Efficient synthesis of 4-amino-4-deoxy-L-arabinose and spacer-equipped 4-amino-4-deoxy-L-arabinopyranosides by transglycosylation reactions

Article abstract of DOI:10.1055/s-0030-1258174

Methyl 4-azido-4-deoxy-l-arabinopyranoside has been synthesized in five steps starting from methyl-d-xylopyranoside in a multigram scale without chromatographic purification in 78% overall yield. The transformation relied on selective tosylation/nosylatio

Full text of DOI:10.1055/s-0030-1258174

PAPER
3143
Efficient Synthesis of 4-Amino-4-deoxy-L-arabinose and Spacer-Equipped
4-Amino-4-deoxy-L-arabinopyranosides by Transglycosylation Reactions
S
ynthesis of Spac
e
e
r-Equippe
r
d
4-A
m
n
ino-4-deoxy-
h
L
-arabinopy
a
anosides rd Müller,a Markus Blaukopf,a Andreas Hofinger,a Alla Zamyatina,a Helmut Brade,b Paul Kosma*a
a
Department of Chemistry, University of Natural Resources and Applied Life Sciences-Vienna, Muthgasse 18, 1190 Vienna, Austria
Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 22, 23845 Borstel, Germany
Fax +43(1)476546059; E-mail: paul.kosma@boku.ac.at
b
Received 21 April 2010
carrier. The conjugation chemistry has to be compatible
with the presence of both amino and carboxylic acid
groups in order to retain the antigenic properties of LPS
core sugars. The selection of conjugation conditions
Abstract: Methyl 4-azido-4-deoxy-b-L-arabinopyranoside has
been synthesized in five steps starting from methyl b-D-xylopyrano-
side in a multigram scale without chromatographic purification in
78% overall yield. The transformation relied on selective tosylation/
nosylation at O-4 followed by acylation, SN2 displacement with so- would also have to take into account the labile phospho-
dium azide, and subsequent deprotection. The methyl 4-azido-4-
deoxy-arabinoside was then converted into allyl, propenyl, w-bro-
mohexyl, and chloroethoxyethyl spacer glycosides by transglycosy-
diester linkages of Ara4N as well as the acid-labile glyco-
sidic linkages of Kdo residues.
In this communication, we describe the preparation of
Ara4N building blocks in multigram amounts as well as
their conversion into a thiol-equipped spacer derivative,
which was coupled to bovine serum albumin (BSA).
lation with the respective alcohols in good yields and fair anomeric
selectivity. Reduction of the azido group and further transforma-
tions of the aglycone afforded w-thiol-containing spacer deriva-
tives. Coupling to maleimide-activated BSA provided a potent
immunogen, which was used to generate murine and rabbit poly-
clonal sera binding to LPS-core epitopes containing 4-amino-4-
deoxy-arabinose residues.
Previously, Ara4N has been prepared from methyl b-D-
xylopyranoside via isopropylidene protection of O-2 and
O-3 followed by triflation of O-4 and subsequent inver-
sion.12,13 Since in our hands the formation of the acetonide
turned out to be unsuitable for large-scale preparation of
the compound, we resorted to regioselective tosylation of
O-4 via the intermediate stannylene acetal.14–18 Thus, re-
action of the commercially available methyl xyloside 1
with dibutyltin oxide and p-toluenesulfonyl chloride in
1,4-dioxane afforded the 4-O-tosylate 2 in 99% yield, fol-
lowed by benzoylation with benzoyl chloride in pyridine,
which gave the known dibenzoate 3 in 92% yield.19
(Scheme 1). As an alternative option, the 4-O-nosylate 4
was prepared from 1 in 99% yield.20 The subsequent con-
version of nosylate 4 into the dibenzoate 5, however, suf-
fered from reduced yields, whereas the acetylation was
smoothly effected in 93% yield to give the 2,3-di-O-
Key words: aminoarabinose, neoglycoconjugate, transglycosyla-
tion, lipopolysaccharide, spacer
4-Amino-4-deoxy-L-arabinose (Ara4N) is an important
sugar constituent of complex glycolipids constituting the
outer leaflet of the cell wall of Gram-negative bacteria.
Specifically, Ara4N has been detected in substoichiomet-
ric up to stoichiometric amounts ester-linked to the 1- and
4¢-phosphate groups of the glucosamine disaccharide
backbone of lipid A, which inserts the lipopolysaccharide
(LPS) chain into the outer membrane.1–3 In addition,
Ara4N residues have been found glycosidically linked to
position 8 of the higher carbon sugar 3-deoxy-D-manno-
oct-2-ulosonic acid (Kdo) as in the core region of Proteus
strains4,5 and also linked to D-glycero-D-talo-oct-2-ul-
osonic acid (Ko) detected in the core region of Burkhold-
eria,6–9 as well as in a Serratia marcescens strain.10 The
incorporation of Ara4N has been implicated in the onset
and maintenance of antibiotic resistance in bacteria by
masking the anionic charges of the phosphate and carbox-
ylic acid groups.11 In order to study the antigenic proper-
ties of Ara4N-substituted LPS domains and develop
monoclonal antibodies for diagnostic applications, a high-
yielding approach to generate sufficient amounts of
Ara4N building blocks was needed. Furthermore, to pro-
duce neoglycoconjugates carrying covalently linked
Ara4N ligands, suitable spacer groups should be intro-
duced allowing for a selective attachment onto the protein
O
O
a,b
d,e
R1O
HO
O
R1O
R2O
HO
HO
OMe
OMe
OMe
OR2
OH
OH
2 R1 = Ts
4 R1 = Ns
3 R1 = Ts, R2 = Bz
5 R1 = Ns, R2 = Bz
6 R1 = Ns, R2 = Ac
1
c
f
N3
N3
NH2
g
h
O
O
O
OMe
OMe
BzO
OMe
HO
HO
OBz
OH
OH
9
8
7
Scheme 1 Reagents and conditions: (a) Bu2SnO, toluene, reflux, 5
h, then TsCl, 1,4-dioxane, r.t., 48 h, 99% yield for 2, or (b) NsCl,
dioxane, 20 h r.t., 99% yield for 4; (c) from 4: NaN3, DMSO, 45 °C,
6 h, 85% yield for 8 (Method B); (d) BzCl, pyridine, r.t., 15 h, 92%
yield for 3, 47% yield for 5; (e) from 4: Ac2O, pyridine, 0 °C, 2 h, 93%
yield for 6; (f) from 3: NaN3, DMSO, 110 °C, 15 h, 93%; (g) 0.1 M
NaOMe, MeOH, r.t., 1 h, 93%; (h) 10% Pd/C, MeOH, H2, r.t., 8 h,
99%.
SYNTHESIS 2010, No. 18, pp 3143–3151
x
x.
x
x
.
2
0
1
0
Advanced online publication: 16.07.2010
DOI: 10.1055/s-0030-1258174; Art ID: P06510SS
© Georg Thieme Verlag Stuttgart · New York

3144
B. Müller et al.
PAPER
Table 1 Conditions and Yields for Transglycosylation Reactions
acetyl-4-O-nosyl derivative 6. The introduction of the 4-
azido group was achieved uneventfully by treatment of 3
with sodium azide in DMSO at 110 °C to furnish methyl
a-L-arabino-pyranoside 7 in 93% yield. Transesterifica-
tion of the benzoate groups under Zemplén conditions
then afforded the 4-azido derivative 8 in 93% yield.
Entry R1 of R1OH
Acid
Temp
(°C)
Time Yield a/b
(h)
4/8
18
(%)
1
2
3
4
5
6
7
Alla
HClb
AcCl
AcCl
60/r.t.
r.t.
55
1:2.0
1:2.3
All
76
In addition, direct conversion of the unprotected nosylate
4 into compound 8 could be achieved in 85% yield. This
three-step approach required a chromatography purifica-
tion thereby limiting a large-scale application.
(CH2)3CH=CH2
r.t.
24
70c 1:3.1
77 1:2.0
72c 1:2.9
(CH2)2O(CH2)2Cl HCl
(CH2)2O(CH2)2Cl AcCl
80/r.t.
r.t.
1/18
22
Reduction of the azide-group of 8 was accomplished by
hydrogenation to furnish methyl 4-amino-4-deoxy-a-L-
arabino-pyranoside 9 in 99% yield.
(CH2)6Br
(CH2)6Br
HCl
70/r.t.
r.t.
4/18
18
72
65
1:3.5
1:3.2
AcCl
The synthesis of 8 could thus be achieved in multigram
amounts via 7 without the need of chromatography and
more than twice overall yield to previous reports.12 The
methyl glycoside 8 constitutes a suitable precursor for fur-
ther transformations into glycosyl donors as well as spacer
glycosides. Whereas acid hydrolysis of the methyl glyco-
side 8 under various conditions suffered from reduced
yields and side reactions, transglycosylation has turned
out as a versatile approach for direct conversion into gly-
cosides.
a All = allyl.
b 1 M solution in Et2O.
c Isolated as 2,3-di-O-acetate.
tions resulted in slightly higher enrichment of the
respective b-anomers (entries 3 and 4). Similarly, the
pent-4-enyl glycosides 14, 15, the (2-chloroethoxy)ethyl
derivatives 16, 17, and the w-bromohexyl glycosides 18
and 19 were obtained in satisfactory yields (Table 1, en-
tries 3–6). The anomeric mixtures of 10/11 and 18/19
could be partially resolved by HPLC-separation, whereas
the acetylated allyl glycosides 12 and 13 were amenable
to separation on a standard silica gel phase. The pent-4-
enyl derivatives 14/15 and the (2-chloroethoxy)ethyl de-
rivatives 16/17 were also isolated as the 2,3-di-O-acetyl
derivatives in order to allow for a facile separation of the
excess alcohol components via their respective acetates.
The assignment of the anomeric configuration of the b-
glycosides 10, 12, 14, 16, and 18 was based on the low-
Thus, an anomeric mixture of allyl arabinosides 10 and 11
was generated from the methyl glycoside precursor 8 by
reaction with excess allyl alcohol and 1 M ethereal HCl at
60 °C (Table 1, entry 1, Scheme 2). The glycosides were
obtained in a combined yield of 55% with a higher propor-
tion of the axially oriented b-glycoside 10 (ratio of 10:11
~2:1).
N3
HO
N3
R2O
R1OH
O
1
field shifted H NMR signals of the anomeric proton (in
O
OMe
a or a + b
R1
the range of d = 5.19–4.85) and the small value of the
homonuclear coupling constant (J1,2 = ~3.6 Hz). In addi-
tion, the values of the optical rotation showed a higher
dextrorotation for the axial glycosides 10, 12, 14, and 18
in comparison to their equatorial counterparts.
OR2
OH
8
10 R1 = β-ΟAll, R2 = H
11 R1 = α-OAll, R2 = H
12 R1 = β-OAll, R2 = Ac
13 R1 = α-OAll, R2 = Ac
b
14 R1 = β-O(CH2)3CH=CH2, R2 = Ac
15 R1 = α-O(CH2)3CH=CH2, R2 = Ac
16 R1 = β-O(CH2)2O(CH2)2Cl, R2 = Ac
17 R1 = α-O(CH2)2O(CH2)2Cl, R2 = Ac
18 R1 = β-O(CH2)6Br, R2 = H
N3
O
a
HO
HO
19 R1 = α-O(CH2)6Br, R2 = H
SH
S
O
22
N3
N3
HO
NH2
O
c
O
18
HO
b
O
R3
OH
HO
O
HO
HO
O
O
20 R3 = SAc
10
23
d
21 R3 = SH
NH2
NHAc
Scheme 2 Reagents and conditions: (a) see Table 1; (b) Ac2O, py-
ridine, 0 °C, 2 h, 95% yield for 12/13; (c) KSAc, DMF, r.t., 15 h, 83%;
(d) 0.2 M NaOH, 1.5 h, r.t., 87%.
O
O
c
d
HO
HO
HO
HO
O
O
24
25
Reaction of the methyl glycoside 8 with acetyl chloride
and excess of allyl alcohol proceeded smoothly at room
temperature and gave a higher product yield (Table 1, en-
try 2) due to less by-product formation. Also, these condi-
Scheme 3 Reagents and conditions: (a) HS(CH2)3SH, hn, MeOH,
r.t., h, 65%; (b) HS(CH2)3SH, aq pyridine, r.t., 15 h, 91%; (c) 10%
Pd/C, MeOH, H2, r.t., 18 h, 97%; (d) Ac2O, pyridine, r.t. 36 h, then
0.1 M NaOMe, MeOH, r.t., 15 h, 95%.
Synthesis 2010, No. 18, 3143–3151 © Thieme Stuttgart · New York

PAPER
Synthesis of Spacer-Equipped 4-Amino-4-deoxy-L-arabinopyranosides
3145
O
BSA
N
NH2
HO
O
O
b
NH2
HO
O
a
O
21
N
BSA
HO
O
HO
(CH2)6S
O
(CH2)6SH
O
27
26
Scheme 4 Reagents and conditions: (a) HS(CH2)3SH, aq pyridine, r.t., 15 h, 80% yield; (b) aq buffer (pH 6.6), 4 °C, 16 h.
In order to introduce reactive end groups for subsequent with cysteamine, cysteine, by epoxide formation, or via
conjugation, the terminal bromide group of the glycoside oxidative cleavage of the double bond. Alternatively, se-
derivative 18 was exchanged for a thioacetyl group by re- lective reduction of the azido group leaving the allyl
action with KSAc in DMF to afford the w-thioacetyl de- group unaffected could be accomplished by reaction of 10
rivative 20 in 83% yield. Finally, the S-acetyl group was with propane-1,3-dithiol in the absence of UV irradiation
hydrolyzed under alkaline conditions and with exclusion in aqueous pyridine to give compound 23 in 91% yield.21
of oxygen to furnish the w-thiol compound 21 with mini-
mal dimer formation (Scheme 2).
As additional haptenic ligands to be used in inhibition
studies, the 4-amino-4-deoxy-derivative 24 and the N-
Notably, the allyl aglycone in compound 10 could also be acetylated derivative 25 were synthesized. Thus, the pro-
diversified with respect to spacer elongation (Scheme 3).
pyl 4-amino-4-deoxy-glycoside 24 was prepared by re-
duction of the 4-azido group with concomitant saturation
of the allyl group by hydrogenation on palladium/charcoal
in 97% yield. The 13C NMR data of the glycoside 24 com-
pare favorably with those reported for arabinoside units in
LPS core and lipid A oligosaccharides, confirming the
configurational assignments.7 The N-acetyl derivative 25
Reaction of 10 with propane-1,3-dithiol under UV irradi-
ation led to chain elongation and provided the thioether
bridged w-thiol derivative 22 in 65% yield (Scheme 3). A
small portion of the respective dimer was also present in
the product mixture. This strategy thus broadens the scope
of using allyl glycosides as precursor of spacer glyco-
sides, which have previously been obtained by reaction
Table 2 Serum Antibody Titers by ELISAa in Rabbits and Mice after Immunization with Ara4N-BSA (27)
Animal
Rabbit
28
BSA
E. coli
Proteus mirabilis Burkh. cepacia
Salmon. enterica
K446b
K446c
K447b
K447c
Mouse
<1000
<1000
<1000
<1000
<1000
<500
<500
<500
<500
<500
8.000
<500
1000
<500
64.000
<500
<500
<500
<500
<500
1.024.000
1.000
256.000
32.000
M1d
M2
M3
M4
M5
M6
M7
M8
M9
M10
512.000
128.000
256.000
<1000
<1000
<1000
<1000
<1000
<1000
<1000
<1000
<1000
<1000
<1000
<100
<100
100
100
100
<100
<100
<100
<100
<100
800
100
<100
100
100
<100
<100
100
<100
<100
800
<100
<100
<100
<100
100
128.000
512.000
256.000
1.024.000
64.000
100
100
100
100
3200
<100
100
200
<100
100
100
<100
100
256.000
100
a Final dilution yielding OD405 >0.2 with immobilized BSA, 27, or LPS.
b Before immunization.
c 56 days after immunization.
d 35 days after immunization.
Synthesis 2010, No. 18, 3143–3151 © Thieme Stuttgart · New York

3146
B. Müller et al.
PAPER
trap. Full conversion was accomplished when 2.74 mL of H2O had
been collected after 5 h. The reaction mixture was cooled to 40 °C
and concentrated. The remaining, slightly yellow solid was dried
under reduced pressure overnight and used without further purifica-
tion. A solution of TsCl (28.95 g, 151.85 mmol) in anhyd 1,4-diox-
ane (110 mL) was added dropwise with cooling to a suspension of
the stannylidene derivative in anhyd 1,4-dioxane (400 mL). The re-
action mixture was stirred for 48 h at r.t. SiO2 (25 g) was added and
the solvent was removed. The remaining, slightly yellowish solid
was purified by flash chromatography first with toluene (1.2 L) fol-
lowed by elution with EtOAc (1.2 L). The EtOAc fraction was con-
centrated until a slightly yellow oil remained, which crystallized
overnight. Crystallization from n-hexane–EtOAc gave 2 (48.16 g,
99%) as colorless needles; mp 128–130 °C (Lit.18 mp 135–136 °C).
was produced in 95% yield via peracetylation of 24 fol-
lowed by Zemplén de-O-acetylation.
For the synthesis of the neoglycoconjugate 27, the 4-azi-
do-4-deoxy spacer derivative 21 was converted into the
corresponding 4-amino compound 26 by reaction with
propane-1,3-dithiol in 81% yield (Scheme 4). The materi-
al contained ca. 15% of the corresponding disulfide and
was directly used for the conjugation step. Coupling of the
thiol-containing ligand to maleimide-activated BSA at pH
7.4 afforded the neoglycoprotein 27. MALDI MS analysis
of the Ara4N-BSA product indicated a high ligand density
of ~34 mol ligand/mol BSA.
1H NMR (400 MHz, CDCl3): d = 7.82 (d, J = 12.0 Hz, 2 Harom), 7.36
(d, J = 12.0 Hz, 2 Harom), 4.36 (dt, J = 4.5, 8.0 Hz, 1 H, H-4), 4.23
(d, J = 6.5 Hz, 1 H, H-1), 4.05 (dd, J = 4.8, 12.2 Hz, 1 H, H-5eq),
3.68 (dt, J = 4.5, 8.0 Hz, 1 H, H-3), 3.49 (s, 3 H, OCH3), 3.35–3.44
(m, 2 H, H-2, H-5ax), 3.04–3.12 (br s, 1 H, 3-OH), 2.81–2.92 (br s,
1 H, 2-OH), 2.45 (s, 3 H, ArCH3).
13C NMR (100 MHz, CDCl3): d = 145.44 (C1-Ar), 132.84 (C4-Ar),
130.01 (C3-, C5-Ar), 128.05 (C2-, C6-Ar), 103.27 (C-1), 77.58 (C-
4), 72.39 (C-2, C-3), 62.05 (C-5), 56.97 (OCH3), 21.70 (ArCH3).
Immunization of rabbits and mice with Ara4N-BSA re-
sulted in a specific response in all animals except one
mouse against the immunizing antigen as measured by
ELISA. The titers reached values of up to 1 million
whereby no reactivity (<1000) was seen with BSA alone
(Table 2). We also tested a panel of bacterial LPS known
to contain Ara4N in a terminal position. No reactivity was
observed with Re-mutant LPS of E. coli, which is not sub-
stituted with Ara4N, and LPS of S. enterica serovar Min-
nesota, which carries Ara4N on the 4¢-phosphate of the
lipid A backbone in nonstoichiometric amounts. Howev-
er, both rabbits developed antibodies against the LPS of P.
mirabilis and B. cepacia whereby the titers against the lat-
ter antigen were significantly higher. From 10 mice only
two (animal M6 and M8) had developed antibodies
against LPS of P. mirabilis and B. cepacia. Interestingly,
animal M8 had a higher titer against P. mirabilis LPS than
against B. cepacia LPS of 3200 and 800, respectively.
Methyl 2,3-Di-O-benzoyl-4-O-p-toluenesulfonyl-b-D-xylopyra-
noside (3)
Benzoyl chloride (2.30 mL, 9.71 mmol) was added dropwise to a
stirred solution of 2 (3.00 g, 9.4 mmol) in anhyd pyridine (15 mL)
at 0 °C. The colorless solution turned pink and a colorless solid pre-
cipitated. The ice-bath was removed and the reaction mixture was
stirred overnight at r.t. The mixture was dissolved in CHCl3 (50 mL)
and washed with H2O (50 mL). The aqueous layer was extracted
with CHCl3 (40 mL), the organic were layers combined, washed
with aq 0.1 M HCl (50 mL) and sat. aq NaHCO3 (50 mL), and dried
(MgSO4). The solution was concentrated and the solid residue was
crystallized from n-hexane–EtOAc to give 3; yield: 4.34 g (92%);
20
colorless solid; mp 138–140 °C (Lit.19 mp 139–141 °C); [a]D
+39.5 (c 0.31, CHCl3).
Maleimide-activated BSA was purchased from Sigma Aldrich.
Known compounds were identified by comparison with reported
melting points as well as 1H and 13C NMR data. Melting points were
determined with a Kofler hot stage microscope and are uncorrected.
Optical rotations were measured with a Perkin-Elmer 243 B pola-
rimeter. [a]D20 Values are given in units of 10–1 degcm2g–1. 1H NMR
spectra were recorded at 297 K with a Bruker DPX instrument op-
erating at 300 MHz or 400 MHz for 1H using CDCl3 as solvent and
TMS as standard, unless otherwise stated. 13C NMR spectra were
measured at 75.47 or 100.62 MHz and referenced to 1,4-dioxane
(d = 67.40). Homo- and heteronuclear 2D NMR spectroscopy was
performed with Bruker standard software. MALDI-TOF-MS ion-
ization spectra were recorded in the positive ion mode, with sinapic
acid as matrix. TLC was performed on Merck precoated plates
(5 × 10 cm, layer thickness 0.25 mm, silica gel 60 F254); spots were
detected by spraying with anisaldehyde/H2SO4. For column chro-
matography, silica gel (0.040–0.063 mm) was used. Concentration
of solutions was performed at reduced pressure at temperatures
<40 °C. Elemental analyses were provided by Dr. J. Theiner, Mik-
roanalytisches Laboratorium, Institut für Physikalische Chemie,
Universität Wien. LPS of Re mutants of E. coli (strain F576), P.
mirabilis (strain R45) and S. enterica serovar Minnesota (strain
R595) and LPS from B. cepacia (strain Ko2b) were obtained by ex-
traction of bacteria with phenol–chloroform–light petroleum ether
as described.22
1H NMR (400 MHz, CDCl3): d = 7.93–7.87 (m, 2 Harom), 7.68 (d,
J = 8.3 Hz, 2 Harom), 7.61 (d, J = 8.3 Hz, 2 Harom), 7.53–7.45 (m, 2
Harom), 7.37–7.28 (m, 4 Harom), 6.95 (d, J = 8.3 Hz, 2 Harom), 5.57 (t,
J = 8.7 Hz, 1 H, H-3), 5.24 (dd, J = 6.8, 8.8 Hz, 1 H, H-2), 4.70–
4.60 (m, 1 H, H-4), 4.58 (d, J = 6.8 Hz, 1 H, H-1), 4.38 (dd, J = 5.1,
J = 12.0 Hz, 1 H, H-5eq), 3.69 (dd, J = 12.0, 8.8 Hz, 1 H, H-5ax),
3.47 (s, 3 H, OCH3), 2.17 (s, 3 H, ArCH3).
13C NMR (100 MHz, CDCl3): d = 165.00, 164.74 (C=O), 144.92,
133.23, 133.17, 132.45, 129.73, 129.71, 128.91, 128.60, 128.26,
128.11, 127.57 (C-Ar), 101.59 (C-1), 74.93 (C-4), 70.73 (C-2, C-3),
62.86 (C-5), 56.91 (OCH3), 21.50 (ArCH3).
Methyl 4-O-4-Nitrobenzenesulfonyl-b-D-xylopyranoside (4)
Compound 4 was prepared according to the procedure described for
2 from the stannylidene intermediate (4.00 g, 10.1 mmol) and nosyl
chloride (2.20 g, 9.9 mmol) in dioxane (30 mL) with stirring for 20
h at r.t. and worked up as described; yield: 3.46 g (99%); colorless
solid; mp 64–67 °C (Lit.20 mp 62–65 °C).
1H NMR (400 MHz, CDCl3): d = 8.40 (d, J = 9.1 Hz, 2 Harom), 8.17
(d, J = 9.1 Hz, 2 Harom), 4.39 (dt, J = 5.4, 9.3 Hz, 1 H, H-4), 4.14
(dd, J = 5.2, 11.5 Hz, 1 H, H-5eq), 4.13 (d, J = 7.4 Hz, 1 H, H-1),
3.57 (app. t, J = 8.9 Hz, 1 H, H-3), 3.51 (s, 3 H, OCH3), 3.43 (dd,
J = 9.8, 11.8 Hz, 1 H, H-5ax), 3.36 (br s, 2 H, 2-OH, 3-OH), 3.22 (dd,
J = 7.4, 9.0 Hz, 1 H, H-2).
Methyl 4-O-p-Toluenesulfonyl-b-D-xylopyranoside (2); Large-
Scale Preparation
Commercially available methyl b-D-xylopyranoside (1; 25.00 g,
152.3 mmol) and Bu2SnO (37.92 g, 152.3 mmol) were suspended in
anhyd toluene (500 mL) and heated to reflux with a Dean–Stark
13C NMR (100 MHz, CDCl3): d = 150.65 (C4-Ar), 141.71 (C1-Ar),
129.40 (C3-, C5-Ar), 124.09 (C2-, C6-Ar), 103.88 (C-1), 79.11 (C-
4), 73.24 (C-2), 72.90 (C-3), 62.88 (C-5), 56.95 (OCH3).
Synthesis 2010, No. 18, 3143–3151 © Thieme Stuttgart · New York

PAPER
Synthesis of Spacer-Equipped 4-Amino-4-deoxy-L-arabinopyranosides
3147
Methyl 2,3-Di-O-benzoyl-4-O-4-nitrobenzenesulfonyl-b-D-xy-
lopyranoside (5)
Anal. Calcd for C20H19N3O6: C, 60.45; H, 4.82; N, 10.57. Found: C,
60.58; H, 4.78; N, 10.36.
Compound 5 was prepared from 4 (1.71 g, 3.89 mmol) and benzoyl
chloride (1.16 mL, 9.71 mmol) in anhyd pyridine (12 mL) with stir-
ring for 4 h at r.t. according to the procedure described for 3. Work-
up as described for 3 gave compound 5; yield: 1.29 g (47%);
colorless crystals; mp 151–152 °C (n-hexane); [a]D +6.7 (c 1.0,
CHCl3).
1H NMR (400 MHz, CDCl3): d = 8.20–7.23 (m, 14 Harom), 5.57 (t,
J = 8.6 Hz, 1 H, H-3), 5.26 (dd, J = 6.8, 8.8 Hz, 1 H, H-2), 4.79 (dt,
J = 5.1 Hz, 1 H, H-4), 4.61 (d, J = 6.8 Hz, 1 H, H-1), 4.17 (dd,
J = 5.1, 12.1 Hz, 1 H, H-5eq), 3.75 (dd, J = 12.1, 8.8 Hz, 1 H, H-5ax),
3.50 (s, 3 H, OCH3).
13C NMR (100 MHz, CDCl3): d = 164.48, 164.92 (C=O), 150.17,
141.30, 133.88, 133.37, 129.71, 129.44, 128.73, 128.33, 128.28,
128.03, 124.21 (C2-, C6-Ar), 101.67 (C-1), 76.57 (C-4), 70.72 (C-
3), 70.60 (C-2), 62.90 (C-5), 57.00 (OCH3).
Synthesis of 7 under Microwave Irradiation: Compound 3 (3.70 g,
7.03 mmol) and NaN3 (0.69 g, 10.6 mmol) were suspended in anhyd
DMSO (10 mL) and heated in a MSL Microwave to 110 °C (max.
250 W). After 30 min, a second portion of NaN3 (200 mg) was add-
ed and heating was continued for 15 h. Cooling to r.t and workup as
described above afforded 7 (2.26 g, 81%) as colorless needles.
20
Methyl 4-Azido-4-deoxy-a-L-arabinopyranoside (8)
Method A, from 7: Azide 7 (47.42 g, 119.3 mmol) was suspended in
anhyd MeOH (300 mL). A 1 M solution of methanolic NaOMe (12
mL) was added and the mixture was stirred for 1 h at r.t. The solu-
tion was made neutral by adding DOWEX 50 H+ resin. The resin
was removed by filtration and the filtrate was concentrated. n-Hex-
ane (200 mL) was added, the suspension was stirred overnight and
the solid product 8 was collected by filtration and dried; yield: 20.97
g (93%); colorless solid; mp 94–95 °C {Lit.12 mp 94–95 °C); [a]D
20
–22.6 (c 0.3, CHCl3); Lit.8 –26.1 (c 0.5, CHCl3)}.
Anal. Calcd for C26H23NO11S: C, 55.75; H, 3.95; N, 2.51. Found: C,
56.01; H, 4.16; N, 2.51.
1H NMR (300 MHz, CDCl3): d = 5.48–5.43 (m, 1 H, 3-OH), 5.25–
5.20 (m, 1 H, 2-OH), 4.04 (d, J = 7.2 Hz, 1 H, H-1), 3.83–3.71 (m,
2 H, H-4, H-5eq), 3.70–3.60 (m, 1 H, H-3), 3.56–3.48 (m, 1 H, H-
5ax), 3.38 (s, 3 H, OCH3), 3.34–3.24 (m, 1 H, H-2).
13C NMR (75 MHz, CDCl3): d = 104.33 (C-1), 72.54 (C-3), 70.66
(C-2), 63.03 (C-5), 61.13 (C-4), 55.84 (OCH3).
Methyl 2,3-Di-O-acetyl 4-O-4-nitrobenzenesulfonyl-b-D-xylopy-
ranoside (6)
Compound 6 was prepared from 4 (3.00 g, 8.6 mmol) and Ac2O (15
mL, 132 mmol) in anhyd pyridine (15 mL) with stirring for 2 h at
r.t. The formed precipitate was filtered off and the filtrate was con-
centrated to 1/3 volume, which led to additional precipitation. Sol-
ids were combined and dried at 40 °C under reduced pressure to
afford 6; yield: 3.4 g (93%); colorless crystals; mp 157 °C (n-hex-
ane); [a]D20 –65.7 (c 0.4, CHCl3).
1H NMR (400 MHz, CDCl3): d = 8.42 (d, J = 8.8 Hz, 2 Harom), 8.11
(d, J = 8.8 Hz, 2 Harom), 5.15 (app. t, J = 8.5 Hz, 1 H, H-3), 4.84 (dd,
J = 7.0, 8.5 Hz, 1 H, H-2), 4.67 (dt, J = 5.1, 8.6 Hz, 1 H, H-4), 4.38
(d, J = 6.8 Hz, 1 H, H-1), 4.17 (dd, J = 5.1, 11.9 Hz, 1 H, H-5eq),
3.51 (dd, J = 12.1, 9.1 Hz, 1 H, H-5ax), 3.46 (s, 3 H, OCH3), 2.03 (s,
3 H, 2-OAc), 1.85 (s, 3 H, 3-OAc).
Method B, from 4: Nosylate 4 (0.200 g, 0.57 mmol) and NaN3 (0.40
g, 6.15 mmol) were suspended in anhyd DMSO (1 mL) and stirred
at 45 °C for 5 h. The reaction mixture was dissolved in CHCl3 (30
mL) and washed with H2O (20 mL), sat. aq NaHCO3 (2 × 20 mL),
and brine (15 mL). The organic layer was dried (MgSO4) and con-
centrated. Purification of the residue by column chromatography (n-
hexane–EtOAc, 3:1) afforded 8; yield: 0.092 g (85%); colorless sol-
id.
Methyl 4-Amino-4-deoxy-a-L-arabinopyranoside (9)
Pd/C (10 mol%) was added to a solution of azide 8 (308 mg, 1.63
mmol) in MeOH (25 mL) and the suspension was stirred at r.t. over-
night under H2 at atmospheric pressure. The catalyst was removed
by filtration over Celite and the filtrate was concentrated and dried
to give 9; yield: 264 mg (99%); colorless syrup; [a]D20 –36.4 (c 0.31,
MeOH).
1H NMR (300 MHz, CDCl3): d = 4.33 (d, J = 6.9 Hz, 1 H, H-1),
3.86 (dd, J = 3.2, 12.4 Hz, 1 H, H-5eq), 3.76 (dd, J = 4.2, 8.8 Hz, 1
H, H-3), 3.71 (dd, J = 2.4, 12.4 Hz, 1 H, H-5ax), 3.56 (s, 3 H, OCH3),
3.52–3.59 (m, 1 H, H-2), 3.08–3.14 (m, 1 H, H-4).
13C NMR (100 MHz, CDCl3): d = 169.50, 169.38 (C=O), 150.95
(ArNO2), 141.75 (C1-Ar), 129.24 (C3-, C5-Ar), 124.52 (C2-, C6-
Ar), 101.47 (C-1), 75.68 (C-4), 70.82 (C-3), 70.64 (C-2), 62.36 (C-
5), 56.90 (OCH3), 20.61 (COCH3), 20.46 (COCH3).
Anal. Calcd for C16H19NO11S: C, 44.34; H, 4.42; N, 3.23. Found: C,
44.00; H, 4.23; N, 3.22.
Methyl 4-Azido-2,3-di-O-benzoyl-4-deoxy-a-L-arabinopyrano-
side (7)
Compound 3 (69.15 g, 131 mmol) and NaN3 (13.7 g, 210.6 mmol)
were suspended in anhyd DMSO (280 mL) and stirred at 90 °C
overnight. The reaction mixture was dissolved in CHCl3 (0.8 L) and
washed with H2O (3 × 400 mL). The combined organic layers were
washed with sat. aq NaHCO3 (400 mL) and brine (400 mL), dried
(Na2SO4), and concentrated. The residual oil was dissolved in
EtOAc (20 mL) and n-hexane was added until a colorless solid pre-
cipitated. The crystals were collected and the filtrate was evaporat-
ed to dryness and to afford an additional crop. Crystallization from
2:1 n-hexane–EtOAc afforded 7; yield: 48.64 g (93%); mp 104–
105 °C (EtOH); [a]D20 +18.6 (c 0.3, CHCl3).
1H NMR (300 MHz, CDCl3): d = 8.06–7.97 (m, 4 Harom), 7.58–7.50
(m, 2 Harom), 7.45–7.38 (m, 4 Harom), 5.58 (dd, J = 5.4, 7.9 Hz, 1 H,
H-2), 5.51 (dd, J = 3.2, 7.9 Hz, 1 H, H-3), 4.60 (d, J = 5.6 Hz, 1 H,
H-1), 4.22–4.14 (m, 2 H, H-4, H-5eq), 3.84–3.75 (m, J = 4.3, 13.8
Hz, 1 H, H-5ax), 3.50 (s, 3 H, OCH3).
13C NMR (75 MHz, CDCl3): d = 103.84 (C-1), 72.07 (C-3), 70.19
(C-2), 65.29 (C-5), 56.88 (OCH3), 49.72 (C-4).
Allyl 4-Azido-4-deoxy-b-L-arabinopyranoside (10) and Allyl 4-
Azido-4-deoxy-a-L-arabinopyranoside (11)
Method A: Azide 8 (1.03 g, 5.44 mmol) was dissolved in anhyd allyl
alcohol (3 mL) and a 1 M solution of HCl in Et2O (2.0 mL) was add-
ed. The solution was stirred for 4 h at 60 °C and for further 18 h at
r.t. The brownish reaction mixture was diluted with EtOAc (30 mL),
neutralized with sat. aq NaHCO3 (30 mL), and washed with brine
(20 mL). The solvent was removed and the remaining yellowish oil
was purified by column chromatography (100 g SiO2, 10 mm, tolu-
ene–EtOAc, 3.5:1). The product-containing fractions were pooled
and concentrated to afford 10.
10
13C NMR (75 MHz, CDCl3): d = 165.63, 165.03 (C=O), 133.57,
133.32, 130.01, 129.80, 129.30, 128.78, 128.48, 128.39, (C-Ar),
101.05 (C-1), 71.47 (C-3), 69.18 (C-2), 61.66 (C-5), 57.56 (C-4),
56.45 (OCH3).
20
Yield: 320 mg (27%); Rf = 0.66 (EtOAc); mp 32–33 °C; [a]D
+192.4 (c 0.4, CHCl3).
1H NMR (400 MHz, CD3OD): d = 5.93–6.04 (m, 1 H, =CH), 5.35
(qd, J = 1.7, 17.2 Hz, 1 H, CH=CH2trans), 5.21 (qd, J = 1.5, 10.4 Hz,
Synthesis 2010, No. 18, 3143–3151 © Thieme Stuttgart · New York

3148
B. Müller et al.
PAPER
1 H, CH=CH2cis), 4.85 (d, J = 3.6 Hz, 1 H, H-1), 4.20 (tdd, J = 1.5,
5.2, 13.0 Hz, 1 H, OCH2), 4.04 (dd, J = 3.6, 9.6 Hz, 1 H, H-3), 4.01–
4.08 (m, 1 H, OCH2), 3.86–3.93 (m, 2 H, H-5eq, H-4), 3.75 (dd,
J = 3.6, 9.7 Hz, 1 H, H-2), 3.62 (dd, J = 2.6, 12.8 Hz, 1 H, H-5ax).
13C NMR (100 MHz, CD3OD): d = 133.37 (=CH), 118.02 (=CH2),
97.70 (C-1), 70.41 (C-3), 69.75 (C-2), 68.74 (OCH2), 61.42 (C-4),
60.85 (C-5).
13
Yield: 10 mg (8%); Rf = 0.45 (n-hexane–EtOAc, 3:1); [a]D20 +26.3
(c 0.2 CHCl3).
1H NMR (300 MHz, CDCl3): d = 5.92–5.78 (m, 1 H, =CH), 5.28
(dq, J = 1.7, 17.2 Hz, 1 H, CH=CH2trans), 5.19 (dq, J = 1.4, 10.4 Hz,
1 H, CH=CH2cis), 5.17 (dd, J = 5.4, 7.9 Hz, 1 H, H-2), 5.07 (dd,
J = 3.6, 7.7 Hz, 1 H, H-3), 4.49 (d, J = 5.4 Hz, 1 H, H-1), 4.28 (ddt,
J = 1.6, 4.8, 13.2 Hz, 1 H, OCH2), 4.05 (dd, J = 5.2, 12.0 Hz, 1 H,
H-5eq), 4.04 (ddt, J = 5.2, 12.0 Hz, 1 H, OCH2), 3.96–3.90 (m, 1 H,
H-4), 3.61 (dd, J = 2.6, 12.1 Hz, 1 H, H-5ax), 2.12 (s, 3 H, COCH3),
2.08 (s, 3 H, COCH3).
Anal. Calcd for C8H13N3O4: C, 44.65; H, 6.09; N, 19.53. Found: C,
44.68; H, 5.79; N, 18.79.
Further elution provided a mixture of 10 and 11; yield: 233 mg
(20%), followed by 11.
13C NMR (75 MHz, CDCl3): d = 170.59 and 169.50 (C=O), 133.43
(=CH), 117.23 (CH=CH2), 98.65 (C-1), 71.06 (C-3), 69.11 (OCH2),
68.63 (C-2), 61.36 (C-5), 56.98 (C-4), 20.78 and 20.63 (COCH3).
11
Yield: 98 mg (8%); Rf = 0.55 (EtOAc); mp 39–42 °C; [a]D20 +61.8
(c 0.51, CHCl3).
ESI-TOF: m/z calcd for C12H17O6N3 + Na [M + Na]+: 322.1010;
found: 322.1024.
1H NMR (400 MHz, CD3OD): d = 5.89–6.00 (m, 1 H, =CH), 5.32
(qd, J = 1.7, 17.3 Hz, 1 H, CH=CH2trans), 5.16 (qd, J = 1.5, 10.4 Hz,
1 H, CH=CH2cis), 4.29 (tdd, J = 1.5, 5.2, 12.9 Hz, 1 H, OCH2), 4.22
(d, J = 7.2 Hz, 1 H, H-1), 4.09 (tq, J = 1.4, 6.3 Hz, 1 H, OCH2), 3.89
(dd, J = 2.7, 12.7 Hz, 1 H, H-5eq), 3.74 (dd, J3,4 = 3.9 Hz, J = 8.8 Hz,
1 H, H-3), 3.76–3.80 (m, 1 H, H-4), 3.58 (dd, J = 1.8, 12.7 Hz, 1 H,
H-5ax), 3.50 (dd, 1 H, J = 7.2, 8.9 Hz, H-2).
13C NMR (75 MHz, CDCl3): d = 133.48 (=CH), 118.21 (=CH2),
101.70 (C-1), 72.60 (C-3), 71.23 (C-2), 69.95 (OCH2), 62.98 (C-5),
60.06 (C-4).
Pent-4-enyl 2,3-Di-O-acetyl-4-azido-4-deoxy-b-L-arabinopyra-
noside (14) and Pent-4-enyl 2,3-Di-O-acetyl-4-azido-4-deoxy-a-
L-arabinopyranoside (15)
Compound 8 (400 mg, 2.11 mmol) and anhyd pent-4-en-1-ol (2.5
mL) was treated with freshly distilled AcCl (0.50 mL) and pro-
cessed as described for the allyl derivatives 10 and 11 (Method B).
Purification of the crude material by column chromatography (n-
hexane–EtOAc, 4:1) afforded first a pure fraction of 14.
14
Anal. Calcd for C8H13N3O4: C, 44.65; H, 6.09; N, 19.53. Found: C,
44.51; H, 6.03; N, 19.22.
Yield: 250 mg (34%); colorless oil; [a]D20 +171.9 (c 0.36, CHCl3).
1H NMR (300 MHz, CDCl3): d = 5.87–5.72 (m, 1 H, =CH), 5.37
(dd, J = 3.8, 10.5 Hz, 1 H, H-3), 5.16 (dd, J = 3.6, 10.5 Hz, 1 H, H-
2), 5.04 (d, J = 3.6 Hz, 1 H, H-1), 5.07–4.95 (m, 2 H, CH=CH2),
4.12–4.07 (m, 1 H, H-4), 3.94 (dd, J = 1.9, 12.6 Hz, 1 H, H-5eq),
3.62–3.73 (m, 2 H, CH2O and H-5ax), 3.45–3.35 (m, 1 H, CH2O),
2.18–2.06 (m, 2 H, CH2), 2.12 (s, 3 H, COCH3), 2.07 (s, 3 H,
COCH3), 1.74–1.63 (m, 2 H, CH2).
13C NMR (75 MHz, CDCl3): d = 170.18 and 170.28 (C=O), 137.76
(=CH), 115.21 (CH=CH2), 96.41 (C-1), 69.39 (C-3), 68.42 (C-2),
67.75 (OCH2), 59.92 (C-5), 59.86 (C-4), 30.09 and 28.49 (CH2),
20.73 (COCH3), 20.59 (COCH3).
Method B: Freshly distilled AcCl (0.5 mL) was added at r.t. to a so-
lution of 8 (400 mg; 2.11 mmol) in anhyd allyl alcohol (2.5 mL).
Upon addition a strong exothermic reaction occurred. After cooling
to r.t., the solution was stirred for 18 h. The slightly yellowish solu-
tion was diluted with EtOAc (30 mL), treated with sat. aq NaHCO3
(2 × 20 mL), washed with brine (20 mL), and dried (MgSO4). The
solvent was removed and the remaining oil was purified by column
chromatography (60 g, 63 mm SiO2, n-hexane–EtOAc, 4:1). The
product-containing fractions were pooled and concentrated to af-
ford 522 mg (76%) 10 and 11 as a 2.3:1 anomeric mixture.
Allyl 2,3-Di-O-acetyl-4-azido-4-deoxy-b-L-arabinopyranoside
(12) and Allyl 2,3-Di-O-acetyl-4-azido-4-deoxy-a-L-arabinopyr-
anoside (13)
ESI-TOF: m/z calcd for C14H21N3O6 [M + Na]+: 350.1323;
found: 350.1328.
A solution of the anomeric mixture 10,11 (100 mg, 0.46 mmol) in
anhyd pyridine (5 mL) and Ac2O (500 mL, 3.9 mmol) was stirred at
r.t. overnight. The solution was co-evaporated with toluene (2 × 20
mL) and concentrated. Purification of the residue on silica gel (tol-
uene–EtOAc, 8:1) afforded first 12 as a colorless oil.
Further elution gave a fraction containing 14 and 15 (208 mg, 28%)
and pure 15.
15
Yield: 70 mg (9%); colorless oil; [a]D20 –14.3 (c 0.21, CHCl3).
1H NMR (300 MHz, CDCl3): d = 5.87–5.72 (m, 1 H, CH=), 5.10–
4.93 (m, 3 H, CH=CH2, H-3), 5.37 (dd, J = 3.8, 10.5 Hz, 1 H, H-3),
5.14 (dd, J = 5.4, 7.7 Hz, 1 H, H-2), 4.44 (d, J = 5.3 Hz, 1 H, H-1),
4.03 (dd, J = 5.3, 12.1 Hz, 1 H, H-5eq), 3.95–3.89 (m, 1 H, H-4),
3.81 (dt, J = 6.3, 9.5 Hz, 1 H, CH2O), 3.62 (dd, J = 2.6, 12.1 Hz, 1
H, H-5ax), 3.44 (dt, J = 6.4, 9.5 Hz, 1 H, CH2O), 2.17–2.06 (m, 2 H,
CH2), 2.12 (s, 3 H, COCH3), 2.07 (s, 3 H, COCH3), 1.72–1.62 (m, 2
H, CH2).
13C NMR (75 MHz, CDCl3): d = 170.10 and 169.15 (C=O), 137.84
(=CH), 114.98 (CH=CH2), 99.78 (C-1), 71.11 (C-3), 68.70 (C-2),
68.33 (OCH2), 61.30 (C-5), 57.02 (C-4), 29.94 and 28.61 (CH2),
20.71 (COCH3), 20.56 (COCH3).
12
Yield: 25 mg (18%); colorless syrup; Rf = 0.55 (n-hexane–EtOAc,
3:1); [a]D20 +187.8 (c 0.4, CHCl3).
1H NMR (300 MHz, CDCl3): d = 5.92–5.79 (m, 1 H, =CH), 5.40
(dd, J = 3.8, 10.5 Hz, 1 H, H-3), 5.30 (dq, 1 H, J = 1.6, 17.2 Hz,
CH=CH2trans), 5.21 (dq, 1 H, J = 1.3, 10.5 Hz, CH=CH2cis), 5.20 (dd,
J = 3.6, 10.5 Hz, 1 H, H-2), 5.09 (d, J = 3.6 Hz, 1 H, H-1), 4.19 (ddt,
1 H, J = 1.5, 5.1, 13.1 Hz, OCH2), 4.11 (m, 1 H, H-4), 4.03–3.94 (m,
2 H, OCH2, H-5eq), 3.67 (dd, J = 2.0, 12.6 Hz, 1 H, H-5ax), 2.12 (s,
3 H, COCH3), 2.09 (s, 3 H, COCH3).
13C NMR (75 MHz, CDCl3): d = 170.22 and 170.17 (C=O), 133.23
(=CH), 117.84 (=CH2), 95.51 (C-1), 69.28 (C-3), 68.56 (OCH2),
68.20 (C-2), 60.01 (C-5), 59.86 (C-4), 20.78 and 20.59 (CH3C=O).
ESI-TOF: m/z calcd for C14H21N3O6 [M + Na]+: 350.1323;
found: 350.1329.
Further elution of the column afforded 13.
Synthesis 2010, No. 18, 3143–3151 © Thieme Stuttgart · New York

PAPER
Synthesis of Spacer-Equipped 4-Amino-4-deoxy-L-arabinopyranosides
3149
2-(2-Chloroethoxy)ethyl 2,3-Di-O-acetyl-4-azido-4-deoxy-b-L-
arabinopyranoside (16) and 2-(2-Chloroethoxy)ethyl 2,3-Di-O-
acetyl-4-azido-4-deoxy-a-L-arabinopyranoside (17)
19
Yield: 108 mg (9%); colorless crystals; mp 47–49 °C; [a]D20 –11 (c
0.8, CHCl3).
Compound 8 (400 mg, 2.11 mmol) and anhyd (2-chloroethoxy)eth-
anol (2.5 mL) were treated with freshly distilled AcCl (0.50 mL)
and processed as described for the allyl derivatives 10 and 11
(Method B). The product-containing fractions were pooled, and
concentrated to afford 430 mg (72%) 16 and 17 as a ~2.9:1 anomer-
ic mixture of colorless oil.
1H NMR (300 MHz, CDCl3): d = 4.26 (d, J = 6.4 Hz, 1 H, H-1),
3.90–3.81 (m, 3 H, H-3, H-4, OCH2), 3.77 (dd, J = 3.8, 12.6 Hz, 1
H, H-5eq), 3.70 (dd, J = 6.4, 7.9 Hz, 1 H, H-2), 3.59 (dd, J = 2.5,
12.6 Hz, 1 H, H-5ax), 3.49 (td, J = 6.6, 9.6 Hz, 1 H, OCH2), 3.41 (t,
J = 6.7 Hz, 2 H, BrCH2), 2.94 (br s, 1 H, OH), 2.54 (br s, 1 H, OH),
1.84–1.76 (m, 2 H, CH2CH2Br), 1.61–1.51 (m, 2 H), 1.45–1.26 (m,
4 H, CH2).
b-Anomer 16
NMR-data were extracted from anomeric mixture.
13C NMR (75 MHz, CDCl3): d = 102.45 (C-1), 72.61 (C-2), 71.38
(C-3), 69.55 (OCH2), 62.54 (C-4), 59.33 (C-5), 33.78 (BrCH2),
32.55, 29.30, 27.82, 25.15 (CH2).
1H NMR (400 MHz, CDCl3): d = 5.41 (dd, J = 3.7, 10.5 Hz, 1 H, H-
3), 5.19 (dd, J = 3.6, 10.5 Hz, 1 H, H-2), 5.11 (d, J = 3.6 Hz, 1 H,
H-1), 4.12–4.09 (m, 1 H, H-4), 4.05 (ddd, J = 1.8, 12.6 Hz, 1 H, H-
5eq), 3.85–3.79 (m, 1 H, CH2O), 3.78–3.74 (m, 2 H, CH2O), 3.71–
3.60 (m, 6 H, CH2, H-5ax), 2.12 (s, 3 H, COCH3), 2.09 (s, 3 H,
COCH3).
13C NMR (100 MHz, CHCl3): d = 170.11 (C=O), 96.48 (C-1),
71.36, 71.34, 70.12, 69.25 (C-3), 68.25 (C-2), 67.37, 59.97 (C-5),
59.89 (C-4), 42.81 (CH2Cl), 20.59 (COCH3), 20.79 (COCH3).
Anal. Calcd for C11H20BrN3O4: C, 39.07; H, 5.96; N, 12.42. Found:
C, 39.36; H, 5.78; N, 12.28.
Method B: Freshly distilled AcCl (0.25 mL) was added at r.t. to a
solution of 8 (200 mg; 1.6 mmol) in 6-bromohexanol (1 mL, 6.6
mmol). Upon addition a strong exothermic reaction occurred. After
cooling to r.t., the solution was stirred for 18 h. The slightly yellow-
ish solution was diluted with EtOAc (50 mL), treated with sat. aq
NaHCO3 (60 mL), washed with brine (40 mL), and dried (Na2SO4).
The solvent was removed and the remaining oil was purified by col-
umn chromatography (50 g, 63 mm SiO2, n-hexane–EtOAc, 4:1).
The product-containing fractions were pooled and concentrated to
afford 238 mg (65%) 18 and 19 as a 3:1 anomeric mixture.
a-Anomer 17
NMR-data were extracted from anomeric mixture.
1H NMR (300 MHz, CDCl3): d = 5.16 (dd, J = 5.5, 8.0 Hz, 1 H, H-
2), 5.07 (dd, J = 3.6, 8 Hz, 1 H, H-3), 4.53 (d, J = 5.6 Hz, 1 H, H-
1), 4.09–4.12 (m, 1 H, H-4), 4.05 (dd, J = 5.1, 12.2 Hz, 1 H, H-5eq),
3.91–3.95 (m, 1 H, H-4), 3.88–3.91 (m, 1 H, CH2O), 3.58–3.77 (m,
6 H), 2.12 (s, 3 H, COCH3), 2.08 (s, 3 H, COCH3).
6-S-Thioacetylhexyl 4-Azido-4-deoxy-b-L-arabinopyranoside
(20)
A solution of KSAc (17 mg, 0.15 mmol) in anhyd DMF (1mL) was
added to a solution of bromide 18 (98 mg, 0.29 mmol) in anhyd
DMF (2 mL) and the solution was stirred at r.t. overnight whereup-
on a colorless solid precipitated.The reaction mixture was diluted
with EtOAc (20 mL) and poured onto H2O (20 mL). The aqueous
layer was extracted with EtOAc (2 × 15 mL). The combined organic
layers were washed with sat. NaHCO3 (40 mL), brine (30 mL), and
dried (MgSO4). Concentration of the solution afforded a slightly
yellow oil, which was further purified by column chromatography
(15.0 g SiO2; n-hexane–EtOAc, 3:2) to furnish 20; yield: 82 mg
(83%); colorless syrup; [a]D20 +133 (c 0.5 CHCl3).
1H NMR (300 MHz, CDCl3): d = 4.86 (d, J = 3.7 Hz, 1 H, H-1),
4.00–3.91 (m, 2 H, H-4, H-5eq), 3.88–3.65 (m, 4 H, H-2, H-3, H-5ax,
OCH2), 3.49–3.39 (m, 1 H, OCH2), 2.86 (t, J = 7.2 Hz, 2 H, SCH2),
2.32 (s, 3 H, CH3COS), 2.17 (br s, 2 H, OH), 1.68–1.51 (m, 4 H,
CH2), 1.46–1.29 (m, 4 H, CH2).
13C NMR (75 MHz, CDCl3): d = 169.22 (C=O), 99.90 (C-1), 71.37,
71.20, 70.33, 68.61 (C-3), 68.25 (C-2), 68.15, 61.60 (C-5), 57.13
(C-4), 42.78 (CH2Cl), 20.74 (COCH3), 20.56 (COCH3).
ESI-TOF: m/z calcd for C13H20ClN3O7 [M + Na]+: 388.0882;
found: 388.0887.
6-Bromohexyl 4-Azido-4-deoxy-b-L-arabinopyranoside (18)
and 6-Bromohexyl 4-azido-4-deoxy-a-L-arabinopyranoside (19)
Method A: 6-Bromohexanol (1.6 mL) was added to a suspension of
compound 8 (700 mg, 3.7 mmol) in 1 M ethereal HCl (1.0 mL) and
heated for 4 h at 70 °C in a 25 mL two-necked flask. The resulting
solution was then stirred overnight at r.t. EtOAc (80 mL) and solid
Na2CO3 (2.5 g) were added and the suspension was filtered. The fil-
trate was washed with brine (100 mL), dried (Na2SO4), and concen-
trated. The residue was purified by column chromatography (120 g
SiO2, n-hexane–EtOAc, 7:3 → 0:1) yielding the pure b-anomer 18.
13C NMR (75 MHz, CDCl3): d = 196.11 (C=O), 98.46 (C-1), 70.82
(C-2), 69.97 (C-3), 68.46 (OCH2), 61.17 (C-4), 60.57 (C-5), 30.66
(CH3), 29.37, 29.23, 28.88, 28.35, 25.60 (CH2).
18
20
Yield: 542 mg (44%); colorless crystals; mp 33–34 °C; [a]D
+132.8 (c 1.3, CHCl3).
Anal. Calcd for C11H20BrN3O4: C, 46.83; H, 6.95; N, 12.60. Found:
C, 46.94; H, 6.96; N, 12.35.
1H NMR (400 MHz, CDCl3): d = 4.87 (d, J = 3.7 Hz, 1 H, H-1),
3.97 (dd, J = 3.8, 9.2 Hz, 1 H, H-3), 3.96–3.92 (m, 1 H, H-4), 3.83
(dd, J = 1.7, 12.5 Hz, 1 H, H-5eq), 3.79 (dd, J = 3.7, 9.2 Hz, 1 H, H-
2), 3.72 (dt, J = 6.8, 9.7 Hz, 1 H, OCH2), 3.69 (dd, J = 2.2, 12.5 Hz,
1 H, H-5ax), 3.46 (dt, J = 6.3, 9.5 Hz, 1 H, OCH2), 3.35 (t, J = 6.7
Hz, 2 H, BrCH2), 2.34 (br s, 2 H, OH), 1.84–1.76 (m, 2 H,
CH2CH2Br), 1.61–1.51 (m, 2 H, CH2), 1.45–1.26 (m, 4 H, CH2).
13C NMR (100 MHz, CDCl3): d = 98.44 (C-1), 70.74 (C-2), 69.93
(C-3), 68.39 (OCH2), 61.19 (C-4), 60.53 (C-5), 33.75 (BrCH2),
32.54, 29.24, 27.83, 25.30 (CH2).
6-Mercaptohexyl 4-Azido-4-deoxy-b-L-arabinopyranoside (21)
Thioacetate 20 (210 mg, 0.629 mmol) was stirred with aq 0.2 M
NaOH (50 mL) under argon at r.t. for 1.5 h. The reaction mixture
was extracted with EtOAc (3 × 40 mL), and the combined organic
layers were dried (MgSO4) and concentrated to give 21 as a color-
less oil, which solidified at r.t.; yield: 169 mg (87%); Rf = 0.14 (n-
hexane–EtOAc, 1:1); [a]D20 +146 (c 0.5, MeOH).
1H NMR (300 MHz, CD3OD): d = 4.77 (d, J = 3.7 Hz, 1 H, H-1),
4.00 (dd, J = 3.8, 9.7 Hz, 1 H, H-3), 3.91–3.84 (m, 2 H, H-4, H-5eq),
3.74 (dd, J = 3.7 Hz, 1 H, H-2), 3.69 (dt, J = 7.2, 9.7 Hz, 1 H,
OCH2), 3.61 (dd, J = 2.6, 12.8 Hz, 1 H, H-5ax), 3.46 (dt, J = 6.3, 9.7
Hz, 1 H, OCH2), 2.52 (t, J = 7.1 Hz, 2 H, SCH2), 1.72–1.55 (m, 4 H,
CH2), 1.50–1.36 (m, 4 H, CH2).
Anal. Calcd for C11H20BrN3O4: C, 39.07; H, 5.96; N, 12.42. Found:
C, 39.44; H, 5.71; N, 12.32.
Further elution gave a fraction containing 18 and 19 (240 mg, 19%)
and pure 19.
Synthesis 2010, No. 18, 3143–3151 © Thieme Stuttgart · New York

3150
B. Müller et al.
PAPER
13C NMR (75 MHz, CD3OD): d = 100.72 (C-1), 70.90 (C-3), 70.55
(C-2), 69.41 (OCH2), 64.07 (C-4), 61.63 (C-5), 35.11 (SCH2),
30.46, 29.16, 26.76, 24.88 (CH2).
13C NMR (75 MHz, CD3OD): d = 100.5 (C-1), 71.1 (OCH2), 70.6
(C-3), 70.2 (C-2), 66.99 (C-3), 63.4 (C-5), 52.3 (C-4), 23.7 (CH2),
10.9 (CH3).
ESI-TOF: m/z calcd as the disulfide C22H40O8N6S2 [M – H]–:
MS: ESI-TOF: m/z calcd for C8H17NO4 [M + H]+: 192.123; found:
579.2276; found: 579.2275.
192.1231.
3-Mercaptopropyl-3-thiopropyl 4-Azido-4-deoxy-b-L-arabino-
pyranoside (22)
Propyl 4-Acetamido-4-deoxy-b-L-arabinopyranoside (25)
A solution of amine 24 (40 mg, 0.209 mmol) in anhyd pyridine (2
mL) and Ac2O (0.5 mL) was stirred for 36 h at r.t. The reaction mix-
ture was diluted with EtOAc (15 mL) and stirred for 15 min with
EtOH (0.5 mL). The organic phase was washed with aq 2 M HCl (8
mL), H2O (10 mL), and sat. aq NaHCO3 (10 mL), and dried
(MgSO4). Concentration afforded a colorless residue (63 mg, 95%).
A portion of the residue (56 mg, 0.176 mmol) was dissolved in an-
hyd MeOH (2 mL) and treated with 0.1 M methanolic NaOMe (1.4
mL) for 15 h at r.t. The solution was neutralized with Dowex 50 H+
resin. The resin was filtered off, washed with MeOH (10 mL), and
the filtrate was concentrated to give 25; yield: 30 mg (95%); color-
less oil; Rf = 0.48 (CHCl3–MeOH, 4:1); [a]D20 +138 (c 0.3, MeOH).
Propane-1,3-dithiol (0.2 mL) was added to a solution of azide 10
(42 mg, 0.19 mmol) in MeOH (1.0 mL) in a quartz flask. The reac-
tion mixture was stirred under UV radiation (254 nm) for 4 h at r.t.
to give full conversion. The solvent was removed and the residue
was purified by chromatography (n-hexane–EtOAc–MeOH,
1:1:0.1) to afford 22 as a colorless oil; yield: 40 mg (65%).
1H NMR (300 MHz, CDCl3): d = 4.88 (d, J = 3.7 Hz, 1 H, H-1),
4.02–3.75 (m, 5 H, H-2, H-3, H-4, H-5eq, OCH2), 3.69 (dd, J = 2.2,
12.6 Hz, 1 H, H-5ax), 3.53 (dt, J = 6.1, 9.9 Hz, 1 H, OCH2), 2.70–
2.57 (m, 6 H, CH2), 1.95–1.81 (m, 4 H, CH2), 1.38 (t, J = 8.0 Hz, 1
H, SH).
1H NMR (300 MHz, CD3OD): d = 5.97 (d, J = 8.7 Hz, 1 H,
NHCOCH3), 4.78 (d, J = 3.3 Hz, 1 H, H-1), 4.20–4.26 (m, 1 H, H-
4), 3.92 (dd, J = 4.4, 9.0 Hz, 1 H, H-3), 3.83 (dd, J = 2.7, 11.8 Hz,
1 H, H-5eq), 3.68 (dd, J = 3.3, 9.2 Hz, 1 H, H-2), 3.59–3.69 (m, 1 H,
OCH2), 3.50 (dd, J = 3.6 Hz, 11.8 Hz, 1 H, H-5ax), 3.37–3.46 (m, 1
H, OCH2), 1.99 (s, 3 H, COCH3), 1.63 (m, J = 7.2 Hz, 2 H, CH2),
0.96 (t, J = 7.4 Hz, 3 H, CH3).
13C NMR (75 MHz, CD3OD): d = 100.43 (C-1), 71.28 (C-1¢), 70.82
(C-3), 69.71 (C-2), 62.51 (C-5), 51.21 (C-4), 23.79 (NHCOCH3),
22.62 (C-2¢), 10.96 (C-3¢).
13C NMR (75 MHz, CDCl3): d = 98.65 (C-1), 70.68 (C-3), 69.89
(C-2), 67.24 (OCH2), 61.17 (C-4), 60.63 (C-5), 33.16, 30.51, 29.09,
29.03, 23.34 (CH2).
ESI-TOF: m/z calcd for C11H20N3O4S2 [M – H]–: 322.0901; found:
322.0905.
Allyl 4-Amino-4-deoxy-b-L-arabinopyranoside (23)
A solution of azide 10 (50 mg, 0.23 mmol) in pyridine–H2O (10:1,
2.2 mL) was purged with argon. Et3N (0.48 mL) and propane-1,3-
dithiol (0.49 mL, 4.65 mmol) were added at r.t. and the mixture was
stirred overnight. The mixture was co-evaporated with toluene (2 ×
10 mL) and the residue was purified on an Isolute Si column (500
mg) using EtOAc as eluent. The first eluate (15 mL) contained re-
sidual propane-1,3-dithiol. Further elution with MeOH and pooling
of the fractions afforded product 23; yield: 40 mg (91%); colorless
syrup; Rf = 0.18 (CHCl3–MeOH–AcOH, 4:1:0.1); [a]D20 +183.6 (c
0.4, MeOH).
ESI-TOF: m/z calcd for C10H19NO5 + Na [M + Na]+: 256.1155;
found: 256.1151.
6-Mercaptohexyl 4-Amino-4-deoxy-b-L-arabinopyranoside (26)
Azide 21 (150 mg, 0.51 mmol) was dissolved in pyridine (5 mL),
deionized H2O (0.6 mL), and Et3N (0.6 mL). The solution was
purged with argon for 5 min and propane-1,3-dithiol (0.6 mL) was
added. Stirring at r.t. overnight gave full conversion. The reaction
mixture was co-evaporated with toluene (2 × 20 mL), and purified
by flash chromatography over SiO2 (6 g) using EtOAc and MeOH
as eluents. The methanolic product fractions were pooled and evap-
orated to dryness to give 26 as a syrup, which was stored under ar-
gon; yield: 110 mg (80%, containing 15% disulfide according to
NMR spectroscopy).
1H NMR (300 MHz, CD3OD): d = 4.76 (d, J = 3.7 Hz, 1 H, H-1),
3.86 (dd, J = 2.7, 11.8 Hz, 1 H, H-5eq), 3.81 (dd, J = 4.4, 9.5 Hz, 1
H, H-3), 3.74–3.66 (m, 1 H, OCH2), 3.67 (dd, J = 3.3, 9.5 Hz, 1 H,
H-2), 3.52–3.39 (m, 2 H, H-5ax, OCH2), 3.06–3.01 (m, 1 H, H-4),
2.52 (t, 2 H, SCH2), 1.78–1.54 (m, 4 H, CH2), 1.53–1.33 (m, 4 H,
CH2).
1H NMR (300 MHz, CD3OD): d = 6.03–5.88 (CH=), 5.32 (dq,
J = 1.7, 17.3 Hz, 1 H, CH=CH2trans), 5.16 (dq, J = 1.5, 10.4 Hz, 1
H, CH=CH2cis), 4.75 (d, J = 3.5 Hz, 1 H, H-1), 4.19 (m, 1 H, OCH2),
4.02 (m, 1 H, OCH2), 3.87 (dd, J = 2.5, 11.9 Hz, 1 H, H-5eq), 3.82
(dd, J = 4.1, 9.2 Hz, 1 H, H-3), 3.68 (dd, J = 3.4, 9.2 Hz, 1 H, H-2),
3.48 (dd, J = 3.1, 11.9 Hz, 1 H, H-5ax), 3.06–3.01 (m, 1 H, H-4).
13C NMR (75 MHz, CD3OD): d = 134.23 (=CH), 116.07
(CH=CH2), 98.33 (C-1), 69.32 (C-3), 68.84 (C-2), 68.27 (OCH2),
62.29 (C-5), 50.96 (C-4).
ESI-TOF: m/z calcd for C8H15NO4 [M + Na]+: 212.0893; found:
212.0898.
Propyl 4-Amino-4-deoxy-b-L-arabinopyranoside (24)
13C NMR (75 MHz, CD3OD): d = 100.56 (C1), 70.69 (C3), 70.24
(C2), 69.34 (OCH2), 63.56 (C5), 52.32 (C4), 35.08 (CH2S), 30.46
(CH2), 29.14 (CH2), 26.73 (CH2), 24.92 (CH2).
A suspension of azide 10 (37.0 mg, 0.53 mmol) and 10% Pd/C (15
mg) in anhyd MeOH (4 mL) was stirred at r.t. under H2 at atmo-
spheric pressure for 18 h. The suspension was filtered over a bed of
Celite and the filtrate was concentrated. The remaining oil was fur-
ther dried under high vacuum yielding the pure product 24; yield:
33 mg (97%); colorless oil; Rf = 0.17 (CHCl3–MeOH–AcOH,
2:1:0.1); [a]D20 +197.5 (c 0.28, MeOH).
BSA-Conjugate 27
According to the general procedure of the commercially available
‘Sigma-Aldrich Maleimide Activated BSA, KLH Conjugatio Kit-
Stock No. MBK-1’: Thiol 26 (5 mg) was dissolved in buffer (0.5
mL; 20 mM sodium phosphate buffer, 100 mM EDTA, and 80 mM
sucrose, pH 6.6) and immediately added to a solution of 5 mg/mL
maleimide-activated BSA (20 mM sodium phosphate buffer, 230
mM NaCl, 2 mM EDTA, and 80 mM sucrose, pH 6.6). The reaction
mixture was purged with argon for 2 min, and stirred at 4 °C over-
night. The reaction mixture was purified over a Sephadex G-25M
column (part of the BSA-Kit), and as eluent 0.01 M PBS-buffer in
double distilled H2O was used. The protein containing fractions
1H NMR (300 MHz, CD3OD): d = 4.75 (d, J = 3.6 Hz, 1 H, H-1),
3.87 (dd, J = 2.6, 11.9 Hz, 1 H, H-5eq), 3.81 (dd, J = 4.2, 9.1 Hz, 1
H, H-3), 3.66 (dd, J = 3.6, 9.0 Hz, 1 H, H-2), 3.69–3.60 (m, 1 H,
OCH2), 3.47 (dd, J = 3.1, 11.9 Hz, 1 H, H-5ax), 3.45–3.36 (m, 1 H,
OCH2), 3.07–3.01 (m, 1 H, H-4), 1.66–1.61 (m, 2 H, CH2), 0.95 (t,
J = 7.4 Hz, 3 H, CH3).
Synthesis 2010, No. 18, 3143–3151 © Thieme Stuttgart · New York

PAPER
Synthesis of Spacer-Equipped 4-Amino-4-deoxy-L-arabinopyranosides
3151
were pooled and lyophilized to afford 1.5 mg of 27 as colorless sol-
id.
Acknowledgment
The authors are grateful to Buko Lindner (Research Center Borstel)
for recording the MALDI MS and Stephan Hann and Madeleine
Dell’mour (BOKU Vienna) for recording the ESI-TOF MS. Finan-
cial support by Fonds zur Förderung der wissenschaftlichen For-
schung (FWF grant P 19295) is gratefully acknowledged.
Immunization of Mice and Rabbits with Ara4N-BSA (27)
Ten mice (Balb/c) were injected on day 0 subcutaneously into four
sites of the back skin with Ara4N-BSA conjugate (50 mg in 200 mL
of PBS) emulsified with an equal volume of Freund’s complete ad-
juvant. The animals received a booster injection intraperitoneally on
day 28 (50 mg in 200 mL of PBS emulsified with an equal volume of
Freund’s incomplete adjuvant) and bled on day 35. From two rab-
bits (chinchilla bastards) preimmune sera were collected on day –2.
On day 0 they were injected with Ara4N-BSA conjugate (100 mg in
1 mL of PBS) emulsified with an equal volume of Freund’s com-
plete adjuvant into the popliteal and elbow lymph nodes. The ani-
mals received a booster injection subcutaneously on day 47 (150 mg
in 1 mL of PBS emulsified with an equal volume of Freund’s in-
complete adjuvant) into 6 sites of the back skin and were bled on
day 56.
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ELISA Using Neoglycoconjugate and LPS Antigens
i) Neoglycoconjugates in carbonate buffer (50 mM, pH 9.2) were
coated onto MaxiSorp microtiter plates (96-well, U-bottom,
NUNC) at 4 C overnight. Antigen solutions were adjusted to
equimolar concentrations based on the amount of ligand present in
the respective glycoconjugate. If not stated otherwise, a volume of
50 mL was used. Plates were washed twice in PBS supplemented
with Tween 20 (0.05%, Bio-Rad) and thimerosal (0.01%, PBS-T)
and were then blocked with PBS-T supplemented with casein
(2.5%, PBS-TC) for 1 h at 37 °C on a rocker platform followed by
two washings. Appropriate antibody dilutions in PBS-TC supple-
mented with 5% BSA (PBS-TCB) were added and incubated for 1
h at 37 °C. After two washings, peroxidase-conjugated goat anti-
mouse IgG or goat-antirabbit IgG (both heavy and light chain spe-
cific; Dianova; diluted 1:1.000 in PBS-TCB) was added and incu-
bation was continued for 1 h at 37 °C. The plates were washed three
times with BPS-T. Substrate solution was freshly prepared and was
composed of 2,2¢-azinobis(3-ethylbenzthiazolinsulfonic acid) di-
ammonium salt (1 mg) dissolved in substrate buffer (0.1 M sodium
citrate, pH 4.5; 1 mL) followed by the addition of H2O2 (25 mL of a
0.1% solution). After 30 min at 37 °C, the reaction was stopped by
the addition of aq oxalic acid (2%) and the plates were read by a mi-
croplate reader (Tecan Sunrise) at 405 nm. Tests were run twice in
quadruplicates with confidence values not exceeding 10%.
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ii) When LPS was used as an antigen in ELISA, microtiter polyvi-
nyl plates (96-well, Falcon 3911; Becton Dickinson) were coated
with LPS of varying concentration (2 to 250 ng/well) diluted in PBS
(pH 7.2) and were incubated overnight at 4 °C. PBS and PBS-con-
taining solutions were supplemented with thimerosal (0.01%). Fur-
ther incubation steps were performed at 37 °C under gentle
agitation. The coated plates were washed four times with PBS and
were blocked for 1 h with PBS supplemented with casein (2.5%,
Sigma; PBS-C; 200 mL per well). Serial serum dilutions in PBS-C
were subsequently added, and the mixture was incubated for 1 h at
37 °C. After washing as described above, secondary antibodies di-
luted in PBS-C (same source and dilution as above) were added. Af-
ter four washings in PBS, the following steps were done as
described for ELISA using neoglycoconjugate antigens.
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Synthesis 2010, No. 18, 3143–3151 © Thieme Stuttgart · New York