Synthesis of a phosphorylated disaccharide fragment of the O-specific polysaccharide of Vibrio cholerae O139, functionalized for conjugation

Article abstract of DOI:10.1002/hlca.200690036

The title disaccharide, 2-(2-(2-[(2-ethoxy-3,4-dioxocyclobut-1-en-1-yl) amino]ethoxy}ethoxy}ethyl 2-O-(3,6-dideoxy-α-L-xylo-hexopyranosyl)-β- D-galactopyranoside cyclic 4,6-(potassium phosphate) (2), was synthesized from the two isomeric linker-equipped galactose acceptors 9 and 10, obtained by phosphorylation of 2-[2-(2-azidoethoxy)ethoxy]ethyl 3-O-benzyl-β-D- galactopyranoside (8). which were glycosylated with ethyl 2,4-di-O-benzyl-3,6- dideoxy-1-thio-β-L-xylo-hexopyranoside (12; Scheme). Mainly the fully protected α-(1 → 2)-linked products 13a and 14a were formed. Catalytic hydrogenolysis/hydrogenation effected global deprotection, thereby removing the chirality at the P-atom. and simultaneously converted the azido group in the linker to an amino group (→15). Final treatment with diethyl squarate (=3,4-diethoxycyclobut-3-ene-1,2-dione) gave target compound 2, amenable for conjugation to proteins.

Full text of DOI:10.1002/hlca.200690036

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Synthesis of a Phosphorylated Disaccharide Fragment of the O-Specific
Polysaccharide of Vibrio cholerae O139, Functionalized for Conjugation
by Bart Ruttens and Pavol Kovácˇ*
National Institutes of Health, NIDDK, LMC, Carbohydrates, Bldg. 8, Rm. B1A25, 8 Center Drive,
Bethesda, MD, 20892-0815, USA
(phone: +1301-496-3569; fax: +1301-402-0589; e-mail: kpn@helix.nih.gov)
The title disaccharide, 2-{2-{2-[(2-ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino]ethoxy}ethoxy}ethyl
2-O-(3,6-dideoxy-a-L-xylo-hexopyranosyl)-b-D-galactopyranoside cyclic 4,6-(potassium phosphate) (2),
was synthesized from the two isomeric linker-equipped galactose acceptors 9 and 10, obtained by phos-
phorylation of 2-[2-(2-azidoethoxy)ethoxy]ethyl 3-O-benzyl-b-D-galactopyranoside (8), which were gly-
cosylated with ethyl 2,4-di-O-benzyl-3,6-dideoxy-1-thio-b-L-xylo-hexopyranoside (12; Scheme). Mainly
the fully protected a-(1 ! 2)-linked products 13a and 14a were formed. Catalytic hydrogenolysis/hydro-
genation effected global deprotection, thereby removing the chirality at the P-atom, and simultaneously
converted the azido group in the linker to an amino group (! 15). Final treatment with diethyl squarate
(=3,4-diethoxycyclobut-3-ene-1,2-dione) gave target compound 2, amenable for conjugation to proteins.
Introduction. – Cholera [1] is an enteric disease caused by Vibrio cholerae. Ingestion
of contaminated water or food can lead to severe diarrhea, dehydration, and even
hypotensive shock and death. Current vaccines give only short-term protection and
produce an inadequate serological response, particularly in young children. Thus, chol-
era is a persistent problem for the developing and, occasionally, the developed world.
Before 1992, only the O1 serogroup was associated with epidemic and pandemic chol-
era, other strains merely caused sporadic illness. Since its appearance in India in 1992,
Vibrio cholerae O139 has emerged as a new serious threat to public health [2]. The lack
of protective immunity against this new strain of the adult population in V. cholerae O1
endemic regions comprises its epidemic and pandemic potential. Research indicates
that 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]. As a result, the main differences between both serotypes are found in the con-
stitution of the cell surface [4]. As opposed to V. cholerae O1, serotype O139 expresses
a capsular polysaccharide (CPS), whose repeating unit is identical to the monomeric O-
chain of the lipooligosaccharide (LOS, Fig. 1) [4][5]. Both the CPS and the O-chain of
the LOS were shown to be virulance factors [4], and a critical level of serum immuno-
globulin G (IgG) antibodies against these surface saccharides is required for immunity
to the parent Vibrio [6].
Carbohydrates, classified as T-cell-independent (TI) antigens, are poor immuno-
gens [7]. They are capable of activating antibody-producing B cells but fail to generate
memory B cells. In contrast, T-cell-dependant (TD) antigens, such as proteins, not only
initiate immunologic memory but also educe a stronger and earlier antibody response.
© 2006 Verlag Helvetica Chimica Acta AG, Zürich

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Fig. 1. Schematic representation of a) the capsular polysaccharide (CPS) and b) the O-specific polysac-
charide (O-PS) of V. cholerae O139. (Colitose=Colp, galactose=Galp, N-acetylglucosamine=Glcp-
NAc, galacturonic acid=GalpA, and N-acetylquinovosamine=QuipNAc).
Avery and Goebel [8] first demonstrated that chemical conjugation to proteins con-
verted carbohydrates into TD antigens, or rather, elicited anti-carbohydrate antibodies
in T-dependant fashion. Although the underlying cellular mechanisms are still not fully
understood, synthetic glycoconjugates have found wide application as carbohydrate
immunogens [9]. Initially, this concept was used mainly to produce vaccines where
the hapten was harvested from pathogens. More recently, the progress in oligo- and
polysaccharide synthesis sparked efforts to prepare conjugate vaccines from synthetic
carbohydrate antigens [10]. Clinical trials with the first synthetic conjugate vaccine
demonstrated the feasibility of a synthetic approach [11].
For V. cholerae O139, only cellular vaccines [12][13] and conjugate vaccines based
on detoxified CPS [14] or LOS [15] have been reported. Compared to these pathogen-
derived vaccines, conjugate vaccines from synthetic O-PS, although more difficult to
obtain, would offer significant advantages when it comes to purity, reproducibility of
preparation, homogeneity, shelf life, and potential side effects. In addition, access to
fragments of the O-PS allows mapping of the epitopes that are crucial for protective
capacity.
Recently, as an extension of our earlier and ongoing work on a conjugate vaccine
for cholera caused by V. cholerae O1 [16], we started a project focused on the develop-
ment of a conjugate vaccine 1 for cholera (Fig. 2) from the synthetic hexasaccharide
that represents the O-antigenic region of the LOS of V. cholerae O139. Oscarson and
co-workers [17] were the first to synthesize fragments of the V. cholerae O139 O-PS,
but the reported tri- and tetrasaccharides lacked the phosphate group and needed fur-
ther derivatization to allow conjugation with a carrier protein. Here, we describe the
first synthesis of a phosphate-containing, V. cholerae O-PS related disaccharide frag-
ment, equipped with a linker functionalized for conjugation.

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Fig. 2. General structure of a conjugate vaccine 1 against V. cholerae O139 and the upstream terminal
disaccharide fragment 2 for conjugation
Because interaction of antigenic oligo- and polysaccharide with antibodies often
occurs through the terminal saccharide units at the upstream [18] end, we concentrated
our initial efforts on the synthesis of disaccharide fragment 2, which is designed for con-
jugation by the squaric acid diester methodology originally developed by Tietze et al.
[19]. It allowed us to research some of the key issues in the preparation of 1, such as
formation of the cyclic phosphate and the stability of the labile colitosidic bond. The
8-amino-3,6-dioxaoctyl (=2-[2-(2-aminoethoxy)ethoxy]ethyl) linker [20] provides
good chemical inertness and, if required, allows length variation without fundamental
changes to the chemistry.
Results and Discussion. – The synthesis of fragment 2 (see Scheme) was based on
results of preliminary experiments aimed at probing cyclic 4,6-phosphate, i.e., 1,3,2-
dioxaphosphorinane oxide, formation, stability, and deprotection. The 1,3,2-dioxaphos-
phorinane 2-oxides with electronegative 2-substituents preferentially adopt chair con-
formations with axially oriented 2-aryloxy or 2-alkoxy groups [21]. However, for the
formation of 1,3,2-dioxaphosphorinane 2-oxides by condensation of a phosphoric diha-
lide (=phosphorodihalogenidate) and a diol, a kinetic preference for the thermody-
namically less stable isomer is generally observed [22], leading to a phosphate isomer
mixture. The final isomer ratio depends on steric interactions in transition intermedi-
ates and the relative stabilities of the chair conformers. Indeed, reaction of methyl
2,3-di-O-benzyl-b-D-galactopyranoside with alkyl or aryl phosphorodichloridate
reagents in the presence of pyridine as base and catalyst (not described in the Exper.

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323
Part) gave virtually theoretical yields of a mixture of isomeric cyclic phosphates. They
showed good acid stability but were unstable under basic conditions. Treatment with
KOH in aqueous solution, e.g., led quickly to deprotection of the cyclic phosphate or
ring opening. The acyclic phosphate was subsequently converted slowly into the depro-
tected cyclic derivative by intramolecular substitution of the phosphate protecting
group, rather than further hydrolyzed to the acyclic dibasic phosphate. These observa-
tions (not described in the Exper. Part) can be explained by the considerable difference
in rates of alkaline hydrolysis of phosphate esters [23] and are consistent with earlier
reports [24]. Due to their base lability, basic reaction conditions such as for deacetyla-
tions and debenzoylations are incompatible with protected phosphates, and isomer for-
mation suggests the phosphate be introduced as late as possible in the synthesis, ideally
just before deprotection. We do not follow the latter advice because it would lenghten
our synthesis and could potentially lead to problems (see further).
The amine function, necessary for conjugation by dialkyl squarate chemistry, was
generated at the end of the synthesis by catalytic reduction of an azide precursor
(Scheme). A common approach to obtain the 8-azido-3,6-dioxaoctan-1-ol (=2-[2-(2-
azidoethoxy)ethoxy]ethanol; 4) linker relies on monosulfonylation of triethylene glycol
(=2,2’-[ethane-1,2-diylbis(oxy)]bis[ethanol]), followed by nucleophilic displacement of
the leaving group with azide ion [19][25]. The major drawback of this approach is the
moderate selectivity for monoactivation of the OH groups in the starting glycol. There-
fore, we opted for direct substitution of the Cl-atom in commercially available 2-[2-(2-
chloroethoxy)ethoxy]ethanol (3), by a modified procedure of Rensen et al. [26]. Since
monitoring of this conversion by TLC (normal or reversed phase) was difficult, it was
done by GC. Purification by destillation led to significant losses due to thermal decom-
position of the azide derivative 4. Equally pure 4 was obtained more efficiently by chro-
matography on silica gel.
The lability of the 3-O-benzyl group under the conditions reported [27] for the prep-
aration of galactosyl chloride 6 from 5 led to considerable fluctuations in yield
(60–80%), especially when working on small scale. Three factors appeared to contrib-
ute to this problem, i.e., the reaction temperature, the workup procedure, and, in par-
ticular, the amount of ZnCl2 catalyst. To make addition of freshly fused ZnCl2 to the
reaction mixture more accurate, the catalyst was added from a stock solution in
AcOH. This ZnCl2 solution has a shelf life of at least 8 months, and we found its activity
superior to the commercially available solutions in Et2O or THF. When the reaction
was carried out at room temperature instead of 608 [27] and the crude mixture was
washed with an ice-cold saturated hydrogen carbonate solution before evaporation, 6
was obtained from 5 [28] in a consistent yield of 85%. Glycosidation of 6 with acceptor
4, followed by debenzoylation of the formed 8-azido-3,6-dioxaoctyl b-D-galactoside 7,
gave triol 8.
The most obvious sequence to convert triol 8 into disaccharide 2 starts with selec-
tive 4,6-protection, followed by O(2) glycosylation and release of the 4,6-diol to intro-
duce the phosphate. In this way, formation of the phosphate is the last step before
deprotection. However, reports that formation of six-membered cyclic phosphates is
favored over polymerization [29] prompted us to explore a more straightforward
approach, namely phosphorylation of 8. From the few commercially available phospho-
ric dihalide reagents, 2,2,2-trichloroethyl phosphorodichloridate was selected, due to

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Scheme. Preparation of a Terminal Upstream Disaccharide Fragment 2 of the O-PS of V. cholerae
O139 Designed for Conjugation by the Squaric Acid Diester Methodology
a) NaN3, NaI, aq. EtOH, 1258. b) ZnCl2, Cl2CH2OMe, CH2Cl2, r.t. c) AgOTf, sym-collidine, CH2Cl2,
r.t. d) K2CO3, MeOH, r.t. e) Cl3CCH2OP(O)Cl2, pyridine, CH2Cl2, ꢀ158. f) 1. K2CO3, MeOH, r.t.; 2.
NaH, BnBr, DMF, 08 to r.t. g) Powdered 4 Å mol. sieves, AgOTf, NIS, sym-collidine, toluene/dioxane
i
3 :1, 08. h) Pd/C, H2 (65 psi), PrOH, potassium phosphate buffer (pH 7), r.t. i) KHCO3, diethyl squa-
rate, abs. EtOH, r.t.
the wide variety of methods for removal of the 2,2,2-trichloroethyl group [30]. From 8
and phosphorylating reagent, a 35 :65 mixture (NMR) of the two phosphate isomers 9

Helvetica Chimica Acta – Vol. 89 (2006)
325
and 10 was obtained in virtually theoretical yield. Concomitant phosphorylation at
HOꢀC(2) can be avoided by portionwise addition of the reagent based on monitoring
by TLC. Phosphorylation this early in the sequence requires separate glycosylation of
phosphates 9 and 10, but it avoids possible problems with deprotection of a temporary
4,6-O protecting group in the presence of the labile colitosidic bond.
The a/b-L ratio of the glycosylation of acceptor 10 with b-L thioglycoside 12 [31]
under various conditions was determined by isolation of both anomers after coupling
(Table). Reasonable a-L-selectivity was observed in CH2Cl2/toluene at room tempera-
ture (Entry 1), yet an improved a-L-directional solvent was found in dioxane/toluene
[32] (Entry 2). Performing the glycosylation at 08 (Entry 3) did not change the a/b-L
ratio, but a cleaner reaction mixture was formed. Since acceptor 10 is insoluble in Et2O,
THF was tested as a substitute for dioxane for coupling at lower temperatures. How-
ever, the reaction in THF/toluene at 08 (Entry 4) was less selective. Iodonium dicolli-
dine triflate (=bis(2,4,6-trimethylpyridine-kN)iodine(1+) trifluoromethanesulfonate;
IDCT) [33] (Entry 5) gave the best anomeric selectivity, but the reaction was sluggish,
and a fraction of the donor remained unchanged, even with a large excess of promoter.
Although the use of the NIS/AgOTf (=N-iodosuccinimide/AgOSO2CF3) promoter sys-
tem led to formation of N-succinimidyl colitoside (=2,5-dioxopyrrolidin-1-yl 3,6-
dideoxy-L-xylo-hexoside) by-products [17], this side reaction was only minor and did
not seem to affect the yield of the glycosylation. Thus, glycosylation of 10 with b-L-thio-
glycoside 12 in dioxane/toluene at 08 in the presence of sym-collidine (=2,4,6-trime-
thylpyridine), powdered molecular sieves, and NIS/AgOTf gave a mixture of 14a
and 14b in 68% and 11% yield, respectively, in an overall yield of 95%, based on con-
sumed acceptor 10. Glycosylation of alcohol 9 with donor 12 under the same conditions
furnished 13a in 76% and 13b in 19% yield. The lower a/b-L ratio and complete con-
sumption of the acceptor suggest a higher reactivity of 9 compared to its phosphate iso-
mer 10. It is noteworthy that b-L-thioglycoside 12 slowly anomerized and decomposed,
even when kept at ꢀ208. Therefore, it is advisable to prepare it from its stable crystal-
line diacetate 11 [31] as required, following the procedure given in the Exper. Part,
which is a slight modification of that reported [31].
Table. Glycosylation of Acceptor 10 with b-L-Donor 12 in the Presence of Powdered 4 Å Molecular Sieves
and sym-Collidine
Entry
Activatora)
Solvent (v/v)
Temperature
a/b-L
1
2
3
4
5
NIS/AgOTf
NIS/AgOTf
NIS/AgOTf
NIS/AgOTf
IDCT
CH2Cl2/toluene 1:1
dioxane/toluene 3 :1
dioxane/toluene 3 :1
THF/toluene 3 :1
r.t.
r.t.
08
08
4 :1
6 :1
6 :1
3 :1
8 :1
dioxane/toluene 3 :1
08 to r.t.
a) NIS=N-Iodosuccinimide, AgOTf=AgOSO2CF3, IDCT=iodonium dicollidine triflate.
Hydrogenolysis/reduction of the benzyl ethers, the trichloroethyl group [34], and
the azide group was accomplished in one step in the presence of Pd/C catalyst, but reac-
tion conditions had to be carefully optimized. Partial decomposition of the disaccharide
caused by HCl generated during cleavage of the trichloroethyl group was observed

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when the reaction was carried out in EtOH/H2O in the absence of a proton scavenger.
In a mixture of EtOH and a potassium phosphate buffer (pH 7), cleavage of the glyco-
sidic bonds was suppressed but the reaction was incomplete. At elevated temperature
(508), required to push the reaction to completion, N-ethylation of the formed amine
i
was observed [35]. When PrOH instead of EtOH was used, N-alkylation did not
occur, but another side reaction of the amine took place, namely transamination.
This led to the formation of the dimer of the desired deprotected disaccharide [36].
The mechanism of dimerization involves dehydrogenation and release of ammonia
[37], suggesting this side reaction could be minimized under higher H2 pressure. Indeed,
at room temperature and 60–70 psi H2 pressure, a reasonably clean reaction was
observed (TLC), and amino derivative 15 was isolated in 65–70% yield. The two phos-
phate isomers 13a and 14a gave the same material 15 (1H-NMR, 13C-NMR, 31P-NMR,
TLC) after deprotection.
Amino derivative 15 displayed an atypical reactivity toward squaric acid diesters
[19] in that nucleophilicity seemed subdued at slightly acidic and neutral pH, possibly
by internal salt formation with the phosphate. This had important consequences for
purification and derivatization. Purification could be accomplished on regular silica
gel without addition of a base to the solvent because, apparently, little protonation of
the amine by the acidic silanol groups occurred. Derivatization required slightly
basic conditions to be efficient. Formation of squarate 2, e.g., was sluggish in a pH 7
potassium phosphate buffer or in anhydrous EtOH, the two classic methods [19]. In
anhydrous EtOH in the presence of solid KHCO3 [38], however, the reaction pro-
ceeded smoothly with smaller excess of reagent. The slightly basic conditions led to
some hydrolysis of the formed monoethyl squarate, but 2 was obtained in excellent
yield (90–95%).
No elemental analyses or optical rotations were collected for the disaccharide phos-
phate salts 15 and 2. 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 spec-
tra, and their purity was verified by TLC and NMR spectroscopy.
This research was supported by the Intramural Research Program of the NIH, NIDDK.
Experimental Part
General. HPLC-Grade solvents were used, and reactions requiring anh. conditions were carried out
under N2 or Ar. A Parr hydrogenator was used for reactions under H2. Dioxane was distilled from Na/
benzophenone, and toluene from Na. N-Iodosuccinimide was recrystallized from dioxane/CCl4 and
stored in a desiccator. Dichloromethyl methyl ether (Cl2CH2OMe), 2,2,2-trichloroethyl phosphorodi-
chloridate (dꢁ1.7 g/ml at 208, density roughly determined by differential weighing of 1 ml of reagent)
and 2-[2-(2-chloroethoxy)ethoxy]ethanol (3) were purchased from Aldrich and used as provided.
Strata-SPE-C18 cartridges were purchased from Phenomenex. The 5% Pd/C catalyst (Escat 103) was a
product of Engelhard Industries. sym-Collidine was stored over KOH pellets, pyridine over CaH2. Con-
version of 3 to 4 was monitored by GC, all other reactions by TLC. Unless stated otherwise, solns. in org.
solvents were dried with anh. Na2SO4 and concentrated at ca. 408/2 kPa. GC: Finnigan GCQ gas chroma-
tograph/mass spectrometer equipped with a ZB-5 column (5% phenyl, 95% dimethyl polysiloxane, 30 m;
1008 to 2808, 108/min). TLC: silica gel 60 glass slides; visualized by heating after treatment with 5%
H2SO4 in EtOH (v/v) or 5% ninhydrine in EtOH (w/v) for primary amines. Column chromatography
(CC): for purification of 6, silica gel 60 was dried overnight at 1608 and cooled under a stream of Ar.

Helvetica Chimica Acta – Vol. 89 (2006)
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M.p.: Kofler hot stage. [a]D: Perkin-Elmer 341 automatic polarimeter. NMR Spectra: Varian Mercury
1
spectrometer, at 300 MHz for H, 75 MHz for 13C, and 121 MHz for 31P; Bruker Avance spectrometer,
1
at 600 MHz for H and 150 MHz for 13C; assignments by first-order analysis and, when feasible, sup-
ported by homonuclear decoupling experiments or homonuclear and heteronuclear 2-dimensional corre-
lation spectroscopy, run with the software supplied with the spectrometers; chemical shifts d in ppm rel.
to SiMe4 as internal standard (1H,13C) or rel. to H3PO4 in D2O as an internal standard (stem coaxial
insert; 31P), coupling constants J in Hz. Arbitrary primed atom numbering (C(1’), C(2’), C(4’), C(5’),
C(6’)) for the 8-azido-3,6-dioxaoctyl spacer, and serial disaccharides numbering with superscripts I
(unit bearing the aglycon) and II; J(P,C) was observed for all phosphates; some 13C-NMR signals of
the squaric acid derivative 2 were split due to the effect of the vinylogous amide group [19].
8-Azido-3,6-dioxaoctan-1-ol (=2-[2-(2-Azidoethoxy)ethoxy]ethanol; 4). A mixture of 2-[2-(2-
chloroethoxy)ethoxy]ethanol (3; (42.015 g, 250 mmol), NaN3 (24.400 g, 375 mmol), NaI (3.750 g, 25
mmol), and 95% aq. EtOH (250 ml) was heated in a closed vessel at 1258. After 24 h (GC monitoring),
the mixture was filtered and the precipitate washed with 95% aq. EtOH (3×100 ml). The combined fil-
trate was evaporated and the residue treated with Et2O. The precipitate was filtered off, washed several
times with Et2O, and the combined filtrate evaporated. The residue was subjected to CC (petroleum
ether/acetone 3 :1): 4 (38.185 g, 87%). Sligthly yellow liquid.
2,4,6-Tri-O-benzoyl-3-O-benzyl-a-D-galactopyranosyl Chloride (6). A soln. of freshly fused ZnCl2 in
AcOH (0.62M; 40 ml, 3.4 mg, 24.8 mmol) was added to a stirred soln. of 5 [28] (2.0 g, 3.355 mmol) in a mix-
ture of CH2Cl2 (6 ml) and Cl2CH2OMe (6 ml). The flask was equipped with a drying tube, and the reac-
tion was monitored by TLC while stirring was continued at r.t. After 2–5 h, the mixture was diluted with
CH2Cl2 (100 ml) and washed with an ice-cold, sat. NaHCO3 soln. (100 ml) and H2O (100 ml). The org.
layer was dried and evaporated at r.t. and the residue subjected to CC (CHCl3/AcOEt 99 :1): 6 (1.713
g, 85%). Foam.
2-[2-(2-Azidoethoxy)ethoxy]ethyl 2,4,6-Tri-O-benzoyl-3-O-benzyl-b-D-galactopyranoside (7).
A
soln. of 6 (1.7 g, 2.833 mmol) and sym-collidine (375 ml, 2.833 mmol) in CH2Cl2 (22.5 ml) was added to
a stirred mixture of AgOTf (800 mg, 3.116 mmol) and 4 (496 mg, 2.833 mmol) in CH2Cl2 (7.5 ml). Stirring
was continued in the dark. After 2 h, the mixture was treated with Et3N, filtered through a Celite pad, and
the solids were washed with CH2Cl2. The combined filtrate (ca. 100 ml) was washed with 10% aq.
Na2S2O3 soln. (100 ml) and H2O (100 ml), dried, and concentrated, and the residue was subjected to
1
CC (hexane/AcOEt 3 :1): 7 (1.771 g, 85%). Colorless oil. [a]2D5 =+251.3 (c=0.515, CHCl3). H-NMR
(CDCl3, 300 MHz): 8.14–8.20 (m, 2 H); 7.97–8.08 (m, 4 H); 7.53–7.64 (m, 3 H); 7.40–7.52 (m, 6 H);
7.04–7.20 (m, 5 H, PhCH2OꢀC(3)); 5.93 (br. d, J=2.8, HꢀC(4)); 5.56 (dd, J=8.1, 10.1, HꢀC(2));
4.72 (d, J=8.1, partial overlap, HꢀC(1)); 4.71 (d, J=12.6, partial overlap, 1 H, PhCH2O); 4.62 (dd,
J=6.9, 11.3, HaꢀC(6)); 4.52 (d, J=12.6, 1 H, PhCH2O); 4.44 (dd, J=6.0, 11.3, HbꢀC(6)); 4.10 (br. t,
J=6.5, HꢀC(5)); 4.00 (dt, J=4.1, 11.3, HaꢀC’(1)); 3.83 (dd, J=3.4, 10.1, HꢀC(3)); 3.75 (ddd, J=3.9,
7.2, 11.3, HbꢀC(1’)); 3.56–3.63 (m, 2 H); 3.42–3.52 (m, 4 H); 3.30–3.37 (m, 2 H); 3.23–3.30 (m, 2 H,
CH2N3). 13C-NMR (CDCl3, 75 MHz): 166.06, 165.78, 165.06 (3 PhCO); 137.13; 133.34; 133.19; 133.0;
130.02 (2 C); 129.86; 129.76 (2 C); 129.66 (2 C); 129.47; 129.26; 128.43 (2 C); 128.39 (2 C); 128.24 (2
C); 128.15 (2 C); 127.83 (2 C); 127.58; 101.53 (C(1)); 76.06 (C(3)); 71.26 (C(5)); 71.08 (C(2)); 70.86
(PhCH2); 70.53, 70.35, 70.22, 69.73, 69.21 (C(1’), C(2’), C(3’), C(4’), C(5’)); 66.57 (C(4)); 62.52 (C(6));
50.45 (CH2N3). ES-TOF-MS (pos.): 757.3 ([M+NH4]+), 762.3 ([M+Na]+). Anal. calc. for C40H41N3O11
(739.27): C 64.94, H 5.59, N 5.68; found: C 65.01, H 5.59, N 5.70.
2-[2-(2-Azidoethoxy)ethoxy]ethyl 3-O-Benzyl-b-D-galactopyranoside (8). Anh. K2CO3 (331 mg,
2.394 mmol) was added to a stirred soln. of 7 (1.770 g, 2.394 mmol) in MeOH (25 ml). When TLC indi-
cated complete debenzoylation (ca. 8 h), the mixture was neutralized with IR-120(H+) resin. The resin
was filtered off and washed with MeOH, the combined filtrate concentrated, and the residue subjected
1
to CC (CH2Cl2/MeOH 97:3): 8 (975 mg, 95%). Colorless oil. [a]2D5 =ꢀ31.5 (c=0.54, CHCl3). H-NMR
(C5D5N, 300 MHz): 7.54–7.62 (m, 2 H); 7.26–7.37 (m, 3 H); 5.03, 4.98 (2d, J=12.1, PhCH2); 4.80 (d,
J=7.7, HꢀC(1)); 4.68 (br. d, J=3.3, HꢀC(4)); 4.57 (dd, J=7.7, 9.3, HꢀC(2)); 4.48 (dd, J=6.3, 11.0, par-
tial overlap, HaꢀC(6)); 4.43 (dd, J=6.1, 11.0, partial overlap, HbꢀC(6)); 4.26 (dt, J=5.0, 10.4, HaꢀC(1’));
3.99 (br. t, J=6.2, partial overlap, HꢀC(5)); 3.93 (dt, J=5.1, 10.4, partial overlap, HbꢀC(1’)); 3.87 (dd,
J=3.3, 9.3, partial overlap, HꢀC(3)); 3.72 (br. t, J=5.1, 2 H); 3.56–3.67 (m, 6 H); 3.33 (br. t, J=5.0,

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Helvetica Chimica Acta – Vol. 89 (2006)
CH2N3). 13C-NMR (C5D5N, 75 MHz): 140.38; 129.03 (2 C); 128.62 (2 C); 128.09; 105.78 (C(1)); 83.55
(C(3)); 77.21 (C(5)); 72.38 (PhCH2); 71.86 (C(2)); 71.27, 71.25, 71.14, 70.61, 69.39 (C(1’), C(2’), C(3’),
C(4’), C(5’)); 67.36 (C(4)); 62.66 (C(6)); 51.38 (CH2N3). ES-TOF-MS (pos.): 450.1 ([M+Na]+). Anal.
calc. for C19H29N3O8 (427.20): C 53.39, H 6.84, N 9.83; found: C 53.68, H 6.95, N 9.73.
(2-[2-(2-Azidoethoxy)ethoxy]ethyl 3-O-benzyl-b-D-galactopyranoside Cyclic [P(R)]-4,6-(2,2,2-Tri-
chloroethyl Phosphate) (9) and 2-[2-(2-Azidoethoxy)ethoxy]ethyl 3-O-Benzyl-b-D-galactopyranoside
Cyclic [P(S)]-4,6-(2,2,2-Trichloroethyl Phosphate) (10). The 2,2,2-trichloroethyl phosphorodichloridate
(425 ml, 2.711 mmol) was added dropwise to a cooled (ꢀ158; ice/acetone bath) and stirred soln. of 8
(965 mg, 2.259 mmol) and pyridine (1.83 ml, 22.590 mmol) in CH2Cl2 (25 ml). After 5 min, TLC indicated
incomplete reaction, and more 2,2,2-trichloroethyl phosphorodichloridate (212 ml, 1.356 mmol) was
added while stirring was continued at ꢀ158. One additional portion of reagent (106 ml, 0.678 mmol)
was required to drive the reaction to completion. The reaction was quenched with MeOH (1 ml), the mix-
ture concentrated, and the residue treated with AcOEt. The precipitate was filtered off and washed with
AcOEt, the combined filtrate concentrated, and the residue chromatographed (hexane/acetone 65 :35 !
1:1): 9 followed by 10. Minor impurities in both fractions were removed by CC (Strata C18 SPE, MeOH/
H2O 1:1): pure 9 (460 mg, 33%) and 10 (854 mg, 61%).
Data for 9: White solid. M.p. 67.8–68.58 (CH2Cl2/Et2O). [a]2D5 =ꢀ3.6 (c=0.56, CHCl3). H-NMR
1
(CDCl3, 600 MHz): 7.36–7.39 (m, 2 Ho); 7.32–7.36 (m, 2 Hm); 7.27–7.31 (m, 1 Hp); 4.79 (br. d, J=3.1,
partial overlap, HꢀC(4)); 4.78, 4.75 (2d, J=12.2, partial overlap, PhCH2); 4.61 (d, J=6.9, CCl3CH2);
4.53 (br. d, J=1.4, partial overlap, HaꢀC(6)); 4.51 (ddd, J=1.4, 12.3, 31.4, partial overlap, HbꢀC(6));
4.40 (d, J=7.9, HꢀC(1)); 4.05 (dt, J=4.3, 11.3, HaꢀC(1’)); 3.96 (dd, J=7.9, 9.7, HꢀC(2)); 3.77 (dt,
J=4.9, 11.3, HbꢀC(1’)); 3.64–3.75 (m, CH2(2’), C(3’), C(4’), C(5’)); 3.55 (br. s, partial overlap, Hꢀ
C(5)); 3.53 (ddd, J=3.3, 4.3, 9.7, partial overlap, HꢀC(3)); 3.40 (dt, J=5.2, 13.2, partial overlap,
CHaN3); 3.37 (dt, J=5.0, 13.2, partial overlap, CHbN3). 13C-NMR (CDCl3, 150 MHz): 137.61; 128.42 (2
C); 127.84; 127.66 (2 C); 103.21 (C(1)); 94.99 (d, J=10.2, CCl3); 77.53 (d, J=7.2, C(3)); 76.73 (d,
J=4.2, CCl3CH2); 76.43 (d, J=7.2, C(4)); 72.02 (PhCH2); 70.79 (d, J=7.7, C(6)); 70.44, 70.40, 70.15,
69.86, 68.37 (C(1’), C/2’), C(3’), C(4’), C(5’)); 69.37 (C(2)); 66.13 (d, J=6.6, C(5)); 50.52 (CH2N3). 31P-
NMR (CDCl3): ꢀ11.18. ES-TOF-MS (pos.): 620, 622, 624 ([M+H]+), 642, 644, 646 ([M+Na]+). Anal.
calc. for C21H29Cl3N3O10P (619.07): C 40.63, H 4.71, N 6.77; found: C 40.70, H 4.58, N 6.69.
Data for 10: Slightly yellow oil. [a]D25 =+9.2 (c=0.53, CHCl3). 1H-NMR (CDCl3, 600 MHz):
7.38–7.41 (m, 2 Ho); 7.33–7.37 (m, 2 Hm); 7.27–7.31 (m, 1 Hp); 4.95 (br. d, J=3.0, HꢀC(4)); 4.81,
4.71 (2d, J=12.0, PhCH2); 4.67 (br. dt, J=2.7, 12.3, HaꢀC(6)); 4.63 (dd, J=1.1, 7.4, CCl3CH2); 4.47
(ddd, J=1.6, 12.3, 20.8, HbꢀC(6)); 4.41 (d, J=7.9, HꢀC(1)); 4.04 (dt, J=4.3, 11.3, HaꢀC(1’)); 3.88
(dd, J=7.9, 9.7, HꢀC(2)); 3.77 (m, HbꢀC(1’)); 3.61–3.72 (m, CH2(2’), CH2(3’), CH2(4’), CH2(5’)); 3.57
(br. d, J=1.1, HꢀC(5)); 3.53 (ddd, J=3.2, 4.3, 9.7, HꢀC(3)); 3.39 (dt, J=4.9, 13.3, partial overlap,
CHaN3); 3.36 (dt, J=5.1, 13.3, partial overlap, CHbN3); 3.15 (br. s, HOꢀC(2)). 13C-NMR (CDCl3, 150
MHz): 137.26; 128.48 (2 C); 127.94; 127.86 (2 C); 103.19 (C(1)); 94.46 (d, J=10.9, CCl3); 77.65 (d,
J=5.3, CCl3CH2); 77.42 (d, J=7.0, C(3)); 74.96 (d, J=5.3, C(4)); 71.62 (PhCH2); 70.49, 70.42, 70.17,
69.91, 68.59 (C(1’), C(2’), C(3’), C(4’), C(5’)); 70.16 (d, J=5.8, partial overlap, C(6)); 69.53 (C(2));
66.53 (d, J=7.0, C(5)); 50.51 (CH2N3). 31P-NMR (CDCl3): ꢀ8.82. ES-TOF-MS (pos.): 620, 622, 624
([M+H]+), 642, 644, 646 ([M+Na]+). Anal. calc. for C21H29Cl3N3O10P (619.07): C 40.63, H 4.71, N
6.77; found: C 40.79, H 4.80, N 6.72.
Ethyl 2,4-Di-O-benzyl-3,6-dideoxy-1-thio-b-L-xylo-hexopyranoside (12). Anh. K2CO3 (125 mg, 0.906
mmol) was added to a stirred soln. of 11 [31](1.0 g, 3.622 mmol) in MeOH (14.5 ml). After 16 h, the mix-
ture was neutralized with IR-120(H+) resin. The resin was filtered off and washed with MeOH, the com-
bined filtrate concentrated, and the residue concentrated from MeCN (3×25 ml), and dried. NaH (60%
dispersion in mineral oil; 435 mg, 10.866 mmol) was added with stirring at 08 to a soln. of the intermediate
diol in DMF (14.5 ml), followed by BnBr (1.3 ml, 10.866 mmol). The cooling was removed and, after 2 h,
MeOH (0.5 ml) was added to destroy excess of BnBr. The mixture was partitioned between Et2O (100
ml) and H2O (100 ml), the aq. phase extracted with Et2O (2×100 ml), the combined org. phase washed
with brine (100 ml), dried, and concentrated, and the residue chromatographed (hexane/AcOEt 95 :5):
12 (1.296 g, 96%). Nearly colorless oil. The a-L-anomer, formed by slow anomerization, can be separated
from 12 by chromatography (hexane/acetone 95 :5).

Helvetica Chimica Acta – Vol. 89 (2006)
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Data for Ethyl 2,4-Di-O-benzyl-3,6-dideoxy-1-thio-a-L-xylo-hexopyranoside: 1H-NMR (CDCl3, 300
MHz): 7.26–7.40 (m, 10 H); 5.50 (br. d, J=4.9, HꢀC(1)); 4.67, 4.47 (2d, J=11.7, PhCH2); 4.62, 4.42 (2d,
J=12.1, PhCH2); 4.24 (dq, J=1.6, 6.6, HꢀC(5)); 4.13 (dt, J=4.7, 12.1, HꢀC(2)); 3.45 (br. s, HꢀC(4));
2.62 (dq, J=7.4, 12.9, partial overlap, 1 H, MeCH2S); 2.54 (dq, J=7.4, 12.9, partial overlap,
1 H, MeCH2S); 2.16 (dddd, J=1.4, 4.5, 4.5, 13.7, HeqꢀC(3)); 1.76 (ddd, J=2.7, 12.1, 13.7, HaxꢀC(3));
1.30 (t, J=7.4, MeCH2S); 1.19 (d, J=6.6, Me(6)). 13C-NMR (CDCl3, 75 MHz): 138.22; 137.99; 128.31
(2 C); 128.23 (2 C); 127.87 (2 C); 127.75 (2 C); 127.65; 127.55; 83.91 (C(1)); 75.31 (C(4)); 71.12, 70.56
(2 PhCH2); 70.43 (C(2)); 66.16 (C(5)); 29.63 (C(3)); 23.45 (MeCH2S); 16.29 (C(6)); 15.00 (MeCH2S).
2-[2-(2-Azidoethoxy)ethoxy]ethyl 3-O-Benzyl-2-O-(2,4-di-O-benzyl-3,6-dideoxy-a-L-xylo-hexopyra-
nosyl)-b-D-galactopyranoside Cyclic [P(R)]-4,6-(2,2,2-Trichloroethyl Phosphate) (13a). Powdered 4 Å
molecular sieves (1.570 g) were added to a stirred soln. of 9 (485 mg, 0.783 mmol), 12 (438 mg, 1.175
mmol), and sym-collidine (260 ml, 1.959 mmol) in 31.5 ml of dioxane/toluene (v/v; 3 :1). After 30 min,
the flask was covered with aluminium foil, AgOTf (353 mg, 1.371 mmol) was added, and the mixture
was cooled to 08. NIS (309 mg, 1.371 mmol) was added (5 portions in 15-min intervals). Stirring was con-
tinued for 1 h at 08, and the mixture was filtered through a Celite pad, directly into. 10% aq. Na2S2O3
soln./sat. aq. NaHCO3 soln. 2 :1 (v/v; 150 ml). The solids were washed with CH2Cl2 (200 ml) and the lay-
ers were separated. The aq. phase was extracted with CH2Cl2 (2×100 ml) and the combined org. layer
dried and evaporated. The residue was subjected to CC (petroleum ether/acetone 4 :1 ! 3 :2): 13b
(138 mg, 19%) followed by 13a (553 mg, 76%).
Data for 13a: Nearly colorless oil. [a]2D5 =ꢀ42.6 (c=0.51, CHCl3). 1H-NMR (CDCl3, 600 MHz):
7.21–7.32 (m, 13 H); 7.14 (m, 2 H); 5.48 (d, J=3.1, HꢀC(1II)); 4.84 (d, J=3.1, HꢀC(4I)); 4.75 (d,
J=11.6, 1 H, PhCH2); 4.56–4.61 (m, 4 H, HꢀC(1I), PhCH2, CCl3CH2); 4.47–4.55 (ddd, J=1.6, 12.2,
26.9, partial overlap, HaꢀC(6I)); 4.53 (m, HbꢀC(6I)); 4.52, 4.33 (2d, J=12.3, PhCH2); 4.49, 4.29 (2d,
J=12.3, PhCH2); 4.35 (dq, J=1.7, 6.5, partial overlap, HꢀC(5II)); 4.21 (dd, J=7.8, 9.4, HꢀC(2I)); 3.99
(ddd, J=4.1, 5.4, 10.5, HaꢀC(1’)); 3.81 (br. dt, J=4.0, 12.2, HꢀC(2II)); 3.77 (m, HꢀC(3I)); 3.70 (ddd,
J=4.2, 6.5, 10.5, HbꢀC(1’)); 3.55–3.67 (m, CH2(2’), CH2(3’), CH2(4’), CH2(5’)); 3.54 (br. s, HꢀC(5I));
3.38 (br. s, HꢀC(4II)); 3.35 (br. t, J=5.1, CH2N3); 2.07 (br. dt, J=4.1, 13.0, HeqꢀC(3II)); 1.84 (dt,
J=2.3, 13.0, HaxꢀC(3II)); 1.15 (d, J=6.5, Me(6II)). 13C-NMR (CDCl3, 150 MHz): 138.25; 138.01;
137.27; 128.31 (2 C); 128.09 (2 C); 128.08 (2 C); 127.69 (2 C); 127.60 (2 C); 127.58; 127.40; 127.38;
126.89 (2 C); 101.59 (C(1I)); 96.40 (C(1II)); 94.89 (d, J=10.3, CCl3); 80.00 (d, J=7.2, C(3I)); 76.64 (d,
J=4.3, CCl3CH2); 75.53 (C(4II)); 75.37 (d, J=7.0, C(4I)); 71.06 (PhCH2); 71.00 (C(2I)); 70.77
(PhCH2); 70.74 (d, J=7.9, partial overlap, C(6I)); 70.57 (PhCH2); 70.43, 70.30, 70.09, 69.80 (C(2’),
C(3’), C(4’), C(5’)); 68.23 (C’(1)); 65.94 (C(5II)); 65.67 (d, J=6.7, C(5I)); 50.46 (CH2N3); 27.23
(C(3II)); 16.28 (C(6II)). 31P-NMR (CDCl3): ꢀ11.25. ES-TOF-MS (pos.): 952.2, 954.2, 956.2 ([M+Na]+).
Anal. calc. for C41H51Cl3N3O13P (929.22): C 52.88, H 5.52, N 4.51; found: C 52.98, H 5.26, N 4.56.
Data for 2-[2-(2-Azidoethoxy)ethoxy]ethyl 3-O-Benzyl-2-O-(2,4-di-O-benzyl-3,6-dideoxy-b-L-xylo-
hexopyranosyl)-b-D-galactopyranoside Cyclic [P(R)]-4,6-(2,2,2-Trichloroethyl Phosphate) (13b): Color-
less oil. 1H-NMR (CDCl3, 600 MHz): 7.42 (m, 2 H); 7.21–7.34 (m, 13 H); 4.88, 4.79 (2d, J=12.0,
PhCH2); 4.84 (d, J=7.6, HꢀC(1II)); 4.80 (br. d, J=3.6, partial overlap, HꢀC(4I)); 4.78, 4.58 (2d,
J=11.9, PhCH2); 4.62 (dd, J=6.6, 11.4, partial overlap, 1 H, CCl3CH2); 4.60 (dd, J=6.9, 11.4, partial
overlap, 1 H, CCl3CH2); 4.59, 4.42 (2d, J=12.0, PhCH2); 4.46–4.55 (ddd, J=1.7, 12.4, 31.6, partial over-
lap, HaꢀC(6I)); 4.52 (m, partial overlap, HbꢀC(6I)); 4.43 (d, J=7.8, partial overlap, HꢀC(1I)); 4.23 (dd,
J=7.8, 9.5, HꢀC(2I)); 3.93 (ddd, J=4.3, 6.3, 10.7, HaꢀC(1’)); 3.53–3.69 (m, HꢀC(3I), HꢀC(2II), Hꢀ
C(5II), HbꢀC(1’), CH2(2’), CH2(3’), CH2(4’), CH2(5’)); 3.51 (br. d, J=1.4, HꢀC(5I)); 3.35 (m, Hꢀ
C(4II), CH2N3); 2.36 (ddd, J=3.1, 4.8, 13.8, HeqꢀC(3II)); 1.48 (ddd, J=2.8, 11.7, 13.8, HaxꢀC(3II)); 1.23
(d, J=6.4, Me(6II)). 13C-NMR (CDCl3, 150 MHz): 139.18; 138.58; 138.15; 128.16 (6 C); 127.80 (2 C);
127.54 (2 C); 127.49 (2 C); 127.44; 127.36; 127.30; 104.10 (C(1II)); 103.46 (C(1I)); 95.01 (d, J=10.8,
CCl3); 77.34 (d, J=7.1, C(4I)); 76.84 (d, J=7.2, partial overlap, C(3I)); 76.71 (d, J=4.2, CCl3CH2);
75.44 (C(4II)); 74.43 (C(2I)); 73.83 (C(2II)); 73.34 (C(5II)); 72.71, 72.43, 71.08 (3 PhCH2); 70.66 (d,
J=7.6, C(6I)); 70.52, 70.39, 70.16, 69.84 (C(2’), C(3’), C(4’), C(5’)); 69.04 (C(1’)); 65.88 (d, J=6.7,
C(5I)); 50.57 (CH2N3); 33.00 (C(3II)); 16.66 (C(6II)). 31P-NMR (CDCl3): ꢀ11.41. ES-TOF-MS (pos.):
902.1, 904.1, 906.1 ([Mꢀ N2 +H]+); 954.1, 956.1, 958.1 ([M+Na]+).

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Helvetica Chimica Acta – Vol. 89 (2006)
2-[2-(2-Azidoethoxy)ethoxy]ethyl 3-O-Benzyl-2-O-(2,4-di-O-benzyl-3,6-dideoxy-a-L-xylo-hexopyra-
nosyl)-b-D-galactopyranoside Cyclic [P(S)]-4,6-(2,2,2-Trichloroethyl Phosphate) (14a). As described for
13a, with molecular sieves (4.120 g), 10 (1.275 g, 2.060 mmol), 12 (1.151 g, 3.090 mmol), sym-collidine
(681 ml, 5.150 mmol), dioxane/toluene 3 :1 (82.5 ml), AgOTf (926 mg, 3.605 mmol), and NIS (811 mg,
3.605 mmol). CC (CH2Cl2/acetone 10 :1 ! 2 :1) gave 14b (210 mg, 11%), then 14a (1.306 g, 68%), fol-
lowed by unreacted 10 (204 mg, 16%).
Data for 14a: Nearly colorless oil. [a]2D5 =ꢀ9.1 (c=0.58, CHCl3). 1H-NMR (CDCl3, 600 MHz):
7.22–7.32 (m, 13 H); 7.12–7.16 (m, 2 H); 5.49 (d, J=3.4, HꢀC(1II)); 4.97 (d, J=3.1, HꢀC(4I)); 4.80,
4.53 (2d, J=11.5, PhCH2); 4.70 (dd, J=7.7, 11.4, 1 H, CCl3CH2); 4.65 (m, partial overlap, HaꢀC(6I));
4.64 (dd, J=7.7, 11.4, partial overlap, 1 H, CCl3CH2); 4.60 (d, J=7.9, HꢀC(1I)); 4.52, 4.33 (2d,
J=12.2, PhCH2); 4.49, 4.28 (2d, J=12.3, PhCH2); 4.48 (ddd, J=1.6, 12.1, ca. 21.0, partial overlap, Hbꢀ
C(6I)); 4.35 (dq, J=1.2, 6.5, partial overlap, HꢀC(5II)); 4.13 (dd, J=7.9, 9.4, HꢀC(2I)); 3.99 (dt,
J=4.8, 10.8, HaꢀC(1’)); 3.77–3.83 (m, HꢀC(3I), HꢀC(2II)); 3.71 (ddd, J=4.9, 6.1, 10.8, HbꢀC(1’));
3.50–3.65 (m, HꢀC(5I), CH2(2’), CH2(3’), CH2(4’), CH2(5’)); 3.38 (br. s, HꢀC(4II)); 3.34 (t, J=5.0,
CH2N3); 2.08 (br. dt, J=3.8, 12.9, HeqꢀC(3II)); 1.83 (dt, J=2.4, 12.9, HaxꢀC(3II)); 1.13 (d, J=6.5,
Me(6II)). 13C-NMR (CDCl3, 150 MHz): 138.28; 138.03; 137.08; 128.38 (2 C); 128.19 (2 C); 128.16 (2
C); 127.74 (2 C); 127.69 (3 C); 127.52; 127.47; 127.17 (2 C); 101.58 (C(1I)); 96.45 (C(1II)); 94.50 (d,
J=10.7, CCl3); 79.93 (d, J=6.9, C(3I)); 77.68 (d, J=5.4, CCl3CH2); 75.51 (C(4II)); 74.28 (d, J=5.6,
C(4I)); 71.13 (C(2I)); 70.93, 70.82, 70.67 (3 PhCH2); 70.55, 70.38, 70.21, 69.93 (C(2’), C(3’), C(4’),
C(5’)); 70.25 (C(2II)); 70.19 (d, J=7.1, partial overlap, C(6I)); 68.38 (C(1’)); 66.06 (d, J=5.0, partial over-
lap, C(5I)); 66.04 (C(5II)); 50.52 (CH2N3); 27.32 (C(3II)); 16.33 (C(6II)). 31P-NMR (CDCl3): ꢀ8.96. ES-
TOF-MS (pos.): 952.1, 954.1, 956.1 ([M+Na]+). Anal. calc. for C41H51Cl3N3O13P (929.22): C 52.88, H
5.52, N 4.51; found: C 53.06, H 5.55, N 4.51.
Data for 2-[2-(2-Azidoethoxy)ethoxy]ethyl 3-O-Benzyl-2-O-(2,4-di-O-benzyl-3,6-dideoxy-b-L-xylo-
hexopyranosyl)-b-D-galactopyranoside Cyclic [P(S)]-4,6-(2,2,2-Trichloroethyl Phosphate) (14b): Nearly
colorless oil. 1H-NMR (CDCl3, 600 MHz): 7.43–7.46 (m, 2 H); 7.20–7.33 (m, 13 H); 4.93 (d, J=3.2,
HꢀC(4I)); 4.84 (d, J=7.6, partial overlap, HꢀC(1II)); 4.83, 4.80 (2d, J=11.9, PhCH2); 4.77, 4.57 (2d,
J=11.8, PhCH2); 4.69 (dd, J=7.6, 11.4, 1 H, CCl3CH2); 4.64 (dt, J=2.8, 12.2, HaꢀC(6I)); 4.61 (dd,
J=7.6, 11.4, partial overlap, 1 H, CCl3CH2); 4.59, 4.41 (2d, J=12.0, PhCH2); 4.47 (ddd, J=1.8, 12.2,
20.8, partial overlap, HbꢀC(6I)); 4.45 (d, J=7.7, partial overlap, HꢀC(1I)); 4.14 (dd, J=7.7, 9.3, Hꢀ
C(2I)); 3.94 (ddd, J=4.3, 5.9, 10.5, HaꢀC(1’)); 3.68 (ddd, J=4.2, 6.0, 10.5, HbꢀC(1’)); 3.50–3.66 (m,
HꢀC(3I), HꢀC(5I), HꢀC(2II), HꢀC(5II), CH2(2’), CH2(3’), CH2(4’), CH2(5’)); 3.34 (br. s, HꢀC(4II));
3.32 (t, J=5.1, CH2N3); 2.36 (ddd, J=3.2, 4.8, 14.0, HeqꢀC(3II)); 1.46 (ddd, J=2.7, 11.5, 14.0, Haxꢀ
C(3II)); 1.23 (d, J=6.4, Me(6II)). 13C-NMR (CDCl3, 150 MHz): 139.12; 138.51; 137.79; 128.18 (2 C);
128.17 (2 C); 128.16 (2 C); 127.91 (2 C); 127.56 (2 C); 127.52 (2 C); 127.50; 127.40; 127.33; 103.91
(C(1II)); 103.33 (C(1I)); 94.58 (d, J=10.6, CCl3); 77.62 (d, J=5.3, CCl3CH2); 76.90 (d, J=7.0, C(3I));
75.91 (d, J=5.3, C(4I)); 75.34 (C(4II)); 74.43 (C(2I)); 73.85 (C(2II)); 73.33 (C(5II)); 72.80, 72.24, 71.08
(3 PhCH2); 70.55, 70.33, 69.86 (C(3’), C(4’), C(5’)); 70.18 (C(2’)); 70.01 (d, J=6.0, C(6I)); 69.01
(C(1’)); 66.18 (d, J=7.0, C(5I)); 50.53 (CH2N3); 32.97 (C(3II)); 16.72 (C(6II)). 31P-NMR (CDCl3):
ꢀ8.56. ES-TOF-MS (pos.): 952.2, 954.2, 956.2 ([M+Na]+).
2-[2-(2-Aminoethoxy)ethoxy]ethyl 2-O-(3,6-Dideoxy-a-L-xylo-hexopyranosyl)-b-D-galactopyrano-
side Cyclic 4,6-(Potassium Phosphate) (15). A mixture of Pd/C (1.2 g), 13a (1.200 g, 1.291 mmol),
iPrOH (60 ml) and potassium phosphate buffer (pH 7, 0.1M; 60 ml) was shaken under H2 (65 psi) until
TLC indicated complete conversion (30–40 h). The catalyst was filtered off and washed with H2O,
and the combined filtrates were evaporated. The residue was treated with 95% aq. EtOH (30 ml), and
the insoluble salts were filtered off and washed with 95% aq. EtOH (3×15 ml). The combined filtrate
was evaporated and the residue subjected to CC (iPrOH/H2O 3 :1) to give, after freeze-drying, 15 (460
mg, 66%). White amorphous solid. 1H-NMR (D2O, 600 MHz): 5.14 (d, J=3.7, HꢀC(1II)); 4.61 (d,
J=7.8, HꢀC(1I)); 4.56 (d, J=3.3, HꢀC(4I)); 4.39 (br. d, J=12.6, HaꢀC(6I)); 4.22–4.30 (m, HꢀC(5II),
HbꢀC(6I)); 4.03–4.08 (m, HaꢀC(1’)); 3.96–4.01 (m, HꢀC(2II)); 3.93 (dt, J=3.2, 9.6, HꢀC(3I));
3.79–3.85 (m, HbꢀC(1’), HꢀC(4II)); 3.65–3.77 (m, HꢀC(2I), HꢀC(5I), CH2(2’), CH2(3’), CH2(4’),
CH2(5’)); 3.16–3.20 (m, CH2NH2); 1.91–1.98 (m, CH2(3II)); 1.13 (d, J=6.6, Me(6II)). 13C-NMR (D2O,
150 MHz): 101.44 (C(1I)); 98.82 (C(1II)); 76.52 (d, J=4.7, C(4I)); 75.53 (C(2I)); 72.24 (d, J=7.4,

Helvetica Chimica Acta – Vol. 89 (2006)
331
C(3I)); 69.89, 69.77, 69.54, 66.57 (C(2’), C(3’), C(4’), C(5’)); 69.05 (C(1’)); 68.61 (C(4II)); 68.36 (d, J=5.5,
C(6I)); 67.25 (d, J=4.5, C(5I)); 66.62 (C(5II)); 63.42 (C(2II)); 39.26 (CH2NH2); 32.86 (C(3II)); 15.68
(C(6II)). 31P-NMR (D2O): ꢀ3.96. ES-TOF-MS (pos.): 504.2 ([MꢀK+2 H]+); 526.2
([MꢀK+Na+H]+). ES-TOF-MS (neg.): 502.2 ([MꢀK]ꢀ).
2-{2-{2-[(2-Ethoxy-3,4-dioxocyclobut-1-en-1-yl)amino]ethoxy}ethoxy}ethyl 2-O-(3,6-Dideoxy-a-L-
xylo-hexopyranosyl)-b-D-galactopyranoside Cyclic 4,6-(Potassium phosphate) (2). Drierite (287 mg,
10–20 mesh) was added to a stirred mixture of 15 (287 mg, 0.530 mmol) and abs. EtOH (5.3 ml).
After 60 min, diethyl squarate (=3,4-diethoxycyclobut-3-ene-1,2-dione; 98 ml, 0.663 mmol) and
KHCO3 (265 mg, 2.652 mmol) were added, and stirring was continued at r.t. until TLC indicated disap-
pearance of 15. The mixture was filtered, the solid washed with abs. EtOH (3×5 ml), the combined fil-
trate evaporated, and the residue subjected to CC (MeCN/H2O 85 :15) to give, after freeze-drying, 2 (324
mg, 92%). White amorphous solid. 1H-NMR (D2O, 600 MHz): 5.18 (m, HꢀC(1II)); 4.75, 4.72 (2q, J=7.1,
partially overlapping, MeCH2O); 4.62, 4.61 (2d, J=8.0, partially overlapping, HꢀC(1I)); 4.58 (br. d,
J=3.4, HꢀC(4I)); 4.42 (br. dt, J=1.7, 11.4, HaꢀC(6I)); 4.29 (m, HꢀC(5II), HbꢀC(6I)); 3.99–4.60 (m,
HꢀC(2II), HaꢀC(1’)); 3.96 (dt, J=3.4, 9.6, HꢀC(3I)); 3.83 (br. s, partial overlap, HꢀC(4II)); 3.78–3.84
(m, HbꢀC(1’), CHaNH); 3.77 (m, HꢀC(5I)); 3.72 (dd, J=8.0, 9.6, partial overlap, HꢀC(2I)); 3.64–3.72
(m, CHbNH, CH2(2’), CH2(3’), CH2(4’), CH2(5’)); 1.92–2.02 (m, CH2(3II)); 1.45, 1.43 (2t, J=7.3, partially
overlapping, MeCH2O); 1.13 (d, J=7.1, Me(6II)). 13C-NMR (D2O, 150 MHz): 189.45, 183.91, 183.88,
177.95, 177.71, 174.40, 174.36 (4 cyclobutene C); 101.94 (C(1I)); 99.20 (C(1II)); 77.0 (d, J=5.0, C(4I));
75.73, 75.68 (C(2I)); 72.85 (d, J=7.2, C(3I)); 71.27, 71.21 (MeCH2); 70.45, 70.43, 70.29, 70.26, 70.08
(C(2’), C(3’), C(4’), C(5’)); 69.38 (C(1’)); 69.14 (C(4II)); 68.81 (d, J=5.6, C(6I)); 67.80 (d, J=4.7,
C(5I)); 67.05 (C(5II)); 63.91 (C(2II)); 44.60, 44.45 (CH2NH); 33.38 (C(3II)); 16.17 (C(6II)); 15.76, 15.70
(MeCH2O). 31P-NMR (D2O): ꢀ3.93. ES-TOF-MS (pos.): 650.2 ([MꢀK+Na+H]+). ES-TOF-MS
(neg.): 626.2 ([MꢀK]ꢀ).
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Received October 20, 2005