Synthesis of terminal disaccharide elements corresponding to the Ogawa and Inaba antigenic determinant from Vibrio cholerae O1

Article abstract of DOI:10.1016/S0008-6215(97)10052-0

Vibrio cholerae O1 LPS terminal mono- and disaccharide elements were synthesized by reduction of the azido group in several 4-amino-4,6-dideoxy- D-mannose mono- and disaccharide derivatives, followed by coupling with 2,4- di-O-acetyl-3-deoxy-L-glycero-tetronic acid in the presence of 2-ethoxy-l- ethoxycarbonyl-1,2-dihydroquinoline. This compound represents a useful model in order to elucidate the size of the epitopes which define Ogawa and Inaba serotypes from Vibrio cholerae O1.

Full text of DOI:10.1016/S0008-6215(97)10052-0

Ž
.
Carbohydrate Research 306 1998 163–170
Synthesis of terminal disaccharide elements
corresponding to the Ogawa and Inaba antigenic
determinant from Vibrio cholerae O1 1
a
a
Adriana Arencibia-Mohar , Alina Ariosa-Alvarez ,
a
a
b
Odalys Madrazo-Alonso , Elba Gonzalez Abreu , Luis Garcıa-Imia ,
´
´
b
a,)
Gustavo Sierra-Gonzalez , Vicente Verez-Bencomo
´
a
Laboratory of Synthetic Antigens, Facultad de Quımica, UniÕersidad de La Habana, Ciudad de La
´
Habana 10400, Cuba
Instituto Finlay de Sueros y Vacunas, AÕe 27 No 1985, La Lisa, A.P. 6017, Ciudad de La Habana
11600, Cuba
b
Received 11 December 1996; accepted in revised form 1 October 1997
Abstract
Vibrio cholerae O1 LPS terminal mono- and disaccharide elements were synthesized by
reduction of the azido group in several 4-amino-4,6-dideoxy-D-mannose mono- and disaccha-
ride derivatives, followed by coupling with 2,4-di-O-acetyl-3-deoxy-L-glycero-tetronic acid in
the presence of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline. This compound represents a
useful model in order to elucidate the size of the epitopes which define Ogawa and Inaba
serotypes from Vibrio cholerae O1. q 1998 Elsevier Science Ltd. All rights reserved.
Keywords: Vibrio cholerae, disaccharide; Ogawa serotype, synthesis; Inaba serotype
Ž
species are linear homopolymers composed of a- 1
1. Introduction
.
Ž
.
™ 2 -linked N- 3-deoxy-L-glycero-tetronyl -per-
Recently, the interest in cholera has been intensi-
fied by the occurrence of epidemics in Latin America
Ž
osamine perosamine 4-amino-4,6-dideoxy-D-man-
.
w
x
nose 4,5 . Furthermore, for the LPS of the Ogawa
serotype 2-O-methyl-perosamine was demonstrated
w x
w x
1 and Asia 2 . Taking into account the structure of
Ž
.
the lipopolysaccharide LPS , Vibrio cholerae caus-
ing cholera in man has been divided into three
serotypes: O1 Ogawa, O1 Inaba, and O139 Bengal
w
x
to occur at the nonreducing terminus 6,7 , suggesting
that its antigenic determinant is associated with the
methyl group.
w
x
2,3 . The LPSs belonging to the Ogawa and Inaba
w x
Several syntheses of the monosaccharide 8 , the
Ž
.
w x
disaccharide with an a- 1™2 linkage 9 and even
w
x
) Corresponding author.
larger fragments 10 containing the 3-deoxy-L-
glycero-tetronamide unit were reported. In all the
previous examples, the amide linkage was formed by
1 Presented at the XVIIth International Carbohydrate
Symposium, Ottawa, Canada, 1994.
0008-6215r98r$19.00 q 1998 Elsevier Science Ltd All rights reserved.

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)
164
A. Arencibia-Mohar et al.rCarbohydrate Research 306 1998 163–170
glycero-tetronic acid, and its use in the synthesis of
all of the possible terminal mono- and disaccharides
from Vibrio cholerae O1 LPS.
2. Results and discussion
3-Deoxy-L-glycero-tetronic acid with a suitable
protective group was prepared from L-malic acid 1 by
the sequence of reactions as described in Scheme 1.
Reaction of 1 with acetyl chloride gave the corre-
sponding anhydride 2, which was treated directly
Scheme 1.
Ž
with excess ethanol to afford the C-1 ester 3 79%
.
direct acylation with the corresponding lactone. Our
over two steps . The C-4 carboxylic function of 3
w
x
strategy 11 is based on the synthesis of a peracety-
lated L-glycero-tetronyl donor and its activation with
2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
was then reduced with diborane in tetrahydrofuran
Ž
.
™5, 86% , but attempts to remove the ethyl ester
by using either acidic or basic conditions proceeded
with a significant decomposition and probably the
formation of several lactonic byproducts. To over-
come this problem, we turned then to the benzyl ester
4 that was obtained in a similar fashion from 2. After
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.
EEDQ for the coupling with perosamine oligosac-
charidic derivatives.
In the present paper we describe full experimental
data for the synthesis of 2,4-di-O-acetyl-3-deoxy-L-
Scheme 2.

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A. Arencibia-Mohar et al.rCarbohydrate Research 306 1998 163–170
165
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.
reduction ™6, 53% over two steps , the resulting
Ž
hydroxyl group was protected by acetylation ™7,
.
83% to avoid lactonization. Hydrogenolysis of 7
afforded 8 in quantitative yield.
Two different 4-amino-4,6-dideoxy-D-mannose
derivatives 12 and 13 were selected for the synthesis
of terminal monosaccharide derivatives as occurring
in V. cholerae O1 LPS and two others 18 and 19 for
the synthesis of corresponding disaccharide elements
Ž
.
Scheme 2 . We reproduced with minor changes the
w
x
previously described 12 route to the perosamine
derivatives 9 and 10. The methylation of 10 with
methyl iodide–sodium hydride in N,N-dimethylform-
amide afforded 11 in 95% yield.
(
.
Fig. 1. Inhibition with synthetic fragments 27 -B- , 25
(
.
(
.
(
.
-x- , 26 -'- , 24 -o- of the reaction between anti-Ogawa
polyclonal serum and the homologous lipopolysaccharide.
w
x
Disaccharide derivative 15 12 was obtained by
condensation of 2-O-acetyl-4-azido-3-O-benzyl-4,6-
hibit the reaction of mouse polyclonal antibodies was
studied by an enzyme-linked immunosorbent assay
w
x
dideoxy-a-D-mannopyranosyl chloride 14 12 and
acceptor 10 in a silver triflate promoted glycosylation
Ž
.
ELISA . The antibodies obtained against the Ogawa
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.
reaction 60% . The presence of a small quantity of a
serotype were absorbed with Inaba LPS in order to
eliminate the fraction of antibodies directed to a
common antigen. The reaction in ELISA between the
remaining fraction and Ogawa LPS was then sepa-
rately inhibited with compounds 24, 25, 26 and 27.
As can be seen from Fig. 1, the reaction was inhib-
ited by the monosaccharide 25 and very strongly by
the disaccharide 27, demonstrating that 27 represents
the epitope recognized by most anti-Ogawa antibod-
ies.
w
x
transacetylated acceptor was also detected 13 . Disa-
ccharide derivative 17 was obtained from 15 after
Ž
.
deacetylation ™16 and methylation with methyl
iodide–sodium hydride in N,N-dimethylformamide
Ž
.
™17, 80% overall yield .
The azido groups in derivatives 9, 11, 16 and 17
were hydrogenated over 5% Pd–C in ethanol for 24 h
to afford the corresponding amines 12, 13, 18 and 19.
The transformation was ascertained by the shielding
13
Ž
of the C-4 C NMR signal 9™12, d 64.1™54.3;
11™13, d 64.2™53.6; 16™18, d 63.8, 63.7™
.
53.6, 53.2; 17™19, d 64.9, 64.8™53.7, 53.6 . It is
3. Experimental
w
x
noteworthy that under these conditions 11 the adja-
cent benzyl group in compounds 11, 16, and 17
General procedures.—Optical rotations were mea-
sured at 25 8C with a POLAMAT A automatic
polarimeter, using a 5-cm 5-mL cell. NMR spectra
were recorded at 25 8C with a Bruker AC-250F
w
x
remained unchanged as recently was also found 14 .
The hydrogenolysis proceeded more cleanly for com-
pound 16 than for the corresponding O-2 acetyl
derivative 15.
1
spectrometer. H and 13C assignments were made on
Condensation of 12, 13, 18 or 19 with 2,4-di-O-
acetyl-3-deoxy-L-glycero-tetronic acid 8 in the pres-
ence of EEDQ in dichloromethane afforded the ex-
the basis of homo- and heteronuclear correlation ex-
Ž .
periments. Chemical shifts
d
are given in ppm
1
relative to the signal for internal Me4Si for H, and
pected amides ™20, 81%; ™21, 85%; ™22, 57%;
indirectly to CDCl3 d 77.03 for 13C. The following
notation was used to define the NMR signals: p for
Ž
Ž
.
.
™23, 75% . Their structures were confirmed by
1
X
NMR spectroscopy which showed in the H NMR
perosamine, pX or just for the second perosamine
spectra one 20, 21 or two 22, 23 doublets at
unit, t and tX for the L-glycero-tetronyl moieties.
Ž
.
Ž
.
Ž
.
5.80–6.50 ppm J 5–6 Hz corresponding to the
NHCO proton and a doublet of doublet correspond-
ing to the tetronamide H-2 at 5.19–5.22 ppm. Hy-
drogenolysis of the benzyl groups of 21, 22, 23 with
5% Pd–C or acid hydrolysis of the isopropylidene
All compounds characterized were purified by col-
Ž
umn chromatography on Kieselgel 60 Fluka, -230
.
mesh ASTM and fractions were monitored by TLC
Ž
.
on Kieselgel 60F254 Merck . Detection was effected
by charring with aq 50% H2SO4 after examination
under UV light. Evaporations were conducted under
Ž
.
groups 20 followed by Zemplen deacetylation af-
forded 24, 25, 26, and 27, respectively.
Ž
.
reduced pressure at 50 8C bath .
Ž .
The ability of synthetic model compounds to in-
S -3-Acetoxy-3-ethoxycarbonyl propionic acid

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166
A. Arencibia-Mohar et al.rCarbohydrate Research 306 1998 163–170
Ž .
Ž
.
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.
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3 .—A solution of L-malic acid 1; 1 g, 7.46 mmol
y51.88 c 1.08, CHCl31; Rf 0.42 1:1 hexane–
Ž
.
.
Ž
.
Ž
in AcCl 6 mL was boiled under reflux for 24 h,
then concentrated and co-concentrated with toluene
EtOAc ; NMR CDCl3 : H, d 7.37–7.25 m, 5 H,
C6H5 , 5.20 dd, 1 H, H-2 , 3.76–3.60 m, 2 H,
.
Ž
.
.
Ž
Ž
.
( )
.
Ž
Ž
4=2 mL to give 2-O-acetyl-L-malic anhydride 2 .
CH2OH , 2.10 s, 3 H, Ac , 2.09–2.00 m, 2 H,
H-3a,b ; 13C, d 170.4 and 170.2 C5O , 135.0,
Ž
.
Ž
.
The residue was immediately dissolved in EtOH 6
mL and stirred for 24 h at room temperature. The
mixture was concentrated and distilled at 80–105 8C
to afford 3 1.20 g, 79% as a low melting solid;
.
Ž
.
Ž
.
128.4, 128.2, and 127.9 C6H5 , 69.4 C-2 , 66.9
Ž
.
Ž
.
Ž
.
Ž
.
PhCH2 , 57.7 CH2OH , 33.6 C-3 , 20.3 CH3CO .
Ž
.
Benzyl 2,4-di-O-acetyl-3-deoxy-L-glycero-tetronate
7 .—To a solution of 6 3.74 g, 14.8 mmol in
1
w x
Ž . Ž .
y20.68 c 1, CHCl3 ; NMR CDCl3 : H, d
Ž .
Ž
.
a
DŽ
Ž
.
Ž
.
Ž
.
5.45 t, 1 H, J2.3 6.0 Hz, H-3 , 4.23 q, 2 H, OCH2 ,
pyridine 50 mL cooled to 0–5 8C was added Ac2O
.
Ž
Ž
.
Ž
Ž
.
2.94 d, 2 H, H-2a,b , 2.15 s, 3 H, Ac , 1.28 t, 3 H,
CH3CH2 ; C, d 174.7 C-1 , 170.0 and 168.7
10 mL . The mixture was stirred for 24 h, concen-
trated and co-concentrated with toluene 3=10 mL .
Column chromatography 1:1 hexane–EtOAc of the
residue afforded 7, isolated as a syrup 3.60 g, 83% ;
13
.
.
Ž
.
Ž
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
C5O , 68.0 C-3 , 61.9 OCH2 , 35.8 C-2 , 20.4
CH3CO , 13.9 CH3 . Anal. Calcd for C8H12O6: C,
.
Ž
.
Ž
.
w x
a
Ž . Ž
y29.78 c 1, CHCl3 ; Rf 0.5 2:1 hexane–
1
46.07; H, 5.93. Found: C, 46.32; H, 5.78.
D
Ž .
.
Ž
.
Ž
S -3-Acetoxy-3-benzyloxycarbonyl propionic acid
EtOAc ; NMR CDCl3 : H, d 7.40–7.30 m, 5 H,
Ž .
.
Ž
.
Ž
.
4 .—To a solution of freshly prepared 2-O-acetyl-
C6H5 , 5.18 s, 2 H, PhCH2 , 5.16 dd, 1 H, H-2 ,
Ž
.
Ž
.
Ž
.
L-malic anhydride 2 3 g, 22.4 mmol in dry CH2Cl2
4.22–4.09 m, 2 H, H-4a,b , 2.13 and 2.00 2 s, each
3 H, 2 Ac , 2.24–1.97 m, 2 H, H-3a,b ; 13C, d
Ž
.
Ž
.
Ž
.
3 mL was added benzyl alcohol 2.3 mL, 2.23
.
Ž
Ž
mmol . The mixture was stirred for 24 h and concen-
trated. Column chromatography 2:1 hexane–EtOAc
of the residue afforded 4, isolated as a syrup 2.5 g,
83% ;
170.6, 170.1, 169.5 C5O , 135.0, 128.5, 128.3, and
128.0 C6H5 , 68.9 C-2 , 67.1 PhCH2 , 59.6 C-4 ,
Ž
.
Ž
.
.
Ž
.
Ž
.
Ž
Ž
.
Ž
.
30.0 C-3 , 20.6 and 20.5 CH3CO . Anal. Calcd for
C15H18O6: C, 61.21; H, 6.16. Found: C, 61.23; H,
6.46.
.
w x
Ž
.
Ž
a
y28.58 c 1, CHCl3 ; Rf 0.5 3:1
D
1
.
Ž
.
CHCl3–acetone ; NMR CDCl3 : H, d 7.35–7.30
Ž
.
Ž
.
Ž
m, 5 H, C6H5 , 5.51 t, 1 H, H-3 , 5.18 s, 2 H,
2,4-Di-O-acetyl-3-deoxy-L-glycero-tetronic acid
8 .—A suspension of 7 0.3 g, 1.09 mmol and 5%
Pd–C 45 mg in EtOH 5 mL was stirred under H2
for 20 h, filtered, and concentrated to yield 8 0.208
.
Ž
.
Ž
.
Ž .
Ž
.
PhCH2 , 2.93 d, 2 H, H-2a,b , 2.12 s, 3 H, Ac ;
13
Ž
.
Ž
.
Ž
.
Ž
.
C, d 174.5 C-1 , 170.0 and 168.6 C5O , 134.9,
Ž
.
Ž
.
Ž
128.6, 128.5, and 128.1 C6H5 , 67.9 C-3 , 67.5
Ž
.
Ž
.
Ž
.
.
.
.
w x
Ž
.
PhCH2 , 35.7 C-2 , 20.4 CH3CO . Anal. Calcd
g, 94% ;
a
y23.28 c 1.12, CHCl3 ; NMR
D
1
Ž
Ž
.
Ž
for C13H14O6: C, 58.64; H, 5.30. Found: C, 58.50; H,
CDCl3 : H, d 7.12 bs, 1 H, COOH , 5.12 dd, 1
H, H-2 , 4.24–4.18 m, 2 H, H-4a,b , 2.27–2.05 m,
2 H, H-3a,b , 2.15 and 2.05 2 s, each 3 H, 2 Ac ;
C, d 173.6 COOH , 171.3 and 170.6 C5O , 68.8
Ž
.
Ž
5.65.
Ž .
.
Ž
.
Ethyl 2-O-acetyl-3-deoxy-L-glycero-tetronate 5 .
—Compound 3 0.2 g, 0.98 mmol was dried in
vacuo for 2 h. A solution of borane tetrahydrofuran
complex 1 M; 3.4 mL, 3.43 mmol was added under
Ar, and the mixture was stirred for 1 h. The reaction
13
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.
Ž
.
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.
Ž
Ž
.
Ž
.
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.
C-2 , 59.8 C-4 , 30.0 C-3 , 20.7 and 20.4
Ž
.
.
CH3CO . Anal. Calcd for C8H12O6: C, 47.06; H,
5.93. Found: C, 47.35; H, 5.90.
Ž
.
was quenched with EtOH 1 mL , and the mixture
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-
Ž
Ž
.
was concentrated and co-concentrated with EtOH 2
methyl-a-D-mannopyranoside 11 .—To a solution of
.
Ž
.
w
x
Ž
.
Ž
.
mL and toluene 2=2 mL to afford 5, isolated as a
10 12 0.05 g, 0.17 mmol in DMF 0.5 mL was
added NaH 8 mg, 0.34 mmol, oil dispersion at 0 8C.
After 30 min, CH3I 13 mL, 0.204 mmol was added
at 0 8C, and the mixture was stirred for an additional
period of 15 min at 25 8C. Then MeOH was added to
destroy the excess of NaH. After concentration, a
Ž
.
w x
Ž
.
Ž
.
syrup 0.16 g, 86% ; a y25.58 c 0.9, CHCl3 ;
1
Ž
.
D Ž
.
.
Ž
Ž
Ž
.
NMR CDCl3 : H, d 5.16 dd, 1 H, H-2 , 4.22 q, 2
.
Ž
H, OCH2 , 3.85–3.65 m, 2 H, CH2OH , 2.15 s, 3
H, Ac , 2.13–2.05 m, 2 H, H-3a,b , 1.28 t, 3 H,
CH3 ; C, d 170.6 C5O , 69.7 C-2 , 61.6 OCH2 ,
.
Ž
Ž
.
.
Ž
Ž
13
.
.
Ž
Ž
.
Ž
.
Ž
.
.
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.
58.2 CH2OH , 33.9 C-3 , 20.6 CH3CO , 14.1
solution of the residue in CHCl3 5 mL was washed
Ž
.
Ž
.
Ž
.
CH3 . Anal. Calcd for C8H14O5: C, 50.51; H, 5.93.
Found: C, 50.20; H, 5.86.
Benzyl 2-O-acetyl-3-deoxy-L-glycero-tetronate 6 .
—Compound 4 0.3 g, 1.12 mmol was reduced as
described for the preparation of 5. Column chro-
with water 2 mL , dried Na2SO4 , and concen-
trated. Column chromatography toluene of the
residue afforded 11, isolated as a syrup 0.049 g,
Ž
.
Ž .
Ž
Ž
.
.
w x
Ž
.
Ž
95% ; a q105.38 c 1.1, CH2Cl2 ; Rf 0.56 3:1
D
1
.
Ž
.
.
Ž
hexane–EtOAc ; NMR CDCl3 : H, d 4.68 s, 1 H,
Ž
.
.
Ž
Ž
.
matography 1:1 hexane–EtOAc of the residue af-
w x
H-1 , 3.71 dd, 1 H, H-3 , 3.50–3.48 m, 2 H, H-2,4 ,
3.47 s, 3 H, OMe , 3.46–3.44 m, 1 H, H-5 , 3.32 s,
Ž
.
Ž
.
Ž
.
Ž
forded 6, isolated as a syrup 0.183 g, 64% ; a
D

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A. Arencibia-Mohar et al.rCarbohydrate Research 306 1998 163–170
167
3 H, OMe , 1.31 d, 3 H, 3 H-6 ; 13C, d 137.7,
X
.
Ž
.
.
Ž
.
Ž
J3,4 9.6 Hz, H-3 , 3.49 m, 1 H, H-5 , 3.47 t, 1 H,
X
Ž
.
Ž
Ž
.
.
Ž
.
Ž
X
X
128.4, 127.9, and 127.8 C6H5 , 98.4 C-1 , 78.2
C-3 , 76.4 C-2 , 71.9 PhCH2 , 66.9 C-5 , 64.2
C-4 , 59.3 C-2–OCH3 , 54.8 C-1–OCH3 , 18.4
J4 ,5 8.5 Hz, H-4 , 3.45 m, 1 H, H-5 , 3.31 s, 3 H,
Ž
Ž
Ž
.
Ž
Ž
.
Ž
.
.
.
.
Ž
.
Ž
OMe , 3.22 t, 1 H, J4,5 9.7 Hz, H-4 , 3.18 s, 3 H,
X
.
Ž
.
.
Ž
.
Ž
.
OMe , 1.31 d, 3 H, 3 H-6 , 1.30 d, 3 H, 3 H-6 ;
C, d 99.9 C-1 , 99.1 C-1 , 78.2 C-3 , 77.0
C-3 , 76.4 C-2 , 73.4 C-2 , 67.7 C-5 , 66.9
C-5 , 64.4 C-4 , 64.1 C-4 , 58.9 C-2–OCH3 ,
13
X
X
.
Ž
.
Ž
.
.
Ž
Ž
.
.
C-6 .
X
X
Ž
Ž
.
.
Ž
.
Ž
Methyl 4-amino-4,6-dideoxy-2,3-O-isopropylidene-
a-D-mannopyranoside 12 .—A solution of 9 12
660 mg, 3.04 mmol in EtOH 5 mL was stirred in
the presence of 5% Pd–C 200 mg under H2. After
X
Ž
.
w x
Ž
.
Ž
.
Ž
.
X
Ž
.
Ž
.
Ž
.
Ž
.
54.9 C-1–OCH3 , 18.6 and 18.5 C-6,6 . Anal.
Calcd for C28H36O7N6: C, 59.14; H, 6.38; N, 14.78.
Found: C, 59.42; H, 6.18; N, 14.85.
Ž
.
Ž
.
24 h, TLC 4:1 EtOAc–MeOH revealed a complete
(
conversion of the starting material into 12, isolated as
Methyl 4-amino-3-O-benzyl-4,6-dideoxy-a-D-
Ž
.
Ž
) (
)
a syrup 492 mg, 83% ; Rf 0.33 1:3 hexane–EtOAc,
mannopyranosyl - 1™2 -4-amino-3-O-benzyl-4,6-
1
.
Ž
Ž
.
Ž
Ž
.
ninhydrine positive ; NMR CDCl3 : H, d 4.88 s, 1
H, J1,2 1.4 Hz, H-1 , 4.06 d, 1 H, H-2 , 3.85 dd, 1
H, J2,3 5.5, J3,4 8.5 Hz, H-3 , 3.50 dq, 1 H, J4,5
10.4, J5,6 6.4 Hz, H-5 , 3.35 s, 3 H, OMe , 2.60 dd,
1 H, H-4 , 1.50 and 1.30 2 s, each 3 H, CMe2 , 1.20
d, 3 H, 3 H-6 ; C, d 109.0 CMe2 , 98.0 C-1 ,
56.5 C-4 , 54.6 OCH3 , 28.0 and 26.1 C CH3 2 ,
17.3 C-6 . Anal. Calcd for C10H19NO4: C, 55.28; H,
8.81. Found: C, 55.63; H, 9.12.
Methyl 4-amino-3-O-benzyl-4,6-dideoxy-2-O-
methyl-a-D-mannopyranoside 13 .—A solution of 11
70 mg, 0.23 mmol in EtOH was hydrogenated as
for the preparation of 12. Work-up then gave 13 48
mg, 75% ; Rf 0.35 1:3 hexane–EtOAc, ninhydrine
dideoxy-a-D-mannopyranoside 18 .—A solution of
16 12 40 mg, 0.079 mmol in EtOH was hydro-
genated as for the preparation of 12. Work-up then
gave 18 30 mg, 83% ; Rf 0.60 1:4 MeOH–EtOAc,
.
.
Ž
w
x
Ž
.
.
Ž
.
Ž
.
Ž
Ž
.
Ž
1
.
Ž
.
Ž
.
Ž
.
ninhydrine positive ; NMR CDCl3 : H, d 7.38–7.20
13
X
Ž
.
Ž
.
.
. x
Ž
.
Ž
.
X
X
m, 10 H, 2 C6H5 , 5.02 d, 1 H, J1 2 2.0 Hz, H-1 ,
X
Ž
Ž
.
Ž
.
w
Ž
Ž
.
Ž
.
4.70 d, 1 H, J1,2 2.0 Hz, H-1 , 4.09 dd, 1 H, H-2 ,
3.90 1 H, H-2 , 3.69–3.67 m, 2 H, H-3,3 , 3.52–
3.51 m, 2 H, H-5,5 , 3.32 s, 3 H, OMe , 2.90 dd,
X
.
Ž
.
Ž
Ž
.
X
Ž
.
.
Ž
X
.
Ž
.
Ž
1 H, H-4 , 2.85 dd, 1 H, H-4 , 1.35 and 1.30 2 d,
each 3 H, 3 H-6,6 ; 13C, d 137.7, 137.5, 128.5,
X
Ž
.
.
X
Ž
.
Ž
.
Ž
.
128.0 and 127.9 C6H5 , 101.0 and 100.2 C-1,1 ,
X
Ž
Ž
.
Ž
.
54.7 OCH3 , 53.6 and 53.2 C-4,4 , 18.2 and 18.0
X
.
Ž
Ž
.
C-6,6 .
1
.
Ž
.
Ž
(
positive ; NMR CDCl3 : H, d 7.40–7.20 m, 5 H,
Methyl 4-amino-3-O-benzyl-4,6-dideoxy-2-O-meth-
.
Ž
.
Ž
) (
)
C6H5 , 4.75 d, 1 H, J1,2 1.1 Hz, H-1 , 4.63 dd, 2
H, PhCH2 , 3.47 s, 3 H, OMe , 3.34 s, 3 H, OMe ,
yl-a-D-mannopyranosyl - 1™2 -4-amino-3-O-ben-
.
Ž
.
Ž
.
Ž
.
zyl - 4,6 - dideoxy - a - D - mannopyranosyde 19 .—A
Ž
.
Ž
Ž
.
2.94 dd, 1 H, J3,4 sJ4,5 s9.6 Hz, H-4 , 1.25 d, 3
solution of 17 52 mg, 0.1 mmol in EtOH was
H, 3 H-6 ; 13C, d 138.1, 128.5, 128.0, and 127.9
.
hydrogenated as for the preparation of 12. Work-up
then gave 19, isolated as a syrup 29 mg, 61% ; Rf
0.43 1:4 MeOH–EtOAc, ninhydrine positive ; NMR
CDCl3 : H, d 7.40–7.25 m, 10 H, 2 C6H5 , 5.02
d, 1 H, J1 2 1.9 Hz, H-1 , 4.73 d, 1 H, PhCH ,
4.70 d, 1 H, PhCH , 4.65 d, 1 H, J1 2 2.0 Hz, H-1 ,
4.55 d, 1 H, PhCH , 3.30 s, 3 H, OMe , 3.29 s, 3
Ž
Ž
Ž
.
Ž
Ž
.
Ž
.
.
Ž
.
Ž
.
C6H5 , 98.6 C-1 , 79.6 C-3 , 76.5 C-2 , 71.4
.
Ž
.
Ž
.
PhCH2 , 59.2 C-2–OCH3 , 54.7 C-1–OCH3 , 53.6
1
.
Ž
.
Ž
Ž
.
Ž
.
C-4 , 18.1 C-6 .
Methyl 4-azido-3-O-benzyl-4,6-dideoxy-2-O-methyl-
X
(
.
Ž
.
.
X
X
) (
)
Ž
Ž
.
.
Ž
X
X
a-D-mannopyranosyl - 1™2 -4-azido-3-O-benzyl-4,6-
Ž
.
Ž
.
Ž
dideoxy-a-D-mannopyranoside 17 .—To a solution
X
w
x
Ž
.
Ž
.
.
Ž
.
of 15 12 90 mg, 1.60 mmol in dry MeOH 1 mL
was added NaOMe to pH 9, and the mixture was
stirred for 30 min. Then the solution was neutralized
with Dowex-50 H resin, filtered, and concentrated
to afford 16. The residue was methylated as for the
preparation of 11 using CH3I 12 mL, 1.9 mmol and
DMF 8 mg, 3.20 mmol, 80% oil dispersion in
Me2 NCHO 1 mL . Column chromatography 20:1
H, OMe , 2.98–2.80 m, 2 H, H-4,4 , 1.33 and 1.29
2 d, each 3 H, 3 H-6,6 ; 13C, d 138.0, 137.9, 128.5,
X
Ž
.
X
Ž
.
Ž
Ž
.
128.0, and 127.8 C6H5 , 100.4 and 99.1 C-1,1 ,
q
Ž
.
Ž
.
.
58.9 C-2–OCH3 , 54.6 C-1–OCH3 , 53.8 and 53.6
X
X
Ž
.
Ž
.
C-4,4 , 18.2 and 18.1 C-6,6 .
Methyl 4- 2,4-di-O-acetyl-3-deoxy-L -glycero-
tetronamido -4,6-dideoxy-2,3-O-isopropylidene-a-D-
Ž
.
(
Ž
.
)
Ž
.
Ž
Ž
.
Ž
.
mannopyranoside 20 .—To a solution of 12 228
.
.
.
Ž
toluene–acetone of the residue afforded 17, isolated
as a syrup 74 mg, 80% ; a
CHCl3 ; Rf 0.56 20:1 toluene–acetone ; NMR
mg, 0.97 mmol and 8 398 mg, 1.94 mmol in
CH2Cl2 4 mL was added EEDQ 479 mg, 1.94
Ž
.
w x
Ž
Ž
Ž
q58.58 c 1.06,
D
.
Ž
.
.
Ž
mmol , and the mixture was stirred for 15 min. Then,
TLC 1:3 hexane–EtOAc showed the absence of 12
Rf 0.33 and a new spot Rf 0.50 . After concentra-
tion, column chromatography 1:1 hexane–EtOAc of
1
Ž
.
Ž
.
.
CDCl3 : H, d 7.37–7.26 m, 10 H, 2 C6H5 , 4.89
X
Ž
.
Ž
.
X
X
Ž
.
Ž
.
d, 1 H, J1 ,2 1.8 Hz, H-1 , 4.70 d, 2 H, PhC H2 ,
4.63 d, 2 H, PhCH2 , 4.49 d, 1 H, J1,2 1.6 Hz,
H-1 , 3.92 dd, 1 H, J2,3 2.4 Hz, H-2 , 3.71 dd, 1 H,
Ž
.
Ž
Ž
.
.
Ž
.
Ž
Ž
the residue afforded 20, isolated as a syrup 335 mg,

(
)
168
A. Arencibia-Mohar et al.rCarbohydrate Research 306 1998 163–170
X
.
w x
Ž
.
Ž
.
Ž
.
81% ; a y37.78 c 1.06, CH2Cl2 ; Rf 0.50 3:1
Hz, H-1p , 4.70 d, 1 H, J1p,2p 1.8 Hz, H-1p , 4.68
D
1
.
Ž
.
Ž
Ž
.
Ž
EtOAc–hexane ; NMR CDCl3 : H, d 6.37 d, 1 H,
NHCO , 5.23 dd, 1 H, H-2t , 4.85 s, 1 H, J1p,2p 1.4
Hz, H-1p , 4.23–4.07 m, 3 H, H-3p,2p,4t , 3.86 dd,
1 H, H-4p , 3.74 dq, 1 H, H-5p , 3.40 s, 3 H, OMe ,
d, 1 H, J 11.3 Hz, PhCH2 , 4.62 d, 1 H, J 11.5
Hz, PhCH , 4.52 d, J 11.5 Hz, PhCH , 4.46 d, 1
H, J 11.3 Hz, PhCH2 , 4.22–3.98 m, 9 H, H-
2p,2p ,3p,4p,4p ,4t,4t , 3.90–3.60 m, 3 H, H-
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
X
X
X
.
Ž
.
Ž
.
.
Ž
X
Ž
.
Ž
.
Ž
.
Ž
2.35–2.16 m, 1 H, H-3t , 2.15 and 2.05 2 s, each 3
3p,5p,5p , 3.30 s, 3 H, OMe , 2.35–2.10 m, 4 H,
X
.
Ž
.
.
Ž
.
H, 2 Ac , 1.53 and 1.33 2 s, each 3 H, CMe2 1.24
H-3t,3t , 2.05–1.90 m, 12 H, 4 Ac , 1.20 and 1.05
d, 3 H, 3 H-6p ; 13C, d 170.8, 169.6, and 169.1
2 d, each 3 H, 3 H-6p,6p ; 13C, d 171.0, 169.7, and
X
Ž
.
Ž
.
X
Ž
.
Ž
.
Ž
.
Ž
Ž
.
.
Ž
.
C5O , 109.6 CMe2 , 98.3 C-1p , 74.7 C-3p ,
74.4 C-2p , 71.1 C-2t , 65.8 C-5p , 59.9 C-4t ,
169.6 C-1t,1t and 4CH3CO , 137.6, 128.6, 128.0,
and 127.8 C6H5 , 101.1 and 100.1 C-1p,1p , 68.1
and 67.8 C-2t,2t , 60.1 and 60.0 C-4t,4t , 55.1
OCH3 , 52.3 and 52.2 C-4p,4p , 31.0 and 30.9
X
Ž
.
Ž
Ž
.
.
.
Ž
.
Ž
Ž
.
Ž
.
.
X
X
Ž
.
Ž
.
.
Ž
55.0 OCH3 , 52.9 C-4p , 30.7 C-3t , 27.5 and 25.9
X
Ž
.
Ž
Ž
.
Ž
Ž
.
Ž
.
2 CH3 , 20.7 CH3CO , 18.2 C-6p . Anal. Calcd
for C18H29O9N: C, 53.59; H, 7.24; N, 3.47. Found:
C, 53.32; H, 7.82; N, 3.22.
Methyl 4- 2,4-di-O-acetyl-3-deoxy-L -glycero-
tetronamido -3-O-benzyl-4,6-dideoxy-2-O-methyl-a-D-
X
.
Ž
.
C-3t,3t , 20.9, 20.8, and 20.6 CH3CO , 18.0 and
X
Ž
.
17.8 C-6p,6p . Anal. Calcd for C43H58O18N2: C,
59.03; H, 6.68; N, 3.20. Found: C, 58.75; H, 6.80; N,
3.29.
(
)
Ž
.
[
(
mannopyranoside 21 .—The reaction was per-
Methyl 4- 2,4-di-O-acetyl-3-deoxy-L -glycero-
Ž
)
formed as for the preparation of 20, using 13 54 mg,
0.19 mmol , 8 0.75 mg, 0.37 mmol , and EEDQ 93
mg, 0.37 mmol in CH2Cl2 1 mL . Column chro-
matography 1:1 hexane–EtOAc of the residue af-
tetronamido -3-O-benzyl-4,6-dideoxy-2-O-methyl-a-D-
.
Ž
.
Ž
] (
)
(
mannopyranosyl - 1™2 -4- 2,4-di-O-acetyl-3-deoxy-L
.
Ž
.
)
-glycero-tetronamido -3-O-benzyl-4,6-dideoxy-a-D-
Ž
.
Ž
Ž
.
mannopyranoside 23 .—The reaction was per-
formed as for the preparation of 20, using 19 70 mg,
0.14 mmol , 8 110 mg, 0.54 mmol , EEDQ 134 mg,
0.54 mmol in CH2Cl2 2 mL . Column chromatog-
.
Ž
forded 21, isolated as a syrup 76 mg, 85% ; mp
w x
Ž
.
.
.
Ž
.
Ž
161.1–163.6 8C; a q3.178 c 0.63, CHCl3 ; Rf
D
0.55 4:1 EtOAc–hexane ; NMR CDCl3 : 1H, d
Ž
.
Ž
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
7.50–7.22 m, 10 H, 2 C6H5 , 5.90 d, 1 H, NHCO ,
5.17 dd, 1 H, H-2t , 4.73 d, 1 H, J1p,2p 1.3 Hz,
H-1p , 4.68–4.45 2 d, each 1 H, PhCH2 , 4.10 m, 2
H, H-4t , 3.95–3.80 m, 3 H, H-3p,4p,5p , 3.59 s, 1
raphy 1:3 hexane–EtOAc of the residue afforded
Ž
.
Ž
Ž
. w x
23, isolated as a syrup 90 mg, 75% ; a y17.88
D
.
Ž
.
.
Ž
Ž
Ž
.
.
c 1, CH2Cl2 ; Rf 0.73 2:1 CH2Cl2–acetone ; NMR
1
.
Ž
Ž
.
Ž
.
CDCl3 : H, d 7.45–7.20 m, 10 H, 2 C6H5 , 5.88
X
.
Ž
.
Ž
.
Ž
H, H-2p , 3.50 and 3.30 2 s, each 3 H, 2 OMe , 2.15
and 5.85 2 d, each 1 H, NH, NH , 5.20 dd, 2 H,
X
X
Ž
.
Ž
.
.
Ž
.
X
X
m, 2 H, H-3t , 2.10 and 2.00 2 s, each 3 H, 2 Ac ,
H-2t,2t , 4.96 d, 1 H, J1p ,2p 2.5 Hz, H-1p , 4.72
d, 1 H, J 11.25 Hz, PhCH , 4.66 d, 1 H, J1p,2p 2.5
1.22 d, 3 H, 3 H-6p ; 13C, d 170.8, 169.5, and 169.2
Ž
.
Ž
.
Ž
Ž
Ž
.
Ž
.
.
Ž
.
C5O , 138.1, 128.4, 128.0, and 127.7 C6H5 , 98.7
Hz, H-1p , 4.66 d, 1 H, J 11.25 Hz, PhCH ,
X
X
X
X
.
Ž
.
Ž
Ž
.
Ž
Ž
.
C-1p , 76.1 C-2p , 75.6 C-3p , 71.1 C-2t and
4.20–4.00 m, 8 H, H-3p ,4p,4p ,2p ,4t,4t , 3.83–
3.60 m, 4 H, H-2p,3p,5p,5p , 3.33 and 3.30 2 s,
each 3 H, 2 OMe , 2.40–2.20 m, 4 H, H-3t,3t ,
X
.
Ž
.
.
Ž
.
Ž
.
Ž
PhCH2 , 67.3 C-5p , 60.0 C-4t , 59.3 C-4p , 59.2
C2–OCH3 , 54.3 C1–OCH3 , 30.9 C-3t , 20.8 and
20.7 2 CH3CO , 18.0 C-6p . Anal. Calcd for
C23H33O9N: C, 59.09; H, 7.11; N, 3.00. Found: C,
58.90; H, 7.35; N, 2.91.
X
Ž
.
Ž
.
.
Ž
.
.
Ž
.
Ž
Ž
.
Ž
.
2.15, 2.08, 2.00, and 1.95 4 s, each 3 H, 4 Ac , 1.29
and 1.19 2 d, each 3 H, 3 H-6p,6p ; 13C, d 171.0,
X
Ž
.
Ž
.
.
169.9, and 169.5 C5O , 138.0, 137.9, 128.5, 128.0,
and 127.6 C6H5 , 100.2 and 99.6 C-1p,1p , 60.1
and 59.9 C-4t,4t , 59.2 C2–OCH3 , 55.1 C1–
OCH3 , 52.4 and 52.1 C-4p,4p , 31.1 and 30.9
C-3t,3t , 20.9, 20.9, and 20.8 CH3CO , 18.0 C-
6p,6p . Anal. Calcd for C44H60O17N2: C, 59.45; H,
6.80; N, 3.15. Found: C, 59.19; H, 7.19; N, 3.21.
Methyl 4- 3-deoxy-L -glycero-tetronamido -4,6-
dideoxy-a-D-mannopyranoside 24 .—A solution of
20 272 mg, 0.67 mmol in aq 90% CF3COOH was
stirred at ambient temperature for 15 min. Then, TLC
3:1 EtOAc-hexane showed the reaction to be com-
plete. The mixture was concentrated and co-con-
centrated with toluene 3=5 mL . To a solution of
X
[
(
Ž
Ž
.
Methyl 4- 2,4-di-O-acetyl-3-deoxy-L-glycero-
tetronamido - 3 - O - benzyl - 4,6 - dideoxy - a - D -
X
)
Ž
.
Ž
.
Ž
X
] (
)
(
.
Ž
.
mannopyranosyl - 1™2 -4- 2,4-di-O-acetyl-3-deoxy-L
X
)
Ž
.
Ž
.
Ž
-glycero-tetronamido -3-O-benzyl-4,6-dideoxy-a-D-
X
Ž
.
.
mannopyranoside 22 .—The reaction was per-
Ž
formed as for the preparation of 20, using 18 70 mg,
0.14 mmol , 8 113 mg, 0.55 mmol , and EEDQ 136
mg, 0.55 mmol in CH2Cl2 2 mL . Column chro-
matography 1:4 hexane–EtOAc of the residue af-
forded 22, isolated as a syrup 300 mg, 57% ; a
q498 c 1, CHCl3 ; Rf 0.33 2:1 CH2Cl2–acetone ;
NMR CDCl3 : H, d 7.40–7.20 m, 10 H, 2 C6H5 ,
.
Ž
.
Ž
(
)
.
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
w x
D
Ž
Ž
.
Ž
.
.
Ž
.
1
.
Ž
Ž
.
Ž
.
Ž
6.11 and 5.91 2 d, each 1 H, 2 NHCO , 5.20 and
5.18 2 dd, each 1 H, H-2t,2t , 4.98 d, 1 H, J1 ,2 1.8
X
Ž
.
Ž
X
X
.
the crude residue in dry MeOH 2 mL was added

(
)
A. Arencibia-Mohar et al.rCarbohydrate Research 306 1998 163–170
169
X
X
Ž
.
Ž
.
Ž
NaOMe to pH 9, and the mixture was stirred for 15
C-2t,2t , 69.3 C-2p , 68.7, 68.7, and 68.4 C-
X
X
X
.
Ž
.
Ž
.
min. Then the solution was neutralized with Dowex-
3p,3p ,5p,5p , 59.0 C-4t,4t , 56.1 OCH3 , 54.2
C-4p , 54.0 C-4p , 37.2 C-3t , 18.1 C-6p,6p .
X
X
X
q
Ž
.
Ž
.
Ž
.
Ž
.
Ž
.
50 H resin, filtered, and concentrated. The residue
[
(
)
was dissolved in water and freeze dried to give 24
Methyl 4- 3-deoxy-L-glycero-tetronamido -4,6-
Ž
Ž
Ž
Ž
.
w x
Ž
.
] (
)
163 mg, 87% ; a q208 c 1, water , lit. q348
dideoxy-2-O-methyl-a-D-mannopyranosyl - 1™2 -4-
D
.
w
x
Ž
.
(
)
water 11,12 ; Rf 0.33 5:1 EtOAc–MeOH ; NMR
D2O : H, d 4.76 s, 1 H, J1p,2p 1.5 Hz, H-1p , 4.35
3-deoxy-L-glycero-tetronamido -4,6-dideoxy-a-D-
mannopyranoside 27 .—The reaction was per-
formed as for the preparation of 25, using 23 30 mg,
1
.
Ž
.
Ž
.
.
Ž
.
Ž
Ž
dd, 1 H, H-2t , 4.00 d, 1 H, H-2p , 3.98–3.88 m, 3
.
Ž
.
Ž
.
Ž
.
Ž
.
H, H-3p,4p,5p , 3.83 t, 2 H, H-4t , 3.46 s, 3 H,
OMe , 2.20–2.02 m, 1 H, H-3ta , 2.02–1.84 m, 1
H, H-3tb , 1.26 d, 3 H, 3 H-6p ; C, d 102.1 C-1p,
JC1,H1 170.7 Hz , 70.4 C-2p , 70.2 C-2t , 69.2
Ž
Ž
34 mmol and 5% Pd–C 15 mg in EtOH 1 mL to
.
Ž
.
Ž
Ž
x
.
w x
Ž
.
afford 27 18 mg, 98% ; a D q1.18 c 1, water , lit.
13
1
.
Ž
.
Ž
w
Ž
.
Ž
X
X
q2.78 10 ; NMR D2O : H, d 5.22 d, 1 H, J1p ,2p
X
.
Ž
.
Ž
.
.
Ž
.
.
1.7 Hz, H-1p , 4.85 d, 1 H, J1p,2p 2.0 Hz, H-1p ,
X
X
.
.
Ž
.
Ž
.
.
Ž
.
Ž
.
Ž
C-3p , 68.4 C-5p , 59.1 C-4t , 56.0 OCH3 , 54.1
C-4p , 37.2 C-3t , 18.1 C-6p .
Methyl 4- 3-deoxy-L -glycero-tetronamido -4,6-
4.35 dd, 2 H, H-2t,2t , 4.15 dd, 1 H, H-3p ,
4.10–3.95 m, 6 H, H-2p,5p ,3p ,4p,4p ,5p , 3.85–
X
X
X
X
Ž
.
Ž
Ž
.
X
X
(
)
Ž
.
Ž
3.71 m, 3 H, H-2p ,4t,4t , 3.55 and 3.45 2 s, each 3
X
Ž
.
.
Ž
.
dideoxy-2-O-methyl-a-D-mannopyranoside 25 .—A
solution of 21 154 mg, 0.33 mmol in EtOH 2 mL
was stirred in the presence of 5% Pd–C 0.1 mg
under H2. After 24 h, TLC 1:4 hexane–EtOAc
revealed a complete conversion of the starting mate-
rial Rf 0.9 into a new spot Rf 0.21 . The mixture
was filtered, concentrated, and dissolved in dry MeOH
H, 2 OMe , 2.15–2.00 m, 2 H, H-3ta,3ta , 2.00–1.85
X
Ž
.
Ž
.
.
.
Ž
.
Ž
m, 2 H, H-3tb,3tb , 1.30 and 1.25 2 d, each 3 H, 3
H-6p,6p ; C, d 178.6 and 178.4 C-1t,1t , 100.2
X
13
X
Ž
.
Ž
.
X
X
Ž
Ž
.
Ž
.
Ž
.
and 100.7 C-1p,1p , 80.1 C-2p , 79.1 C-2p , 70.1
X
X
X
Ž
.
Ž
.
Ž
.
Ž
C-2t,2t , 69.1 C-3p , 68.6 C-3p ,5p,5p , 59.9 C-
X
Ž
.
Ž
.
.
Ž
.
Ž
.
2–OCH3 , 59.0 C-4t,4t , 56.1 C-1–OCH3 , 54.4
X
X
X
Ž
.
Ž
.
Ž
.
Ž
.
C-4p , 54.2 C-4p , 37.1 C-3t,3t , 18.0 C-6p,6p .
Ž
.
1 mL , NaOMe was added to pH 9, and the mixture
was stirred for 15 min. Then the solution was neutral-
Inhibition reaction.—A pool of sera obtained from
male BALBrC mice, immunized with V. cholerae
q
Ž
.
Ž
. w x
ized with Dowex-50 H resin, filtered, and concen-
trated. The residue was dissolved in water and freeze
dried to give 25 0.090 g, 93% ; a q16.68 c
0.96, MeOH ; Rf 0.36 5:1 EtOAc–MeOH ; NMR
Ž
Ž
O1 serotype Ogawa strain E 7946 15 was ab-
sorbed with 0.25 mgrmL of LPS serotype Inaba
Ž
.
w x
Ž
Ž
.
strain 569 B by incubation at 37 8C for 2 h to
D
.
Ž
.
eliminate cross reactions. The strains were from the
1
.
Ž
.
Ž
.
D2O : H, d 5.00 d, 1 H, J1p,2p 1.7 Hz, H-1p , 4.38
Finlay Institute collection Havana, Cuba .
.
Ž
.
Ž
dd, 1 H, H-2t , 4.04 dd, 1 H, H-3p , 3.93–3.81 m,
Compounds 24, 25, 26 and 27 were dissolved in
water, adjusted to the following concentration: 0.063,
0.0316, 0.0157, and 0.0078 mmol, and added to the
pool of absorbed sera diluted 1r1000 in 1% skim
milk in phosphate-buffered saline PBS, pH 7.4 –
Tween 20. The mixture was incubated for 2 h at 37
8C and then analyzed by ELISA. Briefly, the 96-well
flat-bottom microdilution plates Flow Laboratories
were coated with 0.1 mL of LPS 27 mgrmL, from
both serotypes in carbonaterbicarbonate buffer
overnight at 37 8C. After the plates were washed four
times with water–Tween, skim milk was added at a
concentration of 2% in PBS and the plates were kept
at 37 8C for 1 h. After the skim milk solution was
discarded, the samples were added and incubated for
1 h at 37 8C. The plates were washed as described
above, 100 mL of Peroxidase-conjugated rabbit anti-
mouse immunoglobulin Sigma diluted 1r1000 in
1% skim milkrPBS was added and incubated for 1 h
at 37 8C. Then, the plates were washed and 100 mL
of substrate solution H2O2–o-phenylenediamine in
phosphate–citrate buffer, pH 5.0 was added to each
.
Ž
.
.
4 H, H-5p,4p,4t , 3.67 dd, 1 H, H-2p , 3.59 and 3.51
Ž
Ž
.
.
2 s, each 3 H, 2 OMe , 2.20–2.05 m, 1 H, H-3ta ,
Ž
.
Ž
Ž
2.02–1.85 m, 1 H, H-3tb , 1.35 d, 3 H, 3 H-6p ;
13
Ž
.
.
Ž
.
Ž
.
C, d 178.4 C5O , 99.0 C-1p , 80.3 C-2p , 70.2
C-2t , 68.9 C-3p , 68.3 C-5p , 60.1 C-2–OCH3 ,
Ž
.
Ž
.
Ž
.
.
Ž
.
Ž
.
Ž
Ž
.
59.1 C-4t , 56.1 C-1–OCH3 , 54.5 C-4p , 37.2
Ž
.
Ž
.
Ž
.
C-3t , 18.1 C-6p .
Methyl 4- 3-deoxy-L-glycero-tetronamido -4,6-
[
(
)
Ž
] (
)
(
.
dideoxy-a-D-mannopyranosyl - 1™2 -4- 3-deoxy-L-
)
glycero-tetronamido - 4,6-dideoxy-a-D-mannopyrano-
Ž
.
side 26 .—The reaction was performed as for the
preparation of 25, using 22 70 mg, 0.08 mmol and
5% Pd–C 40 mg in EtOH 2 mL to afford 26 35
Ž
.
Ž
.
Ž
.
Ž
.
w x w x
Ž
.
mg, 83% ; a q1.08 c 1, water , lit. 08 9 ; NMR
D
1
X
Ž
.
Ž
.
Ž
D2O : H, d 5.10 s, 1 H, H-1p , 4.85 d, 1 H,
X
.
Ž
.
J1p,2p s2.0 Hz, H-1p , 4.35 dd, 2 H, H-2t,2t , 4.15
X
Ž
.
Ž
Ž
.
dd, 1 H, H-2p , 4.10–3.85 m, 6 H, H-
X
X
X
.
.
Ž
.
.
3p,3p ,2p,4p,5p,5p , 3.80–3.70 m, 2 H, H-4t,4t ,
X
Ž
Ž
3.95 s, 3 H, OMe , 2.15–2.00 m, 2 H, H-3ta,3ta ,
2.00–1.80 m, 2 H, H-3tb,3tb , 1.30 and 1.25 2 d,
each 3 H, 3 H-6p,6p ; C, d 178.5 C-1t,1t , 103.4
and 100.8 C-1p,1p , 79.0 C-2p , 70.4 C-2p , 70.2
X
Ž
.
Ž
Ž
X
13
X
.
Ž
.
.
X
X
Ž
.
Ž
.
Ž
.
well and incubated for 15 min at room temperature in

(
)
170
A. Arencibia-Mohar et al.rCarbohydrate Research 306 1998 163–170
w x
7 T. Ito, T. Higuchi, M. Hirobe, K. Hiramatsu, and T.
the dark. The reaction was stopped with 100 mL 2.5
M H2SO4. The optical density at 450 nm was mea-
Ž
.
Yokota, Carbohydr. Res., 256 1994 113–128.
w x
8 L. Kenne, P. Unger, and T. Wehler, J. Chem. Soc.,
Ž
.
sured in an ELISA reader Multiskan, Titertek .
Ž
.
Perkin Trans., 1 1988 1183–1196; P. Lei, Y. Ogawa,
J.L. Flippen-Anderson, and P. Kovac, Carbohydr.
Ž
.
Res., 275 1995 117–129; M. Gotoh and P. Kovac,
Carbohydr. Res., 268 1995 73–84; M. Gotoh, C.L.
Acknowledgements
Ž
.
Ž
.
Barnes, and P. Kovac, Carbohydr. Res., 260 1994
203–218.
We wish to recognize the financial support of the
w x
9 M. Gotoh and P. Kovac, J. Carbohydr. Chem., 13
Ž
.
Cuban Government, the Finlay Institute Havana and
especially Mrs. Asela Aguiar Sanchez for performing
Ž
.
1994 1193–1213.
´
w
w
x
10 P. Lei, Y. Ogawa, and P. Kovac, Carbohydr. Res.,
elemental analyses and Ing. Jose Fernandez Santana
´
´
Ž
.
279 1995 117–131; P. Lei, Y. Ogawa, and P.
for recording the NMR spectra.
Ž
.
Kovac, Carbohydr. Res., 281 1996 47–60; Y.
Ogawa, P. Lei, and P. Kovac, Bioorganic. Med.
Ž
.
Chem. Lett., 5 1995 2283–2286.
x
11 A. Arencibia-Mohar, O. Madrazo-Alonso, A. Ariosa-
Alvarez, J. Sarracent-Perez, M. Alfonso, J.L. Perez,
M. Ramirez, R. Montes, and V. Verez-Bencomo,
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