Synthesis of C-8 deuterated glycosides of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) related to chlamydial lipopolysaccharides

Article abstract of DOI:10.1007/s007060200029

Methyl glycosides of Kdo and a (2→8)-linked Kdo disaccharide were prepared which contain a deuterium label at C-8 of the reducing unit. The label was introduced in fair diastereoselectivity upon reduction of an aldehyde group using a chiral borane complex derived from N-benzyloxycarbonyl-(S)-proline which produced the 8-(S)-deuterated derivative as the major isomer. Further coupling with a Kdo bromide gave the α-(2→8)-linked disaccharide in good yield. The deprotected disaccharide serves as a model for NMR spectroscopic studies on the side chain conformation of a carbohydrate epitope from the bacterial pathogen Chlamydia.

Full text of DOI:10.1007/s007060200029

Monatshefte fuÈr Chemie 133, 561±570 ꢀ2002)
Synthesis of C-8 Deuterated Glycosides
of 3-Deoxy-D-manno-oct-2-ulosonic Acid
ꢀKdo) Related to Chlamydial
Lipopolysaccharides
Paul Kosma1;Ã, Martina Strobl1, Andreas Ho®nger1, Jens é. Duus2,
Bent O. Petersen2, Klaus Bock2, and Helmut Brade3
1



Institut fur Chemie, Universitat fur Bodenkultur, A-1190 Wien, Austria
Department of Chemistry, Carlsberg Laboratory, DK-2500 Valby, Denmark
Forschungszentrum Borstel, Institut fur Medizin und Biowissenschaften,
D-23845 Borstel, BRD
2
3
Summary. Methyl glycosides of Kdo and a ꢀ2 ! 8)-linked Kdo disaccharide were prepared which
contain a deuterium label at C-8 of the reducing unit. The label was introduced in fair dias-
tereoselectivity upon reduction of an aldehyde group using a chiral borane complex derived from N-
benzyloxycarbonyl-ꢀS)-proline which produced the 8-ꢀS)-deuterated derivative as the major isomer.
Further coupling with a Kdo bromide gave the ꢀ-ꢀ2 ! 8)-linked disaccharide in good yield. The
deprotected disaccharide serves as a model for NMR spectroscopic studies on the side chain con-
formation of a carbohydrate epitope from the bacterial pathogen Chlamydia.
Keywords. Carbohydrate; Reduction; Kdo; Deuterium labeling; Chlamydia.
Introduction
Members of the order Chlamydiales are intracellular parasites of increasing
biomedical importance [1]. In the outer membrane of the cell, the bacteria contain
a lipopolysaccharide ꢀLPS) which constitutes a highly immunogenic epitope and
induces in¯ammatory responses in a host organism [2]. The epitope is composed of
a trisaccharide of 3-deoxy-D-manno-oct-2-ulopyranosylonic acid ꢀKdo) residues of
the sequence ꢀ-Kdop-ꢀ2 ! 8)-ꢀ-Kdop-ꢀ2 ! 4)-ꢀ-Kdop which may be exploited for
the serological diagnosis of chlamydial infections [3]. Previously, the terminal
disaccharide unit of the epitope has been synthesized as an allyl glycoside, and the
crystal structure has been determined [4, 5]. The structural data revealed the
presence of an interglycosidic hydrogen bond extending from the terminal carboxyl
group to OH-7 of the reducing Kdo unit. Although the side chain linkage between
the Kdo moieties inherently confers additional conformational freedom, it
Ã
Corresponding author. E-mail: pkosma@edv2.boku.ac.at

562
P. Kosma et al.
nonetheless is part of a speci®c epitope which is recognized by monoclonal
antibodies. The binding interaction of the disaccharide with Kdo-speci®c
monoclonal antibodies has been thoroughly studied using NOE experiments [6]
and immunochemical methods [7]. Meanwhile, preliminary crystal data have been
obtained from a complex of the disaccharide ligand with the Fab fragment of a
ꢀ2 ! 8)-linked Kdo disaccharide speci®c monoclonal antibody [8].
The determination of the solution conformation of the disaccharide ligand,
however, has been hampered by the fact that the geminal protons at C-8 of the re-
ducing Kdo unit occur at identical chemical shifts in the H NMR spectrum
1
[9, 10]. Hence we have set out to synthesize Kdo glycosides deuterated at C-8 in order
to be able to differentiate between H-8-ꢀR) and H-8-ꢀS) to be used for NOESY and
ROESY experiments and conformational studies of the hydroxymethyl groups of
Kdo-glycosides [11].
Results and Discussion
Synthesis of glycosides
For the synthesis of the deuterated Kdo glycosides, the previously reported 8-O-
trityl-derivative 1 [12] was O-benzylated using O-benzyl trichloroacetimidate=tri-
¯ic acid [13] to give the 7-O-benzyl derivative 2 in 70% yield. Removal of the trityl
group with formic acid in MeCN proceeded smoothly in 84% yield. Subsequent
Swern oxidation [14] of 3 afforded the aldehyde 4 in 87% yield.
Numerous methods have been developed for the stereoselective reduction of
carbonyl groups, which in general are far less selective for aldehydes compared to
keto groups [15]. Low diastereoselectivities were observed in the reduction of
compound 4 with deuterated boranes ꢀTable 1). The outcome of the reaction was
based on the integration values of the H-8-ꢀR) and H-8-ꢀS) signals in the 1H NMR
spectra. The use of deuterated borane complexes prepared from NaBD4 and D-
mannitol, ꢀ-)-menthol, or ꢀR)- and ꢀS)-mandelic acid did not result in selective
formation of the deuterated hydroxymethyl group. The same held true for other
chiral borane complexes derived from phenyl 1,3-dioxaborolidine and an ꢀR)-
proline complex [16, 17]. By contrast, reduction of 4 at À 126ꢀC in THF with a
complex prepared from NaBD4 and N-benzyloxycarbonyl-ꢀS)-proline [17]
proceeded in excellent yield ꢀ99%) and with fair diastereoselectivity ꢀ ꢁ 56% de)
Table 1. Diastereoselectivities observed upon reduction of 4 with deuterated boranes
T=ꢀC
Solvent
Reagent
Ratio 5a:5b
À 70
À 126
À 126
À 126
À 126
À 126
THF
THF
THF
THF
THF
THF
NaBD4, ꢀS)-mandelic acid
NaBD4, N-Fmoc-ꢀR)-proline
NaBD4, N-Fmoc-ꢀS)-proline
NaBD4, N-Cbz-ꢀR)-proline
NaBD4, N-Cbz-ꢀS)-proline
2H-Catecholborane=B-n-butyl-
oxazaborolidine [15]
45:55
50:50
37:63
50:50
22:78 ꢀde ˆ 56)
60:40

Synthesis of C-8 Deuterated Kdo
563
Scheme 1
to give a mixture of 5a and 5b. For the assignment of the H-8-ꢀR) and H-8-ꢀS)
protons, the mixture of diastereoisomers 5a and 5b was subsequently converted
into a cyclic 7,8-O-carbonate ester following quantitative removal of the
benzyl group by hydrogenolysis on 5% Pd±C. Reaction of the resulting diol with
diphosgene=sym-collidine in THF at low temperature gave the 4,5;7,8-di-O-
carbonyl derivatives 6a and 6b in 40% yield ꢀ2 steps). Irradiation of H-6 lead to a
signi®cant nuclear Overhauser enhancement for the H-8 signal observed at
4.62 ppm, whereas the second ꢀminor) H-8 signal at 4.66 ppm remained unaffected.
2
Thus, the major isomer was assigned as the H-8-ꢀS) substituted isomer 6b, since
only H-8 corresponding to H-8-pro-R in the non-labeled compound would be close
to H-6. For comparison, compounds 6a and 6b were fully deprotected by Zemplꢀen
de-O-acylation and subsequent hydrolysis of the methyl ester group with aqueous
1
0.2 M NaOH. The H NMR data of the methyl ketoside 7b are in good agree-
ment with reported data for an 8-ꢀS) deuterated Kdo ammonium salt [18]. In the
ꢀ-pyranose form of the latter compound, H-8-ꢀR) was observed at 3.62 ppm
ꢀJ7,8ꢀR) ˆ 5.8 Hz), which closely matches the corresponding values found for isomer
7b ꢀ3.64 ppm, J7,8ꢀR) ˆ 6.5 Hz).
Glycosylation of 5a and 5b with 2.5 equivalents of Kdo bromide donor 8 using
HgꢀCN)2 in MeNO2 furnished the glycal ester 12 and the disaccharides 9 and 11
in 85% yield ꢀbased on 5, ꢀ=ꢁ ˆ 5:1) which were separated by chromatography.

564
P. Kosma et al.
Crystallization of the anomeric disaccharide mixture afforded the crystalline ꢀ-
ꢀ2 ! 8)-linked disaccharides 9a and 9b. The assignment of the anomeric
con®guration of the terminal Kdo unit was based on the down®eld shift of the H-40
signal ꢀ5.33 ppm) and on the up®eld shift of the H-30e signal ꢀ2.27 ppm). The ꢁ-linked
isomers 11a and 11b displayed the signal of H-30e at 2.42 ppm. Removal of the 7-O-
benzyl group by hydrogenolysis with 5% Pd±C afforded 10a and 10b in quantitative
yield. Subsequent treatment of 10a and 10b with sodium methoxide and aqueous
0.2 M NaOH gave the target disaccharides 13a and 13b in 95% yield after puri®cation
on Bio-Gel P2.
Scheme 2

Synthesis of C-8 Deuterated Kdo
565
NMR spectroscopic analysis of the glycosides
The assignment of the mixture of 13a and 13b was performed from data obtained
at 800 MHz using standard one- and two-dimensional experiments. Even at the
1
high ®eld used severe overlap was observed, and some of the important H±1H
coupling constants could only be obtained from slices of two-dimensional spectra
3
2
acquired with good digital resolution ꢀe.g. J7,8 of the H-8-ꢀS) compound from a
slice of an HSQC spectrum, Fig. 1). The identities of the R and S compounds in the
mixtures were assigned based on the relative signal intensity, with the S compound
as the major component. All chemical shifts except that of H-8 were completely
identical for the two compounds; the corresponding data are reported in Table 2.
Fig. 1. Two-dimensional plot of the 800 MHz HSQC spectrum of 13a and 13b displaying cross-
peaks of the side chain protons
Table 2. NMR parameters for 13a and 13b assigned at 800 MHz for 1H and 201 MHz for 13C ꢀthe only difference
between 13a and 13b is observed for H-8)
Position
ꢂꢀ1H)=ppm J=Hz
ꢂꢀ13C)=ppm
Position
ꢂꢀ1H)=ppm
J=Hz
ꢂꢀ13C)=ppm
MeO
3a
3e
4
3.107
51.3
34.9
1.725
1.952
3.965
3.970
3.547
3.949
12.2
30a
30e
40
50
60
70
80a
80b
1.754
2.006
3.996
3.950
3.571
3.872
3.843
3.606
12.2
5.1, 12.9
3.0
34.9
5.1, 13.0
66.7
66.9
71.9
68.5
65.3
66.6
66.9
72.2
69.9
63.6
5
< 1
7.3
< 1
8.9
6
7
2
H-8 of H-ꢀR) 3.569
2
H-8 of H-ꢀS) 3.547
< 2
5.5
2.8
6.5, 11.8

566
P. Kosma et al.
As expected, a difference in the isotope effect between the two compounds in the
mixture is too small to be observed.
The assignment of the H-8 protons allows for a more thorough analysis of the
conformational preference around the C-7±C-8 linkage of the reducing residue.
Previous studies [6] have been hampered by complete overlap of the H-8-pro-R and
H-8-pro-S protons even at 800 MHz. In the present study, the analysis has solely
focussed on this problem associated with the side chain conformation, as previous
studies have described the overall conformational properties in detail [6, 9].
The major disaccharide 13b is ꢀS)-con®gured at C-8, as determined in com-
pounds 6a and 6b; that is, the proton observed corresponds to H-8-pro-R in a
nondeuterated disaccharide and vice versa. In the published crystal structure of the
corresponding allyl disaccharide [5] the rotamer with synclinal orientation between
the glycosidic oxygen ꢀO20) and both O-7 and C-6 of the reducing residue is
observed. The À sc arrangement for C-6 and ‡ sc arrangement of O-7 also gives
rise to H-7 being placed between the two H-8 protons. Such an arrangement
is expected to give two small three-bond coupling constants [11] of approximately
1 and 3 Hz, as H-8 with an antiperiplanar orientation relative to O-7 is expected
to give the smallest coupling. The proton in this arrangement is the pro-R in
2
the nondeuterated disaccharide and the remaining proton in H-8-ꢀS), which has a
coupling of 5.5 Hz. This indicates that in solution some rotational averaging of
the C-7±C-8 linkage takes place, and this is most likely an averaging between
the rotamer observed in the crystal and the rotamer which places H-8-pro-R
Scheme 3

Synthesis of C-8 Deuterated Kdo
567
antiperiplanar to H-7 [11]. Only such an averaging will give a larger value for H-8
in 2H-8-ꢀS) and still a small value for H-8 in 2H-8-ꢀR). An approximate population
of the sc,sc and sc,ap rotamers of 1:1 would give a coupling constant close to
2
5.5 Hz for H-8 in H-8-ꢀS) ꢀScheme 3).
Unfortunately, no additional information about the conformation could be
obtained from the NOESY spectra, partly due to overlap and partly to very small
NOEs observed in general. No attempts were made to obtain ROESY spectra.
Experimental
General
Melting points were determined with a hot stage and are uncorrected. Optical rotations were
1
measured with a Perkin-Elmer 243 B polarimeter. H NMR spectra were recorded at 297 K with
1
Bruker AC 300F and DPX instruments operating at 300 MHz for H using CDCl3 as solvent and
TMS as internal standard unless stated otherwise. Coupling constants are given in Hz ꢀ®rst order
values). Homo- and heteronuclear 2D NMR spectroscopy was performed with Bruker standard
software. High-®eld NMR was performed in 3 mm tubes at 298 K at 799.96 MHz for 1H and
201.12 MHz for 13C on a Varian UNITY INOVA 800 spectrometer, using acetone ꢀ2.225 ppm) and
1
1,4-dioxane ꢀ67.4 ppm) as secondary standards for H and 13C, respectively.
TLC was performed on E. Merckprecoated plates ꢀ5 Â10 cm, layer thickness 0.25 mm, Silica Gel
60F254); detection was effected by spraying with anisaldehyde-H2SO4. For column chromatography,
silica gel ꢀ40±63 ꢃm) was used. Concentration of solutions was performed at reduced pressure and
below 40ꢀC. Elemental analyses were provided by Dr. J. Theiner, Mikroanalytisches Laboratorium,

Institut fur Physikalische Chemie, University of Vienna; the results were in good agreement with the
calculated values.
Methyl &methyl 7-O-benzyl-4,5-O-carbonyl-3-deoxy-8-O-triphenylmethyl-ꢀ-D-manno-oct-
2-ulopyranosid)-onate ꢀ2; C37H36O9)
A solution of 880 mg ꢀ1.64 mmol) 1 and 1.04 g ꢀ4.1 mmol) benzyl 2,2,2-trichloroacetimidate in
10 cm3 dry CH2Cl2 was treated with 40 mm3 tri¯uoromethane sulfonic acid for 3 h at room temper-
ature. Solid NaHCO3 ꢀ2 g) was added, and stirring was continued for 15 min. The suspension was
diluted with 50 cm3 CH2Cl2 and washed with satd. aq. NaHCO3. The organic phase was dried
ꢀMgSO4) and concentrated. Puri®cation of the residue by ¯ash chromatography on silica gel ꢀtoluene:
EtOAc ˆ 3:1) gave 2 as a colorless syrup.
20
D
Yield: 720 mg ꢀ70%); ‰ꢀŠ ˆ À14ꢀ ꢀc ˆ 1.0, CHCl3); 1H NMR ꢀCDCl3, ꢂ, 300 MHz): 7.48±7.20
ꢀm, 20 H, arom. H), 5.06 ꢀdd, 1 H, J5,4 ˆ 8.8, J5,6 ˆ 1.5 Hz. H-5), 4.98 ꢀt, H-4), 4.86 and 4.62 ꢀAB,
J ˆ 10.6 Hz, OCH2), 4.12 ꢀdd, J6,7 ˆ 9.5 Hz, H-6), 4.00 ꢀddd, J7,8a ˆ 1.6, J7,8b ˆ 4.4 Hz, H-7), 3.77
ꢀs, OMe), 3.64 ꢀdd, J8a,8b ˆ 10.3 Hz, H-8a), 3.29 ꢀdd, H-8b), 2.82 ꢀs, OMe), 2.72 ꢀdd, J3e,4 ˆ 3.7,
J3e,3a ˆ 16.1 Hz, H-3e), 2.02 ꢀdd, J3a,4 ˆ 3.2 Hz, H-3a) ppm.
Methyl &methyl 7-O-benzyl-4,5-O-carbonyl-3-deoxy-ꢀ-D-manno-oct-2-ulopyranosid)-onate
ꢀ3; C18H22O9)
A solution of 52 mg ꢀ0.083 mmol) 2 in 5 cm3 dry CH3CN was stirred with 2 cm3 of a mixture of formic
acid:Et2O ˆ 2:1 ꢀv=v), for 15 h at room temperature and for 2 h at 40ꢀC. Solid NaHCO3 ꢀ1 g) was added,
and the suspension was diluted with 50 cm3 CH2Cl2 and washed with satd. aq. NaHCO3. The organic
phase was dried ꢀMgSO4) and concentrated. Puri®cation of the residue by ¯ash chromatography on
silica gel ꢀtoluene:EtOAc ˆ 1:1) afforded 3 as colorless crystals.

568
P. Kosma et al.
D
Yield: 27 mg ꢀ84%); m.p.: 171±172ꢀC ꢀEtOAc=hexane); ‰ꢀŠ ˆ ‡11ꢀ ꢀc ˆ 1.0, CHCl3); 1H NMR
20
ꢀCDCl3, ꢂ, 300 MHz): 7.40±7.28 ꢀm, 5 arom. H), 5.00 ꢀddd, J4,5 ˆ 8.5 Hz, H-4), 4.95 ꢀdd, J5,4 ˆ 8.5,
J5,6 ˆ 1.5 Hz, H-5), 4.70 and 4.65 ꢀAB, J ˆ 10.9 Hz, OCH2), 4.05 ꢀdd, J6,7 ˆ 9.2 Hz, H-6), 3.95 ꢀm,
H-8a), 3.91 ꢀdt, J7,8a ˆ 2.5, J7,8b ˆ 2.5 Hz, H-7), 3.83 ꢀs, OMe), 3.77 ꢀm, H-8b), 3.28 ꢀs, OMe), 2.72 ꢀdd,
J3e,4 ˆ 3.7, J3e,3a ˆ 16.0 Hz, H-3e), 2.09 ꢀdd, J3a,4 ˆ 3.2 Hz, H-3a), 1.82 ꢀdd, J ˆ 2.9, J ˆ 9.6 Hz,
OH) ppm.
Methyl &methyl 2-O-benzyl-4,5-O-carbonyl-6-deoxy-ꢀ-D-manno-oct-7-ulo-4,7-pyranosid)-onate
ꢀ4; C18H20O9)
A solution of 112 mm3 ꢀ1.3 mmol) freshly distilled oxalyl chloride in 10 cm3 dry CH2Cl2 was cooled
to À 40ꢀC. A solution of 222 mm3 ꢀ2.88 mmol) DMSO in 1 cm3 CH2Cl2 was added under N2, followed
by addition of 230 mg ꢀ0.6 mmol) 3 in 2 cm3 CH2Cl2. After stirring of the solution for 15 min, 0.84 cm3
ꢀ6 mmol) triethylamine in 2 cm3 CH2Cl2 was added, and the temperature was raised to 20ꢀC within
30 min. The suspension was diluted with 50 cm3 CH2Cl2, washed with H2O, and the organic layer was
dried ꢀNa2SO4) and concentrated. The residue was chromatographed on silica gel ꢀtoluene:EtOAc ˆ
1:1) which furnished 4 as colorless crystals.
D
20
Yield: 200 mg ꢀ87%); m.p.: 169±172ꢀC ꢀdec., CH2Cl2=pentane); ‰ꢀŠ ˆ ‡15ꢀ ꢀc ˆ 0.6, CHCl3);
1H NMR ꢀCDCl3, ꢂ, 300 MHz): 9.80 ꢀd, J1,2 ˆ 1.5 Hz, H-1), 7.38±7.35 ꢀm, 5 arom. H), 5.00 ꢀddd,
J6e,5 ˆ 3.6, J6a,5 ˆ 3.1 Hz, H-5), 4.93 ꢀdd, J5,4 ˆ 8.7, J4,3 ˆ 1.5 Hz, H-4), 4.74 and 4.62 ꢀAB, J ˆ
10.9 Hz, OCH2), 4.30 ꢀd, J1,2 ˆ 1.5 Hz, H-2), 4.06 ꢀdd, J3,2 ˆ 9.4 Hz, H-3), 3.83 ꢀs, OMe), 3.19
ꢀs, OMe), 2.81 ꢀdd, J6e,5 ˆ 3.6, J6e,6a ˆ 16.2 Hz, H-6e), 2.02 ꢀdd, J6a,5 ˆ 3.1 Hz, H-6a) ppm.
Methyl &methyl 7-O-benzyl-4,5-O-carbonyl-3-deoxy-8-&R)-[8-2H]-ꢀ-D-manno-oct-2-
ulopyranosid)-onate ꢀ5a; C18H21DO9) and methyl &methyl 7-O-benzyl-4,5-O-carbonyl-
3-deoxy-8-&S)-[8-2H]-ꢀ-D-manno-oct-2-ulopyranosid)-onate ꢀ5b; C18H21DO9)
N-Benzyloxycarbonyl-ꢀS)-proline ꢀ1.3 g, 5.23 mmol) was lyophilized twice from 5 cm3 D2O and
dissolved in 10 cm3 dry THF. NaBD4 ꢀ73 mg, 1.74 mmol) was added, and the solution was stirred
under Ar for 3 h at room temperature. The reaction vessel was cooled to À 126ꢀC ꢀmethyl-
cyclohexane and liquid N2 as coolant), and a solution of 60 mg 4 ꢀ0.158 mmol) in 5 cm3 THF was
added over 30 min. After additional 30 min at À 126ꢀC the reaction was allowed to attain room
temperature. The solution was concentrated and puri®ed by column chromatography ꢀtoluene:
EtOAc ˆ 1:1) which afforded 60 mg of a mixture of 5a and 5b ꢀ99%) as a syrup.
1
Selected H NMR data ꢀCDCl3, ꢂ, 300 MHz): 5a: 3.95 ꢀd, J7,8-ꢀS)-H ˆ 3.0 Hz, H-8-ꢀS)); 5b: 3.76
ꢀd, J7,8-ꢀR)-H ˆ 2.5 Hz, H-8-ꢀR)).
Methyl &methyl 4,5;7,8-di-O-carbonyl-3-deoxy-8-&R)-[8-2H]-ꢀ-D-manno-oct-2-ulopyranosid)-
onate ꢀ6a; C12H13DO10) and methyl &methyl 4,5;7,8-di-O-carbonyl-3-deoxy-8-&S)-[8-2H]-
ꢀ-D-manno-oct-2-ulopyranosid)-onate ꢀ6b; C12H13DO10)
A suspension of 15 mg ꢀ0.04 mmol) 5a and 5b and 20 mg 5% Pd±C in 5 cm3 MeOH was hydrogenated
at atmospheric pressure for 2 h at room temperature. The suspension was ®ltered over a pad of Celite,
and the ®ltrate was evaporated to dryness. A solution of the residue in 5 cm3 pyridine was cooled to
À 20ꢀC. Diphosgene ꢀ15 mm3, 0.12 mmol) was added, and the mixture was stirred for 30 min
at À 20ꢀC. MeOH ꢀ2 cm3) was added, and the solution was coevaporated twice with toluene. The
residue was chromatographed on silica gel ꢀtoluene:EtOAc ˆ 1:3) which afforded a mixture of 6a and
6b as colorless crystals.
D
20
Yield: 5 mg ꢀ40%); m.p.: 154±156ꢀC ꢀEtOAc=pentane); ‰ꢀŠ ˆ ‡57ꢀ ꢀc ˆ 0.2, CHCl3); 1H NMR
ꢀCDCl3, ꢂ, 300 MHz): 5.06 ꢀdt, J5,4 ˆ 8.6, Hz, H-4), 5.00 ꢀm, H-7), 4.85 ꢀdd, J5,6 ˆ 1.7 Hz, H-5), 4.66
ꢀbr d, 0.3 H, H-8-ꢀS)), 4.62 ꢀbr d, 0.7 H, J7,8 ˆ 6.5 Hz, H-8-ꢀR)), 4.12 ꢀdd, J6,7 ˆ 5.1 Hz, H-6), 3.85 ꢀs,
OMe), 3.29 ꢀs, OMe), 2.84 ꢀdd, J3e,3a ˆ 16.2 Hz, H-3e), 2.10 ꢀdd, J3a,4 ˆ 3.4 Hz, H-3a) ppm.

Synthesis of C-8 Deuterated Kdo
569
Sodium &methyl 3-deoxy-8-&R)-[8-2H]-ꢀ-D-manno-oct-2-ulopyranosid)-onateꢀ7a; C9H14DO8Na)
and sodium &methyl 3-deoxy-8-&S)-[8-2H]-ꢀ-D-manno-oct-2-ulopyranosid)-onate
ꢀ7b; C9H14DO8Na)
A solution of 2.5 mg of 6a and 6b in 4 cm3 dry MeOH was stirred with 0.3 cm3 of an 0.1 M methanolic
sodium methoxide solution for 4 h at room temperature. The pH of the solution was adjusted to 7.5 by
‡
adding Dowex-50 H resin. The suspension was ®ltered, and the ®ltrate was concentrated. The residue
was dissolved in 2 cm3 H2O and stirred with 2 cm3 0.2 M NaOH for 12 h at 0ꢀC. The pH of the solution
‡
was adjusted to 7.5 by adding Dowex-50 H resin, the resin was ®ltered off, and the ®ltrate was ly-
ophilized giving 2 mg of 7a and 7b ꢀ100%) as an amorphous solid.
D
20
1
‰ꢀŠ ˆ ‡66ꢀ ꢀc ˆ 0.2, H2O); H NMR ꢀD2O, ꢂ, 300 MHz): 4.02 ꢀddd, J5,4 ˆ 3.0, J4,3e ˆ 5.0,
J4,3a ˆ 11.5 Hz, H-4), 4.00 ꢀdd, J4,5 ˆ 3.0 Hz, H-5), 3.94 ꢀm, H-7), 3.91 ꢀm, 0.3 H, H-8-ꢀS)), 3.64 ꢀdd,
0.7 H, J8,7 ˆ 6.5 Hz, H-8-ꢀR)), 3.55 ꢀdd, J7,6 ˆ 9.0 Hz, H-6), 3.14 ꢀs, OMe), 2.01 ꢀm, H-3e), 1.77 ꢀdd,
J3e,3a ˆ 13.2 Hz, H-3a) ppm.
Methyl &&methyl 4,5,7,8-tetra-O-acetyl-ꢀ-D-manno-oct-2-ulopyranosyl)-onate)-&2 ! 8)-&methyl 7-
O-benzyl-4,5-O-carbonyl-3-deoxy-8-&R)-[8-2H]-ꢀ-D-manno-oct-2-ulopyranosid)-onate
ꢀ9a; C35H43DO20) and methyl &&methyl 4,5,7,8-tetra-O-acetyl-ꢀ-D-manno-oct-2-ulopyranosyl)-
onate)-&2 ! 8)-&methyl 7-O-benzyl-4,5-O-carbonyl-3-deoxy-8-&S)-[8-2H]-ꢀ-D-manno-oct-2-
ulopyranosid)-onate ꢀ9b; C35H43DO20)
A suspension of 65 mg ꢀ0.17 mmol) 5a and 5b, 60 mg ꢀ0.24 mmol) HgꢀCN)2, and 0.3 g molecular sieve
3
3
4 A in 4 cm dry MeNO2 was stirred for 15 min under N2. A solution of 320 mg ꢀ0.66 mmol) 8 in 2 cm
Ê
dry MeNO2 was added dropwise during 30 min at room temperature, and stirring was continued for
12 h. The suspension was diluted with 50 cm3 CH2Cl2 and ®ltered over a pad of Celite. The ®ltrate was
washed sequentially with aq. 5% KI and satd. aq. NaHCO3, dried ꢀMgSO4), and concentrated. The
residue was chromatographed on silica gel ꢀtoluene:EtOAc ˆ 3:2) which furnished ®rst glycal ester
12, followed by 112 mg of the disaccharides 11a,b and 9a,b as a syrup ꢀ85%). Crystallization from
EtOAc= n-pentane furnished 72 mg of 9a,b as colorless crystals.
D
M.p.: 119±126ꢀC; ‰ꢀŠ ˆ ‡51ꢀ ꢀc ˆ 0.5, CHCl3); 1H NMR ꢀCDCl3, ꢂ, 300 MHz): 7.38±7.28 ꢀm, 5
20
arom. H), 5.33 ꢀddd, J4 ,5 ˆ 3.1, J4 ,3 e ˆ 5.0, J4 ,3 a ˆ 11.8 Hz, H-40), 5.30 ꢀbr s, H-50), 5.23 ꢀddd,
0
0
0
0
0
0
J7 ,6 ˆ 10.0, J7 ,8 a ˆ 2.6, J7 ,8 b ˆ 4.4 Hz, H-70), 5.03±4.93 ꢀm, H-4, H-5), 4.80 and 4.63 ꢀAB,
0
0
0
0
0
0
J ˆ 10.7 Hz, OCH2), 4.55 ꢀdd, J8 a,8 b ˆ 12.3 Hz, H-80a), 4.15 ꢀdd, J6 ,5 ˆ 1.3 Hz, H-6 ), 4.14 ꢀdd,
H-80b), 4.00 ꢀm, H-7), 3.93 ꢀdd, ꢁ 0.3 H, J8ꢀS),7 ˆ 1.4 Hz, H-8-ꢀS)), 3.84 ꢀdd, J6,7 ˆ 9.5, J6,5 ˆ 1.4 Hz,
H-6), 3.83 ꢀs, CO2Me), 3.74 ꢀs, CO2Me), 3.65 ꢀdd, ꢁ 0.7 H, J8ꢀR),7 ˆ 6.3 Hz, H-8-ꢀR)), 3.27 ꢀs, OMe),
0
0
0
0
0
2.77 ꢀdd, J3e,4 ˆ 3.8, J3e,3a ˆ 16.0 Hz, H-3e), 2.27 ꢀdd, J3 e,3 a ˆ 12.5 Hz, H-30e), 2.09 ꢀs, Ac), 2.09
0
0
ꢀt, J4 ,3 a ˆ 11.8 Hz, H-30a), 2.06 ꢀdd, J3a,4 ˆ 3.3 Hz, H-3a), 1.99 ꢀs, 3ÂAc) ppm.
0
0
Methyl &&methyl 4,5,7,8-tetra-O-acetyl-ꢀ-D-manno-oct-2-ulopyranosyl)-onate)-&2 ! 8)-&methyl
4,5-O-carbonyl-3-deoxy-8-&R)-[8-2H]-ꢀ-D-manno-oct-2-ulopyranosid)-onate ꢀ10a; C28H37DO20)
and methyl &&methyl 4,5,7,8-tetra-O-acetyl-ꢀ-D-manno-oct-2-ulopyranosyl)-onate)-&2 ! 8)-
&methyl 4,5-O-carbonyl-3-deoxy-8-&S)-[8-2H]-ꢀ-D-manno-oct-2-ulopyranosid)-onate
ꢀ10b; C28H37DO20)
A solution of 68 mg ꢀ0.086 mmol) 9a and 9b in 20 cm3 dry MeOH was stirred with 75 mg 5% Pd±C
under H2 at atmospheric pressure for 2 h at room temperature. The catalyst was ®ltered off, and the
®ltrate was taken to dryness to afford 10a and 10b as a syrup.
D
Yield: 58 mg ꢀquant.); ‰ꢀŠ ˆ ‡65ꢀ ꢀc ˆ 1.2, CHCl3); 1H NMR ꢀCDCl3, ꢂ, 300 MHz): 5.35±5.27
20
ꢀm, 2 H, H-50, H-40), 5.23 ꢀddd, J7 ,6 ˆ 10.0, J7 ,8 a ˆ 2.4, J7 ,8 b ˆ 3.7 Hz, H-70), 5.03 ꢀm, H-4, H-5), 4.54
0
0
0
0
0
0
ꢀdd, J8 a,8 b ˆ 12.4 Hz, H-80a), 4.17 ꢀdd, J7 ,8 b ˆ 3.7 Hz, H-80b), 4.16±4.06 ꢀm, H-7, H-60), 3.90 ꢀdd,
J6,7 ˆ 9.4 Hz, H-6), 3.87±3.72 ꢀm, H-8), 3.87 ꢀs, CO2Me), 3.81 ꢀs, CO2Me), 3.49 ꢀbr s, OH), 3.33
0
0
0
0

570
P. Kosma et al.: Synthesis of C-8 Deuterated Kdo
ꢀs, OMe), 2.73 ꢀdd, J3e,4 ˆ 3.3, J3e,3a ˆ 15.7 Hz, H-3e), 2.20±2.02 ꢀm, H-30e, 30a, 3a), 2.11, 2.08, 2.05,
2.00, 1.99 ꢀ5s, 5ÂAc) ppm.
Methyl &sodium &3-deoxy-ꢀ-D-manno-oct-2-ulopyranosyl)-onate)-&2 ! 8)-&sodium 3-deoxy-8-
&R)-[8-2H]-ꢀ-D-manno-oct-2-ulopyranosid)-onate ꢀ13a) and methyl &sodium &3-deoxy-ꢀ-
D-manno-oct-2-ulopyranosyl)-onate)-&2 ! 8)-&sodium 3-deoxy-8-&S)-[8-2H]-ꢀ-D-manno-
oct-2-ulopyranosid)-onate ꢀ13b; C17H24DO15Na2)
A solution of 10 mg of ꢀ0.013 mmol) 10a and 10b in 5 cm3 dry MeOH was stirred with 0.5 cm3 0.1 M
methanolic sodium methoxide solution for 3 h at room temperature. The pH of the solution was
‡
adjusted to 7.5 by adding Dowex-50 H resin. The suspension was ®ltered, and the ®ltrate was
concentrated. The residue was dissolved in 2 cm3 H2O and stirred with 0.5 cm3 0.2 M NaOH for 15 h at
‡
0ꢀC. The pH of the solution was adjusted to 8.5 by adding Dowex-50 H resin, and the resin was
®ltered off. The ®ltrate was concentrated and puri®ed on a column of Bio-Gel P2 ꢀ100Â2.6 cm,
EtOH:H2O ˆ 5:95) which afforded 13a and 13b as amorphous solids.
Yield: 5.5 mg ꢀ95%); NMR data: see Table 2.
Acknowledgments
Financial support by a grant from FWF ꢀP-13843 CHE) is gratefully acknowledged. The 800 MHz
spectra were obtained using the Varian Unity Inova spectrometer of the Danish Instrument Center for
NMR Spectroscopy of Biological Macromolecules.
References
[1] Moulder JW ꢀ1991) Microbiol Rev 55: 143


[2] Nurminen M, Leinonen M, Saikku P, Makela PH ꢀ1983) Science 220: 1279
[3] Brade H, Brade L, Nano FE ꢀ1987) Proc Natl Acad Sci USA 84: 2508
[4] Kosma P, Schulz G, Brade H ꢀ1988) Carbohydr Res 183: 183
[5] Mikol V, Kosma P, Brade H ꢀ1994) Carbohydr Res 263: 35
[6] Sokolowski T, Haselhorst T, Schef¯er K, Weisemann R, Kosma P, Brade H, Brade L, Peters T
ꢀ1998) J Biomol NMR 12: 123
[7] Fu Y, Baumann M, Kosma P, Brade L, Brade H ꢀ1992) Infect Immun 60: 1314
[8] Nguyen H, Evans SL ꢀpersonal communication)
[9] BockK, Thomsen U, Kosma P, Christian R, Holst O, Brade H ꢀ1992) Carbohydr Res 229: 213
[10] Maaheimo H, Kosma P, Brade L, Brade H, Peters T ꢀ2000) Biochemistry 39: 12778
[11] BockK, Duus J ꢀ1994) J Carbohydr Chem 13: 513
[12] Kosma P, Schulz G, Unger FM ꢀ1984) Carbohydr Res 132: 261
[13] Wessel HP, Iversen T, Bundle DR ꢀ1985) J Chem Soc Perkin Trans I, 2247
[14] Mancuso AJ, Huang SL, Swern D ꢀ1978) J Org Chem 43: 2480
[15] Nishizawa M, Noyori R ꢀ1991) In: Trost BM, Fleming I ꢀeds) Comprehensive Organic Synthesis,
vol 8. Pergamon Press, Oxford, New YorkSeoul Tokyo, p 159
[16] Corey EJ, LinkJO ꢀ1989) Tetrahedron Lett 30: 6275
[17] Hayashi M, Terashima S, Koga K ꢀ1981) Tetrahedron 37: 2797
[18] Hori H, Morita M, Ito H, Nishida Y, Ohrui H, Meguro H ꢀ1990) XVth Int Carbohydr Symposium,
August 12±17, Yokohama, Abstr C030
Received September 17, 2001. Accepted October 17, 2001