Synthesis of phosphorylated, conjugation-ready di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide of V. cholerae O139

Article abstract of DOI:10.1002/ejoc.200700322

The fragments described contain a galactose residue with cyclic 4,6-phosphate and are equipped with an amino-functionalized spacer, allowing further derivatization or direct conjugation to suitable carriers. The core Gal-(1→3)-GlcNAc disaccharide was obtained by condensation of 8-azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-D- glucopyranoside with 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide under Helferich conditions. Reductive opening of the benzylidene acetal, followed by deacetylation and selective benzylation gave 8-azido-3,6-dioxaoctyl 2-acetamido-6-O-benzyl-3-O-(3-O-benzyl-β-D-galactopyranosyl) -2-deoxyβ-D-glucopyranoside, which was regioselectively phosphorylated to give two isomeric 4,6-cyclic 2,2,2-trichloroethyl phosphates. The (R)-phosphate was subjected to catalytic hydrogenation/hydrogenolysis to give the fully deprotected title disaccharide fragment. Glycosylation of the (S)-phosphate diol with 2,4-di-O-benzyl-3,6-dideoxy-α-L-xylo-hexopyranosyl bromide under halide-assisted conditions yielded a mixture of the tetrasaccharide and a trisaccharide, which were readily separable by chromatography. Their hydrogenation/ hydrogenolysis effected global deprotection as well as reduction of the azide, to furnish the deprotected title tri- and tetrasaccharide. Wiley-VCH Verlag GmbH & Co. KGaA, 2007.

Full text of DOI:10.1002/ejoc.200700322

FULL PAPER
DOI: 10.1002/ejoc.200700322
Synthesis of Phosphorylated, Conjugation-Ready Di-, Tri- and Tetrasaccharide
Fragments of the O-Specific Polysaccharide of V. cholerae O139
[a]
[a]
[a]
ˇ
Bart Ruttens, Rina Saksena, and Pavol Kovác*
Keywords: Carbohydrates / Regioselective phosphorylation / Glycosylation / Oligosaccharides / Conjugate vaccine
The fragments described contain a galactose residue with cy- phosphates. The (R)-phosphate was subjected to catalytic hy-
clic 4,6-phosphate and are equipped with an amino-function-
drogenation/hydrogenolysis to give the fully deprotected ti-
alized spacer, allowing further derivatization or direct tle disaccharide fragment. Glycosylation of the (S)-phosphate
conjugation to suitable carriers. The core Gal-(1Ǟ3)-GlcNAc
diol with 2,4-di-O-benzyl-3,6-dideoxy-α-L-xylo-hexopyrano-
disaccharide was obtained by condensation of 8-azido-3,6-
syl bromide under halide-assisted conditions yielded a mix-
ture of the tetrasaccharide and a trisaccharide, which were
readily separable by chromatography. Their hydrogenation/
hydrogenolysis effected global deprotection as well as re-
duction of the azide, to furnish the deprotected title tri- and
tetrasaccharide.
dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-β-
D-glu-
copyranoside with 2,3,4,6-tetra-O-acetyl-α- -galactopyrano-
D
syl bromide under Helferich conditions. Reductive opening
of the benzylidene acetal, followed by deacetylation and se-
lective benzylation gave 8-azido-3,6-dioxaoctyl 2-acetamido-
6-O-benzyl-3-O-(3-O-benzyl-β-D-galactopyranosyl)-2-deoxy-
β- -glucopyranoside, which was regioselectively phosphory-
lated to give two isomeric 4,6-cyclic 2,2,2-trichloroethyl
D
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
immunogenicity of carbohydrates is most noticeable in
young children, whose immune system is not fully devel-
oped. Avery and Goebel first demonstrated that inoculation
with neoglycoproteins, obtained by chemical conjugation of
carbohydrates to proteins, elicited anticarbohydrate anti-
bodies in T-cell dependant fashion, thus not only initiating
immunologic memory but also inducing a stronger and ear-
lier response to a subsequent challenge.[8] These observa-
tions initiated the use of glycoconjugates from cell surface
carbohydrates as experimental vaccines against the parent
microorganism.[9] Initially, haptens were harvested from
pathogens, but recent progress in oligosaccharide synthesis
enabled preparation of conjugate vaccines from synthetic
carbohydrate antigens.[10] Clinical trials with the first syn-
thetic conjugate vaccine provided proof of principle and
demonstrated the feasibility of a synthetic approach.[11]
The O-antigen of V. cholerae O139 is a hexasaccharide
(1, Figure 1) consisting of five different monosaccharides,
two of which are rare deoxysugars.[5b,5c,12] Within a project
focused on the development of a synthetic conjugate vac-
cine for cholera from this hexasaccharide, we prepare frag-
ments of the O-PS, which will aid to identify epitopes that
are crucial for protective capacity. Recently, we reported the
synthesis of a linker-equipped phosphorylated disaccharide
fragment (2, Figure 1),[13] amenable to conjugation with
proteins by squaric acid chemistry.[14] Here, we describe ex-
perimental details of our synthesis of additional phosphor-
ylated fragments, disaccharide 12, trisaccharide 17 and the
tetrasaccharide 16 (Scheme 1), which were not included in
Introduction
Cholera,[1] caused by Vibrio cholerae, is a serious enteric
disease. Its main symptoms are severe diarrhea and dehy-
dration, which can lead to hypotensive shock and death.
Until 1992, V. cholerae O1 was the only strain associated
with epidemic and pandemic cholera. In 1992, a new sero-
type, V. cholerae O139, emerged as an additional threat to
public health in Third World countries because the popula-
tion in V. cholerae O1 endemic regions lacked protective
immunity against the new strain.[2] Serotype O139 has
evolved from serotype O1 through acquisition of new genes
encoding for enzymes involved in the biosynthesis of O-
specific polysaccharides (O-PS),[3] and consequently, the
main differences between the two strains are in the consti-
tution of the cell surface.[4] V. cholerae O139 expresses a
capsular polysaccharide (CPS) whose repeating unit is iden-
tical to the O-PS.[4,5] Both the CPS and the O-PS are viru-
lence factors,[4] and a critical level of serum immunoglobu-
lin G (IgG) antibodies against these cell surface compo-
nents is required for immunity to the parent vibrio.[6]
Carbohydrates are poor immunogens because they acti-
vate antibody-producing B cells in T-cell independent (TI)
fashion and fail to generate memory B cells.[7] The inferior
[a] NIDDK, LBC, Section on Carbohydrates, National Institutes
of Health,
Bethesda, MD 20892-0815, USA
Fax: +1-301-402-0589
E-mail: kpn@helix.nih.gov
4366
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Synthesis of Fragments of the O-Specific Polysaccharide of V. cholerae O139
FULL PAPER
the preliminary report.[15] The compounds are equipped diate 10 to lead to two disaccharide phosphate isomers[17]
with an amino-functionalized spacer allowing direct conju- 11a and 11b, with the remaining free hydroxy groups avail-
gation to proteins, or further derivatization for conjugation able for glycosylation with a colitose donor. Substance 10
by a method of choice.
represents a motif common in human milk and blood group
Lewis oligosaccharides, whose synthesis was first described
by Flowers and Jeanloz.[18] Compound 7, a precursor of
10, is an intermediate for the preparation of several linker-
equipped upstream[19] di-, tri- and tetrasaccharide frag-
ments of the O-antigen of V. cholerae O139.
8-Azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-
2-deoxy-β-d-glucopyranoside (5),[20] the acceptor needed
for the preparation of 7, was synthesized from 2-acetamido-
3,4,6-tri-O-acetyl-2-deoxy-α-d-glucopyranosyl
chloride
(4),[21] according to the same sequence of reactions as de-
scribed by Amvam-Zollo and Sinaÿ,[20] albeit using dif-
ferent reagents and reaction conditions. Crude 4, prepared
as reported by Horton,[21] contains a small amount of 2-
acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-d-glucopyranoses
that co-crystallizes with the chloride.[22] We found chroma-
tographic removal of this impurity prior to crystallization
the most efficient way to obtain pure 4. Condensation of 5
and 2,3,4,6-tetra-O-acetyl-α-d-galactosyl bromide (6) under
Helferich conditions,[23] according to a procedure that is a
slight modification of that described by Khare et al.,[24] gave
disaccharide 7 in good yield (85%, Scheme 1). The disac-
charide was obtained as a crystalline solid after spontane-
ous evaporation of solvent from its solution in CH2Cl2, but
all attempts to crystallize it from common solvents failed.
The synthesis described required opening of the 4,6-O-
benzylidene ring. DeNinno et al. introduced the use of Et3-
SiH/CF3COOH for reductive cleavage of benzylidene ace-
tals in carbohydrates.[25] The method is described as highly
selective and to effect exclusive formation of the 6-O-benzyl
derivatives. However, the reaction has been reported as
poor yielding and sluggish, with concomitant cleavage of
the acetal to form the 4,6-O-deprotected compounds.[26]
Kiessling and co-workers engaged Et3SiH/CF3SO3H as an
alternative reagent,[27] and a more detailed study of the tri-
alkylsilane/acid reducing system showed that substitution
of TFA by TfOH resulted in a more efficient reagent.[28]
Indeed, reductive cleavage of acetal 7 with Et3SiH/TfOH at
–78 °C led to 6-O-benzyl derivative 8, with only a small
amount of the diol (8%). Examination of the NMR spectra
of 8 showed that the compound exists as two conformers,
whose ratio is strongly solvent-dependent (NMR). The
presence of a mixture is most prominent in [D6]benzene (ra-
Figure 1. Structure of the O-antigen of V. cholerae O139 (1) and
of its terminal, disaccharide fragment amenable to conjugation by
squaric acid chemistry.
Oscarson and co-workers have recently reported[16] an al-
ternative preparation of a linker-equipped, phosphorylated
tetrasaccharide fragment of the V. cholerae O139 O-PS. The
two approaches are fully independent. For example, a dif-
ferent colitosyl donor and different phosphorylation strat-
egy were used, and the functionalized linker in the final
tetrasaccharide was also different. The advantage of Os-
carson and co-workers’ approach is that their tetrasaccha-
ride thioglycoside can be used as a glycosyl donor to be
coupled with a GalA-QuiNAc disaccharide acceptor, to
construct the complete hexasaccharide (1, Figure 1). On the
other hand, our strategy allows preparation of several
smaller fragments and requires fewer chemical manipula-
tions with precious oligosaccharides.
Results and Discussion
The synthesis of the tetrasaccharide fragment 16 tio of conformers ca. 80:20, based on integration). The
(Scheme 1) was designed based on the previous[13] observa- amount of the less abundant conformer decreases in [D]-
tion that the 4,6-cyclic 2,2,2-trichloroethyl phosphate was chloroform (ca. 85:15), and it becomes negligible in [D5]-
formed in excellent yield from a galactose derivative with pyridine, [D4]methanol, [D6]dimethyl sulfoxide and [D4]ace-
more than two free hydroxy groups. This selectivity is prob- tic acid (Ͻ5%). At 50 °C in [D]chloroform, the presence of
ably the result of a considerable difference in reactivity of the conformers is hardly noticeable, but interpretation of
the primary and secondary hydroxy groups under the reac- the spectrum was difficult due to line broadening. The diol
tion conditions. Initial attack of the primary hydroxy group byproduct of the reductive cleavage displays similar behav-
on the phosphorus dihalide reagent is followed by intramo- ior, with a conformer ratio of 85:15 in [D]chloroform at
lecular closure to form two stable six-membered ring epi- room temperature. These observations are in accord with a
mers, rather than intermolecular polymerization. Thus, we recent report that oligosaccharides containing N-acetylglu-
expected phosphorylation of linker-equipped key interme- cosamine can adopt unexpected conformations as a result
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B. Ruttens, R. Saksena, P. Kovácˇ
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Scheme 1. Reagents and conditions: (a) (1) NaOMe, MeOH, room temp., (2) Ac2O, room temp.; (b) AcCl, room temp.; (c) 8-azido-3,6-
dioxaoctan-1-ol, AgOTf, tetramethylurea, CH2Cl2, room temp.; (d) anhydr. K2CO3, MeOH, room temp.; (e) PhCH(OMe)2, CSA, CH3CN,
room temp.; (f) Hg(CN)2, MS (4 Å), CH3NO2/benzene (1:1), 40 °C; (g) Et3SiH, CF3SO3H, MS (4 Å), CH2Cl2, –78 °C; (h) (1) Bu2SnO,
toluene, reflux, (2) CsF, BnBr, DMF, room temp.; (i) Cl3CCH2OP(O)Cl2, pyridine, CH2Cl2, –15 °C; (j) (1) Br2, CCl4, room temp., (2)
hex-1-ene, room temp.; (k) Bu4NBr, MS (4 Å), CH2Cl2/DMF (5:1), room temp.; (l) Pd/C, H2, iPrOH/potassium phosphate buffer (pH =
7, 0.1 m), room temp.
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Synthesis of Fragments of the O-Specific Polysaccharide of V. cholerae O139
FULL PAPER
of formation of hydrogen bonds involving the amide 1III-H at δ = 5.50 ppm (J1III
= 3.3 Hz) evidenced forma-
,2III
group.[29] Liao et al. studied the coupling constants for the tion of the α-colitosyl linkage. As expected, downfield shifts
N-acetylglucosamine residue in small oligosaccharides and were observed in the spectrum of tetrasaccharide 14 of sig-
4
found that they were inconsistent with a C1 chair confor- nals for 2II-H and 4I-H (by 0.24 and 0.48 ppm, respectively,
mation in non-hydrogen bond-accepting solvents. However, compared to those in the spectrum of 11b), and the coup-
they did not observe the presence of different conformers. ling constants of the doublets for 1III-H at δ = 5.62 ppm
,2III
,2IV
=
We presume that, in our case, the long polyethylene glycol (J1III
= 3.4 Hz) and 1IV-H at δ = 5.02 ppm (J1IV
spacer slows down formation of the equilibrium between 3.5 Hz) are consistent with the α-linkage of colitose resi-
conformations sufficiently to lead, in solution, to the exis- dues. Unlike in the NMR spectra of trisaccharide 15, coup-
tence of different conformers on the NMR time scale. In ling constants for 1I-H (J1I,2I = 6.7 Hz, JC-1I,H-1I = 166.7 Hz)
[D]chloroform, the 13C NMR spectrum of the major con- in NMR spectra of 14 differ from those usually found for
former of 8 shows a significant upfield shift of the signal a β-glucosamine residue in a 4C1 conformation.[32] They are
for C-4I (by ca. 11 ppm; for numbering of sugar residues, in accord, however, in particular JC-1I,H-1I, with a distorted
see Scheme 1, structure 14) and downfield shifts of signals conformation of the β-glucosamine unit due to the involve-
for C-3I (by ca. 7 ppm) and for C-6I (by ca. 1 ppm) com- ment of the acetamido group in hydrogen-bond forma-
pared to those in the spectra of 7, which is consistent with tion.[29] Bush and co-workers[33] analyzed the conformation
reductive opening of the acetal to form the 6-benzyl ether. of the tetrasaccharide portion of the hexasaccharide repeat-
Additional proof that the intended opening of the benzyl- ing unit of the CPS of V. cholerae O139 by NMR spec-
idene ring had occurred was provided by the cross peak troscopy and molecular modeling. They showed that the
between 4I-H and the HO group observed in the COSY tetrasaccharide adopts a compact and tightly folded confor-
spectra of 8 taken in [D]chloroform and [D6]dimethyl sulf- mation in which the cyclic phosphate is in close contact
oxide. Deacetylation of substance 8 (Ǟ 9), followed by ben- with the colitose residue linked to the β-glucosamine unit.
zylation at the 3-position of the galactose residue (Ǟ 10) Though protected, NMR spectroscopic data found for the
and selective phosphorylation[13] with 2,2,2-trichloroethyl synthetic tetrasaccharide fragment 14 reflect these findings.
phosphorodichloridate, gave a mixture (ca. 1:3.8) of (R)- Deshielding, as a result of the interaction between the phos-
and (S)-(P)-4,6-cyclic 2,2,2-trichloroethyl phosphates 11a phate group and the colitose residue attached to β-GlcNAc,
and 11b, respectively.
leads to a downfield shift (by 7.11 ppm) of the 31P signal
The (S)-phosphate acceptor 11b was subjected to halide- compared to that in the spectrum of trisaccharide 15, and
assisted[30] glycosylation with the colitosyl bromide (13a) downfield shifts of the signals for 3IV-Hax (by 0.09 ppm),
obtained from ethyl 1-thio-β-colitoside[31] (13) by treatment 4IV-H (by 0.1 ppm), 5IV-H (by 0.4 ppm) and 6IV-H (by
with Br2. As expected from the well-known fact that the 0.05 ppm), compared to those for 3III-Hax, 4III-H, 5III-H
4-OH group of the GlcNAc residues is quite unreactive, and 6III-H, respectively.
these glycosylation conditions furnished not only the de-
Unusually high hydrogen pressure (160 psi) was required
sired tetrasaccharide 14, but also the trisaccharide 15, to maintain a practicable reaction rate of hydrogenolysis/
whose deprotected forms are useful haptens for immunolog- hydrogenation of 14 (Ǟ 16) in the presence of palladium-
ical studies. Oscarson and co-workers[16] reported similar on-charcoal catalyst, probably because the aforementioned
formation of a mixture of a trisaccharide and a tetrasaccha- compact conformation impedes interaction of reaction sites
ride, albeit with a better yield of the latter. It is interesting with the catalyst surface. Global deprotection of 11a (Ǟ
to note that this was accomplished with fewer equivalents 12) and 15 (Ǟ 17) with concomitant reduction of the azide
of a colitosyl bromide donor under very similar reaction to an amine was possible to achieve at considerably lower
conditions. It seems that the use of electron-withdrawing hydrogen pressure (70 psi). As reported previously,[13] de-
protecting groups instead of benzyl ethers increased the sta- protection was done in a buffered solution, to protect the
bility of their donor to allow prolonged reaction time (7 d) acid-labile α-colitosyl bonds. Oscarson and co-workers[16]
resulting in more complete conversion. We found that with did not report problems with catalytic hydrogenolysis of the
bromide 13a the reaction was finished within 48 h, and that benzyl groups in their tetrasaccharide. However, they had
the yield of oligosaccharides 14 and 15 depended on the two fewer benzyl groups to remove from the tetrasaccha-
excess of glycosyl donor used. With 5 equiv. of 13a, the gly- ride. The benzoyl groups used instead to protect the 4-OH
cosylation furnished more of the trisaccharide 15 (48%, in groups in the colitose residues were very resistant to base-
addition to 34% of 14), while with 6 equiv. of 13a similar catalyzed deacylation, indicating that this position in the
yields of 14 and 15 (41 and 45%, respectively) were ob- tetrasaccharide is rather unreactive. Furthermore, the p-
tained. Both compounds were fully characterized and their chlorobenzyl ethers at C-2 of the colitosyl residues[16] can
structure was confirmed by NMR spectrioscopy and high- be expected to be more easily cleaved than their non-halo-
resolution mass spectrometry. For trisaccharide 15, the sig- genated counterparts. Finally, it is possible that the buffered
nificant downfield shift of the signal for 2II-H (by 0.32 ppm, reaction medium deactivated the catalyst, thus necessitating
compared to that in the spectrum of 11b) and the COSY the use of substantially higher hydrogen pressure. This
crosspeak between 4I-H and the hydroxy group confirmed seems to be confirmed by the fact that deprotection of 12
the point of attachment of the colitose residue at O-2 in and 15 also required more than atmospheric hydrogen pres-
galactose, while the coupling constant of the doublet for sure to effect the conversion. After chromatography to re-
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B. Ruttens, R. Saksena, P. Kovácˇ
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less stated otherwise, solutions in organic solvents were dried with
anhydrous Na2SO4 and concentrated at ca. 40 °C/2 kPa.
move several less polar byproducts, the deprotected oligo-
saccharides 12, 16 and 17 were obtained in yields of 60%,
55% and 58%, respectively. Extended reaction times led to
lower yields of the desired oligosaccharides and more exten-
sive formation of the byproducts (TLC). This indicates that
these materials were not the products of incomplete depro-
tection, but their nature was not further investigated.
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl Chlo-
ride (4): Prepared from d-glucosamine hydrochloride (3) as de-
scribed by Horton,[21] except that crude 4 was chromatographed
(dry silica gel/crude material: 20:1, hexane/EtOAc: 1:2, sample
loaded as a solution in CH2Cl2) before crystallization, to remove
2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-d-glucopyranose.
8-Azido-3,6-dioxaoctyl 2-Acetamido-4,6-O-benzylidene-2-deoxy-β-
D
-glucopyranoside (5): A solution of 4 (15.68 g, 42.84 mmol) and
tetramethylurea (3.25 mL, 27.13 mmol) in CH2Cl2 (60 mL) was
added slowly (exothermic!) to stirred mixture of AgOTf
Conclusions
a
An efficient synthesis has been developed for the prepa-
ration of three fragments of the O-PS of V. cholerae O139.
The key step is the regioselective phosphorylation of a po-
lyol disaccharide intermediate, which greatly reduces the
number of synthetic steps. The phosphorylated di-, tri- and
tetrasaccharide fragments are equipped with an amino-
functionalized spacer to allow direct conjugation to pro-
teins or further derivatization for conjugation by a method
of choice.
(11.74 g, 45.69 mmol) and 8-azido-3,6-dioxaoctan-1-ol[13] (5.0 g,
28.56 mmol) in CH2Cl2 (40 mL). Stirring was continued in the dark
for 24 h. The mixture was treated with Et3N (8 mL, 57.11 mmol),
filtered through a Celite pad, and the solids were washed with
CH2Cl2. The combined filtrates (ca. 500 mL) were washed with
H2O (500 mL), ice-cold 10% aqueous H2SO4 (500 mL), saturated
NaHCO3 (500 mL) and H2O (500 mL), dried, and concentrated.
The residue (14.82 g) was dissolved in MeOH (100 mL) and anhy-
drous K2CO3 (1.0 g, 7.14 mmol) was added. The mixture was
stirred for 4 h and neutralized with IR-120 (H+) resin. The resin
was filtered off, washed with MeOH, and the combined filtrates
were concentrated.
A solution of the residue in CH3CN
Experimental Section
(3ϫ100 mL) was concentrated to remove residual MeOH, and
dried. A mixture of the residue, CSA (665 mg, 2.86 mmol), α,α-
dimethoxytoluene (8.6 mL, 57.11 mmol) and CH3CN (200 mL)
was stirred at room temp. After 14 h, Et3N (1 mL, 7.14 mmol) was
added and the solvent was evaporated. The residue was chromato-
graphed (CH2Cl2/iPrOH: 95:5Ǟ90:10) to give 5, containing ca. 5%
of the α-anomer (11.615 g, 87%). Crystallization from EtOH gave
pure 5 (8.646 g, 65%). Column chromatography (reversed phase,
MeOH/H2O: 1:1) of the mother liquor, followed by crystallization
from EtOH gave an additional crop of pure 5 (1.505 g, 11%). M.p.
184–187 °C (ref.[20] 193–194 °C). [α]2D5 = –78 [c = 1.63, CHCl3;
ref.[20] –76 (c = 1.6, CHCl3)]. 1H NMR (600 MHz, CDCl3): δ =
2.02 (s, 3 H, COCH3), 3.40 (br. t, J = 5.0 Hz, partial overlap, 2 H,
General Methods: HPLC grade solvents were used, and reactions
requiring anhydrous conditions were carried out under nitrogen or
argon. A Parr mini bench top reactor, series 4560, was used for
reactions under H2. Tetramethylurea was stored over KOH pellets,
pyridine over CaH2. The density of 2,2,2-trichloroethyl phosphor-
odichloridate was determined by differential weighing of 1 mL of
reagent (d ≈ 1.7 g/mL at 20 °C). Compound 6 was purchased from
Sigma. The palladium-on-charcoal catalyst (5%, Escat 147) was a
product of Engelhard Industries. Reversed phase purifications were
done on Analogix SuperFlash-Sepra C18 columns. Reactions were
monitored by thin-layer chromatography (TLC) on silica gel 60
glass slides. Spots were visualized by heating, after spraying with
H2SO4 in EtOH (5% v/v) or ninhydrine in EtOH (5% w/v) for
primary amines. For purification of 4 and 6, silica gel 60 was dried
overnight at 160 °C. Melting points were determined with a Kofler
hot stage. Optical rotations were measured with a Perkin–Elmer
automatic polarimeter, Model 341. NMR spectra were measured
CH2N3), 3.41 (m, partial overlap, 1 H, 5-H), 3.56 (t, J3,4 = J4,5
=
9.2 Hz, 1 H, 4-H), 3.60 (dt, J1,2a = 3.1, J2a,2b = 11.0 Hz, 1 H, 2Јa-
H), 3.62–3.65 (m, 6 H, 3Ј-H, 4Ј-H, 5Ј-H), 3.68 (m, 1 H, 2Јb-H),
3.73 (m, 1 H, 2-H), 3.75 (t, J5,6a = J6a,6b = 10.4 Hz, partial overlap,
1 H, 6a-H), 3.78 (ddd, J1a,2a = 2.8, J1a,2b = 8.7, J1a,1b = 12.0 Hz,
1
at 25 °C with a Varian Mercury spectrometer, at 300 MHz for H,
partial overlap, 1 H, 1Јa-H), 3.86 (br. dt, J3,OH = 2.4, J2,3 = J3,4
=
75 MHz for 13C and 121 MHz for 31P, or with a Bruker Avance
spectrometer, at 600 MHz for 1H and 150 MHz for 13C. Assign-
ments of NMR signals were made by first-order analysis of the
spectra and, when feasible, the assignments were supported by
homonuclear decoupling experiments or homonuclear and hetero-
nuclear 2-dimensional correlation spectroscopy, run with the soft-
ware supplied with the spectrometers. When reporting assignments
of NMR signals, nuclei associated with the spacer are denoted with
a prime (Ј). Sugar residues in listings of signal assignments are seri-
ally numbered by a Roman numeral superscript, beginning with the
one bearing the aglycon (see Scheme 1, 14). 31P-13C coupling was
observed for all phosphates. Chemical shifts are reported relative
to tetramethylsilane (TMS) or acetone[34] as internal standard.
Chemical shifts for 31P NMR spectra are reported relative to
H3PO4 in D2O as an internal standard (stem coaxial insert). No
elemental analyses or optical rotations were collected for the
di-, tri- and tetrasaccharide phosphate salts. The structure of these
compounds follows unequivocally from the mode of synthesis and
the m/z values found in their low- and high-resolution mass spectra,
and their purity was verified by TLC and NMR spectroscopy. Un-
9.5 Hz, 1 H, 3-H), 3.92 (dt, J1b,2a = J1b,2b = 3.2, J1a,1b = 12.0 Hz,
1 H, 1Јb-H), 4.30 (dd, J5,6b = 5.0, J6a,6b = 10.4 Hz, 1 H, 6b-H),
4.66 (d, J1,2 = 8.5 Hz, 1 H, 1-H), 4.86 (d, J3,OH = 2.9 Hz, 1 H, HO-
3), 5.51 (s, 1 H, CHPh), 6.85 (d, J2,NH = 6.6 Hz, 1 H, NH), 7.31–
7.36 (m, 3 H, Ph), 7.47–7.50 (m, 2 H, Ph) ppm. 13C NMR
(150 MHz, CDCl3): δ = 22.97 (COCH3), 50.41 (CH2N3), 58.11 (C-
2), 66.15 (C-5), 68.43 (C-6), 68.85 (C-1Ј), 69.73, 70.26, 70.43 (C-3Ј,
C-4Ј, C-5Ј), 70.86 (C-2Ј), 72.62 (C-3), 81.47 (C-4), 101.68 (CHPh),
101.69 (C-1), 126.28, 128.07, 128.95, 137.08 (Ph), 172.53 (COCH3)
ppm.
2,3,4,6-Tetra-O-acetyl-α-D-galactopyranosyl Bromide (6): Commer-
cial bromide (30 g) was purified by chromatography (hexane/
EtOAc: 3:1) to remove the stabilizer (CaCO3) and other impurities.
Crystallization of the resulting material (25.55 g) from diisopropyl
ether gave 6 (20.67 g), whose purity and structure was confirmed
1
by H NMR spectroscopy.
8-Azido-3,6-dioxaoctyl 2-Acetamido-4,6-O-benzylidene-2-deoxy-3-
O-(2,3,4,6-tetra-O-acetyl-β-
D-galactopyranosyl)-β-D-glucopyrano-
side (7): Powdered anhydrous CaSO4 (30 g) and Hg(CN)2 (9.56 g,
4370
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4366–4375

Synthesis of Fragments of the O-Specific Polysaccharide of V. cholerae O139
FULL PAPER
37.84 mmol) were added to a stirred solution of 5 (8.82 g,
18.92 mmol) in CH3NO2/benzene (1:1; 520 mL). After 1 h, a solu-
tion of 6 (11.67 g, 28.38 mmol) in the same solvent (75 mL) was
added, and the mixture was stirred at 40 °C for 16 h. After filtration
through a Celite pad, the solids were washed with CH2Cl2
(5ϫ250 mL), the filtrate was concentrated and a solution of the
residue in CH2Cl2 (1.5 L) was washed with 10% KBr in brine (1 L),
saturated NaHCO3 (1 L) and H2O (1 L). The organic phase was
dried, and concentrated, and the residue was chromatographed
(CH2Cl2/iPrOH: 100:2.5 and petroleum ether/acetone: 3:2) to give
disaccharide 7 (12.86 g, 85%). A portion was dissolved in CH2Cl2
and the solvent was allowed to evaporate. The solid obtained after
drying under reduced pressure at 40 °C showed m.p. 129.0–
132.5 °C (see Results and Discussion). [α]2D5 = –16.8 (c = 0.51,
CHCl3). 1H NMR (600 MHz, CDCl3): δ = 1.95, 1.96, 2.00, 2.02,
2.12 (5 s, 15 H, 5 CH3CO), 3.24 (m, 1 H, 2I-H), 3.43 (m, 2 H,
= 7.6 Hz, 1 H, NH), 7.24–7.30 (m, 2 H, Ph), 7.30–7.36 (m, 3 H,
Ph) ppm. 13C NMR (150 MHz, CDCl3; major conformer): δ =
20.47, 20.49, 20.57, 20.71, 23.64 (5 CH3CO), 50.53 (CH2N3), 56.85
(C-2I), 61.41 (C-6II), 66.88 (C-4II), 68.48 (C-1Ј), 68.74 (C-2II), 69.43
(C-4I), 69.65 (C-6I), 69.86, 70.35, 70.59, 70.71 (C-2Ј, C-3Ј, C-4Ј, C-
5Ј), 70.76 (C-3II), 70.94 (C-5II), 73.51 (CH2Ph), 75.26 (C-5I), 83.89
(C-3I), 99.71 (C-1I), 101.36 (C-1II), 127.45, 127.52, 128.25, 138.33
(Ph), 169.15, 170.00, 170.09, 170.41, 170.64 (5 CH3CO) ppm. 1H
NMR (600 MHz, [D6]DMSO; major conformer): δ = 1.82, 1.90,
1.98, 2.02, 2.11 (5 s, 15 H, 5 CH3CO), 3.20 (dt, J4,OH = 4.0, J3,4
=
J4,5 = 9.0 Hz, 1 H, 4I-H), 3.35–3.39 (m, 3 H, 5I-H, CH2N3), 3.45–
3.60 (m, 11 H, 1aЈ-H, 6aI-H, 2I-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.64
(br. t, J2,3 = J3,4 = 9.2 Hz, 1 H, 3I-H), 3.71–3.79 (m, 2 H, 1bЈ-H,
6bI-H), 4.01 (dd, J5,6a = 5.9, J6a,6b = 11.2 Hz, 1 H, 6aII-H), 4.10
(dd, J5,6b = 7.3, J6a,6b = 11.2 Hz, 1 H, 6bII-H), 4.21 (br. t, J4,5 ≈ 0,
J5,6a = J5,6b = 6.9 Hz, 1 H, 5II-H), 4.40 (d, J1,2 = 8.3 Hz, 1 H, 1I-
CH2N3), 3.53 (m, 1 H, 5I-H), 3.61 (m, partial overlap, 1 H, 5II-H), H), 4.51, 4.53 (2 d, 2J = 12.1 Hz, partial overlap, 2 H, CH2Ph),
3.62–3.70 (m, 9 H, 4I-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.75 (m, partial 4.52 (d, J4,OH = 3.7 Hz, partial overlap, 1 H, OH), 4.78 (d, J1,2
=
overlap, 1 H, 1Јb-H), 3.78 (t, partial overlap, J5,6a = J6a,6b
=
=
8.1 Hz, 1 H, 1II-H), 4.93 (dd, J1,2 = 8.1, J2,3 = 10.4 Hz, 1 H, 2II-
10.4 Hz, 1 H, 6Ia-H), 3.93 (dd, partial overlap, J5,6a = 5.9, J6a,6b
H), 5.13 (dd, J3,4 = 3.6, J2,3 = 10.4 Hz, 1 H, 3II-H), 4.26 (br. d, J4,5
≈ 0, J3,4 = 3.6 Hz, 1 H, 4II-H), 7.26–7.36 (m, 5 H, Ph), 7.79 (d,
JNH,2 = 9.0 Hz, 1 H, NH) ppm. 13C NMR (150 MHz, [D6]DMSO;
major conformer): δ = 20.30, 20.41 (2 C), 20.42, 22.95 (5 CH3CO),
11.1 Hz, 1 H, 6IIa-H), 3.94 (m, partial overlap, 1 H, 1Јa-H), 4.06
(dd, J5,6b = 7.8, J6a,6b = 11.1 Hz, 1 H, 6IIb-H), 4.31 (dd, J5,6b
4.9, J6a,6b = 10.4 Hz, 1 H, 6Ib-H), 4.60 (t, J2,3 = J3,4 = 9.5 Hz, 1
=
H, 3I-H), 4.74 (d, J1.2 = 8.0 Hz, 1 H, 1II-H), 4.93 (dd, J2,3 = 10.4, 49.97 (CH2N3), 53.83 (C-2I), 61.02 (C-6II), 67.18 (C-4II), 67.90 (C-
J3,4 = 3.4 Hz, 1 H, 3II-H), 5.12 (d, J1,2 = 8.3 Hz, 1 H, 1I-H), 5.17
(dd, J1,2 = 8.0, J2,3 = 10.3 Hz, 1 H, 2II-H), 5.29 (dd, J3,4 = 3.4, J4,5
= 1.0 Hz, 1 H, 4II-H), 5.52 (s, 1 H, CHPh), 6.10 (d, JNH,2 = 7.4 Hz,
1Ј), 68.31 (C-2II), 68.52 (C-4I), 69.22, 69.45, 69.69, 69.83 (C-2Ј, C-
3Ј, C-4Ј, C-5Ј), 69.51 (C-6I), 69.93 (C-5II), 70.39 (C-3II), 72.28
(CH2Ph), 75.14 (C-5I), 82.74 (C-3I), 100.10 (C-1II), 100.51 (C-1I),
1 H, NH), 7.36–7.39 (m, 3 H, arom.), 7.46–7.49 (m, 2 H, arom.) 127.33, 128.20, 138.57 (Ph), 169.01, 169.29, 169.48, 169.89, 169.95
ppm. 13C NMR (150 MHz, CDCl3): δ = 20.49, 20.53, 20.57, 20.69,
23.53 (5 CH3CO), 50.54 (CH2N3), 57.78 (C-2I), 60.92 (C-6II), 65.89
(5 CH3CO) ppm. ES-TOF-MS (pos. ion): m/z = 805.3347 ([M +
Li]+; calcd. 805.3331). C35H50N4O17 (798.3): calcd. C 52.63, H 6.31,
(C-5I), 66.77 (C-4II), 68.69 (C-6I), 68.81 (C-1Ј), 69.23 (C-2II), 70.35 N 7.01; found C 52.81, H 6.42, N 6.99. Next eluted was 8-Azido-
(C-5II), 69.89, 70.40, 70.49, 70.60 (C-2Ј, C-3Ј, C-4Ј, C-5Ј), 71.03 (C-
3II), 76.92 (C-3I), 80.44 (C-4I), 99.86 (C-1I), 100.39 (C-1II), 101.32
(CHPh), 126.00, 128.24, 129.15, 137.06 (Ph), 169.46, 170.09,
170.15, 170.19, 170.71 (5 CH3CO) ppm. ES-TOF-MS (pos. ion):
m/z = 803.3179 ([M + Li]+; calcd. 803.3175). C35H48N4O17
(796.30): calcd. C 52.76, H 6.07, N 7.03; found C 52.59, H 6.08, N
7.01.
3,6-dioxaoctyl 2-Acetamido-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-β-
D
-galactopyranosyl)-β-
D
-glucopyranoside (353 mg, 8%). 1H NMR
(600 MHz, CDCl3; major conformer): δ = 1.98, 2.01, 2.06, 2.10,
2.16 (5 s, 15 H, 5 CH3CO), 2.47 (t, J6,OH = 6.7 Hz, 1 H, 6-OH),
3.35 (m, 1 H, 2I-H), 3.41 (m, 1 H, 5I-H), 3.42–3.46 (m, 2 H,
CH2N3), 3.49 (m, 1 H, 4I-H), 3.63–3.72 (m, 8 H, 2Ј-H, 3Ј-H, 4Ј-H,
5Ј-H), 3.74–3.80 (m, 2 H, 1aЈ-H, 6aI-H), 3.88–3.95 (m, 3 H, 1bЈ-H,
6bI-H, 4-OH), 4.04 (br. ddd, J4,5 = 1.1, J5,6a = 5.9, J5,6b = 7.1 Hz, 1
H, 5II-H), 4.13 (m, 2 H, 6II-H), 4.18 (dd, J = 8.4, J = 10.3 Hz, 1
H, 3I-H), 4.60 (d, J1,2 = 8.0 Hz, 1 H, 1II-H), 4.88 (d, J1,2 = 8.4 Hz,
1 H, 1I-H), 5.02 (dd, J3,4 = 3.5, J2,3 = 10.5 Hz, 1 H, 3II-H), 5.21
(dd, J1,2 = 8.0, J2,3 = 10.5 Hz, 1 H, 2II-H), 5.38 (dd, J4,5 = 1.0, J3,4
= 3.5 Hz, 1 H, 4II-H), 6.27 (d, JNH,2 = 7.8 Hz, 1 H, NH) ppm. 13C
NMR (150 MHz, CDCl3; major conformer): δ = 20.48, 20.51,
20.57, 20.69, 23.56 (5 CH3CO), 50.48 (CH2N3), 56.42 (C-2I), 61.42
(C-6II), 62.75 (C-6I), 66.87 (C-4II), 68.63 (C-1Ј), 68.67 (C-2II),
69.83, 70.31, 70.56, 70.80 (C-2Ј, C-3Ј, C-4Ј, C-5Ј), 69.93 (C-4I),
70.72 (C-3II), 70.93 (C-5II), 75.24 (C-5I), 83.86 (C-3I), 100.09 (C-
1I), 101.34 (C-1II), 169.24, 170.03, 170.11, 170.44, 170.76 (5
8-Azido-3,6-dioxaoctyl 2-Acetamido-6-O-benzyl-2-deoxy-3-O-
(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-β-D-glucopyranoside
(8): Powdered molecular sieves (4 Å, 2.5 g) were added to a stirred
solution of 7 (5.0 g, 6.28 mmol) in CH2Cl2 (50 mL). After 30 min,
the mixture was cooled to –78 °C, and Et3SiH (3.0 mL,
18.84 mmol) and TfOH (1.42 mL, 12.56 mmol) was added. Stirring
was continued at –78 °C, and after 2 h the reaction was quenched
by addition of solid NaHCO3 (5.0 g) and MeOH (25 mL). The mix-
ture was allowed to reach room temperature, filtered through a
Celite pad, and the solids were washed with CH2Cl2. The combined
filtrates (500 mL) were washed with saturated aq. NaHCO3
(250 mL) and H2O (250 mL), dried, and concentrated. The residue
was chromatographed (toluene/EtOH: 9:1Ǟ8:2) to give first 8
(4.343 g, 87%). M.p. 103.5–104.0 °C (from iPrOH). [α]2D5 = +1.7 (c
= 0.54, CHCl3). 1H NMR (600 MHz, CDCl3; major conformer): δ
= 1.98, 2.00, 2.02, 2.09, 2.15 (5 s, 15 H, 5 CH3CO), 3.28 (m, 1 H,
CH3CO) ppm. ES-TOF-MS (pos. ion): m/z
=
715.2882
([M + Li]+; calcd. 715.2862).
8-Azido-3,6-dioxaoctyl 2-Acetamido-6-O-benzyl-2-deoxy-3-O-(β-
galactopyranosyl)-β- -glucopyranoside (9): Anhydrous K2CO3
solution of (4.2 g,
D-
D
2I-H), 3.40–3.52 (m, 4 H, 5I-H, 4I-H, CH2N3), 3.60–3.70 (m, 9 H, (727 mg, 5.26 mmol) was added to
a
8
6aI-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.76 (m, 1 H, 1aЈ-H), 3.80 (s, 1 H, 5.26 mmol) in MeOH (50 mL). The mixture was stirred at room
OH), 3.86 (dd, J5,6b = 1.6, J6a,6b = 10.9 Hz, 1 H, 6bI-H), 3.95 (m,
1 H, 1bЈ-H), 4.01 (m, 1 H, 5II-H), 4.12 (m, 2 H, 6II-H), 4.23 (dd, J =
temperature for 2.5 h, neutralized with IR-120 (H+) resin, the resin
was filtered off, washed with MeOH, and the filtrate was concen-
7.8, J = 10.3 Hz, 1 H, 3I-H), 4.59 (d, J1,2 = 8.0 Hz, partial overlap, 1 trated. The residue was chromatographed (CH2Cl2/10% NH4OH
H, 1II-H), 4.58, 4.61 (2 d, 2J = 12.2 Hz, partial overlap, 2 H, in MeOH: 85:15Ǟ80:20) to give, after freeze-drying, amorphous 9
CH2Ph), 4.86 (d, J1,2 = 8.3 Hz, 1 H, 1I-H), 5.01 (dd, J3,4 = 3.4, J2,3 (3.194 g, 96%). [α]2D5 = –13.8 (c = 0.55, MeOH). 1H NMR
= 10.5 Hz, 1 H, 3II-H), 5.22 (dd, J1,2 = 8.0, J2,3 = 10.5 Hz, 1 H, (600 MHz, D2O): δ = 2.02 (s, 3 H, CH3CO), 3.47 (m, 2 H, CH2N3),
2II-H), 5.37 (dd, J4,5 = 0.9, J3,4 = 3.4 Hz, 1 H, 4II-H), 5.98 (d, JNH,2 3.52 (dd, J1,2 = 7.7, J2,3 = 10.0 Hz, 2II-H, 1 H, partial overlap),
Eur. J. Org. Chem. 2007, 4366–4375
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4371

B. Ruttens, R. Saksena, P. Kovácˇ
FULL PAPER
3.54 (dd, J = 8.5, J = 10.0 Hz, 1 H, 4I-H), 3.60 (m, 1 H, 5I-H), (1 mL), the mixture concentrated, and EtOAc was added to the
3.63 (dd, J3,4 = 3.4, J2,3 = 10.0 Hz, 1 H, 3II-H), 3.64–3.71 (m, 9 H, residue. The precipitate was filtered off, washed with EtOAc and
5II-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.71–3.79 (m, 5 H, 3I-H, 6aI-H, 6II- the combined filtrates were concentrated. The residue was chro-
H, 1aЈ-H), 3.83 (dd, J1,2 = 8.5, J2,3 = 10.4 Hz, 1 H, 2I-H), 3.89– matographed (CH2Cl2/MeOH: 95:5) to give 11a and 11b, in that
3.93 (m, 2 H, 6bI-H, 4II-H), 3.95 (ddd, J1b,2a = 3.1, J1b,2b = 5.6,
order. Further purification of both compounds by reversed phase
J1a,1b = 11.7 Hz, 1 H, 1bЈ-H), 4.41 (d, J1,2 = 7.7 Hz, 1 H, 1II-H), chromatography (CH3CN/H2O: 2:3Ǟ1:1) gave 11a (136 mg, 18%)
2
4.57 (d, J1,2 = 8.5 Hz, 1 H, 1I-H), 4.62, 4.64 (2 d, J = 11.8 Hz, 2 as an oil, and 11b (521 mg, 68%) as a white solid.
H, CH2Ph), 7.38–7.46 (m, 5 H, Ph) ppm. 13C NMR (150 MHz,
Data for 11a: [α]2D5 = –9.03 (c = 0.54, CHCl3). 1H NMR (600 MHz,
D2O): δ = 25.01 (CH3CO), 52.87 (CH2N3), 57.24 (C-2I), 63.79 (C-
CDCl3): δ = 2.00 (s, 3 H, CH3CO), 3.40 (m, 2 H, CH2N3), 3.45–
6II), 71.29 (C-4II), 71.74 (C-6I), 71.76 (C-4I), 71.82 (C-1Ј), 71.98,
3.57 (m, 5 H, 5II-H, 4I-H, 5I-H, 2I-H, 3II-H), 3.60–3.67 (m, 8 H,
72.33, 72.42, 72.43 (C-2Ј, C-3Ј, C-4Ј, C-5Ј), 73.44 (C-2II), 75.25 (C-
2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.70 (dd, J5,6a = 5.7, J6a,6b = 10.8 Hz, 1
3II), 75.99 (CH2Ph), 77.01 (C-5I), 78.04 (C-5II), 85.16 (C-3I), 103.59
H, 6aI-H), 3.77 (ddd, J1a,2a = 4.3, J1a,2b = 6.6, J1a,1b = 11.8 Hz, 1
(C-1I), 106.31 (C-1II), 131.07, 131.18, 131.48, 140.04 (Ph), 177.37
H, 1aЈ-H), 3.85 (br. d, J4,OH = 0.7 Hz, partial overlap, 1 H, OHI),
(CH3CO) ppm. ES-TOF-MS (pos. ion): m/z = 637.2905 ([M +
3.85 (dd, J5,6b = 2.3, J6a,6b = 10.8 Hz, partial overlap, 1 H, 6bI-H),
Li]+; calcd. 637.2908). C27H42N4O13 (630.3): calcd. C 51.42, H 6.71,
3.94 (br. dt, J1b,2a = J1b,2b = 4.0, J1a,1b = 11.8 Hz, 1 H, 1bЈ-H),
N 8.88; found C 51.18, H 6.69, N 8.89.
3.96–4.01 (m, 2 H, 3I-H, 2II-H), 4.21 (br. d, J2,OH = 2.6 Hz, 1 H,
8-Azido-3,6-dioxaoctyl 2-Acetamido-6-O-benzyl-3-O-(3-O-benzyl-β-
-galactopyranosyl)-2-deoxy-β- -glucopyranoside (10): A mixture
OHII), 4.33 (d, J1,2 = 7.8 Hz, 1 H, 1II-H), 4.40–4.49 (m, 2 H, 6II-
H), 4.57, 4.60 (2 d, 2J = 12.1 Hz, partial overlap, 2 H, CH2Ph),
D
D
2
of 9 (3.0 g, 4.76 mmol), Bu2SnO (1.185 g, 4.76 mmol) and toluene
(150 mL) was heated under reflux in a Soxhlet apparatus contain-
ing molecular sieves (4 Å). After 6 h, the mixture was cooled to
room temperature, CsF (1.45 g, 9.52 mmol) was added, and the
solvent was evaporated under reduced pressure. The residue was
dissolved in DMF (50 mL) and BnBr (1.15 mL, 9.52 mmol) was
added to the stirred solution. After 20 h, the mixture was concen-
trated and the residue was partitioned between CH2Cl2 (500 mL)
and brine (500 mL). The aqueous phase was extracted with CH2Cl2
(2ϫ250 mL), the combined organic layers were dried, concen-
trated, and the residue was chromatographed (CH2Cl2/MeOH:
100:0Ǟ95:5) to give 10 (2.753 g, 80%). [α]2D5 = –1.90 (c = 0.59,
4.58 (dd, JH,P = 7.0, J = 11.2 Hz, partial overlap, 1 H, CH2CCl3),
2
4.60 (dd, JH,P = 6.9, J = 11.2 Hz, partial overlap, 1 H, CH2CCl3),
4.74 (d, 2J = 12.1 Hz, partial overlap, 1 H, CH2Ph), 4.75 (br. d, J3,4
2
= 3.8 Hz, partial overlap, 1 H, 4II-H), 4.79 (d, J = 12.3 Hz, 1 H,
CH2Ph), 4.87 (d, J1,2 = 8.4 Hz, 1 H, 1I-H), 6.71 (d, J2,NH = 7.6 Hz,
1 H, NH), 7.25–7.38 (m, 10 H, 2 Ph) ppm. 13C NMR (150 MHz,
CDCl3): δ = 23.50 (CH3CO), 50.54 (CH2N3), 56.02 (C-2I), 66.26
(d, JC,P = 6.5 Hz, C-5II), 68.63 (C-1Ј), 69.40 (C-2II), 69.46 (C-4I),
69.82, 70.35, 70.45, 70.56 (C-2Ј, C-3Ј, C-4Ј, C-5Ј), 69.90 (C-6I),
70.74 (d, JC,P = 7.4 Hz, C-6II), 72.27, 73.44 (2 CH2Ph), 74.87 (C-
5I), 76.78 (d, JC,P = 7.1 Hz, partial overlap, C-4II), 76.84 (d, JC,P
5.4 Hz, partial overlap, CH2CCl3), 77.52 (d, JC,P = 7.0 Hz, C-3II),
=
MeOH). 1H NMR (600 MHz, CD3OD): δ = 1.98 (s, 3 H, CH3CO), 84.69 (C-3I), 94.85 (d, JC,P = 9.5 Hz, CCl3), 100.14 (C-1I), 104.07
3.35–3.38 (m, 3 H, 3II-H, CH2N3), 3.45 (t, J3,4 = J4,5 = 9.8 Hz,
(C-1II), 127.51, 127.65, 127.76, 127.85, 128.30, 128.46, 137.78,
partial overlap, 1 H, 4I-H), 3.44–3.52 (m, 2 H, 5II-H, 5I-H), 3.60– 138.29 (2 Ph), 172.30 (CH3CO) ppm. 31P NMR (121 MHz,
3.66 (m, 8 H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.67–3.73 (m, 5 H, 2II-H, CDCl3): δ = –10.61 ppm. ES-TOF-MS (pos. ion): m/z = 919.2075
3I-H, 6aI-H, 6aII-H, 1aЈ-H), 3.74–3.78 (m, 2 H, 2I-H, 6bII-H), 3.87 ([M + Li]+; calcd. 919.2079). C36H48Cl3N4O15P (912.19): calcd. C
(dd, J5,6b = 1.6, J6a,6b = 11.0 Hz, 1 H, 6bI-H), 3.93 (m, 1 H, 1bЈ-
H), 4.00 (br. d, J3,4 = 3.2 Hz, 1 H, 4II-H), 4.30 (d, J1,2 = 7.8 Hz, 1
H, 1II-H), 4.56 (d, J1,2 = 8.3 Hz, 1 H, 1I-H), 4.59 (s, 2 H, CH2Ph),
4.67, 4.76 (2 d, 2J = 11.8 Hz, 2 H, CH2Ph), 7.24–7.29 (m, 2 H, Ph),
7.30–7.37 (m, 6 H, Ph), 7.42–7.45 (m, 2 H, Ph) ppm. 13C NMR
(150 MHz, CD3OD): δ = 23.23 (CH3CO), 51.72 (CH2N3), 56.23
(C-2I), 62.46 (C-6II), 67.12 (C-4II), 70.01 (C-1Ј), 70.69 (C-4I), 70.85
(C-6I), 71.06, 71.51, 71.53, 71.65 (C-2Ј, C-3Ј, C-4Ј, C-5Ј), 71.74 (C-
2II), 72.64, 74.50 (CH2Ph), 76.63 (C-5I), 76.97 (C-5II), 81.99 (C-3II),
85.02 (C-3I), 102.42 (C-1I), 105.51 (C-1II), 128.62, 128.64, 128.81,
129.07, 129.27, 129.36, 139.70, 139.82 (2 Ph), 174.21 (CH3CO)
ppm. ES-TOF-MS (pos. ion): m/z = 727.3398 ([M + Li]+; calcd.
727.3378). C34H48N4O13 (720.3): calcd. C 56.66, H 6.71, N 7.77;
found C 56.83, H 6.76, N 7.64.
47.30, H 5.29, N 6.13; found C 47.44, H 5.44, N 6.12.
Data for 11b: M.p. 166–168 °C (from iPrOH; dec.). [α]2D5 = –2.54 (c
= 0.50, CHCl3). 1H NMR (600 MHz, CDCl3): δ = 2.01 (s, 3 H,
CH3CO), 3.37–3.53 (m, 5 H, 4I-H, 5I-H, 3II-H, CH2N3), 3.59–3.69
(m, 10 H, 2I-H, 6aI-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.71 (br. s, 1 H,
OHI), 3.73 (br. d, J2,OH = 2.3 Hz, 1 H, OHII), 3.80 (ddd, J1a,2a
=
3.1, J1a,2b = 8.2, J1a,1b = 12.1 Hz, partial overlap, 1 H, 1aЈ-H), 3.81–
3.86 (m, 2 H, 3I-H, 6bI-H), 3.87 (ddd, J2,OH = 2.3, J1,2 = 7.7, J2,3
= 9.8 Hz, 1 H, 2II-H), 3.93 (br. dt, J1b,2a = J1b,2b = 3.4, J1a,1b
=
12.1 Hz, 1 H, 1bЈ-H), 4.33 (d, J1,2 = 7.7 Hz, 1 H, 1II-H), 4.41 (ddd,
J5,6a = 1.5, J6a,6b = 12.6, J6a,P = 20.1 Hz, 1 H, 6aII-H), 4.58 (d, J
2
= 12.0 Hz, partial overlap, 1 H, CH2Ph), 4.58 (dd, JH,P = 7.9, 2J =
11.3 Hz, partial overlap, 1 H, CH2CCl3), 4.60 (d, 2J = 12.0 Hz,
2
partial overlap, 1 H, CH2Ph), 4.61 (dd, JH,P = 7.6, J = 11.3 Hz,
8-Azido-3,6-dioxaoctyl 2-Acetamido-6-O-benzyl-3-O-[3-O-benzyl-β-
partial overlap, 1 H, CH2CCl3), 4.65 (ddd, J = 2.3, J = 3.7, J6a,6b
2
D
-galactopyranosyl-(R)-(P)-4,6-cyclic 2,2,2-trichloroethyl phos-
phate]-2-deoxy-β- -glucopyranoside (11a) and 8-Azido-3,6-dioxaoc-
tyl 2-Acetamido-6-O-benzyl-3-O-[3-O-benzyl-β- -galactopyranosyl-
(S)-(P)-4,6-cyclic 2,2,2-trichloroethyl phosphate]-2-deoxy-β- -gluco-
= 12.6 Hz, 1 H, 6bII-H), 4.70 (d, J = 12.0 Hz, 1 H, CH2Ph), 4.78
2
D
(d, J1,2 = 8.5 Hz, partial overlap, 1 H, 1I-H), 4.79 (d, J = 12.0 Hz,
D
partial overlap, 1 H, CH2Ph), 4.90 (br. d, J3,4 = 3.1 Hz, 1 H, 4II-
H), 6.70 (d, J2,NH = 7.8 Hz, 1 H, NH), 7.26–7.38 (m, 10 H, 2 Ph)
ppm. 13C NMR (150 MHz, CDCl3): δ = 23.41 (CH3CO), 50.47
(CH2N3), 55.81 (C-2I), 66.73 (d, JC,P = 7.2 Hz, C-5II), 68.54 (C-1Ј),
69.40 (C-4I), 69.52 (C-2II), 69.66 (C-6I), 69.71, 70.26, 70.58, 70.98
(C-2Ј, C-3Ј, C-4Ј, C-5Ј), 69.85 (d, JC,P = 5.9 Hz, C-6II), 71.85, 73.54
(2 CH2Ph), 75.01 (d, JC,P = 5.5 Hz, partial overlap, C-4II), 75.05
(C-5I), 77.14 (d, JC,P = 6.9 Hz, partial overlap, C-3II), 77.73 (d, JC,P
= 5.0 Hz, CH2CCl3), 85.78 (C-3I), 94.35 (d, JC,P = 10.1 Hz, CCl3),
100.55 (C-1I), 103.91 (C-1II), 127.55, 127.59, 127.99, 128.08, 128.31,
128.56, 137.17, 138.15 (2 Ph), 172.28 (CH3CO) ppm. 31P NMR
D
pyranoside (11b): 2,2,2-Trichloroethyl phosphorodichloridate
(160 μL, 1.0 mmol) was added dropwise at –15 °C and with stirring
to a solution of 10 (600 mg, 0.83 mmol) and pyridine (675 μL,
8.33 mmol) in CH2Cl2 (8.5 mL). After 5 min, when TLC (reversed
phase, CH3CN/H2O: 1:1) indicated incomplete reaction, more
2,2,2-trichloroethyl phosphorodichloridate (160 μL, 1.0 mmol) was
added while stirring was continued at –15 °C. One additional por-
tion of reagent (160 μL, 1.0 mmol) was required to drive the reac-
tion to completion. The reaction was quenched with MeOH
4372
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© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2007, 4366–4375

Synthesis of Fragments of the O-Specific Polysaccharide of V. cholerae O139
FULL PAPER
(121 MHz, CDCl3): δ = –8.71 ppm. ES-TOF-MS (pos. ion): m/z =
919.2057 ([M + Li]+; calcd. 919.2079). C36H48Cl3N4O15P (912.19):
calcd. C 47.30, H 5.29, N 6.13; found C 47.14, H 5.39, N 6.14.
70.44 (CH2PhE), 70.66 (CH2PhA), 70.86 (C-2III), 71.06 (C-2IV),
71.12, 71.21, 71.35 (CH2PhB, CH2PhC, CH2PhD), 72.39 (C-4I),
73.21 (CH2PhF), 74.00 (d, JC,P = 4.4 Hz, C-4II), 74.79 (C-5I), 75.47
(C-4III), 76.69 (C-4IV), 77.64 (d, JC,P = 2.8 Hz, CH2CCl3), 78.13
(C-3I), 79.96 (d, JC,P = 7.6 Hz, C-3II), 94.59 (d, JC,P = 7.2 Hz,
CCl3), 95.73 (JC,H = 172.8 Hz, C-1III), 96.22 (JC,H = 167.0 Hz, C-
1IV), 98.86 (JC,H = 166.7 Hz, C-1I), 101.98 (JC,H = 158.8 Hz, C-
1II), 127.29, 127.37, 127.38, 127.45, 127.52, 127.58, 127.60, 127.68,
127.75, 127.76, 127.81, 127.94, 128.17, 128.23 (2 C), 128.24, 128.29,
128.44, 136.86, 138.13, 138.15, 138.17, 138.40, 138.95 (6 Ph),
170.59 (CH3CO) ppm. 31P NMR (121 MHz, CDCl3): δ = –2.30
ppm. ES-TOF-MS (pos. ion): m/z = 1539.5226 ([M + Li]+; calcd.
1539.5217). C76H92Cl3N4O21P (1532.51): calcd. C 59.47, H 6.04, N
3.65; found C 59.77, H 6.13, N 3.72. Next eluted was 8-Azido-3,6-
Ethyl 2,4-Di-O-benzyl-3,6-dideoxy-1-thio-β-L-xylo-hexopyranoside
(13): This compound was prepared as described.[31] When prepared
on large scale, it solidified spontaneously. M.p. 32–33 °C (from di-
isopropyl ether/hexane). [α]2D5 = +71.8 (c = 1, CHCl3). C22H28O3S
(372.5): calcd. C 70.93, H 7.58; found C 70.86, H 7.54.
2,4-Di-O-benzyl-3,6-dideoxy-α-L-xylo-hexopyranosyl Bromide (13a):
Br2 (1.28 mL, 25.0 mmol) was added to a solution of 13[31] (4.656 g,
12.50 mmol) in CCl4 (50 mL). Occasionally, the mixture was
shaken gently manually and, after 5 min, hex-1-ene (6.5 mL,
52.08 mmol) was added. The mixture was concentrated and a solu-
tion of the residue in CCl4 was concentrated (twice). Crude 13a
was used immediately in the next step without purification.
dioxaoctyl [2,4-Di-O-benzyl-3,6-dideoxy-α-
(1Ǟ2)-3-O-benzyl-β- -galactopyranosyl-(S)-(P)-4,6-cyclic 2,2,2-tri-
chloroethyl phosphate-(1Ǟ3)]-2-acetamido-6-O-benzyl-2-deoxy-β-
L-xylo-hexopyranosyl-
D
D-
8-Azido-3,6-dioxaoctyl 2,4-Di-O-benzyl-3,6-dideoxy-α-
pyranosyl-(1Ǟ4)-[2,4-di-O-benzyl-3,6-dideoxy-α-
pyranosyl-(1Ǟ2)-3-O-benzyl-β- -galactopyranosyl-(S)-(P)-4,6-
cyclic 2,2,2-trichloroethyl phosphate-(1Ǟ3)]-2-acetamido-6-O-
benzyl-2-deoxy-β- -glucopyranoside (14): A solution of 13a in
L-xylo-hexo-
glucopyranoside (15) (1.15 g, 45%; after further purification by
chromatography with CH2Cl2/MeOH: 25:1Ǟ15:1). [α]2D5 = –9.02 (c
L
-xylo-hexo-
1
D
= 0.55, CHCl3). H NMR (600 MHz, CDCl3): δ = 1.17 (d, J5,6
=
6.6 Hz, 3 H, 6III-H), 1.75 (dt, J3,4 = 1.9, J2,3 = J = 12.4 Hz, 1 H,
3III-Hax), 1.96 (s, 3 H, CH3CO), 2.07 (dt, J2,3 = J3,4 = 3.9, 2J =
12.9 Hz, 1 H, 3III-Heq), 3.22 (m, 1 H, 2I-H), 3.31–3.39 (m, 2 H,
CH2N3), 3.42 (br. t, J3,4 = J4,5 = 9.2 Hz, 1 H, 4I-H), 3.46 (br. s, 1
H, OHI), 3.48–3.54 (m, 2 H, 4III-H, 5I-H), 3.58–3.68 (m, 10 H, 6aI-
H, 5II-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.68–3.76 (m, 2 H, 1aЈ-H, 3II-
H), 3.79–3.85 (m, 2 H, 6bI-H, 2III-H), 3.92 (m, 1 H, 1bЈ-H), 4.16–
2
D
CH2Cl2 (15 mL) was added to a stirred mixture of 11b (1.9 g,
2.08 mmol), Bu4NBr (2.686 g, 8.33 mmol) and powdered molecular
sieves (4 Å, 4.5 g) in CH2Cl2/DMF (2:1, 15 mL). Stirring was con-
tinued at room temperature for 36 h before MeOH (5 mL) was
added. After 30 min, the mixture was diluted with CH2Cl2
(100 mL), filtered through a Celite pad, and the solids were washed
with CH2Cl2 (200 mL). The combined filtrates (ca. 500 mL) were
washed with saturated aq. NaHCO3 (500 mL) and H2O (500 mL),
dried, and concentrated. The residue was chromatographed (tolu-
ene/acetone: 50:50Ǟ0:100) to give first 14 (1.308 g, 41%; after fur-
ther purification by chromatography with CH2Cl2/MeOH:
19:1Ǟ16:1). [α]2D5 = –36.75 (c = 0.55, CHCl3). 1H NMR (600 MHz,
4.22 (m, 2 H, 5III-H, 2II-H), 4.26 (d, J = 12.3 Hz, partial overlap,
2
1 H, CH2PhA), 4.29 (br. t, J2,3 = J3,4 = 9.5 Hz, partial overlap, 1
H, 3I-H), 4.38 (d, 2J = 12.0 Hz, 1 H, CH2PhB), 4.43 (d, 2J =
12.3 Hz, partial overlap, 1 H, CH2PhA), 4.45 (m, 1 H, 6aII-H), 4.50
(d, 2J = 12.0 Hz, partial overlap, 1 H, CH2PhB), 4.51 (d, 2J =
11.5 Hz, partial overlap, 1 H, CH2PhC), 4.55–4.66 (m, 6 H, 1II-H,
6bII-H, CH2CCl3, OCH2PhD), 4.75 (d, 2J = 11.5 Hz, 1 H,
CDCl3): δ = 1.20 (d, J5,6 = 6.5 Hz, 3 H, 6III-H), 1.25 (d, J5,6
=
CH2PhC), 4.82 (d, J1,2 = 8.2 Hz, 1 H, 1I-H), 4.98 (br. d, J3,4
=
6.6 Hz, 3 H, 6IV-H), 1.71 (dt, J3,4 = 2.2, J2,3 = 2J = 12.8 Hz, partial
overlap, 1 H, 3III-Hax), 1.73 (s, 3 H, CH3CO), 1.80 (dt, J3,4 = 2.2,
2.5 Hz, 1 H, 4II-H), 5.50 (d, J1,2 = 3.3 Hz, 1 H, 1III-H), 6.07 (d,
J2,NH = 7.4 Hz, 1 H, NH), 7.12–7.34 (m, 20 H, 4 Ph) ppm. 13C
NMR (150 MHz, CDCl3): δ = 16.17 (C-6III), 23.60 (CH3CO), 27.58
(C-3III), 50.49 (CH2N3), 56.98 (C-2I), 66.40 (d, JC,P = 7.9 Hz, C-
5II), 66.61 (C-5III), 68.45 (C-1Ј), 69.38 (C-4I), 69.58 (C-6I), 69.76,
70.28, 70.50, 70.55 (C-2Ј, C-3Ј, C-4Ј, C-5Ј), 69.87 (d, JC,P = 6.2 Hz,
C-6II), 70.50 (CH2PhA), 70.73 (C-2III), 71.02 (CH2PhC), 71.28
(CH2PhB), 71.64 (C-2II), 73.40 (CH2PhD), 74.56 (d, JC,P = 5.4 Hz,
C-4II), 75.29 (C-5I), 75.63 (C-4III), 79.47 (d, JC,P = 6.4 Hz, C-3II),
80.59 (C-3I), 94.40 (d, JC,P = 9.9 Hz, CCl3), 96.56 (JC,H = 172.8 Hz,
C-1III), 100.05 (JC,H = 161.0 Hz, C-1I), 101.33 (JC,H = 159.3 Hz, C-
1II), 127.17, 127.38, 127.42, 127.43, 127.44, 127.62, 127.73, 128.15,
128.16, 128.16, 128.17, 128.35, 136.97, 138.16, 138.22, 138.33 (4
Ph), 171.02 (CH3CO) ppm. 31P NMR (121 MHz, CDCl3): δ =
–9.41 ppm. ES-TOF-MS (pos. ion): m/z = 1229.3718 ([M + Li]+;
calcd. 1229.3648). C56H70Cl3N4O18P (1222.4): calcd. C 54.93, H
5.76, N 4.58; found C 54.67, H 5.78, N 4.58.
2
2
J2,3 = J = 12.7 Hz, 1 H, 3IV-Hax), 2.12 (dt, J2,3 = J3,4 = 3.8, J =
13.0 Hz, partial overlap, 1 H, 3III-Heq), 2.15 (dt, J2,3 = J3,4 = 3.8,
2J = 12.7 Hz, partial overlap, 1 H, 3IV-Heq), 3.25 (br. q, J = 7.0 Hz,
1 H, 2I-H), 3.33 (t, J = 5.1 Hz, 2 H, CH2N3), 3.47 (br. s, 1 H, 4III
-
H), 3.53 (br. s, 1 H, 5II-H), 3.54–3.65 (m, 11 H, 3II-H, 5I-H, 4IV-H,
2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.69 (dd, J5,6a = 2.9, J6a,6b = 10.7 Hz, 1
H, 6aI-H), 3.71–3.79 (m, 2 H, 1Ј-H), 3.82–3.88 (m, 2 H, 2III-H, 2IV-
H), 3.89 (dd, J5,6b = 3.8, J6a,6b = 10.7 Hz, partial overlap, 1 H, 6bI-
H), 3.99 (t, J3,4 = J4,5 = 8.4 Hz, 1 H, 4I-H), 4.11 (t, J1,2 = J2,3
=
8.8 Hz, 1 H, 2II-H), 4.20 (br. q, J5,6 = 6.5 Hz, 1 H, 5III-H), 4.33 (d,
2J = 11.9 Hz, 1 H, CH2PhA), 4.35–4.45 (m, 6 H, 3I-H, 6aII-H,
CH2PhB, CH2PhC, CH2PhD), 4.45 (d, 2J = 11.8 Hz, partial overlap,
1 H, CH2PhC), 4.46 (d, 2J = 12.1 Hz, partial overlap, 1 H,
CH2PhA), 4.47 (d, 2J = 11.8 Hz, 1 H, CH2PhE), 4.48 (d, 2J =
15.5 Hz, partial overlap, 1 H, CH2PhF), 4.51 (d, 2J = 12.2 Hz, 1 H,
CH2PhF), 4.52 (d, 2J = 12.1 Hz, 1 H, CH2PhD), 4.62–4.68 (m, 3 H,
6bII-H, 1II-H, 5IV-H), 4.62 (dd, JH,P = 10.2, 2J = 11.5 Hz, partial 8-Amino-3,6-dioxaoctyl 2-Acetamido-2-deoxy-3-O-(β-
D-galactopyr-
2
overlap, 1 H, CH2CCl3), 4.68 (dd, JH,P = 9.1, J = 11.5 Hz, partial
anosyl-4,6-cyclic potassium phosphate)-β-D-glucopyranoside (12):
overlap, 1 H, CH2CCl3), 4.77 (d, 2J = 11.8 Hz, 1 H, CH2PhE), 4.97
(d, J3,4 = 3.3 Hz, 1 H, 4II-H), 5.02 (d, J1,2 = 3.5 Hz, 1 H, 1IV-H),
Pd/C (540 mg) was added to a mixture of 11a (540 mg, 0.59 mmol),
iPrOH (21 mL) and potassium phosphate buffer (21 mL; pH = 7,
0.1 m). The mixture was stirred at room temperature in a Parr reac-
tor under H2 (160 psi). After 2 d, when TLC showed complete con-
5.06 (d, J1,2 = 6.7 Hz, 1 H, 1I-H), 5.62 (d, J1,2 = 3.4 Hz, 1 H, 1III
-
H), 6.35 (d, J2,NH = 7.0 Hz, 1 H, NH), 7.16–7.32 (m, 30 H, 6 Ph)
ppm. 13C NMR (150 MHz, CDCl3): δ = 16.28 (C-6IV), 16.39 (C- version, the mixture was filtered through a Celite pad, the catalyst
6III), 23.17 (CH3CO), 27.24 (C-3IV), 28.07 (C-3III), 50.64 (CH2N3), was washed with MeOH and H2O, and the filtrate was concen-
58.11 (C-2I), 66.51 (C-5IV), 66.64 (C-5III), 67.02 (d, JC,P = 5.7 Hz, trated. The residue was purified by normal phase chromatography
C-5II), 68.05 (C-1Ј), 68.29 (C-6I), 69.97, 70.49, 70.51, 70.76 (C-2Ј, (MeOH/H2O:100:0Ǟ10:1) to give, after freeze-drying, 12 (218 mg,
C-3Ј, C-4Ј, C-5Ј), 70.01 (C-2II), 70.23 (d, JC,P = 4.5 Hz, C-6II),
60%). H NMR (600 MHz, D2O): δ = 1.99 (s, 3 H, CH3CO), 3.18
1
Eur. J. Org. Chem. 2007, 4366–4375
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4373

B. Ruttens, R. Saksena, P. Kovácˇ
FULL PAPER
(br. t, J = 5.1 Hz, 2 H, CH2NH2), 3.45 (ddd, J5,6b = 2.3, J5,6a
=
was stirred at room temperature in a Parr reactor under H2 (160
psi). After 3 d, TLC showed complete conversion. The mixture was
filtered through a Celite pad, the catalyst was washed with MeOH
5.6, J4,5 = 10.0 Hz, 1 H, 5I-H), 3.52 (dd, J3.4 = 8.5, J4,5 = 10.0 Hz,
1 H, 4I-H), 3.56 (dd, J1,2 = 7.9, J2,3 = 10.0 Hz, 1 H, 2II-H), 3.63–
3.70 (m, 6 H, 3 CHЈ2O), 3.71–3.77 (m, 7 H, 3I-H, 5II-H, 6aI-H, 3II- and H2O, and the filtrate was concentrated. The residue was puri-
H, 1aЈ-H, CHЈ2O), 3.81 (dd, J1,2 = 8.4, J2,3 = 10.4 Hz, 1 H, 2I-H), fied by reversed phase chromatography (H2O/MeOH: 100:0Ǟ97:3)
3.89 (dd, J5,6b = 2.3, J6a,6b = 12.4 Hz, 1 H, 6bI-H), 3.98 (ddd, J1b,2a
to give, after freeze-drying, 17 (70 mg, 58%). H NMR (600 MHz,
1
2
= 3.2, J1b,2b = 5.9, J1a,1b = 11.6 Hz, 1 H, 1bЈ-H), 4.22 (ddd, J5,6a
=
D2O): δ = 1.15 (d, J5,6 = 6.7 Hz, 3 H, 6III-H), 1.80 (dt, J = 2.8, J
1.7, J6a,6b = 12.8, J6a,P = 22.3 Hz, 1 H, 6aII-H), 4.38 (br. d, J6a,6b
= 12.8 Hz, 1 H, 6bII-H), 4.48 (d, J1,2 = 7.9 Hz, 1 H, 1II-H), 4.55
(d, J1,2 = 8.4 Hz, 1 H, 1I-H), 4.56 (br. d, J3,4 = 3.5 Hz, 1 H, 4II-
H) ppm. 13C NMR (150 MHz, D2O): δ = 22.42 (CH3CO), 39.25
= 12.9 Hz, 1 H, 3III-Hax), 1.91 (br. dt, J = 4.0, J = 12.9 Hz, 1 H,
2
3III-Heq), 2.03 (s, 3 H, CH3CO), 3.20 (t, J = 5.0 Hz, 2 H, CH2NH2),
3.47–3.51 (m, 2 H, 4I-H, 5I-H), 3.63–3.79 (m, 14 H, 2I-H, 6aI-H,
2II-H, 5II-H, 4III-H, 4 CHЈ2O, 1aЈ-H), 3.89–3.95 (m, 3 H, 3I-H, 6bI-
(CH2NH2), 54.57 (C-2I), 60.80 (C-6I), 66.48, 69.63, 69.76, 69.84 (C- H, 3II-H), 3.96–4.02 (m, 2 H, 2III-H, 1bЈ-H), 4.23 (br. q, J5,6
=
=
2Ј, C-3Ј, C-4Ј, C-5Ј), 67.47 (d, JC,P = 4.7 Hz, C-5II), 68.34 (d, JC,P
= 5.4 Hz, C-6II), 68.68 (C-4I), 69.18 (C-1Ј), 69.93 (C-2II), 71.15 (d,
JC,P = 7.4 Hz, C-3II), 75.50 (C-5I), 76.08 (d, JC,P = 5.1 Hz, C-4II),
82.69 (C-3I), 101.12 (C-1I), 103.30 (C-1II), 174.62 (CH3CO) ppm.
31P NMR (121 MHz, D2O): δ = –3.75 ppm. ES-TOF-MS (neg.
ion): m/z = 575.1836 ([M – K]–; calcd. 575.1853).
6.7 Hz, 1 H, 5III-H), 4.31 (ddd, J5,6a = 1.5, J6a,6b = 12.7, J6a,P
22.0 Hz, 1 H, 6aII-H), 4.40 (br. d, J6a,6b = 12.7 Hz, 1 H, 6bII-H),
4.43 (d, J1,2 = 8.5 Hz, 1 H, 1I-H), 4.57 (d, J3,4 = 3.4 Hz, 1 H, 4II-
H), 4.71 (d, J1,2 = 7.7 Hz, 1 H, 1II-H), 5.08 (d, J1,2 = 3.7 Hz, 1 H,
1III-H) ppm. 13C NMR (150 MHz, D2O): δ = 18.12 (C-6III), 24.96
(CH3CO), 35.55 (C-3III), 41.93 (CH2NH2), 57.48 (C-2I), 63.43 (C-
6I), 65.95 (C-2III), 69.04 (C-5III), 69.18, 71.96, 72.36, 72.52, 72.55
(C-1Ј, C-2Ј, C-3Ј, C-4Ј, C-5Ј), 70.00 (d, JC,P = 5.0 Hz, C-5II), 70.97
(d, JC,P = 5.3 Hz, C-6II), 71.26, 71.28 (C-4III, C-4I or C-5I), 74.75
(d, JC,P = 7.2 Hz, C-3II), 78.19 (C-4I or C-5I), 78.52 (C-2II), 79.30
(d, JC,P = 4.9 Hz, C-4II), 80.97 (C-3I), 101.92 (C-1III), 102.85 (C-
1II), 104.59 (C-1I), 176.56 (CH3CO) ppm. 31P NMR (121 MHz,
D2O): δ = –3.73 ppm. ES-TOF-MS (neg. ion): m/z = 705.2466
([M – K]–; calcd. 705.2483).
8-Amino-3,6-dioxaoctyl 3,6-Dideoxy-α-
(1Ǟ4)-[3,6-dideoxy-α- -xylo-hexopyranosyl-(1Ǟ2)-β-
pyranosyl-4,6-cyclic potassium phosphate-(1Ǟ3)]-2-acetamido-2-de-
oxy-β- -glucopyranoside (16): Pd/C (1.2 g) was added to a mixture
L
-xylo-hexopyranosyl-
L
D
-galacto-
D
of 14 (1.2 g, 0.78 mmol), iPrOH (47 mL) and potassium phosphate
buffer (47 mL; pH = 7, 0.1 m). The mixture was stirred at room
temperature in a Parr reactor under H2 (160 psi). After 24 h, when
TLC showed complete conversion, the mixture was filtered through
a Celite pad, the catalyst was washed with MeOH and H2O, and
the filtrate was concentrated. The residue was purified by reversed
phase chromatography (H2O/MeOH: 100:0Ǟ95:5) to give, after
Acknowledgments
1
freeze-drying, 16 (378 mg, 55%). H NMR (600 MHz, D2O): δ =
1.19 (d, J5,6 = 6.7 Hz, partial overlap, 3 H, 6III-H), 1.19 (d, J5,6
=
This research was supported by the Intramural Research Program
of the National Institutes of Health (NIH), and the National Insti-
tute of Diabetes and Digestive and Kidney (NIDDK).
6.8 Hz, partial overlap, 3 H, 6IV-H), 1.80–1.89 (m, 3 H, 3III-H, 3IV-
Heq), 2.02 (s, 3 H, CH3CO), 2.05 (m, 1 H, 3IV-Hax), 3.15 (br. t, J
= 5.0 Hz, 2 H, CH2NH2), 3.47 (br. dt, J = 3.0, J = 9.8 Hz, 1 H,
5I-H), 3.59 (dd, J1,2 = 7.9, J2,3 = 9.7 Hz, partial overlap, 1 H, 2II-
H), 3.60 (br. s, partial overlap, 1 H, 5II-H), 3.62–3.73 (m, 10 H, 4I-
H, 1aЈ-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Ј-H), 3.76 (br. t, J3,4 = 3.5 Hz, 1 H,
4III-H), 3.81 (dd, J1,2 = 8.5, J2,3 = 10.6 Hz, 1 H, 2I-H), 3.86 (dd,
J5,6a = 3.7, J6a,6b = 12.7 Hz, partial overlap, 1 H, 6aI-H), 3.88 (m,
1 H, 3II-H), 3.91–4.01 (m, 4 H, 2IV-H, 2III-H, 6bI-H, 1bЈ-H), 4.03
(dd, J3,4 = 9.3, J2,3 = 10.6 Hz, 1 H, 3I-H), 4.21 (br. t, J = 3.6 Hz,
1 H, 4IV-H), 4.28 (br. q, J5,6 = 6.7 Hz, 1 H, 5III-H), 4.30–4.41 (m,
2 H, 6II-H), 4.39 (d, J1,2 = 8.5 Hz, partial overlap, 1 H, 1I-H), 4.54
(d, J3,4 = 3.8 Hz, 1 H, 4II-H), 4.69 (d, J1,2 = 7.9 Hz, 1 H, 1II-H),
4.75 (br. q, J5,6 = 6.8 Hz, 1 H, 5IV-H), 4.89 (d, J1,2 = 3.8 Hz, 1 H,
1IV-H), 4.97 (d, J1,2 = 3.8 Hz, 1 H, 1III-H) ppm. 13C NMR
(150 MHz, D2O): δ = 15.61 (C-6III), 15.72 (C-6IV), 22.33 (CH3CO),
32.68 (C-3IV), 32.84 (C-3III), 39.35 (CH2NH2), 55.86 (C-2I), 59.46
(C-6I), 63.41 (C-2IV), 63.66 (C-2III), 66.27 (C-5III), 66.93, 69.76,
70.00, 70.05 (C-2Ј, C-3Ј, C-4Ј, C-5Ј), 66.96 (C-5IV), 67.53 (d, JC,P
= 4.9 Hz, C-5II), 68.55 (C-4IV), 68.85 (d, JC,P = 4.2 Hz, partial over-
lap, C-6II), 68.87 (C-4III), 69.32 (C-1Ј), 72.50 (d, JC,P = 7.0 Hz, C-
3II), 72.60 (C-4I), 75.44 (C-3I), 75.63 (C-5I), 76.32 (C-2II), 76.60 (d,
JC,P = 5.2 Hz, C-4II), 97.90 (C-1IV), 99.68 (C-1III), 101.19 (C-1II),
101.97 (C-1I), 173.99 (CH3CO) ppm. 31P NMR (121 MHz, D2O):
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Published Online: July 5, 2007
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