Synthesis of neoglycoconjugates containing 4-amino-4-deoxy- l -arabinose epitopes corresponding to the inner core of burkholderia and proteus lipopolysaccharides

Article abstract of DOI:10.1002/ejoc.201101171

Disaccharides that contain 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and D-glycero-D-talo-oct-2-ulosonic acid (Ko) substituted at the 8-position by 4-amino-4-deoxy-β-L-arabinopyranosyl (Ara4N) residues have been prepared. Coupling an N-phenyltrifluoroacetimidate-4-azido-4-deoxy-L-arabinosylglycosyl donor to acetyl-protected allyl glycosides of Kdo and Ko afforded anomeric mixtures of disaccharide products in 74 and 90 % yield, respectively, which were separated by chromatography. Further extension of an intermediate Ara4N-(1→8)-Kdo disaccharide acceptor, which capitalized on a regioselective glycosylation with a Kdo bromide donor under Helferich conditions, afforded the branched trisaccharide α-Kdo-(2→4)[β-L- Ara4N-(1→8)]-α-Kdo derivative. Deprotection of the protected di- and trisaccharide allyl glycosides was accomplished by TiCl₄-promoted benzyl ether cleavage followed by the removal of ester groups and reduction of the azido group with thiol or Staudinger reagents, respectively. The reaction of the anomeric allyl group with 1,3-propanedithiol under radical conditions afforded the thioether-bridged spacer glycosides, which were efficiently coupled to maleimide-activated bovine serum albumin. The neoglycoconjugates serve as immunoreagents with specificity for inner core epitopes of Burkholderia and Proteus lipopolysaccharides.

Full text of DOI:10.1002/ejoc.201101171

FULL PAPER
DOI: 10.1002/ejoc.201101171
Synthesis of Neoglycoconjugates Containing 4-Amino-4-deoxy- -arabinose
L
Epitopes Corresponding to the Inner Core of Burkholderia and Proteus
Lipopolysaccharides
Markus Blaukopf,[a] Bernhard Müller,[a] Andreas Hofinger,[a] and Paul Kosma*[a]
Keywords: Biomimetic synthesis / Glycoconjugates / Glycosylation / Glycolipids / Lipopolysaccharide / Carbohydrates
Disaccharides that contain 3-deoxy-
acid (Kdo) and -glycero- -talo-oct-2-ulosonic acid (Ko) sub-
stituted at the 8-position by 4-amino-4-deoxy-β- -arabinopy-
ranosyl (Ara4N) residues have been prepared. Coupling an
N-phenyltrifluoroacetimidate-4-azido-4-deoxy- -arabinosyl-
glycosyl donor to acetyl-protected allyl glycosides of Kdo and
Ko afforded anomeric mixtures of disaccharide products in
74 and 90% yield, respectively, which were separated by
chromatography. Further extension of an intermediate
Ara4N-(1Ǟ8)-Kdo disaccharide acceptor, which capitalized
on a regioselective glycosylation with a Kdo bromide donor
under Helferich conditions, afforded the branched trisaccha-
D
-manno-oct-2-ulosonic
ride α-Kdo-(2Ǟ4)[β-L-Ara4N-(1Ǟ8)]-α-Kdo derivative. De-
D
D
protection of the protected di- and trisaccharide allyl glyco-
sides was accomplished by TiCl4-promoted benzyl ether
cleavage followed by the removal of ester groups and re-
duction of the azido group with thiol or Staudinger reagents,
respectively. The reaction of the anomeric allyl group with
1,3-propanedithiol under radical conditions afforded the
thioether-bridged spacer glycosides, which were efficiently
coupled to maleimide-activated bovine serum albumin. The
neoglycoconjugates serve as immunoreagents with specific-
ity for inner core epitopes of Burkholderia and Proteus lipo-
polysaccharides.
L
L
ble for severe and fatal infections in cystic fibrosis sufferers.
The incorporation of Ara4N in the lipid A and core regions
has been implicated in the development of antibiotic resis-
tance in bacteria by masking the anionic charges of the
phosphate and carboxylic acid groups, which counteracts
the effects of cationic antimicrobial peptides.[12] Previously,
antibodies raised against neoglycoconjugates containing the
Ko-(2Ǟ4)-Kdo disaccharide did not bind to the LPS of
Burkholderia, which could be because of steric hindrance
by the terminal Ara4N residue, which masks Ko and Kdo
epitopes.[13] In order to study the antigenic properties of
Ara4N-substituted inner core LPS units and develop poly-
and monoclonal antibodies for diagnostic applications, we
have set out to synthesize the central Ara4N-Kdo/Ko units
that correspond to part of the structures of the Burkholderia
and Proteus LPS. Herein we present results pertaining to
the development of new Ara4N glycosyl donors, assembly
of the di- and trisaccharide units, and conversion into the
corresponding neoglycoconjugates. An additional feature
relates to the orthogonal utilization of allyl and benzyl pro-
tecting groups, which considerably adds to the repertoire of
protecting group strategies in oligosaccharide syntheses.
Introduction
4-Amino-4-deoxy-l-arabinose (Ara4N) constitutes an
important sugar component within the outer membrane of
Gram-negative bacteria. Thus, Ara4N residues occur in
substoichiometric to stoichiometric amounts ester-linked to
the 1- and 4Ј-phosphate groups of the glucosamine disac-
charide backbone of lipid A, which anchors the lipopoly-
saccharide (LPS) chain to the outer membrane.[1–3] In ad-
dition, Ara4N residues have also been detected glycosidi-
cally-linked to the LPS core sugar 3-deoxy-d-manno-oct-2-
ulosonic acid (Kdo), which provides the linkage unit to the
lipid A domain. In particular, Ara4N has been found in a
β-(1Ǟ8) linkage to Kdo in the inner core region of many
Proteus strains and in a Providencia strain.[4–6] Bacteria of
the genus Proteus are important pathogens that cause noso-
comial wound infections and urinary tract infections. Simi-
larly, Ara4N has been detected linked to the 8-position of
the Kdo-isosteric sugar d-glycero-d-talo-oct-2-ulosonic acid
(Ko) in the core region of Burkholderia[7–10] and in a Serra-
tia marcescens strain.[11] Burkholderia species are responsi-
[a] Department of Chemistry,
University of Natural Resources and Life Sciences,
Muthgasse 18, 1190 Vienna, Austria
Results and Discussion
Fax: +43-1-47654-6055
E-mail: paul.kosma@boku.ac.at
Multigram amounts of methyl 4-azido-4-deoxy-α-l-arab-
inopyranoside (1) have been generated from commercial
methyl β-d-xylopyranoside.[14] Benzyl groups were selected
as nonparticipating protecting groups at the 2- and 3-posi-
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Eur. J. Org. Chem. 2012, 119–131
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M. Blaukopf, B. Müller, A. Hofinger, P. Kosma
FULL PAPER
tions, and 1 was converted into the 2,3-di-O-benzyl glyco- Gratifyingly, the reaction of 12 with tert-butyldimethylsilyl
side 2 by reaction with benzyl bromide/NaH in N,N-di- chloride (tBDMSCl) in dry acetonitrile in the presence of
methylformamide (DMF) in 93% yield (Scheme 1). In or- diazabicyclo[2.2.2]octane (DABCO) afforded the 8-O-silyl
der to generate hemiacetal 3, which was the precursor for derivative 13, which was acetylated with acetic anhydride/
the armed glycosyl donors 4–7, hydrolysis of 2 was ac- 4-(dimethylamino)pyridine (DMAP) in pyridine to furnish
complished by treatment with 2 m HCl in acetic acid at the 3,4,5,7-tetra-O-acetyl Ko glycosyl acceptor 14 in 62%
65 °C, and lactol 3 was isolated after flash chromatography overall yield. The structural assignment of 14 was based on
in 61% yield. As fluoride glycosyl donors have served as the low-field chemical shifts observed for protons H-3, H-
1
efficient donors in numerous glycoside syntheses,[15] includ- 4, H-5, and H-7 in the H NMR spectrum. Removal of the
ing coupling to 8-O-silylated Kdo derivatives,[16] donor 4 8-O-tBDMS groups from 10 and 14 was effected by treat-
was prepared by the reaction of 3 with diethylaminosulfur ment with 2% HF in acetonitrile to give the alcohols 11
trifluoride (DAST) in CH2Cl2 at –20 °C to afford an anom- and 15, respectively, in near quantitative yields. As the 7-O-
eric mixture of 4-azido-4-deoxy-l-arabinopyranosyl fluo- acetyl group is prone to migration to the 8-position upon
ride (4) in ca. 99% yield with preferential formation of the contact with silica gel, the crude products of 11 and 15 were
equatorial isomer (α/β ratio ca. 2:1). In addition, the tri- used for the subsequent glycosylation step.
chloroacetimidate donor 5 (α/β ratio ca. 3:1) was prepared
from the reaction of 3 with trichloroacetonitrile/K2CO3 at
room temperature in 78% yield.[17] Similarly, conversion of
3 into the N-phenyltrifluoroacetimidate derivative 6 (α/β ra-
tio ca. 7:1) was effected by treatment of 3 with N-phenyltri-
fluoroacetimidoyl chloride/K2CO3 in acetone in 84%
yield.[18] In order to exploit the stabilization of a putative
oxacarbenium ion intermediate in the glycosylation step by
Scheme 2. Reagents and conditions for the synthesis of Kdo and
Ko glycosyl acceptors. (a) 2% HF, MeCN, room temp., 2 h, ca.
the intramolecular participation of a 4-acylamino moiety,
the 4-deoxy-4-trifluoroacetamido derivative 7 was also pre-
pared.[19,20] The azido group of 4 was subjected to a Staud-
inger reaction with triphenylphosphane and trifluoroacetic
anhydride in CH2Cl2, which gave a moderate yield (47%)
of the corresponding 4-deoxy-4-trifluoroacetamidoarabino-
syl fluoride derivative 7 together with the byproducts 8 and
9 (35% combined yield), which were removed by column
chromatography. The anomeric mixtures of 5, 6, and 7 were
resolved by silica gel chromatography.
quant.; (b) tBDMSCl, DABCO, MeCN, room temp., 15 h, then (c)
Ac2O, DMAP, pyridine, room temp., 12 h, 62%.
The glycosylation conditions were elaborated with the
Kdo acceptor derivatives 10 and 11. Coupling reactions of
10 and 4 in the presence of 1.1 equiv. of BF3·Et2O as the
promoter in CH2Cl2 and acetonitrile, respectively, afforded
low yields of glycosides (Table 1, Entries 1 and 2). Em-
ploying a large excess of promoter provided good yields of
the glycosides with a very moderate stereoselectivity in
favor of the cis-configured β-l-Ara4N glycosides (Entries 3
and 4). The trimethylsilyl triflate (TMSOTf)-promoted cou-
pling reaction of 7 with 15 did not improve the stereochemi-
cal outcome of the reaction and gave a 55% yield of disac-
charide (Entry 5). Coupling of 5 with 11 also gave a modest
yield of products (Entry 6), whereas less reactive, armed 6
led to good yields of Ara4N-Kdo and Ara4N-Ko glycosides
albeit with no improvement in anomeric selectivity (Entry
7).[24] The disaccharide mixture generated from the reaction
of 11 with 6 was partially resolved by chromatography,
which allowed the separation of the equatorially-linked α-
Scheme 1. Reagents and conditions for the synthesis of Ara4N gly-
cosyl donors. (a) BnBr, NaH, DMF, room temp., 1 h, 93%; (b)
AcOH, 2 m HCl, 65 °C, 15 h, 61%; (c) DAST, CH2Cl2, –20 °C, 2 h,
99%; (d) Cl3CCN, K2CO3, CH2Cl2, room temp., 12 h, 78%;
(e) F3C(Cl)C=NPh, K2CO3, CH2Cl2, room temp., 12 h, 84%; (f)
(Ph)3P, (F3CCO)2O, CH2Cl2, room temp., 1 h, 47% for 7.
Table 1. Glycosylation conditions and product yields.[a]
Entry Donor Acceptor Promotor Solvent
Yield ax/eq
[%]
ratio[b]
For the straightforward assembly of the (1Ǟ8)-linked di-
saccharide units, the peracetylated 8-O-tert-butyldimethylsi-
lyl allyl glycoside derivatives of Kdo (10) and Ko (14) were
chosen as glycosyl acceptors that allow direct coupling with
fluoride donors and provide access to the respective alcohol
by reaction with fluoride ions. Although the Kdo derivative
11 had previously been used for the preparation of the
Chlamydia-related 2Ǟ8 interlinked Kdo disaccharide,[21] re-
gioselective silylation of the known[22,23] Ko allyl glycoside
1
2
3
4
5
6
7
8
4
4
4
4
7
5
6
6
10
10
10
14
15
11
11
15
BF3·Et2O CH2Cl2
BF3·Et2O MeCN
BF3·Et2O[c] CH2Cl2
BF3·Et2O[c] Et2O
17
21
71
80
55
38
74
90
2:1
1:2
1.4:1
1.5:1
1.2:1
1.2:1
1.6:1
1.6:1
TMSOTf CH2Cl2
TMSOTf CH2Cl2
TMSOTf CH2Cl2
TMSOTf CH2Cl2
[d]
[a] Reactions were performed at –25 °C. [b] From integration of the
anomeric 1H NMR signals. [c] 10 equiv. of promoter. [d] Performed
methyl ester derivative 12 had to be established (Scheme 2). at –78 °C.
120
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Synthesis of Neoglycoconjugates
l-glycoside 18 (Scheme 3). The assignment of the anomeric order to prevent formation of a 4Ј-acetamido product.
configuration of the Ara4N residue in 18 was based on the Thus, 21 was subjected to Zemplén deacetylation followed
value of the coupling constant J1Ј,2Ј of ca. 6.2 Hz, which by hydrolysis of the Kdo methyl ester group to furnish the
indicated a 1,2-trans-glycosidic linkage. The attachment site 4Ј-azido-4Ј-deoxy-disaccharide derivative 22 in 95% yield,
was identified by the low-field-shifted C-8 Kdo signal at which was purified by chromatography on BioGel P2. The
67.35 ppm in the 13C NMR spectrum.
reduction of the azido group of 22 was accomplished by
treatment with dithiothreitol (DTT)[26] in aqueous pyridine/
diisopropylamine, which left the allyl group intact and pro-
vided the 4Ј-amino-4Ј-deoxy-l-arabinopyranosyl derivative
23. Purification was performed by ion-exchange chromatog-
raphy on Dowex WX50 (H+ form) eluted with 1 m aqueous
NH3 followed by desalting on BioGel P2 to give the β-l-
Ara4N-(1Ǟ8)-Kdo allyl glycoside as the ammonium salt 23
in 60% yield.
In order to produce neoglycoconjugates with covalently-
linked Ara4N-K(d)o ligands, suitable spacer groups were
introduced by using the remaining allylic function. The con-
jugation chemistry has to be compatible with the presence
of both amino and carboxylic acid groups in order to retain
the antigenic properties of these LPS inner core sugars.
Hence, frequently-employed chain-elongation methods by
addition of cysteamine or ω-mercaptoalkanoic acid deriva-
tives to the allyl aglycon were not feasible.[27,28] Conjugation
Scheme 3. Reagents and conditions for the synthesis of Ara4N-
(1Ǟ8)-Kdo disaccharide derivatives. (a) TMSOTf (0.05 equiv.), conditions also have to take into account the acid-labile gly-
CH2Cl2, –25 °C, 1 h, 74%; (b) 0.1 m NaOMe, MeOH, room temp.,
4 h, 30% for 20, 69% for 17; (c) Ac2O, DMAP, pyridine, room
conjugation chemistry was envisaged to provide chemose-
temp., 24 h, 80%; (d) TiCl4, CHCl3, 0 °C, 12 h, then Ac2O, DMAP,
cosidic linkages of the Kdo residues. Hence, thiol-based
lective ligation to the protein matrix.[29] The introduction of
pyridine, room temp., 24 h, 68%; (e) 0.1 m NaOMe, MeOH, then
the thiol-based spacer moiety was effected by UV-mediated
addition of 1,3-propanedithiol to the allylic aglycon to give
the thioether-bridged thiol 24 in 63% yield.[14] The resulting
material contained a small fraction of the corresponding
dimer and was immediately used for the preparation of the
0.2 m NaOH, H2O, room temp., 3 h, 98%; (f) dithiothreitol, aq.
diisopropylamine, room temp., 2 h; then Dowex H+, 1 m aq. NH3,
60%; (g) HS(CH2)3SH, H2O, hν, room temp., 1 h, BioGel P-2,
63%.
Subsequently, the 7-substituted byproduct 19 Ϫ arising glycoconjugate.
from acetyl migration to the 8-position in 11 Ϫ was re-
The corresponding Ara4N-Ko glycoside was prepared in
moved by Zemplén deacetylation of the mixture of 16 and a similar way using the conditions established for the
19 to give 17 and 20 in 69 and 30% yield, respectively. Ara4N-Kdo derivatives (Scheme 4). Thus, glycosylation of
Based on the amount of 11, the overall yield of desilylation, 15 with 6 proved to be highly efficient and gave a 90% yield
glycosylation, and isolation of 17 was 36%. The glycosyla- of disaccharide products (Table 1, Entry 8). Again, a minor
tion sites were assigned from the chemical shifts of C-8 in amount of the α-linked product 27 was formed, which was
the 13C NMR spectrum of 17 (70.64 ppm) and C-7 in that separated from the mixture of 25 and 28 by chromatog-
of 20 (81.87 ppm). In addition, a diagnostic HMBC corre- raphy. Final purification of the target disaccharide 25 was
lation from the anomeric proton of the Ara4N residue to achieved by deacetylation with NaOMe, which produced a
C-7 of the Kdo unit was observed in 20. The cis configura- separable mixture of the triol derivatives 26 (68%) and 29
tion of the arabinopyranosyl units was corroborated by the (30%). The overall yield for the preparation of 26 from 15
small value of the coupling constant J1Ј,2Ј of ca. 3.6 Hz. The was 38%. Subsequent reacetylation of the β-(1Ǟ8)-linked
triol derivative 17 was reacetylated to afford pure 16 in 80% disaccharide 26 afforded 25 in 94% yield. Similar to the
yield. Deprotection of 16 was accomplished by a Lewis Ara4N-Kdo intermediate 16, the benzyl groups of 25 were
acid-promoted cleavage of the 2Ј-O- and 3Ј-O-benzyl cleaved by the action of TiCl4 followed by acetylation to
groups in the presence of TiCl4.[25] This transformation was furnish the hexa-O-acetyl disaccharide 30 in 89% yield.
accompanied by minor hydrolysis of the glycosidic linkages. Zemplén deacetylation and hydrolysis of the Ko methyl es-
The intermediate 2Ј,3Ј-diol was subsequently acetylated to ter group gave 31 in 97% yield. The 4Ј-azido group of 31
afford the penta-O-acetyl derivative 21 in 68% overall yield. was reduced with dithiothreitol to produce the 4Ј-amino
1
The H NMR spectroscopic data for 21 compare favorably glycoside 32 in 69% yield, which was transformed into the
with those reported for a related 4Ј-acetamido-4Ј-deoxy de- thio-spacer derivative 33 in 80% yield.
rivative, which was prepared by chemical transformation of
Proceeding towards the branched inner core unit related
a disaccharide isolated from the Proteus mirabilis LPS.[4a] to the Proteus mirabilis LPS, the Ara-(1Ǟ8)-Kdo interme-
Prior to reduction of the 4-azido group of 21, the acetyl diate 17 was subjected to a regioselective glycosylation with
groups were removed by transesterification in methanol in the known Kdo bromide donor 34.[30] The outcome of the
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121

M. Blaukopf, B. Müller, A. Hofinger, P. Kosma
FULL PAPER
Scheme 4. Reagents and conditions for the synthesis of Ara4N-
(1Ǟ8)-Ko disaccharide derivatives. (a) see Table 1; (b) 0.1 m Na-
OMe, MeOH, room temp., 4 h 68% for 26, 30% for 29; (c) Ac2O,
DMAP, pyridine, room temp., 24 h, 94%; (d) TiCl4, CHCl3, 0 °C,
6 h, then Ac2O, DMAP, pyridine, room temp., 24 h, 89%; (e) 0.1 m
NaOMe, MeOH, room temp., 4 h, then 0.2 m NaOH, 3 h, 97%; (f)
dithiothreitol, iPr2N/H2O, room temp., 12 h, then DOWEX (H+),
1 m aq. NH3, 69%; (g) HS(CH2)3SH, H2O, hν, room temp., 1 h,
BioGel P-2, 80%.
Scheme 5. Reagents and conditions for the synthesis of Ara4N-
(1Ǟ8)-[Kdo-(2Ǟ4)]Kdo trisaccharide derivatives. (a) Hg(CN)2/
HgBr2, MeNO2, room temp., 14 h, 19% for 35, 5.5% for 38; (b)
Ac2O, DMAP, pyridine, room temp., 12 h, 75%; c) TiCl4, CHCl3,
room temp., 13 h, then Ac2O, DMAP, pyridine, room temp., 24 h,
68%; d) 0.1 m NaOMe, MeOH, room temp., 4 h, then 0.1 m aq.
NaOH, room temp., 3 h 96%; (e) Me3P, THF/0.1 m NaOH, room
Helferich glycosylation procedure was dependant on the
solvent system. With CH2Cl2, the reaction gave a low yield
and prevailing formation of the (1Ǟ7)-linked product,
whereas nitromethane afforded a better yield, regio-, and
anomeric selectivity. The excess amount of 34 was limited temp., 4 h, 99%.
to avoid formation of the (2Ǟ7)-linked trisaccharide 38 and
NMR spectroscopic data for the allyl glycosides 23, 32,
tetrasaccharide byproducts. Separation of 38 and the glycal
ester byproduct 37 was achieved by column chromatog-
raphy followed by HPLC (Scheme 5). Acetylation of 35 af-
forded the hexa-O-acetyl derivative 36 in 75% yield. A
small amount of the β-(2Ǟ4)-linked trisaccharide isomer
and 41 are in full agreement with data measured for LPS–
core oligosaccharides.[8]
Conjugation
1
was visible in the H NMR spectrum of 36, which was re-
Chain elongation of trisaccharide 41 with 1,3-propanedi-
thiol produced the spacer derivative 42 in excellent yield
moved in the next step. Deprotection was performed by
TiCl4-promoted hydrolysis of the benzyl protecting groups
followed by acetylation to afford the branched octa-O-ace-
tyl trisaccharide derivative 39 in 68% yield. Remarkably,
both of the acid-labile Kdo units were stable during the
Lewis acid treatment, and only minor amounts of the hy-
drolysis products were observed. The ester groups were un-
blocked by transesterification with NaOMe followed by sa-
ponification with aqueous NaOH to give the branched tri-
saccharide 40, which was isolated as the disodium salt in
96% yield. The assignment of the structure of 40 was based
on the 13C NMR spectrum, which showed low-field-shifted
signals for C-4 (69.23 ppm) and C-8 (70.51 ppm) ac-
companied by high-field-shifted signals of the adjacent car-
bon atoms (C-7 68.52, C-3 33.94, C-5 64.92 ppm). Re-
duction of the azido group by treatment with dithiothreitol
finally produced the 4ЈЈ-amino derivative 41 albeit with the
formation of a minor byproduct, which was not fully re-
moved by chromatography. Hence, reduction of the azido
group was accomplished by treatment of 40 with trimeth-
ylphosphane to afford the 4ЈЈ-amino derivative 41 in 99%
yield.
Scheme 6. Reagents and conditions for the synthesis of neoglyco-
conjugates 43–45. (a) HS(CH2)3SH, H2O, hν, NH3, room temp.,
1 h 99%; (b) room temp., 2 h.
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Synthesis of Neoglycoconjugates
solution of 1[14] (2.01 g, 10.6 mmol) in dry DMF (40 mL) at 0 °C
and stirred for 10 min. Benzyl bromide (3.04 mL, 28.6 mmol) in
DMF (10 mL) was added over 5 min, and the solution was stirred
at room temp. for 1 h. The reaction was quenched by addition of
methanol (5 mL). The solution was diluted with CH2Cl2 (50 mL)
and washed with water and brine. The organic phase was dried
(MgSO4) and concentrated. The residue was dried in vacuo to give
crude 2 (3.65 g, 93%) as a yellowish syrup. [α]2D0 = +29.4 (c = 0.24,
CHCl3). 1H NMR (300 MHz, CDCl3): δ = 7.41–7.25 (m, 10 H,
Ph), 4.85–4.66 (m, 4 H, CH2Ph), 4.22 (d, J1,2 = 6.75 Hz, 1 H, 1-
H), 3.95 (dd, J5a,4 = 3.3, J5a,5b = 12.4 Hz, 1 H; 5a-H), 3.79–3.74
(m, 1 H, 4-H), 3.70–3.61 (m, 2 H, 2-H, 3-H), 3.51 (s, 3 H, OCH3),
3.43 (dd, J5b,4 = 1.8 Hz, 1 H, 5b-H) ppm. 13C NMR (CDCl3,
100 MHz): δ = 138.33, 137.71, 128.42, 128.31, 127.96, 127.85,
127.81 and 127.66 (C-Ar), 104.39 (C-1), 79.36 (C-3), 78.57 (C-2),
74.92 (CH2Ph), 72.82 (CH2Ph), 62.98 (C-5), 58.57 (C-4), 56.77
(OCH3) ppm.
(Scheme 6). Conjugation of the ligands to maleimide-acti-
vated bovine serum albumin (BSA) was achieved by incu-
bation with the thiol spacer-equipped derivatives 24, 33,
and 42 to furnish the neoglycoconjugates 43–45, which were
purified by chromatography on BioGel P2.[31] The high li-
gand-to-protein ratios were based on MALDI-TOF analy-
sis (5.4 mol/mol for 43, 16.5 mol/mol for 44, and 13.6 mol/
mol for 45). Immunochemical results with polyclonal hu-
man and murine sera will be published elsewhere.
Conclusions
The synthesis of neoglycoconjugates that contain inner
core LPS epitopes composed of 4-amino-4-deoxy-l-arabi-
nose residues glycosidically-linked to Kdo and Ko has been
accomplished by a straightforward strategy that used a ben-
zylated N-phenyl-trifluoroacetimidate Ara4N donor and
acetylated allyl glycoside acceptors. Selective cleavage of the
benzyl protecting groups in the presence of the allylic agly-
con with TiCl4 allows the subsequent deprotection of the
4-Azido-2,3-di-O-benzyl-4-deoxy-L-arabinopyranose (3): A solution
of 2 (29.15 g, 79 mmol) in acetic acid (290 mL) and HCl (73 mL,
2 m) was kept at 65 °C for 15 h. The brownish solution was diluted
with toluene (450 mL) and cooled to 0 °C. NaHCO3 (125 g) was
slowly added at 0 °C until CO2 formation had ceased. The organic
anomeric center or transformation of the remaining ter- layer was then washed with saturated aqueous NaHCO3 and NaCl
solutions, dried (MgSO4), and concentrated. The residue was dis-
solved in n-hexane/EtOAc, silica gel was added, and the solvent
was removed in vacuo. The remaining solid was subjected to flash
chromatography on silica gel (toluene/EtOAc, 3:1) to afford 3
(17.21 g, 61%) as a colorless solid. M.p. 62–63 °C. [α]2D0 = +49.6 (c
= 0.28, CHCl3). 1H NMR for β-anomer (400 MHz, CDCl3): δ =
7.38–7.27 (m, 10 H, Ph), 5.13 (d, J1,2 = 3.9 Hz, 1 H, 1-H), 4.77–
4.66 (m, 4 H, CH2Ph), 3.96 (dd, J5a,4 = 2.4, J5a,5b = 11.8 Hz, 1 H,
5a-H), 3.95 (dd, J3,4 = 3.7, J3,2 = 7.8 Hz, 1 H, 3-H), 3.82–3.78 (m,
1 H, 4-H), 3.73 (dd, 1 H, 2-H), 3.69 (dd, J5b,4 = 4.8 Hz, 1 H, 5b-
H) ppm. 13C NMR (CDCl3, 100 MHz): δ = 137.64–136.70 (m, Cq,
Ar), 128.89–128.68 (m, C-Ar), 92.00 (C-1), 76.11 (C-3), 76.02 (C-
2), 73.75 (CH2Ph), 73.04 (CH2Ph), 60.90 (C-5), 58.39 (C-4) ppm.
1H NMR for α-anomer (400 MHz, CDCl3): δ = 7.38–7.27 (m, 10
H, Ph), 4.81 (d, J1,2 = 3.9 Hz, 1 H, 1-H), 4.77–4.56 (m, 4 H,
CH2Ph), 4.02 (dd, J5a,4 = 7.9, J5a,5b = 11.8 Hz, 1 H, 5a-H), 3.84
(dd, J3,4 = 3.2, J3,2 = 5.8 Hz, 1 H, 3-H), 3.80–3.75 (m, 1 H, 4-H),
3.61 (dd, J5b,4 = 3.4 Hz, 1 H, 5b-H), 3.57 (dd, 1 H, 2-H) ppm. 13C
NMR (CDCl3, 100 MHz): δ = 137.64–136.70 (m, Cq, Ph), 128.89–
128.68 (m, CH, Ph), 94.40 (C-1), 77.50 (C-3), 75.79 (C-2), 73.87
(CH2Ph), 73.42 (CH2Ph), 59.04 (C-5), 56.05 (C-4) ppm.
C19H21N3O4 (355.40): calcd. C 64.21, H 5.96, N 11.82; found C
64.22, H 5.79, N 11.66.
minal alkene for spacer elongation by Michael addition of
the thiol moieties. The spacer ligands are useful probes for
the preparation of glyconanoparticles and glycoarrays, and
the neoglycoconjugates serve as specific immunoreagents.
Experimental Section
General: Maleimide-activated BSA was purchased from Sigma Ald-
rich. Known compounds were identified by comparison with re-
ported melting points as well as 1H and 13C NMR spectroscopic
data. Melting points were determined with a Kofler hot stage
microscope. Optical rotations were measured with a Perkin–Elmer
243 B Polarimeter. [α]2D0 values are given in units of 10–1degcm2 g–1.
1H NMR spectra were recorded at 297 K with a Bruker DPX in-
strument operating at 300, 400, or 600 MHz for 1H with CDCl3 as
the solvent and Me4Si as the standard, unless stated otherwise. 13
C
NMR spectra were measured at 75.47, 100.62, or 150.9 MHz and
referenced to 1,4-dioxane (67.40 ppm). Homo- and heteronuclear
2D NMR spectroscopy was performed with Bruker standard soft-
ware. MALDI-TOF-MS were recorded in the positive ion mode
with sinapic acid as matrix. HPLC–HRMS analysis was carried
out with H2O/MeCN solutions (concentration 1 mg/L) with an
HTC PAL system autosampler (CTC Analytics AG), an Agilent
1100/1200 HPLC with binary pumps, degasser, and column ther-
mostat (Agilent Technologies, Waldbronn, Germany), and Agilent
6210 ESI-TOF mass spectrometer (Agilent Technologies, Palo
Alto, U.S.). The mass spectrometer was previously tuned with Ag-
ilent tune mix and further reference masses were added to provide
a mass accuracy below 2 ppm. Data analysis was performed with
Mass Hunter software (Agilent Technologies). TLC was performed
with Merck precoated plates (5ϫ10 cm, layer thickness 0.25 mm,
silica gel 60F254); spots were detected by spraying with anisal-
dehyde/H2SO4. Silica gel (0.040–0.063 mm) was used for column
chromatography. Concentration of solutions was performed at re-
duced pressure at temperatures Ͻ 40 °C. Elemental analyses were
performed by Dr. J. Theiner, Mikroanalytisches Laboratorium, In-
stitut für Physikalische Chemie, Universität Wien.
4-Azido-2,3-di-O-benzyl-4-deoxy-L-arabinopyranosyl Fluoride (4):
Diethylaminosulfur trifluoride (111 μL, 0.84 mmol) was slowly
added to a solution of 3 (250 mg, 0.70 mmol) in dry CH2Cl2 (5 mL)
at –20 °C. After stirring for 2 h at –20 °C, MeOH (0.5 mL) was
added and stirring was continued for a further 10 min. The solution
was diluted with CH2Cl2 (50 mL), washed with saturated aqueous
NaHCO3, dried (MgSO4), and concentrated. The crude yellow oil
was purified by column chromatography on silica gel (hexane/
EtOAc, 10:1) to give an anomeric mixture of 4 (250 mg, 99%) as a
colorless liquid. 1H NMR (600 MHz, CDCl3): δ = 7.43–7.27 (m,
20 H, 4ϫ Ph), 5.55 (dd, J1β,F = ca. 53.1, J1β,2β = ca. 2.6 Hz, 1 H,
1β-H), 5.35 (dd, J1α,F = ca. 50.4, J1α,2α = 2.6 Hz, 1 H, 1α-H), 4.89–
4.56 (m, 8 H, 4ϫ OCH2), 4.23–4.18 (m, 1 H, 5aα-H), 4.06 (dd, J3,2
= 9.7, J3,4 = 3.7, 1 H, 3β-H), 3.96–3.92 (m, 2 H, 4β-H, 5aβ-H),
3.89 (ddd, J2,F = 25.0 Hz, 2β-H), 3.85–3.82 (m, 1 H, 3α-H), 3.97–
3.67 (m, 4 H, 2α-H, 4α-H, 5bα-H, 5bβ-H) ppm. 13C NMR
(150 MHz, CDCl3): δ = 137.78, 137.65, 137.30, 137.07 (4ϫ C-Ar),
Methyl 4-Azido-2,3-di-O-benzyl-4-deoxy-α-
L-arabinopyranoside (2):
Sodium hydride (60% in oil, 1.06 g, 26.5 mmol) was added to a
Eur. J. Org. Chem. 2012, 119–131
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
123

M. Blaukopf, B. Müller, A. Hofinger, P. Kosma
128.62–127.82 (12ϫ C-Ar), 106.56 (d, J1,F = 223.7 Hz, C-1β), 128.49, 128.07, 128.07, 128.06 and 127.91 (C-Ar), 124.28 (N-Ph
FULL PAPER
1
1
2
106.37 (d, J1,F = 224.8 Hz, C-1α), 76.55 (C-3β), 75.46 (d, J2β,F
=
=
=
Cp), 119.38 (N-Ph Co), 78.04 (C-3), 75.33 (C-2), 74.51 and 72.95
(2ϫ OCH2), 62.50 (C-5) and 59.91 (C-4) ppm.
2
23.9 Hz, C-2β), 75.34 (C-3α), 73.99 (OCH2), 73.92 (d, J2α,F
3
29.8 Hz, C-2α), 73.59, 73.14, 72.79 (3ϫ OCH2), 62.54 (d, J1,F
2,3-Di-O-benzyl-4-deoxy-4-trifluoroacetamido-α-
Fluoride (7a) and 2,3-Di-O-benzyl-4-deoxy-4-trifluoroacetamido-β-
-arabinopyranosyl Fluoride (7b): Trifluoroacetic anhydride
L-arabinopyranosyl
3.6 Hz, C-5β), 59.78 (d,3J1,F = 3.2 Hz, C-5α), 59.59 (C-4β), 55.03
(C-4α) ppm. HRMS (ESI-TOF): calcd. for C19H20FN3O3
[M + Na]+ 380.1381; found 380.1377.
L
(193 μL, 1.39 mmol) was added to a solution of 4 (250 mg,
0.69 mmol) in dry CH2Cl2 (10 mL). Triethylamine (193 μL,
1.39 mmol) and a solution of PPh3 (365 mg, 1.39 mmol) in dry tol-
uene (1 mL) were added slowly. The solution was stirred for 1 h at
room temp., and diluted with CH2Cl2 (50 mL). The solution was
washed with saturated aqueous NaHCO3 solution, dried (MgSO4),
and concentrated. The residue was purified by column chromatog-
raphy on silica gel (hexane/EtOAc, 9:1). Pooling of the fractions
containing the least polar compound gave 2,3-di-O-benzyl-4,5-di-
deoxy-l-threo-pent-4-enopyranosyl fluoride (8) as a colorless syrup
(28 mg, 9%). 1H NMR (400 MHz, CDCl3): δ = 7.38–7.26 (m, 10
H, Ph-H), 6.18 (ddd, J5,4 = 6.2, J5,3 = 1.7 Hz, 1 H, 5-H), 5.60 (d,
J1,F = 49.5, J1,2 = 1.9 Hz, 1 H, 1-H), 5.02 (dd, J3,4 = 2.5 Hz, 1 H,
4-H), 4.78 and 4.66 (2ϫ d, each 2 H, CH2Ph), 4.33 (ddd, 1 H, 3-
H) and 3.81 (ddd, J2,F = 24, J3,2 = 7.8 Hz, 1 H, 2-H) ppm. 13C
NMR (HSQC): δ = 139.05 (C-5), 130.36–124.91 (C-Ar), 104.22
(C-1), 102.56 (C-4), 76.01 (C-2), 73.14 (CH2Ph), 72.32 (C-3), 71.7
(CH2Ph) ppm.
4-Azido-2,3-di-O-benzyl-4-deoxy-α-
aceteimidate (5a) and 4-Azido-2,3-di-O-benzyl-4-deoxy-β-
pyranosyl Trichloroacetimidate (5b): Compound
L-arabinopyranosyl Trichloro-
L-arabino-
3
(200 mg,
0.56 mmol) was dissolved in dry CH2Cl2 (5 mL). K2CO3 (285 mg,
2.06 mmol) was added followed by trichloroacetonitrile (282 μL,
2.81 mmol) and the suspension was stirred for 12 h at room temp.
The suspension was filtered, the filtrate was concentrated, and the
residue was chromatographed over deactivated silica gel (contain-
ing 1% triethylamine). Elution of the column (hexane/EtOAc,
3:1+1% triethylamine) afforded 5b as the fastest moving isomer
1
(55 mg, 19%). [α]2D0 = +71 (c = 1.2 CHCl3). H NMR (300 MHz,
CDCl3): δ = 8.58 (br. s, 1 H, NH), 7.39–7.26 (m, 10 H, 2ϫ Ph),
6.41 (d, J1,2 = 2.9 Hz, 1 H, 1-H), 4.85–4.73 (m, 4 H, 2ϫ CH2Ph),
4.11 (dd, J2,3 = 9.5 Hz, 1 H, 2-H), 4.06 (dd, J3,4 = 2.8 Hz, 1 H, 3-
H), 3.97 (dd, J5a,5b = 12.9, J5a,4 = 1.8 Hz, 1 H, 5a-H), 3.96 (br. s,
1 H, 4-H), 3.77 (dd, J5b,4 = 2.5 Hz, 1 H, 5b-H) ppm. 13C NMR
(75 MHz, CDCl3): δ = 161.09 (C=NH), 138.05, 137.74, 128.45,
128.35, 127.87, 127.67, 127.47 (C-Ar), 95.09 (C-1), 76.13 (C-2),
75.51 (C-3), 73.22, 73.16 (2ϫ CH2Ph), 63.05 (C-5), 59.77 (C-4)
ppm.
Further elution of the column afforded 2,3-di-O-benzyl-4-deoxy-d-
glycero-pent-3-enopyranosyl fluoride (9) as
a colorless syrup
1
(77 mg, 26%). H NMR (400 MHz, CDCl3): δ = 7.39–7.26 (m, 10
H, Ph-H), 5.65 (d, 1 H, J1,F = 52.7, J1,2 = 3.3 Hz, 1-H), 4.92–4.73
(m, 5 H, 4-H, 2ϫ CH2Ph), 4.53 (dq, 1 H, J5a,5b = 14.8 Hz, 5a-H),
Further elution of the column gave 5a as a colorless amorphous
solid (166 mg, 59%). [α]2D0 = +12 (c = 1.6, CHCl3). 1H NMR
(300 MHz, CDCl3): δ = 8.63 (br. s, 1 H, NH), 7.38–7.28 (m, 10 H,
2ϫ Ph), 5.88 (d, J1,2 = 4.8 Hz, 1 H, H-1), 4.83–4.62 (m, 4 H, 2ϫ
CH2Ph), 4.14 (dd, J5a,5b = 11.7, J5a,4 = 6.0 Hz, 1 H, 5a-H), 3.93
(dd, J2,3 = 6.1 Hz, 1 H, 2-H), 3.83 (dd, J3,4 = 3.2 Hz, 1 H, 3-H),
3.81 (m, 1 H, 4-H), 3.67 (dd, J5b,4 = 3.2 Hz, 1 H, 5b-H). 13C NMR
(75 MHz, CDCl3): δ = 161.27 (C=NH), 137.48, 137.33, 128.59–
127.85 (C-Ar), 96.76 (C-1), 77.39 (C-3), 75.10 (C-2), 74.27, 72.77
(2ϫ CH2Ph), 62.08 (C-5), 56.76 (C-4) ppm.
4.22 (ddd, 1 H, J5b,4 = 3.4 Hz, 5b-H) and 4.16 (m, 1 H, J2,F
18.7 Hz, 2-H). 13C NMR (100 MHz, CDCl3): δ = 148.94 (C-3),
138.05, 136.53 and 128.58–127.39 (C-Ar), 105.13 (d, J1,F
=
=
226.3 Hz, C-1), 94.55 (C-4), 73.66 (OCH2), 70.73 (d, J2,F = 23.5 Hz,
C-2), 69.21 (CH2Ph) and 61.20 (d, J5,F = 5.0 Hz, C-5).
Continued elution furnished 7a (87 mg, 29%) as a colorless syrup.
[α]2D0 = –105.5 (c = 0.3, CHCl3). H NMR (400 MHz, CDCl3): δ =
1
7.42–7.19 (m, 10 H, Ph-H), 6.49 (d, JNH,4 = 8.3 Hz, 1 H, NH), 5.48
(d, J1,F = 49.5 Hz, 1 H, 1-H), 4.69–4.55 (m, 3 H, 1.5ϫ CH2Ph),
4-Azido-2,3-di-O-benzyl-4-deoxy-α-
L-arabinopyranosyl N-Phenyltri-
4.48 (m, 1 H, 4-H), 4.31 (d, 1 H, 0.5ϫ CH2Ph), 3.92 (dt, J5a,5b
=
fluoroacetimidate (6a) and 4-Azido-2,3-di-O-benzyl-4-deoxy-β-
L-ar-
10.6 Hz, 1 H, 5a-H), 3.76 (ddd, 1 H, 2-H), 3.71–3.66 (m, 2 H, 3-
H, 5b-H) ppm. 13C NMR (100 MHz, CDCl3): δ = 136.72, 136.56,
128.85, 128.73, 128.22, 128.03, 127.60, 127.43 (C-Ar), 105.05 (d,
J1,F = 225.3 Hz, C-1), 73.09 (CH2Ph), 72.58 (C-3), 71.83 (CH2Ph),
70.34 (d, J2,F = 32.3 Hz, C-2), 58.07 (d, J5,F = 3.0 Hz, C-5) and
44.04 (C-4) ppm. HRMS (ESI-TOF): calcd. for C21H21F4NO4 [M +
Na]+ calcd: 450.1299; found 450.1304.
abinopyranosyl N-Phenyltrifluoroacetimidate (6b): A suspension of
3 (1 g, 2.81 mmol), K2CO3 (780 mg, 5.63 mmol), and N-phenyl-
2,2,2-trifluoroacetimidoyl chloride (780 mg, 5.63 mmol; prepared
according to ref.[32]) in acetone (10 mL) was stirred for 12 h at room
temp. The suspension was filtered. The filtrate was concentrated
and the residue was chromatographed with silica gel. Elution with
hexane/EtOAc (20:1) afforded 6a (510 mg, 34%) as a colorless
1
syrup. [α]2D0 = +48 (c = 0.5, CHCl3). H NMR (400 MHz, CDCl3):
Finally, elution of the column gave 7b (55 mg, 18%) as a syrup.
[α]2D0 = +12.2 (c = 0.3, CHCl3). H NMR (400 MHz, CDCl3): δ =
1
δ = 7.40–7.22 (m, 12 H, Ph-H), 7.08 [t, J = 7.6 Hz, 1 H, N-Ph (H-
p)], 6.73 [d, 2 H, N-Ph (2ϫ o-H)], 6.40 (br. s, 1 H, 1-H), 4.86–4.67
(m, 4 H, 2ϫ CH2Ph), 4.10–3.97 (m, 2 H, 2-H, 3-H), 3.97–3.69 (m,
3 H, 4-H, 5a-H, 5b-H) ppm. 13C NMR (100 MHz, CDCl3): δ =
143.48 (N-Ph Cq), 137.87, 137.71, 128.70, 128.48, 128.42, 127.91,
127.81, 127.77, 127.52 (C-Ar), 124.25 (N-Ph C-p), 119.41 (N-Ph C-
o), 76.44 (C-2), 75.31 (C-3), 73.63 and 73.24 (CH2Ph), 62.95 (C-5)
and 59.62 (C-4) ppm.
7.40–7.27 (m, 10 H, Ph-H), 6.53 (br. s, 1 H, NH), 5.48 (dd, J1,F
=
52.4, J1,2 = 2.7 Hz, 1 H, 1-H), 4.85–4.59 (m, 4 H, 2ϫ CH2Ph), 4.47
(m, 1 H, 4-H), 4.04 (dd, J5a,4 = 2.3, J5a,5b = 12.9 Hz, 1 H, 5a-H),
4.03 (dd, J3,2 = 9.2 Hz, 1 H, 3-H), 3.92 (dd, J5b,4 = 2.4 Hz, 1 H,
5b-H) and 3.76 (ddd, J2,F = 23.5 Hz, 1 H, 2-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 137.52, 137.06, 128.59, 128.55, 128.18,
128.11, 128.08, 127.93 (C-Ar), 105.74 (d, J1,F = 228.3 Hz, C-1),
74.43 (C-3), 74.42 (J2,F = 23.6 Hz, C-2), 73.88 and 72.28 (2ϫ
CH2Ph), 61.58 (d, J5,F = 5.0 Hz, C-5), and 48.16 (C-4) ppm.
HRMS (ESI-TOF): calcd. for C21H21F4NO4 [M + Na]+ 450.1299;
found 450.1292.
Further elution gave 6b (752 mg, 50%) as a colorless syrup. [α]2D0
=
+89 (c = 1.1, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.35–7.25
(m, 12 H, Ph-H), 7.09 [t, J = 7.4 Hz, 1 H, N-Ph (H-p)], 6.79 [d, 2
H, N-Ph (2ϫ o-H)], 5.75 (br. s, 1 H, 1-H), 4.75–4.65 (m, 4 H, 2ϫ
CH2Ph), 4.05 (br. s, 1 H, 5a-H), 3.88 (br. s, 1 H, 2-H), 3.78 (br. s,
2 H, 3-H, 4-H), 3.54 (br. s, 1 H, 5b-H) ppm. 13C NMR (100 MHz,
Methyl (Allyl 3,4,5,7-tetra-O-acetyl-8-O-tert-butyldimethylsilyl-
D-
glycero-α- -talo-oct-2-ulopyranoside)onate (14): solution of
D
A
CDCl3): δ = 143.49 (N-Ph Cq), 137.39, 137.32, 128.70, 128.51, methyl (allyl 3,4,5,7,8-penta-O-acetyl-d-glycero-α-d-talo-oct-2-ulo-
124
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 119–131

Synthesis of Neoglycoconjugates
pyranoside)onate (250 mg, 0.48 mmol) in dry MeOH (5 mL) was
stirred with methanolic NaOMe (0.25 mL, 0.1 m) for 3 h at room
temp. The solution was deionized with Dowex 50 (H+) cation-ex-
change resin, filtered, and the filtrate was concentrated. The residue
was dried and dissolved in dry acetonitrile (5 mL). DABCO
(65 mg, 0.58 mmol) and (tert-butyl)chlorodimethylsilane (87 mg,
0.58 mmol) were added. The mixture was stirred at room temp. for
15 h. The suspension was concentrated and the residue was purified
by flash chromatography (EtOAc). The product was taken up in
dry pyridine (5 mL) and cooled to –10 °C. A catalytic amount of
DMAP was added followed by a mixture of pyridine/Ac2O (1:1,
4 mL). The solution was stirred for 12 h at room temp., cooled to
0 °C, and dry MeOH (3 mL) was added. Stirring was continued at
0 °C for a further 15 min. The solution was coevaporated several
times with toluene, and the residue was taken up in chloroform
(50 mL). The organic layer was washed with saturated aqueous
NaHCO3 solution and dried (MgSO4). Concentration left a syrup,
which was purified on a column of silica gel (toluene/EtOAc, 5:1
adding triethylamine (50 μL). The suspension was diluted with
CH2Cl2 (20 mL), filtered through a short plug of Celite®, and
washed with CH2Cl2. The filtrate was washed with saturated aque-
ous NaHCO3, dried (MgSO4), and concentrated. The crude mate-
rial (2 g) was directly applied to column chromatography (toluene/
EtOAc, 3:1), which gave a mixture of 16/19 (550 mg, 52%) and 18
(230 mg, 22%) as a colorless syrup. [α]2D0 = +45 (c = 0.5, CHCl3).
1H NMR (400 MHz, CDCl3): δ = 7.36–7.27 (m, 10 H, Ph-H), 5.75
(m, 1 H, –CH=), 5.33 (dd, J4,5 = 3.0, J6,5 = 1.4 Hz, 1 H, 5-H), 5.29
(ddd, J4,3e = 5.0, J4,3a = 12.0 Hz, 1 H, 4-H), 5.24–5.18 [m, 2 H, 7-
H, =CH2(trans)], 5.12 [dq, 1 H, =CH2(cis)], 4.74–4.63 (m, 4 H, 2ϫ
CH2Ph), 4.32 (d, J1Ј,2Ј = 6.2 Hz, 1 H, 1Ј-H), 4.10 (dd, J8a,7 = 2.4,
J8a,8b = 11.2 Hz, 1 H, 8a-H), 4.06 (dd, J6,7 = 9.6 Hz, 1 H, 6-H),
4.00 (m, 1 H, OCH2), 3.94 (dd, J5Јa,5Јb = 12.1, J4Ј,5Јa = 4.1 Hz, 1
H, 5Јa-H), 3.87 (m, 1 H, OCH2), 3.78 (s, 3 H, CO2CH3), 3.77–3.72
(m, 2 H, 8b-H, 4Ј-H), 3.68–3.62 (m, 2 H, 2Ј-H, 3Ј-H), 3.42 (dd,
J4Ј,5Јb = 2.2 Hz, 1 H, 5Јb-H), 2.18 (ddd, J3e,3a = 12.7 Hz, 1 H, 3e-
H), 2.09–2.04 (m, 4 H, 3a-H, COCH3), 1.97 (s, 3 H) and 1.90 (s, 3
+ 1% triethylamine) to afford 14 (179 mg, 62%) as a colorless H, 2ϫ COCH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 170.46–
1
syrup. [α]2D0 = +26 (c = 1.8, CHCl3). H NMR (300 MHz, CDCl3):
δ = 5.89–5.75 (m, 1 H, –CH=), 5.52 (dd, J3,4 = 3.7, J3,5 = 0.9 Hz,
1 H, 3-H), 5.43 (t, J4,5 = 3.7 Hz, 1 H, 4-H), 5.35 (ddd, J5,6 = 1.7 Hz,
169.79 (3ϫ OCCH3), 167.77 (C-1), 138.07, 137.61 (2ϫ C-Ar),
133.28 (–CH=), 128.44, 128.37, 127.90, 127.80, 127.78 and 127.71
(6ϫ C-Ar), 117.26 (=CH2), 102.85 (C-1Ј), 98.53 (C-2), 78.99 (C-
3Ј), 77.71 (C-2Ј), 74.63 and 72.71 (2ϫ OCH2Ph), 68.83 (C-7), 68.73
(C-6), 67.34 (C-8), 66.41 (C-4), 64.77 (OCH2), 64.49 (C-5), 62.38
(C-5Ј), 57.94 (C-4Ј), 52.62 (OCH3), 31.93 (C-3), 20.84–20.67 (3ϫ
OCCH3) ppm. HRMS (ESI-TOF): calcd. for C37H45N3O14 [M +
1 H, 5-H), 5.27 [dq, 1 H, =CH2(trans)], 5.21–5.15 (m, 2 H, =CH2cis
,
7-H), 4.32 (dd, J6,7 = 9.9 Hz, 1 H, 6-H), 4.23 (dq, 1 H, OCH2),
4.06 (dd, J8a,8b = 11.9, J8a,7 = 2.2 Hz, 1 H, 8a-H), 3.89–3.80 (m, 2
H, OCH2, 8b-H), 3.75 (s, 3 H, CO2CH3), 2.05 (s 3 H), 2.04 (s, 3
H), 1.99 (s, 3 H) and 1.96 (s, 3 H, 4ϫ COCH3), 0.87 [s, 9 H, HCOO–]– 800.2884; found 800.2890.
C(CH3)3], 0.04 (s, 3 H) and 0.01 [s, 3 H, Si(CH3)2] ppm. 13C NMR
Methyl
(1Ǟ8)-(allyl 3-deoxy-α-
Methyl
(1Ǟ7)-(allyl 3-deoxy-α-
4-Azido-2,3-di-O-benzyl-4-deoxy-β-
L
-arabinopyranosyl-
-manno-oct-2-ulopyranoside)onate (17) and
-arabinopyranosyl-
-manno-oct-2-ulopyranoside)onate (20): A
(75 MHz, CDCl3):
δ = 170.34, 169.79, 169.36, 168.95 (4ϫ
D
COCH3), 166.01 (C-1), 132.59 (–CH=), 117.54 (=CH2), 99.08 (C-
2), 70.15 (C-7), 67.66 (C-3), 67.02 (C-6), 66.16 (C-4), 65.08 (OCH2),
64.20 (C-5), 61.12 (C-8), 52.61 (CO2CH3), 25.84 [C(CH3)3], 20.80,
20.65, 20.52, 20.49 (4ϫ COCH3) and 18.43 [C(CH3)3] ppm. HRMS
(ESI-TOF): calcd. for C26H42O13Si [M + Na]+ 613.2287; found
613.2280.
4-Azido-2,3-di-O-benzyl-4-deoxy-β-L
D
solution of 16/19 (550 mg, 0.727 mmol) in dry MeOH (5 mL) was
stirred with NaOMe (0.5 mL, 0.1 m) for 4 h at room temp. The
solution was neutralized by addition of Dowex 50 (H+) cation-ex-
change resin and filtered. The filtrate was concentrated. Purifica-
tion of the residue by column chromatography (toluene/EtOAc,
1:2) first afforded 20 (138 mg, 30%). [α]2D0 = +107 (c = 0.9, CHCl3).
1H NMR (400 MHz, CDCl3): δ = 7.43–7.29 (m, 10 H, Ph-H), 5.85
(m, 1 H, –CH=), 5.25 [dq, 1 H, =CH2(trans)], 5.14 [dq, 1 H,
=CH2(cis)], 5.00 (d, J1Ј,2Ј = 3.6 Hz, 1 H, 1Ј-H), 4.98–4.65 (m, 4 H,
2ϫ CH2Ph), 4.03 (dd, J3Ј,2Ј = 9.8, J3Ј,4Ј = 3.5 Hz, 1 H, 3Ј-H), 4.00–
3.62 (m, 14 H, 2Ј-H, 4Ј-H, 4-H, 5-H, 5Јa-H, 5Јb-H, 7-H, 8a-H, 8b-
H, OCH2CO2CH3), 3.61 (dd, J6,5 = 1.3, J6,7 = 8.5 Hz, 1 H, 6-H),
2.14 (dd, J3e,3a = 12.8, J3e,4 = 4.9 Hz, 1 H, 3e-H) and 1.72 (dd, J3a,4
= 11.8 Hz, 1 H, 3a-H) ppm. 13C NMR (100 MHz, CDCl3): δ =
168.41 (C-1), 137.26 and 136.38 (C-Ar), 133.55 (–CH=), 128.71,
128.66, 128.50, 128.17, 128.01 and 127.80 (C-Ar), 116.90 (=CH2),
100.80 (C-1Ј), 98.89 (C-2), 81.87 (C-7), 77.18 (C-3Ј), 75.49 (C-2Ј),
75.22 and 72.32 (2ϫ CH2Ph), 71.07 (C-6), 65.30 (C-4), 64.72 (C-
5), 64.49 (OCH2), 63.74 (C-8), 61.53 (C-5Ј), 59.35 (C-4Ј), 52.51
(CO2CH3) and 35.26 (C-3) ppm.
Desilylation of 10: A solution of 10 (736 mg, 1.38 mmol) was
treated with a solution of 2% HF in MeCN (0.6 mL) in dry aceto-
nitrile (5 mL) at room temp. for 2 h. Solid NaHCO3 was added to
the solution and stirring was continued for 20 min. The suspension
was filtered, and the filtrate was concentrated, coevaporated with
toluene three times, and thoroughly dried to give 11 (578 mg, 99%)
as a syrup. The material was immediately used for the subsequent
glycosylation step.
Desilylation of 14: A solution of 14 (550 mg, 0.93 mmol) was
treated with a solution of 2% HF in MeCN (0.6 mL) in dry aceto-
nitrile (10 mL) at room temp. for 2 h. Solid NaHCO3 was added
to the solution and stirring was continued for 20 min. The suspen-
sion was filtered, and the filtrate was concentrated, coevaporated
with toluene three times, and thoroughly dried to give 15 (443 mg,
99%) as a syrup. The material was immediately used for the subse-
quent glycosylation step.
Further elution of the column with EtOAc/EtOH (2:1) gave 17
Methyl
(1Ǟ8)-(allyl 4,5,7-tri-O-acetyl-3-deoxy-α-
side)onate (16) and Methyl 4-Azido-2,3-di-O-benzyl-4-deoxy-α-
arabinopyranosyl-(1Ǟ8)-(allyl 4,5,7-tri-O-acetyl-3-deoxy-α-
4-Azido-2,3-di-O-benzyl-4-deoxy-β-
L
-arabinopyranosyl- (316 mg, 69%) as a colorless syrup. [α]2D0 = +78.6 (c = 0.36, CHCl3).
D-manno-oct-2-ulopyrano-
1H NMR (400 MHz, CDCl3): δ = 7.34–7.22 (m, 10 H, Ph-H), 5.73
(m, 1 H, –CH=), 5.16 [dq, 1 H, =CH2(trans)], 5.03 [dq, 1 H,
=CH2(cis)], 4.79 (d, J1Ј,2Ј = 3.6 Hz, 1 H, 1Ј-H), 4.82–4.58 (m, 4 H,
CH2Ph), 3.99–3.78 (m, 9 H, 2Ј-H, 3Ј-H, 4Ј-H, 4-H, 5-H, 7-H, 8a-
H, OCH2), 3.71 (dd, J5Јa,4Ј = 1.7, J5Јa,5Јb = 12.5 Hz, 1 H, 5Јa-H),
3.69 (s, 3 H, CO2CH3), 3.69 (dd, J8a,7 = 3.5, J8a,8b = 11.5 Hz, 1 H,
8b-H), 3.53 (dd, J5Јb,4Ј = 2.2 Hz, 1 H, 5Јb-H), 3.48 (dd, J6,5 = 0.9,
J6,7 = 7.8 Hz, 1 H, 6-H), 2.11 (dd, J3e,3a = 12.8, J3e,4 = 5.3 Hz, 1
H, 3e-H), 1.80 (dd, J3a,4 = 11.3 Hz, 1 H, 3a-H) ppm. 13C NMR
(100 MHz, CDCl3): δ = 168.53 (C-1), 137.59 and 137.52 (C-Ar),
L-
D
-
manno-oct-2-ulopyranoside)onate (18): A solution of 11 (578 mg,
obtained by desilylation of 10) in dry CH2Cl2 (5 mL) was added to
a suspension of 6 (1.45 g, 2.76 mmol) in dry CH2Cl2 (20 mL) with
4 Å molecular sieves. The suspension was stirred for 2 h at –15 °C
under Ar. A solution of TMSOTf (12.5 μL, 69 μmol) in dry CH2Cl2
(2 mL) was slowly added and stirring was continued for 1 h with
gradual warming to room temp. The reaction was quenched by
Eur. J. Org. Chem. 2012, 119–131
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
125

M. Blaukopf, B. Müller, A. Hofinger, P. Kosma
FULL PAPER
133.67 (–CH=), 128.53, 128.28, 128.17, 128.00 and 127.73 (C-Ar),
116.77 (=CH2), 101.06 (C-1Ј), 98.82 (C-2), 77.15 (C-3Ј), 75.97 (C-
2Ј), 74.46 and 72.51 (2ϫ CH2Ph), 71.74 (C-6), 70.64 (C-8), 69.04
(C-7), 66.57 (C-5), 65.63 (C-4), 64.27 (OCH2), 60.86 (C-5Ј), 59.64
(C-4Ј), 52.57 (CO2CH3) and 34.98 (C-3) ppm. HRMS (ESI-TOF):
5Јb-H), 2.22 (ddd, J3e,3a = 12.5, J3e,4 = 4.9 Hz, 1 H, 3e-H), 2.09 (t,
J3a,4 = 11.8 Hz, 1 H, 3a-H), 2.13 (s, 3 H), 2.12 (s, 3 H), 2.07 (s, 3
H), 2.00 (s, 3 H) and 1.97 (s, 3 H, 5ϫ CH3CO) ppm. 13C NMR
(150 MHz, CDCl3): δ = 170.47, 170.13, 170.09, 169.94, and 169.56
(5ϫ OCCH3), 167.71 (C-1), 133.25 (–CH=), 117.22 (=CH2), 98.58
calcd. for C31H39N3O11 [M + HCOO–]– 674.2567; found 674.2570. (C-2), 97.29 (C-1Ј), 69.13 (C-2Ј), 69.09 (C-3Ј), 68.17 (C-6), 68.10
(C-7), 66.59 (C-4), 65.82 (C-8), 64.79 (OCH2), 64.38 (C-5), 60.05
(C-5Ј), 59.61 (C-4Ј), 52.7 (CO2CH3), 31.95 (C-3), 20.73, 20.71,
Acetylation of 17: A solution of 17 (230 mg, 0.35 mmol) in dry
pyridine (3 mL) was stirred with acetic anhydride (2 mL) in pyr-
idine (1 mL) with a catalytic amount of DMAP for 24 h at room
temp. The solution was cooled to 0 °C, methanol (3 mL) was
20.66, 20.55, 20.53 (5ϫ OCCH3) ppm. HRMS (ESI-TOF): calcd.
for C27H37N3O16 [M + Na]+ 682.2066; found 682.2075.
added, and stirring was continued for 30 min. The solution was
coevaporated with toluene. The residue was directly applied to col-
Sodium 4-Azido-4-deoxy-β-
L-arabinopyranosyl-(1Ǟ8)-(allyl 3-de-
oxy-α- -manno-oct-2-ulopyranoside)onate (22): A solution of 21
D
umn chromatography (toluene/EtOAc, 5:1) to give 16 (241 mg, (33 mg, 0.045 mmol) in dry methanol (2 mL) was stirred with meth-
80%) as a colorless syrup. [α]2D0 = +83 (c = 0.52, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 7.47–7.27 (m, 10 H, Ph-H), 5.80 (m, 1 H,
–CH=), 5.36–5.34 (m, 1 H, 5-H), 5.32 (ddd, 1 H, 4-H), 5.25 [dq, 1
H, =CH2(trans)], 5.20 (m, 1 H, 7-H), 5.09 [dq, 1 H, =CH2(cis)],
anolic NaOMe (0.1 mL, 0.1 m) for 4 h at room temp. The solution
was neutralized with Dowex 50 (H+) cation-exchange resin. The
suspension was filtered, and the filtrate was concentrated. A solu-
tion of the residue in water (2 mL) was then adjusted to pH 12
4.89 (d, J1Ј,2Ј = 3.3 Hz, 1 H, 1Ј-H), 4.77–4.64 (m, 4 H, 2ϫ CH2Ph), with aqueous NaOH (ca. 2 mL, 0.2 m) and stirred at room temp.
4.21 (dd, J6,5 = 1.3 Hz, 1 H, 6-H), 4.15–4.09 (m, 1 H, OCH2), 3.91–
3.84 (m, 4 H, 8a-H, 8b-H, 3Ј-H, OCH2), 3.82 (dd, J2Ј,1Ј = 3.3, J2Ј,3Ј
for 3 h. The solution was neutralized with Dowex 50 (H+) cation-
exchange resin. The resin was removed by filtration and the filtrate
= 12.5 Hz, 1 H, 2Ј- H), 3.82 (m, 1 H, 4Ј-H), 3.79 (s, 3 H, CO2CH3), was lyophilized to give 22 (20 mg, 98%) as a colorless syrup. [α]2D0
3.72 (dd, J5Јa,5Јb = 12.3, J4Ј,5Јa = 1.9 Hz, 1 H, 5Јa-H), 3.57 (dd,
J4Ј,5Јb = 2.8 Hz, 1 H, 5Јb-H), 2.21 (ddd, J3e,3a = 12.5 Hz, 1 H, 3e- (m, 1 H, –CH=), 5.31–5.24 [dq, 1 H, =CH2(trans)], 5.14–5.08 [dq,
= +112 (c = 0.47, MeOH). 1H NMR (400 MHz, CD3OD): δ = 5.96
H), 2.05 (t, 1 H, 3a-H), 2.08 (s, 3 H), 1.99 (s, 3 H), and 1.92 (s, 3
1 H, =CH2(cis)], 4.83 (d, J1Ј,2Ј = 3.6 Hz, 1 H, 1Ј-H), 4.06 (ddd, J7,8a
H, 3ϫ COCH3) ppm. 13C NMR (100 MHz, CDCl3): δ = 170.50, = 5.2, J7,8b = 3.3, J7,6 = 7.9 Hz, 1 H, 7-H), 4.02 (dd, J3Ј,4Ј = 3.9,
169.81 and 169.61 (3ϫ OCCH3), 167.81 (C-1), 138.21, 137.94 (2ϫ J2Ј,3Ј = 9.7 Hz, 1 H, 3Ј-H), 3.99–3.94 (m, 4 H, 4-H, 5-H, OCH2),
C-Ar), 133.52 (–CH=), 128.5–127.5 (10ϫ C-Ar), 117.60 (=CH2),
98.51 (C-2), 98.45 (C-1Ј), 76.95 (C-3Ј), 75.96 (C-2Ј), 73.61 and
3.92 (dd, J5Јa,5Јb = 12.6 Hz, 1 H, 5Јa-H), 3.89 (dd, J8a,8b = 10.7 Hz,
1 H, 8a-H), 3.83 (dt, J4Ј,5Јa = 1.9 Hz, 1 H, 4Ј-H), 3.73 (dd, J2Ј,1Ј
=
73.13 (2ϫ OCH2Ph), 69.38 (C-5), 68.39 (C-6), 66.47 (C-4), 65.73 3.6, J2Ј,3Ј = 9.7 Hz, 1 H, 2Ј-H), 3.69 (dd, 1 H, 8b-H), 3.60 (dd, J6,5
(C-8), 65.01 (OCH2), 64.50 (C-7), 60.77 (C-5Ј), 59.80 (C-4Ј), 52.63 = 0.9 Hz, 1 H, 6-H), 3.59 (dd, 1 H, 5Јb-H), 2.09 (ddd, J3e,3a = 12.5,
(OCH3), 31.96 (C-3), 20.78, 20.78, 20.72 (3ϫ OCCH3) ppm. J3e,4 = 5.0 Hz, 1 H, 3e-H), 1.84 (dd, J3a,4 = 11.5 Hz, 1 H, 3a-H)
HRMS (ESI-TOF): calcd. for C37H45N3O14 [M + HCOO–]– ppm. 13C NMR (100 MHz, CD3OD): δ = 175.83 (C-1), 136.37 (–
800.2884; found 800.2890.
CH=), 116.25 (=CH2), 101.79 (C-2), 101.66 (C-1Ј), 73.55 (C-6),
71.59 (C-8), 70.73 (d.i. C-2Ј, C-3Ј), 69.69 (C-7), 68.67 (C-5), 67.85
(C-4), 65.15 (OCH2), 64.00 (C-4Ј), 61.87 (C-5Ј) and 36.24 (C-3)
ppm. HRMS (ESI-TOF): calcd. for C27H37N3O16 [M + Na]+
458.1381; found 458.1387.
Methyl
2,3-Di-O-acetyl-4-azido-4-deoxy-β-
L
-arabinopyranosyl-
(1Ǟ8)-(allyl 4,5,7-tri-O-acetyl-3-deoxy-α-
D-manno-oct-2-ulopyrano-
side)onate (21): A solution of 16 (56 mg, 0.074 mmol) in dry chloro-
form (5 mL) was stripped with argon. A solution of TiCl4 (17.2 μL,
0.156 mmol) in chloroform (1 mL) was added slowly at 0 °C. After
1 h, additional TiCl4 (17.2 μL) was added until the TLC showed
the complete consumption of starting material and the appearance
of a lower migrating compound (Rf 0.18, EtOAc/toluene, 1:1).
Ethyl ether (10 mL) and saturated aqueous NaHCO3 solution
(5 mL) were added with caution and the solution was stirred for
30 min. The organic layer was dried (MgSO4), and concentrated.
The residue was purified by flash chromatography over silica gel
(toluene/EtOAc, 1:1) to give the crude debenzylated product. A
solution of this residue in pyridine (2 mL) and a catalytic amount
of DMAP was cooled to 0 °C. A solution of acetic anhydride in
pyridine (2 mL, 2:1) was added slowly and the reaction was stirred
for 24 h at room temp. Dry MeOH (2 mL) was added at 0 °C and
stirring was continued for 30 min. The solution was coevaporated
with toluene five times and concentrated. The residue was purified
by column chromatography (toluene/EtOAc, 4:1) to furnish 21
(33 mg, 68%) as a colorless syrup. [α]2D0 = +72 (c = 0.3, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 5.93 (m, 1 H, –CH=), 5.36–5.30
Sodium 4-Amino-4-deoxy-β-L-arabinopyranosyl-(1Ǟ8)-(allyl 3-de-
oxy-α- -manno-oct-2-ulopyranoside)onate (23): Compound 22
D
(20 mg, 0.045 mmol) was dissolved in a solution of H2O/diisopro-
pylamine (2 mL, 3:1) and stripped with argon in a light-protected
flask. Dithiothreitol (29.8 mg, 0.183 mmol) was added and the
solution was stirred for 2 h at room temp. under argon. Another
portion of dithiothreitol (29.8 mg) was added to complete the con-
version. The solution was diluted with H2O (2 mL) and washed
with chloroform (2 mL) five times. The organic phases were reex-
tracted with water. The combined aqueous phases were stripped
with argon until the solution became clear and was lyophilized.
The residue was dissolved in H2O (1 mL) and rinsed through a
column of Dowex 50 (H+) cation-exchange resin. The column was
washed with water (20 mL) and the product was eluted with ammo-
nia (20 mL, 1 m). The appropriate fractions were pooled and lyo-
philized to give 23 (12 mg, 60%) as a white amorphous solid.
1
[α]2D0 = +83 (c = 0.29, H2O). H NMR (400 MHz, D2O, pD 7.5): δ
= 5.93 (m, 1 H, –CH=), 5.35–5.28 [dq, 1 H, =CH2(trans)], 5.23–
5.18 [dq, 1 H, =CH2(cis)], 4.99 (d, J1Ј,2Ј = 3.7 Hz, 1 H, 1Ј-H), 4.11–
4.03 (m, 3 H, 3Ј-H, 4-H, 7-H), 4.01 (br. s, 1 H, 5-H), 3.99 (dd,
[m, 3 H, 4-H, 5-H, =CH2(trans)], 5.23 (dd, J2Ј,1Ј = 3.7, J2Ј,3Ј
10.5 Hz, 1 H, 3Ј-H), 5.20 [dq, 1 H, =CH2(cis)], 5.15 (ddd, J7,8a
=
=
3.3, J7,8b = 2.2, J7,6 = 9.8 Hz, 1 H, 7-H), 5.15 (dd, J2Ј,1Ј = 3.7, J2Ј,3Ј J5Јa,5Јb = 11.5, J5Јa,4Ј = 2.0 Hz, 1 H, 5Јa-H), 3.94–3.78 (m, 4 H, 8a-
= 10.5 Hz, 1 H, 2Ј-H), 5.12 (d, J1Ј,2Ј = 3.7 Hz, 1 H, 1Ј-H), 4.19 (dd,
J6,5 = 1.3 Hz, 1 H, 6-H), 4.16–4.12 (ddt, 1 H, OCH2), 4.09 (dt,
J4Ј,5Јa = 1.8 Hz, 1 H, 4Ј-H), 4.00–3.96 (ddt, 1 H, OCH2), 3.95 (dd,
J8a,8b = 12.9 Hz, 1 H, 8a-H), 3.90 (dd, J5Јa,5Јb = 12.6 Hz, 1 H, 5Јa-
H), 3.81 (s, 3 H, CO2CH3), 3.80 (dd, 1 H, 8b-H), 3.66 (dd, 1 H,
H, 8b-H, OCH2), 3.74 (dd, J2Ј,1Ј = 3.7, J2Ј,3Ј = 9.9 Hz, 1 H, 2Ј-H),
3.68 (dd, 1 H, 5Јb-H), 3.61 (dd, J6,5 = 0.9, J6,7 = 9.3 Hz, 1 H, 6-
H), 3.49 (br. s, 1 H, 4Ј-H), 2.03 (dd, J3e,3a = 13.0, J3e,4 = 4.8 Hz, 1
H, 3e-H), 1.77 (t, J3a,4 = 13.0 Hz,1 H, 3a-H) ppm. 13C NMR
(100 MHz, D2O): δ = 176.00 (C-1), 134.60 (–CH=), 118.50 (=CH2),
126
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 119–131

Synthesis of Neoglycoconjugates
100.80 (C-2), 99.92 (C-1Ј), 72.33 (C-6), 70.84 (C-8), 68.90, 68.53
102.56 (C-1Ј), 99.14 (C-2), 78.89 (C-3Ј), 77.50 (C-2Ј), 74.60 and
(C-2Ј, C-3Ј), 67.23 (C-7), 67.03 (C-4), 66.79 (C-5), 65.19 (OCH2), 72.66 (CH2Ph), 68.71 (C-7), 68.15 (C-6), 67.54 (C-3), 66.91 (C-8),
60.07 (C-5Ј), 52.30 (C-4Ј) and 34.92 (C-3) ppm. HRMS (ESI-TOF):
66.00 (C-4), 65.09 (OCH2), 63.97 (C-5), 62.10 (C-5Ј), 57.72 (C-4Ј),
52.66 (CO2CH3), 20.74, 20.70, 20.55, and 20.51 (4ϫ OCCH3) ppm.
HRMS (ESI-TOF): calcd. for [M + HCOO–]– 858.2938; found
858.2946.
calcd. for C16H27NO11 [M + H]+ 410.1657; found 410.1659.
Ammonium
4-Amino-4-deoxy-β-L-arabinopyranosyl-(1Ǟ8)-[3-(3-
mercaptopropylthio)propyl
3-deoxy-α-
D-manno-oct-2-ulopyrano-
side]onate (24): Propane-1,3-dithiol (0.1 mL) was added to a solu-
tion of 23 (2.6 mg, 6.3 μmol) in MeOH (2 mL) and stripped with
argon in a quartz flask. The reaction mixture was stirred under UV
radiation (254 nm) for 1 h at room temp. to give full conversion.
The solution was diluted with H2O (2 mL) and washed with chloro-
form (2 mL) five times. The organic phases were reextracted with
water, and the combined aqueous phases were stripped with argon
until the solution became clear. The solution was lyophilized, and
the residue was purified on BioGel P-2 (5% aq. EtOH) to give 24
(2.1 mg, 63%) as a white solid. [α]2D0 = +76.5 (c = 0.2, H2O). 1H
NMR (400 MHz, D2O): δ = 5.04 (d, J1Ј,2Ј = 3.7 Hz, 1 H, 1Ј-H),
Methyl
(1Ǟ8)-(allyl
Methyl
4-Azido-2,3-di-O-benzyl-4-deoxy-β-
-glycero-α- -talo-oct-2-ulopyranoside)onate (26) and
4-Azido-2,3-di-O-benzyl-4-deoxy-β- -arabinopyranosyl-
-glycero-α- -talo-oct-2-ulopyranoside)onate (29):
L-arabinopyranosyl-
D
D
L
(1Ǟ7)-(allyl
D
D
A
solution of 25/28 (430 mg, 0.528 mmol) in dry MeOH (15 mL) was
stirred at room temp. for 4 h with NaOMe (1 mL, 0.1 m) at pH 10.
The solution was neutralized with Dowex 50 (H+) cation-exchange
resin and filtered. The filtrate was concentrated and purified by
column chromatography (toluene/EtOAc, 1:3) to give 29 (101 mg,
30%) as the fastest migrating compound. [α]2D0 = +78 (c = 1.3,
CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.41–7.27 (m, 10 H, Ph-
H), 5.82 (m, 1 H, –CH=), 5.24 [dq, 1 H, =CH2(trans)], 5.16 [dq, 1
H, =CH2(cis)], 4.93 (d, 1 H, CH2Ph), 4.90 (d, J1Ј,2Ј = 3.2 Hz, 1 H,
1Ј-H), 4.81–4.66 (m, 3 H, 1.5ϫ CH2Ph), 4.02–3.76 (m, 15 H, 3-H,
4-H, 5-H, 7-H, 8a-H, 8b-H, 2Ј-H, 3Ј-H, 4Ј-H, 5Јa-H, CO2CH3,
OCH2), 3.70 (dd, J5Јa,5Јb = 12.3, J5Јa,4Ј = 1.7 Hz, 1 H, 5Јb-H) and
3.64 (dd, J6,7 = 8.5, J6,5 = 1.4 Hz, 1 H, 6-H) ppm. 13C NMR
(100 MHz, CHCl3): δ = 167.44 (C-1), 137.16 and 136.47 (C-Ar),
132.87 (–CH=), 128.84–127.78 (C-Ar), 117.48 (=CH2), 101.08 (C-
2), 100.68 (C-1Ј), 81.61 (C-7), 76.83 (C-3Ј), 75.36 (C-2Ј), 75.32 and
72.20 (CH2Ph), 72.14 (C-3), 70.59 (C-6), 67.91 (C-5), 65.45 (C-4),
64.89 (OCH2), 63.44 (C-8), 61.67 (C-5Ј), 59.25 (C-4Ј) and 52.58
(CO2CH3) ppm.
4.19–4.05 (m, 5 H, 3Ј-H, 4-H, 5-H, 5Јa-H, 7-H), 3.93 (dd, J8a,8b
=
10.6, J8a,7 = 2.7 Hz, 1 H, 8a-H), 3.86 (dd, J8b,7 = 7.5 Hz, 1 H, 8b-
H), 3.80 (dd, J2Ј,3Ј = 9.8 Hz, 1 H, 2Ј- H), 3.72 (dd, J5Јb,5Јa = 12.9,
J5Јb,4Јb = 2.2 Hz, 1 H, 5Јb-H), 3.61 (dd, J6,7 = 9.2 Hz, 1 H, 6-H),
3.53–3.49 (m, 2 H, 4Ј-H, OCH2), 3.38–3.36 (m, 1 H, OCH2), 2.73–
2.64 (m, 6 H, 3ϫ CH2), 2.06 (dd, J3e,3a = 13.1, J3e,4 = 5.0 Hz, 1 H,
3e-H), 1.95–1.87 (m, 4 H, 2ϫ CH2), 1.80 (t, J4,3a = 13.1 Hz, 1 H,
3a-H) ppm. 13C NMR (100 MHz, D2O): δ = 176.28 (C-1), 101.05
(C-2), 99.83 (C-1Ј), 72.77 (C-6), 71.29 (C-8), 69.12 (C-2Ј), 68.73 (C-
3Ј), 67.87 (C-7), 67.23 (C-4), 67.70 (C-5), 62.56 (OCH2), 60.65
(C-5Ј), 52.38 (C-4Ј), 35.22 (C-3), 33.58 (OCH2CH2CH2S),
30.50 (OCH2CH2CH2S), 29.57 (SCH2CH2CH2SH), 28.92
(SCH2CH2CH2SH), and 23.62 (SCH2CH2CH2SH) ppm. HRMS
(ESI-TOF): calcd. for C19H35NO11S2 [M – H]– 516.1579; found
516.1576.
Further elution of the column with EtOAc/EtOH (2:1) gave 26 as
a colorless solid (230 mg, 68%). [α]2D0 = +71.6 (c = 0.4, CHCl3): 1H
NMR (600 MHz, CDCl3): δ = 7.39–7.27 (m, 10 H, Ph-H), 5.79 (m,
1 H, –CH=), 5.22 [dq, 1 H, =CH2(trans)], 5.11 [dq, 1 H,
=CH2(cis)], 4.99 (d, J1Ј,2Ј = 3.5 Hz, 1 H, 1Ј-H), 4.79–4.69 (m, 2 H,
2ϫ CH2Ph), 4.08 (dd, 1 H, 5-H), 4.07 (m, 1 H, 7-H), 4.02 (m, 1
H, OCH2), 3.97 (d, 1 H, 3-H), 3.96 (dd, J3Ј,2Ј = 10.0, J3Ј,4Ј = 3.8 Hz,
1 H, 3Ј-H), 3.88 (dt, J4Ј,5Јa = 1.8 Hz, 1 H, 4Ј-H), 3.86 (dd, 1 H, 2Ј-
H), 3.86 (m, 1 H, 8a-H), 3.82 (dd, J5Јa,5Јb = 12.4 Hz, 1 H, 5Јa-H),
3.79 (s, 3 H, CO2CH3), 3.76 (dd, J8b,8a = 11.4 Hz, 1 H, 8b-H), 3.76–
3.72 (m, 2 H, 4-H, OCH2), 3.59 (dd, 1 H, 5Јb-H) and 3.58 (dd, J6,7
= 8.2 Hz, 1 H, 6-H) ppm. 13C NMR (100 MHz, CHCl3): δ = 169.49
(C-1), 138.14 and 138.00 (C-Ar), 133.17 (–CH=), 128.64, 128.57,
128.14, 128.04 and 127.74 (C-Ar), 117.80 (=CH2), 101.92 (C-2),
99.03 (C-1Ј), 77.29 (C-3Ј), 76.24 (C-2Ј), 73.82 and 72.88 (CH2Ph),
71.87 (C-6), 70.32 (C-3), 68.98 (C-8), 68.33 (C-7), 67.25 (C-5), 66.18
(C-4), 65.45 (OCH2), 60.75 (C-5Ј), 59.96 (C-4Ј) and 53.31
(CO2CH3) ppm. HRMS (ESI-TOF): calcd. for [M + NH4]+
663.2872; found 663.2875.
Methyl
(1Ǟ8)-(allyl 3,4,5,7-tetra-O-acetyl-
pyranoside)onate (25) and Methyl 4-Azido-2,3-di-O-benzyl-4-deoxy-
α- -arabinopyranosyl-(1Ǟ8)-(allyl 3,4,5,7-tetra-O-acetyl-
glycero-α- -talo-oct-2-ulopyranoside)onate (27): A solution of 15
4-Azido-2,3-di-O-benzyl-4-deoxy-β-L-arabinopyranosyl-
D
-glycero-α-
D
-talo-oct-2-ulo-
L
D-
D
(443 mg, 0.93 mmol) was taken up in dry CH2Cl2 (5 mL) and
added to a suspension of 6 (970 mg, 1.86 mmol) in dry CH2Cl2
(5 mL) containing molecular sieves 4 Å. The suspension was stirred
under argon at –15 °C for 2 h. A solution of TMSOTf (8.4 μL,
0.04 mmol) dissolved in dry CH2Cl2 (5 mL) was slowly added. Af-
ter 1 h, triethylamine (50 μL) was added slowly. The suspension was
filtered through a short plug of Celite, and the Celite bed was
washed with CH2Cl2. The filtrate was washed with saturated aque-
ous NaHCO3 solution, dried (MgSO4), filtered, and concentrated.
The crude mixture was directly applied to column chromatography
(toluene/EtOAc, 3:1) to give a mixture of 25/28 (430 mg, 57%) and
27 (284 mg, 37%) as a colorless syrup. [α]2D0 = +24 (c = 0.8, CHCl3).
1H NMR (600 MHz, CDCl3): δ = 7.38–7.27 (m, 10 H, Ph-H), 5.69 Acetylation of 26: A solution of 26 (230 mg, 0.35 mmol) in dry
(m, 1 H, –CH=), 5.49 (dd, J3,4 = 3.2, J3,5 = 1.3 Hz, 1 H, 3-H),
5.32–5.27 (m, 3 H, 4-H, 5-H, 7-H), 5.19 [dq, 1 H, =CH2(trans)],
5.12 [dq, 1 H, =CH2(cis)], 4.73–4.63 (m, 4 H, 2ϫ CH2Ph), 4.38 (d,
J1Ј,2Ј = 5.5 Hz, 1 H, 1Ј-H), 4.15 (dd, J6,5 = 1.5, J6,7 = 9.7 Hz, 1 H,
6-H), 4.10 (dd, J8a,7 = 2.3, J8a,8b = 11.4 Hz, 1 H, 8a-H), 4.06 (m, 1
pyridine (3 mL) was stirred with acetic anhydride (2 mL) in pyr-
idine (1 mL) with a catalytic amount of DMAP for 24 h at room
temp. The solution was cooled to 0 °C, methanol (3 mL) was
added, and stirring was continued for 30 min. The solution was
coevaporated with toluene. The residue was directly applied to col-
H, OCH2), 3.96 (dd, J5Јa,4Ј = 4.5, J5Јa,5Јb = 12.4 Hz, 1 H, 5Јa-H), umn chromatography (toluene/EtOAc, 5:1) to give 25 (273 mg,
1
3.88 (dd, J8b,7 = 3.9 Hz, 1 H, 8b-H), 3.79–3.72 (m, 5 H, 4Ј-H,
CO2CH3, OCH2), 3.68 (dd, J3Ј,2Ј = 7.6, J3Ј,4Ј = 3.4 Hz, 1 H, 3Ј-H), (400 MHz, CDCl3): δ = 7.40–7.27 (m, 10 H, Ph-H), 5.76 (m, 1 H,
3.63 (d, 1 H, 2Ј-H), 3.43 (dd, J4Ј,5Јb = 2.4 Hz, 1 H, 5Јb-H), 2.05 (s, –CH=), 5.50 (dd, J3,4 = 3.6, J3,5 = 1.0 Hz, 1 H, 3-H), 5.33 (ddd,
94%) as a colorless syrup. [α]2D0 = +31 (c = 1.7, CHCl3). H NMR
3 H), 2.03 (s, 3 H), 1.93 (s, 3 H) and 1.92 (s, 3 H, 4ϫ CH3CO) J7,6 = 9.9, J7,8a = 3.3, J7,8b = 1.9 Hz, 1 H, 7-H), 5.30–5.26 (m, 2 H,
ppm. 13C NMR (100 MHz, CHCl3): δ = 170.29–168.97 (4ϫ
CH3CO), 165.86 (C-1), 137.97 and 137.61 (C-Ar), 132.47 (–CH=),
4-H, 5-H), 5.23 [dq, 1 H, =CH2(trans)], 5.10 [dq, 1 H, =CH2(cis)],
4.97 (d, J1Ј,2Ј = 2.9 Hz, 1 H, 1Ј-H), 4.78–4.63 (m, 4 H, 2ϫ CH2Ph),
128.48, 128.45, 127.95, 127.84 and 127.67 (C-Ar), 117.80 (=CH2), 4.31 (dd, J6,5 = 1.8 Hz, 6-H), 4.21 (dq, 1 H, OCH2), 3.98 (dd, J8a,8b
Eur. J. Org. Chem. 2012, 119–131
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
127

M. Blaukopf, B. Müller, A. Hofinger, P. Kosma
FULL PAPER
= 12.8 Hz, 1 H, 8a-H), 3.90 (dd, 1 H, 8b-H), 3.86–3.78 (m, 4 H,
1
(c = 0.4, MeOH). H NMR (600 MHz, D2O): δ = 5.84 (m, 1 H, –
2Ј-H, 3Ј-H, 4Ј-H, OCH2), 3.73 (dd, J5Јa,5Јb = 12.3 Hz, 1 H, 5Јa-H), CH=), 5.26–5.21 [dq, 1 H, =CH2(trans)], 5.15–5.12 [dq, 1 H,
3.73 (s, 3 H, CO2CH3), 3.58 (dd, 1 H, 5Јb-H), 2.04 (s, 3 H), 2.03 =CH2(cis)], 4.89 (d, J1Ј,2Ј = 3.7 Hz, 1 H, 1Ј-H), 4.09 (ddd, J7,6
(s, 3 H), 1.97 (s, 3 H) and 1.93 (s, 3 H, 4ϫ CH3CO) ppm. 13C
8.8 Hz, 1 H, 7-H), 4.01–3.97 (m, 2 H, 3Ј-H, 5-H), 3.92–3.88 (m, 4
=
NMR (100 MHz, CDCl3): δ = 170.31, 169.44, 169.20 and 168.93 H, 3-H, 4-H, 4Ј-H, OCH2), 3.85–3.82 (m, 2 H, 5Јa-H, 8a-H), 3.75–
(4ϫ OCCH3), 165.94 (C-1), 138.13, 137.86 (2ϫ Ph-Cq), 132.84 3.70 (m, 3 H, 2Ј-H, 8b-H, OCH2), 3.65 (dd, J5Јb,5Јa = 12.9 Hz, 1 H,
(–CH=), 128.53, 128.48, 127.98, 127.87, 127.79, 127.60 (C-Ar),
118.09 (=CH2), 99.04 (C-2), 98.33 (C-1Ј), 76.69 (C-2Ј), 75.99 (C-
3Ј), 73.71 and 73.07 (2ϫ CH2Ph), 69.57 (C-7), 67.80 (C-6), 67.52
5Јb-H) and 3.58 (dd, 1 H, 6-H) ppm. 13C NMR (150 MHz, D2O):
δ = 174.04 (C-1), 134.25 (–CH=), 118.29 (=CH2), 102.66 (C-2),
101.33 (C-1Ј), 72.41 (C-3), 72.14 (C-6), 70.58 (C-8), 69.54 (C-2Ј),
(C-3), 66.03 (C-4), 65.41 (OCH2), 64.97 (C-8), 63.87 (C-5), 60.75 69.40 (C3Ј), 68.85 (C-5), 68.50 (C-7), 67.09 (C-4), 65.03 (OCH2),
(C-5Ј), 59.69 (C-4Ј), 52.62 (CO2CH3), 20.79, 20.67, 20.52 and 20.48
(4ϫ OCCH3) ppm. HRMS (ESI-TOF): calcd. for C37H45N3O14 [M
+ HCOO–]– 858.2938; found 858.2953.
63.08 (C-4Ј) and 61.21 (C-5Ј) ppm. HRMS (ESI-TOF): calcd. for
C16H25N3O16 [M – H]– 450.1365; found 450.1366.
Ammonium 4-Amino-4-deoxy-β-L-arabinopyranosyl-(1Ǟ8)-(allyl D-
Methyl
4-Azido-2,3-di-O-acetyl-4-deoxy-β-L-arabinopyranosyl-
glycero-α- -talo-oct-2-ulopyranoside)onate (32): A solution of 31
D
(1Ǟ8)-(allyl 3,4,5,7-tetra-O-acetyl-
D
-glycero-α-
D
-talo-oct-2-ulopyr-
(20 mg, 0.045 mmol) in H2O/diisopropylamine (3:1, 2 mL) was
stripped with argon in a light protected flask. Dithiothreitol
(29.8 mg, 0.183 mmol) was added, and the solution was stirred for
2 h at room temp. under argon. Another portion of dithiothreitol
(29.8 mg) was added to complete the conversion. The solution was
diluted with H2O (2 mL) and washed with chloroform (2 mL) five
times. The organic phases were reextracted with water and the com-
bined aqueous phases were stripped with argon until the solution
became clear again. The material obtained upon lyophilization was
dissolved in H2O (1 mL) and transferred to a packed column of
Dowex 50 (H+) cation-exchange resin. The column was washed
anoside)onate (30): A solution of 25 (169 mg, 0.207 mmol) in dry
chloroform (10 mL) was stripped with argon. A solution of TiCl4
(100 μL, 0.913 mmol) in chloroform (2 mL) was added slowly at
0 °C. After 1 h, additional TiCl4 (100 μL) was added until TLC
showed the complete consumption of starting material to a lower
running sport (Rf = 0.13; EtOAc/toluene, 1:1). Ethyl ether (20 mL)
and saturated aqueous NaHCO3 solution (10 mL) were added, and
the solution was stirred for 30 min. The layers were separated, and
the organic phase was dried (MgSO4) and concentrated. The resi-
due was purified by flash chromatography on silica gel (toluene/
EtOAc, 1:1). The crude product was taken up in pyridine (3 mL), with water (20 mL) under TLC control. The product was then
cooled to 0 °C, and a catalytic amount of DMAP and a solution
of acetic anhydride (2 mL) in pyridine (1 mL) was added slowly.
The solution was stirred for 24 h at room temp. Dry MeOH (3 mL)
was added slowly at 0 °C and the solution was stirred for an ad-
ditional 30 min. The solution was concentrated, coevaporated with
toluene five times, and directly subjected to column chromatog-
raphy. Elution with toluene/EtOAc, 4:1 afforded 30 (133 mg,
0.185 mmol, 89%) as a colorless syrup. [α]2D0 = +48 (c = 0.57,
eluted with ammonia (20 mL, 1 m) and lyophilized to give 32
(12 mg, 60%) as a white amorphous solid. [α]2D0 = +83 (c = 0.29,
H2O). 1H NMR (400 MHz, D2O at pD 7.5): δ = 5.93 (m, 1 H,
–CH=), 5.35–5.28 [dq, 1 H, =CH2(trans)], 5.23–5.18 [dq, 1 H,
=CH2(cis)], 4.99 (d, J1Ј,2Ј = ca. 3.7 Hz, 1 H, 1Ј-H), 4.11–4.03 (m, 3
H, 3Ј-H, 4-H, 7-H), 4.01 (br. s, 1 H, 5-H), 3.99 (dd, J5Јa,5Јb = 11.5,
J5Јa,4Ј = 2.0 Hz, 1 H, 5Јa-H), 3.94–3.78 (m, 4 H, 8a-H, 8b-H,
OCH2), 3.74 (dd, J2Ј,1Ј = 3.7, J2Ј,3Ј = 9.9 Hz, 1 H, 2Ј-H), 3.68 (dd,
1 H, 5Јb-H), 3.61 (dd, J6,5 = 0.9, J6,7 = 9.3 Hz, 1 H, 6-H), 3.49 (br.
s, 1 H, 4Ј-H), 2.03 (dd, J3e,3a = 13.0 Hz, 1 H, 3e-H), 1.77 (t, 1 H,
1
CHCl3). H NMR (600 MHz, CDCl3): δ = 5.89 (m, 1 H, –CH=),
5.54 (dd, 1 H, J3,5 = 0.85 Hz, J3,4 = 3.7 Hz, 3-H), 5.38 (t, 1 H, J4,5
= 3.7 Hz, 4-H), 5.33–5.28 [m, 3 H, 5-H, 7-H, =CH2(trans)], 5.23 3a-H) ppm. 13C NMR (100 MHz, D2O): δ = 176.00 (C-1), 134.60
(dd, 1 H, J3Ј,4Ј = 3.9, J2Ј,3Ј = 10.4 Hz, 2Ј-H), 5.21 [dq, 1 H, (–CH=), 118.50 (=CH2), 100.80 (C-2), 99.92 (C-1Ј), 72.33 (C-6),
=CH2(cis)], 5.15 (dd, 1 H, J3Ј,2Ј = 10.3, J3Ј,4Ј = 3.6 Hz, 3Ј-H), 5.13 70.84 (C-8), 68.90, 68.53 (C-2Ј, C-3Ј), 67.23 (C-7), 67.03 (C-4),
(d, 1 H, J1Ј,2Ј = 3.7 Hz, 1Ј-H), 4.26 (dd, 1 H, J6,5 = 1.9, J6,7
9.8 Hz, 6-H), 4.23–4.19 (ddt, 1 H, OCH2), 4.08 (dt, 1 H, J4Ј,3Ј
=
=
66.79 (C-5), 65.19 (OCH2), 60.07 (C-5Ј), 52.30 (C-4Ј), 34.92 (C-3)
ppm. HRMS (ESI-TOF): calcd. for C16H27NO11 [M + H]+
3.6, J4Ј,5Јa = 1.8 Hz, 4Ј-H), 4.04 (dd, 1 H, J8a,8b 13.0 Hz, 8a-H), 410.1657; found 410.1659.
3.93–3.89 (ddt, 1 H, OCH2), 3.90 (dd, 1 H, J5Јa,5Јb = 12.7 Hz, 5Јa-
Ammonium
4-Amino-4-deoxy-β-L-arabinopyranosyl-(1Ǟ8)-[3-(3-
H), 3.85 (dd, 1 H, J8b,7 = 2.2 Hz, 8b-H), 3.75 (s, 3 H, CO2CH3),
3.67 (dd, 1 H, 5Јb-H), 2.12 (s, 3 H), 2.11 (s, 3 H), 2.04 (s, 6 H),
1.99 (s, 3 H) and 1.96 (s, 3 H, 6ϫ COCH3) ppm. 13C NMR
(150 MHz, CDCl3): δ = 132.25 (–CH=), 117.49 (=CH2), 99.22 (C-
2), 97.36 (C-1Ј), 68.99 (d.i., C-2Ј, C-7), 68.21 (C-3Ј), 67.80 (C-6),
67.55 (C-3), 66.11 (C-4), 65.66 (C-8), 65.17 (OCH2), 63.77 (C-5),
60.11 (C-5Ј), 59.60 (C-4Ј), 52.72 (CO2CH3), 20.79, 20.61, 20.51,
20.49, 20.49 (6ϫ OCCH3) ppm. HRMS (ESI-TOF): calcd. for
C29H39N3O18 [M + HCOO–]– 762.2211; found 762.2224.
mercaptopropylthio)propyl
D
-glycero-α- -talo-oct-2-ulopyranoside]-
D
onate (33): Propane-1,3-dithiol (0.1 mL) was added to a solution
of 32 (2.5 mg, 5.8 μmol) in MeOH (2 mL) and stripped with argon
in a quartz flask. The solution was stirred under UV radiation
(254 nm) for 1 h. The reaction mixture was diluted with H2O
(2 mL) and washed with chloroform (2 mL) five times. The organic
phases were reextracted with water and the combined aqueous
phases were stripped with argon until the solution become clear.
The aqueous phase was lyophilized, and the residue was purified
on BioGel P-2 to give 33 (2.5 mg, 80%) as a colorless solid. [α]2D0
= +46 (c = 0.2, H2O). 1H NMR (400 MHz, D2O): δ = 5.06 (d, J1Ј,2Ј
Sodium 4-Azido-4-deoxy-β-
cero-α- -talo-oct-2-ulopyranoside)onate (31):
(10 mg, 0.013 mmol) in dry MeOH (1 mL) was adjusted to pH 10
with NaOMe (0.1 mL, 0.1 m) and stirred for 4 h at room temp. The 8.8 Hz, 1 H, 7-H), 4.13–4.05 (m, 2 H, 5-H, 5Јa-H), 4.08 (dd, J2Ј,3Ј
solution was neutralized with Dowex 50 (H+) cation-exchange
= 9.9, J3Ј,4Ј = 5.3 Hz, 1 H, 3Ј-H), 4.01–3.95 (m, 3 H, 3-H, 4-H, 8a-
resin. The suspension was filtered, and the filtrate was concen- H), 3.92 (dd, J8b,8a = 10.6 Hz, 1 H, 8b-H), 3.82 (dd, 1 H, 2Ј-H),
L-arabinopyranosyl-(1Ǟ8)-(allyl
D-gly-
D
A
solution of 30
= 3.6 Hz, 1 H, 1Ј-H), 4.26 (ddd, J8b,7 = 7.3, J8a,7 = 2.8, J6,7
=
trated. A solution of the residue in water (1 mL) was stirred with
aqueous NaOH (2 mL, 0.2 m) at pH 12 for 3 h at room temp. The
solution was neutralized with Dowex 50 (H+) cation-exchange
resin. The resin was removed by filtration, and the filtrate was lyo-
philized to give 31 as a white solid (6.3 mg, 97%). [α]2D0 = +102.7
3.70 (dd, J5Јb,5Јa = 12.8, J5Јb,4Јb = 2.3 Hz, 1 H, 5Јb-H), 3.64 (dd, 1
H, 6-H), 3.60–3.53 (m, 1 H, OCH2), 3.46 (br. m, 1 H, 4Ј-H), 3.38–
3.31 (m, 1 H, OCH2), 2.73–2.62 (m, 6 H, 3ϫ CH2) and 1.95–1.83
(m, 4 H, 2ϫ CH2) ppm. 13C NMR (100 MHz, D2O): δ = 175.07
(C-1), 102.83 (C-2), 99.86 (C-1Ј), 72.73 (C-6), 72.71 (C-3), 71.16
128
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© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 119–131

Synthesis of Neoglycoconjugates
(C-8), 69.09 (C-2Ј, C-5), 68.72 (C-3Ј), 68.24 (C-7Ј), 67.34 (C-4),
62.69 (OCH2), 61.26 (C-5Ј), 51.23 (C-4Ј), 33.54 (OCH2CH2CH2S),
= 170.77, 170.28, 169.91 and 169.63 (4ϫ OCCH3), 168.23 (C-1Ј),
168.04 (C-1), 137.72 (C-Ar), 137.64 (C-Ar), 133.61 (–CH=),
30.42 (OCH2CH2CH2S), 29.46 (SCH2CH2CH2SH), 28.83 129.00–125.27 (C-Ar), 116.81 (=CH2), 100.11 (C-1ЈЈ), 98.50, 97.68
(SCH2CH2CH2SH) and 23.59 (SCH2CH2CH2SH) ppm. HRMS
(ESI-TOF): calcd. for C19H35NO12S2 [M – H]– 532.1528; found
532.1523.
(C-2, C-2Ј), 77.10 (C-3ЈЈ), 76.08 (C-2ЈЈ), 74.41, 72.63 (2ϫ
OCH2Ph), 71.35 (C-6), 71.32 (C-8), 69.26 (C-6Ј), 68.67 (C-4), 67.80
(C-7Ј), 67.44 (C-7), 66.11 (C-4Ј), 64.28 (C-5Ј), 64.18 (OCH2), 63.55
(C-5), 61.33 (C-8Ј), 60.93 (C-5ЈЈ), 59.77 (C-4ЈЈ), 53.04, 52.50 (2ϫ
CO2CH3), 32.98 (C-3Ј), 32.25 (C-3), 20.70, 20.70, 20.61 and 20.59
(4ϫ OCCH3) ppm. HRMS (ESI-TOF): calcd. for C48H61N3O22 [M
+ Na]+ 1054.3639; found 1054.3641.
Dimethyl 4-Azido-2,3-di-O-benzyl-4-deoxy-β-
(1Ǟ8)-[(4,5,7,8-O-tetra-O-acetyl-3-deoxy-
pyranosyl)onate-(2Ǟ4)]-(allyl 3-deoxy-α-
pyranoside)onate (35) and Dimethyl 4-Azido-2,3-di-O-benzyl-4-de-
oxy-β- -arabinopyranosyl-(1Ǟ8)-[(4,5,7,8-O-tetra-O-acetyl-3-de-
oxy- -manno-oct-2-ulopyranosyl)onate-(2Ǟ7)]-(allyl 3-deoxy-α-
manno-oct-2-ulopyranoside)onate (38): A suspension of 17 (88 mg,
0.139 mmol), Hg(CN)2 (23.3 mg, 0.092 mmol), HgBr2 (16.6 mg, oct-2-ulopyranoside)onate (36): Acetic anhydride (0.2 mL) was
L
-arabinopyranosyl-
-manno-oct-2-ulo-
-manno-oct-2-ulo-
D
D
L
Dimethyl 4-Azido-2,3-di-O-benzyl-4-deoxy-β-
(1Ǟ8)-[(4,5,7,8-O-tetra-O-acetyl-3-deoxy-α-
pyranosyl)onate-(2Ǟ4)](allyl 5,7-di-O-acetyl-3-deoxy-α-
L
-arabinopyranosyl-
-manno-oct-2-ulo-
-manno-
D
D
-
D
D
0.046 mmol), and 4 Å molecular sieves in dry nitromethane
(10 mL) was stirred at room temp. for 2 h. A solution of 34
(67.5 mg, 0.14 mmol) in MeNO2 (5 mL) was added over 2 h using
a syringe pump. After 6 h, additional portions of 34 (34 mg,
added to a solution of 35 (36 mg, 0.034 mmol) in dry pyridine
(4 mL) and a catalytic amount of DMAP at 0 °C. The solution was
stirred for 12 h at room temp. and the reaction was quenched by
the addition of dry MeOH (1 mL) at 0 °C. The solution was con-
0.007 mmol), Hg(CN)2 (12.0 mg, 0.046 mmol), and HgBr2 (8.3 mg, centrated and coevaporated several times with toluene. The residue
0.023 mmol) were added and stirring was continued for 8 h. The
suspension was diluted with EtOAc (50 mL) and filtered through a
short plug of Celite®. The Celite bed was washed with EtOAc, and
the combined filtrates were washed with aqueous KI solution
(15%), saturated aqueous NaHCO3 solution, and dried (MgSO4).
The organic phase was concentrated, and the residue was directly
applied to a column of silica gel. Elution with toluene/EtOAc (1:1)
first afforded 38 (8 mg, 5.5%) as a colorless syrup. [α]2D0 = +106 (c
was dissolved in CHCl3, washed with saturated aqueous NaHCO3
solution and dried (MgSO4). The solution was concentrated and
the residue was purified by column chromatography on silica gel
(toluene/EtOAc, 1:1) to afford 36 (29 mg, 75%) as a colorless syrup.
[α]2D0 = +63.5 (c = 0.2, CHCl3). H NMR (400 MHz, CDCl3): δ =
1
7.36–7.26 (m, 10 H, Ar-H), 5.75 (m, 1 H, –CH=), 5.35 (br. s, 1 H,
5Ј-H), 5.22 (br. s, 1 H 5-H), 5.21–55.12 [m, 4 H, 4Ј-H, 7Ј-H, 7-H,
=CH2(trans)], 5.05 [dq, 1 H, =CH2(cis)], 4.86 (dd, J1ЈЈ,2ЈЈ = 3.3 Hz,
1 H, 1ЈЈ-H), 4.78 (dd, J8Јb,8Јa = 12.3, J8Јa,7Ј = 2.6 Hz, 1 H, 8aЈ-H),
4.75–4.64 (m, 5 H, 2ϫ OCH2Ph, 4-H), 4.14 (dd, J6Ј,7Ј = 9.4, J6Ј,5Ј
1
= 0.9, CHCl3). H NMR (400 MHz, CDCl3): δ = 7.41–7.25 (m, 10
H, Ar-H), 5.86 (m, 1 H, –CH=), 5.39–5.36 (br. s, 1 H, 5Ј-H), 5.30–
5.22 [m, 3 H, 4Ј-H, 7Ј-H, =CH2(trans)], 5.12 [dq, 1 H, =CH2(cis)], = 1.5 Hz, 1 H, 6Ј-H), 4.08 (m, 1 H, OCH2), 4.05 (dd, J6,7 = 9.7,
5.01 (dd, J1ЈЈ,2ЈЈ = 3.9 Hz, 1 H, 1ЈЈ-H), 4.86–4.64 (m, 4 H, 2ϫ J6,5 = 1.2 Hz, 1 H, 6-H), 4.01 (dd, J8Јb,7Ј = 3.8 Hz, 1 H, 8Јb-H),
OCH2Ph), 4.60 (dd, J8Јb,8Јa = 11.5, J8Јb,7Ј = 2.8 Hz, 1 H, 8aЈ-H), 3.89 (dd, J3ЈЈ,2ЈЈ = 9.1, J3ЈЈ,4ЈЈ = 3.6 Hz, 1 H, 3ЈЈ-H), 3.88–3.78 (m,
4.33 (dd, J6Ј,7Ј = 9.4, J6Ј,5Ј = 1.4 Hz, 1 H, 6Ј-H), 4.19 (m, 1 H, 7- 10 H, 2ЈЈ-H, 4ЈЈ-H, 8a-H, 8b-H, 2ϫ CO2CH3), 3.71 (dd, J5ЈЈb,5ЈЈa
H), 4.09–3.98 (m, 4 H, 3ЈЈ-H, 4ЈЈ-H, 8Јb-H, OCH2), 3.88 (dd, J3ЈЈ,2ЈЈ = 12.3, J5ЈЈb,4ЈЈ = 2.0 Hz, 1 H, 5ЈЈa-H), 3.56 (dd, J5ЈЈb,4ЈЈ = 3.0 Hz,
= 9.0 Hz, 1 H, 2ЈЈ-H), 3.86–3.70 (m, 13 H, 4-H, 5-H, 5ЈЈa-H, 6-H, 1 H, 5ЈЈb-H), 2.13 (ddd, J3e,3a = 12.6, J3e,4 = 5.1 Hz, 1 H, 3e-H),
8a-H, 8b-H, 2ϫ CO2CH3, OCH2), 3.65 (dd, J5ЈЈb,5ЈЈa = 12.3 Hz, 1
H, 5ЈЈb-H), 2.19 (dd, J3e,3a = 12.9, J3e,4 = 4.9 Hz, 1 H, 3e-H), 2.13–
2.11 (t, J3Јa,4Ј = J3Јa,3Јe = 12.7 Hz, 1 H, 3Јa-H), 2.08–1.92 (m, 20 H,
3a-H, 3Јe-H, 6ϫ COCH3) ppm. 13C NMR (100 MHz, CDCl3): δ
= 170.68, 170.43, 170.33, 170.10, 169.65 and 169.54 (6ϫ OCCH3),
167.85 and 167.04 (C-1Ј, C-1), 138.35 (C-Ar), 137.98 (C-Ar),
133.58 (–CH=), 128.45, 128.42, 127.90, 127.81, 127.76 and 127.68
(C-Ar), 117.29 (=CH2), 98.59 and 97.62 (C-2, C-2Ј), 98.52 (C-1ЈЈ),
1.88 (m, 14 H, 3Јa-H, 3Јe-H, 4ϫ COCH3) and 1.46 (dd, J4,3a
=
11.8 Hz, 1 H, 3a-H) ppm. 13C NMR (100 MHz, CDCl3): δ =
170.80, 170.37, 169.87 and 169.63 (4ϫ OCCH3), 168.41 (C-1Ј),
168.09 (C-1), 137.94 (C-Ar), 137.84 (C-Ar), 133.68 (–CH=),
128.42–127.58 (C-Ar), 116.78 (=CH2), 99.23 (C-2Ј), 98.82 (C-1ЈЈ), 76.93 (C-3ЈЈ), 75.80 (C-2ЈЈ), 73.39, 73.12 (2ϫ OCH2Ph), 69.37 (C-
98.70 (C-2), 77.72 (C-3ЈЈ), 75.59 (C-2ЈЈ), 74.46 (C-7), 74.12 and
72.62 (2ϫ OCH2Ph), 73.58 (C-6), 70.07 (C-6Ј), 67.76 (C-4), 67.57
6Ј), 69.29 (C-6), 68.84 (C-7), 67.92 (C-7Ј), 66.56 (C-4), 66.35 (C-
4Ј), 66.10 (C-8), 65.12 (C-5), 64.83 (OCH2), 64.35 (C-5Ј), 61.35 (C-
(C-7Ј), 66.53 (C-8), 65.93 (C-5), 65.84 (C-4Ј), 64.64 (OCH2), 64.23 8Ј), 60.85 (C-5ЈЈ), 59.73 (C-4ЈЈ), 52.73 and 52.56 (2ϫ CO2CH3),
(C-5Ј), 63.00 (C-8Ј), 60.85 (C-5ЈЈ), 59.52 (C-4ЈЈ), 53.14 and 52.41
(2ϫ CO2CH3), 34.68 (C-3Ј), 32.31 (C-3), 20.75, 20.70, 20.65 and
20.59 (4ϫ OCCH3) ppm.
34.04 (C-3), 31.57 (C-3Ј), 20.82–20.54 (6ϫ OCCH3) ppm. HRMS
(ESI-TOF): calcd. for C53H66N3O26 [M + HCOO–]– 1160.3940;
found 1160.3958.
Continued elution of the column furnished a small amount of the
β-(2Ǟ4)-linked trisaccharide (2 mg, 1.4%) followed by 35 (27 mg,
19%) as a colorless oil. [α]2D0 = +55 (c = 0.4, CHCl3). 1H NMR
(400 MHz, CDCl3): δ = 7.42–7.27 (m, 10 H, Ar-H), 5.74 (m, 1 H,
–CH=), 5.40–5.37 (br. s, 1 H, 5Ј-H), 5.29 (ddd, J4Ј,5Ј = 3.0, J4Ј,3Јe
= 4.7, J4Ј,3Јa = 12.2 Hz, 1 H, 4Ј-H), 5.20 (dt, 1 H, 7Ј-H), 5.16 [dq,
1 H, =CH2(trans)], 5.06 [dq, 1 H, =CH2(cis)], 4.86–4.65 (m, 6 H,
2ϫ OCH2Ph, 1ЈЈ-H, 8aЈ-H), 4.28 (ddd, 1 H, 4-H), 4.17 (dd, J6Ј,7Ј
= 9.4, J6Ј,5Ј = 1.0 Hz, 1 H, 6Ј-H), 4.14–4.07 (br. s, 1 H, 7-H), 4.03
Dimethyl
(1Ǟ8)-[(4,5,7,8-O-tetra-O-acetyl-3-deoxy-α-
pyranosyl)onate-(2Ǟ4)]-(allyl 5,7-di-O-acetyl-3-deoxy-α-
oct-2-ulopyranoside)onate (39): solution of 36 (27 mg,
0.024 mmol) in chloroform (3 mL) was stripped with argon. A
solution of TiCl4 (5.8 μL, 0.053 mmol) in chloroform (1 mL) was
added slowly at 0 °C. After stirring for 12 h at room temp., ad-
ditional TiCl4 (2.9 μL, 0.026 mmol) was added until TLC showed
the complete consumption of starting material to a lower migrating
spot. Ethyl ether (5 mL) and saturated aqueous NaHCO3 solution
4-Azido-2,3-di-O-acetyl-4-deoxy-β-
L
-arabinopyranosyl-
-manno-oct-2-ulo-
-manno-
D
D
A
(dd, J8Јb,8Јa = 12.4, J8Јb,7Ј = 3.6 Hz, 1 H, 8Јb-H), 3.98 (dd, J3ЈЈ,2ЈЈ
=
9.6, J3ЈЈ,4ЈЈ = 3.5 Hz, 1 H, 3ЈЈ-H), 3.96–3.74 (m, 13 H, OCH2, 2ЈЈ- (5 mL) were added and the solution was stirred for 30 min at room
H, 4ЈЈ-H, 5ЈЈa-H, 8a-H, 8b-H, 2ϫ CO2CH3), 3.59 (dd, J5ЈЈb,5ЈЈa
=
temp. The layers were separated and the organic phase was dried
(MgSO4) and concentrated. The crude product was taken up in
pyridine (3 mL), and a catalytic amount of DMAP and acetic anhy-
dride (0.3 mL) were added slowly. The solution was stirred for 24 h
12.3, J5ЈЈb,4ЈЈ = 2.1 Hz, 1 H, 5ЈЈb-H), 3.43 (dd, 1 H, 6-H), 2.28 (ddd,
J3e,3a = 12.4, J3e,4 = 4.8 Hz, 1 H, 3e-H), 2.16–1.92 (m, 15 H, 3a-H,
3Јa-H, 3Јe-H, 4ϫ COCH3) ppm. 13C NMR (100 MHz, CDCl3): δ
Eur. J. Org. Chem. 2012, 119–131
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
129

M. Blaukopf, B. Müller, A. Hofinger, P. Kosma
FULL PAPER
at room temp. and processed as described for 36. The residue was
purified by column chromatography (toluene/EtOAc, 50:1) to af-
ford 39 (17 mg, 68%) as a colorless syrup. [α]2D0 = +96 (c = 0.4,
solution was diluted with H2O (2 mL) and washed with chloroform
(2 mL) five times. The organic phases were reextracted with water.
The combined aqueous phases were stripped with argon until the
solution became clear again. The solution was lyophilized, and the
1
CHCl3). H NMR (600 MHz, CDCl3): δ = 5.88 (m, 1 H, –CH=),
5.35–5.33 (br. s, 1 H, 5Ј-H), 5.25 [dq, 1 H, =CH2(trans)], 5.21–5.16 residue was purified by gel chromatography (BioGel P-2) and lyo-
(m, 2 H, 3ЈЈ-H, 4Ј-H), 5.15–5.10 [m, 5 H, 1ЈЈ-H, 2ЈЈ-H, 5-H, 7Ј-H, philized to give 41 (2.8 mg, 99%) as a colorless amorphous solid.
1
=CH2(cis)], 5.01 (dt, J7,6 = 9.8, J7,8a = 2.3 Hz, 1 H, 7-H), 4.80 (dd,
J8Јa,7Ј = 2.4, J8Јa,8Јb = 12.2 Hz, 1 H, 8Јa-H), 4.72 (ddd, J4,5 = 3.0,
[α]2D0 = +46.5 (c = 0.4, H2O). H NMR (600 MHz, D2O): δ = 5.93
(m, 1 H, –CH=), 5.32 [dq, 1 H, =CH2(trans)], 5.22 [dq, 1 H,
J4,3e = 4.8, J4,3a = 11.9 Hz, 1 H, 4-H), 4.15 (dq, 1 H, OCH2), 4.13 =CH2(cis)], 4.98 (d, J1ЈЈ,2ЈЈ = 3.7 Hz, 1 H, 1ЈЈ-H), 4.22 (ddd, J4,5
(dd, J6Ј,5Ј = 1.1, J6Ј,7Ј = 9.4 Hz, 1 H, 6Ј-H), 4.11–4.08 (br. s, 1 H, 2.6, J4,3e = 5.2, J4,3a = 12.0 Hz, 1 H, 4-H), 4.10 (dd, J3ЈЈ,2ЈЈ = 9.9,
4ЈЈ-H), 4.05 (dd, J6,5 = 1.1, J6,7 = 9.9 Hz, 1 H, 6-H), 3.97 (dd, J8Јb,7Ј J3ЈЈ,4ЈЈ = 4.7 Hz, 1 H, 3ЈЈ-H), 4.07–4.02 (m, 3 H, 4Ј-H, 5-H, 7-H),
= 3.9, J8Јb,8Јa = 12.3 Hz, 1 H, 8Јb-H), 3.93 (dd, J8a,7 = 2.8, J8a,8b 4.00 (br. s, 1 H, 5Ј-H), 3.94–3.90 (m, 3 H, 7Ј-H, 8Јa-H, OCH2),
13.1 Hz, 1 H, 8a-H), 3.92 (dq, 1 H, OCH2), 3.88 (dd, J5ЈЈa,4ЈЈ = 0.6, 3.83 (dq, 1 H, OCH2), 3.81 (dd, J8a,8b = 10.9, J8a,7 = 6.0 Hz, 1 H,
J5ЈЈa,5ЈЈb = 12.2 Hz, 1 H, 5ЈЈa-H), 3.82 and 3.81 (2ϫ CO2CH3), 3.79 8a-H), 3.77 (dd, J8b,7 = 3.0 Hz, 1 H, 8b-H), 3.72 (dd, J5ЈЈb,5ЈЈa
=
=
=
(dd, J8b,7 = 1.8 Hz, 1 H, 8b-H), 3.64 (dd, J5ЈЈb,4ЈЈ = 1.9 Hz, 1 H,
5ЈЈb-H), 2.21–1.94 (m, 28 H, 3e-H, 3eЈ-H, 3a-H, 3aЈ-H, 8ϫ
COCH3) ppm. 13C NMR (150 MHz, CDCl3): δ = 170.69, 170.44,
170.42, 170.31, 170.13, 170.11, 169.64 and 169.49, (8ϫ COCH3),
167.81 (C-1Ј), 167.04 (C-1), 133.29 (–CH=), 116.84 (=CH2), 98.61,
12.9, J5ЈЈb,4ЈЈ = 1.0 Hz, 1 H, 5ЈЈb-H), 3.71 (dd, 1 H, 2ЈЈ-H), 3.68
(dd, J8Јb,8Јa = 13.0 Hz, 1 H, 8Јb-H), 3.62 (br. s, 1 H, 4ЈЈ-H), 3.59
(dd, J6Ј,7Ј = 9.8 Hz, 1 H, 6Ј-H), 3.57 (dd, J6,7 = 9.5 Hz, 1 H, 6-H),
2.12 (dd, J3Јe,3Јa = 13.1, J3Јe,4Ј = 5.1 Hz, 1 H, 3Јe-H), 2.00 (dd, J3e,3a
= 13.6 Hz, 1 H, 3e-H), 1.95 (dd, J3a,4 = 12.4 Hz, 1 H, 3a-H), 1.78
97.31 (C-2, C-2Ј), 97.15 (C-1ЈЈ), 69.39 (C-6), 69.36 (C-7), 69.12 (C- (dd, 1 H, 3Јa-H) ppm. 13C NMR (150 MHz, D2O): δ = 175.97 and
3ЈЈ), 68.12 (C-6Ј), 68.06 (C-7Ј), 67.85 (C-2ЈЈ), 66.34 (C-4Ј), 66.24
(C-4), 65.14 (C-8), 64.95 (C-5), 64.61 (OCH2), 64.28 (C-5Ј), 61.33
(C-8Ј), 59.97 (C-5ЈЈ), 59.53 (C-4ЈЈ), 52.66 and 52.63 (2ϫ CO2CH3),
175.06 (C-1, C-1Ј), 134.07 (–CH=), 117.13 (=CH2), 100.20 and
99.32 (C-2Ј, C-2), 99.15 (C-1ЈЈ), 72.40 (C-6Ј), 71.41 (C-6), 70.00 (C-
8), 70.63 (C-7Ј), 68.50 (C-4), 68.17 (C-2ЈЈ), 67.86 (C-7), 66.27 (C-
34.11 (C-3), 31.39 (C-3Ј), 20.82–20.54 (8ϫ COCH3) ppm. HRMS 5Ј), 66.13 (C-4Ј), 65.92 (C-3ЈЈ), 64.20 (C-5), 64.03 (OCH2), 63.18
(ESI-TOF): calcd. for C43H58N3O28 [M + HCOO–]– 1064.3212;
found 1064.3205.
(C-8Ј), 57.97 (C-5ЈЈ), 51.66 (C-4ЈЈ), 34.51 (C-3Ј) and 33.21 (C-3)
ppm. HRMS (ESI-TOF): calcd. for C24H39NO18 [M + H]+
630.2240; found 630.2233.
Disodium 4-Azido-4-deoxy-β-L-arabinopyranosyl-(1Ǟ8)-[(3-deoxy-
α- -manno-oct-2-ulopyranosyl)onate-(2Ǟ4)]-(allyl 3-deoxy-α-
D
D
-
Disodium 4-Amino-4-deoxy-β-
α- -manno-oct-2-ulopyranosyl)onate-(2Ǟ4)]-[3-(3-mercaptopropyl-
thio)propyl 3-deoxy-α- -manno-oct-2-ulopyranoside]onate (42): Pro-
L-arabinopyranosyl-(1Ǟ8)-[(3-deoxy-
manno-oct-2-ulopyranoside)onate (40): A solution of 39 (5 mg,
0.004 mmol) in dry methanol (1 mL) was stirred with NaOMe
(0.1 mL, 0.1 m) for 4 h at room temp. The solution was neutralized
with Dowex 50 (H+) cation-exchange resin. The resin was removed
by filtration, and the filtrate was concentrated. A solution of the
residue in water (1 mL) was treated with aqueous NaOH (2 mL,
0.1 m) at room temp. for 3 h. The solution was neutralized with
Dowex 50 (H+) cation-exchange resin. The resin was removed by
filtration, and the filtrate was lyophilized to give 40 (3 mg, 96%)
as a white foamy solid. [α]2D0 = +112.6 (c = 0.3, H2O). 1H NMR
D
D
pane-1,3-dithiol (50 μL) was added to a solution of 41 (2.5 mg,
6.1 μmol) in MeOH (2 mL) and stripped with argon in a quartz
flask. The solution was stirred under UV radiation (254 nm) for
1 h to give full conversion. The reaction mixture was diluted with
H2O (2 mL) and extracted with chloroform (2 mL) five times. The
organic phases were reextracted with water, and the combined
aqueous phases were stripped with argon until the solution became
clear. The solution was lyophilized, and the residue was desalted
(600 MHz, D2O): δ = 5.87 (m, 1 H, –CH=), 5.26 [dq, 1 H, on BioGel P-2 (size 28ϫ1 cm) to give 42 as a white solid (2.8 mg,
1
=CH2(trans)], 5.15 [dq, 1 H, =CH2(cis)], 4.86 (d, J1ЈЈ,2ЈЈ = 3.7 Hz,
1 H, 1ЈЈ-H), 4.08 (ddd, J4,5 = 2.7, J4,3e = 5.0, J4,3a = 11.9 Hz, 1 H,
4-H), 4.00–3.94 (m, 4 H, 3ЈЈ-H, 4Ј-H, 5-H, 7-H), 3.94–3.92 (br. s,
1 H, 5Ј-H), 3.91 (dt, J4ЈЈ,3ЈЈ = 4.0 Hz, 1 H, 4ЈЈ-H), 3.89–3.85 (m, 2
99%). [α]2D0 = +45 (c = 0.3, H2O). H NMR (600 MHz, D2O): δ =
4.98 (br. s, 1 H, 1ЈЈ-H), 4.16–3.93 (m, 9 H, 3ЈЈ-H, 4-H, 4Ј-H, 5-H,
5Ј-H, 5ЈЈa-H, 7-H, 7Ј-H, 8Јa-H), 3.87–3.81 (m, 2 H, 8a-H, 8b-H),
3.80–3.77 (dd, 1 H, 2ЈЈ-H), 3.72 (dd, 1 H, 8Јb-H), 3.64–3.56 (m, 2
H, 7Ј-H, 8Јa-H), 3.83 (dq, 1 H, OCH2), 3.81 (dd, J5ЈЈa,5ЈЈb = 12.8, H, 5ЈЈb-H, 6-H), 3.54–3.51 (m, 1 H, 6Ј-H), 3.47–3.43 [br. s, 1 H,
J5ЈЈa,4ЈЈb = 1.6 Hz, 1 H, 5ЈЈa-H), 3.77 (dd, J8a,8b = 10.9, J8a,7
5.6 Hz, 1 H, 8a-H), 3.73 (dq, 1 H, OCH2), 3.69 (dd, J2ЈЈ,3ЈЈ
=
=
OCH2(CH2)2S], 3.34–3.29 [m, 2 H, 4ЈЈ-H, OCH2(CH2)2S], 2.71–
2.59 (m, 6 H, 3ϫ CH2), 2.13 (dd, 1 H, 3Јe-H), 2.04–1.79 (m, 6 H,
3e-H, 3a-H, 2ϫ CH2), 1.71 (t, 1 H, 3Јa-H) ppm. HRMS (ESI-
TOF): calcd. for C27H47NO18S2 [M + H]+ 738.2307; found
738.2304.
10.0 Hz, 1 H, 2ЈЈ-H), 3.66–3.62 (m, 3 H, 5ЈЈb-H, 8Јb-H, 8b-H), 3.52
(dd, J6,7 = 9.2 Hz, 1 H, 6-H), 3.51 (dd, J6Ј,7Ј = 8.5 Hz, 1 H, 6Ј-H),
2.04 (dd, J3Јe,3Јa = 13.2, J3Јe,4Ј = 4.8 Hz, 1 H, 3Јe-H), 1.90 (dd, J3e,3a
= 13.1, J3e,4 = 5.0 Hz, 1 H, 3e-H), 1.83 (t, 1 H, 3a-H) and 1.67 (t,
J3Јa,4Ј = 13.1 Hz, 1 H, 3Јa-H) ppm. 13C NMR (150 MHz, D2O): δ
= 176.67 (C-1Ј), 176.57 (C-1), 134.76 (–CH=), 117.82 (=CH2),
100.89 (C-2Ј), 100.28 (C-1ЈЈ), 100.04 (C-2), 73.09 (C-6Ј), 71.95 (C-
6), 70.63 (C-7Ј), 70.51 (C-8), 69.50 (C-2ЈЈ), 69.45 (C-3ЈЈ), 69.23 (C-
4), 68.52 (C-7), 66.99 (C-5Ј), 66.64 (C-4Ј), 64.92 (C-5), 64.70
(OCH2), 63.87 (C-8Ј), 63.09 (C-4ЈЈ), 61.20 (C-5ЈЈ), 35.24 (C-3Ј) and
33.94 (C-3) ppm. HRMS (ESI-TOF): calcd. for C24H36N3O18 [M –
H]– 654.1999; found 654.2005.
Synthesis of Neoglycoconjugates 43–45: Thiol 24 (2.1 mg) was dis-
solved in a freshly prepared conjugation buffer [0.5 mL, 20 mm so-
dium phosphate buffer, 100 mm ethylenediaminetetraacetic acid
(EDTA), 80 mm sucrose, pH 6.6] and immediately added to a solu-
tion of 5 mg/mL maleimide-activated BSA (20 mm sodium phos-
phate buffer, 230 mm NaCl, 2 mm EDTA, 80 mm sucrose, pH 6.6).
The reaction mixture was purged with argon for 2 min and stirred
at room temp. for 2 h. The reaction mixture was purified over a
Sephadex G-25M column (0.01 m phosphate-buffered saline in
Disodium 4-Amino-4-deoxy-β-L-arabinopyranosyl-(1Ǟ8)-[(3-deoxy- double distilled H2O as eluent). The protein-containing fractions
α- -manno-oct-2-ulopyranosyl)onate-(2Ǟ4)]-(allyl 3-deoxy-α-
D
D
-
were pooled, lyophilized, and purified again over a P-2 gel column
(30ϫ1 cm, water) to afford 44 (18.4 mg) as a colorless solid. Neo-
glycoconjugate 45 was prepared from 33 (2.0 mg) by this procedure.
Yield of 45: 20 mg. Neoglycoconjugate 43 was synthesized from 42
manno-oct-2-ulopyranoside)onate (41): A solution of 40 (3 mg,
4.5 μmol) in THF/0.1 m NaOH (1:2, 1 mL) was stirred with tri-
methylphosphane (1.3 μL, 18.3 μmol) for 4 h at room temp. The
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Synthesis of Neoglycoconjugates
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Received: August 9, 2011
Published Online: November 16, 2011
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