Efficient chemical synthesis of both anomers of ADP L-glycero- and D-glycero-D-manno-heptopyranose

Article abstract of DOI:10.1016/S0008-6215(03)00319-7

A series of anomeric phosphates and ADP-activated L-glycero- and D-glycero-D-manno-heptopyranoses has been prepared in high overall yields, which provided model compounds and substrates in the elucidation of biosynthetic pathways and glycosyl transfer reactions of nucleotide-activated bacterial heptoses. The α-anomers of the heptosyl phosphates were obtained in high yield and selectivity using the phosphoramidite procedure, whereas the β-phosphates were formed preferentially employing acylation of reducing heptoses with diphenyl phosphorochloridate. An efficient route to the formation of the nucleotide diphosphate sugars was elaborated by coupling of the O-acetylated phosphates with AMP-morpholidate followed by alkaline deprotection to furnish ADP-L- and D-glycero-α-D-manno-heptose in 84 and 89% yield, respectively. Deacetylation of the O-acetylated β-configured ADP heptoses was conducted at strictly controlled conditions (-28°C at pH 10.5) to suppress formation of cyclic heptose-1,2-phosphodiesters with concomitant release of AMP. Isolation of the unstable β-configured ADP-heptoses by anion-exchange chromatography and gel-filtration afforded ADP L- and D-glycero-β-D-manno-heptose in high yields.

Full text of DOI:10.1016/S0008-6215(03)00319-7

Carbohydrate Research 338 (2003) 2571Á2589
/
www.elsevier.com/locate/carres
Efficient chemical synthesis of both anomers of ADP
-glycero- -manno-heptopyranose
L-glycero- and
D
D
Alla Zamyatina,a Sabine Gronow,b Michael Puchberger,a Andrea Graziani,a
Andreas Hofinger,a Paul Kosmaa,*
a Institute of Chemistry, University of Agricultural Sciences, Muthgasse 18, A-1190 Vienna, Austria
b Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-23845 Borstel, Germany
Received 11 March 2003; accepted 11 June 2003
Abstract
A series of anomeric phosphates and ADP-activated
L-glycero- and D-glycero-D-manno-heptopyranoses has been prepared in
high overall yields, which provided model compounds and substrates in the elucidation of biosynthetic pathways and glycosyl
transfer reactions of nucleotide-activated bacterial heptoses. The a-anomers of the heptosyl phosphates were obtained in high yield
and selectivity using the phosphoramidite procedure, whereas the b-phosphates were formed preferentially employing acylation of
reducing heptoses with diphenyl phosphorochloridate. An efficient route to the formation of the nucleotide diphosphate sugars was
elaborated by coupling of the O-acetylated phosphates with AMP-morpholidate followed by alkaline deprotection to furnish ADP-
L
- and
D
-glycero-a-
D
-manno-heptose in 84 and 89% yield, respectively. Deacetylation of the O-acetylated b-configured ADP
28 8C at pH 10.5) to suppress formation of cyclic heptose-1,2-
heptoses was conducted at strictly controlled conditions (ꢀ
/
phosphodiesters with concomitant release of AMP. Isolation of the unstable b-configured ADP-heptoses by anion-exchange
chromatography and gel-filtration afforded ADP
# 2003 Elsevier Ltd. All rights reserved.
L- and
D-glycero-b-
D-manno-heptose in high yields.
Keywords: Heptose; ADP heptose; Lipopolysaccharide; Sugar nucleotides; Sugar phosphates
1. Introduction
may prevent the assembly of a functionally intact
bacterial cell wall by inhibition of its biosynthesis.
Heptoses of the L-glycero-D-manno configuration are
common constituents of the inner core-region of LPS
and have been found in Salmonella, Escherichia coli,
Shigella, Klebsiella, Proteus, Pseudomonas, Neisseria,
Vibrio, Bordetella, Yersinia, Campylobacter and many
1.1. Occurrence of glycero-D-manno-heptoses
Lipopolysaccharides (LPS) are structurally complex
amphipathic and microheterogeneous glycolipids which
are essential components of the outer membrane of
Gram-negative bacteria.1 Furthermore, LPS are media-
tors of numerous immunological and pathophysiologi-
cal effects in bacterial infections and are thus responsible
for high mortality rates associated with septic shock.
Due to an increasing resistance of many bacterial strains
against conventional antibiotics, considerable effort is
being devoted to the development of novel drugs, which
other pathogenic bacteria.2
heptoses*up to now always present in the a-anomeric
configuration*have also been found in the outer core,
L-Glycero-D-manno-
/
/
e.g., of E. coli K-12, Haemophilus ducreyi, Haemophilus
influenzae and Proteus penneri strains3Á6 and have also
been identified in a few cases in repeating units of O-
antigens, such as in Yokenella regensburgei and Pseu-
domonas cepacia strains.7,8
Similar to the L-glycero-D-manno forms, D-glycero-D-
manno-heptoses are present as a-pyranosides in inner
core oligosaccharides, as illustrated in LPS from Rho-
dococcus gelatinosus, Yersinia enterocolitica, Coxiella
burnetii, Mannheimia haemolytica, Aeromonas hydro-
* Corresponding author. Tel.: ꢁ
1-360066059.
E-mail address: pkosma@edv2.boku.ac.at (P. Kosma).
/
43-1-360066055; fax: ꢁ43-
/
0008-6215/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00319-7

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A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á2589
/
phila and Vibrio salmonicida.9Á14 Moreover,
D
-glycero-
biosynthetic pathways involving a kinase/phosphatase
sequence (instead of the mutase step) and utilizing
D-manno-heptoses are frequently found as side chain
units in the outer core of Proteus strains,15 H. influenzae
different anomers of
D-glycero-D-manno-heptose 1-
LPS,16 but also as short, a-(10
/
3 or 0
/2)-linked
phosphates were unambiguously established (Fig. 1).29
The biosynthesis of GDP heptose has recently been
shown to be also valid for the GDP derivative of 6-
deoxy-heptose in Yersinia pseudotuberculosis.30
oligomeric side chains attached to the core units of
Helicobacter pylori LPS,17 and Klebsiella pneumoniae
LPS.18
O-Antigens from Vibrio cholerae strains harbor
D
-
For the full elucidation of the biosynthesis of these
nucleotide-activated heptoses, well defined synthetic
compounds were needed to serve as biochemical probes.
Herein a high yield synthesis of both anomeric forms of
glycero-D-manno-heptoses in both anomeric configura-
tions. Thus, the O-antigen polysaccharide from ser-
ogroup O:3 contains a-heptosyl residues as constituent
of a tetrasaccharide repeating unit,19 whereas serogroup
O:21 contains a b-linked heptosyl moiety.20 In addition,
D
-glycero-
D
-manno-heptose 1-phosphate*
/
occurring as
and ADP
-manno-heptose 1 and 3 as well as ADP
-manno-heptose 2 and 4 (Fig. 2) as substrates
intermediates in both biosynthetic pathways*
/
b-linked
D
-glycero-D-manno-heptoses and 6-deoxy-D-
L
-glycero-
D
D-
manno-heptoses are present in the O-antigen from
Plesiomonas shigelloides O54:H2.21
glycero-
D
or substrate analogs for bacterial heptosyl transferases is
described. A preliminary account of this work has been
published previously.31
In the S-layer glycoprotein glycan from a Gram-
positive microorganism, Aneurinibacillus thermoaerophi-
lus DSM 10155, a disaccharide repeating unit 0
/
3)-D-
had
glycero-b- -manno-Hepp-(104)-a- -Rhap-(10
D
/
L
/
been identified, substantiating similarities of S-layer
glycans to Gram-negative LPS structures.22 Very re-
cently, a glycoprotein mediating adhesion as an auto-
transporter protein in enterotoxigenic E. coli strains has
been found which is substituted on average with 19
heptose residues. The heptose is transferred to the
2. Results and discussion
Since glycosyl phosphates are key intermediates in the
biosynthetic pathways, an efficient route for the stereo-
selective preparation of both anomeric forms of the
heptosyl phosphates was required. Among other routes,
glycosyl phosphates have been prepared by glycosyla-
tion of phosphoric acid diester derivatives with an
activated glycosyl donor or by reaction of a free
hydroxyl group at the anomeric centre with an activated
phosphate precursor.32 Following the latter approach,
protein by a glycosyl transferase*/an autotransporter
heptosyl transferase (AAH)*in the presence of ADP-
/
glycero-manno-heptose.23 The heptosyl transferase has
been cloned and partially characterized. A second
glycoprotein, termed TibA has been identified in enter-
otoxigenic E. coli, which acts as an adhesin and whose
carbohydrate modification is introduced upon the
action of a similar heptosyl transferase.24
the
L-glycero-D
-manno-heptopyranose derivative 633
was prepared by anomeric O-de-acetylation of the
peracetate 5 using ammonium acetate/N,N-diisopropy-
lethylamine (DIPEA)/DMF in high yield, thereby
avoiding the use of the equally effective but toxic
hydrazinium acetate (Scheme 1).34 The 2,3,4,6,7-penta-
1.2. Biosynthesis of nucleotide activated glycero-
manno-heptoses
D-
O-acetyl-D-glycero-D-manno-heptopyranose 16 was si-
The concept of the biosynthetic pathway leading to
nucleotide-activated heptose dates back to the funda-
mental work of Eidels and Osborn who proposed a
sequence of enzymatic reactions starting from sedohep-
tulose 7-phosphate, which is first converted by the
milarly obtained in 95% yield from the peracetate
derivative 15 (Scheme 2). Treatment of 6 with bis(ben-
zyloxy)(diisopropylamino)phosphine35 with 1H-tetra-
zole as catalyst, and subsequent oxidation with
tBuOOH, afforded the a-anomeric phosphotriester 7
/
action of an isomerase to yield
D
-glycero-D-manno-
as the major isomer (a/b ratio of the product mixture ꢀ
heptose 7-phosphate.25 A postulated phosphomutase
would then furnish the anomeric heptosyl phosphate,
which is transformed into a nucleotide activated deri-
vative. Later, Kontrohr and Kocsis provided evidence
that substrates of LPS core heptosyl transferases corre-
9:1) in 85% isolated yield after separation from the b-
phosphate 9 (9%) (Scheme 1).
Separation of the anomers was also achieved at the
stage of the heptosyl phosphites, which may be utilized
as potential glycosyl donors. The phosphites were
subsequently separately oxidised to give the phosphates
in similar overall yield.
By using the phosphoramidite approach, the equili-
brium could be shifted from the thermodynamically
preferred formation of a-phosphite towards the kineti-
cally controlled generation of b-product in the presence
of an excess of 4-N,N-dimethylaminopyridine (DMAP)
spond to ADP derivatives of
heptose.26 In the final step, an epimerase effects the
inversion of configuration at C-6 to furnish ADP
D- and L-glycero-D-manno-
L
-
glycero-D-manno-heptose. Based on previous findings
by Valvano27 and the complete elucidation of the
biosynthesis of GDP-heptose in the Gram-positive
microorganism A. thermoaerophilus DSM 10155,28 two

A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á
/2589
2573
Fig. 1. General biosynthetic pathway for ADP- and GDP-heptose.
by very slow addition of dibenzyl phosphorotetrazoli-
dite prepared in situ. After oxidation with tert-BuOOH
and chromatographic separation, the a- and b-config-
ured heptosyl phosphotriesters 7 and 9 were obtained in

2574
A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á2589
/
Scheme 1. (i) N,N-Diisopropylethylamine, NH4OAc, DMF,
95%; (ii) bis(benzyloxy)(diisopropylamino)phosphine, 1H-tet-
razole (3.5% in MeCN), CH2Cl2, then tBuOOH (to furnish
85% of 7 and 9% of 9) or 4-N,N-dimethylaminopyridine,
dibenzyl phosphorotetrazolidite (soln in 1:1 CH2Cl2Á
/
CH3CN), CH2Cl2, then tBuOOH (to furnish 56% of 7 and
34% of 9) or ClP(O)(OPh)2, DMAP, CH2Cl2 (to furnish 9% of
8 and 84% of 10); (iii) Pd/C, H2, MeOH, Et3N, 99% (for
deprotection of 7 and 9) or PtO2, H2, MeOH, 98% (for
Fig. 2. Structures of ADP L-glycero- and D-glycero-D-manno-
heptoses 1Á4.
/
deprotection of 8 and 10); (iv) 7:3:1 MeOHÁ
/
waterÁ
/
Et3N, 97Á
/
98%.
56 and 34% yield, respectively (Scheme 1). This proce-
dure was also applied to the synthesis of a- and b-
phosphates in the D,D-series (Scheme 2).
For the selective synthesis of the b-anomeric heptosyl
phosphates, the higher nucleophilicity of the equato-
rially oriented 1-OH group in 6 was exploited in a direct
acylation step using diphenyl phosphorochloridate in
the presence of excess of DMAP.36 Thus, 10 was formed
in 84% yield, whereas the a-anomer 8 was obtained in
9% yield. Comparable results were observed for the
6.4Á
/
6.6 Hz for a-anomers and 7.7Á7.8 Hz for b-
/
configured heptosyl phosphates. The anomeric config-
uration of the phosphates was unambiguously estab-
lished on the basis of the values of the heteronuclear
coupling constant JC-1,H-1, which was 185Á
phosphates and 163Á165 Hz for b-anomers. The assign-
ment of the anomeric configuration of heptosyl phos-
phates could also be substantiated from the chemical
/
187 Hz for a-
/
shift of H-5, which is 0.31Á0.37 ppm upfield for the b-
/
synthesis of the
which were isolated in 15 and 81% yield, respectively.
D-glycero-D-manno isomers 18 and 20,
anomers in comparison to the corresponding a-counter-
parts. The facile cleavage of the phosphate protecting
groups of benzyl-protected phosphotriesters 7, 9 in the
L,D-series (Scheme 1) and 17, 19 in the D,D-series
(Scheme 2) by catalytic hydrogenation in the presence
of Pd/C afforded penta-O-acetyl-heptosyl phosphates
1
The H NMR data of benzyl-protected heptosyl phos-
photriesters 7, 9, 17 and 19 are summarised in Table 1.
The 1H NMR spectra show a doublet of doublets for H-
1, in which the coupling constant JH-1,P is in the range

A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á
/2589
2575
Removal of the acetyl groups of the O-acetylated
heptosyl phosphates with triethylamine in MeOHÁ
water furnished the L,D-heptopyranosyl phosphates 12
/
and 14 as well as the D,D-configured compounds 22 and
,
24 as triethylammonium salts in near quantitative yields
respectively. The NMR data of all four heptosyl
phosphates are in full agreement with the assigned
structures (Tables 2 and 3). The downfield shifts (Table
2) of H-5 for a-anomers 12 and 22 (3.92 and 3.83 ppm)
in comparison to the related shifts of the b-phosphates
14 and 24 (3.38 and 3.40 ppm) and the measurement of
the heteronuclear coupling constant (JC-1,H-1
ꢀ
/
170 Hz
for 12 and 22 160 Hz for 14 and 24) confirmed
,
and ꢀ
/
the assignment of the anomeric configuration. The 13C
NMR data displayed downfield shifts for C-3 at 73.74
and 73.54 ppm for b-phosphates 14 and 24 (Table 3).
The data of the previously reported
manno-heptosyl phosphate compare favorably with
those recorded for compound 22.37
D
-glycero-a-
D-
Both anomeric phosphates of
heptose have been used in the elucidation of the
biosynthesis of nucleotide-activated heptoses wherein
D-glycero-D-manno-
,
only the b-phosphate 24 serves as a substrate for the
adenylyl transfer reaction to yield ADP heptose with
retention of configuration. Conversely, in the activating
step leading to the formation of GDP heptose, only the
a-anomer 22 is accepted by the enzyme.28,29
One of the most frequently used methods for the
stereospecific chemical synthesis of sugar nucleoside
diphosphates employs the coupling of the reducing
sugar phosphate with nucleoside monophosphomorpho-
Scheme 2. (i) N,N-Diisopropylethylamine, NH4OAc, DMF,
95%; (ii) 4-N,N-dimethylaminopyridine, dibenzyl phosphor-
lidate.38 The yields
, however, are mostly moderate even
after prolonged reaction times, although improvements
have been reported.39,40 Following the procedure devel-
oped for the synthesis of GDP-Fucose,41 the target
otetrazolidite (soln in 1:1 CH2Cl2ÁCH3CN), CH2Cl2, then
/
tBuOOH (to furnish 58% of 17 and 34% of 19) or
ClP(O)(OPh)2, DMAP, CH2Cl2 (to furnish 9% of 18 and
84% of 20); (iii) Pd/C, H2, MeOH, Et3N, 99% (for deprotection
of 17 and 19) or PtO2, H2, MeOH, 98% (for deprotection of 18
compound ADP
1) was initially prepared in 15% yield by the coupling of
4-morpholine N N?-dicyclohexylcarboxamidinium salt
L
-glycero-a-D-manno-heptose 1 (Fig.
and 20); (iv) 7:3:1 MeOHÁ
/
waterÁEt3N, 98%.
/
,
of adenosine monophosphomorpholidate (AMP-mor-
pholidate) 25 to a-heptosyl phosphate 12. The major
difficulty was encountered in the poor solubility of
heptosyl phosphate 12 (as triethyl- or tri-n-butylammo-
11, 13 and 21, 23 in quantitative yields. Confirmation of
1
their anomeric purity was provided by H NMR data
(Table 1) and on the basis of the measurement of
heteronuclear coupling constants JC-1,H-1 which were
determined from the proton-coupled HMQC spectra
nium salt) in anhydrous pyridine
for the phosphomorpholidate coupling
,
the preferred medium
leading to
,
and revealed ꢀ
/
180 Hz for a-configured phosphates and
heterogeneous reaction conditions. A homogeneous
solution in anhydrous pyridine was obtained using tri-
ꢀ
/
164 Hz for b-anomers.
Catalytic hydrogenation of the phenyl-protected b-
phosphate 10 in the L,D-series (Scheme 1) and 20 in the
D,D-series (Scheme 2) in the presence of PtO2 led to
n-octylammonium salt of 12
not significantly increase the yield (25%) of the dipho-
sphate forming reaction probably due to micelle-like
, which, however, also did
,
partial (ꢀ
/
5Á10%) deacetylation of the resulting penta-
/
aggregate formation, rendering the phosphate moiety
less accessible to the phosphomorpholidate. As an
alternative to sluggishly reactive heptosyl phosphate
12, its penta-O-acetyl derivative 11 was used in the
coupling with AMP-morpholidate 25 (Scheme 3). The
advantageous application of acylated sugar intermedi-
ates in the synthesis of sugar-diphosphates has been
O-acetyl-heptosyl phosphates 13 and 23. The latter were
either purified by chromatography or directly used as
partially deacetylated mixtures in the following coupling
with AMP-morpholidate, which did not exert any
negative influence on the yield of the diphosphate
forming reaction.

Table 1
1H NMR data of acetylated heptosyl phosphates (in CDCl3 for 7, 9, 17, 19 and in D2O for 11, 13, 21 and 23) a
H-1
H-2
H-3
H-4
H-5
H-6
H-7a
H-7b
CH2
CH3CO
7
9
5.59 (dd) 5.25 (bs) 5.23 (dd) 5.25 (t)
4.11(dd) 5.26 (m)
3J3,4 9.5 3J4,5 9.5 3J5,6 2.8 3J6,7a 3.9
3J6,7b 8.4
4.22 (dd)
2J7a,7b 12.0
4.09 (dd) 5.90 (2 d, 4 H)
2JH,P 9.5
2.17, 2.12, 2.03, 2.0, 1.99 (5 s)
3J1,2
B
/
1
3J1,P 6.4
5.38 (dd) 5.45 (dd) 5.02 (dd) 5.30 (t)
3
3.76 (dd) 5.28 (ddd) 4.27 (dd)
2J7a,7b 11.6
4.09 (dd) 5.07 (d, 2 H)
5.03, 5.01 (2 d)
2JH,P 8.3
2.16, 2.10, 2.23, 2.20, 1.98 (5 s)
2.22, 2.18, 2.10, 2.07, 2.02 (5 s)
2.26, 2.18, 2.07, 2.09, 2.01 (5 s)
2.15, 2.10, 2.04, 2.0, 1.94 (5 s)
2.15, 2.11, 2.06, 2.03, 2.01 (5 s)
2.14, 2.10, 2.08, 2.04, 1.98 (5 s)
2.09, 2.0, 1.99, 1.95, 1.88 (5 s)
3J1,2 1.1 3J2,3 3.3 3J3,4 10.1 J4,5 10.1 J5,6 2.4 3J6,7a 5.1
3
3J1,P 7.7
3J6,7b 7.8
11 5.52 (dd) 5.36 (dd) 5.43 (dd) 5.21 (t)
3
4.48 (dd) 5.38 (ddd) 4.32 (dd)
2J7a,7b 12.1
4.40 (dd)
3J1,2 1.8 3J2,3 3.2 3J3,4 10.1 J4,5 10.1 J5,6 1.9 3J6,7a 3.9
3J1,P 7.8
3
3J6,7b 8.4
13 5.44 (dd) 5.54 (dd) 5.34 (dd) 5.15 (t)
3J1,2
4.17 (dd) 5.35 (m)
3J2,3 3.0 3J3,4 10.0 J4,5 10.0 J5,6 1.8 3J6,7a 4.0
4.35 (dd)
2J7a,7b 12.0
4.44 (dd)
3
3
B
/
1
3J1,P 9.0
3J6,7b 8.0
17 5.58 (dd) 5.20 (dd) 5.28 (dd) 5.31 (t)
4.16 (dd) 5.18 (ddd) 4.34 (dd)
2J7a,7b 12.0
3J6,7b 7.8
3.85 (dd) 5.27 (ddd) 4.43 (dd)
2J7a,7b 12.1
3J6,7b 7.0
4.37 (dd) 5.21 (ddd) 4.26 (dd)
2J7a,7b 12.2
4.20 (dd) 5.10 (d, 2 H), 5.09 (d, 2 H)
2JH,P 8.8
3J1,2 2.0 3J2,3 2.6 3J3,4 9.7 3J4,5 9.7 3J5,6 2.2 3J6,7a 3.8
3J1,P 6.6
2
4.27 (dd) 5.12 (d, 2 H), 5.06 (d, 2 H), JH,P 7.8
19 5.49 (dd) 5.44 (dd) 5.06 (dd) 5.27 (t)
3J1,2 1.4 3J2,3 3.2 3J3,4 9.5 3J3,4 9.5 3J5,6 3.7 3J6,7a 3.6
3J1,P 7.8
21 5.44 (dd) 5.26 (dd) 5.34 (dd) 5.29 (t)
4.41 (dd)
4.32 (dd)
3J1,2 1.8 3J2,3 2.7 3J3,4 9.6 3J4,5 9.6 3J5,6 2.8 3J6,7a 4.0
3J1,P 7.6
3J6,7b 7.3
4.00 (dd) 5.14 (ddd) 4.20 (dd)
2J7a,7b 12.2
23 5.29 (dd) 5.37 (dd) 5.17 (dd) 5.13 (t)
3J1,2 1.3 3J2,3 2.8 3J3,4 9.5 3J4,5 9.5 3J5,6 2.5 3J6,7a 4.0
3J1,P 8.8
3J6,7b 7.0
a
Coupling constants (in Hz) are first order values.

A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á
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Table 2
1H NMR data of heptosyl phosphates 12, 14, 22 and 24 in D2O
H-1
H-2
H-3
H-4
H-5
H-6
H-7a
H-7b
12 5.33(dd)
3J1,2 1.5
3.93(m)
n.d. a
3.95(m)
n.d. a
3.90(m)
3J4,5 9.5
3.81(m)
3J5,6 1.6
4.0(ddd)
3J6,7a 7.9
3J6,7b 6.1
3.73(dd)
2J7a,7b 11.4
3.59(dd)
3J1,P 6.6
14 5.08(dd)
3J1,2 0.9
4.01(dd)
3J2,3 3.2
3.70(dd)
3J3,4 9.7
3.80(dd)
3J4,5 9.6
3.38(dd)
3J5,6 1.9
3.96(ddd)
3J6,7a 5.2
3J6,7b 6.6
3.75(dd)
2J7a,7b 11.5
3.70(dd)
3.65(dd)
3J1,P 8.5
22 5.27(dd)
3J1,2 1.7
3.87(dd)
3J2,3 2.8
3.82 (dd)
3.67(dd)
3J4,5 9.5
3.83(dd)
3J5,6 1.8
3.92(ddd)
3J6,7a 7.1
3J6,7b 5.0
3.70(dd)
2J7a,7b 12.1
3J1,P 7.9
3J3,4 9.3
24 4.99(dd)
3J1,2 1.0
3.92(dd)
3J2,3 2.6
3.59(m)
3.59(m)
3.40(m)
3.95(ddd)
3J6,7a 6.6
3J6,7b 4.2
3.70Á/3.68 (m)
3.70Á3.68 (m)
/
3J1,P 8.6
a
n.d. not determined.
Table 3
13C NMR data of heptosyl phosphates in D2O
Carbon
C-1
12
14
22
24
96.15
2J1,P 5.5
71.52
96.03
2J1,P 3.8
72.06
96.06
2J1,P 5.3
71.19
96.01
2J1,P 4.0
71.70
C-2
3J2,P 9.7
71.20
66.85
3J2,P 6.0
73.74
66.66
3J2,P 7.6
70.92
68.44
3J2,P 6.0
73.54
67.92
C-3
C-4
C-5
C-6
C-7
71.53
69.02
62.53
75.70
69.51
63.11
73.48
72.88
62.60
77.72
72.01
62.22
previously reported.42 The reaction proceeded rapidly to
give approx 70% of desired product 26 within 1 day (as
judged by TLC), then slowed down and needed two
more days to be driven to completion. The acetylated
ADP
L-glycero-a-
D-manno-heptose 26 was isolated in
91% yield as triethylammonium salt by anion-exchange
chromatography.
Coupling of the other three O-acetylated heptosyl
phosphates 13, 21, 23 to AMP-morpholidate 25 gave,
after isolation and purification by anion-exchange
chromatography, similar and reproducible yields (85Á
/
95%) of adenosine heptosyl diphosphates 27, 28 and 31,
respectively (Schemes 3 and 4). The structures of the
acetylated ADP-L- and D-glycero-D-manno-heptopyra-
Scheme 3. (i) 4?-Morpholine-N,N?-dicyclohexylcarboxamidi-
nium salt of 25, pyridine, anion exchange on BioRad (Q) 5 mL
noses 26, 27, 28 and 31 were unambiguously confirmed
by 1H (Table 4) and 31P NMR experiments. Removal of
the acetyl groups from the heptosyl moiety of the a-
configured nucleoside diphosphates 26 and 27 with
aqueous ammonia or an aqueous methanolic solution
of Et3N at pH 12 furnished the ADP-heptoses 1 and 2,
respectively, in quantitative yields. The overall yield for
cartridge (/
HCOꢀ2 form), elution 0.010
waterÁ
/
0.2 M TEAB, 91% for
Et3N, 98%.
26 and 86% for 27; (ii) 7:3:1 MeOHÁ
/
/
the preparation of 1 starting from 5 was 71%. Cleavage
of the acetate groups from b-configured sugar nucleo-
tide 28 using these conditions, however, resulted in the

2578
A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á2589
/
NMR spectra of heptose 2-phosphate 30 were in
agreement with previously reported data.43
Cleavage of the diphosphate bond and cyclisation
under neighbouring group participation of the hydroxyl
group at C-2 of the heptose moiety was found to occur
also under milder reaction conditions [MeOH/water/
Et3N, pH 10.5 at 4 8C or 2 M triethylammonium
bicarbonate (TEAB) buffer, pH 10, 25 8C] and the cyclic
phosphate 29 was the major product after the deacetyla-
tion step. Careful adjustment of the conditions for the
basic hydrolysis of the acetate groups of b-configured
ADP-heptoses 28 and 31, however, finally resulted in
the isolation of 3 and 4 in 85Á
on acetylated heptosyl phosphates 13 and 23. When
deacetylation was conducted at ꢀ28 8C in MeOHÁ0.1-
M aq TEABÁEt3N (pH 10Á11) for 20 h followed by
immediate neutralisation with Dowex Hꢁ-resin, the
formation of only 5Á10% of cyclic phosphates 29 and
/90% overall yields, based
/
/
/
/
/
32 was observed (Scheme 4).
The ADP-heptoses 3 and 4 were eventually purified
by anion-exchange chromatography on Bio-Rad High Q
resin using a linear TEAB gradient, and by subsequent
gel chromatography on Superdex Peptide using the same
buffer to remove residual AMP. Purified ADP-
cero- and -manno-heptoses may be stored
-glycero-b-
in a frozen solution (pH 5, triethylammonium salt) or
after lyophilisation, for several months at ꢀ20 8C
L-gly-
D
D
/
without noticeable decomposition. In solution, dipho-
sphate 3 (as triethylammonium salt at pD 5) slowly
Scheme 4. (i) 4?-Morpholine-N,N?-dicyclohexylcarboxamidi-
nium salt of 25, pyridine, anion exchange on BioRad (Q) 5 mL
decomposed at room temperature (ꢀ/30% in 10 days,
cartridge (
28 and 95% for 31; (ii) 4:3:0.05 0.1 M aq TEABÁ
Et3N, ꢀ28 8C; anion exchange on BioRad (Q) 5 mL cartridge
(HCO2Oꢀ form), elution 0.010
0.2 M TEAB; gel filtration on
Superdex Peptide HR 10/30 Pharmacia column, elution 0.1 M
TEAB; Dowex 50 (Hꢁ-form), than Et3N; (iii) 7:3:1 MeOHÁ
waterÁEt3N, 85% for 29 and 80% for 32; (iv) silica gel, 10:3:3
MeOHÁwaterÁ25% aq NH4OH, 85%.
/
HCOꢀ2 form), elution 0.010
/0.2 M TEAB, 92% for
Fig. 3).
The structure and purity of all four isomers of ADP-
heptose was unambiguously confirmed by NMR data.
/
MeOHÁ
/
/
1
1
Homonuclear H,1H-COSY- and heteronuclear H,13C-
HMQC- and 1H,31P-HMQC-experiments allowed the
complete assignment of all proton (Table 5) and carbon
(Table 6) signals of the carbohydrate residues.
/
/
/
/
/
Decoupled 31P NMR spectra showed two doublets (at
ꢀ
/
10.8 ppm and in the range of ꢀ
/
12.7 to ꢀ/13.2 ppm)
with a coupling constant JP,P
ꢀ
/
21 Hz for each
formation of the b-heptosyl 1,2-cyclic phosphate 29
along with a minor amount of heptose 2-phosphate 30
and 1-phosphate 14 with release of AMP at the expense
of b-heptose nucleoside diphosphate (Scheme 4). The
1,2-cyclic phosphate 29 was isolated by anion-exchange
chromatography, and the compound was found to be
unstable under extended contact with silica gel to
furnish 2-phosphate 30 predominantly. The formation
of the five membered ring of the cyclic phosphodiester
29 was evident from the large heteronuclear coupling
compound; the signals of the diphosphodiester group
were correlated to the anomeric proton of the heptose
and to H-5 of the ribose, respectively. The 13C NMR
spectra of 1, 2, 3 and 4 clearly showed a doublet signal
for C-1 at around 96.5Á
constants JC-1,P were in the range of 5.8Á
anomers and 4.1Á4.8 Hz for the b-counterparts. The
signals of C-2 of the heptose residues were also coupled
to phosphorus with coupling constants of 7Á
10 Hz. 13C
/97.0 ppm. The coupling
/
6.0 Hz for a-
/
/
NMR signals of C-5 and C-4 of ribose appeared as
doublets with heteronuclear coupling constants JC,P
constant JH-1,P
ꢀ
/
25 Hz, the 31P NMR chemical shift of
1
29 (d 17.8), the results of an H,31P HMQC experiment
and the downfield shift of C-2 in 13C NMR-spectrum (d
79.5). Additional proof was provided by subsequent
alkaline (1 M NaOH) hydrolysis of 29 which gave a 1:1
mixture of 1-phosphate 14 and 2-phosphate 30. The
4.5Á/5.8 and JC,P
ꢀ9 Hz respectively. The anomeric
/
proton coupling constants of 1 and 2 (JH-1,H-2 1.9 and
2.0 and JH-1,P 7.4 and 7.9 Hz) are characteristic of a-
anomers, whereas the corresponding values of 3 and 4
(JH-1,H-2 1.0, JH-1,P 8.5 and 8.7 Hz) confirm the b-

Table 4
1HgNMR data of acetylated ADP-heptoses 26, 27, 28 and 31 in D2O
H-1
H-2
H-3
H-4
H-5
H-6
H-7a
H-7b
CH3CO
Ade
26 Hepp
5.60(dd)
3J1,2
5.39(dd)
3J2,3 3.3
5.33(dd)
3J3,410.1
5.13(t)
3J4,510.1
4.42(dd)3
J5,6 1.6
5.21(m)
4.35(m)
4.31(m)
2.18, 2.12, 2.00, 1.96, 1.94 (5 s)
1
3J1,P 7.7
Ribf
6.15(d)
3J1,2 5.5
4.70(dd)
3J2,3 4.8
4.51(dd)
3J3,4 4.1
4.39(m)
4.23(m, 2 H)
8.60, 8.27
27 Hepp
5.58(dd)
3J1,2 1.9
3J1,P 8.2
5.34(m)
5.27(m)
5.11(t)
3J4,5 10.0
4.35(dd)
3J5,6 4.0
5.21(m)
3J6,7a 4.5
3J6,7b 7.8
4.29(dd)
2J7a,7b 12.1
4.17(dd)
4.26(dd)
4.40(dd)
2.13, 2.08, 2.03, 2.02, 1.93 (5 s)
2.07, 2.01, 1.92, 1.88, 1.83 (5 s)
2.20, 2.11, 2,10, 2.06, 1.99 (5 s)
Ribf
6.12(d)
3J1,2 5.4
4.70(dd)
3J2,3 5.0
4.49(dd)
3J3,4 4.2
4.36(m)
4.24(m, 2 H)
8.56, 8.26
8.50, 8.29
8.59, 8.36
28 Hepp
5.47(dd)
3J1,2
5.54(dd)
3J2,3 3.0
5.12(dd)
3J3,4 10.0
5.07(t)
3J4,5 10.0
3.86(dd)
3J5,6 1.8
5.20(ddd)
3J6,7a 4.9
3J6,7b 8.6
4.38(dd)
2J7a,7b 12.2
1
3J1,P 9.3
Ribf
6.15(dd)
3J1,2 5.9
4.70(dd)
3J2,3 4.9
4.50(dd)
3J3,4 3.8
4.38(m)
4.22(m, 2H)
31 Hepp
5.51(dd)
3J1,2 1.2
3J1,P 11.8
5.52(bs)
3J2,3 2.8
5.16(dd)
3J3,4 9.6
5.23(dd)
3J4,5 9.4
3.97(dd)
3J5,6 3.0
5.20(m)
3J6,7a 4.1
3J6,7b 7.3
4.40(dd)
2J7a,7b 12.0
Ribf
6.16(dd)
3J1,2 5.7
4.70(dd)
3J2,3 4.9
4.51(dd)
3J3,4 3.7
4.38(m)
4.23(m, 2 H)

2580
A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á2589
/
1
Fig. 3. 300 MHz H NMR spectrum and proton assignments of the heptose unit of purified ADP
L-glycero-b-D-manno-heptose 3
(bottom line). Upon standing at room temperature at pH 5 for 10 days, the 1,2-cyclophosphate 29 was formed (insert), which is
readily identified by the large heteronuclear coupling constant JH-1,P displayed by the anomeric proton (asterix).
anomeric configuration (Table 5). The upfield-shifted
signals at 3.37 and 3.45 ppm attributed to H-5 of 3 and
4, respectively, are also consistent with the b-manno
configuration of the heptose residue.
85Á90% yield of the title compounds for the coupling
/
and deprotection step. Whereas the ADP-a-heptoses 1
and 2 are stable to either mildly acidic (pH 3) or strongly
basic conditions (up to pH 13), the substrates of
enterobacterial heptosyltransferases*
ADP -glycero- and -glycero- -manno-heptoses 3 and
4*are highly susceptible to hydrolysis of the dipho-
sphate linkage.
/
the b-configured
The a- and b-anomeric configurations were also
confirmed using ‘gated decoupled’ 13C NMR measure-
L
D
D
/
ments (JC-1,H-1
ꢀ
/
173 Hz for a-anomers 1 and 2 and ꢀ
/
163 Hz for b-derivatives 3 and 4). The downfield shifts
of heptose C-3 and C-5 for b-anomers 3 and 4 (at 73.34
and 73.31 ppm for C-3 and at 75.66 and 77.64 ppm for
C-5, respectively) in comparison with the a-dipho-
sphates 1 and 2 (C-3 at 70.81 and 70.75 ppm and C-5
at 72.70 and 74.35 ppm, respectively) fully confirm the
structural assignments. Furthermore, the a-anomers of
the heptose 1-phosphates as well as ADP heptoses
showed dextrorotation in contrast to their b-configured
analogs.
3. Experimental
3.1. General methods
Column chromatography was performed on Silica Gel
60 (230Á400 mesh, E. Merck). Anion-exchange chro-
/
matography was performed on BioRad anion-exchange
resin (Pharmacia). Reactions were monitored by TLC
on Silica Gel 60 F254 HPTLC precoated glass plates with
2.5 cm concentration zone (E. Merck), unless stated
otherwise; spots were visualised by spraying with
In conclusion, the chemical synthesis of both, a- and
b-configured sugar nucleotides ADP-L- and
D-manno-heptopyranose was performed in a high-yield
D-glycero-
procedure using acetyl-protected heptosyl phosphates in
the coupling step to AMP-morpholidate followed by
base-catalysed deacetylation. The merit of this proce-
dure pertains to its simplicity and reproducibility giving
anisaldehydeÁH2SO4; phosphorus-containing com-
/
pounds were additionally detected with a molybdate
solution (0.02 M solution of ammonium cerium(IV)sul-

A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á
/2589
2581
Table 5
1H NMR data of ADP-heptoses 1,2,3 and 4 in D2O
H-1
H-2
H-3
H-4
H-5
H-6
H-7a
H-7b
Adenine
1 Hepp
5.51(dd)
3J1,2 1.9
3J1,P 7.4
4.02(dd)
3J2,3 3.2
3.92(m)
3.85(m)
3.83(m)
3.99(ddd)
3J6,7a 6.2
3J6,7b 7.1
3.73(dd)
2J7a,7b 11.6
3.64(dd)
8.51(H-8)
8.26(H-2)
Ribf
6.14(d)
3J1,2 6.0
4.70(m)
3J2,3 5.1
4.52(dd)
3J3,4 3.5
4.39(m)
4.22(m, 2 H)
2 Hepp
5.56(dd)
3J1,2 2.0
3J1,P 7.9
4.09(dd)
3J2,3 3.2
3.94(dd)
3J3,4 9.3
3.83(t)
3J4,5 9.3
3.97
3J5,6 3.0
4.05(ddd)
3J6,7a 4.0
3J6,7b 6.5
3.83(dd)
2J7a,7b 12.1
3.78(dd)
3.71(dd)
3.75(m)
8.56(H-8)
8.29(H-2)
Ribf
6.19(dd)
3J1,2 6.0
4.70(m)
3J2,3 5.1
4.59(dd)
3J3,4 3.5
4.46(m)
4.28(m, 2 H)
3 Hepp
5.23(dd)
3J1,2 1.0
3J1,P 8.5
4.07(dd)
3J2,3 3.2
3.67(dd)
3J3,4 9.7
3.82(t)
3J4,5 9.7
3.37(dd)
3J5,6 1.7
3.95(ddd)
3J6,7a 6.0
3J6,7b 5.0
3.75(dd)
2J7a,7b 12.3
8.60(H-8)
8.37(H-2)
Ribf
6.18(d)
3J1,2 5.8
4.70(m)
3J2,3 5.2
4.55(dd)
3J3,4 3.5
4.42(m)
4.25(m, 2 H)
4 Hepp
5.21(dd)
3J1,2 1.0
3J1,P 8.7
4.07(dd)
3J2,3 3.3
3.63(dd)
3J3,4 9.4
3.69(t)
3J4,5 9.4
3.45(dd)
3J5,6 3.3
3.99(m)
3.75(m)
8.53(H-8)
8.29(H-2)
Ribf
6.16(d)
3J1,2 5.8
4.73(m)
3J2,3 4.9
4.54(dd)
3J3,4 3.8
4.41(m)
4.23(m, 2 H)
fate dihydrate and ammonium molybdate(VI)tetrahy-
drate in aqueous H2SO4), adenosine-containing com-
pounds were detected by examination under UV light.
Concentration of solutions was performed at diminished
Table 6
13C NMR data of ADP-heptoses in D2O a
1
2
3
4
pressure at temperatures
B30 8C. Triethylamine,
/
C-1 Hepp
97.23
2J1,P 6.0
87.55
97.08
2J1,P 5.8
87.48
96.51
2J1,P 4.8
88.26
96.52
2J1,P 4.1
87.61
CH2Cl2, dry pyridine were purchased from E. Merck,
and were dried by refluxing with CaH2 (5 g/L) for 16 h,
then distilled and stored under argon. Toluene was
distilled from phosphorus pentaoxide and redistilled
from CaH2. The liquids were stored over molecular
sieves 0.4 nm. DMF was stirred with CaH2 (5 g/L) for 16
h at 20 8C, distilled under reduced pressure and stored
over activated molecular sieves 0.3 nm. Triethylammo-
nium bicarbonate (TEAB) buffer, was prepared by
passing a stream of CO2-gas through a cooled (ice-
water bath) solution of Et3N (2 M) in de-ionized water
until a near neutral solution (pH 7.5) was obtained.
Ribf
C-2 Hepp
70.92
70.74
71.37
71.22
3J2,P 10
74.98
153.05
3J2,P 8.7
74.96
153.48
3J2,P 7.1
75.23
152.20
3J2,P 9.9
75.01
152.68
Ribf
Ade
C-3 Hepp
Ribf
70.81
71.06
70.75
71.09
73.34
70.99
73.31
71.09
C-4 Hepp
Ribf
66.58
84.59
3J4,P 9.0
149.76
67.89
84.60
3J4,P 9.2
149.78
66.28
84.80
3J4,P 9.0
149.19
67.78
84.65
3J4,P 9.1
149.73
Ade
Optical rotations were measured with a PerkinÁElmer
/
243 B polarimeter. [a]D20 values are given in units of 10ꢀ1
deg cm3 gꢀ1. NMR-spectra were recorded at 297 K in
D2O as solvent (unless stated otherwise) with a Bruker
DPX 300 spectrometer (1H at 300.13 MHz, 13C at 75.47
MHz and 31P at 121.50 MHz) using standard Bruker
NMR software. 1H NMR spectra were referenced either
to tetramethylsilane or 2,2-dimethyl-2-silapentane-5-sul-
fonic acid. 13C NMR spectra were referenced to 1,4-
dioxane (d 67.40), 31P NMR spectra to 85% aq H3PO4
in separate experiments prior to measurements. Ele-
C-5 Hepp
Ribf
72.70
65.87
74.35
65.91
75.66
65.82
77.64
65.91
2J5,P 5.0
119.20
2J5,P 5.0
119.27
2J5,P 5.8
119.13
2J5,P 4.5
119.30
Ade
C-6 Hepp
Ade
69.24
155.98
72.89
156.23
69.31
156.55
72.74
156.15
C-7 Hepp
C-8 Ade
63.10
140.62
62.65
140.48
62.98
142.34
62.30
140.83
a
Recorded at pD 5.

2582
A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á2589
/
mental analyses were provided by Dr J. Theiner,
Mikroanalytisches Laboratorium, Institut fur Physika-
¨
1 Hz, CH2Ph), 4.93 (dd, 1 H, JH,P 8.5 Hz, JH,H
CH2Ph), 4.94 (dd, 1 H, JH,P 8.5 Hz, JH,H
B
/
1 Hz,
1 Hz,
B
/
lische Chemie, Universitat Wien. MALDI-TOF-MS-
¨
CH2Ph), 4.19 (dd, 1 H, J6,7a 4.6, J7a,7b 12.2 Hz, H-7a),
4.11 (dd, 1 H, J6,7b 7.8 Hz, H-7b), 4.10 (dd, 1 H, H-5),
2.16, 2.11, 2.02, 1.99 and 1.98 (5 s, each 3 H; 5 CH3);
Further elution afforded dibenzyl (2,3,4,6,7-penta-O-
ionisation spectra were recorded on a Dynamo (Thermo
BioAnalysis) instrument in the positive or negative ion
mode using MeCN or water, both with 2% 2,5-
dihydroxybenzoic acid as matrix.
acetyl-
as a syrup. Yield: 25 mg (0.039 mmol, 9%): Rf 0.38 (1:2
n-hexaneÁ
ether); 31P NMR (CDCl3): d 141.20; 1H
NMR (CDCl3): d 7.27Á7.36 (m, 10 H, 2 C6H5), 5.43
(dd, 1 H, J2,3 3.4 Hz, H-2), 5.30 (t, 1 H, J3,4 J4,5 10.1
L-glycero-b-
D-manno-heptopyranosyl)phosphite
3.2. 2,3,4,6,7-Penta-O-acetyl-
heptopyranose (6)
L
-glycero-D-manno-
/
/
ꢃ
/
A soln of heptopyranosyl peracetate 533a (300 mg, 0.65
mmol) in a mixture of dry DMF (5 mL) and N,N-
diisopropylethylamine (1 mL) was stirred with ammo-
nium acetate (200 mg); crystals were prewashed with dry
ether (50 mL) and dried for 10 min under diminished
pressure) for 16 h at room temperature (rt). The reaction
mixture was decanted from the undissolved crystals of
ammonium acetate, diluted with CH2Cl2 (300 mL) and
Hz, H-4), 5.27 (ddd, 1 H, J5,6 2.5 Hz, H-6), 5.17 (dd, 1
H, J1,2 1.0, J1,P 8.0 Hz, H-1), 5.03 (dd, 1 H, H-3), 4.90
(dd, 2 H, JH,P 8.5 Hz, JH,H
B1 Hz, CH2Ph), 4.87 (dd, 2
/
H, JH,P 8.5 Hz, JH,H 1 Hz, CH2Ph), 4.29 (dd, 1 H, J6,7a
B
/
4.9, J7a,7b 11.6 Hz, H-7a), 4.12 (dd, 1 H, J6,7b 7.8 Hz, H-
7b), 3.69 (dd, 1 H, H-5), 2.16, 2.11, 2.02, 1.99 and 1.98 (5
s, each 3 H; 5 CH3).
Procedure without isolation of phosphites from
heptose pentaacetate 6 (195 mg, 0.46 mmol): the
reaction mixture containing phosphite triesters was
cooled to 0 8C and a soln of tert-BuOOH (150 mL of
80% soln in di-tert-butyl peroxide) in CH2Cl2 (1 mL)
was added gradually over a period of 20 min, the
reaction mixture was warmed to rt and stirred for 3 h.
The reaction mixture was diluted with CH2Cl2 (200 mL)
washed with satd aq NaHCO3 (2ꢂ20 mL), water (10
/
mL), dried (MgSO4) and concentrated under diminished
pressure. The residue was purified by silica gel chroma-
tography (3:1 tolueneÁEtOAc) to give amorphous 5
/
(260 mg, 95%). NMR data were in accordance with the
previously published data.33b
3.3. Dibenzyl (2,3,4,6,7-penta-O-acetyl-
manno-heptopyranosyl) phosphate (7) and dibenzyl
(2,3,4,6,7-penta-O-acetyl- -glycero-b- -manno-
heptopyranosyl) phosphate (9)
L
-glycero-a-D-
and successively washed with 1 M aq TEAB (pH 8, 2ꢂ
/
20 mL), water (20 mL) and brine (20 mL). The organic
phase was dried (cotton) and concentrated. The a- and
b-phosphates were separated by chromatography on
L
D
silica gel (3:1 n-hexaneÁ
fractions were pooled, concentrated and purified by a
second chromatography (1:1 tolueneÁEtOAc) to give 7
(268 mg, 85%) as a syrup, Rf 0.6 (ether) or 0.6 (1:2
tolueneÁ
EtOAc); [a]2D0 198 (c 1.0, CHCl3); 13C
NMR(CDCl3): 170.44, 170.15, 169.61, 169.45,
/
ether0/ether). Appropriate
3.3.1. Method A. Heptose pentaacetate 6 (180 mg, 0.43
mmol) and bis(benzyloxy)(diisopropylamino)phosphine
(190 mg, 0.55 mmol) were dried by repeated evapora-
/
tions with dry toluene (3ꢂ10 mL) and then under
/
/
ꢁ
/
diminished pressure for 10 h. The flask was fitted with a
septum cap, flushed with N2 and charged with CH2Cl2
(5 mL). A soln of 1H-tetrazole in MeCN (1 mmol, 2
mL) was added and the mixture was stirred at rt for 2 h
under N2 atmosphere. Monitoring of the reaction by
TLC and 31P NMR spectroscopy showed the formation
of intermediate phosphite triesters. The reaction mixture
was diluted with CH2Cl2 (200 mL) and the soln was
d
169.40 (5 C, CO, Ac), 135.13, 135.05 (2 C, C6H5, Bn),
128.83, 128.70, 128.37, 128.27 (10 C, C6H5, Bn), 95.22
(C-1, J1,P 5.5 Hz), 70.68 (C-5), 70.17, 70.10 (2 C, JC,P
B1
/
Hz, CH2, Bn), 68.79 (C-2, J2,P 11.6 Hz), 68.38 (C-3),
66.88 (C-6), 64.12 (C-4), 62.89 (C-7), 20.71, 20.63, 20.52
(5 C, CH3, Ac); JC-1,H-1 185 Hz; 31P NMR (CDCl3): d
ꢀ2.1. Anal. Calcd for C31H37O15P (680.6): C, 54.71; H,
5.48; P, 4.55. Found: C, 54.76; H, 5.37; P, 4.66.
Further elution gave 9 (28 mg, 9%) as a syrup, Rf 0.4
/
washed with 0.5 M aq TEAB (2ꢂ20 mL), water (20
mL), brine (20 mL), dried (cotton plug) and concen-
trated. The anomeric phosphites were isolated by
/
(ether) or 0.5 (1:2 tolueneÁ
/
EtOAc); [a]2D0
ꢀ238 (c 0.4,
/
chromatography on silica gel (3:1 n-hexaneÁ
ether) to provide dibenzyl (2,3,4,6,7-penta-O-acetyl-
glycero-a- -manno-heptopyranosyl)phosphite as faster-
eluting isomer. Yield: 242 mg (0.36 mmol, 85%); Rf 0.45
(1:2 n-hexaneÁ
ether); [a]2D0 268 (c 1.0, CHCl3); 31P
NMR (CDCl3): d 140.35; H NMR (CDCl3): d 7.25Á
/
ether0
/
CHCl3); 13C NMR (CDCl3): d 170.82, 170.64, 170.36,
170.10, 169.82 (5 C, CO, Ac), 135.64, 135.18 (2 C, C6H5,
Bn), 129.10, 129.01, 128.56, 128.44 (10 C, C6H5, Bn),
95.14 (C-1, J1,P 2.8 Hz), 73.79 (C-5), 71.10 (C-3), 70.21,
L
-
D
/
ꢁ
/
1
70.22 (2 C, JC,P
66.95 (C-6), 64.41 (C-4), 62.46 (C-7), 21.10, 20.98, 20.88
B1 Hz, CH2), 68.90 (C-2, J2,P 8.5 Hz),
/
/
7.37 (m, 10 H, 2 C6H5), 5.47 (dd, 1 H, J1,2 1.8, J1,P 8.1
Hz, H-1), 5.34 (dd, 1 H, J2,3 3.3 Hz, H-3), 5.27 (t, 1 H,
(5 C, CH3, Ac); JC-1,H-1 163 Hz; 31P NMR (CDCl3): d
ꢀ
/
2.1; Anal. Calcd for C31H37O15P (680.6): C, 54.71; H,
J3,4
6), 5.22 (dd, 1 H, H-2), 4.90 (d, 2 H, JH,P 8.0 Hz, JH,H
ꢃJ4,5 10.1 Hz, H-4), 5.24 (ddd, 1 H, J5,6 2.2 Hz, H-
/
B
/
5.48. Found: C, 54.80; H, 5.68.

A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á
/2589
2583
3.3.2. Method B. Heptose pentaacetate 6, bis(benzylox-
y)(diisopropylamino)phosphine and DMAP were sepa-
rately dried by repeated evaporations with dry toluene
150.55, 150.45 (2 C, Ph), 130.48, 126.32, 120.56, 120.49,
120.42 (10 C, C6H5), 96.57 (C-1, J1,P 5.6 Hz), 71.53 (C-
5), 69.20 (C-2, J2,P 11.2 Hz), 68.73 (C-3), 67.20 (C-6),
64.46 (C-4), 62.89 (C-7), 21.13, 21.04, 20.96 (5 C, CH3,
Ac); JC-1,H-1180 Hz. Anal. Calcd for C29H33O15P
(652.5): C, 53.38; H, 5.10. Found: C, 53.62; H, 5.36.
Further elution gave 10 (52 mg, 84%), Rf 0.5 (ether)
(3ꢂ10 mL each). A solution of dibenzyl phosphorote-
/
trazolidite was prepared in situ by adding a soln of 1H-
tetrazole in CH3CN (2 mL, 1 mmol) to a stirred soln of
bis(benzyloxy)(diisopropylamino)phosphine (173 mg,
0.5 mmol) in CH2Cl2 (2 mL) under N2. A clear soln of
or 0.6 (1:2 tolueneÁ
1H NMR (CDCl3): d 7.40Á
(dd, 1 H, J1,2 1.2, J1,P 7.0 Hz, H-1), 5.54 (dd, 1 H, J2,3 3.3
Hz, H-2), 5.34 (t, 1 H, J3,4 J4,5 10.1 Hz, H-4), 5.29
/
EtOAc); [a]2D0
7.15 (m, 10 H, 2 C6H5), 5.58
ꢀ248 (c 0.9, CHCl3);
/
dibenzyl phosphorotetrazolidite in 1:1 CH2Cl2Á
/MeCN
/
(4 mL) (which was decanted from the precipitated
diisopropylammonium tetrazolide) was added dropwise
during 2 h to the stirred solution of 6 (155 mg, 0.37
mmol) and 4-N,N-dimethylaminopyridine (98 mg, 0.8
mmol) in CH2Cl2 (5 mL) through a rubber septum with
a syringe under N2. The reaction mixture was cooled to
0 8C, and a soln of tert-BuOOH (120 mL of 80% soln in
di-tert-butyl peroxide) in CH2Cl2 (1 mL) was added
gradually over 20 min, the reaction mixture was warmed
to rt and stirred for 3 h, diluted with CH2Cl2 (200 mL)
ꢃ
/
(ddd, 1 H, J5,6 2.4 Hz, H-6), 5.07 (dd, 1 H, H-3), 4.28
(dd, 1 H, J6,7a 5.3, J7a,7b 11.5 Hz, H-7a), 4.12 (dd, 1 H,
J6,7b 7.7 Hz, H-7b), 3.84 (dd, 1 H, H-5), 2.14, 2.15, 2.04,
2.03, 2.0 (5 s, each 3 H, 5 CH3); 13C NMR (CDCl3): d
170.38, 170.16, 169.78, 169.70, 169.41 (5 C, CO, Ac),
150.23, 150.15 (2 C, Ph), 129.90Á120.19 (12 C, C6H5),
/
95.34 (C-1, J1,P 5.6 Hz), 73.46 (C-5), 70.56 (C-3), 68.21
(C-2, J2,P 8.7 Hz), 66.55 (C-6), 63.95 (C-4), 61.98 (C-7),
20.64, 20.45 (5 C, CH3, Ac); JC-1,H-1 167 Hz. Anal. Calcd
for C29H33O15P (652.5): C, 53.38; H, 5.10. Found: C,
53.30; H, 5.11.
and successively washed with 1 M aq TEAB (pH 8, 2ꢂ
/
20 mL), water (20 mL) and brine (20 mL). The organic
phase was dried (cotton) and concentrated. The phos-
photriesters were isolated in the same fashion as
described in method A, which furnished first 7 (140
mg, 56%) and finally 9 (85 mg, 34%).
3.5. 2,3,4,6,7-Penta-O-acetyl-
heptopyranosyl phosphate (monotriethylammonium salt)
(11)
L
-glycero-a-D-manno-
3.4. Diphenyl (2,3,4,6,7-penta-O-acetyl-
manno-heptopyranosyl) phosphate (8) and diphenyl
(2,3,4,6,7-penta-O-acetyl- -glycero-b- -manno-
heptopyranosyl) phosphate (10)
L
-glycero-a-D-
A soln of 7 (68 mg, 0.10 mmol) in dry MeOH (20 mL)
was hydrogenated in the presence of 10% Pd/C (20 mg)
for 10 h at atmospheric pressure. After completion of
the reaction, the catalyst was removed by filtration
through a pad of Celite and washed with MeOH (20
mL). The free phosphate was neutralised by addition of
Et3N (0.2 mmol). The filtrate was concentrated and the
residue was lyophilised from water (10 mL) to give the
monotriethylammonium salt of 11 (59 mg, 99%) as a
white fluffy solid which was used in the next step
L
D
Diphenyl phosphorochloridate and 6 were dried by
coevaporation with dry toluene (2ꢂ10 mL each), and
/
dried under diminished pressure. To a stirred soln of 6
(40 mg, 0.095 mmol) and 4-N,N-dimethylaminopyridine
(56 mg, 0.46 mmol) in CH2Cl2 (3 mL), a soln of diphenyl
phosphorochloridate (25 mL, 0.12 mmol) in CH2Cl2 (2
mL) was added dropwise during 2 h at ambient
temperature under N2. The reaction mixture was diluted
with CH2Cl2 (100 mL), washed successively with 1 M aq
without further purification. Rf 0.5 (60:35:6:1 CHCl3Á
MeOHÁ25% aq NH4OHÁ 108 (c 1.0,
water); [a]2D0
water); 31P NMR: d ꢀ2.0; 13C NMR: d 174.28, 173.58,
/
/
/
ꢁ
/
/
TEAB buffer (2ꢂ
mL), the organic phase was dried (cotton) and concen-
trated. The residue was fractionated by chromatography
/
20 mL), water (10 mL) and brine (20
173.52, 173.15, 172.98 (5 C, CO), 93.65 (d, C-1, J1,P 5.1
Hz), 70.75 (d, C-2, J2,P 10.6 Hz), 69.82 (C-3), 69.58 (C-
5), 68.29 (C-6), 65.43 (C-4), 63.91 (C-7), 47.09 (CH2,
Et3N), 20.54, 20.51, 20.45 (5 C, CH3), 8.63 (CH3, Et3N);
on silica gel (3:1 n-hexaneÁ
fractions were pooled, concentrated and purified by a
second chromatography (49:107:3 tolueneÁEtOAc)
which gave 8 as faster eluting isomer. Yield: 5 mg
(9%), syrup, Rf 0.8 (ether) or 0.7 (1:2 tolueneÁEtOAc);
188 (c 0.7, CHCl3); H NMR (CDCl3): d 7.43Á
/
ether0/ether). Appropriate
JC-1,H-1 178 Hz. Anal. Calcd for C23H39NO15P×
/H2O
/
/
(618.5): C, 44.66; H, 6.68; N, 2.26. Found: C, 44.85; H,
6.56; N, 2.11.
/
1
[a]2D0
ꢁ
/
/
3.6.
(monotriethylammonium salt) (12)
L-Glycero-a-D-manno-heptopyranosyl phosphate
7.20 (m, 10 H, 2 C6H5), 5.90 (dd, 1 H, J1,2 1.2, J1,P 7.0
Hz, H-1), 5.40 (dd, 1 H, J2,3 3.1, J3,4 10.0 Hz, H-3), 5.36
(m, 1 H, H-4), 5.35 (m, 1 H, H-2), 5.3 (ddd, 1 H, J6,7a
6.1, J6,7b 8.3 Hz, H-6), 4.29 (dd, 1 H, J5,6 2.0, J4,5 9.0 Hz,
H-5), 4.16 (m, 2 H, H-7a, H-7b), 2.20, 2.14, 2.25, 2.22
and 1.95 (5 s, each 3 H, 5 Ac). 13C NMR (CDCl3): d
170.80, 170.55, 170.11, 169.91, 169.83 (5 C, CO, Ac),
A soln of 11 (25 mg, 0.04 mmol) in 7:3:1 MeOHÁ
/
waterÁ
/
Et3N (2 mL, pH 12) was stirred at ambient temperature
for 3 h. The reaction mixture was concentrated and
lyophilised from water (2ꢂ/20 mL) to give 15 mg (0.038
mmol, 97%) of 12 as a white amorphous solid; Rf 0.2

2584
A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á2589
/
(5:7:2:2 CHCl3Á
218 (c 1.0, water); 31P NMR: d ꢀ
47.72 (CH2, Et3N), 9.26 (CH3, Et3N), other signals see
Table 3; JC-1,H-1 173 Hz. Anal. Calcd for C13H29NO10P×
/
MeOHÁ
/
25% aq NH4OHÁ
/
water); [a]2D0
3.9. 2,3,4,6,7-Penta-O-acetyl-
heptopyranose (16)
D
-glycero-D-manno-
ꢁ
/
/
0.4; 13C NMR: d
/
Compound 16 was prepared from heptosyl peracetate
1533 (110 mg, 0.24 mmol) in the same fashion as
described for 6. Yield: 95 mg (95%); [a]D20
H2O (408.2): C, 38.24; H, 7.65. Found: C, 38.90; H,
7.41.
ꢁ278 (c 0.6,
/
CHCl3); 1H NMR (CDCl3): d 5.40 (dd, 1 H, J2,3 3.4, J3,4
9.6 Hz, H-3), 5.29 (dd, 1 H, J4,5 9.7 Hz, H-4), 5.23 (m, 3
H, H-1, H-2, H-6), 4.39 (dd, 1 H, J6,7 3.6, J7a,7b 12.0 Hz,
H-7a), 4.28 (dd, 1 H, J6,7b 4.8 Hz, H-7b), 4.21 (dd, 1 H,
J5,6 2.8 Hz, H-5), 3.18 (d, 1 H, OH), 2.18, 2.11, 2.09 and
2.02 (4 s, 15 H, 5 Ac). Anal. Calcd for C17H24O12
(420.37): C, 48.57; H, 5.75. Found: C, 48.52; H, 6.07.
3.7. 2,3,4,6,7-Penta-O-acetyl-
heptopyranosyl phosphate (monotriethylammonium salt)
(13)
L
-glycero-b-D-manno-
3.7.1. Method A. Compound 13 was prepared from 9
(78 mg, 0.11 mmol) in the same manner as described for
the synthesis of 11 from 7. Yield: 68 mg (99%); Rf 0.4
(60:35:6:1 CHCl3Á
/
MeOHÁ
/
25% aq NH4OHÁ
/
water);
1.5; 13C
3.10. Dibenzyl (2,3,4,6,7-penta-O-acetyl-
manno-heptopyranosyl) phosphate (17) and dibenzyl
(2,3,4,6,7-penta-O-acetyl- -glycero-b- -manno-
heptopyranosyl) phosphate (19)
D
-glycero-aD-
[a]2D0 318 (c 1.0, water); 31P NMR: d ꢀ
ꢀ
/
/
NMR (D2O): d 174.27, 173.72, 173.59, 173.53, 172.93 (5
C, CO), 93.98 (d, C-1, J1,P 3.2 Hz), 72.50 (C-5), 71.52
(C-3), 70.46 (d, C-2, J2,P 6.3 Hz), 67.96 (C-6), 65.38 (C-
4), 63.74 (C-7), 47.10 (CH2, Et3N), 20.55, 20.51, 20.48,
20.47, 20.36 (5 C, CH3), 8.64 (CH3, Et3N); JC-1,H-1 164
Hz. Anal. Calcd for C23H39NO15P×
43.40; H, 6.81; N, 2.20. Found: C, 43.21; H, 5.97; N,
2.32.
D
D
Compounds 17 and 19 were prepared from heptosyl
pentaacetate 16 (110 mg, 0.26 mmol) in the fashion
described for the synthesis of 7 and 9 using method B.
Yield for 17 (103 mg, 0.15 mmol, 58%); syrup, Rf 0.6
/2 H2O (636.6): C,
(ether); [a]2D0
ꢁ
42.58 (c 1.0, CHCl3); 31P NMR (CDCl3):
/
d ꢀ
/
2.7; 13C NMR (CDCl3): d 170.94, 170.31, 170.11,
3.7.2. Method B. A soln of 10 (40 mg, 0.06 mmol) in dry
MeOH (20 mL) was hydrogenated in the presence of
PtO2 (10 mg) for 10 h at atmospheric pressure. After
addition of Et3N (0.12 mmol), the catalyst was removed
by filtration through a pad of Celite and washed with
MeOH (20 mL). The combined filtrates were concen-
170.05, 169.83, (5 C, CO, Ac), 135.63, 135.54 (2 C,
C6H5), 129.21, 129.14, 129.11, 128.57, 128.37 (10 C,
C6H5), 95.19 (C-1, J1,P 5.4 Hz), 72.33 (C-5), 70.42 (1 C,
JC,P 5.5 Hz, CH2Ph), 70.24 (1 C, JC,P 5.5 Hz, CH2Ph),
70.09 (C-6), 68.96 (C-2, J2,P 10.8 Hz), 68.69 (C-3), 66.21
(C-4), 61.90 (C-7), 21.12, 21.09, 20.98 (5 C, CH3, Ac);
JC-1,H-1 187 Hz. Anal. Calcd for C31H37O15P (680.6): C,
54.71; H, 5.48. Found: C, 54.80; H, 5.26.
trated. The partially deacetylated (ꢀ
/
5Á10% according
/
to 1H NMR data) phosphate 13 may be used in the next
steps without further purification. For characterisation,
crude 13 was purified by flash chromatography on silica
Yield for 19: 60 mg (34%), syrup, Rf 0.45 (ether); [a]D20
ꢀ
/
16.58 (c 1.0, CHCl3); 31P NMR (CDCl3): d ꢀ
2.14;
/
gel (30:20:10
/
25:25:1 CHCl3Á
/
MeOHÁ
/
water). Appro-
13C NMR (CDCl3): d 171.24, 170.82, 170.15, 170.0,
169.65, (5 C, CO, Ac), 135.94, 135.65 (2 C, C6H5Ph),
129.14, 129.04, 129.01, 128.93, 128.40, 128.36 (10 C,
C6H5Ph), 95.20 (C-1, J1,P 7.3 Hz), 74.38 (C-5), 70.29 (1
C, JC,P 7.7 Hz, CH2Ph), 69.93 (1 C, JC,P 8.5 Hz,
CH2Ph), 69.85, 69.82 (C-3, C-6), 68.64 (C-2, J2,P 12.4
Hz), 65.94 (C-4), 61.52 (C-7), 21.21, 21.12, 20.91 (5 C,
CH3, Ac); JC-1,H-1 165 Hz. MALDI-TOF-MS: m/z:
priate fractions were concentrated to 10 mL volume, the
solution was cooled to 0 8C and the pH was adjusted to
4.5 with Dowex 50 (Hꢁ) resin. The resin was removed
by filtration, the total eluate was made neutral by
addition of Et3N, concentrated to 5 mL volume (result-
ing in a pH of ꢀ5) at 25 8C and lyophilised to give the
monotriethylammonium salt of 13 (35 mg, 93%) as a
white fluffy solid.
/
704.97 [Mꢁ
Na]ꢁ.
/
3.8.
(triethylammonium salt) (14)
L
-Glycero-b-
D
-manno-heptopyranosyl phosphate
3.11. Diphenyl (2,3,4,6,7-penta-O-acetyl-
manno-heptopyranosyl) phosphate (18) and diphenyl
(2,3,4,6,7-penta-O-acetyl- -glycero-b- -manno-
heptopyranosyl) phosphate (20)
D
-glycero-a-D-
D
D
Compound 14 was prepared from 13 (32 mg, 0.05 mmol)
as described for the synthesis of 12. Yield: 21 mg (0.05
mmol, 98%); Rf 0.15 (5:7:2:2 CHCl3Á
NH4OHÁ
water); [a]2D0 118 (c, 0.4, water); 31P NMR:
d ꢀ
2.0, 13C NMR: d 47.57 (CH2, Et3N), 9.07 (CH3,
Et3N), other signals see Table 3; JC-1,H-1 161 Hz. Anal.
Calcd for C13H29NO10P×0.5 H2O (399.4): C, 39.10; H,
7.57. Found: C, 38.89; H, 8.15.
/
MeOHÁ
/
25% aq
Compounds 18 and 20 were prepared from 16 (19.3 mg,
0.046 mmol) in the same manner as described for the
synthesis of 8 and 10.
/
ꢀ
/
/
Yield for 18: 4.5 mg (15%); Rf 0.55 (4:1 etherÁ
/
1
/
hexane); [a]2D0
ꢁ398 (c 0.4, CHCl3); H NMR (CDCl3):
/
d 7.40Á7.20 (m, 10 H, 2 C6H5), 5.85 (dd, 1 H, J1,2 2.1,
/

A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á
/2589
2585
J1,P 6.4 Hz, H-1), 5.38 (dd, 1 H, J2,3 3.1, J3,4 9.7 Hz, H-
3), 5.37 (t, 1 H, J4,5 9.7 Hz, H-4), 5.30 (dd, 1 H, H-2),
5.16 (ddd, 1 H, J6,7a 4.1, J6,7b 7.8 Hz, H-6), 4.38 (dd, 1
H, J7a,7b 12.2 Hz, H-7a), 4.24 (dd, 1 H, J5,6 2.4, J4,5 9.8
Hz, H-5), 4.22 (dd, 1 H, H-7b), 2.15, 2.11, 2.03, 2.01 (5 s,
each 3 H; 5 CH3); 13C NMR (CDCl3): d 170.52, 169.97,
169.65, 169.42 (5 C, CO, Ac), 150.30, 149.98 (2 C, Ph),
129.96, 125.87, 125.81, 120.29, 120.22, 120.15 (10 C, Ph),
96.75 (C-1, J1,P 5.9 Hz), 72.35 (C-5), 69.59 (C-6), 68.45
(C-2, J2,P 12.0 Hz), 68.21 (C-3), 65.72 (C-4), 61.36 (C-7),
20.68, 20.57, 20.54 (5 C, CH3, Ac); JC-1,H-1 177 Hz.
H2O (408.4): C, 38.24; H, 7.65; N, 3.43. Found: C,
38.39; H, 7.93; N, 3.62.
3.14. 2,3,4,6,7-Penta-O-acetyl-
heptopyranosyl phosphate (monotriethylammonium salt)
(23)
D
-glycero-b-D-manno-
3.14.1. Method A. Compound 23 was prepared from 19
(30 mg, 0.05 mmol) in the same way as described for the
synthesis of 11 from 7. Yield: 29 mg (98%); Rf 0.33
(60:35:6:1 CHCl3Á
/
MeOHÁ
/
25% aq NH4OHÁ
/
water);
1.6; 13C
MALDI-TOF-MS: m/z: 675.88 [Mꢁ
Yield for 20: 24.4 mg (81%), Rf 0.45 (4:1 etherÁ
hexane); [a]2D0 5.68 (c 1.2, CHCl3); 1H NMR
(CDCl3): d 7.38Á7.09 (m, 10 H, 2 C6H5), 5.59 (dd, 1
/
Na]ꢁ.
[a]2D0 1.48 (c 1.0, water); 31P NMR: d ꢀ
ꢀ
/
/
/
NMR: d 174.18, 173.54, 173.35, 173.25, 172.95 (5 C,
CO), 93.64 (d, C-1, J1,P 3.3 Hz), 73.09 (C-5), 71.63 (C-3),
70.90 (C-6), 70.13 (d, C-2, J2,P 6.2 Hz), 66.98 (C-4),
62.35 (C-7), 47.09 (CH2, Et3N), 20.70, 20.60, 20.56,
20.47, 20.38 (5 C, CH3), 8.63 (CH3, Et3N); JC-1,H-1 164
ꢀ
/
/
H, J1,2 1.7, J1,P 7.2 Hz, H-1), 5.37 (dd, 1 H, J2,3 3.3 Hz,
H-2), 5.22 (ddd, 1 H, J5,6 4.6 Hz, H-6), 5.19 (t, 1 H,
J3,4
ꢃ/J4,5 8.7 Hz, H-4), 5.0 (dd, 1 H, H-3), 4.32 (dd, 1 H,
Hz. MALDI-TOF-MS: m/z: 523.54 [Mꢁ
Na]ꢁ.
/
J6,7a 3.5, J7a,7b 12.2 Hz, H-7a), 4.12 (dd, 1 H, J6,7b 6.5
Hz, H-7b), 3.80 (dd, 1 H, H-5), 2.03, 2.02, 1.99, 1.98,
1.93 (5 s, each 3 H; 5 CH3); 13C NMR (CDCl3): d
170.478, 169.82, 169.69, 169.65, 169.52 (5 C, CO, Ac),
3.14.2. Method B. Compound 23 was prepared from 20
(8 mg, 0.012 mmol) in the same fashion as described for
the synthesis of 13 from 10. Yield: 5.8 mg (93%).
150.28, 150.0 (2 C, Ph), 129.88Á120.11 (10 C, Ph), 94.70
/
(C-1, J1,P 4.7 Hz), 74.01 (C-5), 69.63 (C-3), 67.38 (C-2,
J2,P 8.1 Hz), 69.47 (C-6), 65.85 (C-4), 61.30 (C-7), 20.82,
20.69, 20.54, 20.47 (5 C, CH3, Ac); JC-1,H-1 169 Hz.
Anal. Calcd for C29H33O15P (652.5): C, 53.38; H, 5.10.
Found: C 53.36; H 5.43.
3.15.
(triethylammonium salt) (24)
D
-Glycero-b-D-manno-heptopyranosyl phosphate
Compound 24 was prepared from 23 (25 mg, 0.04 mmol)
in the way described for the synthesis of 12. Yield: 16 mg
(98%); Rf 0.1 (5:7:2:2 CHCl3Á
/
MeOHÁ25% aq
/
3.12. 2,3,4,6,7-Penta-O-acetyl- -glycero-a- -manno-
heptopyranosyl phosphate (monotriethylammonium salt)
(21)
D
D
NH4OHÁ
/
water); [a]2D0
ꢀ
358 (c 0.7, water); 31P NMR:
/
d 1.3; 13C NMR: d 47.07 (CH2, Et3N), 8.62 (CH3,
Et3N), other signals see Table 3; JC-1,H-1 160 Hz.
MALDI-TOF-MS: m/z: 335.71 [Mꢁ .
2Na]2ꢁ
/
Compound 21 was prepared from 17 (56 mg, 0.08 mmol)
in the same fashion as described for the synthesis of 11
3.16. Adenosine 5?-(2,3,4,6,7-penta-O-acetyl-
a- -manno-heptopyranosyl)diphosphate
(triethylammonium salt) (26)
L
-glycero-
from 7. Yield: 47.5 mg (99%); Rf 0.4 (60:35:6:1 CHCl3Á
MeOHÁ25% aq NH4OHÁ 398 (c 0.8,
water); [a]2D0
water); 31P NMR: d ꢀ1.7; 13C NMR (D2O): d 174.17,
/
D
/
/
ꢁ
/
/
173.49, 173.37, 173.17, 173.08 (5 C, CO), 93.30 (d, C-1,
J1,P 4.8 Hz), 70.93 (C-6), 70.13 (C-5), 70.12 (d, C-2, J2,P
9.9 Hz), 70.03 (C-3), 67.01 (C-4), 62.41 (C-7), 47.09
(CH2, Et3N), 20.72, 20.61, 20.57, 20.53, 20.48 (5 C,
CH3), 8.63 (CH3, Et3N); JC-1,H-1182 Hz. Anal. Calcd for
Pentaacetyl heptosyl phosphate 11 (25 mg, 0.042 mmol)
was made anhydrous by repeated dissolution in dry
pyridine and evaporation of solvent (4ꢂ10 mL). After
/
each evaporation step, dry N2 was flushed into the
rotary evaporator. AMP-morpholidate (4?-morpholine-
N,N?-dicyclohexylcarboxamidinium salt) 25 (44 mg,
0.06 mmol) was dissolved in dry pyridine and evapo-
rated to dryness and the process was repeated three
times with exclusion of moisture under N2. Both
components were finally dissolved in pyridine and
combined in a one-neck round-bottom flask under N2
atmosphere. The reaction mixture was repeatedly eva-
C23H39NO15P×
/
1.5 H2O (627.6): C, 44.02; H, 6.75; N,
2.23. Found: C, 43.96; H, 6.54; N, 2.49.
3.13.
(triethylammonium salt) (22)
D
-Glycero-a- -manno-heptopyranosyl phosphate
D
Compound 22 was prepared from 21 (19 mg, 0.03 mmol)
in the way as described for the synthesis of 12. Yield: 12
porated from pyridine (3ꢂ10 mL) and flushed with dry
/
mg (98%); Rf 0.15 (5:7:2:2 CHCl3Á
NH4OHÁ
water); [a]2D0 208 (c 0.7, water); lit.37
(c 0.5, 1:1 waterÁ
THF); 31P NMR: d 0.5; 13C NMR: d
47.51 (CH2, Et3N), 9.04 (CH3, Et3N), other signals see
Table 3; JC-1,H-1 170 Hz. Anal. Calcd for C13H29NO10P×
/
MeOHÁ
/
25% aq
N2. A final amount of pyridine (10 mL) was added to
give a clear solution. About 70% of pyridine was
removed by concentration and the reaction vessel was
sealed under N2. The solution was vigorously stirred and
the progress of the reaction was monitored by TLC-
/
ꢁ
/
ꢁ
/
338
/
/

2586
A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á2589
/
analysis (35:20:2:2 CHCl3Á
water). The reaction was generally complete within 2Á
/
MeOHÁ
/
25% aq NH4OHÁ
/
3.17. Adenosine 5?-(2,3,4,6,7-penta-O-acetyl-
a- -manno-heptopyranosyl)diphosphate
(triethylammonium salt) (27)
D
-glycero-
D
/
3 days as judged by the appearance of a major UV- and
Pi-positive spot of ADP-Hep (always as two spots with
DRf approx 0.2 corresponding to two different salt
forms of 26: ammonium salt with Rf 0.3 and 4-morpho-
line-N,N?-dicyclohexylcarboxamidinium salt with Rf
0.5). The reaction was stopped by evaporation of
pyridine. The diphosphate 26 was isolated using silica
gel or anion-exchange chromatography.
Compound 27 was prepared from 21 (20 mg, 0.033
mmol) as described for the synthesis of 26 and purified
using method A. Yield 28 mg (0.03 mmol, 86%); Rf 0.4
(35:20:2:2 CHCl3Á
48 (c 0.5, water); 31P NMR: d ꢀ
Rib), ꢀ13.5 (d, PHep).
/
MeOHÁ
/
25% NH4OHÁ
/
water); [a]2D0
ꢁ
P
/
/
10.5 (d, JP,P 24.5 Hz;
/
3.18. Adenosine 5?-(2,3,4,6,7-penta-O-acetyl-
b- -manno-heptopyranosyl)diphosphate
(triethylammonium salt) (28)
L
-glycero-
D
3.16.1. Purification
using
anion-exchange
chromatography (Method A). The crude reaction pro-
ducts were dissolved in 10 mL water and the solution
was allowed to slowly adsorb on a resin bed of BioRad
anion-exchange column (prepacked cartridge 5 mL,
Compound 28 was prepared from 13 (30 mg, 0.05 mmol)
in the same fashion as described for the synthesis of 26,
and isolated using method A. Yield 43 mg (92%); Rf
HCO2
ꢀ-form) connected to an FPLC-system. The col-
/
0.30 (35:20:2:2 CHCl3Á
water); [a]2D0 198 (c 0.5, water); 31P NMR: d ꢀ
(d, JP,P 19.0 Hz; PRib), ꢀ13.4 (d, PHep).
/
MeOHÁ
/
25% aq NH4OHÁ
/
umn was operated at 0.7 mL/min, fractions (1 mL) were
collected. The column was washed first with water (20
mL) and then developed with a linear gradient of TEAB
ꢀ
/
/10.6
/
buffer, pH 8 (0.010
/
0.25 M). The eluate was monitored
3.19.
cyclophosphate (triethylammonium salt) (29)
L
-Glycero-b-D-manno-heptopyranosyl 1,2-
at 280 nm, 26 was eluted at a concentration of 0.15 M
TEAB. The fractions containing ADP-Hep were pooled,
concentrated to 10 mL vol, the solution was cooled to
0 8C, and the pH was adjusted to 4.5 with Dowex 50
(Hꢁ) resin. The resin was removed by filtration, the
total eluate was made neutral by addition of Et3N,
concentrated to 5 mL vol at 25 8C and lyophilised to
give 26 as white solid. Yield: 36 mg (91%); Rf 0.37
A soln of 28 (9 mg, 0.01 mmol) in 7:3:1 MeOHÁ
/
waterÁ
/
Et3N (pH 12, 2 mL) was stirred at ambient temperature
for 3 h. The reaction mixture was diluted with water (20
mL), concentrated to a vol of 10 mL, and lyophilised to
furnish a mixture of monotriethylammonium salts of 29
and AMP. The residue was dissolved in water (5 mL)
and packed on a strong anion-exchange resin (5 mL Bio-
Rad Econo-pack Q cartridge, HCOꢀ3 form). A gradient
(35:20:2:2 CHCl3Á
[a]2D0 88 (c 1.0, water); 31P NMR: d ꢀ
25.2 Hz; PRib), ꢀ
13.9 (d, PHep); 13C NMR: d 174.13,
/
MeOHÁ
/
25% aq NH4OHÁ/water);
ꢁ
/
/10.5 (d, JP,P
0.01 M TEAB (pH 7.5)0/0.2 M TEAB was used, the
/
eluate was monitored at 280 nm and checked by TLC
for the presence of 29. Fractions containing 29 were
pooled and lyophilised to give, after desalting on a
173.56, 173.42, 172.84, 172.79 (5 C, CO), 153.59 (C-
6Ade), 149.85 (C-2Ade), 149.13 (C-4Ade), 141.45 (C-8Ade),
118.93 (C-5Ade), 94.16 (C-1Hep, J1,P 4.9 Hz), 87.87 (C-
1Rib), 84.19 (C-4Rib, J4,P 9.2 Hz), 75.18 (C-2Rib), 70.58
(C-3Hep), 69.91 (C-2Hep, J2,P 11.4 Hz), 69.73 (C-5Hep),
68.27 (C-6Hep), 65.55 (C-5Rib, J5,P 4.5 Hz), 65.32 (C-
4Hep), 64.09 (C-7Hep), 47.07 (CH2, Et3N), 20.52, 20.47,
20.42, 20.36, 20.33 (5 C, CH3, Ac), 8.62 (CH3, Et3N).
BioGel P-2 column (2ꢂ
/
50 cm), 29 as a white solid (3
MeOHÁ25% aq
water); H NMR: d 5.53 (dd, 1 H, J1,2 1.9,
mg, 85%): Rf 0.6 (5:7:2:2 CHCl3Á
/
/
1
NH4OHÁ
/
J1,P 25.2 Hz, H-1), 4.62 (m, 1 H, H-2), 4.01 (ddd, 1 H, H-
6), 3.88 (m, 2 H, H-3, H-4), 3.71 (dd, 1 H, J6,7a 7.8, J7a,7b
11.6 Hz, H-7a), 3.70 (dd, 1 H, J6,7b 6.3 Hz, H-7b), 3.40
(m, 1 H, H-5), 3.20 (m, 6 H, CH2, Et), 1.28 (t, 9 H, CH3,
Et); 31P NMR: d 17.8; 13C NMR: d 97.9 (C-1), 79.5 (C-
2), 74.2 (C-5), 72.5 (C-3), 69.6 (C-6), 66.5 (C-4), 63.5 (C-
7).
3.16.2. Purification
procedure
using
silica
gel
chromatography (Method B). The reaction mixture
containing 26 was fractionated on a silica gel column
using stepwise gradient 60:40:1:10
/
50:50:1:1 CHCl3Á
/
3.20.
(ammonium salt) (30)
L
-Glycero-D-manno-heptopyranose 2-phosphate
MeOHÁ25% aq NH4OHÁwater as eluent. Fractions
/
/
containing ADP-Hep were combined, concentrated to
dryness, redissolved in water, cooled to 0 8C, and the pH
was adjusted to 4.5 with Dowex 50 (Hꢁ) resin. The resin
was removed by filtration, the filtrate was adjusted to
pH 6 with Et3N, concentrated at 25 8C to about 5 mL
and lyophilised to furnish 26 (28 mg, 72%).
A suspension of 29 (6.5 mg, 0.022 mmol) and silica gel
(0.5 g) in 10:3:3 MeOHÁ
/
waterÁ25% aq NH4OH (5 mL)
/
was stirred for 4 h at rt. Silica gel was separated on a
filter and the filtrate was concentrated. Chromatogra-
phy of the residue on silica gel (10:12:1:10
/
10:12:3:3

A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á
/2589
2587
CHCl3Á
phosphate 30 (5.5 mg, 85%) as a white solid. Rf 0.2
(5:7:2:2 CHCl3ÁMeOHÁ25% aq NH4OHÁ
water); 1H
NMR: d 5.28 (d, 0.7 H, J1a,2 1.5 Hz, H-1a), 4.81 (d, 0.3
H, J1b,2 1 Hz, 0.3 H, H-1b), 4.39 (dd, 0.3 H, J2b,3 3.0,
J2b,P 7.7 Hz, 0.3 H, H-2b), 4.29 (ddd, J2a,3 2.9, J2a,P 8.5
Hz, H-2a), 4.05Á3.90 (m, 1 H, H-6a,b), 3.89 (t, 1 H,
J4,5 J3,4 9.5 Hz, H-4a,b), 3.82 (dd, 0.7 H, H-3a), 3.81
(dd, 0.3 H, H-3b), 3.73 (dd, 0.7 H, J5a,6 1.5 Hz, H-5a),
3.68 (dd, 0.7 H, J6,7a-a 6.7 Hz, 0.7 H, H-7a-a), 3.60Á3.70
/
MeOHÁ
/
25% aq NH4OHÁ
/
water) afforded
Calcd for C17H26N5O16Pꢀ2 ; [Mꢀ
/
H]ꢀ: 618.10. Anal.
2.5 H2O (765.6): C, 36.08; H,
Calcd for C23H42N6O16P2×
/
/
/
/
6.19; N, 10.98. Found: C, 36.39; H, 5.88; N, 10.44.
B
/
3.23.2. Method B. A solution of 26 (9 mg, 0.01 mmol) in
1:5 25% aq NH4OHÁwater (pH 12, 2 mL) was kept at
/
/
4 8C for 48 h, the reaction mixture was diluted with
water (30 mL), concentrated to 20 mL vol and
lyophilised to give ammonium salt of 1 (7 mg, 0.0097
mmol, 98%) as white solid.
ꢃ
/
/
(m, 0.3 H, H-7b-a, H-7b-b), 3.67 (dd, 0.7 H, J6,7a-b 5.5,
J7a-a,7a-b 12.0 Hz, H-7a-b), 3.33 (dd, 0.3 H, J5b,6 1.6, J5b,4
9.8 Hz, H-5b); 31P NMR: d 5.1 (b-anomer), 3.5 (a-
anomer); 13C NMR (D2O): d 95.7 (C-1b), 94.2 (C-1a),
76.0 (C-2b), 75.0 (C-2a), 74.5 (C-5b), 73.0 (C-5a), 69.6
(C-6), 68.0 (C-4), 63.8 (C-7).
3.24. Adenosine 5?-(
heptopyranosyl) diphosphate (2) (monotriethylammonium
salt)
D
-glycero-a-D-manno-
Compound 2 was prepared from 27 (12 mg, 0.013 mmol)
as described for 1 using method A to give 2 (9 mg, 0.012
3.21. Adenosine 5?-(2,3,4,6,7-penta-O-acetyl-
b- -manno-heptopyranosyl)diphosphate
(triethylammonium salt) (31)
D
-glycero-
mmol, 98%) as a white solid. Rf 0.32 (10:12:3:3 CHCl3Á
MeOHÁ25% aq NH4OHÁ 4.58 (c 1.0,
water); [a]2D0
water); 31P NMR: d ꢀ
10.8 (d, JP,P 20.8 Hz; PRib), ꢀ
13.2 (d, PHep); JC-1,H-1 173 Hz. MALDI-TOF-MS: m/z
618.42 [Mꢀ
H]ꢀ. Calcd for C17H26N5O16Pꢀ2 ; [Mꢀ
H]ꢀ: 618.10. Anal. Calcd for C23H42N6O16P2×
0.5 H2O
/
D
/
/
ꢁ
/
/
/
Compound 31 was prepared from 23 (15 mg, 0.025
mmol) in the same way as described for the synthesis of
26, and isolated using method A. Yield: 22 mg (95%); Rf
/
/
/
(729.6): C, 37.87; H, 5.94; N, 11.52. Found: C, 38.08; H,
0.35 (35:20:2:2 CHCl3Á
[a]2D0 48 (c 1.0, water); 31P NMR: d ꢀ
17.8 Hz; PRib), ꢀ13.6 (d, PHep).
/
MeOHÁ
/
25% NH4OHÁ
/
water);
6.09; N, 11.07.
ꢀ
/
/10.6 (d, JP,P
/
3.25. Adenosine 5?-(
L-glycero-b-D-manno-
heptopyranosyl)diphosphate (monotriethylammonium
salt) (3)
3.22.
cyclophosphate (monotriethylammonium salt) (32)
D
-Glycero-b- -manno-heptopyranosyl 1,2-
D
A solution of adenosine 5?-diphospho-heptose 28 (10
Compound 32 was obtained from 31 (7 mg, 0.008 mmol)
in the same way as described for 29. Yield 2.0 mg (0.006
mg, 0.011 mmol) in 4:3:0.05 0.1 M aq TEABÁ
/
MeOHÁ
/
Et3N (3 mL) was kept at ꢀ28 8C for 24 h. The reaction
/
mmol, 80%); Rf 0.6 (5:7:2:2 CHCl3Á
/
MeOHÁ
/
25% aq
water; 1H NMR: d 5.52 (dd, 1 H, J1,2 2.2, J1,P
mixture was diluted with water (20 mL), the pH was
adjusted to 4.7 with Dowex 50 (Hꢁ-form) at 0 8C. The
resin was filtered off, washed excessively with water, the
combined filtrates were concentrated to 20 mL vol and
lyophilised. The mixture contained approx 95% of 3 (Rf
NH4OHÁ
/
24.8 Hz, H-1), 4.61 (m, 1 H, H-2), 4.06 (ddd, 1 H, J5,6
2.0 Hz, H-6), 3.84 (m, 2 H, H-3, H-4), 3.81 (dd, 1 H,
J6,7a 4.2, J7a,7b 11.9 Hz, H-7a), 3.74 (dd, 1 H, J6,7b 7.2
Hz, H-7b), 3.49 (m, 1 H, H-5), 3.20 (m, 6 H, CH2, Et),
1.28 (t, 9 H, CH3, Et); 31P NMR: d 17.5; 13C NMR
(D2O): d 97.7 (C-1), 79.1 (C-2), 76.3 (C-5), 72.5 (C-6),
72.2 (C-3), 67.4 (C-4), 62.4 (C-7).
0.6, 3: 2 CH3CNÁ0.1 M NH4HCO3) and 5% of 29 (Rf
/
1
0.7) and AMP (Rf 0.5) (based on H NMR data). The
residue was redissolved in 10 mL water and applied on a
strong anion-exchange resin (5 mL Bio-Rad Econo-pack
Q cartridge, HCOꢀ2
/
-form). A gradient 0.01 M TEAB0
/
3.23. Adenosine 5?-(
L-glycero-a-
D-manno-
0.2 M TEAB was used and the eluate was monitored at
280 nm. Fractions containing 3 (Rf 0.7, PEI-Cellulose,
0.25 M TEAB, visualisation under UV-light) and AMP
(Rf 0.58) were pooled, and diluted with water to a total
vol of 20 mL. The pH of the solution was adjusted to 5
with Dowex 50 (Hꢁ) at 0 8C, the resin was removed by
filtration and the filtrate was lyophilised. The residue
was purified by gel filtration on Superdex Peptide HR
10/30 column (Pharmacia), connected to FPLC-system,
using 0.05 M TEAB as eluent, at 0.2 mL/min elution
rate. The fractions were checked for the absence of
AMP by TLC (3, Rf 0.3 and AMP, Rf 0.45; HPTLC
heptopyranosyl) diphosphate (monotriethylammonium
salt) (1)
3.23.1. Method A. A soln of 26 (17 mg, 0.018 mmol) in
7:3:1 MeOHÁ
/
waterÁEt3N (pH 12, 2 mL) was stirred at
/
ambient temperature for 3 h. The reaction mixture was
diluted with water (20 mL), concentrated to a volume of
10 mL and lyophilised to give 1 (13 mg, 98%) as a white
solid. Rf 0.27 (10:12:3:3 CHCl3Á
NH4OHÁ
water); [a]2D0 4.58 (c 1.0, water); 31P NMR:
d ꢀ10.7 (d, JP,P 21.1 Hz; PRib), ꢀ13.1 (d, PHep); JC-1,H-
174 Hz. MALDI-TOF-MS: m/z 618.31 [Mꢀ
H]ꢀ.
/
MeOHÁ
/
25% aq
/
ꢁ
/
/
/
/
Cellulose F, 5:9:1:3 CHCl3Á
/
MeOHÁ
/
waterÁ0.3 M
/
1

2588
A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á2589
/
8. Knirel, Y. A.; Tanatar, N. V.; Soldatkina, M. A.;
Shashkov, A. S.; Zakharova, I. Y. Bioorg. Khim. 1988,
TEAB, visualisation under UV-light). Fractions con-
taining pure 3 were pooled, concentrated to 10 mL vol,
the soln was cooled to 0 8C and the pH was adjusted to
4.5 with Dowex 50 (Hꢁ) resin. The resin was removed
by filtration, the filtrate was adjusted to pH 6 with Et3N
at 0 8C, concentrated to a vol of 5 mL at 25 8C and
lyophilised to furnish the monotriethylammonium salt
of 3 (7 mg, 94%) as a white solid. Rf 0.28 (Cellulose F,
14, 77Á
9. Masoud, H.; Mayer, H.; Kontrohr, T.; Holst, O.; Weck-
esser, J. Syst. Arch. Microbiol. 1991, 12, 222Á227.
10. (a) Muller-Loennies, S.; Rund, S.; Ervela, E.; Skurnik, M.;
/
81.
/
¨
¨
Holst, O. Eur. J. Biochem. 1999, 261, 19Á
(b) Oertelt, C.; Lindner, B.; Skurnik, M.; Holst, O. Eur.
Biochem. 2001, 268, 554Á564;
/
24;
/
HPTLC, 5:9:1:3 CHCl3Á
[a]2D0 318 (c 0.3, water); JC-1,H-1 163 Hz; 31P NMR: d
10.8 (d, JP,P 20.8 Hz; PRib), ꢀ12.8 (d, PHep).
MALDI-TOF-MS (neg. mode): m/z 618.63 [Mꢀ
H]ꢀ.
Anal. Calcd for C17H26N5O16Pꢀ2 ; [MꢀH]ꢀ: 618.10.
/
MeOHÁ
/
waterÁ
/
0.3 M TEAB);
(c) Radziejewska-Lebrecht, J.; Shashkov, A.; Stroobant,
V.; Wartenberg, K.; Warth, C.; Mayer, H. Eur. J.
ꢀ
/
ꢀ
/
/
Biochem. 1994, 221, 343Á
/351.
/
11. Toman, R.; Skultety, L. Carbohydr. Res. 1996, 283, 175Á
/
/
185.
12. Brisson, J.-R.; Crawford, E.; Uhrin, D.; Khieu, N. H.;
Perry, M. B.; Severn, W. B.; Richards, J. C. Can. J. Chem.
3.26. Adenosine 5?-(
heptopyranosyl)diphosphate (monotriethylammonium
salt) (4)
D
-glycero-b-D-manno-
2002, 80, 949Á
13. Michon, F.; Shaw, D.; Banoub, J. H. Eur. J. Biochem.
1984, 145, 107Á114.
14. Edebrink, P.; Jansson, P.-E.; Bogwald, J.; Hoffman, J.
Carbohydr. Res. 1996, 287, 225Á245.
15. (a) Vinogradov, E.; Radziejewska-Lebrecht, A.; Kaca, W.
Eur. J. Biochem. 2000, 267, 262Á268;
(b) Vinogradov, E.; Sidorczyk, Z. Carbohydr. Res. 2000,
326, 185Á193;
(c) Vinogradov, E.; Sidorczyk, Z. Carbohydr. Res. 2002,
337, 835Á840.
16. Rahman, M. M.; Gu, X.-X.; Tsai, C.-M.; Kolli, V. S. K.;
Carlson, R. W. Glycobiology 1999, 9, 1371Á1380.
17. (a) Aspinall, G. O.; Monteiro, M. A. Biochemistry 1996,
35, 2498Á2504;
/
963.
/
/
Compound 4 was prepared from 31 (12 mg, 0.013 mmol)
as described for 3. Yield 9 mg (95%); Rf 0.3 (HPTLC
/
Cellulose F, 5:9:1:3 CHCl3Á
TEAB); [a]2D0 228 (c 0.3, water); JC-1,H-1 163 Hz; 31P
NMR: d ꢀ10.8 (d, JP,P 20.9 Hz; PRib) and ꢀ12.7 (d,
/
MeOHÁ
/
waterÁ
/
0.3 M
ꢀ
/
/
/
/
P
Hep); MALDI-TOF-MS (neg. mode): m/z 618.51 [Mꢀ
/
/
H]ꢀ. Anal. Calcd for C17H26N5O16P2ꢀ; [Mꢀ
618.10.
/
H]ꢀ:
/
/
(b) Knirel, Y. A.; Kocharova, N. A.; Hynes, S. O.;
Widmalm, G.; Andersen, L. P.; Jansson, P.-E.; Moran,
Acknowledgements
A. P. Eur. J. Biochem. 1999, 266, 123Á131;
/
This work was supported by a grant from FWF
(P14978-MOB). The authors thank Dr F. Altmann
and Dr A. Potthast for the MALDI-TOF MS data.
(c) Monteiro, M. A.; Appelmelk, B. J.; Rasko, D. A.;
Moran, A. P.; Hynes, S. O.; MayLean, L. L.; Chan, K. H.;
St. Michael, F.; Logan, S. M.; O’Rourke, J.; Lee, A.;
Taylor, D. E.; Perry, M. B. Eur. J. Biochem. 2000, 267,
305Á
/
320.
18. Susskind, M.; Brade, L.; Brade, H.; Holst, O. J. Biol.
¨
Chem. 1998, 273, 7006Á7017.
/
References
19. Chowdhury, T. A.; Jansson, P.-E.; Lindberg, B.; Lindberg,
J.; Gustafsson, B.; Holme, T. Carbohydr. Res. 1991, 215,
1. Raetz, C. R. H.; Whitfield, C. Annu. Rev. Biochem. 2002,
303Á
20. Ansari, A. A.; Kenne, L.; Lindberg, B.; Gustafsson, B.;
Holme, T. Carbohydr. Res. 1986, 150, 213Á219.
/
314.
71, 635Á700.
/
2. (a) Holst, O. In Endotoxin in Health and Disease; Brade,
H.; Opal, S. M.; Vogel, S. N.; Morrison, D. C., Eds. (and
references cited therein); Marcel Dekker: New York,
/
21. (a) Niedziela, T.; Lukasiewicz, J.; Jachymek, W.; Dzie-
ciatkowska, M.; Lugowski, C.; Kenne, L. J. Biol. Chem.
Basel, USA, 1999; pp 115Á
therein);
(b) Wilkinson, S. G. Prog. Lipid Res. 1996, 35, 283Á
3. Holst, O.; Zahringer, U.; Brade, H.; Zamojski, A.
/
154 (and references cited
2002, 277, 11653Á
(b) Czaja, J.; Jachymek, W.; Niedziela, T.; Lugowski, C.;
Aldova, E.; Kenne, L. Eur. J. Biochem. 2000, 267, 1672Á
/
11663;
/
343.
/
¨
1679.
22. Kosma, P.; Wugeditsch, T.; Christian, R.; Zayni, S.;
Carbohydr. Res. 1991, 215, 323Á
4. Melaugh, W.; Phillips, N. J.; Campagnari, A.; Karalus, R.;
Gibson, B. W. J. Biol. Chem. 1992, 267, 13434Á13439.
5. Mansson, M.; Hood, D. W.; Moxon, E. R.; Schweda, E.
K. H. Eur. J. Biochem. 2003, 270, 610Á624.
6. Vinogradov, E.; Sidorczyk, Z.; Knirel, Y. A. Carbohydr.
Res. 2002, 337, 643Á649.
7. Jachymek, W.; Niedziela, T.; Petersson, C.; Lugowski, C.;
Czaja, J.; Kenne, L. Biochem 1999, 38, 11788Á11795.
/
335.
Messner, P. Glycobiology 1995, 5, 791Á
/796.
/
23. Benz, I.; Schmidt, M. A. Mol. Microbiol. 2001, 40, 1403Á
/
1413.
24. (a) Moormann, C.; Benz, I.; Schmidt, M. A. Infect.
/
Immun. 2002, 70, 2264Á
(b) Lindenthal, C.; Elsinghorst, E. A. Infect. Immun. 1999,
67, 4084Á4091.
/
2270;
/
/
/

A. Zamyatina et al. / Carbohydrate Research 338 (2003) 2571Á
/2589
2589
25. Eidels, J.; Osborn, M. J. J. Biol. Chem. 1974, 249, 5642Á
/
34. Excoffier, G.; Gagniere, D.; Utille, J.-P. Carbohydr. Res.
1975, 39, 368Á373.
35. Bannwarth, W.; Trzeciak, A. Helv. Chim. Acta 1987, 70,
175Á186.
36. (a) Sabesan, S.; Neira, S. Carbohydr. Res. 1992, 223, 169Á
/
5648.
26. Kocsis, B.; Kontrohr, T. J. Biol. Chem. 1984, 259, 11858Á
/
/
11860.
/
27. Heinrichs, D. E.; Valvano, M. A. In Endotoxin in Health
and Disease; Brade, H.; Opal, S. M.; Vogel, S. N.;
Morrison, D. C., Eds. (and references cited therein);
185;
(b) Inage, M.; Chaki, H.; Kusumoto, S.; Shiba, T. Chem.
Lett. 1982, 1281Á
37. Paulsen, H.; Pries, M.; Lorentzen, J. P. Liebigs Ann.
Chem. 1994, 389Á397.
38. (a) Roseman, S.; Distler, J. J.; Moffatt, J. G.; Khorana, H.
G. J. Am. Chem. Soc. 1961, 83, 659Á663;
(b) Moffatt, J. G.; Khorana, H. G. J. Am. Chem. Soc.
1954, 80, 3756Á3761;
(c) Adelhorst, K.; Whitesides, G. M. Carbohydr. Res.
1993, 242, 69Á76.
39. Wittmann, V.; Wong, C.-H. J. Org. Chem. 1997, 62,
2144Á2147.
40. Yuasa, H.; Palcic, M. M.; Hindsgaul, O. Can. J. Chem.
1995, 73, 2190Á2195.
41. (a) Nunez, H. A.; O’Connor, J. V.; Rosevear, P. R.;
Barker, R. Can. J. Chem. 1981, 59, 2086Á2095;
(b) Gokhale, U. B.; Hindsgaul, O.; Palcic, M. M. Can. J.
Chem. 1990, 68, 1063Á1071.
42. (a) Warren, C. D.; Jeanloz, R. W. Biochemistry 1972, 11,
2565Á2572;
(b) Baisch, G.;
383Á391;
(c) Liemann, S.; Klaffke, W. Liebigs Ann. Chem. 1995,
1779Á1787;
(d) Naundorf, A.; Natsch, S.; Klaffke, W. Tetrahedron
Lett. 2000, 41, 189Á192.
43. Grzeszczyk, B.; Holst, O.; Zamojski, A. Carbohydr. Res.
1996, 290, 1Á15.
/
1284.
Marcel Dekker: New York, 1999; pp 305Á
references cited therein).
28. Kneidinger, B.; Graninger, M.; Puchberger, M.; Kosma,
P.; Messner, P. J. Biol. Chem. 2001, 276, 20935Á20944.
/
330 (and
/
/
/
29. (a) Kneidinger, B.; Marolda, C. L.; Graninger, M.;
Zamyatina, A.; McArthur, F.; Kosma, P.; Valvano, M.
/
A.; Messner, P. J. Biol. Chem. 2002, 184, 363Á
(b) Valvano, M. A.; Messner, P.; Kosma, P. Microbiol.
2002, 148, 1979Á1989;
/369;
/
/
(c) Gronow, S.; Oertelt, C.; Ervela, E.; Zamyatina, A.;
¨
Kosma, P.; Skurnik, M.; Holst, O. J. Endotoxin Res. 2001,
/
7, 263Á
30. Pacinelli, E.; Wang, L.; Reeves, P. R. Infect. Immun. 2002,
70, 3271Á3276.
/270.
/
/
/
31. Zamyatina, A.; Gronow, S.; Oertelt, C.; Puchberger, M.;
Brade, H.; Kosma, P. Angew. Chem., Int. Ed. 2000, 39,
/
4150Á4153.
/
32. (a) Heskamp, B. M.; Broxterman, H. J. G.; van der Marel,
/
G. A.; van Boom, J. H. J. Carbohydr. Chem. 1996, 15,
¨
Ohrlein, R. Bioorg. Med. Chem. 1997, 5,
.
611Á/622;
/
(b) Garcia, B. A.; Gin, D. Y. Org. Lett. 2000, 2, 2135Á
/
2138 (and references cited therein).
33. (a) Brimacombe, J. S.; Kabir, A. K. M. S. Carbohydr. Res.
/
1986, 162, 329Á
(b) Nepogod’ev, S. A.; Backinowsky, L. V.; Grzeszczyk,
B.; Zamojski, A. Carbohydr. Res. 1994, 254, 43Á60.
/334;
/
/
/