A short synthesis of D-glycero-D-manno-heptose 7-phosphate

Article abstract of DOI:10.1016/j.carres.2005.10.003

D-glycero-D-manno-Heptopyranose 7-phosphate-an intermediate in the biosynthesis of nucleotide-activated heptoses-has been prepared in good overall yield from benzyl 5,6-dideoxy-2,3-O-isopropylidene-α-D-lyxo-(Z)-hept-5- enofuranoside by a short-step synthe

Full text of DOI:10.1016/j.carres.2005.10.003

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 2808–2811
Note
A short synthesis of D-glycero-D-manno-heptose 7-phosphate
Hacer Guzlek, Andrea Graziani and Paul Kosma*
¨
Department of Chemistry, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
Received 21 September 2005; accepted 12 October 2005
Available online 2 November 2005
´
´
Dedicated to Andras Liptak on the occasion of his 70th birthday
Abstract—D-glycero-D-manno-Heptopyranose 7-phosphate—an intermediate in the biosynthesis of nucleotide-activated heptoses—
has been prepared in good overall yield from benzyl 5,6-dideoxy-2,3-O-isopropylidene-a-D-lyxo-(Z)-hept-5-enofuranoside by a
short-step synthesis. Phosphitylation using the phosphoramidite procedure followed by in situ oxidation afforded the corresponding
7-O-phosphotriester derivative in high yield. Subsequent osmylation proceeded in good diastereoselectivity (4:1) to furnish the D-
glycero-D-manno-configured derivative, which was separated from the L-glycero-L-gulo-isomer by chromatography. Hydrogenolysis
led to simultaneous removal of the benzyl and isopropylidene groups and afforded the target compound in high yield, which serves
as a substrate of bacterial heptose 7-phosphate kinases.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Heptose; Heptose phosphate; Lipopolysaccharide; Capsular polysaccharide
Heptoses of the D-glycero-D-manno- and L-glycero-D-
manno-configuration are common constituents of the
core region of numerous bacterial lipopolysaccharides
and have also been detected in various O-antigenic
chains.1 In the assembly of the core region of enterobac-
terial LPS, ADP L-glycero-b-D-manno-heptose serves as
a substrate of heptosyl transferases.2 In addition, GDP
D-glycero-a-D-manno-heptopyranose has been identified
as the substrate for the bacterial glycosyltransferase
involved in the biosynthesis of the S-layer glycoprotein
glycan in Aneurinobacillus thermoaerophilus and has been
proposed as the intermediate leading to GDP
6-deoxy-D-manno-heptose in Yersinia pseudotuberculo-
sis and to related heptoses and 6-deoxy-heptoses in Burk-
holderia pseudomallei and Campylobacter capsular
polysaccharide biosynthesis.3,4 In both biosynthetic
pathways, D-glycero-D-manno-heptopyranose 7-phos-
phate is an essential precursor, which by the action of
the respective kinases encoded by the genes hldE and
hddA, respectively, either furnishes the b- or the a-linked
D-glycero-D-manno-heptopyranose 1,7-bisphosphate.5
For a detailed kinetic analysis of the bifunctional hepto-
kinase/ADP heptose transferase reaction of hldE, the
authentic substrate was needed.6 Previously, the 7-phos-
phate derivative of the L-glycero-D-manno-heptopyra-
nose had been prepared7 and recently reaction of an
a-diazoketone mannopyranoside derivative with dibenz-
yl phosphoric acid afforded a diastereoisomeric mixture
of D-glycero- and L-glycero-D-manno-heptopyranose
7-phosphates.8 Since in the preparation of D-glycero-D-
manno-heptopyranose following the procedure elabo-
rated by Brimacombe, a terminal hydroxy group is
present for further modification, we set out for a short-
step synthesis from precursor derivative 1.9 Compound 1
is prepared in five high-yielding steps from commercially
available 2,3:5,6-di-O-isopropylidene mannofuranose.
Hence, the previously described benzyl D-lyxo-hepte-
nofuranoside 1 was subjected to phosphitylation using
the phosphoramidite method.10 Treatment of 1 with
bisbenzyloxy-N,N-diisopropylaminophosphine/1H-tetra-
zole and subsequent oxidation with tert-BuOOH gave
7-O-phosphotriester derivative 2 in 65% yield. Catalytic
osmylation of the double bond in the presence of
N-methyl-morpholine-N-oxide afforded a ꢀ4:1 mixture
*
Corresponding author. Tel.: +43 1 36006 6055; fax: +43 1 36006
6059; e-mail: paul.kosma@boku.ac.at
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.10.003

H. Gu€zlek et al. / Carbohydrate Research 340 (2005) 2808–2811
2809
O
P
OH
O
O
O
O
HO
i)
O
O
O
O
O
O
ii)
BnO
BnO
O
P
O
OBn
BnO
OBn
OH
OBn
OBn
1
2
3
O
P
OH
OH
+
O
O
OH
HO
iii)
OH
O
O
O
BnO
BnO
O
P
HO
HO
O
OH
OH
OBn
5
4
Scheme 1. Reagents and conditions: (i) Bisbenzyloxy-N,N-diisopropylaminophosphine, 1H-tetrazole (3.5% in CH3CN), 2 h, CH2Cl2, then tBuOOH,
15 h, rt, 65% for 2; (ii) OsO4, NMMO, 2:1 dioxane–water, 5 h, rt, 52% for 4; (iii) 10% Pd/C, H2, MeOH, 12 h, rt, 81% for 5.
of the diastereoisomers 3 and 4 in 71% yield, which were
separated by silica gel chromatography (Scheme 1).
Assignments of the new stereogenic centres at C-5 and
C-6 were based on the comparison of the NMR data
of 3 and 4 with those of the non-phosphorylated deriv-
atives.11 In addition, the values of the coupling con-
stants J4,5 indicated a cis orientation for H-4 and H-5
for compound 3 (J4,5 2.5 Hz) and a trans configuration
for the D-glycero-D-manno-isomer 4 (J4,5 7.0 Hz).
Catalytic hydrogenation of 4 in the presence of 10%
Pd/C resulted in concomitant cleavage of the isopropyl-
idene group and after treatment with triethylamine pro-
duced the triethylammonium salt of heptosyl phosphate
5 in 81% yield following desalting on a PD-10 Sephadex
G-25 column. The 1H and 13C NMR data of 5 (Table 1)
were in full agreement with the structural assignments
and revealed a ratio of 65:35 of the a/b pyranose forms.
The presence of the phosphomonoester moiety in com-
pound 5 was evident from the chemical shift of the 31P
NMR signal at 2.18 ppm and its location was ascer-
tained by the downfield-shifted 13C NMR signal of
C-7 (66.26 and 66.14 ppm, respectively) as well as
the heteronuclear 13C/31P spin–spin coupling observed
for C-7 and C-6. Moreover, the spectra were identical
to those of the material obtained by HPLC-purification
of the reaction mixture of the enzymatic conversion
of sedoheptulose 7-phosphate in the thermophilic
bacterium A. thermoaerophilus, thereby confirming the
Table 1. NMR dataa of D-glycero-D-manno-heptose 7-phosphate 5
Atom H/C/P (ppm)
1
2
3
4
5
6
7a/7b
a-Pyranose
1H
J (Hz)
13C
31P
5.16
1.7
94.85
3.90
2.9
71.35c
3.81
3.80
n.d.
68.34
3.86
n.d.
72.90
4.15
n.d.
71.78
4.07/3.96
n.d.
66.46
2.18
n.d.b
71.29c
JC,P (Hz)
8.9
5.05
b-Pyranose
1H
J (Hz)
13C
31P
4.86
1.1
94.77
3.92
3.2
71.61
3.62
9.5
74.06
3.73
9.8
68.05
3.45
3.3
76.79
4.15
n.d.
71.55
4.07/3.96
n.d.
66.26
2.18
JC,P (Hz)
9.0
5.16
Other signals
CH3CH2N
1H
13C
3.19
47.49
1.27
9.04
a 13C NMR data are based on HMQC and HMBC-assignments.
b n.d.: not determined.
c Assignments may be reversed.

2810
H. Gu€zlek et al. / Carbohydrate Research 340 (2005) 2808–2811
structure of the biosynthetic intermediate.3a Except for
the signal of C-6, the 13C NMR signals of the heptopyr-
anosyl unit are nearly identical to those of the related
L-glycero-D-manno-heptose 7-phosphate, thus fully con-
firming the structural assignments.7
In conclusion, the method reported herein constitutes
a short and efficient strategy for the synthesis of heptose
7-phosphates.
phase was dried (MgSO4) and concentrated. The residue
was purified by column chromatography (1:1, toluene–
diethyl ether) to give 2 as a colourless syrup (815 mg,
65%); ½aꢁD +26 (c 1.0, CHCl3); H NMR (CDCl3): d
7.36–7.25 (m, 15H, Ph), 5.85 (dd, 1H, J6,5 10.8, J5,4
1.3 Hz, H-5), 5.79 (ddd, 1H, J6,7a = J6,7b 2.3 Hz, H-6),
5.05 (s, 1H, H-1), 5.09–4.94 (m, 4H, CH2Ph), 4.71–
20
1
4.59
(m,
5H,
H-4,
H-7a,
H-7b,
H-2,
H-3), 4.65 and 4.47 (AB system, 2H, JA,B 11.9 Hz,
CH2Ph), 1.43 and 1.27 [s, each 3H, C(CH3)2]; 13C
NMR (CDCl3): d 137.22 (Ph), 135.83 and 135.74 (C-5,
C-6), 128.71–127.42 (Ph), 112.56 [C(CH3)2], 105.32
(C-1), 85.29 (C-3), 81.35 (C-2), 75.61 (C-4), 69.31,
69.24 and 69.05 (CH2Ph), 63.55 (JC,P 5.3 Hz, C-7),
26.04 and 24.77 [C(CH3)2]; 31P NMR (CDCl3): d 0.05.
Anal. Calcd for C31H35O8P: C, 65.72; H, 6.23. Found:
C, 65.47; H, 6.60.
1. Experimental
1.1. General
All solvents were purified and dried by standard proce-
dures. Column chromatography was performed on Silica
Gel 60 (230–400 mesh, E. Merck). Desalting was per-
formed on PD-10 desalting column containing Sepha-
dexTM G-25. Analytical TLC was performed using Silica
Gel 60 F254 HPTLC plates with 2.5 cm concentration
zone (E. Merck). Spots were detected by treatment with
anisaldehyde–H2SO4. Optical rotations were measured
with a Perkin–Elmer 243 B polarimeter. NMR spectra
were recorded at 297 K in D2O and CDCl3 with a Bruker
DPX 300 spectrometer (1H at 300.13 MHz, 13C at
75.47 MHz and 31P at 121.50 MHz) using standard Bru-
ker NMR software. 1H NMR spectra were referenced to
tetramethylsilane or 2,2-dimethyl-2-silapentane-5-sul-
fonic acid. 13C NMR spectra were referenced to chloro-
form for solns in CDCl3 (d 77.00) or dioxane (d 67.40)
for solns in D2O. 31P NMR spectra were referenced
externally to 85% aq H3PO4 (d 0.0). ESIMS data were
obtained on a Waters Micromass Q-TOF Ultima Global
instrument.
1.3. Benzyl 7-O-[bis(benzyloxy)phosphoryl]-2,3-O-isoprop-
ylidene-L-glycero-b-L-gulo-heptofuranoside (3) and benzyl
7-O-[bis(benzyloxy)phosphoryl]-2,3-O-isopropylidene-D-
glycero-a-D-manno-heptofuranoside (4)
A soln of 2 (815 mg, 1.44 mmol) and NMMO (388.7 mg,
2.88 mmol) in 2:1 dioxane–water (10 mL) and acetone
(3 mL) was stirred at rt. Then osmium tetraoxide
(1.5 mL, 0.12 mmol; 2% in water) was transferred into
the flask and the mixture was stirred for 5 h. The soln
was then diluted with CHCl3 (150 mL). After treatment
with ice-cold 5 M HCl (6 mL) the mixture was vigorously
shaken with 45% aq Na2S2O5 (9 mL) and water. The
organic phase was dried (MgSO4) and concentrated to
a syrupy residue (580 mg, 71%). D-glycero-a-D-manno-
Heptofuranoside (4) and the L-glycero-b-L-gulo diaste-
reoisomer 3 were separated by chromatography on silica
gel (2:3 n-hexane–EtOAc) to give 445 mg of 4 as colour-
less crystals, mp 82–83 ꢁC (hexane–EtOAc), Rf 0.62;
1.2. Benzyl 7-O-[bis(benzyloxy)phosphoryl]-5,6-dideoxy-
2,3-O-isopropylidene-a-D-lyxo-(Z)-hept-5-enofuranoside
(2)
20
½aꢁD +40 (c 0.9, CHCl3); 1H NMR (CDCl3): d 7.37–
7.26 (m, 15H, Ph), 5.12 (s, 1H, H-1), 5.10–5.04 (m, 4H,
3
3
Compound 1 (680 mg, 2.22 mmol) and bisbenzyloxy-
N,N-diisopropylaminophosphine (1.87 mL, 5.55 mmol)
were dried by repeated evaporation with dry toluene
(4 · 10 mL) and then under diminished pressure for 5 h.
Then CH2Cl2 (6 mL) was added to the sample. The flask
was charged with a soln of 1H-tetrazole (467 mg,
6.66 mmol) in dry MeCN (3 mL) and stirred at room
temperature for 2 h under Ar. Monitoring of the reac-
tion by TLC showed the formation of phosphite triesters
(7:3 toluene–EtOAc). The reaction mixture was cooled
to 0 ꢁC and a soln of t-BuOOH (617 lL, 3.33 mmol,
80% soln in di-tert-butyl peroxide) was gradually added.
The soln was stirred for 15 h at room temperature and
the solvent was evaporated using a stream of argon.
The residue was dissolved in 2:1 diethyl ether–EtOAc
(50 mL) and washed sequentially with satd NaHCO3–
water (until pH 9 was reached) and brine. The organic
POCH2Ph), 4.91 (dd, 1H, J3,2 6.0, J3,4 3.8 Hz, H-3),
4.70 (s, 2H, CH2Ph), 4.63 (d, 1H, H-2), 4.61 (dd, 1H,
3
J7a,7b 11.8, J7a,6 5.0 Hz, H-7a), 4.45 (m, 1H, H-7b),
3
3
4.16 (dd, 1H, J4,5 7.0 Hz, H-4), 4.01 (t, 1H, J5,6
3
7.0 Hz, H-5), 3.92 (m, 1H, J6,7b 3.0 Hz, H-6), 3.78 (br
s, OH), 1.44 and 1.30 [2s, 6H, C(CH3)2]; 13C NMR
(CDCl3): d 137.09 (Cquart. Ph), 128.65–126.99 (Ph),
112.70 [C(CH3)2], 105.46 (C-1), 84.73 (C-2), 80.44
(C-3), 79.03 (C-4), 72.87 (d, JC6,P 4.9 Hz, C-6), 69.75
(d, POCH2Ph), 69.35, 69.25 and 69.17 (C-5, C-7,
POCH2Ph), 65.36 (CH2Ph), 25.92 and 24.45 [C(CH3)2];
31P NMR (CDCl3):
d
1.14. Anal. Calcd for
C31H37O10P: C, 61.99; H, 6.21. Found: C, 61.46; H,
6.26.
Further elution furnished 3 as a syrup (115 mg), Rf
20
0.57; ½aꢁD +43 (c 1.3, CHCl3). 1H NMR (CDCl3): d
7.36–7.26 (m, 15H, Ph), 5.16 (s, 1H, H-1), 5.11–5.04

H. Gu€zlek et al. / Carbohydrate Research 340 (2005) 2808–2811
2811
(dd, 4H, POCH2Ph), 4.82 (dd, 1H, 3J3,2 5.9, 3J3,4 3.6 Hz,
cial support by the Austrian Science Fund (FWF-Grant
P14978-MOB) is gratefully acknowledged.
H-3), 4.66 (d, 1H, H-2), 4.67 and 4.49 (AB-system, 2H,
3
JA,B 11.7 Hz, CH2Ph), 4.33 (dd, 1H, J7a,7b 11.0, J7a,6
3
2.6 Hz, H-7a), 4.28 (dd, 1H, J4,5 2.5 Hz, H-4), 4.23
3
(dd, 1H, J7b,6 5.0 Hz, H-7b), 3.99 (dd, 1H, H-5), 3.89
References
(m, 1H, H-6), 2.60 (br s, OH), 1.47 and 1.30 [2s, 6H,
C(CH3)2]; 13C NMR (CDCl3): d 137.42, 135.84, 135.72
(Cquart. Ph), 128.76–128.02 (Ph), 112.91 [C(CH3)2],
104.88 (C-1), 85.54 (C-2), 81.87 (C-3), 76.65 (C-4),
70.96 (d, JC6,P 4.9 Hz, C-6), 69.85, 69.82, 69.78 and
69.73 (C-5, C-7, POCH2Ph), 69.23 (CH2Ph), 25.77 and
24.09 [C(CH3)2]; 31P NMR (CDCl3): d 1.36. Anal. Calcd
for C31H37O10P: C, 61.99; H, 6.21. Found: C, 61.48; H,
6.37.
1. (a) Holst, O. In Endotoxin in Health and Disease; Brade,
H., Opal, S. M., Vogel, S. N., Morrison, D. C., Eds.;
Marcel Dekker: New York, Basel, USA, 1999; pp 115–
154, and references cited therein; (b) Wilkinson, S. G.
Prog. Lipid Res. 1996, 35, 283–343.
2. (a) Zamyatina, A.; Gronow, S.; Oertelt, C.; Puchberger,
M.; Brade, H.; Kosma, P. Angew. Chem., Int. Ed. 2000, 39,
4150–4153; (b) Gronow, S.; Oertelt, C.; Ervela¨, E.;
Zamyatina, A.; Kosma, P.; Skurnik, M.; Holst, O. J.
Endotoxin Res. 2001, 7, 263–270; (c) Zamyatina, A.;
Gronow, S.; Puchberger, M.; Graziani, A.; Hofinger, A.;
Kosma, P. Carbohydr. Res. 2003, 338, 2571–2589.
3. (a) Kneidinger, B.; Graninger, M.; Puchberger, M.;
Kosma, P.; Messner, P. J. Biol. Chem. 2001, 276, 20935–
20944; (b) Graziani, A.; Zamyatina, A.; Kosma, P.
Carbohydr. Res. 2004, 339, 147–151.
4. (a) Pacinelli, E.; Wang, L.; Reeves, P. R. Infect. Immun.
2002, 70, 3271–3276; (b) Karlyshev, A. V.; Champion, O.
L.; Churcher, C.; Brisson, J.-R.; Jarrell, H. C.; Gilbert,
M.; Brochu, D.; St. Michael, F.; Li, J.; Wakarchuk, W.
W.; Goodhead, I.; Sanders, M.; Stevens, K.; White, B.;
Parkhill, J.; Wren, B. W.; Szymanski, C. M. Mol.
Microbiol. 2005, 55, 90–103.
5. (a) Kneidinger, B.; Marolda, C. L.; Graninger, M.;
Zamyatina, A.; McArthur, F.; Kosma, P.; Valvano, M.
A.; Messner, P. J. Biol. Chem. 2002, 184, 363–369; (b)
Valvano, M. A.; Messner, P.; Kosma, P. Microbiology
2002, 148, 1979–1989.
1.4. D-glycero-D-manno-Heptopyranose 7-phosphate
(triethylammonium salt) (5)
A soln of 4 (125 mg, 0.21 mmol) in dry MeOH (5.5 mL)
was stirred at room temperature in the presence of 10%
Pd/C (45 mg) for 12 h under hydrogen at atmospheric
pressure. After completion of the reaction, the catalyst
was removed by filtration through Celite and washed
with MeOH. The filtrate was concentrated, redissolved
in water (4 mL, the compound was acidic: pH 1) and
stirred for 5 h at room temperature. Then the pH was
adjusted to 5.5 by adding triethylamine (200 lL) and
the soln was lyophilized to give 5 as amorphous mate-
rial. Final purification was achieved by elution on a
PD-10 column (water) to furnish 66 mg (81%) of 5.
6. MacArthur, F.; Andersson, C. E.; Loutet, S.; Mowbray, S.
L.; Valvano, M. A. J. Bacteriol. 2005, 187, 5292–5300.
7. Grzeszczyk, B.; Holst, O.; Zamojski, A. Carbohydr. Res.
1996, 290, 1–15.
8. Read, J. A.; Ahmed, R. A.; Tanner, M. E. Org. Lett. 2005,
7, 2457–2460.
20
½aꢁD +6.5 ! +9.7 (c 0.85, water); Rf 0.36 (10:10:3
MeOH–CHCl3–water); NMR data see Table 1. ESI-
MS: 291.0508 [MH]+; Calcd for C7H15O10P: 291.0476.
9. Brimacombe, J. S.; Kabir, A. K. M. S. Carbohydr. Res.
1986, 152, 329–334.
10. Bannwarth, W.; Trzeciak, A. Helv. Chim. Acta 1987, 70,
175–186.
Acknowledgements
Authors are grateful to Andreas Hofinger for providing
NMR spectra and Daniel Kolarich, for MS data. Finan-
11. Brimacombe, J. S.; Kabir, A. K. M. S. Carbohydr. Res.
1986, 150, 35–51.