pyridine, room temperature, 2 h,

87% over 2 steps.

TBDMSOTf (TBDMS = tert-butyldimethylsilyl) gave sim- Table 4. Conditions for the synthesis of tetrasaccharide 50 from

acceptor 27.

ilar results, whereas when BF₃·OEt₂was used as a promoter

in CH₂Cl₂, the conversion was slow, and degradation oc-

curred. When the best conditions identified for TCA 40

were used with PTFA donor 41, rearrangement of the do-

nor was not observed, and the coupling reaction was signifi-

cantly improved (Table 4, Entry 2). The fully protected

BCDA tetrasaccharide 50 was obtained in 63% yield. How-

ever, some unreacted acceptor remained, despite the use of

1.5 equiv. of donor, and the selectivity was only moderate,

since α isomer 51 was also formed (9%).

Entry Donor

(equiv.)

Promotor

Solvent

T [°C]

50/51

[%]

1

2

3

4

40 (1.2)

41 (1.5)

TMSOTf

toluene

CH₂Cl₂

–10

–78

n.d.^[a]/15

63/9

75/–

48 (1.2) NIS/TMSOTf CH₂Cl₂–70 Ǟ –55

48 (1.5) NIS/TMSOTf CH₂Cl₂–70 Ǟ –60

80/–

[a] n.d. = not determined.

glycoside^[53]46 (89%). Zemplén deacetylation of this com-

In an attempt to overcome the poor outcome of the imid- pound and subsequent benzylation of the resulting triol^[54]

ate-based [B + CDA] glycosylation, thiophenyl β-glycoside (i.e., 47) under controlled conditions gave the expected tri-

48 – prepared from a peracylated precursor (Scheme 4) – benzyl analog (i.e., 48; 77%). In addition, NIS/TfOH-medi-

was investigated as an alternative donor. Thus, treatment of ated hydrolysis of the thioglycosidic linkage in 48 provided

β-tetraacetate 35 with thiophenol and BF₃·OEt₂gave thio- another access to hemiacetal 39 (77%). Interestingly, under

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O-Antigen from a Shigella flexneri Serotype 6

the conditions used (CH₂Cl₂/H₂O), α-(1↔1)-β-linked disac- cluding its O-acetylation pattern, is consistent with pub-

charide 49 (NMR spectroscopic data for 1-H and 1Ј-H: δ = lished data available at the start of this study. In contrast,

1

5.30 ppm, J_1,2= 8.8 Hz, and δ = 5.44 ppm, J_1,2

=

the extent of the non-O-acetylated population was unexpec-

3.8 Hz) – arising from the glycosylation of hemiacetal 39 ted. It suggests that the S. flexneri strain MDC 2924-71 be-

with thioglycoside 48 – was also isolated, albeit in a mini- longs to type 6 and not to type 6a.^[15]This distinction was

mal amount (3%). Even though this hydrolysis step may be recently introduced to differentiate between SF6 strains di-

improved, this route to hemiacetal 39 was found to be less verging in terms of their O-acetylation ratios.^[15]As a direct

efficient than that involving the allyl glycoside intermediate consequence, the SF6 O-Ag used in this study resembles

(53% compared with 74% over four steps starting from tet- that of E. coli O147 more than anticipated. Whether this

raacetate 35). The preparation of thioglycoside 46 from the O-acetylation pattern has any influence on pathogenicity

crude material resulting from the two-step N-trichloroacet- and/or antigenicity remains to be established. To help estab-

ylation/per-O-acetylation of d-galactosamine hydrochloride lish this, di- to tetrasaccharides bearing a residue A at the

34 was attempted so as to reduce the number of synthetic reducing end were synthesized starting from benzyl (allyl

steps (Scheme 4). Thus, a mixture of α/β-furanose (43/44) 2,3-di-O-benzyl-β-d-galactopyranosid)uronate (9) by step-

and α/β-pyranose isomers (45/35), in a ratio of 7%:11% and wise chain extension with monosaccharide donors. All the

51%:31%, respectively, was treated with thiophenol and glycosylation steps were optimized to reach 80% yield for

BF₃·OEt₂. Thioglycoside 46 was isolated in a satisfactory the formation of the B–C linkage, and 92 and 96% yields

64% yield over three steps. Undoubtedly, this route is better for the formation of the D–A and C–D linkages, respec-

than the previous one (five steps, 38%).

tively. While thioglycoside 48, used in CH₂Cl₂, was the pre-

Glycosylation of trisaccharide alcohol 27 with thioglyco- ferred precursor to residue B (80%), the use of imidate do-

side donor 48 was attempted next (Scheme 5). In contrast nors in toluene gave better results with rhamnosyl donors

to previous observations involving imidate donors 40 and representing residues C and D. Newly described PTFA do-

41, when thioglycoside 48 was used in combination with the nors 16 and 24 were used advantageously to facilitate puri-

NIS/TMSOTf activator system at –70 Ǟ –55 °C in CH₂Cl₂, fication, without adversely affecting the yield of glycosyl-

the pure β-glycosylation product 50 was isolated in a grati- ation products 17 and 26. The synthetic strategy was de-

fying 75% yield (Table 4, Entry 3). The use of a larger ex- signed such that O-acetylation at 3_Cwould be performed at

cess of donor 48 (1.5 vs. 1.2 equiv., Table 4, Entry 4) re- an advanced stage, so providing easy access to both the 3_C-

sulted in a slight increase in yield (80 vs. 75%) while main- OH and 3_C-OAc tri- (2 and 3) and tetrasaccharides (4 and

taining an excellent β selectivity. To our satisfaction, the 5), respectively.

product of α-glycosylation (51) was not detected under

these conditions, while the formation of traces of the sil- 5 indicates that a backbone conformational change may al-

ylated acceptor (i.e., 52) was occasionally noticed. ready be present within one RU-long O-Ag fragments, since

Interestingly, ¹H NMR analysis of tetrasaccharides 4 and

As planned, the one-step Pd/C-catalyzed hydrogenolysis the signal of the anomeric proton of the amino sugar in the

of the benzyl protecting groups, allyl reduction, and con- structural element β-d-GalpNAc-(1Ǟ2)-α-l-Rhap (BC) is

comitant hydrodechlorination of the 2_B-trichloroacetamide shifted upfield by 0.25 ppm upon O-acetylation at O-3 of

moiety in glycosylation product 50 was best performed in the vicinal rhamnosyl residue, consistent with the upfield

THF/H₂O to give the target BCDA propyl glycoside 4 in chemical shift of Ͼ 0.2 ppm observed for the signal of 1_D-

59% yield after RP-HPLC purification. Alternatively, oxi- H in the O-Ag of SF6 strain MDC 2924-71 (vide infra).

dative cleavage of the PMB ether in tetrasaccharide 50 and This contrasts with the behavior of several other S. flexneri

subsequent acetylation of the released hydroxy group pro- O-Ags in which the conformation of the polymer backbone

vided the 3_C-O-acetyl analog 54 (87% over two steps). A was essentially independent of the substituents.^[55]

solution of the latter tetrasaccharide in THF/H₂O was simi-

larly treated with Pd/C under hydrogen to give the

Experimental Section

B

_3AcCDA-Pr tetrasaccharide 5 in 53% yield after RP-

HPLC purification. In this case, the regioisomer resulting

from acetyl migration onto the vicinal hydroxy group was

not isolated.

Bacteria and Cultivation: For the selection of invasive bacteria, the

SF6 strain MCDC 2924-71 (Institut Pasteur, Centre National de

Référence des Entérobactéries) was grown on Congo Red agar

plate. A Congo-Red-positive colony was selected and cultured in

Trypticase Soy Broth (TCS) medium for 30 min at 37 °C with shak-

ing. Then, TCS agar Petri dishes (about 200 with a 100 mm dia-

meter) were filled each with 2 mL of the bacterial subculture and

left at 37 °C overnight. Thereafter, bacteria were recovered by using

physiological water (2 mL per Petri dish). The resulting bacterial

solution (about 400 mL) was centrifuged in glass tubes at 7000 rpm

for 10 min, and then the pellet was washed with physiological

water. The bacterial pellet was resuspended in acetone (250 mL)

and centrifuged at 7000 rpm for 10 min. This step was repeated

once. The acetone-treated bacteria were left under the hood over-

Conclusions

SF6 was recently identified as an essential serotype for

inclusion in a broad-coverage Shigella vaccine preparation.

In the search for a synthetic carbohydrate-based SF6 immu-

nogen, we first analyzed the composition of the O-Ag from

strain MDC 2924-71, which was identified as a representa-

tive strain amongst those available at the Institut Pasteur.

The structure of the RU of strain MDC 2924-71 O-Ag, in- night for drying and mashed to obtain about 5 g of powder.

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4093

L. A. Mulard et al.

FULL PAPER

Preparation of LPS Material: To obtain the LPS crude extract,

powdered bacteria (5 g) were resuspended in glass tubes containing

distilled water (90 mL) heated to 68 °C. A 68 °C-heated solution of

phenol (90% in distilled water; 90 mL) was added, and the suspen-

sion was incubated for 30 min at 68 °C (water bath) with shaking

time to time. After being kept on ice for 10 min, the solution was

centrifuged at 7000 rpm at 4 °C for 45 min. The aqueous upper

layer was recovered and kept on ice. The phenol treatment was

repeated once. The aqueous phases recovered from the two centri-

fugations were combined (about 150 mL in total), and dialyzed

against tap water for 3 d. The dialyzed solution kept in the dialysis

tubing was then concentrated six times by using Aquacide III (Cal-

biochem). The concentrated solution was recovered from the dialy-

sis tubing, dialyzed in distilled water overnight, and then centri-

fuged at 5000 rpm for 15 min. The supernatant was freeze-dried to

give the crude LPS extract (1.5–2 g). To purify the LPS, the freeze-

dried crude extract was suspended at a concentration of 3% (w/v)

in sterile distilled water and centrifuged at 25400 rpm (Beckman

SW41 rotor, about 80000 g) at 4 °C for 7 h. The pellet was resus-

pended in distilled water and centrifuged at 29100 rpm for 3 h. This

step was repeated once. The pellet was then resuspended in distilled

water and centrifuged at 3000 rpm for 10 min. The supernatant,

which contained the purified LPS, was freeze-dried to give a white

powder (70–80 mg). The purified LPS was tested in SDS-PAGE

followed by silver staining.

squared sine-bell functions were applied in both dimensions. The

carbonyl resonances were assigned by using a band-selective con-

stant-time ¹H,¹³C-HMBC experiment^[22]over a spectral region of

6.0 ppm in the direct dimension and 9.0 ppm in the indirect dimen-

sion, with 2048ϫ256 data points and 224 scans per t₁increment.

A 60 ms delay for the evolution of long-range couplings, and a

selective ¹³C excitation pulse (Q3 Gaussian cascade) of 2.5 ms cen-

tered at the carbonyl resonances were used. Zero-filling was per-

formed to 4096ϫ2048 points. Prior to Fourier transformation, 90°-

shifted squared sine-bell functions were applied in both dimensions,

1

and the spectrum was processed in magnitude mode. A 2D H,¹H-

NOESY experiment with a zero-quantum suppression filter^[60]was

recorded over a spectral width of 6.0 ppm, with 2048ϫ512 data

points and 16 scans per t₁increment. A mixing time of 150 ms

was used. A 40 kHz broad and 20 ms long adiabatically smoothed

CHIRP pulse was used during the zero-quantum suppression, ac-

companied by a gradient pulse with a strength of 4% of the maxi-

mum. Prior to Fourier transformation, zero-filling was performed

to 4096ϫ2048 points, and a 90°-shifted squared sine-bell function

was applied in both dimensions. DPFGSE CSSF-NOESY experi-

ments^[24]with two zero-quantum suppression filters were per-

formed to selectively excite the protons of the N-acetyl and O-

acetyl methyl groups. The spectra were acquired with 43008 data

points, 256 scans per increment by using a mixing time of 150 ms

and a total recycle time of 5 s. A 39 Hz broad and 22.5 ms long

Gaussian 180° pulse with truncation at 1% of the total height was

used for selective excitation. WURST inversion pulses were used

for the adiabatic sweeps in the zero-quantum filters, accompanied

by a gradient pulse of 3.2% of the maximum. The increment Δ was

2 ms in all the cases, and t_maxwas 36 ms for the signal at δ =

2.207 ppm and 16 ms for the signals at δ = 2.086 and 2.042 ppm.

Zero-filling to 65k data points and an exponential weighting func-

tion by using a line-broadening factor of 3 Hz were applied prior

Preparation of Lipid-Free Polysaccharide: Lipid-free polysaccharide

(PS) was obtained by subjecting the LPS (30 mg) to weak acidic

hydrolysis in AcOH (1%; pH = 3; 6 mL) at 100 °C for 90 min.^[56]

The lipid A was removed by centrifugation at 15000 g at 4 °C for

20 min. The clear supernatant was cooled to 0 °C and stirred, while

NaOH (1 m) was added very slowly until pH = 5. The solution was

dialyzed against distilled water for 3 d, then it was purified by size

exclusion chromatography on a HiLoad^TM16/60 Superdex^TM30

column (GE healthcare) using an ÄKTA^TMpurifier system (GE

healthcare).

to Fourier transformation. Additionally, spectra for the ¹H and ¹³

C

NMR chemical shift assignment of the PS from E. coli O147^[16]

were recorded in D₂O (5.5 mg in 0.55 mL) at pD = 5 and 19 °C

with a Bruker Avance 500 MHz spectrometer equipped with a

5 mm Z-Gradient (53.0 Gcm^–1) TCI (¹H/¹³C/¹⁵N) CryoProbe and

with a Bruker Avance III 600 MHz spectrometer equipped with a

5-mm inverse Z-gradient (55.7 Gcm^–1) TXI (¹H/¹³C/¹⁵N) probe.

The assignments were performed by using 1D and 2D NMR ex-

NMR Experiments: Spectra for the ¹H and ¹³C NMR chemical

shift assignment of the SF6 PS were recorded in D₂O (1 mg in

0.18 mL, 3 mm NMR tube) at pD = 6 and 17 °C by using a Bruker

Avance III 700 MHz spectrometer equipped with a 5 mm Z-Gradi-

ent (53.0 Gcm^–1) TCI (¹H/¹³C/¹⁵N) CryoProbe. Chemical shifts are

reported in ppm relative to external sodium 3-trimethylsilyl-

(2,2,3,3-²H₄)propanoate (TSP, δ_H= 0.00 ppm) or 1,4-dioxane in

1

periments such as H, ¹³C, H,¹³C-HSQC, H,¹H-TOCSY (t_mix

=

40 and 120 ms) and ¹H,¹³C-BS-CT-HMBC. Chemical shifts were

referenced relative to TSP and 1,4-dioxane as described above.

1

D₂O (δ_C= 67.40 ppm). H NMR spectra were recorded with 34k

data points over a spectral width of 12 ppm, with 128 scans and a

repetition time of 12.0 s. Zero-filling to 128k data points and an

exponential weighting function using a line-broadening factor of

Chemical Synthesis: Anhydrous solvents – including toluene (Tol),

dichloromethane, 1,2-dichloroethane (DCE), tetrahydrofuran

(THF), N,N-dimethylformamide (DMF), methanol (MeOH), and

pyridine – were supplied over molecular sieves, and were used as

received. Other solvents referred to in the text are abbreviated as

Chex (cyclohexane), EtOAc (ethyl acetate), and MeCN (acetoni-

trile), in addition to acetone. Reactions requiring anhydrous condi-

1

0.3 Hz were applied prior to Fourier transformation. H chemical

1

shift assignments were performed by using H,¹H-TOCSY experi-

ments^[57]recorded over 6.0 ppm with 2048ϫ256 data points and 8

scans per t₁increment, using the States-TPPI method. An MLEV-

17 spin-lock of 10 kHz and four different mixing times (20, 40, 80,

and 120 ms) were used. Zero-filling was performed to 4096ϫ1024 tions were run under argon (Ar) by using dried glassware; 4 Å mo-

points. Prior to Fourier transformation, 90°-shifted squared sine-

bell functions were applied in both dimensions. ¹³C chemical shifts

were assigned by using multiplicity-edited ¹H,¹³C-HSQC experi-

ments.^[58]The experiments were recorded with 1024ϫ512 data

points and 16 scans per t₁increment over a spectral region of

6.0 ppm for ¹H and 110 ppm for ¹³C by using the echo/antiecho

method. Adiabatic pulses^[59]were used for ¹³C inversion (smoothed

CHIRP, 20%, 80 kHz, 500 μs, Q = 5.0) and refocusing (composite

smoothed CHIRP, 80 kHz, 2.0 ms). Prior to Fourier transforma-

tion, forward linear prediction to 1024 points in the F₁dimension

and zero-filling to 4096ϫ4096 points were performed; 90°-shifted

lecular sieves (4 Å MS) and 4 Å acid-washed molecular sieves (4 Å

AW 300 MS) were activated before use by heating under high vac-

uum. Analytical thin-layer chromatography (TLC) was performed

with silica gel 60 F₂₅₄0.25 mm pre-coated aluminium foil plates.

Compounds were visualized by using UV₂₅₄and/or orcinol

(1 mgmL^–1) in H₂SO₄(10% aq.) with charring. Flash column

chromatography was carried out by using silica gel (Merck, particle

size 40–63 μm, unless otherwise indicated). Reverse-phase (RP)

HPLC was carried out by using a Kromasil 5 μm C18 100 Å

10ϫ250 mm semi-preparative column, eluting with a 0–20% linear

gradient of MeCN in TFA (0.08% aq.) over 20 min at a flow rate

4094

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Eur. J. Org. Chem. 2013, 4085–4106

O-Antigen from a Shigella flexneri Serotype 6

of 5.5 mLmin^–1. Analytical RP-HPLC analysis of the final com-

benzyl bromide (2.97 mL, 25.0 mmol, 2.0 equiv.) and KHCO₃

(4.75 g, 47.5 mmol, 4.0 equiv.) were added. The reaction mixture

was stirred under Ar for 16 h. After that time, TLC (Chex/EtOAc,

7:3) showed the conversion of the intermediate acid into a major

less polar product. The reaction was quenched by addition of

MeOH, and the volatiles were evaporated. The residue was dis-

solved in EtOAc, the resulting solution was washed with water and

brine, dried with anhydrous Na₂SO₄, and filtered, and the filtrate

was concentrated. The residue was purified by flash chromatog-

raphy (Chex/EtOAc, 9:1 to 6:4) to give uronate 9 (4.69 g, 74%) as

a white solid. The analytical data were as published.^[34]

pounds (λ

= 215 nm) used a Symmetry 3.5 μm C₁₈100 Å

2.1ϫ100 mm analytical column eluting with a 0–35% linear gradi-

ent of MeCN in TFA (0.01 n aq.) over 20 min at a flow rate of

0.3 mLmin^–1. Nuclear magnetic resonance (NMR) spectra were re-

corded at 303 K by using a Bruker Avance spectrometer equipped

with a BBO probe at 400 MHz (¹H) and 100 MHz (¹³C). Spectra

were recorded in deuterated chloroform (CDCl₃), dimethyl sulfox-

ide ([D₆]DMSO), and water (D₂O). Elucidations of chemical struc-

tures were based on ¹H, COSY, TOCSY, DEPT-135, decoupled

DEPT-90, HSQC, decoupled HSQC, ¹³C, decoupled ¹³C, and

HMBC spectra. Signals are reported as s (singlet), d (doublet), t

(triplet), dd (doublet of doublets), q (quadruplet), sext (sextuplet),

dt (doublet of triplets), dq (doublet of quadruplets), ddd (doublet

of doublet of doublets), and m (multiplet). The signals can also be

described as broad (prefix b), pseudo (prefix p), overlapped (suffix

_o), or partially overlapped (suffix _po). Chemical shifts are reported

in ppm (δ) relative to the residual solvent peak [CHCl₃, DMSO,

and DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) at δ = 7.28/

77.0, 2.50/39.5, and 0.00/0.00 ppm for the ¹H and ¹³C spectra,

respectively], and coupling constants are given in Hertz (Hz). Of

the two magnetically non-equivalent geminal protons at C-6, the

one resonating at lower field is denoted 6a-H, and the one at higher

field is denoted 6b-H. Interchangeable assignments are marked

with an asterisk. Sugar residues are labelled according to the label-

ling of the RU of the SF6 O-Ag and identified by a subscript in

the listing of signal assignments. LC-MS and HRMS spectra were

recorded in the positive-ion electrospray ionisation (ESI⁺) mode

with a Waters Q-TOFmicro mass spectrometer coupled to an Al-

liance HPLC. Solutions were prepared in 1:1 MeCN/H₂O contain-

ing 0.1% formic acid.

Benzyl (2,3,4-Tri-O-acetyl-α-

L-rhamnopyranosyl)-(1Ǟ4)-(allyl 2,3-

di-O-benzyl-β- -galactopyrosid)uronate (11): A suspension of ac-

D

ceptor 9 (1.00 g, 1.98 mmol), donor^[39]10 (1.12 g, 2.58 mmol,

1.3 equiv.), and freshly activated 4 Å AW 300 MS (1.5 g) in anhy-

drous CH₂Cl₂(19.8 mL) was stirred at room temperature under Ar

for 1 h. The reaction mixture was cooled to –40 °C and stirred for

15 min, and then TMSOTf (18 μL, 198 μmol, 0.05 equiv.) was

added. Stirring was continued at that temperature for 1 h, and the

bath temperature was allowed to reach room temperature. TLC

(Chex/EtOAc, 7:3) showed the conversion of acceptor 9 into a less

polar product. The reaction was quenched with Et₃N, filtered, and

concentrated. The residue was purified by flash chromatography

(Chex/EtOAc, 8:2 to 6:4) to give disaccharide 11 (1.41 g, 91%) as

1

a white foam. R_f= 0.16 (Chex/EtOAc, 7:3). H NMR (400 MHz,

CDCl₃): δ = 7.44–7.24 (m, 15 H, H_Ar), 5.98 (m, 1 H, CH=_All), 5.52

(dd, J_1,2= 1.8 Hz, J_2,3= 3.3 Hz, 1 H, 2_D-H), 5.41–5.32 (m, 2 H,

3_D-H, =CH_2All), 5.32 (d, J = 12.2 Hz, 1 H, H_CO2Bn), 5.22 (m, J_cis

= 10.5, J_gem= 1.3 Hz, 1 H, =CH_2All), 5.16 (d, 1 H, H_CO2Bn), 5.04

(d, 1 H, 1_D-H), 5.03 (dd, J_3,4= 10.0, J_4,5= 9.8 Hz, 1 H, 4_D-H),

4.98 (d, J = 10.8 Hz, 1 H, H_Bn), 4.80 (d, J = 10.8 Hz, 1 H, H_Bn),

4.78 (d, J = 11.7 Hz, 1 H, H_Bn), 4.67 (d, J = 11.7 Hz, 1 H, H_Bn),

4.53 (m, 1 H, H_All), 4.42 (d, J_1,2= 7.8 Hz, 1 H, 1_A-H), 4.35 (bd, 1

H, 4_A-H), 4.18 (m, 1 H, H_All), 4.07 (bs, 1 H, 5_A-H), 3.98 (dq, 1 H,

5_D-H), 3.88 (dd, J_2,3= 9.6 Hz, 1 H, 2_A-H), 3.54 (dd, J_3,4= 3.0 Hz,

1 H, 3_A-H), 2.07, 2.06, 1.99 (3 s, 9 H, H_Ac), 1.21 (d, J_5,6= 6.3 Hz,

3 H, 6_D-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 170.3, 169.7

(3 C, CO_Ac), 167.3 (C-6_A), 138.4, 137.9, 135.0 (3 C, C_IVAr), 133.9

Allyl 2,3-Di-O-Benzyl-β-D

-galactopyranoside (7):^[36]NaH (60% in

mineral oil; 4.67 g, 116.8 mmol, 4.0 equiv.) was slowly added to a

solution of diol 6^[35](9.0 g, 29.2 mmol) in anhydrous DMF

(102 mL) stirred at 0 °C. After stirring at this temperature for

30 min, benzyl bromide (13.9 mL, 116.8 mmol, 4.0 equiv.) was

added, keeping the temperature close to 0 °C. The reaction mixture

was stirred for 2 h, while the bath temperature reached ambient

temperature. After this time, TLC (Chex/EtOAc, 6:4) showed the

complete transformation of the starting material into a less polar

product. The reaction was quenched by adding MeOH at 0 °C, and

the volatiles were evaporated. The residue was dissolved in EtOAc,

and the resulting solution was washed with water and brine, dried

with anhydrous Na₂SO₄, filtered and concentrated. The crude ma-

terial was suspended in AcOH (80% aq.; 146 mL) and heated at

80 °C for 2 h, after which time, TLC (Chex/EtOAc, 1:1) showed the

conversion of the starting material to a major more polar product.

The volatiles were evaporated and co-evaporated twice with tolu-

ene. The residue was purified by flash chromatography (Chex/

EtOAc, 1:1 to 3:7) to give diol 7 (10.42 g, 89%) as a white solid.

The analytical data were as published.^[36]

(CH=_All), 128.7–127.7 (15 C, C_Ar), 117.6 (=CH_2All), 102.6 (¹J_CH

=

154.0 Hz, C-1_A), 99.7 (¹J_CH= 170.4 Hz, C-1_D), 80.4 (C-3_A), 78.8

(C-2_A), 77.2 (C-4_A), 75.6 (C_Bn), 73.5 (C_Bn), 73.4 (C-5_A), 73.0 (C-

4_D), 70.6 (CH_2All), 69.8 (C-2_D), 69.0 (C-3_D), 67.3 (C_CO2Bn), 67.1

(C-5_D), 21.0, 20.9, 20.8 (3 C, CH_3Ac), 17.6 (C-6_D) ppm. HRMS

(ESI⁺): calcd. for C₄₂H₄₉O₁₄[M + H]⁺777.3123; found 777.3004;

calcd. for C₄₂H₄₈O₁₄Na [M + Na]⁺799.2942; found 799.2759.

Propyl

α-L-Rhamnopyranosyl-(1Ǟ4)-β-D-galactopyranosiduronic

Acid (1): Pd/C (10%; 150 mg) was added to a stirred solution of

disaccharide 11 (218 mg, 0.28 mmol) in EtOAc (14 mL). The sus-

pension was stirred under hydrogen for 16 h. After this time, MS

analysis of the crude reaction mixture indicated the presence of a

single compound, with a molecular weight corresponding to that

of intermediate uronic acid 12. The reaction mixture was filtered,

and the solvents were evaporated to dryness. The residue was dis-

solved in THF/H₂O (1:1, 8.4 mL), and treated with NaOH (1 m

aq.; 4.21 mL, 4.21 mmol, 15 equiv.) for 24 h. The reaction was

quenched with Dowex-H⁺resin, and the suspension was filtered.

Evaporation of the volatiles, freeze drying, and purification of the

crude material by preparative RP-HPLC gave disaccharide 1

(55.1 mg, 51% over two steps) as a white solid following extensive

freeze-drying. t_R= 7.6 min. ¹H NMR (400 MHz, D₂O): δ = 5.06

(d, J_1,2= 1.7 Hz, 1 H, 1_D-H), 4.37 (d, J_1,2= 7.9 Hz, 1 H, 1_A-H),

4.33 (d, J_4,5= 1.2 Hz, 1 H, 5_A-H), 4.28 (dd, J_3,4= 3.0 Hz, 1 H, 4_A-

H), 4.01 (dd, J_2,3= 3.3 Hz, 1 H, 2_D-H), 3.82 (dt_po, J = 6.9, J =

Benzyl (Allyl 2,3-Di-O-benzyl-β-D

-galactopyranosid)uronate^[34](9):

TEMPO (390 mg, 2.50 mmol, 0.2 equiv.) and BAIB (10.05 g,

31.2 mmol, 2.5 equiv.) were added to a vigorously stirred solution

of diol 7 (5.00 g, 12.49 mmol) in CH₂Cl₂/H₂O (2:1, 62 mL) at room

temperature. After 1 h, TLC (CH₂Cl₂/MeOH, 9:1) showed the con-

version of the starting material into the more polar acid 8.^[37]The

reaction was quenched by the addition of NaHSO₃(10% aq.), the

mixture was diluted with CH₂Cl₂, and the phases were separated.

The aqueous phase was acidified with HCl (10% aq.) and re-ex-

tracted twice with CH₂Cl₂. The combined organic extracts were

washed with brine, filtered through a phase separator, and concen-

trated. Crude acid 8 was dissolved in dry DMF (240 mL), then

Eur. J. Org. Chem. 2013, 4085–4106

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4095

L. A. Mulard et al.

9.9 Hz, 1 H, OCH_2Pr), 3.78 (dd_po, J_2,3= 10.0 Hz, 1 H, 3_A-H), 3.69 1 H, 4-H), 2.81–2.69 (m, 4 H, CH_2Lev), 2.19 (s, 3 H, CH_3Lev), 1.34

FULL PAPER

(dd, J_3,4= 9.8 Hz, 1 H, 3_D-H), 3.57–3.49 (m, 2 H, 5_D-H, OCH_2Pr),

3.49 (dd, 1 H, 2_A-H), 3.31 (pt, J_4,5= 9.7 Hz, 1 H, 4_D-H), 1.56 (sext,

(d, 3 H, 6-H) ppm. ¹³C NMR (100 MHz, CDCl₃, partial): δ = 206.1

(CO_Lev), 171.6 (CO_2Lev), 143.3, 138.1, 137.6 (3 C, C_IVAr), 128.8–

127.9 (13 C, C_Ar), 119.4 (2 C, C_Ar), 94.0 (C-1), 79.2 (C-4), 77.3 (C-

3), 75.7, 72.1 (2 C, C_Bn), 70.4 (C-5), 67.7 (C-2), 38.0 (COCH_2Lev),

29.9 (CH_3Lev), 28.0 (CO₂CH_2Lev), 18.0 (C-6) ppm.

2 H, CH_2Pr), 1.16 (d, J_5,6= 6.2 Hz, 3 H, 6_D-H), 0.84 (t, 3 H, CH_3Pr

)

ppm. ¹³C NMR (100 MHz, D₂O): δ = 171.5 (C-6_A), 102.4 (¹J_CH

=

162.7 Hz, C-1_A), 101.4 (¹J_CH= 172.1 Hz, C-1_D), 76.6 (C-4_A), 73.0

(C-3_A), 72.8 (C-5_A), 72.3 (OCH_2Pr), 71.8 (C-4_D), 70.2 (C-2_D), 70.1

(C-2_A), 69.9 (C-3_D) 69.1 (C-5_D), 22.1 (CH_2Pr), 16.5 (C-6_D), 9.6

(CH_3Pr) ppm. HRMS (ESI⁺): calcd. for C₁₅H₂₆O₁₁Na [M + Na]⁺

405.1373; found 405.1366.

Benzyl (3,4-Di-O-benzyl-2-O-levulinoyl-α-

L-rhamnopyranosyl)-

(1Ǟ4)-(allyl 2,3-di-O-benzyl-β- -galactopyranosid)uronate (17)

D

Route 1: A suspension of acceptor 9 (300 mg, 0.60 mmol), acetate

-rhamnopyranose 15 (432 mg, 0.89 mmol, 1.5 equiv.), and freshly activated 4 Å AW

(15): Hemiacetal 13^[42](3.18 g, 7.19 mmol) and DMAP (88 mg, 300 MS (450 mg) in anhydrous CH₂Cl₂(5.9 mL) was stirred at

719 μmol, 0.1 equiv.) were dissolved in dry pyridine under Ar, and room temperature under Ar for 1 h. The reaction mixture was

the solution was cooled to 0 °C. Acetic anhydride (13.6 mL, cooled to –10 °C, and TMSOTf (14 μL, 83 μmol, 0.14 equiv.) was

1-O-Acetyl-3,4-di-O-benzyl-2-O-levulinoyl-α/β-

L

143.7 mmol) was added dropwise, and the reaction mixture was

stirred for 2 h, while the bath reached room temperature. After that

time, TLC (Chex/EtOAc, 6:4) showed the transformation of the

starting material into a mixture of α- and β-anomeric acetates. The

added. The reaction mixture was stirred for 1 h, while the cooling

bath was allowed to reach room temperature. TLC (Tol/acetone,

9:1) showed the conversion of acceptor 9 into a less polar product.

The reaction was quenched by the addition of Et₃N, the solids were

volatiles were evaporated and co-evaporated twice with toluene. filtered, and the filtrate was concentrated. The residue was purified

The residue was purified by flash chromatography (Chex/EtOAc,

7:3 to 6:4) to give a 4.5:1 α/β mixture of acetate 15 (3.33 g, 96%)

as a yellow oil. Data for the α isomer: R_f= 0.26 (Chex/EtOAc, 6:4).

¹H NMR (400 MHz, CDCl₃): δ = 7.39–7.29 (m, 10 H, H_Ar), 6.02

(d, J_1,2= 1.9 Hz, 1 H, 1-H), 5.36 (dd, J_2,3= 3.4 Hz, 1 H, 2-H), 4.94

(d, J = 10.9 Hz, 1 H, H_Bn), 4.72 (d, J = 11.2 Hz, 1 H, H_Bn), 4.66

(d, 1 H, H_Bn), 4.56 (d, 1 H, H_Bn), 3.94 (dd, J_3,4= 9.4 Hz, 1 H, 3-

H), 3.82 (dq, J_4,5= 9.5, J_5,6= 6.2 Hz, 1 H, 5-H), 3.48 (pt, 1 H, 4-

H), 2.84–2.69 (m, 4 H, CH_2Lev), 2.19 (s, 3 H, CH_3Lev), 2.09 (s, 3 H,

CH_3Ac), 1.35 (d, 3 H, 6-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ

= 205.8 (CO_Lev), 171.7 (CO_2Lev), 168.4 (CO_2Ac), 138.2, 137.8 (2 C,

C_IVAr), 128.4–127.8 (10 C, C_Ar), 91.0 (C-1), 79.5 (C-4), 77.6 (C-3),

75.5, 71.8 (2 C, C_Bn), 70.0 (C-5), 68.1 (C-2), 38.0 (COCH_2Lev), 29.7

(CH_3Lev), 28.0 (CO₂CH_2Lev), 20.8 (CH_3Ac), 18.0 (C-6) ppm. Data

for the β isomer: R_f= 0.35 (Chex/EtOAc, 6:4). ¹H NMR (400 MHz,

CDCl₃): δ = 7.39–7.29 (m, 10 H, H_Ar), 5.74 (d, J_1,2= 0.9 Hz, 1 H,

1-H), 5.61 (dd, J_2,3= 3.3 Hz, 1 H, 2-H), 4.94 (d, J = 10.9 Hz, 1 H,

by flash chromatography (Tol/acetone, 95:5) to give disaccharide

17 (472 mg, 89%) as a colourless oil.

Route 2: A suspension of acceptor 9 (300 mg, 595 μmol), TCA

14^[42](419 mg, 713 μmol, 1.2 equiv.), and freshly activated 4 Å AW

300 MS (450 mg) in anhydrous toluene (5.9 mL) was stirred at

room temperature under Ar for 1 h. The reaction mixture was co-

oled to –10 °C, and TMSOTf (5 μL, 30 μmol, 0.05 equiv.) was

added. The reaction mixture was stirred for 30 min, while the cool-

ing bath was allowed to reach room temperature. TLC (Tol/acet-

one, 9:1) indicated the absence of acceptor 9 and the presence of a

less polar product. The reaction was quenched by the addition of

Et₃N. The resulting mixture was filtered, and the filtrate was con-

centrated. The residue was purified by flash chromatography (Tol/

acetone, 97:3 to 96:4) to give disaccharide 17 (519 mg, 94%) as a

colourless oil.

Route 3: A suspension of acceptor 9 (375 mg, 743 μmol), PTFA 16

(547 mg, 892 μmol, 1.2 equiv.), and freshly activated 4 Å AW 300

MS (560 mg) in anhydrous toluene (7.4 mL) was stirred at room

temperature under Ar for 1 h. The reaction mixture was cooled to

–10 °C, and TMSOTf (5 μL, 30 μmol, 0.05 equiv.) was added. The

reaction mixture was stirred for 30 min, while the cooling bath was

allowed to reach room temperature. TLC (Tol/acetone, 9:1) showed

the absence of acceptor 9 and the presence of a less polar product.

The reaction was quenched by addition of Et₃N. The resulting mix-

ture was filtered, and the filtrate was concentrated. The residue

was purified by flash chromatography (Tol/acetone, 95:5) to give

disaccharide 17 (663 mg, 96%) as a colourless oil. R_f= 0.47 (Tol/

H_Bn), 4.72 (d, J = 11.2 Hz, 1 H, H_Bn), 4.66 (d, 1 H, H_Bn), 4.52 (d,

1 H, H_Bn), 3.71 (dd, J_3,4= 9.0 Hz, 1 H, 3-H), 3.53 (dq, J_4,5= 9.4,

J_5,6= 6.1 Hz, 1 H, 5-H), 3.45 (pt, 1 H, 4-H), 2.84–2.69 (m, 4 H,

CH_2Lev), 2.20 (s, 3 H, CH_3Lev), 2.12 (s, 3 H, CH_3Ac), 1.39 (d, 3 H,

6-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 206.03 (CO_Lev), 172.2

(CO_2Lev), 168.8 (CO_2Ac), 138.2, 137.5 (2 C, C_IVAr), 128.4–127.8 (10

C, C_Ar), 91.2 (C-1), 79.6 (C-3), 79.3 (C-4), 75.4 (C_Bn), 72.8 (C-

5), 71.5 (C_Bn), 67.8 (C-2), 38.0 (COCH_2Lev), 29.7 (CH_3Lev), 28.0

(CO₂CH_2Lev), 20.7 (CH_3Ac), 17.9 (C-6) ppm. HRMS (ESI⁺): calcd.

for C₂₇H₃₂O₈Na [M + Na]⁺507.1995; found 507.1999.

3,4-Di-O-benzyl-2-O-levulinoyl-α/β-L-rhamnopyranosyl N-Phenyltri-

1

fluoroacetimidate (16): Hemiacetal 13 (3.22 g, 7.3 mmol) was dis-

solved in acetone (24.7 mL). N-Phenyltrifluoroacetimidoyl chloride

(3.02 g, 14.6 mmol, 2.0 equiv.) followed by Cs₂CO₃(2.61 g,

8.0 mmol, 1.1 equiv.) were added to the solution. The mixture was

stirred at room temperature for 2 h. After this time, TLC (Chex/

EtOAc, 6:4) showed the transformation of the starting material

into a less polar product. The reaction mixture was filtered and

concentrated. The residue was purified by flash chromatography

(Chex/EtOAc, 75:25 to 65:35 + 1% Et₃N) to give a 4:1 α/β mixture

acetone, 9:1). H NMR (400 MHz, CDCl₃): δ = 7.41–7.23 (m, 25

H, H_Ar), 5.99 (m, 1 H, CH=_All), 5.55 (dd, J_1,2= 2.1, J_2,3= 3.3 Hz,

1 H, 2_D-H), 5.36 (m, J_trans= 17.2, J_gem= 1.5 Hz, 1 H, =CH_2All),

5.28 (d, J = 12.3 Hz, 1 H, H_CO2Bn) 5.24–5.20 (m, 2 H, 1_D-H,

=CH_2All), 5.12 (d, 1 H, H_CO2Bn), 4.94 (d, J = 11.0 Hz, 1 H, H_Bn),

4.91 (d, J = 11.2 Hz, 1 H, H_Bn), 4.79 (d, J = 11.0 Hz, 1 H, H_Bn),

4.77 (d, J = 12.1 Hz, 1 H, H_Bn), 4.74 (d, J = 12.1 Hz, 1 H, H_Bn),

4.64 (d, J = 11.1 Hz, 1 H, H_Bn), 4.61 (d, J = 11.2 Hz, 1 H, H_Bn),

4.50 (m, 1 H, H_All), 4.45 (d, J = 11.0 Hz, 1 H, H_Bn), 4.43–4.39 (m,

of PTFA 16 (4.27 g, 95%) as a yellow oil. Data for the α isomer: 2 H, 4_A-H, 1_A-H), 4.17 (m, 1 H, H_All), 4.05 (bs, 1 H, 5_A-H), 3.88

R_f= 0.50 (Chex/EtOAc, 6:4). ¹H NMR (400 MHz, CDCl₃): δ = (dd, J_3,4= 9.4 Hz, 1 H, 3_D-H), 3.80–3.71 (m, 2 H, 2_A-H, 5_D-H),

7.39–7.26 (m, 12 H, H_Ar), 5.13 (m, 1 H, H_Ar), 6.86–6.82 (m, 2 H,

3.56 (dd, J_2,3= 9.7, J_3,4= 2.9 Hz, 1 H, 3_A-H), 3.37 (pt, J_4,5

=

H_Ar), 6.13 (bs, 1 H, 1-H), 5.48 (dd, J_1,2= 2.0, J_2,3= 3.3 Hz, 1 H,

9.4 Hz, 1 H, 4_D-H), 2.72–2.62 (m, 4 H, CH_2Lev), 2.16 (s, 3 H,

2-H), 4.94 (d, J = 10.8 Hz, 1 H, H_Bn), 4.73 (d, J = 11.1 Hz, 1 H, CH_3Lev), 1.30 (d, J_5,6= 6.2 Hz, 3 H, 6_D-H) ppm. ¹³C NMR

H_Bn), 4.66 (d, 1 H, H_Bn), 4.60 (d, 1 H, H_Bn), 3.99 (dd, J_3,4= 9.3 Hz,

1 H, 3-H), 3.90 (dq, J_4,5= 9.5, J_5,6= 6.2 Hz, 1 H, 5-H), 3.51 (pt,

(100 MHz, CDCl₃): δ = 206.4 (CO_Lev), 171.6 (CO_2Lev), 167.4 (C-

6_A), 138.8, 138.3, 138.2, 137.9, 135.0 (5 C, C_IVAr), 133.9 (CH=_All),

4096

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Eur. J. Org. Chem. 2013, 4085–4106

O-Antigen from a Shigella flexneri Serotype 6

128.6–127.5 (25 C, C_Ar), 117.6 (=CH_2All), 102.6 (¹J_CH= 161.3 Hz,

C-1_A), 98.9 (¹J_CH= 172.7 Hz, C-1_D), 80.8 (C-3_A), 79.7 (C-4_D), 78.3

1 H, 3_A-H), 3.47 (pt, J_4,5= 9.3 Hz, 1 H, 4_D-H), 2.43 (bs, 1 H,

OH), 1.32 (d, J_5,6= 6.2 Hz, 3 H, 6_D-H) ppm. ¹³C NMR (100 MHz,

(C-2_A), 77.8 (C-3_D), 75.3, 75.1 (2 C, C_Bn), 73.7 (C-4_A), 73.6 (C-5_A), CDCl₃): δ = 167.3 (C-6_A), 138.8, 138.4, 138.2, 137.8, 135.1 (5 C,

73.1, 71.7 (2 C, C_Bn), 70.6 (CH_2All), 69.0 (C-2_D), 68.3 (C-5_D), 67.4

(C_CO2Bn), 38.1 (COCH_2Lev), 29.9 (CH_3Lev), 28.2 (CO₂CH_2Lev), 18.1

(C-6_D) ppm. HRMS (ESI⁺): calcd. for C₅₅H₆₀O₁₃Na [M + Na]⁺

951.3932; found 951.3846.

C_IVAr), 134.0 (CH=_All), 128.6–127.5 (25 C, C_Ar), 117.5 (=CH_2All),

102.7 (¹J_CH= 159.4 Hz, C-1_A), 100.4 (¹J_CH= 172.7 Hz, C-1_D), 81.2

(C-3_A), 79.9 (C-4_D), 79.6 (C-3_D), 78.4 (C-2_A), 75.2, 75.0 (2 C, C_Bn),

73.7 (C-5_A), 73.5 (C-4_A), 73.3, 72.2 (2 C, C_Bn), 70.6 (CH_2All), 68.7

(C-2_D), 68.1 (C-5_D), 67.3 (C_CO2Bn), 18.1 (C-6_D) ppm. HRMS

(ESI⁺): calcd. for C₅₀H₅₄O₁₁Na [M + Na]⁺853.3564; found

853.3478.

(3,4-Di-O-benzyl-2-O-levulinoyl-β-L-rhamnopyranosyl)(trichloro-

acetyl)amine (19): A suspension of acceptor 9 (3.00 g, 5.95 mmol),

TCA 14 (4.54 g, 7.73 mmol, 1.3 equiv.), and freshly activated 4 Å

AW 300 MS (4.5 g) in anhydrous CH₂Cl₂(59.5 mL) was stirred at

room temperature under Ar for 1 h. The reaction mixture was co-

oled to –40 °C, and TMSOTf (54 μL, 0.30 mmol, 0.05 equiv.) was

added. The reaction mixture was stirred for 30 min, while the cool-

ing bath was allowed to reach room temperature. TLC (Tol/ace-

tone, 9:1) indicated the absence of acceptor 9 and the presence of

a less polar product. The reaction was quenched by the addition

of Et₃N. The resulting mixture was filtered, and the filtrate was

concentrated. The residue was purified by flash chromatography

(Tol/EtOAc, 95:5 to 9:1) to give, in order of elution, glycosylamide

19 (530 mg) and disaccharide 17, contaminated with trichloroacet-

amide. A second purification by flash chromatography (CH₂Cl₂/

Allyl 4-O-Benzyl-2-O-levulinoyl-3-O-p-methoxybenzyl-α-L-rhamno-

pyranoside (21): Levulinic acid (7.81 mL, 76.2 mmol, 2.0 equiv.),

DCC (15.7 g, 76.2 mmol, 2.0 equiv.), and DMAP (931 mg,

7.6 mmol, 0.2 equiv.) were added to a solution of alcohol 20^[45]

(15.8 g, 38.1 mmol) in anhydrous CH₂Cl₂(150 mL). The solution

was stirred under Ar for 2 h. After that time, TLC (Tol/EtOAc,

9:1) showed the conversion of the starting alcohol into a less polar

product. The reaction mixture was filtered through a pad of Celite.

The filtrate was diluted with CH₂Cl₂, and washed successively with

HCl (10 % aq.), NaHCO₃(satd. aq.), and brine, then passed

through a phase-separator filter, and concentrated. The residue was

purified by flash chromatography (Chex/EtOAc, 8:2 to 7:3) to give

EtOAc) gave pure 17 (3.86 g, 70%) as a colourless oil. Data for 19: 21 (19.5 g, quantitative) as a yellow oil. R_f= 0.24 (Tol/EtOAc, 9:1).

R_f= 0.23 (Tol/EtOAc, 9:1). ¹H NMR (400 MHz, CDCl₃): δ = 7.54 ¹H NMR (400 MHz, CDCl₃): δ = 7.39–7.24 (m, 7 H, H_Ar), 6.88–

(d, J_NH,1= 8.8 Hz, 1 H, NH), 7.39–7.29 (m, 10 H, H_Ar), 5.59 (dd,

6.84 (m, 2 H, H_ArPMB), 5.89 (m, 1 H, CH=_All), 5.39 (dd, J_1,2= 1.8,

J_1,2= 1.3, J_2,3= 3.3 Hz, 1 H, 2-H), 5.32 (dd, 1 H, 1-H), 4.93 (d, J J_2,3= 3.4 Hz, 1 H, 2-H), 5.29 (m, J_trans= 17.2, J_gem= 1.7 Hz, 1 H,

= 11.1 Hz, 1 H, H_Bn), 4.74 (d, J = 11.4 Hz, 1 H, H_Bn), 4.65 (d, 1

H, H_Bn), 4.53 (d, 1 H, H_Bn), 3.74 (dd, J_3,4= 9.1 Hz, 1 H, 3-H),

3.55 (dq, J_4,5= 9.3, J_5,6= 6.2 Hz, 1 H, 5-H), 3.38 (pt, 1 H, 4-H),

=CH_2All), 5.21 (m, J_cis= 10.4 Hz, 1 H, =CH_2All), 4.92 (d, J =

10.9 Hz, 1 H, H_Bn), 4.78 (d, 1 H, 1-H), 4.63 (d, J = 10.8 Hz, 1 H,

H_Bn), 4.62 (d, 1 H, H_Bn), 4.46 (d, 1 H, H_Bn), 4.16 (m, 1 H, H_All),

2.85–2.64 (m, 4 H, CH_2Lev), 2.19 (s, 3 H, CH_3Lev), 1.37 (d, 3 H, 6- 4.02–3.94 (m, 2 H, 3-H, H_All), 3.78 (dq, J_4,5= 9.5, J_5,6= 6.3 Hz, 1

H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 206.8 (CO_Lev), 172.0

(CO_2Lev), 161.5 (NHCO), 138.2, 137.4 (2 C, C_IVAr), 128.4–127.8 (10

C, C_Ar), 92.1 (CCl₃), 80.2 (C-3), 79.2 (C-4), 78.4 (¹J_CH= 154.3 Hz,

C-1), 75.4 (C_Bn), 74.0 (C-5), 71.8 (C_Bn), 68.9 (C-2), 38.2

H, 5-H), 3.81 (s, 3 H, CH_3PMB), 3.40 (pt, 1 H, 4-H), 2.82–2.67 (m,

4 H, CH_2Lev), 2.19 (s, 3 H, CH_3Lev), 1.33 (d, 3 H, 6-H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 206.2 (CO_Lev), 172.1 (CO_2Lev), 159.2

(C_IVArPMB), 138.5 (C_IVAr), 133.6 (CH=_All), 130.3 (C_IVArPMB), 130.0–

(COCH_2Lev), 29.6 (CH_3Lev), 28.2 (CO₂CH_2Lev), 18.1 (C-6) ppm. 127.7 (7 C, C_Ar), 117.5 (=CH_2All), 113.7 (2 C, C_ArPMB), 96.8 (C-1),

HRMS (ESI⁺): calcd. for C₂₇H₃₀Cl₃NO₇Na [M + Na]⁺608.0986;

found 608.0972.

80.0 (C-4), 79.7 (C-3), 75.4 (C_Bn), 71.3 (C_PMB), 69.2 (C-2), 68.0

(C_All), 67.7 (C-5), 55.1 (CH_3PMB), 38.0 (COCH_2Lev), 29.8 (CH_3Lev),

28.2 (CO₂CH_2Lev), 18.0 (C-6) ppm. HRMS (ESI⁺): calcd. for

C₂₉H₃₆Cl₃O₈Na [M + Na]⁺535.2308; found 535.2281.

Benzyl (3,4-Di-O-benzyl-α-

L-rhamnopyranosyl)-(1Ǟ4)-(allyl 2,3-di-

O-benzyl-β- -galactopyranosid)uronate (18): Acetic acid (5.6 mL)

D

and then hydrazine monohydrate (326 μL, 6.73 mmol, 5.0 equiv.)

were slowly added to a stirred solution of disaccharide 17 (1.25 g,

1.35 mmol) in dry pyridine (8.3 mL) at 0 °C under Ar. The reaction

mixture was stirred at room temperature for 1.5 h. After this time,

TLC (Chex/EtOAc, 7:3) showed the complete transformation of

the starting material into a less polar product. The volatiles were

evaporated and co-evaporated twice with toluene. The residue was

dissolved in CH₂Cl₂and washed with water. The aqueous phase

was re-extracted twice with CH₂Cl₂, and the combined organic ex-

tracts were washed with brine, passed through a phase-separator

filter, and concentrated. The residue was purified by flash

chromatography (Chex/EtOAc, 8:2 to 7:3) to give alcohol 18

(1.05 g, 90%) as a pale yellow oil. R_f= 0.35 (Chex/EtOAc, 7:3). ¹H

NMR (400 MHz, CDCl₃): δ = 7.42–7.27 (m, 25 H, H_Ar), 6.01 (m,

4-O-Benzyl-2-O-levulinoyl-3-O-p-methoxybenzyl-α/β-L-rhamnopyr-

anose (22): (1,5-Cyclooctadiene)bis(methyldiphenylphosphane)irid-

ium hexafluorophosphate (658 mg, 0.78 mmol, 0.03 equiv.) was dis-

solved in anhydrous THF (130 mL), and hydrogen was bubbled

through the solution for 15 min (H-cube, full H₂mode). The re-

sulting yellow solution was concentrated to dryness. The residue

was dissolved in anhydrous THF (130 mL), and the resulting solu-

tion was poured into a solution of allyl rhamnoside 21 (13.29 g,

25.9 mmol) in anhydrous THF (130 mL). The mixture was stirred

under Ar at room temperature for 2 h. A solution of iodine

(13.16 g, 51.85 mmol, 2.0 equiv.) in THF/H₂O (4:1, 156 mL) was

added, and the mixture was stirred at room temperature for 1 h.

TLC (Chex/EtOAc, 7:3) showed the conversion of the intermediate

compound into a more polar product. The reaction was quenched

with sodium bisulfite (10% aq.). The mixture was concentrated to

a third of its volume, and the aqueous phase was extracted three

times with CH₂Cl₂. The organic extracts were combined, washed

with brine, dried by passing through a phase-separator filter, and

concentrated to dryness. The residue was purified by flash

chromatography (Chex/EtOAc, 1:1) to give hemiacetal 22 (11.46 g,

90%) as a yellow oil (α/β, 3.5:1). Data for the α isomer: R_f= 0.18

(Chex/EtOAc, 6:4). ¹H NMR (400 MHz, CDCl₃): δ = 7.39–7.24

(m, 7 H, H_Ar), 6.87–6.83 (m, J = 8.7 Hz, 2 H, H_ArPMB), 5.38 (dd,

J_1,2= 1.7, J_2,3= 3.3 Hz, 1 H, 2-H), 5.13 (d, 1 H, 1-H), 4.92 (d, J

1 H, CH=_All), 5.39 (d_o, J_1,2= 1.7 Hz, 1 H, 1_D-H), 5.38 (m, J_trans

=

17.2, J_gem= 1.6 Hz, 1 H, =CH_2All), 5.32 (d, J = 12.2 Hz, 1 H,

H_CO2Bn), 5.25 (m, J_cis= 10.5 Hz, 1 H, =CH_2All), 5.14 (d, 1 H,

H_CO2Bn), 4.96 (d, J = 11.0 Hz, 1 H, H_Bn), 4.90 (d, J = 11.3 Hz, 1

H, H_Bn), 4.80 (d_po, J = 11.9 Hz, 1 H, H_Bn), 4.76 (d_po, J = 10.9 Hz,

1 H, H_Bn), 4.75 (d_po, J = 11.9 Hz, 1 H, H_Bn), 4.67–4.64 (m, 3 H,

H_Bn), 4.54 (m, 1 H, H_All), 4.49 (dd, 1 H, 4_A-H), 4.43 (d, J_1,2

7.8 Hz, 1 H, 1_A-H), 4.23–4.16 (m, 2 H, 2_D-H, H_All), 4.08 (d, J_4,5

=

1.2 Hz, 1 H, 5_A-H), 3.89 (dd, J_2,3= 3.2, J_3,4= 9.0 Hz, 1 H, 3_D-H),

3.80–3.71 (m, 2 H, 2_A-H, 5_D-H), 3.58 (dd, J_2,3= 9.8, J_3,4= 3.0 Hz,

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L. A. Mulard et al.

FULL PAPER

= 10.9 Hz, 1 H, H_Bn), 4.63 (d, J = 10.9 Hz, 1 H, H_Bn), 4.62 (d, 1

9.6 Hz, 1 H, 3-H), 3.90 (dq, J_4,5= 9.1, J_5,6= 6.1 Hz, 1 H, 5-H),

H, H_Bn), 4.46 (d, 1 H, H_Bn), 4.03–3.95 (m, 2 H, 3-H, 5-H), 3.81 (s,

3.83 (s, 3 H, CH_3PMB), 3.49 (pt, 1 H, 4-H), 2.82–2.69 (m, 4 H,

3 H, CH_3PMB), 3.40 (pt, J_3,4= 9.3, J_4,5= 9.5 Hz, 1 H, 4-H), 2.93– CH_2Lev), 2.21 (s, 3 H, CH_3Lev), 1.32 (d, 3 H, 6-H) ppm. ¹³C NMR

2.61 (m, 4 H, CH_2Lev), 2.19 (s, 3 H, CH_3Lev), 1.32 (d, J_5,6= 6.3 Hz, (100 MHz, CDCl₃, partial): δ = 206.2 (CO_Lev), 171.8 (CO_2Lev),

3 H, 6-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 206.3 (CO_Lev), 159.4 (C_IVArPMB), 143.3, 138.1 (2 C, C_IVAr), 130.0–119.4 (13 C,

172.1 (CO_2Lev), 159.3 (C_IVArPMB), 138.6 (C_IVAr), 130.3 (C_IVArPMB), 12C_Ar, C_IVArPMB), 113.8 (2 C, C_ArPMB), 93.9 (C-1), 79.2 (C-4), 76.9

129.8–127.6 (7 C, C_Ar), 113.8 (2 C, C_ArPMB), 92.4 (¹J_CH= 171.0 Hz,

C-1), 80.0 (C-4), 77.2 (C-3), 75.3 (C_Bn), 71.3 (C_PMB), 69.6 (C-2), (CH_3PMB), 38.0 (COCH_2Lev), 29.9 (CH_3Lev), 28.0 (CO₂CH_2Lev),

(C-3), 75.6 (C_Bn), 71.7 (C_PMB), 70.4 (C-5), 67.8 (C-2), 55.3

67.7 (C-5), 55.2 (CH_3PMB), 38.1 (COCH_2Lev), 29.8 (CH_3Lev), 28.2

(CO₂CH_2Lev), 18.1 (C-6) ppm. Data for the β isomer: R_f= 0.18

(Chex/EtOAc, 6:4). ¹H NMR (400 MHz, CDCl₃): δ = 7.39–7.24

(m, 7 H, H_Ar), 6.87–6.83 (m, J = 8.7 Hz, 2 H, H_ArPMB), 5.53 (dd,

J_1,2= 1.0, J_2,3= 3.3 Hz, 1 H, 2-H), 4.78 (bs, 1 H, 1-H), 4.68 (d, J

= 10.9 Hz, 1 H, H_Bn), 4.65–460 (2d_o, 2 H, H_Bn), 4.45 (d, J =

10.9 Hz, 1 H, H_Bn), 3.81 (s, 3 H, CH_3PMB), 3.64 (dd, J_3,4= 9.1 Hz,

1 H, 3-H), 3.42–3.38 (dq_o, 1 H, 5-H), 3.40 (pt, J_4,5= 9.4 Hz, 1 H,

4-H), 2.82–2.67 (m, 4 H, CH_2Lev), 2.21 (s, 3 H, CH_3Lev), 1.37 (d,

J_5,6= 6.1 Hz, 3 H, 6-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ =

206.5 (CO_Lev), 172.6 (CO_2Lev), 159.4 (C_IVArPMB), 138.4 (C_IVAr),

130.3 (C_IVArPMB), 129.8–127.6 (7 C, C_Ar), 113.9 (2 C, C_ArPMB), 93.1

(¹J_CH= 158.2 Hz, C-1), 79.7 (C-3), 79.5 (C-4), 75.3 (C_Bn), 71.7 (C-

5), 71.2 (C_PMB), 70.2 (C-2), 55.1 (CH_3PMB), 38.6 (COCH_2Lev), 29.8

(CH_3Lev), 28.2 (CO₂CH_2Lev), 18.0 (C-6) ppm. HRMS (ESI⁺): calcd.

for C₂₆H₃₂O₈Na [M + Na]⁺495.1995; found 495.2004.

18.0 (C-6) ppm.

(4-O-Benzyl-2-O-levulinoyl-3-O-p-methoxybenzyl-β-L-rhamnopyr-

anosyl)(trichloroacetyl)amine (25): A mixture of acceptor 18 (0.30 g,

361 μmol), TCA 23 (334 mg, 542 μmol, 1.5 equiv.), and freshly acti-

vated 4 Å powdered MS (750 mg) in anhydrous toluene (10.8 mL)

was stirred at room temperature under Ar for 1 h. The reaction

mixture was cooled to –10 °C, and TMSOTf (3.3 μL, 18 μmol,

0.05 equiv.) was added. The reaction mixture was stirred at that

temperature for 20 min. TLC (Chex/EtOAc, 6:4) showed the con-

version of acceptor 18 into a more polar product. The reaction was

quenched by the addition of Et₃N. The resulting suspension was

filtered, and the filtrate was concentrated. The residue was purified

by flash chromatography (Tol/EtOAc, 95:5 to 85:15) to give, in or-

der of elution, glycosylamide 25 (59 mg) followed by trisaccharide

26 (391 mg, 84 %) as a pale yellow oil. Data for 25: ¹H NMR

(400 MHz, CDCl₃): δ = 7.60 (d, J_NH,1= 8.8 Hz, 1 H, NH), 7.41–

7.18 (m, 7 H, H_Ar), 6.88–6.84 (m, 2 H, H_ArPMB), 5.57 (dd, J_1,2

1.1, J_2,3= 3.2 Hz, 1 H, 2-H), 5.31 (dd, 1 H, 1-H), 4.91 (d, J =

11.1 Hz, 1 H, H_Bn), 4.67 (d, J = 10.9 Hz, 1 H, H_Bn), 4.62 (d, 1 H,

=

4-O-Benzyl-2-O-levulinoyl-3-O-p-methoxybenzyl-α/β-L-rhamnopyr-

anosyl Trichloroacetimidate (23): Hemiacetal 22 (10.0 g,

21.16 mmol) was dissolved in anhydrous DCE (42 mL) and stirred

under Ar. Trichloroacetonitrile (10.6 mL, 105.8 mmol, 5.0 equiv.)

and DBU (0.95 mL, 6.3 mmol, 0.3 equiv.) were added at room tem-

perature. After 20 min, TLC showed the conversion of the starting

material into a less polar product. The reaction mixture was puri-

fied directly by flash chromatography (Chex/EtOAc, 7:3 to 1:1 +

1% Et₃N) to give TCA 23 (12.66 g, 97%) as a pale yellow oil (α/β,

7.5:1). Data for the α isomer: R_f= 0.46 (Chex/EtOAc, 8:2). ¹H

NMR (400 MHz, CDCl₃): δ = 8.67 (s, 1 H, NH), 7.41–7.26 (m, 7

H_Bn), 4.45 (d, 1 H, H_Bn), 3.82 (s, 3 H, CH_3PMB), 3.72 (dd, J_3,4=

9.1 Hz, 1 H, 3-H), 3.54 (dq, J_4,5= 9.4, J_5,6= 6.1 Hz, 1 H, 5-H),

3.35 (pt, 1 H, 4-H), 2.92–2.63 (m, 4 H, CH_2Lev), 2.21 (s, 3 H,

CH_3Lev), 1.37 (d, 3 H, 6-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ

= 206.9 (CO_Lev), 172.0 (CO_2Lev), 161.5 (NHCO), 159.5 (C_IVPMB),

138.2 (C_IVAr), 129.8 (2 C, C_Ar) 129.6 (C_IVAr), 128.4–127.6 (5 C,

C_Ar), 113.9 (2 C, C_ArPMB), 92.0 (CCl₃), 79.9 (C-3), 79.2 (C-4), 78.4

(C-1), 75.3 (C_Bn), 74.0 (C-5), 71.4 (C_Bn), 69.0 (C-2), 55.3 (CH_3PMB),

38.2 (COCH_2Lev), 29.6 (CH_3Lev), 28.2 (CO₂CH_2Lev), 18.1 (C-6)

ppm. HRMS (ESI⁺): calcd. for C₂₈H₃₂Cl₃NO₈Na [M + Na]⁺

638.1091; found 638.1140.

H, H_Ar), 6.89–6.84 (m, J = 8.7 Hz, 2 H, H_ArPMB), 6.19 (d, J_1,2

=

1.9 Hz, 1 H, 1-H), 5.47 (dd, J_2,3= 3.3 Hz, 1 H, 2-H), 4.94 (d, J =

10.9 Hz, 1 H, H_Bn), 4.66 (d, J = 10.9 Hz, 1 H, H_Bn), 4.65 (d, 1 H,

H

_Bn), 4.51 (d, 1 H, H_Bn), 3.98 (dd, J_3,4= 9.6 Hz, 1 H, 3-H), 3.94

Benzyl (4-O-Benzyl-3-O-p-methoxybenzyl-2-O-levulinoyl-α-

rhamnopyranosyl)-(1Ǟ2)-(3,4-di-O-benzyl-α- -rhamnopyranosyl)-

(1Ǟ4)-(allyl 2,3-di-O-benzyl-β- -galactopyranosid)uronate (26)

L-

(dq, J_4,5= 9.4 Hz, 1 H, 5-H), 3.82 (s, 3 H, CH_3PMB), 3.50 (pt, 1 H,

4-H), 2.84–2.72 (m, 4 H, CH_2Lev), 2.22 (s, 3 H, CH_3Lev), 1.36 (d,

J_5,6= 6.2 Hz, 3 H, 6-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ =

206.3 (CO_Lev), 171.9 (CO_2Lev), 160.1 (C=NH), 159.4 (C_IVArPMB),

138.1 (C_IVAr), 130.0–127.9 (8 C, 7 C_Ar, C_IVArPMB), 113.8 (2 C,

C_ArPMB), 95.1 (C-1), 90.8 (CCl₃), 79.2 (C-4), 76.6 (C-3), 75.6 (C_Bn),

71.5 (C_PMB), 70.7 (C-5), 67.8 (C-2), 55.3 (CH_3PMB), 38.0

(COCH_2Lev), 29.9 (CH_3Lev), 28.1 (CO₂CH_2Lev), 18.0 (C-6) ppm.

L

D

Route 1: A mixture of acceptor 18 (0.30 g, 361 μmol), TCA 23

(267 mg, 433 μmol, 1.2 equiv.), and freshly activated 4 Å powdered

MS (750 mg) in anhydrous toluene (10.8 mL) was stirred at room

temperature under Ar for 1 h. The reaction mixture was cooled to

–10 °C, and TMSOTf (3.3 μL, 18 μmol, 0.05 equiv.) was added.

The reaction mixture was stirred at that temperature for 20 min.

TLC (Chex/EtOAc, 6:4) showed the conversion of acceptor 18 into

a more polar product. The reaction was quenched by the addition

of Et₃N. The resulting suspension was filtered, and the filtrate was

concentrated. The residue was purified by flash chromatography

(Tol/EtOAc, 95:5 to 85:15) to give trisaccharide 26 (426 mg, 92%)

as a pale yellow oil.

4-O-Benzyl-3-O-p-methoxybenzyl-2-O-levulinoyl-α/β-L-rhamnopyr-

anosyl N-Phenyltrifluoroacetimidate (24): Hemiacetal 22 (5.0 g,

10.58 mmol) was dissolved in acetone (36 mL). N-Phenyltrifluo-

roacetimidoyl chloride (4.39 g, 21.2 mmol, 2.0 equiv.) and Cs₂CO₃

(3.79 g, 11.6 mmol, 1.1 equiv.) were added to the solution. The mix-

ture was stirred at room temperature for 4 h. After that time, TLC

(Chex/EtOAc, 6:4) showed the complete transformation of the

starting material into a less polar product. The reaction mixture

was filtered and concentrated. The residue was purified by flash

chromatography (Chex/EtOAc, 9:1 to 7:3 + 1% Et₃N) to give a 5:1

α/β mixture of PTFA 24 (6.70 g, 98%) as a pale yellow oil. Data

for the α isomer: R_f= 0.48 (Chex/EtOAc, 6:4). ¹H NMR (400 MHz,

Route 2: A mixture of acceptor 18 (0.30 g, 361 μmol), PTFA 24

(279 mg, 433 μmol, 1.2 equiv.), and freshly activated 4 Å powdered

MS (750 mg) in anhydrous toluene (10.8 mL) was stirred at room

temperature under Ar for 1 h. The reaction mixture was cooled to

–10 °C, and TMSOTf (3.3 μL, 18 μmol, 0.05 equiv.) was added.

CDCl₃): δ = 7.42–6.81 (m, 14 H, H_Ar), 6.17 (bs, 1 H, 1-H), 5.46 The reaction mixture was stirred at that temperature for 20 min.

(bs, 1 H, 2-H), 4.94 (d, J = 10.6 Hz, 1 H, H_Bn), 4.70–4.62 (m, 2 H,

H_Bn), 4.53 (d, J = 10.6 Hz, 1 H, H_Bn), 3.98 (dd, J_2,3= 3.3, J_3,4

TLC (Chex/EtOAc, 6:4) showed the conversion of acceptor 18 into

a more polar product. The reaction was quenched with Et₃N. The

=

4098

www.eurjoc.org

Eur. J. Org. Chem. 2013, 4085–4106

O-Antigen from a Shigella flexneri Serotype 6

resulting mixture was filtered, and the filtrate was concentrated. H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 167.3 (C-6_A), 159.2

The residue was purified by flash chromatography (Chex/EtOAc,

(C_IVPMB) 139.0, 138.6, 138.5, 137.8, 135.2 (6 C, C_IVAr), 134.1

(CH=_All), 130.2 (C_IVAr), 129.5–127.3 (32 C, C_Ar), 117.4 (=CH_2All),

7:3 to 6:4) to give trisaccharide 26 (415 mg, 89%) as a pale yellow

1

oil. R_f= 0.32 (Chex/EtOAc, 6:4). H NMR (400 MHz, CDCl₃): δ 114.0 (2 C, C_ArPMB), 102.7 (¹J_CH= 161.1 Hz, C-1_A), 100.8 (¹J_CH

=

= 7.43–7.17 (m, 32 H, H_Ar), 6.86 (d, J = 8.5 Hz, 2 H, H_ArPMB),

5.99 (m, 1 H, CH=_All), 5.52 (dd, J_1,2= 1.9, J_2,3= 3.1 Hz, 1 H, 2_C-

169.0 Hz, C-1_D), 100.2 (¹J_CH= 171.9 Hz, C-1_C), 80.5 (C-3_A), 80.2

(2 C, C-4_C, C-4_D), 79.4 (2 C, C-3_C, C-3_D), 78.4 (C-2_A), 75.3, 75.2

H), 5.36 (m, J_trans= 17.2, J_gem= 1.6 Hz, 1 H, =CH_2All), 5.33 (bs_o, (3 C, 2 C_Bn, C-2_D), 74.9 (C_Bn), 73.7 (C-5_A), 73.6 (C-4_A), 72.7, 72.3,

1 H, 1_D-H), 5.27 (d, J = 12.2 Hz, 1 H, H_CO2Bn), 5.22 (m, J_cis

=

71.7 (3 C, C_Bn), 70.5 (CH_2All), 68.8 (C-2_C), 68.6 (C-5_D), 67.9 (C-

5_C), 67.3 (C_CO2Bn), 55.2 (CH_3PMB), 18.2 (C-6_D), 17.8 (C-6_C) ppm.

HRMS (ESI⁺): calcd. for C₇₁H₇₈O₁₆Na [M + Na]⁺1210.5222;

found 1210.5117; calcd. for C₇₁H₇₉O₁₆K [M + H + K]²⁺613.2502;

found 613.2374.

10.5 Hz, 1 H, =CH_2All), 5.12 (d, 1 H, H_CO2Bn), 4.94 (d_po, J =

11.1 Hz, 1 H, H_Bn), 4.92–4.88 (m_o, 3 H, 1_C-H, 2 H_Bn), 4.80 (d, J =

12.1 Hz, 1 H, H_Bn), 4.75 (d, J = 12.1 Hz, 1 H, H_Bn), 4.70–4.57 (m,

6 H, H_Bn), 4.50 (m_po, 1 H, H_All), 4.48 (d, J = 10.9 Hz, 1 H, H_Bn),

4.39 (m_o, 1 H, 4_A-H), 4.38 (d_o, J_1,2= 7.8 Hz, 1 H, 1_A-H), 4.17 (m,

1 H, H_All), 4.11 (pt, 1 H, 2_D-H), 4.02 (d, J_4,5= 1.0 Hz, 1 H, 5_A-

H), 3.94 (dd, J_3,4= 9.3 Hz, 1 H, 3_C-H), 3.86 (dd_po, J_2,3= 2.9, J_3,4

= 9.4 Hz, 1 H, 3_D-H), 3.82 (dq, 1 H, 5_C-H), 3.78 (s, 3 H, CH_3PMB),

3.75–3.64 (m, 2 H, 2_A-H, 5_D-H), 3.51 (dd, J_2,3= 9.8, J_3,4= 2.8 Hz,

Benzyl (4-O-Benzyl-2-O-levulinoyl-α-

(3,4-di-O-benzyl-α- -rhamnopyranosyl)-(1Ǟ4)-(allyl 2,3-di-O-

benzyl-β- -galactopyranosid)uronate (28)

L-rhamnopyranosyl)-(1Ǟ2)-

L

D

Route 1: Water (0.2 mL) and DDQ (27 mg, 120 μmol, 3.0 equiv.)

were added to a solution of trisaccharide 26 (49 mg, 38 μmol) in

CH₂Cl₂(1.8 mL). The reaction mixture was stirred at room tem-

perature for 3 h. After that time, TLC (Tol/EtOAc, 8:2) showed the

transformation of the starting material into a more polar product.

The reaction was quenched with NaHCO₃(satd. aq.). The reaction

mixture was diluted with water and CH₂Cl₂, and the aqueous phase

was extracted three times with CH₂Cl₂. The combined extracts were

washed with brine, passed through a phase-separator filter, and

concentrated. The residue was purified by flash chromatography

(Tol/EtOAc, 8:2) to give alcohol 28 (20 mg, 45%) as a yellow oil.

1 H, 3_A-H), 3.42 (pt, J_4,5= 9.5 Hz, 1 H, 4_D-H), 3.37 (pt, J_4,5

=

9.5 Hz, 1 H, 4_C-H), 2.77–2.66 (m, 4 H, CH_2Lev), 2.18 (s, 3 H,

CH_3Lev), 1.28 (d, J_5,6= 6.2 Hz, 3 H, 6_D-H), 1.20 (d, J_5,6= 6.2 Hz,

3 H, 6_C-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 206.1 (CO_Lev),

171.7 (CO_2Lev), 167.3 (C-6_A), 159.2 (C_IVPMB) 138.9, 138.6, 138.4,

137.8, 135.1 (6 C, C_IVAr), 134.0 (CH=_All), 130.3 (C_IVAr), 129.8–

127.4 (32 C, C_Ar), 117.4 (=CH_2All), 113.7 (2 C, C_ArPMB), 102.7

(¹J_CH= 159.4 Hz, C-1_A), 100.0 (¹J_CH= 174.9 Hz, C-1_D), 99.2 (¹J_CH

= 172.5 Hz, C-1_C), 80.5 (C-3_A), 80.1 (C-4_C), 79.9 (C-4_D), 79.3 (C-

3_D), 78.4 (C-2_A), 77.4 (C-3_C), 75.4, 75.3 (2 C, C_Bn), 75.1 (C-2_D),

75.0 (C_Bn), 73.6 (C-5_A), 73.4 (C-4_A), 72.7, 72.1, 71.2 (3 C, C_Bn),

70.6 (CH_2All), 69.1 (C-2_C), 68.6 (C-5_D), 68.2 (C-5_C), 67.3 (C_CO2Bn),

55.1 (CH_{3 P M B}), 38.2 (COCH_{2 L e v}), 29.8 (CH_{3 L e v}), 28.3

(CO₂CH_2Lev), 18.1 (C-6_D), 17.9 (C-6_C) ppm. HRMS (ESI⁺): calcd.

for C₇₆H₈₄O₁₈Na [M + Na]⁺1307.5555; found 1307.5554.

Route 2: Water (0.2 mL) and CAN (68 mg, 120 μmol, 3.0 equiv.)

were added to a solution of trisaccharide 26 (53 mg, 41 μmol) in

MeCN (1.8 mL). The reaction mixture was stirred at room tem-

perature for 1.5 h. After that time, TLC (Tol/EtOAc, 8:2) showed

the transformation of the starting material into a more polar prod-

uct. The reaction was quenched with NaHCO₃(satd. aq.). The re-

action mixture was diluted with water and CH₂Cl₂, and the aque-

ous phase was extracted three times with CH₂Cl₂. The combined

extracts were washed with brine, passed through a phase-separator

filter, and concentrated. The residue was purified by flash

chromatography (Tol/EtOAc, 9:1 to 8:2) to give alcohol 28 (34 mg,

71 %) as a yellow oil. R_f= 0.21 (Tol/EtOAc, 8:2). ¹H NMR

(400 MHz, CDCl₃): δ = 7.41–7.19 (m, 30 H, H_Ar), 6.00 (m, 1 H,

CH=_All), 5.40–5.33 (m, 3 H, 2_C-H, =CH_2All, 1_D-H), 5.28 (d, J =

12.2 Hz, 1 H, H_CO2Bn), 5.23 (m, J_cis= 10.5, J_gem= 1.3 Hz, 1 H,

=CH_2All), 5.12 (d, 1 H, H_CO2Bn), 4.94 (d, J = 11.1 Hz, 1 H, H_Bn),

4.92–4.87 (m, 3 H, 1_C-H, 2 H_Bn), 4.81 (d, J = 12.1 Hz, 1 H, H_Bn),

4.75 (d_po, J = 10.9 Hz, 1 H, H_Bn), 4.70 (d, J = 11.2 Hz, 1 H, H_Bn),

4.66 (d_po, J = 12.5 Hz, 1 H, H_Bn), 4.63 (d_po, J = 11.2 Hz, 1 H,

H_Bn), 4.61 (bs_o, 2 H, H_Bn), 4.51 (m, 1 H, H_All), 4.41 (d_o, 1 H, 4_A-

Benzyl (4-O-Benzyl-3-O-p-methoxybenzyl-α-

(1Ǟ2)-(3,4-di-O-benzyl-α- -rhamnopyranosyl)-(1Ǟ4)-(allyl 2,3-di-

O-benzyl-β- -galactopyranosid)uronate (27): Acetic acid (11.3 mL)

L-rhamnopyranosyl)-

L

D

and hydrazine monohydrate (492 μL, 10.1 mmol, 5.0 equiv.) were

slowly added to a stirred solution of fully protected 26 (2.60 g,

2.0 mmol) in dry pyridine (16.8 mL) at 0 °C under Ar. The reaction

mixture was stirred at room temperature for 30 min. After that

time, TLC (Tol/EtOAc, 9:1) showed the complete transformation

of the starting material into a more polar product. The volatiles

were evaporated and co-evaporated twice with toluene. The residue

was dissolved in CH₂Cl₂and washed with water. The aqueous

phase was re-extracted twice with CH₂Cl₂, and the combined or-

ganic extracts were washed with brine, passed through a phase-

separator filter, and concentrated. The residue was purified by flash

chromatography (Tol/EtOAc, 9:1 to 7:3) to give alcohol 27 (2.23 g,

93 %) as a white foam. R_f= 0.10 (Tol/EtOAc, 9:1). ¹H NMR H), 4.38 (d_o, J_1,2= 7.9 Hz, 1 H, 1_A-H), 4.22–4.13 (m, 2 H, H_All

,

(400 MHz, CDCl₃): δ = 7.45–7.16 (m, 32 H, H_Ar), 6.89 (d, J =

8.6 Hz, 2 H, H_ArPMB), 6.00 (m, 1 H, CH=_All), 5.37 (m, J_trans= 17.3,

J_gem= 1.6 Hz, 1 H, =CH_2All), 5.35 (bs_o, 1 H, 1_D-H), 5.28 (d, J =

3_C-H), 4.11 (pt, 1 H, 2_D-H), 4.01 (s, 1 H, 5_A-H), 3.86 (dd_po, J_2,3

2.9, J_3,4= 9.5 Hz, 1 H, 3_D-H), 3.82 (dq_po, J_4,5= 9.4 Hz, 1 H, 5_C-

H), 3.74–3.64 (m, 2 H, 2_A-H, 5_D-H), 3.53 (dd, J_2,3= 9.8, J_3,4

=

12.1 Hz, 1 H, H_CO2Bn), 5.23 (m, J_cis= 10.5 Hz, 1 H, =CH_2All), 5.13 2.7 Hz, 1 H, 3_A-H), 3.46 (pt, J_4,5= 9.4 Hz, 1 H, 4_D-H), 3.31 (pt,

(d, 1 H, H_CO2Bn), 5.02 (d, J_1,2= 1.4 Hz, 1 H, 1_C-H), 4.94 (d_po, J =

10.9 Hz, 1 H, H_Bn), 4.90 (d_po, J = 11.2 Hz, 1 H, H_Bn), 4.89 (d_po, J

= 10.9 Hz, 1 H, H_Bn), 4.81 (d, J = 12.1 Hz, 1 H, H_Bn), 4.75 (d, J

J_3,4= 9.4 Hz, 1 H, 4_C-H), 2.89–2.57 (m, 4 H, CH_2Lev), 2.22 (s, 3

H, CH_3Lev), 1.28 (d, J_5,6= 6.2 Hz, 3 H, 6_D-H), 1.21 (d, J_5,6

6.3 Hz, 3 H, 6_C-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 207.2

=

= 10.9 Hz, 1 H, H_Bn), 4.70–4.59 (m, 7 H, H_Bn), 4.51 (m, 1 H, H_All), (CO_Lev), 172.3 (CO_2Lev), 172.2 (C-6_A), 138.9, 138.6, 138.5, 138.4,

4.40 (m_o, 1 H, 4_A-H), 4.39 (d_o, J_1,2= 7.7 Hz, 1 H, 1_A-H), 4.17 (m_po137.8, 135.0 (6 C, C_IVAr), 134.0 (CH=_All), 128.7–127.4 (30 C, C_Ar),

1 H, H_All), 4.15 (m_o, 1 H, 2_D-H), 4.12 (m, 1 H, 2_C-H), 4.03 (d, J_4,5117.6 (=CH_2All), 102.6 (¹J_CH= 156.5 Hz, C-1_A), 99.8 (¹J_CH

= 0.9 Hz, 1 H, 5_A-H), 3.91–3.86 (m, 2 H, 3_C-H, 3_D-H), 3.84 (dq_po

172.4 Hz, C-1_D), 99.1 (¹J_CH= 170.6 Hz, C-1_C), 81.8 (C-4_C), 80.6

1 H, 5_C-H), 3.79 (s, 3 H, CH_3PMB), 3.76–3.66 (m, 2 H, 2_A-H, 5_D- (C-3_A), 80.0 (C-4_D), 79.3 (C-3_D), 78.4 (C-2_A), 75.4, 75.3 (2 C, C_Bn),

,

=

,

H), 3.52 (dd, J_2,3= 9.8, J_3,4= 2.8 Hz, 1 H, 3_A-H), 3.45 (pt_po, J_4,5

= 9.5 Hz, 1 H, 4_C-H), 3.41 (pt, J_3,4= J_4,5= 9.6 Hz, 1 H, 4_D-H),

1.30 (d, J_5,6= 6.2 Hz, 3 H, 6_D-H), 1.21 (d, J_5,6= 6.2 Hz, 3 H, 6_C-

75.2 (2 C, C-2_D, C_Bn), 73.6 (C-5_A), 73.0 (C-4_A), 72.9 (C-2_C), 72.7,

72.2 (2 C, C_Bn), 70.7 (C-3_C), 70.6 (CH_2All), 68.6 (C-5_D), 68.0 (C-

5_C), 67.4 (C_CO2Bn), 38.4 (CH_2Lev), 29.9 (CH_3Lev), 28.3 (CH_2Lev),

Eur. J. Org. Chem. 2013, 4085–4106

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4099

L. A. Mulard et al.

FULL PAPER

18.2 (C-6_D), 17.9 (C-6_C) ppm. HRMS (ESI⁺): calcd. for C₆₈H₇₇O₁₇

[M + H]⁺1165.5161; found 1165.5172; calcd. for C₆₈H₇₆O₁₇Na [M

+ Na]⁺1187.4980; found 1187.5001.

dried by passing through a phase-separator filter, and concentrated

to dryness. The residue was purified by flash chromatography (Tol/

EtOAc, 9:1 to 8:2) to give alcohol 30 (318 mg, 93%), slightly con-

taminated by 2_C-acetate 31, as a white foam. R_f= 0.38 (Tol/EtOAc,

8:2). ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.38–7.12 (m, 30 H,

H_Ar), 5.94 (m, 1 H, CH=_All), 5.35 (d_o, J_OH,2= 5.2 Hz, 1 H, 2_C-OH),

5.34 (m_po, J_gem= 1.5 Hz, 1 H, =CH_2All), 5.22 (d, J_1,2= 1.4 Hz, 1

H, 1_D-H), 5.20–5.15 (m, 2 H, H_CO2Bn, =CH_2All), 4.99 (d, J =

12.2 Hz, 1 H, H_CO2Bn), 4.96 (dd, J_2,3= 3.2, J_3,4= 9.7 Hz, 1 H, 3_C-

H), 4.86 (d, J_1,2= 1.2 Hz, 1 H, 1_C-H), 4.82–4.77 (m, 2 H, H_Bn),

4.73 (d, J = 12.3 Hz, 1 H, H_Bn), 4.68–4.55 (m, 6 H, H_Bn), 4.49 (d,

J_1,2= 7.8 Hz, 1 H, 1_A-H), 4.47 (d, J = 11.9 Hz, 1 H, H_Bn), 4.45

(bs, 1 H, 5_A-H), 4.40 (dd, 1 H, 4_A-H), 4.33 (m, 1 H, H_All), 4.13–

Benzyl (3-O-Acetyl-4-O-benzyl-2-O-levulinoyl-α-

osyl)-(1Ǟ2)-(3,4-di-O-benzyl-α- -rhamnopyranosyl)-(1Ǟ4)-(allyl

2,3-di-O-benzyl-β- -galactopyranosid)uronate (29): Water (2.2 mL)

L-rhamnopyran-

L

D

and CAN (1.57 g, 2.86 mmol, 4.0 equiv.) were added to a solution

of trisaccharide 26 (920 mg, 716 μmol) in MeCN (21.5 mL). The

reaction mixture was stirred at room temperature for 30 min. After

that time, TLC (Tol/EtOAc, 8:2) showed the complete transforma-

tion of the starting material into alcohol 28. The reaction was

quenched with NaHCO₃(satd. aq.). The reaction mixture was di-

luted with water and CH₂Cl₂, and the aqueous phase was extracted

three times with CH₂Cl₂. The combined extracts were washed with

brine, passed through a phase-separator filter, and concentrated to

give the intermediate alcohol. The crude oil was dissolved in pyr-

idine (18 mL). Acetic anhydride (9.45 mL) and DMAP (12.2 mg,

72 μmol, 0.1 equiv.) were added at room temperature. After 2 h,

TLC (Tol/EtOAc, 8:2) indicated that conversion of intermediate 28

into a less polar product was complete. The volatiles were evapo-

rated and co-evaporated three times with toluene. The residue was

purified by flash chromatography (Tol/EtOAc, 9:1 to 85:15) to give

acetate 29 (762 mg, 88%) as a white foam. R_f= 0.41 (Tol/EtOAc,

4.06 (m, 2 H, H_All, 2_D-H), 3.96 (m, 1 H, 2_C-H), 3.75 (dd, J_2,3

=

9.8, J_3,4= 2.7 Hz, 1 H, 3_A-H), 3.73–3.66 (m, 2 H, 5_C-H, 3_D-H),

3.57–3.50 (m, 2 H, 4_C-H, 5_D-H), 3.45 (dd, 1 H, 2_A-H), 3.39 (pt,

J_3,4= J_4,5= 9.4 Hz, 1 H, 4_D-H), 2.04 (s, 3 H, CH_3Ac), 1.15 (d, J_5,6

= 6.1 Hz, 3 H, 6_D-H), 1.11 (d, J_5,6= 6.2 Hz, 3 H, 6_C-H) ppm.

¹³C NMR (100 MHz, [D₆]DMSO): δ = 170.4 (CO_Ac), 167.9 (C-6_A),

139.0, 138.9, 138.8, 138.7, 135.1 (6 C, C_IVAr), 134.9 (CH=_All),

129.4–127.8 (30 C, C_Ar), 117.1 (=CH_2All), 102.1 (C-1_A), 101.6 (C-

1_C), 100.1 (C-1_D), 80.6 (C-3_A), 80.0 (C-4_D), 79.3 (C-3_D), 78.6 (C-

4_C), 78.5 (C-2_A), 74.8, 74.7, 74.6 (3 C, C_Bn), 74.4 (C-3_C), 74.1 (C-

4_A), 73.8 (C-2_D), 73.1 (C-5_A), 72.1, 71.4 (2 C, C_Bn), 70.0 (CH_2All),

68.4 (C-2_C), 68.3 (C-5_D), 68.2 (C-5_C), 66.9 (C_CO2Bn), 21.6 (CH_3Ac),

18.5 (C-6_D), 18.1 (C-6_C) ppm. HRMS (ESI⁺): calcd. for

C₆₅H₇₂O₁₆Na [M + Na]⁺1131.4718; found 1131.4695.

1

8:2). H NMR (400 MHz, CDCl₃): δ = 7.44–7.14 (m, 30 H, H_Ar),

5.99 (m, 1 H, CH=_All), 5.48 (dd, J_1,2= 1.8, J_2,3= 3.4 Hz, 1 H, 2_C-

H), 5.40–5.33 (m, 3 H, =CH_2All, 1_D-H, 3_C-H), 5.29 (d, J = 12.2 Hz,

1 H, H_CO2Bn), 5.23 (m, J_cis= 10.5, J_gem= 1.5 Hz, 1 H, =CH_2All),

5.12 (d, 1 H, H_CO2Bn), 4.93 (d_po, J = 10.0 Hz, 1 H, H_Bn), 4.91 (d,

J = 10.2 Hz, 1 H, H_Bn), 4.86 (d, 1 H, 1_C-H), 4.80 (d, J = 11.8 Hz,

1 H, H_Bn), 4.74 (d, J = 10.6 Hz, 1 H, H_Bn), 4.71–4.60 (m, 5 H,

H_Bn), 4.58 (d, J = 11.9 Hz, 1 H, H_Bn), 4.51 (m, 1 H, H_All), 4.44

(dd, 1 H, 4_A-H), 4.40 (d, J_1,2= 7.6 Hz, 1 H, 1_A-H), 4.17 (m, 1 H,

Propyl α-

β- -galactopyranosiduronic acid (2) and Methyl α-

osyl-(1Ǟ2)-α- -rhamnopyranosyl-(1Ǟ4)-(propyl β-

L

-Rhamnopyranosyl-(1Ǟ2)-α-

L

-rhamnopyranosyl-(1Ǟ4)-

-Rhamnopyran-

-galactopyr-

D

L

D

anosid)uronate (32): Pd/C (10%; 100 mg) was added to a stirred

solution of trisaccharide 27 (100 mg, 84 μmol) in MeOH (5.1 mL).

The suspension was stirred under hydrogen for 1 d. After this time,

MS analysis indicated a molecular weight corresponding to that of

the target trisaccharide. The reaction mixture was filtered. Evapo-

ration of the volatiles, freeze-drying, and purification of the crude

material by preparative RP-HPLC gave, in order of elution, trisac-

charide 2 (30.8 mg, 69%) and methyl ester 32 (2.1 mg, 5%), both

as white solids after repeated freeze-drying.

H_All), 4.12 (pt, 1 H, 2_D-H), 4.04 (d, J_4,5= 1.0 Hz, 1 H, 5_A-H), 3.88

(dq_po, J_4,5= 9.5 Hz, 1 H, 5_C-H), 3.83 (dd, J_2,3= 2.9, J_3,4= 9.4 Hz,

1 H, 3_D-H), 3.72 (dd_po, J_2,3= 9.8 Hz, 1 H, 2_A-H), 3.67 (dq_po, J_4,5

= 9.5 Hz, 1 H, 5_D-H), 3.56 (pt_po, 1 H, 4_D-H), 3.54 (dd_po, J_3,4

=

2.9 Hz, 1 H, 3_A-H), 3.45 (pt, 1 H, 4_C-H), 2.85–2.50 (m, 4 H,

CH_2Lev), 2.22 (s, 3 H, CH_3Lev), 2.01 (s, 3 H, CH_3Ac), 1.31 (d, J_5,6

= 6.2 Hz, 3 H, 6_D-H), 1.16 (d, J_5,6= 6.2 Hz, 3 H, 6_C-H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 205.9 (CO_Lev), 171.4 (CO_2Lev), 169.6

(CO_Ac), 167.3 (C-6_A), 139.0, 138.7, 138.5, 138.2, 137.7, 135.1 (6 C,

1

Data for Compound 2: t_R= 8.5 min. H NMR (400 MHz, D₂O): δ

= 5.24 (d, J_1,2= 1.5 Hz, 1 H, 1_D-H), 4.88 (d, J_1,2= 1.6 Hz, 1 H,

1_C-H), 4.38 (d, J_1,2= 8.0 Hz, 1 H, 1_A-H), 4.34 (d, J_4,5= 1.2 Hz, 1

C

_IVAr), 134.0 (CH=_All), 129.5–127.2 (30 C, C_Ar), 117.4 (=CH_2All),

102.7 (¹J_CH= 159.5 Hz, C-1_A), 99.6 (¹J_CH= 175.8 Hz, C-1_D), 99.1

(¹J_CH= 171.9 Hz, C-1_C), 81.0 (C-3_A), 80.1 (C-4_D), 79.5 (C-3_D),

78.9 (C-4_C), 78.5 (C-2_A), 75.4 (C-2_D), 75.3, 75.2, 75.0 (3 C, C_Bn),

73.7 (C-5_A), 72.9 (C_Bn), 72.5 (C-4_A), 72.3 (C_Bn), 71.8 (C-3_C), 70.6

(CH_2All), 70.4 (C-2_C), 68.9 (C-5_D), 68.0 (C-5_C), 67.3 (C_CO2Bn), 37.8

(COCH_2Lev), 29.9 (CH_3Lev), 28.0 (CO₂CH_2Lev), 20.9 (CH_3Ac), 18.0

(C-6_D), 17.8 (C-6_C) ppm. HRMS (ESI⁺): calcd. for C₇₀H₇₉O₁₈[M

+ H]⁺1207.5266; found 1207.5286; calcd. for C₇₀H₈₂NO₁₈[M +

NH₄]⁺1224.5532; found 1224.5576; calcd. for C₇₀H₇₈O₁₈Na [M +

Na]⁺1229.5085; found 1229.5131.

H, 5_A-H), 4.27 (dd, J_3,4= 3.0 Hz, 1 H, 4_A-H), 4.01 (dd, J_2,3

=

3.2 Hz, 1 H, 2_D-H), 3.98 (dd, J_2,3= 3.4 Hz, 1 H, 2_C-H), 3.86–3.75

(m, 3 H, 3_A-H, 3_D-H, OCH_2Pr), 3.70 (dd, J_3,4= 9.8 Hz, 1 H, 3_C-

H), 3.62 (dq, J_4,5= 9.5, J_5,6= 6.2 Hz, 1 H, 5_C-H), 3.57–3.51 (m, 2

H, 5_D-H, OCH_2Pr), 3.50 (dd, J_2,3= 10.0 Hz, 1 H, 2_A-H), 3.36 (2pt_o,

J_3,4= J_4,5= 9.7 Hz, 2 H, 4_D-H, 4_C-H), 1.57 (sext, J = 7.3 Hz, 2 H,

CH_2Pr), 1.18 (d, 3 H, 6_C-H), 1.17 (d, J_5,6= 6.2 Hz, 3 H, 6_D-H),

0.85 (t, 3 H, CH_3Pr) ppm. ¹³C NMR (100 MHz, D₂O): δ = 174.2

(C-6_A), 104.9 (¹J_CH= 163.4 Hz, C-1_A), 104.7 (¹J_CH= 171.2 Hz, C-

1_C), 102.6 (¹J_CH= 174.0 Hz, C-1_D), 80.9 (C-2_D), 79.2 (C-4_A), 75.5

(C-3_A), 75.4 (C-5_A), 74.9 (OCH_2Pr), 74.7, 74.6 (2 C, C-4_D, C-4_C),

72.8 (C-2_A), 72.6 (2 C, C-2_C, C-3_C), 72.4 (C-3_D) 71.8 (C-5_D), 71.6

Benzyl (3-O-Acetyl-4-O-benzyl-α-

di-O-benzyl-α- -rhamnopyranosyl)-(1Ǟ4)-(allyl 2,3-di-O-benzyl-β-

-galactopyranosid)uronate (30): Acetic acid (3.4 mL) and then hy-

L-rhamnopyranosyl)-(1Ǟ2)-(3,4-

L

D

(C-5_C), 24.7 (CH_2Pr), 19.3, 19.2 (2 C, C-6_D, C-6_C), 12.2 (CH_3Pr

)

drazine monohydrate (74 μL, 1.53 mmol, 5.0 equiv.) were slowly

added to a stirred solution of 29 (0.37 g, 0.31 mmol) in dry pyridine

(5.1 mL) at 0 °C under Ar. The reaction mixture was stirred at

room temperature for 1.5 h. After that time, TLC (Tol/EtOAc, 8:2)

showed the complete transformation of the starting material into

a more polar product. After addition of CH₂Cl₂and water, the two

phases were separated and the aqueous phase was re-extracted with

CH₂Cl₂. The combined organic extracts were washed with brine,

ppm. HRMS (ESI⁺): calcd. for C₂₁H₃₆O₁₅Na [M + Na]⁺551.1952;

found 551.1940.

1

Data for Methyl Ester 32: H NMR (400 MHz, D₂O): δ = 5.18 (d,

J_1,2= 1.7 Hz, 1 H, 1_D-H), 4.85 (d, J_1,2= 1.6 Hz, 1 H, 1_C-H), 4.37

(d, J_4,5= 0.7 Hz, 1 H, 5_A-H), 4.34 (d, J_1,2= 8.0 Hz, 1 H, 1_A-H),

4.28 (dd, J_3,4= 2.8 Hz, 1 H, 4_A-H), 3.97 (dd, J_2,3= 3.0 Hz, 1 H,

2_D-H), 3.93 (dd, 1 H, 2_C-H), 3.79 (dt, J = 6.8, J = 9.8 Hz, 1 H,

4100

www.eurjoc.org

Eur. J. Org. Chem. 2013, 4085–4106

O-Antigen from a Shigella flexneri Serotype 6

OCH_2Pr), 3.76–3.72 (m, 2 H, 3_D-H, 3_A-H), 3.72 (s_o, 3 H, CH_3Me), 3_D), 71.8 (C-5_D), 71.7 (C-5_C), 71.1 (C-3_C), 24.7 (CH_2Pr), 22.9

3.66 (dd, J_2,3= 3.4, J_3,4= 9.8 Hz, 1 H, 3_C-H), 3.59 (dq, J_3,4= 9.5,

J_5,6= 6.2 Hz, 1 H, 5_C-H), 3.50 (dt, J = 6.8 Hz, 1 H, OCH_2Pr), 3.45

(dd, J_2,3= 9.9 Hz, 1 H, 2_A-H), 3.34 (m_o, 1 H, 5_D-H), 3.32 (2t_o, J_3,4

= J_4,5= 9.6 Hz, 2 H, 4_C-H, 4_D-H), 1.53 (sext, J = 7.1 Hz, 2 H,

CH_2Pr), 1.15 (d, J_5,6= 6.0 Hz, 6 H, 6_C-H, 6_D-H), 0.81 (t, J = 7.4 Hz,

3 H, CH_3Pr) ppm. ¹³C NMR (100 MHz, D₂O): δ = 172.8 (C-6_A),

105.1 (¹J_CH= 162.3 Hz, C-1_A), 104.6 (¹J_CH= 171.5 Hz, C-1_C),

102.8 (¹J_CH= 173.7 Hz, C-1_D), 80.8 (C-2_D), 79.7 (C-4_A), 75.7 (C-

5_A), 75.4 (C-3_A), 75.0 (OCH_2Pr), 74.6, 72.5 (2 C, C-4_C, C-4_D), 72.8

(C-2_A), 72.6 (2 C, C-2_C, C-3_C), 72.2 (C-3_D), 71.8 (C-5_D), 71.6 (C-

5_C), 55.7 (CH_3Me), 24.7 (CH_2Pr), 19.5, 19.3 (2 C, C-6_D, C-6_C), 12.2

(CH_3Pr) ppm. HRMS (ESI⁺): calcd. for C₂₂H₃₈O₁₅Na [M + Na]⁺

565.2108; found 565.2087.

(CH_3Ac) 19.2 (2 C, C-6_D, C-6_C), 12.2 (CH_3Pr) ppm. HRMS (ESI⁺):

calcd. for C₂₃H₃₈O₁₆Na [M + Na]⁺593.2057; found 593.2051.

Allyl 3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-galacto-

pyranoside (36): A mixture of β-acetate 35^[50](2.83 g, 5.74 mmol),

allyl alcohol (0.78 mL, 11.5 mmol, 2.0 equiv.), and freshly activated

4 Å powdered MS (2.8 g) in anhydrous CH₂Cl₂(57 mL) was stirred

at room temperature under Ar for 1 h. TMSOTf (1.10 mL,

5.74 mmol, 1.0 equiv.) was added at room temperature, and stirring

was continued for 14 h at this temperature. TLC (Tol/MeCN, 7:3)

showed the complete conversion of donor 35 into a very slightly

less polar product. The reaction was quenched with Et₃N. The re-

sulting mixture was filtered and concentrated. The residue was

purified by flash chromatography (Chex/EtOAc, 6:4) to give allyl

galactopyranoside 36 (2.60 g, 92%) as a white foam. R_f= 0.42 (Tol/

Propyl 3-O-Acetyl-α-

osyl-(1Ǟ4)-β- -galactopyranosiduronic Acid (3) and Propyl 2-O-

Acetyl-α- -rhamnopyranosyl-(1Ǟ2)-α- -rhamnopyranosyl-(1Ǟ4)-β-

-galactopyranosiduronic Acid (33): Pd/C (10%; 150 mg) was added

L-rhamnopyranosyl-(1Ǟ2)-α-L-rhamnopyran-

D

MeCN, 7:3). ¹H NMR (400 MHz, CDCl₃): δ = 6.69 (d, J_NH,2

=

L

8.6 Hz, 1 H, NH), 5.87 (m, 1 H, CH=_All), 5.42 (dd, J_3,4= 3.4, J_4,5

= 1.0 Hz, 1 H, 4-H), 5.36 (dd, J_2,3= 11.3 Hz, 1 H, 3-H), 5.31 (m,

J_trans= 17.3, J_gem= 1.5 Hz, 1 H, =CH_2All), 5.23 (m, J_cis= 10.4 Hz,

1 H, =CH_2All), 4.78 (d, J_1,2= 8.3 Hz, 1 H, 1-H), 4.39 (m, 1 H,

D

to a stirred solution of trisaccharides 30 and 31 (10:1; 169 mg,

0.15 mmol) in THF/H₂O (4:1, 7.6 mL). The suspension was stirred

under hydrogen for 20 h. After this time, MS analysis indicated a

molecular weight corresponding to that of the target trisaccharide.

The reaction mixture was filtered. Evaporation of the volatiles,

freeze-drying, and purification by preparative RP-HPLC gave, in

order of elution, first trisaccharide 3 (67.8 mg, 78%) and then re-

gioisomer 33 (1.8 mg, 2 %), both as white foams after repeated

freeze-drying.

H_All), 4.22 (dd, J_5,6a= 6.7, J_6a,6b= 11.3 Hz, 1 H, 6a-H), 4.13 (dd_po

,

J_5,6b= 6.8 Hz, 1 H, 6b-H), 4.16–4.08 (m, 2 H, H_All, 2-H), 3.96

(pdt, 1 H, 5-H), 2.19, 2.08, 2.02 (3 s, 9 H, CH_3Ac) ppm. ¹³C NMR

(100 MHz, CDCl₃): δ = 170.4, 170.3, 170.1 (3 C, CO_Ac), 162.0

(NHCO), 133.1 (CH=_All), 118.3 (=CH_2All), 99.5 (C-1), 92.3 (CCl₃),

71.3 (C-5), 70.4 (CH_2All), 69.3 (C-3), 66.7 (C-4), 61.3 (C-6), 53.2

(C-2), 20.7, 20.5 (3 C, CH_3Ac) ppm. HRMS (ESI⁺): calcd. for

C₁₇H₂₂Cl₃NO₉Na [M + Na]⁺512.0258; found 512.0239.

1

Data for Compound 3: t_R= 9.0 min. H NMR (400 MHz, D₂O): δ

= 5.29 (d, J_1,2= 1.4 Hz, 1 H, 1_D-H), 4.90 (dd_o, J_2,3= 3.3, J_3,4

9.9 Hz, 1 H, 3_C-H), 4.90 (d, J_1,2= 1.8 Hz, 1 H, 1_C-H), 4.38 (d, J_1,2

= 8.0 Hz, 1 H, 1_A-H), 4.35 (d, J_4,5= 1.2 Hz, 1 H, 5_A-H), 4.28 (dd,

=

Allyl 3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-galacto-

pyranoside (38): Allyl glycoside 36 (2.02 g, 4.11 mmol) was dis-

solved in anhydrous MeOH (20.6 mL), and NaOMe (0.5 m in

MeOH; 2.47 mL, 1.24 mmol, 0.3 equiv.) was added. The solution

was stirred at room temperature for 2 h. TLC (Chex/EtOAc, 6:4)

showed the conversion of the starting material into a more polar

product. The reaction was quenched with Dowex-H⁺resin, the

mixture was filtered, and the solvents were evaporated to give inter-

mediate triol 37 quantitatively as a white solid. The crude material

was dissolved in anhydrous DMF (41 mL), and the solution was

cooled to –10 °C. Benzyl bromide (4.4 mL, 37.0 mmol, 9.0 equiv.)

and NaH (60% in oil, 0.99 g, 24.7 mmol, 6.0 equiv.) were success-

ively added. The reaction mixture was stirred under Ar with the

temperature kept below 0 °C. After 1.5 h, TLC (Tol/EtOAc, 9:1)

showed the conversion of the triol into a major less polar com-

pound. The reaction was quenched by adding the minimum

amount of MeOH. The solution was diluted with EtOAc and

washed with water and brine. The organic phase was dried with

anhydrous Na₂SO₄, filtered, and concentrated. The residue was

purified by flash chromatography (Tol/EtOAc, 95:5 to 9:1) to give

J_3,4= 2.8 Hz, 1 H, 4_A-H), 4.12 (dd, 1 H, 2_C-H), 4.03 (dd, J_2,3

=

3.1 Hz, 1 H, 2_D-H), 3.83 (dt_po, J = 6.8, J = 9.8 Hz, 1 H, OCH_2Pr),

3.81–3.76 (m, 2 H, 3_A-H, 3_D-H), 3.74 (dq, J_4,5= 9.6, J_5,6= 6.2 Hz,

1 H, 5_C-H), 3.59–3.52 (m, 3 H, 4_C-H, 5_D-H, OCH_2Pr), 3.50 (dd,

J_2,3= 10.0 Hz, 1 H, 2_A-H), 3.41 (pt, J_3,4= J_4,5= 9.7 Hz, 1 H, 4_D-

H), 2.09 (s, 3 H, CH_3Ac), 1.56 (sext, 2 H, CH_2Pr), 1.21 (d, 3 H, 6_C-

H), 1.18 (d, J_5,6= 6.2 Hz, 3 H, 6_D-H), 0.84 (t, 3 H, CH_3Pr) ppm.

¹³C NMR (100 MHz, D₂O): δ = 176.2 (CO_Ac), 174.1 (C-6_A), 104.9

(¹J_CH= 162.9 Hz, C-1_A), 104.3 (¹J_CH= 172.3 Hz, C-1_C), 102.5

(¹J_CH= 173.4 Hz, C-1_D), 81.3 (C-2_D), 79.3 (C-4_A), 76.2 (C-3_C),

75.5 (C-3_A), 75.4 (C-5_A), 74.9 (OCH_2Pr), 74.5 (C-4_D), 72.8 (C-2_A),

72.4, 72.3 (2 C, C-3_D, C-4_C), 71.9 (C-5_D), 71.6 (C-5_C), 70.7 (C-2_C),

24.7 (CH_2Pr), 23.1 (CH_3Ac), 19.3, 19.2 (2 C, C-6_D, C-6_C), 12.2

(CH_3Pr) ppm. HRMS (ESI⁺): calcd. for C₂₃H₃₈O₁₆Na [M + Na]⁺

593.2057; found 593.2044.

1

Data for Compound 33: t_R= 9.4 min. H NMR (400 MHz, D₂O):

δ = 5.25 (d, 1 H, 1_D-H), 5.11 (d, J_1,2= 1.7 Hz, 1 H, 2_C-H), 4.91

(d, 1 H, 1_C-H), 4.34 (d, J_1,2= 7.9 Hz, 1 H, 1_A-H), 4.25 (s, 1 H, 5_A- fully protected 38 (2.24 g, 86%) as a white solid. R_f= 0.44 (Tol/

H), 4.24 (dd, 1 H, 4_A-H), 3.99 (dd, J_1,2= 1.8, J_2,3= 2.9 Hz, 1 H,

2_D-H), 3.87 (dd, J_2,3= 3.4, J_3,4= 9.8 Hz, 1 H, 3_C-H), 3.80 (dt_po, J

EtOAc, 9:1). ¹H NMR (400 MHz, CDCl₃): δ = 7.41–6.26 (m, 15

H, H_Ar), 6.92 (d, J_NH,2= 7.0 Hz, NH), 5.88 (m, 1 H, CH=_All), 5.27

= 6.8, J = 9.7 Hz, 1 H, OCH_2Pr), 3.77 (dd_o, 1 H, 3_D-H), 3.74 (dd_po

,

(m, J_trans= 17.3, J_gem= 1.6 Hz, 1 H, =CH_2All), 5.18 (m, J_cis=

J_3,4= 3.0 Hz, 1 H, 3_A-H), 3.68 (dq, J_3,4= 9.6 Hz, 1 H, 5_C-H), 3.44

(dq_po, 1 H, 5_D-H), 3.50 (dt_o, 1 H, OCH_2Pr), 3.46 (dd, J_2,3= 9.9 Hz,

10.4 Hz, 1 H, 1 H,=CH_2All), 4.99 (d, J_1,2= 8.1 Hz, 1 H, 1-H), 4.92

(d, J = 11.6 Hz, 1 H, H_Bn), 4.68 (d, J = 11.4 Hz, 1 H, H_Bn), 4.62

1 H, 2_A-H), 3.38 (t, 1 H, 4_C-H), 3.34 (t, J_3,4= J_4,5= 9.8 Hz, 1 H, (d, 1 H, H_Bn), 4.56 (d, 1 H, H_Bn), 4.52 (d, J = 11.9 Hz, 1 H, H_Bn),

4_D-H), 2.05 (s, 3 H, CH_3Ac), 1.53 (sext, J = 7.2 Hz, 2 H, CH_2Pr), 4.47 (d, 1 H, H_Bn), 4.35 (m_o, 1 H, H_All), 4.32 (dd, J_2,3= 10.9, J_3,4

1.19 (d, J_5,6= 6.2 Hz, 3 H, 6_C-H) 1.16 (d, J_5,6= 6.2 Hz, 3 H, 6_D- = 2.8 Hz, 1 H, 3-H), 4.09 (m, 1 H, H_All), 4.04 (d, 1 H, 4-H), 3.85

H), 0.81 (t, J = 7.4 Hz, 3 H, CH₃) ppm. ¹³C NMR (100 MHz,

D₂O): δ = 175.8 (CO_Ac), 174.6 (C-6_A), 104.9 (¹J_CH= 161.4 Hz, C-

(ddd, 1 H, 2-H), 3.72–3.62 (m, 3 H, 5-H, 6a-H, 6b-H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 161.9 (NHCO), 138.4, 137.9, 137.5

1_A), 102.3 (¹J_CH= 175.9 Hz, C-1_C), 101.8.1 (¹J_CH= 174.6 Hz, C- (3 C, C_IVAr), 133.7 (CH=_All), 126.6–127.6 (15 C, C_Ar), 117.8

1_D), 81.5 (C-2_D), 79.2 (C-4_A), 75.7 (C-5_A), 75.6 (C-3_A), 75.0 (C-

2_C), 74.8 (2 C, C-4_C*, OCH_2Pr), 74.6 (C-4_D*), 72.8 (C-2_A), 72.2 (C-

(=CH_2All), 98.2 (¹J_CH= 162.9 Hz, C-1), 92.6 (CCl₃), 77.2 (C-3),

74.7, 73.6 (2 C, C_Bn), 73.5 (C-5), 72.4 (C_Bn), 72.3 (C-4), 70.3

Eur. J. Org. Chem. 2013, 4085–4106

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4101

L. A. Mulard et al.

FULL PAPER

(CH_2All), 68.6 (C-6), 56.3 (C-2) ppm. HRMS (ESI⁺): calcd. for

4.37 (d, J = 11.4 Hz, 1 H, H_Bn), 4.44 (d, J = 12.1 Hz, 1 H, H_Bn),

4.33–4.25 (m, 3 H, 2 H_Bn, 3-H), 4.13 (m, 1 H, 5-H), 3.96 (ddd, J_2,3

= 10.8 Hz, 1 H, 2-H), 3.91 (d, J_3,4= 2.3 Hz, 1 H, 4-H), 3.77 (bd,

1 H, 4Ј-H), 3.66 (dd, J_3,4= 2.4 Hz, 1 H, 3Ј-H), 3.63 (m, 1 H, 5Ј-

C₃₂H₃₄Cl₃NO₆Na [M + Na]⁺656.1349; found 656.1321.

3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-α/β-

anose (39) and (3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-α-

-galactopyranosyl)-(1↔1)-3,4,6-tri-O-benzyl-2-deoxy-2-trichloro-

acetamido-β- -galactopyranoside (49)

D-galactopyr-

H), 3.53–3.46 (m, 2 H, 6a-H, 6aЈ-H), 3.38 (dd, J_5,6b= 5.3, J_6a,6b

=

D

9.0 Hz, 1 H, 6bЈ-H), 3.29 (dd, J_5,6b= 4.2, J_6a,6b= 9.6 Hz, 1 H, 6b-

H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 162.5 (2 C, NHCO,

NHCOЈ), 138.4–137.4 (6 C, C_IVAr), 128.8–127.5 (30 C, C_Ar), 95.3

(C-1), 93.2 (C-1Ј), 92.8, 92.6 (2 C, CCl₃), 77.8 (C-3Ј), 76.6 (C-3)

74.6, 74.2, 73.9 (3 C, C_Bn), 73.8 (C-5Ј), 73.5 (C_Bn), 72.3 (2 C, C-4Ј,

C_Bn), 72.2 (C_Bn), 72.1 (C-4), 71.0 (C-5), 70.1 (C-6), 68.1 (C-6Ј), 55.7

( C - 2 ) , 5 0 . 6 ( C - 2 Ј ) p p m . H R M S ( E S I ⁺) : c a l c d . f o r

C₅₈H₅₈Cl₆N₂O₁₁Na [M + Na]⁺1191.2069; found 1191.1906.

D

Route 1: (1,5-Cyclooctadiene)bis(methyldiphenylphosphane)irid-

ium hexafluorophosphate (82 mg, 100 μmol, 0.03 equiv.) was dis-

solved in anhydrous THF (17 mL), and hydrogen was bubbled

through the solution for 15 min (H-cube, full H₂mode). The re-

sulting yellow solution was concentrated to dryness. The residue

was dissolved in anhydrous THF (17 mL), and the resulting solu-

tion was poured into a solution of allyl glycoside 38 (2.14 g,

3.36 mmol) in anhydrous THF (17 mL). The mixture was stirred

under Ar at room temperature for 2 h. TLC (Tol/EtOAc, 9:1) indi-

cated the conversion of the starting glycoside into a less polar inter-

mediate. A solution of iodine (1.71 g, 6.73 mmol, 2.0 equiv.) in

THF/H₂O (4:1, 20 mL) was added, and the mixture was stirred

at room temperature for 1 h. TLC (Tol/EtOAc, 9:1) showed the

conversion of the intermediate compound into a more polar prod-

uct. The reaction was quenched with sodium bisulfite (10% aq.).

The mixture was concentrated to a third of its volume, and the

aqueous phase was extracted three times with CH₂Cl₂. The organic

phases were combined, washed with brine, dried by passing

through a phase-separator filter, and concentrated to dryness. The

residue was purified by flash chromatography (Chex/EtOAc, 75:25

to 7:3) to give hemiacetal 39 (1.86 g, 93%) as a yellow solid.

3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-α/β-D-galactopyr-

anosyl Trichloroacetimidate (40):^[52]CCl₃CN (834 μL, 8.3 mmol,

5.0 equiv.) and DBU (75 μL, 500 μmol, 0.3 equiv.) were successively

added to a solution of hemiacetal 39 (990 mg, 1.66 mmol) in anhy-

drous DCE (8.3 mL). The mixture was stirred at room temperature

under Ar for 1 h. TLC (Chex/EtOAc, 8:2) indicated the complete

conversion of the starting hemiacetal into a less polar product. Fol-

lowing concentration to a third of its volume (reduced pressure,

room temperature), the reaction mixture was directly purified by

flash chromatography (Chex/EtOAc, 8:2 + 1% Et₃N) to give a 9:1

α/β mixture of trichloroacetimidate 40 (1.16 g, 94%) as a pale yel-

low oil. The analytical data were as published.^[52]

3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-α/β-

anosyl N-Phenyltrifluoroacetimidate (41) and 2-Trichloromethyl-

(3,4,6-tri-O-benzyl-1,2-dideoxy-α- -galactopyrano)-[2,1-d]-2-ox-

D-galactopyr-

Route 2: NIS (737 mg, 3.28 mmol, 1.5 equiv.) and TfOH (48 μL,

0.55 mmol, 0.25 equiv.) were added at 0 °C to a solution of thio-

phenyl glycoside 48 (1.50 g, 2.18 mmol) in CH₂Cl₂(22 mL) and

water (2.2 mL). The reaction mixture was stirred at that tempera-

ture for 0.5 h. TLC (Chex/EtOAc, 7:3) showed the conversion of

the starting material into a less polar intermediate. The reaction

was quenched by the addition of Et₃N. The organic phase was

washed with brine, dried by passing through a phase-separator fil-

ter, and concentrated to dryness. The residue was purified by flash

chromatography (Chex/EtOAc, 75:25 to 7:3) to give, in order of

elution, first α-(1↔1)-β-linked disaccharide 49 (84 mg, 3%) as a

yellow oil, and then target hemiacetal 39 (998 mg, 77%) as a 9:1 α/

β mixture, isolated as a white solid.

D

azoline (42): Hemiacetal 39 (1.85 g, 3.11 mmol) was dissolved in

acetone (31 mL). N-Phenyltrifluoroacetimidoyl chloride (1.29 g,

6.22 mmol, 2.0 equiv.) and Cs₂CO₃(1.12 g, 3.42 mmol, 1.1 equiv.)

were added to the solution. The mixture was stirred at room tem-

perature for 4 h. After that time, TLC (Chex/EtOAc, 8:2) indicated

the complete transformation of the starting material into a less

polar product. The reaction mixture was filtered, and the filtrate

was concentrated. The residue was purified by flash chromatog-

raphy (Chex/EtOAc, 85:15 to 7:3 + 1% Et₃N) to give, in order of

elution, first oxazoline 42 (83 mg, 5%) and then PTFA 41 (2.10 g,

88%), both as pale yellow oils.

Data for Donor 41: R_f= 0.44 (Chex/EtOAc, 8:2). ¹H NMR

(100 MHz, CDCl₃): δ = 7.61–7.09 (m, 18 H, H_Ar), 6.77 (d, J =

7.6 Hz, 2 H, H_Ar), 6.48 (d_o, J_NH,2= 7.7 Hz, 1 H, NH), 6.47 (bs_o, 1

H, 1-H), 4.97 (d, J = 11.4 Hz, 1 H, H_Bn), 4.76 (d_po, J = 11.9 Hz, 1

H, H_Bn), 4.73 (m, 1 H, 2-H), 4.64 (d, 1 H, H_Bn), 4.57–4.48 (m, 3

Data for α-Hemiacetal 39: R_f= 0.23 (Tol/EtOAc, 7:3). ¹H NMR

(400 MHz, CDCl₃): δ = 7.40–6.25 (m, 15 H, H_Ar), 6.81 (d, J_NH,2

=

8.9 Hz, 1 H, NH), 5.38 (pt, J_OH,1= J_1,2= 3.5 Hz, 1 H, 1-H), 4.97

(d, J = 11.6 Hz, 1 H, H_Bn), 4.72 (d, J = 11.9 Hz, 1 H, H_Bn), 4.65

(m, 1 H, 2-H), 4.57 (d, 1 H, H_Bn), 4.56 (d, 1 H, H_Bn), 4.53 (d, J =

12.2 Hz, 1 H, H_Bn), 4.45 (d, 1 H, H_Bn), 4.15 (pt, 1 H, 5-H), 3.98

(bd, 1 H, 4-H), 3.80 (dd, J_2,3= 10.8, J_3,4= 2.5 Hz, 1 H, 3-H), 3.67

H, H_Bn), 4.20 (bs, 1 H, 4-H), 4.07 (pt, 1 H, 5-H), 3.88 (dd, J_2,3

=

11.0, J_3,4= 1.4 Hz, 1 H, 3-H), 3.72 (pt, J_5,6a= 7.8 Hz, 1 H, 6a-H),

3.62 (dd, J_5,6b= 5.7, J_6a,6b= 9.1 Hz, 1 H, 6b-H) ppm. ¹³C NMR

(100 MHz, CDCl₃): δ = 161.8 (NHCO), 143.1, 138.0, 137.7, 137.0

(4 C, C_IVAr), 129.4–119.3 (20 C, C_Ar), 94.3 (¹J_CH= 162.9 Hz, C-1),

92.3 (CCl₃), 75.5 (C-3), 74.8, 73.7 (2 C, C_Bn), 72.5 (C-5), 71.6 (C-

4), 71.3 (C_Bn), 68.1 (C-6), 50.6 (C-2) ppm.

(dd, J_OH,2= 1.6 Hz, 1 H, 1-OH), 3.62 (dd, J_5,6a= 6.8, J_6a,6b

=

9.6 Hz, 1 H, 6a-H), 3.50 (dd, J_5,6b= 5.8 Hz, 1 H, 6b-H) ppm. ¹³C

NMR (100 MHz, CDCl₃): δ = 161.7 (NHCO), 138.1, 137.5 (3 C,

C_IVAr), 128.6–127.5 (15 C, C_Ar), 92.7 (CCl₃), 91.6 (C-1), 76.8 (C-

3), 74.4, 73.6 (2 C, C_Bn), 72.5 (C-4), 71.8 (C_Bn), 69.9 (C-5), 69.5

(C-6), 51.4 (C-2) ppm. HRMS (ESI⁺): calcd. for C₂₉H₃₀Cl₃NO₆Na

[M + Na]⁺616.1036; found 616.1080.

Data for Oxazoline 42: R_f= 0.46 (Chex/EtOAc, 8:2). ¹H NMR

(400 MHz, CDCl₃): δ = 7.46–7.27 (m, 15 H, H_Ar), 6.28 (d, J_1,2

=

Data for Disaccharide 49: R_f= 0.44 (Tol/EtOAc, 7:3). ¹H NMR

6.6 Hz, 1 H, 1-H), 4.95 (d, J = 11.6 Hz, 1 H, H_Bn), 4.92 (d, J =

(400 MHz, CDCl₃): δ = 7.50 (d, J_NH,2= 6.7 Hz, 1 H, NHЈ), 7.29– 12.2 Hz, 1 H, H_Bn), 4.77 (d, 1 H, H_Bn), 4.64 (d, 1 H, H_Bn), 4.51 (d,

7.11 (m, 30 H, H_Ar), 6.69 (d, J_NH,2= 9.5 Hz, 1 H, NH), 5.44 (d,

J_1,2= 3.8 Hz, 1 H, 1Ј-H), 5.30 (d, J_1,2= 8.8 Hz, 1 H, 1-H), 4.84

(d, J = 11.6 Hz, 1 H, H_Bn), 4.79 (d, J = 11.6 Hz, 1 H, H_Bn), 4.63

(ddd, J_2,3= 10.6 Hz, 1 H, 2Ј-H), 4.58 (d, J = 11.8 Hz, 1 H, H_Bn),

4.50 (d, J = 11.6 Hz, 1 H, H_Bn), 4.48 (d, J = 11.6 Hz, 2 H, H_Bn),

4.43 (d, J = 11.7 Hz, 1 H, H_Bn), 4.38 (d, J = 12.1 Hz, 1 H, H_Bn),

J = 11.8 Hz, 1 H, H_Bn), 4.64 (d, 1 H, H_Bn), 4.40 (pt, 1 H, 2-H),

4.07 (dt, J_4,5= 1.7, J_5,6a= J_5,6b= 6.5 Hz, 1 H, 5-H), 3.98 (pt, 1 H,

4-H), 3.66 (d, 2 H, 6a-H, 6b-H), 3.54 (dd, J_2,3= 7.5, J_3,4= 2.6 Hz,

1 H, 3-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 162.2 (NCO),

138.2, 137.9, 137.7 (3 C, C_IVAr), 128.4–127.7 (15 C, C_Ar), 106.8 (C-

1), 92.3 (CCl₃), 79.3 (C-3), 74.4 (C_Bn), 73.6 (C-5), 73.5, 72.0 (2 C,

4102

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Eur. J. Org. Chem. 2013, 4085–4106

O-Antigen from a Shigella flexneri Serotype 6

C_Bn), 71.6 (C-4), 68.2 (C-6), 67.3 (C-2) ppm. HRMS (ESI⁺): calcd. Phenyl 3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-1-thio-β-

D-

for C₂₉H₂₈Cl₃NO₅Na [M + Na]⁺598.0931; found 598.0910.

galactopyranoside (48): A solution of thioglycoside 46 (5.00 g,

9.21 mmol) in anhydrous MeOH (100 mL) was treated with

MeONa (0.5 m in MeOH; 311 μL, 1.44 mmol, 0.2 equiv.). The solu-

tion was stirred at room temperature under Ar for 2 h. TLC (Chex/

EtOAc, 6:4, CH₂Cl₂/MeOH, 8:2) showed the complete conversion

of thioglycoside 46 into a more polar product. The reaction was

quenched with Dowex-H⁺resin. The resin was filtered, and the

filtrate was concentrated to give crude phenyl 2-deoxy-2-tri-

chloroacetamido-1-thio-β-d-galactopyranoside^[54](47, 3.83 g)

quantitatively. This residue was dissolved in anhydrous DMF

(74 mL), the solution was cooled to –10 °C, and benzyl bromide

(6.6 mL, 55.3 mmol, 6.0 equiv.) and NaH (60 % in oil; 2.21 g,

55.3 mmol, 6.0 equiv.) were successively added. The mixture was

stirred under Ar keeping the temperature below 0 °C. After 1.5 h,

TLC (Tol/EtOAc, 95:5) showed the conversion of triol 47 into a

major less polar compound. The reaction was quenched with the

minimum amount of MeOH. The reaction mixture was diluted

with EtOAc, and washed with water and brine. The organic phase

was dried with anhydrous Na₂SO₄, filtered, and concentrated. The

residue was purified by flash chromatography (Tol/EtOAc, 98:2 to

95:5) to give target tri-O-benzyl derivative 48 (4.90 g, 77%) as a

white solid. R_f= 0.48 (Tol/EtOAc, 95:5). ¹H NMR (400 MHz,

CDCl₃): δ = 7.56–7.22 (m, 2 H, H_Ar), 7.40–7.20 (m, 18 H, H_Ar),

6.84 (d, J_NH,2= 7.5 Hz, 1 H, NH), 5.30 (d, J_1,2= 10.2 Hz, 1 H, 1-

H), 4.90 (d, J = 11.4 Hz, 1 H, H_Bn), 4.68 (d, J = 11.2 Hz, 1 H,

H_Bn), 4.58 (d, 1 H, H_Bn), 4.54 (d, 1 H, H_Bn), 4.53 (d, J = 11.8 Hz,

1 H, H_Bn), 4.48 (d, 1 H, H_Bn), 4.28 (dd, J_2,3= 10.5, J_3,4= 2.7 Hz,

1 H, 3-H), 4.09 (d, 1 H, 4-H), 3.94 (pdt, 1 H, 2-H), 3.78–3.68 (m,

3 H, 5-H, 6a-H, 6b-H) ppm. ¹³C NMR (100 MHz, CDCl₃): δ =

161.7 (NHCO), 138.4, 137.8, 137.3 (3 C, C_IVAr), 132.8 (2 C, C_Ar),

132.2 (C_IVAr), 130.0–127.6 (18 C, C_Ar), 92.5 (CCl₃), 84.3 (C-1),

78.2 (C-3), 77.6 (C-5), 74.5, 73.6 (2 C, C_Bn), 72.4 (C-4), 72.3

(CBn), 68.4 (C-6), 53.7 (C-2) ppm. HRMS (ESI⁺): calcd. for

C₃₅H₃₄Cl₃NO₅SNa [M + Na]⁺708.1121; found 708.1128.

Phenyl 3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-1-thio-β-D-

galactopyranoside (46)^[53]

Route 1: Thiophenol (3.2 mL, 31.3 mmol, 2 equiv.) and BF₃·OEt₂

(12.0 mL, 93.9 mmol, 6 equiv.) were added at room temperature to

a solution of β-acetate 35 (7.71 g, 15.6 mmol) in anhydrous CH₂Cl₂

(150 mL). The mixture was stirred at this temperature for 2.5 h

under Ar. TLC (Chex/EtOAc, 7:3) showed the complete conversion

of the starting material into a less polar product. The reaction was

quenched with NaHCO₃(satd. aq.). CH₂Cl₂was added, and the

phases were separated. The organic phase was washed with H₂O

and brine, then dried by passing through a phase-separator filter,

and concentrated to dryness. The residue was purified by flash

chromatography (Chex/EtOAc, 9:1 to 1:1) to give thioglycoside 46

(7.43 g, 87%) as a white solid.

Route 2: NaOMe (25% in MeOH, 76.9 mL, 336.1 mmol, 3.0 equiv.)

was slowly added to a solution of d-galactosamine hydrochloride

(34; 24.16 g, 112.0 mmol) in anhydrous MeOH (482 mL) stirred at

0 °C under Ar. After 15 min, trichloroacetic anhydride (30.7 mL,

168.1 mmol, 1.5 equiv.) was added dropwise, keeping the reaction

temperature close to 0 °C. The reaction mixture was stirred for 2 h,

until TLC (iPrOH/H₂O/NH₃, 4:1:0.5) showed the complete conver-

sion of the starting material into a less polar product. The reaction

was quenched with Dowex-H⁺resin, the resin was removed by fil-

tration, and the volatiles were evaporated to dryness. The residue

was dissolved in anhydrous pyridine (224 mL), and acetic anhy-

dride (100 mL) was slowly added at 0 °C over 30 min. The reaction

mixture was stirred at room temperature for 16 h. After this time,

TLC (CH₂Cl₂/EtOAc, 9:1) indicated that the intermediate had been

converted into less polar compounds corresponding to the α and β

anomers of the furanose (43, 7% and 44, 11%) and pyranose forms

(45, 51% and 35, 31%) of the peracylated galactosamine, as ascer-

tained by NMR spectroscopy. The solvents were evaporated and

co-evaporated three times with toluene. The residue was dissolved

in CH₂Cl₂(500 mL), and the resulting solution was washed with

water (150 mL), HCl (10 % aq.; 150 mL), NaHCO₃(satd. aq.;

100 mL) and brine (3ϫ 50 mL). The organic phase was dried by

passing through a phase-separator filter, and concentrated to dry-

ness. Thiophenol (23.0 mL, 224 mmol, 2.0 equiv.) and BF₃·OEt₂

(42.8 mL, 336.1 mmol, 3.0 equiv.) were added at room temperature

to a solution of the residue in anhydrous CH₂Cl₂(390 mL). The

mixture was stirred at this temperature under Ar for 16 h. TLC

(Tol/EtOAc, 7:3) showed the conversion of the intermediates into

a major more polar product. The reaction was quenched with

NaHCO₃(satd. aq.; 200 mL). CH₂Cl₂(500 mL) was added, and

the phases were separated. The organic phase was washed with

H₂O (150 mL) and brine (150 mL), then dried by passing through

a phase-separator filter, and concentrated to dryness. The residue

was purified by flash chromatography (Chex/EtOAc, 9:1 to 1:1) to

give thioglycoside 46 (39.0 g, 64%) as a white solid. R_f= 0.35 (Tol/

EtOAc, 7:3). ¹H NMR (400 MHz, CDCl₃): δ = 7.57–7.32 (m, 5 H,

H_Ar), 6.75 (d, J_NH,2= 9.1 Hz, 1 H, NH), 5.42 (d, 1 H, 4-H), 5.32

(dd, J_2,3= 10.6, J_3,4= 3.2 Hz, 1 H, 3-H), 4.88 (d, J_1,2= 10.3 Hz, 1

The analytical data for triol 47 were as published.^[54]

Benzyl (3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-β-

lactopyranosyl)-(1Ǟ2)-(4-O-benzyl-3-O-p-methoxybenzyl-α-

rhamnopyranosyl)-(1Ǟ2)-(3,4-di-O-benzyl-α- -rhamnopyranosyl)-

(1Ǟ4)-(allyl 2,3-Di-O-benzyl-β- -galactopyranosid)uronate (50) and

Benzyl (3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-α-

galactopyranosyl)-(1Ǟ2)-(4-O-benzyl-3-O-p-methoxybenzyl-α-

rhamnopyranosyl)-(1Ǟ2)-(3,4-di-O-benzyl-α-

(1Ǟ4)-(allyl 2,3-di-O-benzyl-β- -galactopyranosid)uronate (51)

D-ga-

L

-

L

D

-

L

-rhamnopyranosyl)-

D

Route 1: A mixture of acceptor 27 (300 mg, 253 μmol), thioglyco-

side 48 (260 mg, 379 μmol, 1.5 equiv.), and freshly activated 4 Å

powdered MS (750 mg) in anhydrous CH₂Cl₂(2.9 mL) was stirred

at room temperature under Ar for 1 h. The reaction mixture was

cooled to –78 °C, then NIS (114 mg, 505 μmol, 2.0 equiv.) and

TMSOTf (4.6 μL, 25 μmol, 0.1 equiv.) were added. The reaction

mixture was stirred for 30 min, while the bath was allowed to reach

–60 °C. TLC (Tol/EtOAc, 9:1) indicated the absence of acceptor 27

and the presence of a less polar product. The reaction was

quenched by the addition of Et₃N. The resulting suspension was

filtered and concentrated. The residue was purified by flash

chromatography (Tol/EtOAc, 98:2 to 92:8) to give tetrasaccharide

50 (356 mg, 80%) as a white foam. Traces of silylated acceptor 52

were also recovered.

H, 1-H), 4.20 (dd_o, 1 H, 6a-H), 4.19 (ddd_o, 1 H, 2-H), 4.16 (dd_po

,

J_5,6b= 6.1, J_6a,6b= 11.4 Hz, 1 H, 6b-H), 3.99 (dt, J_4,5= 0.9 Hz, 1 H,

5-H), 2.15, 2.06, 2.00 (3 s, 9 H, CH3_Ac) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ = 170.4, 170.3, 170.0 (3 C, CO), 161.7 (NHCO), 133.3

(2 C, C_Ar), 132.1 (C_IVAr), 129.0, 128.4 (3 C, C_Ar), 92.3 (CCl₃), 86.7

Route 2: A mixture of acceptor 27 (203 mg, 168 μmol), PTFA 41

(C-1), 74.7 (C-5), 70.6 (C-3), 66.9 (C-4), 61.7 (C-6), 51.4 (C-2), 20.7, (194 mg, 253 μmol, 1.5 equiv.), and powdered 4 Å MS (500 mg) in

20.6, 20.5 (3 C, CH_{3 A c}) ppm. HRMS (ESI⁺): calcd. for anhydrous CH₂Cl₂(3.4 mL) was stirred at room temperature under

C₂₀H₂₂Cl₃NO₈SNa [M + Na]⁺564.0029; found 564.0087.

Ar for 1 h. The suspension was cooled to –78 °C, and TMSOTf

Eur. J. Org. Chem. 2013, 4085–4106

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4103

L. A. Mulard et al.

FULL PAPER

(1.5 μL, 8 μmol, 0.05 equiv.) was added. The reaction mixture was

stirred at this temperature for 20 min, and Et₃N was added since

TLC (Tol/EtOAc, 9:1) had shown the presence of a new major com-

pound, whereas neither acceptor nor donor remained. The solids

were removed by filtration, and the filtrate was concentrated to

dryness. The residue was purified by flash chromatography (Tol/

(CH=_All), 130.4 (C_IVAr), 129.3–127.4 (47 C, C_Ar), 117.5 (=CH_2All),

113.7 (2 C, C_ArPMB), 102.7 (¹J_CH= 160.2 Hz, C-1_A), 99.7 (¹J_CH

=

172.0 Hz, C-1_D), 98.3 (¹J_CH= 172.4 Hz, C-1_C), 96.0 (¹J_CH

175.2 Hz, C-1_B), 92.9 (CCl₃), 80.5 (C-4_C), 80.2 (2 C, C-3_A, C-4_D),

79.4 (C-3_D), 78.5 (C-2_A), 77.2 (2 C, C-3_C, C-3_B), 75.4, 75.3 (2 C,

C_Bn), 75.1 (C-2_D), 74.9, 74.5 (2 C, C_Bn), 73.6 (C-5_A), 73.6, 73.1 (2

EtOAc, 95:5 to 85:15) to give, in order of elution, the product of C, C_Bn), 72.8 (C-5_B), 72.4 (2 C, C-2_C, C_Bn), 72.1 (C-4_B), 71.2 (C_Bn),

α-glycosylation 51 (19 mg, 9%) and desired tetrasaccharide 50

(186 mg, 63%), both as pale yellow foams.

Data for β-Glycoside 50: R_f= 0.38 (Tol/EtOAc, 9:1) ¹H NMR

70.9 (CH_2All), 70.6 (C_Bn), 70.1 (C-4_A), 68.6 (2 C, C-6_B, C-5_D), 68.3

(C-5_C), 67.3 (C_CO2Bn), 55.1 (CH_3PMB), 50.9 (C-2_B), 18.2, 18.1 (2 C,

C-6_D, C-6_C) ppm. HRMS (ESI⁺): calcd. for C₁₀₀H₁₀₆Cl₃NO₂₁Na

[M + Na]⁺1784.6221; found 1784.6265.

(100 MHz, CDCl₃): δ = 7.41–7.05 (m, 47 H, H_Ar), 6.91 (d, J_NH,2

=

7.5 Hz, 1 H, NH), 6.83 (d, J = 8.6 Hz, 2 H, H_ArPMB), 5.97 (m, 1

DataforSilylatedAcceptor52:¹HNMR(400 MHz,CDCl₃):δ=7.39–

H, CH=_All), 5.35 (m, J_trans= 17.3, J_gem= 1.6 Hz, 1 H, =CH_2All), 7.20 (m, 32 H, H_Ar), 6.85 (m, J = 8.6 Hz, 2 H, H_ArPMB), 5.98 (m,

5.26 (bs, 1 H, 1_D-H), 5.25 (d_po, 1 H, H_CO2Bn), 5.21 (m, J_cis1 H, CH=_All), 5.35 (m, J_trans= 17.3, J_gem= 1.6 Hz, 1 H, =CH_2All),

10.5 Hz, 1 H, =CH_2All), 5.10 (d, J = 12.2 Hz, 1 H, H_CO2Bn), 4.98– 5.32 (d, J_1,2= 1.8 Hz, 1 H, 1_D-H), 5.27 (d, 1 H, H_CO2Bn), 5.21 (m,

=

4.92 (m, 4 H, 1_B-H, 1_C-H, 2 H_Bn), 4.90 (d_po, J = 10.9 Hz, 1 H,

_Bn), 4.84 (d_po, J = 11.1 Hz, 1 H, H_Bn), 4.76 (d, J = 12.1 Hz, 1 H,

J_cis= 10.5 Hz, 1 H, =CH_2All), 5.11 (d, J = 12.3 Hz, 1 H, H_CO2Bn),

4.92 (d, J = 10.8 Hz, 1 H, H_Bn), 4.90 (2d_o, J = 11.0 Hz, 2 H, H_Bn),

4.83 (d, J_1,2= 1.8 Hz, 1 H, 1_C-H), 4.81 (d, J = 12.1 Hz, 1 H, H_Bn),

4.73 (d, J = 10.9 Hz, 1 H, H_Bn), 4.71–4.57 (m, 7 H, H_Bn), 4.50 (m,

1 H, H_All), 4.39 (bd_po, 1 H, 4_A-H), 4.38 (d_po, J_1,2= 7.7 Hz, 1 H,

1_A-H), 4.16 (m_po, 1 H, H_All), 4.13 (dd_o, J_2,3= 3.0 Hz, 1 H, 2_D-H),

4.09 (dd, 1 H, 2_C-H), 4.02 (d, J_4,5= 1.0 Hz, 1 H, 5_A-H), 3.85 (dd,

J_3,4= 9.4 Hz, 1 H, 3_D-H), 3.80–3.73 (m, 5 H, 3_C-H, 5_C-H,

CH_3PMB), 3.73–3.67 (m, 2 H, 2_A-H, 5_D-H), 3.53 (pt_po, J_3,4= 9.4,

H

H_Bn), 4.72 (d_po, J = 10.5 Hz, 1 H, H_Bn), 4.71–4.67 (m, 2 H, H_Bn),

4.65–4.55 (m, 3 H, H_Bn), 4.58–4.45 (m, 6 H, 5 H_Bn, H_All), 4.36 (d_o,

J_1,2= 7.7 Hz, 1 H, 1_A-H), 4.34 (bd_o, 1 H, 4_A-H), 4.27 (d, J =

11.6 Hz, 1 H, H_Bn), 4.22 (d, J = 11.6 Hz, 1 H, H_Bn), 4.18–4.10 (m,

2 H, 2_B-H, H_All), 4.08 (pt, 1 H, 2_C-H), 4.03–3.96 (m, 4 H, 5_A-H,

3_B-H, 4_B-H, 2_D-H), 3.87 (dd, J_2,3= 3.0, J_3,4= 9.6 Hz, 1 H, 3_C-H),

3.81 (dd_po, J_2,3= 3.0, J_3,4= 9.5 Hz, 1 H, 3_D-H), 3.79 (dq_po, 1 H,

5_C-H), 3.71 (s, 3 H, CH_3PMB), 3.70–3.59 (m, 3 H, 2_A-H, 6a_B-H, 5_D- J_4,5= 9.5 Hz, 1 H, 4_C-H), 3.52 (dd_o, J_2,3= 9.9, J_3,4= 2.9 Hz, 1 H,

H), 3.51–3.42 (m, 3 H, 3_A-H, 5_B-H, 4_C-H), 3.34 (pt_po, J_4,5= 9.2 Hz, 3_A-H), 3.40 (pt, J_4,5= 9.6 Hz, 1 H, 4_D-H), 1.29 (d, J_5,6= 6.2 Hz,

1 H, 4_D-H), 3.31 (m_o, 1 H, 6b_B-H), 1.28 (d, J_5,6= 6.2 Hz, 3 H, 6_D- 3 H, 6_D-H), 1.19 (d, J_5,6= 6.2 Hz, 3 H, 6_C-H), 0.09 (s, 9 H, SiMe₃)

H), 1.18 (d, J_5,6= 6.2 Hz, 3 H, 6_C-H) ppm. ¹³C NMR (100 MHz, ppm. ¹³C NMR (100 MHz, CDCl₃): δ = 167.3 (C-6_A), 159.0

CDCl₃): δ = 167.3 (C-6_A), 161.6 (NHCO), 159.4 (C_IVPMB), 138.9, (C_IVPMB), 138.9–135.1 (6 C, C_IVAr), 134.0 (CH=_All), 130.8 (C_IVAr),

138.8, 138.7, 138.6, 138.5, 137.8, 137.7, 135.1 (9 C, C_IVAr), 134.1

129.4–127.4 (32 C, C_Ar), 117.4 (=CH_2All), 113.6 (2 C, C_ArPMB),

(CH=_All), 130.7 (C_IVAr), 129.8–127.3 (47 C, C_Ar), 117.4 (=CH_2All), 102.7 (C-1_A), 102.5 (C-1_C), 100.3 (C-1_D), 80.5 (C-3_A), 80.3, 80.2 (2

113.9 (2 C, C_ArPMB), 102.7 (¹J_CH= 162.0 Hz, C-1_A), 101.3 (¹J_CHC, C-4_C, C-4_D), 79.4, 79.2 (2 C, C-3_C, C-3_D), 78.5 (C-2_A), 75.3, 75.1

= 173.5 Hz, C-1_C), 100.7 (¹J_CH= 162.4 Hz, C-1_B), 100.4 (¹J_CH

175.4 Hz, C-1_D), 92.9 (CCl₃), 80.9 (C-4_C), 80.3 (C-3_A), 80.2 (C-4_D),

=

(2 C, C_Bn), 75.0 (C-2_D), 74.9 (C_Bn), 73.7 (C-5_A), 73.4 (C-4_A), 72.6,

72.3, 72.1 (3 C, C_Bn), 70.6 (CH_2All), 70.4 (C-2_C), 68.8, 68.6 (2 C,

79.2 (C-3_C), 78.9 (C-3_D), 78.8 (C-2_D), 78.4 (C-2_A), 75.8 (C-5_A), C-5_C, C-5_D), 67.3 (C_CO2Bn), 55.2 (CH_3PMB), 18.2 (C-6_D), 17.9 (C-

75.6, 75.3 (2 C, C_Bn), 75.2 (C-2_C), 74.9 (2 C, C_Bn), 74.1 (C-4_A), 73.7

(C-3_B), 73.4 (C_Bn), 73.3 (C-5_B), 72.7, 72.5 (2 C, C_Bn), 72.4 (C-4_B),

72.3, 71.6 (2 C, C_Bn), 70.5 (CH_2All), 68.5 (C-5_D), 68.3 (C-5_C), 68.0

(C-6_B), 67.3 (C_CO2Bn), 55.4 (C-2_B), 55.2 (CH_3PMB), 18.2 (C-6_D),

17.8 (C-6_C) ppm. HRMS (ESI⁺): calcd. for C₁₀₀H₁₀₆Cl₃NO₂₁Na

[M + Na]⁺1784.6221; found 1784.6198.

6_C), 0.4 (3 C, SiMe₃) ppm.

Benzyl (3,4,6-Tri-O-benzyl-2-deoxy-2-trichloroacetamido-β-D-ga-

lactopyranosyl)-(1Ǟ2)-(3-O-acetyl-4-O-benzyl-α-

osyl)-(1Ǟ2)-(3,4-di-O-benzyl-α- -rhamnopyranosyl)-(1Ǟ4)-(allyl

2,3-di-O-benzyl-β- -galactopyranosid)uronate (54): Water (1.6 mL)

and CAN (870 mg, 1.59 mmol, 4.0 equiv.) were added to a solution

L-rhamnopyran-

L

D

Data for α Anomer 51: R_f= 0.40 (Tol/EtOAc, 9:1). ¹H NMR of tetrasaccharide 50 (700 mg, 397 μmol) in MeCN (15.9 mL). The

(400 MHz, CDCl₃): δ = 7.42–7.17 (m, 47 H, H_Ar), 6.83–6.76 (m, 3

H, NH, 2 H_ArPMB), 5.98 (m, 1 H, CH=_All), 5.35 (m, J_trans= 17.3,

J_gem= 1.6 Hz, 1 H, =CH_2All), 5.27 (bd, J_1,2= 1.2 Hz, 1 H, 1_D-H),

reaction mixture was stirred at room temperature for 30 min. After

that time, TLC (Tol/EtOAc, 9:1) showed the complete transforma-

tion of the starting material into a more polar product. The reac-

5.26 (d, J = 12.2 Hz, 1 H, H_CO2Bn), 5.22 (m, J_cis= 10.5 Hz, 1 H, tion was quenched with NaHCO₃(satd. aq.). The reaction mixture

=CH_2All), 5.10 (d, 1 H, H_CO2Bn), 5.02 (d, J = 11.4 Hz, 1 H, H_Bn),

4.94 (d, J = 10.9 Hz, 1 H, H_Bn), 4.90 (d, J = 11.2 Hz, 1 H, H_Bn),

was diluted with water and CH₂Cl₂, and the aqueous phase was

extracted three times with CH₂Cl₂. The combined extracts were

washed with brine, passed through a phase-separator filter, and

concentrated. The resulting crude oil was dissolved in pyridine

4.85 (d_po, J = 10.8 Hz, 1 H, H_Bn), 4.84 (bs_o, 1 H, 1_C-H), 4.79 (d_po

,

J = 12.7 Hz, 1 H, H_Bn), 4.75 (d_po, J = 12.7 Hz, 1 H, H_Bn), 4.74 (d,

J = 10.9 Hz, 1 H, H_Bn), 4.69 (d_po, J = 11.6 Hz, 1 H, H_Bn), 4.68– (20 mL), and excess acetic anhydride (3.75 mL) and DMAP

4.45 (m, 11 H, 9 H_Bn, H_All, 2_B-H), 4.43 (d, J_1,2= 3.5 Hz, 1 H, 1_B- (48 mg, 397 μmol, 1.0 equiv.) were added to the solution whilst stir-

H), 4.40–4.34 (m, 4 H, H_Bn, 1_A-H, 4_A-H, 5_B-H), 4.15 (m, 1 H,

H_All), 4.11 (bd, 1 H, 4_B-H), 4.07 (pt, 1 H, 2_C-H), 4.05 (pt, 1 H, 2_D-

ring at room temperature. After 14 h, TLC (Tol/EtOAc, 9:1) indi-

cated that the intermediate alcohol had been converted into a less

polar product. The volatiles were evaporated and co-evaporated

H), 4.00 (d, J_4,5= 1.0 Hz, 1 H, 5_A-H), 3.85 (dd, J_2,3= 2.8, J_3,4

=

9.5 Hz, 1 H, 3_C-H), 3.81 (dd, J_2,3= 2.8, J_3,4= 9.4 Hz, 1 H, 3_D-H), three times with toluene. The residue was purified by flash

3.75 (dd, J_2,3= 10.6, J_3,4= 2.0 Hz, 1 H, 3_B-H), 3.72–3.66 (6 H, 5_C- chromatography (Tol/EtOAc, 95:5 to 9:1) to give monoacetylated

H, 2_A-H, 6a_B-H, CH_3PMB), 3.64 (dq_po, J_4,5= 9.6 Hz, 1 H, 5_D-H), tetrasaccharide 54 (582 mg, 87% over two steps) as a white foam.

3.52–3.46 (m, 2 H, 3_A-H, 6b_B-H), 3.29 (pt, 1 H, 4_D-H), 3.17 (pt, R_f= 0.38 (Tol/EtOAc, 9:1). ¹H NMR (400 MHz, CDCl₃): δ = 7.41–

J_4,5= 9.4 Hz, 1 H, 4_C-H), 1.24 (d, J_5,6= 6.1 Hz, 3 H, 6_D-H), 1.07

(d, J_5,6= 6.2 Hz, 3 H, 6_C-H) ppm. ¹³C NMR (100 MHz, CDCl₃):

δ = 167.4 (C-6_A), 161.5 (NHCO), 159.1 (C_IVArPMB), 138.8, 138.7,

138.5, 138.4, 137.9, 137.9, 137.8, 137.7, 135.0 (9 C, C_IVAr), 134.0

7.04 (m, 45 H, H_Ar), 7.02 (d, J_NH,2= 7.0 Hz, 1 H, NH), 5.98 (m,

1 H, CH=_All), 5.36 (dd_po, J_2,3= 3.1 Hz, 1 H, 3_C-H), 5.35 (m, J_gem

= 1.6 Hz, 1 H, =CH_2All), 5.32 (d, J_1,2= 1.6 Hz, 1 H, 1_D-H), 5.26

(d, J = 12.2 Hz, 1 H, H_CO2Bn), 5.22 (m, J_cis= 10.5 Hz, 1 H,

4104

www.eurjoc.org

Eur. J. Org. Chem. 2013, 4085–4106

O-Antigen from a Shigella flexneri Serotype 6

=CH_2All), 5.09 (d, 1 H, H_CO2Bn), 5.03 (d_po, J_1,2= 8.4 Hz, 1 H, 1_B-

H), 5.01 (d_po, J_1,2= 2.0 Hz, 1 H, 1_C-H), 4.89 (d_po, J = 10.8 Hz, 1

H, H_Bn), 4.88 (d, J = 11.1 Hz, 2 H, 2 H_Bn), 4.77 (d, J = 11.8 Hz,

1 H, H_Bn), 4.70 (d, J = 11.2 Hz, 2 H, H_Bn), 4.66 (d, J = 11.2 Hz,

gen for 2 d. After this time, MS analysis indicated a molecular

weight corresponding to that of the target tetrasaccharide. The re-

action mixture was filtered. Evaporation of the volatiles, freeze-

drying, and purification by preparative RP-HPLC gave tetrasac-

1 H, H_Bn), 4.62–4.52 (m_po, 7 H, H_Bn), 4.50 (m_po, 1 H, H_All), 4.40– charide 5 (45.3 mg, 53%) as a white solid following repeated freeze-

1

4.35 (m, 4 H, 1_A-H, 4_A-H, 3_B-H, 2_C-H), 4.26 (d, J = 11.6 Hz, 1 H,

H_Bn), 4.20 (d, J = 11.6 Hz, 1 H, H_Bn), 4.16 (m, 1 H, H_All), 4.05 (d,

drying. t_R= 9.0 min. H NMR (400 MHz, D₂O): δ = 5.23 (d, J_1,2

= 1.3 Hz, 1 H, 1_D-H), 5.03 (d, J_1,2= 1.7 Hz, 1 H, 1_C-H), 4.92 (dd,

J_3,4= 2.4 Hz, 1 H, 4_B-H), 4.01 (d, J_4,5= 0.9 Hz, 1 H, 5_A-H), 4.00 J_2,3= 3.0, J_3,4= 10.0 Hz, 1 H, 3_C-H), 4.33 (d, J_1,2= 8.0 Hz, 1 H,

(m, 1 H, 2_D-H), 3.92–3.82 (m, 2 H, 2_B-H, 5_C-H), 3.80 (dd, J_2,3

2.9, J_3,4= 9.5 Hz, 1 H, 3_D-H), 3.70–3.58 (m, 3 H, 2_A-H, 6a_B-H,

=

1_A-H), 4.31 (bs_o, 1 H, 5_A-H), 4.30 (d_po, J_1,2= 8.3 Hz, 1 H, 1_B-H),

4.22 (dd, J_3,4= 2.8, J_4,5= 1.1 Hz, 1 H, 4_A-H), 4.12 (pt, 1 H, 2_C-

5_D-H), 3.56–3.47 (m, 4 H, 3_A-H, 5_B-H, 4_C-H, 4_D-H), 3.35 (dd, J_5.6bH), 3.99 (dd, J_2,3= 3.1 Hz, 1 H, 2_D-H), 3.83–3.70 (m, 5 H, 4_B-H,

= 4.5, J_6a,6b= 8.3 Hz, 1 H, 6b_B-H), 2.17 (s, 3 H, H_Ac), 1.29 (d, J_5,6OCH_2Pr, 2_B-H, 3_A-H, 3_D-H), 3.70–3.57 (m, 4 H, 5_C-H, 6a_B-H, 6b_B-

= 6.2 Hz, 3 H, 6_D-H), 1.14 (d, J_5,6= 6.2 Hz, 3 H, 6_C-H) ppm. ¹³C

H, 3_B-H), 3.52–3.46 (m, 3 H, 5_B-H, OCH_2Pr, 5_D-H), 3.45 (dd_po, J_2,3

NMR (100 MHz, CDCl₃): δ = 170.7 (CO_Ac) 167.2 (C-6_A), 161.5 = 10.0 Hz, 1 H, 2_A-H), 3.40 (pt_po, J = 9.3, J = 9.8 Hz, 1 H, 4_C-H),

(NHCO), 139.0, 138.9, 138.6, 138.5, 138.4, 137.8, 137.7, 137.6, 3.35 (pt_po, J = 9.6, J = 9.8 Hz, 1 H, 4_D-H), 2.09 (s, 3 H, CH_3Ac),

135.1 (9 C, C_IVAr), 134.1 (CH=_All), 129.6–127.2 (45 C, C_Ar), 117. 4 1.97 (s, 3 H, CH_3NHAc), 1.56 (sext, J = 7.4 Hz, 2 H, CH_2Pr), 1.16

(=CH_2All), 102.6 (¹J_CH= 156.6 Hz, C-1_A), 101.3 (¹J_CH= 176.3 Hz, (d, J_5,6= 6.3 Hz, 3 H, 6_D-H), 1.14 (d, J_5,6= 6.2 Hz, 3 H, 6_C-H),

C-1_C), 100.0 (¹J_CH= 171.1 Hz, C-1_D), 99.6 (¹J_CH= 163.3 Hz, C-

1_B), 92.9 (CCl₃), 80.7 (C-3_A), 80.2 (C-4_D), 79.6 (C-4_C), 79.0 (C-3_D),

78.5 (C-2_A), 76.7 (C-3_B), 76.1 (C-2_D), 75.4, 75.3 (2 C, C_Bn), 75.2

(C-2_C), 75.1, 75.0 (2 C, C_Bn), 73.6 (C-5_A), 73.4 (C-3_C), 73.3 (C_Bn),

0.80 (t, 3 H, CH_3Pr) ppm. ¹³C NMR (400 MHz, D₂O): δ = 177.1

(NHCO), 176.1 (CO_Ac), 174.1 (C-6_A), 105.8 (¹J_CH= 163.2 Hz, C-

1_B), 104.9 (¹J_CH= 161.8 Hz, C-1_A), 103.6 (¹J_CH= 174.7 Hz, C-1_C),

102.4 (¹J_CH= 173.6 Hz, C-1_D), 81.4 (C-2_D), 79.3 (C-4_A), 79.0 (C-

73.1 (C-4_A), 73.0 (C-5_B), 72.7 (C_Bn), 72.6 (C-4_B), 72.5, 71.8 (2 C, 2_C), 77.4 (C-5_B), 75.4 (C-3_A), 75.3 (C-5_A), 75.2 (C-3_C), 74.8

_Bn), 70.5 (CH_2All), 68.8 (C-5_D), 68.2 (C-5_C), 67.6 (C-6_B), 67.3 (OCH_2Pr), 74.4 (C-4_D), 72.8 (C-3_B), 72.7 (C-2_A), 72.6 (C-4_C), 72.2

C

(C_CO2Bn), 56.4 (C-2_B), 21.3 (CH_3Ac), 18.0 (C-6_D), 17.7 (C-6_C) ppm. (C-3_D), 71.8 (C-5_D), 71.7 (C-5_C), 70.2 (C-4_B), 63.4 (C-6_B), 55.0 (C-

HRMS (ESI⁺): calcd. for C₉₄H₁₀₁Cl₃NO₂₁[M + H]⁺1684.5931; 2_B), 24.9 (CH_3NHAc), 24.7 (CH_2Pr), 23.1 (CH_3Ac), 19.2, 19.1 (2 C,

found 1684.5983; calcd. for C₉₄H₁₀₀Cl₃NO₂₁Na [M + Na]⁺

1706.5751; found 1706.5928.

C-6_D, C-6_C), 12.1 (CH_3Pr) ppm. HRMS (ESI⁺): calcd. for

C₃₁H₅₁NO₂₁Na [M + Na]⁺796.2852; found 796.2866.

Propyl 2-Acetamido-2-deoxy-β-

D

-galactopyranosyl-(1Ǟ2)-α-

L

-

Supporting Information (see footnote on the first page of this arti-

cle): Copies of the NMR spectra (¹H, ¹³C, COSY and HSQC) of

all new compounds, including key intermediates and final products.

rhamnopyranosyl-(1Ǟ2)-α- -rhamnopyranosyl-(1Ǟ4)-β-

L

D-galacto-

pyranosiduronic Acid (4): Pd/C (10 %; 200 mg) was added to a

stirred solution of tetrasaccharide 50 (254 mg, 140 μmol) in THF/

H₂O (4:1, 10.2 mL). The suspension was stirred under hydrogen for

2 d. After this time, MS analysis indicated a single molecular

weight corresponding to that of the target tetrasaccharide. The re-

action mixture was filtered. Evaporation of the volatiles, freeze-

drying, and purification of the crude material by preparative RP-

HPLC gave tetrasaccharide 4 (61.8 mg, 59%) as a white foam fol-

Acknowledgments

We thank F. Thouron (PMM) for LPS preparation, C. Ganneau

(CB) for analytical HPLC, Y.-M. Coïc (CB) for LC-MS analyses,

and F. Bonhomme (CNRS UMR 3523) for HRMS recording. This

work was supported by grants from the Institut Pasteur including

the Vasant and Kusum Joshi Fellowship and Bourses Roux (fellow-

ships to C. G.), the Ministère de l’Education Nationale, de la Re-

cherche et de la Technologie (MENRT) (PhD fellowship to P. C.),

the Swedish Research Council, and The Knut and Alice Wallenberg

Foundation. The research leading to these results has received

funding from the European Commission Seventh Framework Pro-

gram (FP7/2007–2013) under Grant agreement No. 215536 and

No. 261472-STOPENTERICS.

1

lowing repeated freeze-drying. t_R= 8.5 min. H NMR (400 MHz,

D₂O): δ = 5.24 (d, J_1,2= 1.4 Hz, 1 H, 1_D-H), 5.08 (d, J_1,2= 1.5 Hz,

1 H, 1_C-H), 4.55 (d, J_1,2= 8.4 Hz, 1 H, 1_B-H), 4.38 (d, J_1,2= 8.0 Hz,

1 H, 1_A-H), 4.35 (d, J_4,5= 1.2 Hz, 1 H, 5_A-H), 4.27 (dd, J_3,4

=

2.8 Hz, 1 H, 4_A-H), 4.05 (dd, J_2,3= 3.0 Hz, 1 H, 2_C-H), 4.02 (dd,

J_2,3= 3.1 Hz, 1 H, 2_D-H), 3.87–3.74 (m, 6 H, 4_B-H, OCH_2Pr, 2_B-

H, 3_A-H, 3_D-H, 3_C-H), 3.74–3.63 (m, 3 H, 6a_B-H, 6b_B-H, 3_B-H),

3.63–3.51 (m, 4 H, 5_B-H, 5_C-H, OCH_2Pr, 5_D-H), 3.50 (dd, J_2,3

=

9.9 Hz, 1 H, 2_A-H), 3.34 (pt, J_3,4= J_4,5= 9.8 Hz, 1 H, 4_D-H), 3.27

(pt, J_3,4= J_4,5= 9.7 Hz, 1 H, 4_C-H), 1.97 (s, 3 H, CH_3NHAc), 1.57

(sext, J = 7.4 Hz, 2 H, CH_2Pr), 1.20–1.15 (m, 6 H, 6_C-H, 6_D-H),

0.84 (t, 3 H, CH_3Pr) ppm. ¹³C NMR (100 MHz, D₂O): δ = 177.7

(NHCO), 174.2 (C-6_A), 105.7 (¹J_CH= 163.2 Hz, C-1_B), 104.9 (¹J_CH

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= 161.7 Hz, C-1_A), 103.6 (¹J_CH= 175.8 Hz, C-1_C), 102.5 (¹J_CH

174.7 Hz, C-1_D), 81.2 (C-2_C), 81.1 (C-2_D), 79.3 (C-4_A), 77.6 (C-5_B),

75.5 (C-3_A), 75.4 (C-5_A), 74.9 (2 C, C-4_C, OCH_2Pr), 74.5 (C-4_D),

73.4 (C-3_B), 72.8 (C-2_A), 72.3 (2 C, C-3_D, C-3_C), 71.8 (C-5_D), 71.7

=

(C-5_C), 70.3 (C-4_B), 63.5 (C-6_B), 55.4 (C-2_B), 25.0, 24.7 (2 C, CH_2Pr

,

CH_3NHAc), 19.2 (2 C, C-6_D, C-6_C), 12.2 (CH_3Pr) ppm. HRMS

(ESI⁺): calcd. for C₂₉H₄₉NO₂₀Na [M + Na]⁺754.2745; found

754.2722.

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Vaccine 2010, 28, 2231–2235.

Propyl 2-Acetamido-2-deoxy-β-

acetyl-α- -rhamnopyranosyl)-(1Ǟ2)-α-

-galactopyranosiduronic Acid (5): Pd/C (10%; 150 mg) was added

to a stirred solution of tetrasaccharide 54 (187 mg, 110 μmol) in

THF/H₂O (4:1, 8.4 mL). The suspension was stirred under hydro-

D

-galactopyranosyl-(1Ǟ2)-(3-O-

L

L-rhamnopyranosyl-(1Ǟ4)-β-

D

Eur. J. Org. Chem. 2013, 4085–4106

www.eurjoc.org

4105

L. A. Mulard et al.

FULL PAPER

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Structural Studies of the O-Acetyl-Containing O-Antigen from a Shigella flexneri Serotype 6 Strain and Synthesis of Oligosaccharide Fragments Thereof

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