A convenient synthesis of GDP D-glycero-α-D-manno-heptopyranose

Article abstract of DOI:10.1016/j.carres.2003.09.012

GDP D-glycero-α-D-manno-Heptopyranose has been prepared in good overall yield from 2,3,4,6,7-penta-O-acetyl-D-glycero-D-manno-heptopyranose by a short-step synthesis. Phosphitylation using the phosphoramidite procedure afforded the α-anomer in high selectivity. Subsequent oxidation and partial deprotection gave the acetylated phosphate derivative, which was subjected to the coupling reaction with GMP-morpholidate to furnish the acetylated heptose nucleoside diphosphate in good yield. De-O-acetylation and final purification afforded the target GDP D-glycero-α-D-manno- heptopyranose, which serves as the substrate of the heptosyl transferase in Aneurinibacillus thermoaerophilus DSM 10155 and occurs as an intermediate in the biosynthesis of GDP 6-deoxy-heptose in Yersinia pseudotuberculosis.

Full text of DOI:10.1016/j.carres.2003.09.012

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 147–151
Note
A convenient synthesis of GDP
D
-glycero-a-
D
-manno-heptopyranose
Andrea Graziani, Alla Zamyatina and Paul Kosma*
Institute of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
Received 6 August 2003; accepted 9 September 2003
Abstract—GDP
D
-glycero-a-
D-manno-Heptopyranose has been prepared in good overall yield from 2,3,4,6,7-penta-O-acetyl-D-
glycero- -manno-heptopyranose by a short-step synthesis. Phosphitylation usingthe phosphoramidite procedure afforded the
D
a-anomer in high selectivity. Subsequent oxidation and partial deprotection gave the acetylated phosphate derivative, which was
subjected to the couplingreaction with GMP-morpholidate to furnish the acetylated heptose nucleoside diphosphate in good yield.
De-O-acetylation and final purification afforded the target GDP
D
-glycero-a-D-manno-heptopyranose, which serves as the substrate
of the heptosyl transferase in Aneurinibacillus thermoaerophilus DSM 10155 and occurs as an intermediate in the biosynthesis of
GDP 6-deoxy-heptose in Yersinia pseudotuberculosis.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Keywords: Heptose; GDP-heptose; Sugar nucleotide; Lipopolysaccharide; Surface-layer glycoprotein
Heptoses of the
D
-glycero-
D
-manno- configuration are
surface-layer glycoprotein glycan in Aneurinibacillus
thermoaerophilus DSM 10155.5 The genes encoding the
biosynthesis of the nucleotide-activated heptose in this
Gram-positive thermophilic bacterium have been
cloned, overexpressed in E. coli and the gene products
have structurally and functionally been characterized.6
common constituents in the core region of numerous
bacterial lipopolysaccharides and have also been de-
tected in various O-antigenic chains.1 For example,
O-antigens from Vibrio cholerae serogroup O:3 and O:21
contain
units,2;3 and b-linked
6-deoxy- -manno-heptose have been detected in the
D
-glycero-
D
-manno-heptose in the repeating
D
-glycero- -manno-heptose and
D
Thus GDP
D
-glycero-a-D-manno-heptopyranose has
D
been identified as the substrate for the bacterial glyco-
syltransferase involved in the assembly of the S-layer
glycoprotein glycan in A. thermoaerophilus (Scheme 1).
O-antigen from Plesiomonas shigelloides O54:H2.4 Fur-
thermore, b-heptopyranosyl residues are present in the
Scheme 1. Biosynthetic steps and genes for GDP-activated heptoses in A. thermoaerophilus and Y. pseudotuberculosis.
* Correspondingauthor. Tel.: +43-1-36006-6055; fax: +43-1-36006-6059; e-mail: pkosma@edv2.boku.ac.at
0008-6215/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.09.012

148
A. Graziani et al. / Carbohydrate Research 339 (2004) 147–151
Scheme 2. Reagents and conditions: (i) Bis(benzyloxy)-N,N-diisopropylaminophosphine, 1H-tetrazole (3.5% in MeCN), 2 h, CH2Cl2, then t-BuOOH,
12 h, rt, 65% for 2; (ii) 10% Pd/C, H2, MeOH, Et3N, 10 h, rt, 98% for 3; (iii) 40-morpholine-N,N0-dicyclohexylcarboxamidinium salt of GMP, pyridine,
3 days, rt, anion exchange on BioRad (Q) 5 mL cartridge (HCOÀ2 -form), elution 0.01 fi 0.2 M TEAB, 65% for 5; (iv) 7:3:1 MeOH–water–Et3N, 3 h, rt,
98% for 6.
Furthermore, a similar pathway leadingto GDP 6-de-
oxy- -manno-heptose has been identified in Yersinia
efficiency of the nucleoside diphosphate formation.13
The couplingof 3 with GMP-morpholidate was carried
out in pyridine under strictly anhydrous conditions for
D
pseudotuberculosis and the genes encoding the common
steps of the biosynthesis of these nucleotide heptoses
display a high degree of sequence homology.7 Whereas
ADP heptoses involved in the biosynthesis of the lipo-
polysaccharide core units of E. coli are present in the
labile b-anomeric configuration, GDP heptose occurs in
the significantly more stable a-anomeric linkage.8;9
In extension of previous work on the synthesis of
both anomeric forms of ADP heptoses, we report herein
on the chemical synthesis of GDP heptose to be used as
a biochemical probe.10
3 days.14 The acetylated GDP
D
-glycero-a-D-manno-
heptose 5 was isolated in 65% yield after anion-exchange
chromatography. Removal of the acetyl groups with
triethylamine in MeOH–water produced GDP heptose 6
as the triethylammonium salt in 98% yield. The pres-
ence of the diphosphate in compound 6 was confirmed
by the chemical shift of the 31P NMR signals at )11.2
(phosphate attached to ribose) and )13.6 ppm (phos-
phate linked to heptose unit), respectively. The 1H
and 13C NMR data of 6 (Fig. 1) were in full agree-
ment with those found for the native nucleotide-
activated sugar in A. thermoaerophilus, thereby
The previously described
D-glycero-D-manno-
heptopyranose11 1 was subjected to phosphitylation
12
6
usingthe phosphoramidite method.
Treatment of 1
confirmingthe structure of the bacterial GDP heptose.
In addition, the 13C NMR signals of the heptopyr-
anosyl unit are nearly identical (within 0.05 ppm)
with bis(benzyloxy)-N,N-diisopropylaminophosphine/
1H-tetrazole and subsequent oxidation with t-BuOOH
gave the a-phosphate 2 in 65% yield. The minor amount
(5%) of b-anomer 4 was removed by chromatography.
Catalytic hydrogenation in the presence of 10% Pd/C
and treatment with triethylamine produced the known11
triethylammonium salt of penta-O-acetyl-heptosyl
phosphate 3. The partially protected derivative 3 is fairly
soluble in pyridine (Scheme 2) and thereby increases the
to those of the related ADP
D
-glycero-a-D-manno-
heptose, thus fully confirmingthe structural assing-
ments.11
In conclusion, the method reported herein together
with the previously described ADP heptose synthesis
constitutes a flexible and efficient strategy for the syn-
thesis of purine-nucleotide heptoses.

A. Graziani et al. / Carbohydrate Research 339 (2004) 147–151
149
Figure 1. Contour plot of the HMQC spectrum of GDP heptose 6 with 300 MHz 1H NMR projection. The spectrum was recorded at 300 K for a
sample at pD 6.3.
1. Experimental
Bruker DPX 300 spectrometer (1H at 300.13 MHz, 13C
at 75.47 MHz and 31P at 121.50 MHz) usingstandard
1
1.1. General
Bruker NMR software. H NMR spectra were refer-
enced to tetramethylsilane or 2,2-dimethyl-2-silapen-
tane-5-sulfonic acid. 13C NMR spectra were referenced
to chloroform for solutions in CDCl3 (d 77.00) or di-
oxane (d 67.40) for solutions in D2O. 31P NMR spectra
were referenced externally to 85% aq H3PO4 (d 0.0).
MALDI-TOFMS spectra were recorded on a Dynamo
(Thermo BioAnalysis) instrument usingwater and 2,5-
dihydroxybenzoic acid as matrix. TOF-ESMS spectra
were recorded on a Waters QTOF-ES-MS instrument.
All solvents were purified and dried by standard pro-
cedures. Column chromatography was performed on
Silica Gel 60 (230–400 mesh, E. Merck). Anion-ex-
change chromatography was performed on a strong
anion-exchange resin (BioRad, 5 mL cartridge, HCO2À
form). Size-exclusion chromatography was performed
on Sephadex G-25 M. The anion-exchange column was
connected to the FPLC system (Pharmacia) and the
eluates were monitored at 280 nm. Analytical TLC was
performed usingsilica gel 60 F
HPTLC plates with
1.2. Dibenzyl (2,3,4,6,7-penta-O-acetyl-
a- -manno-heptopyranosyl) phosphate (2) and
dibenzyl (2,3,4,6,7-penta-O-acetyl- -glycero-
b- -manno-heptopyranosyl) phosphate (4)
D
-glycero-
254
2.5 cm concentration zone (E. Merck). Spots were de-
tected by treatment with anisaldehyde–H2SO4; guano-
sine-containingcompounds were also detected by UV
light examination. Triethylammonium bicarbonate
(TEAB) buffer was prepared by passinga stream of
CO2-gas through a cooled solution of triethylamine
(2 M) in de-ionized water until a near neutral solution
(pH 7.5) was obtained. Optical rotations were measured
with a Perkin–Elmer 243 B polarimeter. NMR spectra
were recorded at 297 K in D2O and CDCl3 with a
D
D
D
Heptose pentaacetate 1 (60 mg, 0.14 mmol) and bis-
(benzyloxy)-N,N-diisopropylaminophosphine (117 lL,
0.35 mmol) were dried by repeated evaporations with
dry toluene (3 · 10 mL) and then under diminished
pressure for 10 h. Then the flask was charged
with CH2Cl2 (3 mL), a soln of 1H-tetrazole (30 mg,

150
A. Graziani et al. / Carbohydrate Research 339 (2004) 147–151
0.43 mmol) in dry MeCN (1.5 mL) was added and the
mixture was stirred at room temperature for 2 h under
N2. Monitoringof the reaction by TLC showed the
formation of intermediate phosphite triesters (2.5:7.5,
n-hexane–diethyl ether). The reaction mixture contain-
ingphosphite triesters was cooled to 0 ꢁC and a soln of
t-BuOOH (35 lL of an 80% soln in di-tert-butyl per-
oxide) in CH2Cl2 (1.5 mL) was gradually added
(ꢀ20 min). The reaction mixture was warmed to room
temperature and stirred for 12 h. The solvent was
1H, 3J5;6 2.5 Hz, H-5), 4.26 (dd, 1H, 3J7b;6 7.6 Hz, H-7b),
3.16 (q, 6H, NCH2), 2.15, 2.11, 2.09, 2.04, 1.99 (5s, 15H,
5Ac), 1.24 (t, 9H, CH3, Et3N).
1.4. Guanosine 50-(
diphosphate (triethylammonium salt) (6)
D
-glycero-a- -manno-heptopyranosyl)
D
Pentaacetyl heptosyl phosphate 3 (20 mg, 0.0334 mmol)
was made anhyd by repeated dissolution in dry pyridine
and evaporation (4 · 10 mL). After each evaporation
step, dry N2 was flushed into the rotary evaporator.
Guanosine-50-monophosphate morpholidate (40-mor-
pholine-N,N0-dicyclohexylcarboxamidinium salt) (36 mg,
0.0496 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 com-
ponents were finally dissolved in pyridine and combined
in a one-neck round-bottom flask under N2-atmosphere.
The reaction mixture was repeatedly evaporated from
pyridine (3 · 10 mL) and flushed with dry N2. A final
amount of pyridine (10 mL) was added to give a clear
solution. About 70% of pyridine was removed by con-
centration and the reaction vessel was sealed under N2.
The solution was vigorously stirred and the progress of
the reaction was monitored by TLC-analysis (35:20:2:2,
CHCl3–MeOH–25% aq NH4OH–H2O). The reaction
was kept for 3 days and progress was monitored by the
appearance of a major UV-positive spot of GDP-Hep.
The reaction was stopped by evaporation of pyridine.
The diphosphate 5 was isolated usinganion-exchange
chromatography. The crude reaction products were
dissolved in 10 mL water and the soln was allowed to
slowly adsorb on a resin bed of anion-exchange column
(BioRad, 5 mL cartridge, HCOÀ2 -form) connected to an
FPLC-system. The column was operated at 0.7 mL/min,
fractions (1 mL) were collected. The column was washed
first with water and was then developed with a linear
gradient of TEAB buffer. The eluate was monitored at
280 nm, 5 was eluted at a concentration of ꢀ0.15 M
TEAB. The fractions containingGDP-Hep were pooled,
concentrated to 10 mL vol, the soln was cooled to 0 ꢁC,
and the pH was adjusted to 4.5 with Dowex 50 (Hþ)
resin. The resin was removed by filtration, the total
eluate was made neutral by addition of Et3N, concen-
trated to 5 mL vol at 25 ꢁC and lyophilized to give
evaporated usinga stream of N . The residue was re-
2
dissolved in diethyl ether and washed sequentially with
saturated aq NaHCO3–water and brine. The organic
phase was dried (Na2SO4) and concentrated. The a- and
b-phosphates were separated by chromatography on
silica gel (1:9, n-hexane–diethyl ether) to give 2 (62 mg,
65%) as a syrup. Rf 0.40; analytical data were similar to
the previously described compound.11 1H NMR
3
(CDCl3): d 7.37–7.33 (m, 10H, 2Ph), 5.57 (dd, 1H, J1;2
1.7, 3J1;P 6.5 Hz, H-1), 5.30 (t, 1H, 3J4;3 ¼ 3J4;5 9.8 Hz, H-
4), 5.28 (d, 1H, 3J3;2 3.3 Hz, H-3), 5.21–5.14 (m, 2H, H-2,
3
H-6), 5.09 (d, 2H, JH;P 8.5 Hz, CH2Ph), 5.08 (d, 2H,
3
2
3JH;P 8.5 Hz, CH2Ph), 4.33 (dd, 1H, J7a;6 4.0, J7a;7b
3
12.0 Hz, H-7a), 4.19 (dd, 1H, J7b;6 7.7 Hz, H-7b), 4.15
(m, 1H, H-5), 2.13, 2.09, 2.03, 1.99 and 1.93 (5s, 15H,
5Ac).
Further elution gave 4 (4.8 mg, 5%) as a syrup. Rf
0.24; 1H NMR (CDCl3): d 7.37–7.29 (m, 10H, 2Ph), 5.47
3
3
3
(dd, 1H, J1;2 1.5, J1;P 7.8 Hz, H-1), 5.42 (dd, 1H, J2;1
1.5, 3J2;3 3.3 Hz, H-2), 5.26 (t, 1H, 3J4;3 ¼ 3J4;5 9.3 Hz, H-
4), 5.25 (m, 1H, H-6), 5.10 (d, 2H, 3JH;P 8.0 Hz, CH2Ph),
5.05 (d, 2H, 3JH;P 8.0 Hz, CH2Ph), 5.04 (dd, 1H, 3J3;4 9.3,
3J3;2 3.3 Hz, H-3), 4.41 (dd, 1H, 3J7a;6 3.6, 2J7a;7b 12.0 Hz,
3
H-7a), 4.25 (dd, 1H, J7b;6 7.0 Hz, H-7b), 4.15 (dd, 1H,
3J5;6 3.7 Hz, H-5), 2.13, 2.10, 2.04, 2.01 and 1.99 (5s,
15H, 5Ac).
1.3. 2,3,4,6,7-Penta-O-acetyl-
heptopyranosyl phosphate (triethylammonium salt) (3)
D
-glycero-a- -manno-
D
A soln of 2 (23 mg, 0.034 mmol) in dry MeOH
(7 mL) was hydrogenated in the presence of 10% Pd/C
(7 mg) for 10 h at atmospheric pressure. After comple-
tion of the reaction, the catalyst was removed by filtra-
tion through a pad of Celite and washed with MeOH.
The combined filtrates were neutralized by addition of
Et3N (0.1 mL) and concentrated. The residue was lyo-
philized from water to give the triethylammonium salt of
3 (20 mg, 98%) as a white fluffy solid which was used in
the next step without further purification. Rf 0.34
guanosine 50-(2,3,4,6,7-penta-O-acetyl-
D
-glycero-a-D-
manno-heptopyranosyl) diphosphate (triethylammo-
nium salt) (5) as a solid. Yield: 20.7 mg(65%); Rf 0.37
(35:20:2:2, CHCl3–MeOH–25% aq NH4OH–water); 31P
2
1
NMR: d )11.0 (d, JP;P 19 Hz; PRib), )14.2 (d, PHep); H
NMR (D2O): d 8.11 (br s, 1H, H-8Gua), 5.91 (d, 1H, 3J1;2
1
(35:20:2:2, CHCl3–MeOH–25% aq NH4OH–water); H
NMR (D2O): d 5.42 (dd, 1H, 3J1;P 7.5, 3J1;2 2.0 Hz, H-1),
5.38 (dd, 1H, 3J2;3 2.9, 3J3;4 9.8 Hz, H-3), 5.30 (t, 1H, 3J4;5
9.8 Hz, H-4), 5.26 (br s, 1H, H-2), 5.25 (ddd, 1H, H-6),
5.8 Hz, H-1Rib), 5.58 (dd, 1H, J1;2 1.8, J1;P 7.5 Hz, H-
Hep), 5.36 (m, 1H, H-2Hep), 5.35–5.28 (m, 2H, H-3Hep
H-4Hep), 5.22 (m, 1H, H-6Hep), ꢀ4.73 (H-2Rib, under-
neath the water signal), 4.49 (dd, 1H, J3;4 5.1, J3;2
3
3
1
,
3
2
3
3
4.43 (dd, 1H, J7a;6 3.4, J7a;7b 12.0 Hz, H-7a), 4.40 (dd,

A. Graziani et al. / Carbohydrate Research 339 (2004) 147–151
151
3.8 Hz, H-3Rib), 4.37–4.15 (m, 6H, H-4Rib, H-5Hep
,
H-5a,bRib, H-7a,bHep), 3.18 (q, 12H, CH2, Et3N), 2.16,
2.11, 2.05, 1.98, 1.90 (5s, 15H, 5Ac) and 1.27 (t, 18H,
CH3, Et3N).
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A soln of 5 (20.7 mg, 0.022 mmol) in 7:3:1 MeOH–
water–Et3N (2 mL) was stirred at ambient temperature
for 3 h at pH 12. The reaction mixture was diluted with
water and purified on a size-exclusion column (Sephadex
G-25 M, PD-10 column, Pharmacia). The eluate was
concentrated and lyophilized to give 6 (17 mg, 98%) as a
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20
D
NH4OH–water); ½aꢁ +4.6 (c 0.2, water); 31P NMR: d
2
1
)11.2 (d, JP;P 20.9 Hz, PRib), )13.6 (d, PHep); H NMR
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6.2 Hz, H-1Rib), 5.48 (dd, 1H, J1;2 2.0, J1;P 7.8 Hz, H-
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3
3
3
1
3
3
(dd, 1H, J3;4 3.1, J3;2 5.2 Hz, H-3Rib), 4.33 (m, 1H, H-
4
2
Rib), 4.18 (m, 1H, H-5Rib), 4.02 (dd, 1H, 3J2;3 3.4 Hz, H-
Hep), 4.00 (ddd, 1H,3J6;5 3.3, J6;7a 4.1, J6;7b 6.5 Hz,
3
3
H-6Hep), 3.91 (dd, 1H, 3J5;4 9.3 Hz, H-5Hep), 3.87 (dd, 1H,
3J3;4 9.3 Hz, H-3Hep), 3.76 (t, 1H, H-4Hep), 3.76 (dd, 1H,
3J7a;7b 12.1 Hz, H-7aHep), 3.72 (dd, 1H, H-7bHep), 3.19 (q,
ꢀ9H, CH2, Et3N), 1.27 (t, ꢀ13.5H, CH3, Et3N); 13C
NMR (D2O): d 160.78 (C-6Gua), 152.44 (C-4Gua), 136.8
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86.71 (C-1Rib), 84.23 (C-4Rib, 3J4;P 9.0 Hz), 74.32 (C-2Rib
C-5Hep), 72.86 (C-6Hep), 71.18 (C-3Rib), 70.72 (C-2Hep
3J2;P ꢀ9.0 Hz), 70.71 (C-3), 67.88 (C-4Hep), 66.04 (C-5Rib
,
,
,
2J5;P 5.8 Hz), 62.61 (C-7Hep), 47.07 (CH2, Et3N), 9.16
(CH3, Et3N); QTOF-ES-MS: m=z ¼ 636:029 [M+H]þ.
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Acknowledgements
Authors are grateful to Andreas Hofinger for providing
NMR spectra and for financial support by FWF (grant
P14978-MOB).
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