Chemoenzymatic synthesis of ADP-d-glycero-β-d-manno-heptose and study of the substrate specificity of HldE

Article abstract of DOI:10.1016/j.bmc.2013.12.019

An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-d-glycero-β-d-manno-heptose (ADP-d, d-heptose) was reported using chemically synthesized d, d-heptose-7-phosphate and the ADP-d, d-heptose biosynthetic enzymes HldE and GmhB M

Full text of DOI:10.1016/j.bmc.2013.12.019

Bioorganic & Medicinal Chemistry 22 (2014) 1139–1147
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chemoenzymatic synthesis of ADP-D-glycero-b-D-manno-heptose
and study of the substrate specificity of HldE
Tiehai Li a, , Liuqing Wen a, , Adriel Williams a, Baolin Wu a, Lei Li a, Jingyao Qu a, Jeffrey Meisner a,
Zhongying Xiao a, Junqiang Fang b, Peng George Wang a,b,
⇑
a Department of Chemistry, Center for Therapeutics and Diagnostics, Georgia State University, Atlanta, GA 30303, USA
b National Glycoengineering Research Center, Shandong University, Jinan, Shandong 250100, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient one-pot three enzymes strategy for chemoenzymatic synthesis of ADP-D-glycero-b-D-manno-
Received 7 October 2013
Revised 3 December 2013
Accepted 12 December 2013
Available online 19 December 2013
heptose (ADP-
D, D-heptose) was reported using chemically synthesized D, D-heptose-7-phosphate and the
ADP- -heptose biosynthetic enzymes HldE and GmhB. Moreover, the result of investigating substrate
D, D
specificity of the kinase action of HldE revealed that HldE had highly restricted substrate specificity
towards structurally modified heptose-7-phosphate analogs.
Published by Elsevier Ltd.
Keywords:
Chemoenzymatic
Synthesis
Analogs
Specificity
1. Introduction
chain. The heptoses with a glycero-D-manno configuration are
common constituents of the inner core oligosaccharides of many
gram-negative bacteria such as Escherichia coli, Salmonella, Pseudo-
monas, Neisseria and other pathogenic strains of bacteria.5 In many
strains, heptoses are expressed in the inner core region of LPS as
branched trisaccharides or disaccharides. Heptoses in LPS are sig-
nificant in bestowing a hydrophilic character in the cell envelope
to keep out hydrophobic residues. Heptoses also contribute to cell
envelope stability through the interacting of their phosphate
groups with divalent cations.6,7 The core oligosaccharide region
of LPS is largely conserved amongst gram-negative bacteria, with
Lipopolysaccharides (LPS) are important outer membrane com-
ponents of gram-negative bacteria. Being exposed to the surface,
LPS are responsible for many pathophysiological responses to bac-
terial infections and are potent endotoxins responsible for high
rates of mortality due to septic shock.1 LPS consist of three main
parts: an endotoxic lipid A comprising an acylated b-(1?6) linked
glucosamine disaccharide with bisphosphorylation, a core oligo-
saccharide, and an O-antigenic component specific to each bacte-
rial species.1 The core oligosaccharide region can be further
divided into an inner core oligosaccharide region consisting of
most strains consisting of 3-deoxy-
D-manno-octulosonic acid
3-deoxy-
cero-b- -manno-heptose (
heptose ( -heptose) residues with the L, D
D
-manno-octulosonic acid (Kdo) residues as well as
-heptose) or -glycero-b- -manno-
-heptose residues more
D-gly-
(Kdo) and -heptose or
D
,
D
L, D
-heptose (Fig. 1).1,2
D
D,
D
L
D
The biosynthesis of the nucleotide activated heptose precursors
L,
D
for assembly of LPS has been extensively studied.1 These nucleotide
abundant; and an outer core region that can contain heptoses, hex-
oses, or hexosamines (Fig. 1).1,2 The inhibition of the biosynthesis
of these carbohydrates, or their assembly, could be exploited for
novel antimicrobial drug design.3,4
activated heptoses mainly include ADP-
heptose (ADP- -heptose), ADP- -glycero-b-
(ADP- -heptose), and a less common GDP- -glycero-
no-heptopyranose (GDP- -heptose has been
-heptose).8 GDP-
D
-glycero-b-
-manno-heptose
-man-
D-manno-
D,
D
L
D
L
,
D
D
a-D
D,
D
D, D
Heptoses are important seven carbon sugars that occur in
numerous structural variations in several domains of the bacterial
cell envelope. These heptoses manifest themselves in cell envelope
anchored oligo- and polysaccharides in a wide variety of conforma-
tions and stereo chemistries, both in the pyranose ring and side
described in bakers’ yeast and identified as the substrate for the
bacterial glycosyltransferase involved in the assembly of the S-layer
glycoprotein glycan in Aneurinibacillus thermoaerophilus.8,9 ADP-
D,
D
-heptose and ADP-
from Shigella sonnei and Salmonella minnesota mutants.10,11 Hep-
tosyltransferases from E. coli can accept ADP- -heptose and
ADP- -heptose as substrates for core oligosaccharide assem-
bly.1,12 The biosynthetic pathway of ADP-
-heptose initiates
with the formation of sugar sedoheptulose-7-phosphate by the
L, D-heptose have been isolated and identified
D
, D
L
, D
⇑
Corresponding author. Tel.: +1 404 413 3591; fax: +1 404 413 5505.
E-mail address: pwang11@gsu.edu (P.G. Wang).
L/D, D
These authors contributed equally to this work.
0968-0896/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.12.019

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T. Li et al. / Bioorg. Med. Chem. 22 (2014) 1139–1147
Figure 1. The structure of LPS.
transketolase (TktA, EC 2.2.1.1) which catalyzes the reaction of
xylulose-5-phosphate with ribose-5-phosphate (Fig. 2).13,14 Sedo-
the by-product (1,2-cyclic phosphate heptose) with release of
AMP.12 Herein, we reported an efficient chemoenzymatic approach
heptulose-7-phosphate is then converted into
D
-glycero-
D
-manno-
to synthesis of ADP-D, D-heptose based on its biosynthetic pathway.
heptose-7-phosphate by keto-aldose isomerase called GmhA (EC
5.3.1.28), followed by anomeric phosphorylation by the kinase
activity of HldE (EC 2.7.1.167) exclusively forming the b-anomer,
Furthermore, using substrate analogs, we revealed highly
restricted substrate specificity of the kinase action of HldE.
namely,
D-glycero-b-D-manno-heptose-1,7-bisphosphate. HldE
2. Results and discussion
comprises two independently functional domains: an N-terminal
region with homology to the ribokinase superfamily and a C-termi-
nal region with homology to the cytidylytransferase superfamily.6
2.1. Chemoenzymatic synthesis of ADP-
heptose
D-glycero-b-D-manno-
The ADP-
ation at C-7 of
the phosphatase (GmhB, EC 3.1.3.82) and adenylylation of the
resulting -glycero-b-manno-heptose-1-phosphate by the second
activity of HldE (EC 2.7.7.70). Epimerization at C-6 by the epimerase
(HldD, EC 5.1.3.20) produces ADP-
-heptose.13,15,16 Hep-
tosyltranferases use this product as the substrate and incorporate
it into LPS assembly. ADP- -heptose has also been shown to be
D
,
D
-heptose is generated by the sequential dephosphoryl-
D
-glycero-b- -manno-heptose-1,7-bisphosphate by
D
D
,
D
-Heptose-7-phosphate 2 was chemically synthesized as
illustrated in Scheme 1. First, -mannose 9 as the starting material
was subjected to benzylation at the anomeric carbon using benzyl
alcohol and acetyl chloride to give benzyl -mannopyranoside
D
D
a-D
10 in 81% yield.18 Subsequently, the primary hydroxyl of com-
pound 10 was selectively silylated using t-butyldiphenylsilyl
(TBDPS) chloride to afford the 6-O-TBDPS ether 11 in 82% yield.
Benzylation of compound 11 using benzyl bromide and NaH in
DMF gave the benzyl ether 12 in 93% yield. Desilylation with tetra-
butylammonium fluoride (TBAF) in THF afforded free 6-hydroxyl
compound 13 in 93% yield. The primary hydroxyl of compound
13 was subjected to Dess–Martin oxidation to provide the desired
aldehyde, which can be directly used for Wittig reaction without
further purification to afford alkene 14 in 61% yield over two
steps.19,20 Dihydroxylation of alkene 14 with OsO4 and N-methyl-
morpholine N-oxide (NMNO) gave a 5:1 mixture of diastereomers
L,
D
D, D
a substrate for these heptosyltransferases, but with much lower
efficiency.12
Chemical synthesis of ADP-
reaction steps, low yields, tedious separations and purification
steps.12,17 For example, the synthesis of penta-acetyl glycero-b-
manno-heptose-1-phosphate is accompanied by the formation of
the -anomer (penta-acetyl glycero- -manno-heptose-1-phos-
L/D, D-heptose suffers from lengthy
D
-
a
a-D
phate), which must be separated from the desired b-anomer prod-
ucts.13 This process of separation is time-consuming and must be
done utilizing laborious separation techniques. Moreover, removal
of acetyl groups from protected ADP-heptose leads to formation of
that can be separated by silica gel column to give predominant
D,
D-heptose 15 with 63% separated yield. The stereochemical
Figure 2. The biosynthetic pathway of ADP-L/D, D-heptose.

T. Li et al. / Bioorg. Med. Chem. 22 (2014) 1139–1147
1141
Scheme 1. Chemoenzymatic synthesis of ADP-D, D-manno-heptose. Reagents and conditions: (a) BnOH, AcCl, 50 °C, 81%; (b) TBDPSCl, imidazole, DMF, 82%; (c) NaH, BnBr,
DMF, 93%; (d) TBAF, THF, 92%; (e) Dess–Martin reagent, DCM; Ph3PCH3Br, n-BuLi, THF, 61% (two steps); (f) OsO4, NMNO, acetone/H2O 9:1, 63%; (g) TBDPSCl, imidazole, DMF,
74%; (h) NaH, BnBr, TBAI, THF, 93%; (i) TBAF, THF, 92%; (j) dibenzyl N, N-diisopropyl phosphoramidite, 1H-tetrazole, m-CPBA, 82%; (k) Pd(OH)2/C, H2, MeOH, 92%; (l) HldE,
GmhB, ATP, inorganic phosphatase, 42%.
outcome of this dihydroxylation was in accordance with Kishi’s
empirical rule.21 Selective silylation of the primary hydroxyl of
compound 15 gave the primary silyl ether 16 using TBDPSCl and
imidazole in 74% yield. Benzylation of compound 16 using benzyl
bromide and NaH in THF with tetrabutylammonium iodide (TBAI)
as phase transfer catalyst gave the benzyl ether 17 in 94% yield.
TBDPS group was removed with TBAF to give free 7-hydroxyl
compound 18 in 92% yield. Phosphorylation of compound 18
in the presence of dibenzyl N,N-diisopropyl phosphoramidite and
1H-tetrazole, followed by in situ m-CPBA oxidation gave the
compound 19.22 The final deprotection of benzyl groups was
achieved using Pearlman’s catalyst (Pd(OH)2/C) under H2 to afford
functional group modifications on these positions were synthe-
sized. Thus, compound 16 was subjected to Mitsunobu reaction
in the presence of diisopropyl azodicarboxylate (DIAD), PPh3 and
p-nitrobenzoic acid to give p-nitrobenzyl ester, with inversion of
configuration at the C-6, which was then treated with base
(K2CO3) to afford compound 21.20 Compound 21 was benzylated
at the 6-OH position to afford compound 22. Conversion of com-
pound 16 to the corresponding thionocarbonate 25 using phenyl
chlorothionoformate and 4-(dimethylamino)-pyridine (DMAP),
followed by radical deoxygenation in the presence of tributylst-
annane and 2, 2-azobisisobutyronitrile (AIBN) gave compound
28.24,25 Based on similar procedures, compounds 22 and 26
were subjected to deprotection of silylation (TBDPS), phosphoryla-
tion of free hydroxyl in C-7 and deprotection of benzyl groups
the desired
D,D
-heptose-7-phosphate 2 in 92% yield.
-heptose-7-phosphate) of HldE
With the natural substrate (
D, D
in hand, the bifunctional enzyme (HldE) and phosphatase (GmhB)
by hydrogenation resulting in the desired analogs
5 and
from E. coli were overexpressed, and purified as previously
6. -Heptose-7-sulfate 7 was prepared by a two-step synthesis:
D, D
reported.13,15
D
,
D-Heptose-7-phosphate was incubated with HldE,
sulfonation of intermediate 18 with sulfur trioxide/pyridine com-
plex gave compound 29, followed by deprotection of benzyl groups
by hydrogenation to afford 7.26–28
GmhB, inorganic pyrophosphatase and ATP, followed by inorganic
pyrophosphatase. Inorganic pyrophosphatase, an enzyme (EC
3.6.1.1) that catalyzed the conversion of one molecule of pyrophos-
phate to two phosphate ions, contributed to promoting the chem-
ical equilibrium of adenylylation for completion of the reaction.
2.3. Substrate specificity of HldE
The resulting sugar nucleotide (ADP-
D
,
D-heptose) can be separated
With the D, D-heptose-7-phosphate analogs in hand, activity
from the other reaction components by Bio-Gel P-2 column with
42% separated yield. The NMR spectroscopic data of the product
assays of HldE towards these analogs were performed. The result-
ing products were analyzed by ESI HRMS. Compound 2, the natural
was in good agreement with chemically synthesized ADP-
D,
D
-hep-
substrate of HldE, was converted to D-glycero-D-manno-b-heptose
tose by Paul Kosma and co-workers.12
1, 7-bisphosphate. Treatment of the heptose diastereomers 3 with
HldE yielded no products according to mass spectrometry analysis.
This is most likely due to the absence of the C-7 phosphate group
on the substrate. Compound 4, mannose 6-phosphate, did not yield
2.2. Preparation of D, D-heptose-7-phosphate analogs
To determine the substrate specificity of the kinase activity of
HldE, we designed and synthesized five -heptose-7-phosphate
the hypothesized product of
after HldE treatment because of missing the C-7. The desired
products were not detected by treatment of HldE with -hep-
tose-7-phosphate 5 and -heptose-7-sulfate 7. Interestingly,
6-deoxy-glycero- -manno-b-heptose 7-phosphate 6 showed weak
reactivity with HldE (TLC analysis showed that the starting mate-
rial 6 did not almost decrease and no desired product be found,
but MS can detect the product with very low abundance compared
with the starting material 6). The lack of any functional group on
C-6, but the presence of the C-7 phosphate group provided insight
into the importance of the C-7 phosphate group for enzyme bind-
ing and function. Based on the reactivity of the substrate analogs
assays, we concluded that the kinase action of HldE had exclusive
substrate specificity towards structurally modified heptose-7-
D-manno-b-heptose 1, 6-bisphosphate
D, D
analogs (Scheme 2). Similar to the synthesis of N-acetyl-hexosa-
mine 1-phosphate using N-acetylhexosamine 1-kinase,23,24 we
hypothesized that the enzyme HldE would directly phosphorylate
heptose diastereomers 3 at the anomeric carbon to give heptose
1-phosphate. Therefore, heptose diastereomers 3 were synthesized
by hydrogenation of compound 20 in 92% yield. Based on the
similarity of glycero-D-manno-heptose and regular D-mannose,
commercially available mannose 6-phosphate 4 was hypothesized
to be a potential substrate of HldE. In order to investigate the
L, D
D
, D
D
importance of the phosphate group on the C-7 of
phosphate, and also the importance of the stereochemistry or
group attached to C-6 of -heptose-7-phosphate, analogs with
D, D-heptose-7-
D, D

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T. Li et al. / Bioorg. Med. Chem. 22 (2014) 1139–1147
Scheme 2. Synthesis of
D,
D
-heptose-7-phosphate analogs. Reagents and conditions: (a) Pd(OH)2/C, H2, 3: 91%; 5: 90%; 6: 92%; 7: 92%; (b) DIAD, PPh3, p-nitrobenzoic acid,
THF; K2CO3, MeOH/DCM, 81% (two steps); (c) NaH, BnBr, TBAI, THF, 91%; (d) TBAF, THF, 92%; (e) dibenzyl N, N-diisopropyl phosphoramidite, 1H-tetrazole, t-BuOOH, 24: 81%;
28: 82%; (f) phenyl chlorothionoformate, DMAP,CH3CN, 60 °C, 72%; (g) (C4H9)3SnH; AIBN, 60 °C, 63%; (h) SO3/Pry, Pyridine, 89%.
phosphate analogs, especially, the stereo-configuration around the
6-hydroxyl and 7-phosphate group were showed to be critical for
enzyme’s functions.
(3.31 ppm for 1H NMR, 49.00 ppm for 13C NMR) and D2O
(4.79 ppm for 1H NMR). Coupling constants were reported in hertz.
High-resolution mass spectra (HRMS) were obtained on a Varian
QFT-ESI mass spectrometer.
3. Conclusion
4.2. Synthesis
In summary, an efficient chemoenzymatic synthesis of ADP-
-heptose was successfully performed using an improved chemical
synthesis of the important intermediate -heptose-7-phosphate
D,
4.2.1. Benzyl
a
-
D
-mannopyranoside (10)
-mannose 9 (10.0 g, 55.5 mmol) in 80 mL benzyl
D
A solution of
D
D, D
alcohol containing 4 mL acetyl chloride was heated at 50 °C for 2 h
and then cooled to room temperature. Benzyl alcohol was removed
under high vacuum pump at 75 °C. The residue was then titrated
with ethyl acetate to form precipitate. The precipitate was col-
lected by filtration and washed with ethyl acetate to give a white
solid 10 (12.1 g, 81%). 1H NMR (CD3OD, 400 MHz): d 3.61–3.66
(m, 2H), 3.71–3.75 (m, 2H), 3.84–3.87 (m, 2H), 4.52 (d,
J = 11.6 Hz, 1H), 4.75 (d, J = 11.6 Hz, 1H), 4.84 (s, 1H); 13C NMR
(CD3OD, 100 MHz): d 62.93, 68.63, 69.87, 72.19, 72.63, 74.86,
100.65, 128.76, 129.11, 129.38, 139.00. HRMS: m/z calcd for
and one-pot three enzymes reaction. This will contribute to studies
of LPS assembly with heptosyltranferases. Moreover, the specificity
study of the kinase action of HldE revealed that it had exclusive
substrate specificity towards structurally modified heptose-
7-phosphate analogs. This work will be important in our
understanding of what substrates and functional groups play roles
in recognition by enzyme, which in turn can aid in the develop-
ment of inhibitors of the kinase for antimicrobial drug design.
4. Experimental
C
13H19O6 [M+H]+ 271.1176, found 271.1173.
4.1. General procedures
4.2.2. Benzyl 6-O-tetra-butyldiphenylsilyl-a-D-
mannopyranoside (11)
All reagents were purchased from commercial sources and were
used without further purification. All solvents were available with
commercially dried or freshly dried and distilled prior to use. Reac-
tions were monitored by Thin Layer Chromatography (TLC) using
silica gel GF254 plates with detection by short wave UV fluores-
cence (k = 254 nm) and staining with 10% phosphomolybdic acid
in EtOH or p-anisaldehyde solution (ethanol/p-anisaldehyde/acetic
acid/sulfuric acid 135:5:4:1.5), followed by heating on a hot plate.
Column chromatography was conducted by silica gel (200–
300 mesh) with ethyl acetate and hexane or ethyl acetate (or
dichloromethane) and methanol as eluent. 1H NMR and 13C NMR
were recorded with Bruker AV 400 spectrometer at 400 MHz (1H
NMR), 100 MHz (13C NMR) using CDCl3, CD3OD and D2O as sol-
vents. Chemical shifts were reported in d (ppm) from CDCl3
(7.26 ppm for 1H NMR, 77.00 ppm for 13C NMR), CD3OD
Compound 10 (3.24 g, 12.0 mmol) was added to flask with
imidazole (1.8 g, 26.4 mmol) and dissolved in DMF (10.0 mL) at
0 °C. TBDPSCl (3.74 mL, 14.4 mmol) was added to reaction mixture
in a dropwise manner. The reaction mixture was then stirred at
room temperature for 6 h. TLC analysis showed complete conver-
sion of starting material to a major product (CH2Cl2/MeOH 10:1,
Rf = 0.67). The reaction was quenched with MeOH and concen-
trated in vacuum. The residue was dissolved with CH2Cl2, and
the organic layer was washed with 5% hydrochloric acid, followed
by water, dried and filtrated. The filtrate was concentrated in vac-
uum and purified by silica gel column (hexane/ethyl acetate 1:1) to
afford a syrup 11 (5.0 g, 82%). 1H NMR (CD3OD, 400 MHz): d 1.06 (s,
9H), 3.58 (t, J = 9.6 Hz, 1H), 3.73–3.80 (m, 2H), 3.84–3.88 (m, 2H),
4.07 (d, J = 10.8 Hz, 1H), 4.54 (d, J = 10.8 Hz, 1H), 4.81
(d, J = 11.6 Hz, 1H), 7.27–7.44 (m, 11H), 7.73–7.77 (m, 4H); 13C

T. Li et al. / Bioorg. Med. Chem. 22 (2014) 1139–1147
1143
NMR (CD3OD, 100 MHz): d 20.13, 27.36, 65.41, 68.89, 69.49, 72.09,
72.86, 75.45, 100.29, 128.70, 128.72, 128.78, 129.19, 129.38,
(15 mL), and extracted with ethyl acetate. The organic layer was
washed with water and brine, followed by drying and filtration.
The filtrate was concentrated in vacuum to give a crude product,
which was purified by silica gel column (hexane/ethyl acetate
20:1) to afford a syrup 14 (655 mg, 61%). 1H NMR (CDCl3,
400 MHz): d 3.89–3.94 (m, 2H), 4.08 (dd, J = 2.8 Hz, J = 9.2 Hz,
1H), 4.24–4.27 (m, 1H), 4.55 (d, J = 12.0 Hz, 1H), 4.73–4.90 (m,
6H), 4.97 (d, J = 10.8 Hz, 1H), 5.05 (s, 1H), 5.42 (d, J = 10.4 Hz,
1H), 5.59 (d, J = 17.2 Hz, 1H), 6.13–6.22 (m, 1H), 7.38–7.46 (m,
20H); 13C NMR (CDCl3, 100 MHz): d 69.03, 72.58, 72.88, 73.30,
75.08, 75.29, 78.93, 79.94, 97.42, 118.16, 127.59, 127.71, 127.84,
127.96, 128.17, 128.40, 128.48, 135.58, 137.38, 138.34, 138.53,
138.68; ESI HRMS: m/z calcd for C35H36O5Na [M+Na]+ 559.2460,
found 559.2466.
130.76, 136.79, 138.85. HRMS: m/z calcd for
C29H36O6SiNa
[M+Na]+ 531.2173, found 531.2159.
4.2.3. Benzyl 6-O-tetra-butyldiphenylsilyl-2,3,4-O-tribenzyl-a-D-
mannopyranoside (12)
Compound 11 (3.9 g, 7.7 mmol) was dissolved in 10 mL DMF
and benzyl bromide (5.5 mL, 46.2 mmol) was added quickly.
Sodium hydride (60% dispersion in mineral oil, 1.85 g, 46.2 mmol)
was then added slowly at 0 °C. The reaction mixture was then stir-
red at room temperature overnight. TLC analysis showed complete
conversion of starting material to a major product (hexane/ethyl
acetate 20:1, Rf = 0.23). The reaction was quenched with MeOH
and concentrated in vacuum. The residue was dissolved with
CH2Cl2, and the organic layer was washed with water, followed
by drying and filtration. The filtrate was concentrated in vacuum
and purified by silica gel column (hexane/ethyl acetate 20:1) to
afford a syrup 12 (5.58 g, 93%). 1H NMR (CDCl3, 400 MHz): d 1.23
(s, 9H), 3.91 (dd, J = 3.6 Hz, J = 9.6 Hz, 1H), 3.99 (s, 1H), 4.07–4.16
(m, 3H), 4.24 (t, J = 9.6 Hz, 1H), 4.59 (d, J = 11.6 Hz, 1H),
4.73–4.85 (m, 5H), 4.92 (d, J = 12.4 Hz, 1H), 5.06–5.10 (m, 2H),
7.32–7.50 (m, 26H), 7.88–7.93 (m, 4H); 13C NMR (CDCl3,
100 MHz): d 19.44, 26.91, 63.43, 68.61, 72.37, 72.79, 73.51, 74.99,
75.30, 75.40, 80.52, 96.86, 127.58, 127.65, 127.78, 128.06, 128.10,
128.37, 128.43, 128.47, 129.62, 135.76, 136.03, 137.45, 138.57,
138.63, 138.71; ESI HRMS: m/z calcd for C50H54O6NaSi [M+Na]+
801.3580, found 801.3574.
4.2.6. Benzyl 2,3,4-O-tri-benzyl-D-glycero-a-D-manno-
heptopyranoside (15)
To a solution compound 14 (1.0 g, 1.86 mmol) in acetone/water
(9:1, 20 mL) at 0 °C was added NMNO (435.8 mg, 3.72 mmol) and
OsO4 (12.7 mg, 0.05 mmol). The reaction mixture was then stirred
at room temperature for 4 h. TLC analysis showed complete con-
version of starting material to a major product 15 (hexane/ethyl
acetate 1:1, Rf = 0.39, the diastereomer Rf = 0.30). The reaction
was quenched with a saturated solution of NaHSO3. After stirring
for addition 15 min, the reaction mixture was extracted with ethyl
acetate and the organic layer was washed with H2O and brine,
dried with anhydrous Na2SO4 and filtrated. The filtrate was con-
centrated in vacuum and purified by silica gel column (hexane/
ethyl acetate 3:2) to afford a syrup 15 (669 mg, 63%). 1H NMR
(CDCl3, 400 MHz): d 2.73 (br, 1H, –OH), 3.72 (d, J = 3.6 Hz, 1H),
3.78–3.81 (m, 1H), 3.91–3.98 (m, 3H), 4.08 (t, J = 3.6 Hz, 1H), 4.13
(dd, J = 2.8 Hz, J = 9.2 Hz, 1H), 4.20 (t, J = 9.2 Hz, 1H), 4.54 (d,
J = 11.6 Hz, 1H), 4.68–4.86 (m, 6H), 5.00 (s, 1H), 5.15 (d,
J = 10.4 Hz, 1H), 7.40–7.46 (m, 20H); 13C NMR (CDCl3, 100 MHz):
d 62.97, 69.00, 71.63, 71.97, 72.71, 72.85, 74.57, 75.00, 76.69,
80.46, 96.95, 127.70, 127.80, 127.87, 127.97, 128.19, 128.41,
128.52, 137.02, 137.70, 138.02, 138.11; ESI HRMS: m/z calcd for
4.2.4. Benzyl 2,3,4-O-tribenzyl-a-D-mannopyranoside (13)
To a solution of 12 (4.0 g, 5.1 mmol) in THF (20 mL) was added
1 M TBAF in THF solution (10.2 mL, 10.2 mmol). The reaction mix-
ture was then stirred at room temperature overnight. TLC analysis
showed complete conversion of starting material to a major prod-
uct (hexane/ethyl acetate 5:1, Rf = 0.11). The reaction mixture was
concentrated in vacuum and purified by silica gel column (hexane/
ethyl acetate 4:1) to afford a syrup 13 (2.54 g, 92%). 1H NMR
(CDCl3, 400 MHz): d 2.47 (br, 1H), 3.86 (dd, J = 3.6 Hz, J = 8.8 Hz,
1H), 3.93–3.96 (m, 3H), 4.11–4.19 (m, 2H), 4.55 (d, J = 12.0 Hz,
1H), 4.73–4.81 (m, 5H), 4.88 (d, J = 12.4 Hz, 1H), 5.05–5.09 (m,
2H), 7.37–7.50 (m, 20H); 13C NMR (CDCl3, 100 MHz): d 62.26,
69.10, 72.29, 72.58, 72.91, 74.89, 75.27, 80.20, 97.55, 127.58,
127.64, 127.72, 127.82, 127.87, 128.10, 128.38, 128.42, 128.45,
137.19, 138.21, 138.43, 138.50; ESI HRMS: m/z calcd for
C
35H42NO7 [M+NH4]+ 588.2961, found 588.2939.
4.2.7. Benzyl 7-O-tetra-butyldiphenylsilyl-2,3,4-O-tri-benzyl-
glycero- -manno-heptopyrano-side (16)
To solution of 15 (467 mg, 0.82 mmol) and imidazole
D-
a
-D
a
(55.8 mg, 1.8 mmol) in 10 mL DMF was added TBDPSCl (0.25 mL,
0.98 mmol) at 0 °C. The reaction mixture was then stirred at room
temperature for 6 h. TLC analysis showed complete conversion of
starting material to a major product (hexane/ethyl acetate 8:1,
Rf = 0.22). The reaction was quenched with MeOH and concen-
trated in vacuum. The residue was added DCM, and the organic
layer was washed with water, dried and filtrated. The filtrate was
concentrated in vacuum and purified by silica gel column (hex-
ane/ethyl acetate 8:1) to afford a syrup 16 (491 mg, 74%). 1H
NMR (CDCl3, 400 MHz): d 1.18 (s, 9H), 3.01 (d, J = 3.6 Hz, 1 H,
-OH), 3.88 (m, 1H), 3.94–3.99 (m, 2H), 4.03–4.14 (m, 3H), 4.22
(dd, J = 3.6 Hz, J = 6.8 Hz, 1H), 4.50 (t, J = 12.0 Hz, 1H), 4.62 (d,
J = 10.4 Hz, 1H), 4.69–4.80 (m, 5H), 4.97–5.00 (m, 2H), 7.27–7.50
(m, 26H), 7.77–7.82 (m, 4H); 13C NMR (CDCl3, 100 MHz): d 19.34,
27.00, 64.88, 69.04, 71.88, 72.21, 72.66, 73.40, 74.63, 74.81,
75.63, 80.39, 97.06, 127.60, 127.67, 127.78, 127.91, 128.39,
129.76, 133.48, 133.57, 135.71, 137.30, 138.32, 138.43; ESI HRMS:
m/z calcd for C51H56O7NaSi [M+Na]+ 831.3693, found 831.3671.
C
34H36O6Na [M+Na]+ 563.2410, found 563.2419.
4.2.5. Benzyl 2,3,4-O-tribenzyl-6,7-dideoxy-a-D-hept-6-
enopyranoside (14)
To a solution of 13 (1.08 g, 2.0 mmol) in CH2Cl2 15 mL at 0 °C
was added 0.3 M Dess–Martin reagent in CH2Cl2 solution
(13.3 mL, 4.0 mmol). The reaction mixture was then stirred at
room temperature for 40 min. The reaction was quenched with
an aqueous solution of sodium sulfite (1 M, 10 mL), followed by a
saturated solution sodium bicarbonate (10 mL). The reaction
mixture was further stirred for 15 min and aqueous layer was
extracted with CH2Cl2 (2 ꢀ 30 mL), and the combined organic lay-
ers were dried and concentrated in vacuum to afford the crude
aldehyde to directly use for next step without further purification.
A solution of methyltriphenylphosphonium bromide (857 mg,
2.4 mmol) in 20 mL THF was cooled to ꢁ40 °C and n-BuLi
(1.0 mL, 2.5 mmol, 2.5 M in hexane) was added. The reaction mix-
ture was stirred at ꢁ40 °C for 30 min and a solution of the crude
aldehyde in THF (5.0 mL) was added. The reaction mixture was
stirred for an additional 1 h at ꢁ40 °C. Then the reaction was
warmed up slowly to room temperature over a period of 4 h before
it was quenched with a saturated solution of ammonium chloride
4.2.8. Benzyl 7-O-tetra-butyldiphenylsilyl-2,3,4,6-O-tetra-
benzyl-D-glycero-a-D-manno-heptopyranoside (17)
To
a solution 16 (447 mg, 0.55 mmol), TBAI (20 mg,
0.055 mmol) and BnBr (0.13 mL, 1.1 mmol) in 10 mL THF at 0 °C
was slowly added NaH (60% dispersion in mineral oil, 44 mg,

1144
T. Li et al. / Bioorg. Med. Chem. 22 (2014) 1139–1147
1.1 mmol). The reaction mixture was then stirred at room temper-
ature overnight. TLC analysis showed complete conversion of start-
1H), 3.77–3.80 (m, 2H), 4.06–4.12 (m, 2H), 4.19–4.23 (m, 2H),
5.06 (s, 1H); 13C NMR (CD3OD, 100 MHz): d 68.93 (d, JC–P = 5.3 Hz),
69.88, 72.30, 72.58, 73.02, 73.14 (d, JC–P = 7.7 Hz), 95.78; HRMS: m/
z calcd for C7H14O10P [MꢁH]ꢁ 289.0325, found 289.0328.
ing material to
a major product (hexane/ethyl acetate 8:1,
Rf = 0.53). The reaction mixture was filtered, the filtrate was con-
centrated in vacuum and purified by silica gel column (hexane/
ethyl acetate 15:1) to afford a syrup 17 (465 mg, 94%). 1H NMR
(CDCl3, 400 MHz): d 1.27 (s, 9H), 4.00 (s, 1H), 4.12–4.22 (m, 5H),
4.35 (t, J = 9.2 Hz, 1H), 4.61 (d, J = 11.6 Hz, 1H), 4.70 (d,
J = 10.8 Hz, 1H), 4.81–4.83 (m, 3H), 4.87–5.13 (m, 6H), 7.26–7.27
(m, 2H), 7.38–7.57 (m, 29H), 7.85–7.91 (m, 4H); 13C NMR (CDCl3,
100 MHz): d 19.24, 27.00, 65.14, 68.90, 72.31, 72.70, 72.85, 73.15,
74.75, 74.94, 75.29, 80.61, 81.36, 97.10, 127.30, 127.40, 127.56,
127.65, 127.72, 127.78, 127.86, 127.94, 128.24, 128.28, 128.35,
128.40, 128.44, 129.62, 133.71, 135.71, 135.82, 137.50, 138.54,
138.61, 139.24; ESI HRMS: m/z calcd for C58H66O7Si [M+NH4]+
916.4609, found 916.4592.
4.2.12. Adenosine 50-diphosphate-
heptopyranose (Tris-salt) (1)
D
-glycero-b-
D-manno-
A solution (7.0 mL) containing 10 mM D, D-heptose-7-phosphate
2 (20 mg, 0.07 mmol), 25 mM ATP (0.175 mmol), 5 mM MgCl2
(0.15 mmol) and 20 mM Tris–HCl buffer (pH 8.0), and the pH of
the solution was adjusted to 8.0 by the addition of 1 M NaOH, fol-
lowed by adding 3.0 mg HldE, 3.0 mg phosphatese GmhB, and
3.0 mg inorganic pyrophosphatase into the reaction mixture, incu-
bating for 12 h at 37 °C. The reaction mixture was removed the
proteins using Millipore (Amicon Ultra-4, 10 000 MWCO). Then
the reaction mixture was added alkaline phosphatase (100 units)
incubating for 12 h at 37 °C. The resulting solution was purified
by Bio-Gel P-2 column. The product fractions were lyophilized to
4.2.9. Benzyl 2,3,4,6-O-tetra-benzyl-D-glycero-a-D-manno-
heptopyranoside (18)
give ADP-D, D
-heptose 1 as white powder (18 mg, 42%). 1H NMR
To a solution of 17 (360 mg, 0.4 mmol) in 10 mL THF was added
1 M TBAF in THF solution (0.6 mL, 0.6 mmol). The reaction mixture
was then stirred at room temperature overnight. TLC analysis
showed complete conversion of starting material to a major prod-
uct (hexane/ethyl acetate 3:1, Rf = 0.27). The reaction mixture was
concentrated in vacuum and purified by silica gel column (hexane/
ethyl acetate 3:1) to afford a syrup 18 (243 mg, 92%).1H NMR
(CDCl3, 400 MHz): d 2.45 (br, 1 H, -OH), 3.77–3.82 (m, 1H), 3.91–
3.94 (m, 1H), 4.06–4.17 (m, 3H), 4.54 (d, J = 12.0 Hz, 1H), 4.66–
4.74 (m, 5H), 4.80–4.89 (m, 3H), 5.02–5.04 (m, 2H), 7.31–7.44
(m, 25H); 13C NMR (CDCl3, 100 MHz): d 62.16, 69.08, 72.04,
72.23, 72.72, 72.80, 74.81, 74.93, 78.81, 80.47, 97.06, 127.71,
127.78, 127.81, 127.92, 128.12, 128.43, 128.49, 137.31, 138.23,
138.25, 138.37, 138.43; ESI HRMS: m/z calcd for C42H44O7Na
[M+Na]+ 683.2985, found 683.3006.
(D2O, 400 MHz): d 3.35 (dd, J = 2.8 Hz, J = 9.6 Hz, 1H), 3.65–3.70
(m, 4H), 3.91–3.92 (m, 1H), 3.98 (m, 1H), 4.15–4.16 (m, 2H), 4.32
(m, 1H), 4.44 (dd, J = 3.6 Hz, J = 4.4 Hz, 1H), 4.74 (m, 1H), 5.11 (d,
J = 8.8 Hz, 1H), 6.05 (d, J = 5.6 Hz, 1H), 8.15 (s, 1H), 8.41 (s, 1H);
13C NMR (D2O, 100 MHz): d 62.75, 66.37 (d, JC–P = 5.2 Hz), 68.18,
71.45, 71.62, 72.77, 73.73, 75.38, 78.05, 84.90 (d, JC–P = 8.9 Hz),
88.00, 96.94, 119.69, 140.94, 150.16, 153.90, 156.64; ESI HRMS:
m/z calcd for C17H26N5O16P2 [MꢁH]ꢁ 618.0850, found 618.0845.
4.2.13. D, L-glycero-D-manno-heptose (3)
To a solution of 20 (114 mg, 0.2 mmol) in MeOH (4.0 mL) was
added Pd(OH)2 on carbon (100 mg). The reaction was stirred in
H2 atmosphere (50 psi) for 4 h. The reaction mixture was filtered
and washed MeOH. The filtrate was concentrated to give a syrup
3 (38 mg, 90%). ESI HRMS: m/z calcd for C7H14O7Na [M+Na]+
233.0637, found 233.0637.
4.2.10. Benzyl 7-O-dibenzyl-phosphono-2,3,4,6-O-tetra-benzyl-
D
-glycero-
a
-
D
-manno-heptopyranoside (19)
4.2.14. Benzyl 7-O-tetra-butyldiphenylsilyl-2,3,4-O-tri-benzyl-
glycero- -manno-heptopyranoside (21)
To solution 16 (435 mg, 0.538 mmol), PPh3 (283 mg,
1.08 mmol) and p-NO2C6H4COOH in THF (20 mL) was added DIAD
(213 L, 1.08 mmol). The reaction mixture was then stirred at
L-
A mixture of compound 18 (220 mg, 0.33 mmol), 4 Å molecular
a
a
-D
sieves (200 mg) and 1H-tetrazole (3.7 mL, 0.45 M in CH3CN,
1.67 mmol) in anhydrous DCM (10 mL) was stirred 30 min at room
temperature. Then (BnO)2PN(i-Pr)2 (330
lL, 1.0 mmol) was added.
l
The reaction mixture was stirred at room temperature for another
2 h. Subsequently, the reaction mixture was cooled ꢁ20 °C,
m-CPBA (374.3 mg 1.67 mmol) was added, and slowly warmed
up room temperature. TLC analysis showed complete conversion
of starting material to a major product (hexane/ethyl acetate 3:1,
Rf = 0.23). The reaction was quenched by addition of 0.2 mL trieth-
ylamine and filtered. The filtrate was concentrated in vacuum and
purified by silica gel flash chromatography (hexane/ethyl acetate
room temperature for 5 h. TLC analysis showed complete conver-
sion of starting material to a major product (hexane/ethyl acetate
8:1, Rf = 0.36). The reaction mixture was concentrated in vacuum
and purified by silica gel column (hexane/ethyl acetate 15:1) to af-
ford a syrup (474 mg). Then the solution of the syrup (474 mg,
0.49 mmol) and K2CO3 (82 mg, 0.59 mmol) in 11 mL MeOH/DCM
(10:1) was stirred at room temperature for 4 h. TLC analysis
showed complete conversion of starting material to a major prod-
uct (hexane/ethyl acetate 4:1, Rf = 0.30). The reaction mixture was
concentrated in vacuum, and then the crude was dissolved with
ethyl acetate, and washed with water and brine. The organic layer
was dried with Na2SO4, followed by filtration, the filtrate was con-
centrated in vacuum and purified by silica gel column (hexane/
ethyl acetate 5:1) to afford a syrup (351 mg, 81%, two steps).
1H NMR (CDCl3, 400 MHz): d 1.18 (s, 9H), 2.47 (d, J = 8.4 Hz, 1 H,
-OH), 3.88 (dd, J = 7.6 Hz, J = 9.6 Hz, 1H), 3.95–3.98 (m, 2H), 4.02
(d, J = 9.6 Hz, 1H), 4.14 (dd, J = 1.2 Hz, J = 9.2 Hz, 1H), 4.21 (dd,
J = 7.6 Hz, J = 14.4 Hz, 1H), 4.36 (t, J = 9.6 Hz, 1H), 4.48 (d,
J = 12.0 Hz, 1H), 4.73–4.82 (m, 4H), 4.86–4.89 (m, 2H), 5.02 (s,
1H), 5.13 (d, J = 11.6 Hz, 1H), 7.27–7.29 (m, 2H), 7.37–7.53 (m,
24H), 7.78–7.79 (m, 4H); 13C NMR (CDCl3, 100 MHz): d 19.13,
26.78, 64.58, 68.62, 69.43, 70.55, 72.25, 72.69, 74.40, 74.60,
75.13, 80.28, 96.97, 127.43, 127.51, 127.64, 127.74, 128.24,
128.30, 129.63, 133.18, 133.45, 135.42, 135.50, 136.87, 138.17,
4:1) to afford
a
syrup 19 (249 mg, 82%). 1H NMR (CDCl3,
400 MHz): d 3.90 (m, 1H), 4.01 (d, J = 10.0 Hz, 1H), 4.06–4.19 (m,
3H), 4.39–4.44 (m, 1H), 4.52 (d, J = 12.0 Hz, 1H), 4.67–4.88 (m,
8H), 4.97 (d, J = 10.4 Hz, 1H), 5.02–5.12 (m, 6H), 7.29–7.42 (m,
35H); 13C NMR (CDCl3, 100 MHz): d 67.96 (d, JC–P = 5.3 Hz), 68.95,
69.11 72.17, 72.30, 72.68, 72.73, 74.62, 74.84, 78.40, 78.44
(d, JC–P = 7.0 Hz), 80.44, 97.00, 127.43, 127.50, 127.59,
127.71, 127.75, 127.78, 127.85, 127.88, 128.21, 128.33, 128.40,
128.47, 137.22, 138.25, 138.28, 138.36; ESI HRMS: m/z calcd for
C
56H57O10NaP [M+Na]+ 943.3587, found 943.3586.
4.2.11. 7-Phosphate- -glycero-b- -manno-heptose (2)
D
D
To a solution of 19 (184 mg, 0.2 mmol) in MeOH (4 mL) was
added Pd(OH)2 on carbon (100 mg). The reaction was stirred in
H2 atmosphere (50 psi) for 4 h. The reaction mixture was filtered
and washed with MeOH. The filtrate was concentrated to give a
syrup 2 (53 mg, 92%).1H NMR (CD3OD, 400 MHz): 3.35–3.38 (m,

T. Li et al. / Bioorg. Med. Chem. 22 (2014) 1139–1147
1145
138.51, 138.68; ESI HRMS: m/z calcd for C51H57O7Si [M+H]+
809.3868, found 809.3861.
127.81, 127.84, 127.87, 128.19, 128.23, 128.25, 128.32, 128.45,
128.48, 135.60, 135.67, 137.61, 137.99, 138.14, 138.20, 138.63; ESI
HRMS: m/z calcd for C56H58O10P [M+H]+ 921.3762, found 921.3750.
4.2.15. Benzyl 7-O-tetra-butyldiphenylsilyl-2,3,4,6-O-tetra-
benzyl-
To a solution 21 (160 mg, 0.2 mmol), TBAI (3.7 mg, 0.01 mmol)
and BnBr (47 L, 0.4 mmol) in THF (10 mL) at 0 °C was slowly
L
-glycero-
a
-
D
-manno-heptopyranoside (22)
4.2.18. 7-O-Phosphono-L-glycero-D-manno-heptopyranosyl
phosphate (5)
l
To a solution of 24 (80 mg, 0.087 mmol) in MeOH (4.0 mL) was
added Pd(OH)2 on carbon (100 mg). The reaction was stirred in H2
atmosphere (50 pis) for 4 h. The reaction mixture was filtered and
washed with MeOH. The filtrate was concentrated to give a syrup 5
(22 mg, 90%). 1H NMR (D2O, 400 MHz): d 3.30–3.34 (m, 1H), 3.73–
3.81 (m, 2H), 3.87 (m, 1H), 3.94–4.00 (m, 2H), 4.10–4.16 (m, 1H),
5.13 (s, 1H); 13C NMR (D2O, 100 MHz): d 65.23, 66.44 (d, JC–P = 7.7 -
Hz), 69.66 (d, JC–P = 6.4 Hz), 70.30, 72.35, 73.26, 93.32; HRMS: m/z
calcd for C7H14O10P [MꢁH]ꢁ 289.0330, found 289.0333.
added NaH (60% dispersion in mineral oil, 16 mg, 0.4 mmol). The
reaction mixture was then stirred at room temperature overnight.
TLC analysis showed complete conversion of starting material to a
major product (hexane/ethyl acetate 8:1, Rf = 0.38). The reaction
mixture was filtered, the filtrate was concentrated in vacuum
and purified by silica gel column (hexane/ethyl acetate 15:1) to
afford a syrup 22 (164 mg, 91%). 1H NMR (CDCl3, 400 MHz): d
1.19 (s, 9H), 3.95 (m, 1H), 4.10–4.16 (m, 5H), 4.33 (t, J = 9.6 Hz,
1H), 4.35–4.45 (m, 2H), 4.52 (d, J = 12.0 Hz, 1H), 4.72 (s, 2H),
4.76–4.86 (m, 4H), 5.02 (d, J = 11.6 Hz, 1H), 5.15 (s, 1H), 7.30–
7.55 (m, 31H), 7.71–7.82 (m, 4H); 13C NMR (CDCl3, 100 MHz): d
19.05, 26.77, 62.78, 68.48, 70.85, 71.95, 72.21, 72.56, 74.19,
74.24, 74.28, 76.75, 80.68, 96.46, 127.21, 127.24, 127.27, 127.34,
127.41, 127.62, 127.66, 127.72, 127.78, 128.13, 128.21, 128.25,
129.63, 129.67, 133.23, 133.33, 135.44, 135.59, 137.01, 138.21,
138.37, 138.48, 138.95; ESI HRMS: m/z calcd for C58H63O7Si
[M+H]+ 899.4338, found 899.4320.
4.2.19. Benzyl-7-O-tetra-butyldiphenylsilyl-6-O-
(phenorythiocarbonyl)-2,3,4-O-tri-benzyl-D-glycero-a-D-
manno-heptopyranoside (25)
To the mixture of compound 16 (380 mg, 0.47 mmol), DMAP
(103.8 mg, 0.85 mmol) and anhydrous acetonitrile (8 mL) was
added phenyl chlorothionoformate (98 lL, 0.71 mmol), and the
solution was stirred at 60 °C for 5 h at Ar atmosphere. TLC analysis
showed complete conversion of starting material to a major prod-
uct (hexane/ethyl acetate 4:1, Rf = 0.46). Then the reaction mixture
was cooled room temperature and filtered. The precipitate was
washed with ethyl acetate. The filtrate was concentrated in vac-
uum, and purified by silica gel flash chromatography (hexane/ethyl
acetate 15:1) to afford a syrup 25 (320 mg, 72%). 1H NMR (CDCl3,
400 MHz): d 1.23 (s, 9H), 3.93 (m, 1H), 4.09 (d, J = 8.8 Hz, 1H),
4.17 (t, J = 9.6 Hz, 1H), 4.27–4.29 (m, 2H), 4.36 (dd, J = 8.0 Hz,
J = 10.8 Hz, 1H), 4.53 (d, J = 12.0 Hz, 1H), 4.67 (d, J = 10.8 Hz, 1H),
4.73–4.77 (m, 3H), 4.82–4.85 (m, 2H), 4.98 (d, J = 10.8 Hz, 1H),
5.04 (s, 1H), 6.33 (m, 1H), 7.13–7.15 (m, 2H), 7.38–7.51 (m, 27H),
7.84–7.89 (m, 6H); 13C NMR (CDCl3, 100 MHz): d 19.13, 26.74,
61.75, 68.83, 71.52, 72.16, 72.56, 74.83, 74.87, 74.96, 80.25,
84.51, 96.76, 121.91, 126.27, 127.51, 127.60, 127.63, 127.66,
127.76, 127.90, 127.94, 128.23, 128.36, 129.34, 129.54, 129.61,
129.64, 134.71, 135.57, 135.60, 136.92, 138.02, 138.16, 138.27,
153.39, 194.59; ESI HRMS: m/z calcd for C58H60O8SSiNa [M+Na]+
967.3670, found 967.3649.
4.2.16. Benzyl 2,3,4,6-O-tetra-benzyl-L-glycero-a-D-manno-
heptopyranoside (23)
To a solution of 22 (158 mg, 0.176 mmol) in THF 10 mL was
added 1 M TBAF in THF solution (352 L, 0.352 mmol). The reaction
l
mixture was then stirred at room temperature overnight. TLC anal-
ysis showed complete conversion of starting material to a major
product (hexane/ethyl acetate 2:1, Rf = 0.43). The reaction mixture
was concentrated in vacuum and purified by silica gel column (hex-
ane/ethyl acetate 3:1) to afford a syrup 23 (107 mg, 92%). 1H NMR
(CDCl3, 400 MHz): d 2.39 (br s, 1H, –OH), 3.92–3.97 (m, 4H), 4.03–
4.09 (m, 2H), 4.33 (t, J = 9.6 Hz, 1H), 4.50–4.55 (m, 2H), 4.64–4.71
(m, 3H), 4.75–4.79 (m, 3H), 4.87 (d, J = 11.6 Hz, 1H), 5.02 (d,
J = 11.2 Hz, 1H), 5.09 (s, 1H), 7.28–7.46 (m, 25H); 13C NMR (CDCl3,
100 MHz): d 62.29, 69.01, 71.92, 72.02, 72.39, 73.20, 74.33, 74.54,
75.93, 80.31, 97.04, 127.44, 127.59, 127.65, 127.75, 128.17,
128.24, 128.36, 136.99, 138.12, 138.19, 138.32, 138.47; ESI HRMS:
m/z calcd for C42H44O7Na [M+Na]+ 683.2979, found 683.2969.
4.2.20. Benzyl-7-O-tetra-butyldiphenylsilyl-2,3,4-O-tri-benzyl-
4.2.17. Benzyl 7-O-dibenzyl-phosphono-2,3,4,6-O-tetra-benzyl-
6-deoxy-glycero-a-D-manno-heptopyranoside (26)
L
-glycero-
a
-
D
-manno-heptopyranoside (24)
A solution of 25 (284 mg, 0.3 mmol), Tributylstannane (1.2 mL,
1.0 M in hexane, 1.2 mmol) and AIBN (24.6 mg, 0.15 mmol) in dry
toluene (10 mL) was stirred at 60 °C for 4 h in Ar atmosphere. TLC
analysis showed complete conversion of starting material to a ma-
jor product (hexane/ethyl acetate 8:1, Rf = 0.50). Then the reaction
mixture was cooled room temperature and concentrated in vac-
uum, and purified by silica gel flash chromatography (hexane/ethyl
acetate 20:1) to afford a syrup 26 (150 mg, 63%). 1H NMR (CDCl3,
400 MHz): d 1.18 (s, 9H), 1.89–1.94 (m, 1H), 2.36–2.43 (m, 1H),
3.86 (t, J = 9.2 Hz, 1H), 3.97–3.99 (m, 2H), 4.07–4.13 (m, 3H), 4.52
(d, J = 12.0 Hz, 1H), 4.73–4.88 (m, 6H), 4.98 (s, 1H), 5.10 (d,
J = 11.6 Hz, 1H), 7.39–7.50 (m, 25H), 7.80–7.84 (m, 5H); 13C NMR
(CDCl3, 100 MHz): d 19.14, 26.83, 35.01, 60.45, 68.41, 72.18,
72.70, 74.72, 75.08, 79.04, 80.38, 96.59, 127.55, 127.72, 127.76,
127.83, 128.25, 129.45, 129.54, 135.46, 135.52, 137.15, 138.19,
138.53, 138.64; ESI HRMS: m/z calcd for C51H57O6Si [M+H]+
793.3919, found 793.3896.
A mixture of compound 23 (108 mg, 0.163 mmol), 4 Å molecular
sieves (200 mg) and 1H-tetrazole (1.8 mL, 0.45 M in CH3CN,
0.815 mmol) in anhydrous DCM (10 mL) was stirred 30 min at room
temperature. Then (BnO)2PN(i-Pr)2 (161 lL, 0.489 mmol) was
added. The reaction mixture was stirred at room temperature for
another 2 h. Subsequently, the reaction mixture was cooled
ꢁ20 °C, t-BuOOH (163
lL, 5.0 M in decane, 0.815 mmol) was added,
slowly warmed up room temperature. TLC analysis showed com-
plete conversion of starting material to a major product (hexane/
ethyl acetate 2:1, Rf = 0.36). The reaction was quenched by addition
of 0.2 mL triethylamine and filtered. The filtrate was concentrated
in vacuum and purified by silica gel flash chromatography
(hexane/ethyl acetate 4:1) to afford a syrup 24 (121 mg, 81%).
1H NMR (CDCl3, 400 MHz):
d
3.86–3.88 (m, 2H), 4.03
(dd, J = 2.8 Hz, J = 9.2 Hz, 1H), 4.15 (t, J = 6.8 Hz, 1H), 4.24–4.30
(m, 2H), 4.37–4.42 (m, 3H), 4.50 (d, J = 11.6 Hz, 1H), 4.63–4.70
(m, 3H), 4.75–4.78 (m, 3H), 4.96 (d, J = 11.2 Hz, 1H), 5.02–5.12 (m,
5H), 7.25–7.37 (m, 35H); 13C NMR (CDCl3, 100 MHz): d 65.66 (d, JC–
-
4.2.21. Benzyl 2,3,4,6-O-tri-benzyl-6-deoxy-glycero-a-D-manno-
P = 6.2 Hz), 68.86, 69.23 (d, JC–P = 3.4 Hz), 69.29 (JC–P = 3.1 Hz),
70.77, 71.91, 72.37, 72.78, 73.90, 74.16, 74.50, 74.83 (d, JC–P = 8.7
Hz), 80.51, 96.86, 127.40, 127.44, 127.48, 127.51, 127.68, 127.74,
heptopyranoside (27)
To a solution of 26 (172 mg, 0.217 mmol) in THF 10 mL was
added 1 M TBAF in THF solution (434 L, 0.434 mmol). The
l

1146
T. Li et al. / Bioorg. Med. Chem. 22 (2014) 1139–1147
reaction mixture was then stirred at room temperature overnight.
TLC analysis showed complete conversion of starting material to a
major product (hexane/ethyl acetate 4:1, Rf = 0.65). The reaction
mixture was concentrated in vacuum and purified by silica gel col-
umn (hexane/ethyl acetate 8:1) to afford a syrup 27 (111 mg, 92%).
1H NMR (CDCl3, 400 MHz): d 1.87–1.96 (m, 1H), 2.18–2.21 (m, 1H),
2.44 (br s, 1H), 3.82–3.96 (m, 5H), 4.01 (dd, J = 2.4 Hz, J = 9.2 Hz,
1H), 4.48 (d, J = 12.0 Hz, 1H), 4.68–4.83 (m, 6H), 4.92 (s, 1H), 5.03
(d, J = 11.2 Hz, 1H), 7.35–7.40 (m, 20H); 13C NMR (CDCl3,
100 MHz): d 33.80, 60.77, 68.90, 71.43, 72.08, 72.78, 74.66, 75.26,
78.22, 80.07, 96.98, 127.49, 127.57, 127.62, 127.77, 127.97,
128.28, 128.37, 136.97, 138.06, 138.25, 138.31; ESI HRMS: m/z
calcd for C51H57O7Si [M+H]+ 555.2741, found 555.2738.
4.39 (d, J = 11.6 Hz, 1H), 4.50–4.56 (m, 5H), 4.62–4.80 (m, 5H),
5.06 (s, 1H), 7.11–7.32 (m, 25H); 13C NMR (CDCl3, 100 MHz): d
68.96, 71.97, 72.12, 72.53, 73.61, 74.55, 74.76, 80.20, 97.04,
127.43, 127.66, 127.85, 128.06, 128.21, 128.24, 128.35, 137.38,
137.76, 138.23; HRMS: m/z calcd for
C42H43O10
S
[MꢁH]ꢁ
739.2582, found 739.2561.
4.2.25. 7-O-Sulfo-D-glycero-D-manno-heptopyranoside (7)
To a solution of 29 (150 mg, 0.2 mmol) in 6.0 mL t-BuOH/H2O
(5:1) was added Pd(OH)2/C on carbon (100 mg). The reaction was
stirred in H2 atmosphere (50 psi) for 4 h. The reaction mixture
was filtered and washed with t-BuOH/H2O (1:2). The filtrate was
concentrated to give a syrup 7 (54 mg, 92%). 1H NMR (D2O,
400 MHz): d 3.67 (dd, J = 5.2 Hz, J = 10.8 Hz, 1H), 3.80–3.89 (m,
2H), 3.94 (t, J = 8.4 Hz, 1H), 4.16–4.17 (m, 2H), 4.25–4.26 (m, 1H),
5.18 (s, 1H); 13C NMR (D2O, 100 MHz): d 68.30, 68.63, 68.96,
4.2.22. Benzyl-7-O-dibenzyl-phosphono-2,3,4-O-tri-benzyl-6-
deoxy-glycero-a-D-manno-heptopyranoside (28)
A mixture of compound 27 (95 mg, 0.17 mmol), 4 Å molecular
sieves (100 mg) and 1H-tetrazole (1.9 mL, 0.45 M in CH3CN,
0.85 mmol) in anhydrous DCM (10 mL) was stirred 30 min at room
71.19, 72.32, 74.92, 93.14. ESI HRMS: m/z calcd for C7H13O10
S
[MꢁH]ꢁ 289.0235, found 289.0238.
temperature. Then (BnO)2PN(i-Pr)2 (168 lL, 0.51 mmol) was
added. The reaction mixture was stirred at room temperature for
another 2 h. Subsequently, the reaction mixture was cooled
4.3. HldE assays for
D
,
D
-heptose-7-phosphate analogs
-heptose-7-phosphate 2 and its
A mixture containing 10 mM
D, D
ꢁ20 °C, t-BuOOH (170
lL, 5.0 M in decane, 0.85 mmol) was added,
analogues (3,4,5,6,7), 15 mM ATP, 10 mM MgCl2 and 1 mg/mL HldE
in 20 mM Tris–HCl buffer (pH 8.0), and the pH of the solution was
adjusted to 8.0 by the addition of 1 M NaOH. After incubation at
37 °C for 12 h, the mixture was briefly boiled and centrifuged to
remove protein. The supernatant was subjected to analysis by ESI
HRMS.
slowly warmed up room temperature. TLC analysis showed com-
plete conversion of starting material to a major product (hexane/
ethyl acetate 2:1, Rf = 0.34). The reaction was quenched by addition
of 0.2 mL triethylamine and filtered. The filtrate was concentrated
in vacuum and purified by silica gel flash chromatography (hex-
ane/ethyl acetate 4:1) to afford a syrup 28 (114 mg, 82%). 1H
NMR (CDCl3, 400 MHz): d 1.84–1.89 (m, 1H), 2.32–2.40 (m, 1H),
3.75 (t, J = 9.6 Hz, 1H), 3.83–3.87 (m, 2H), 3.98 (dd, J = 2.4 Hz,
J = 9.2 Hz, 1H), 4.25–4.30 (m, 2H), 4.38 (d, J = 12.0 Hz, 1H), 4.66–
4.80 (m, 6H), 4.88 (s, 1H), 4.98–5.04 (m, 5H), 7.28–7.40 (m, 30H);
Acknowledgements
This work was supported by the National Institutes of Health
(NIH grant R01 GM085267), and National Basic Research Program
of China (973 Program, No. 2012CB822102).
13C NMR (CDCl3, 100 MHz):
d 32.54 (d, JC–P = 7.4 Hz), 64.42
(d, JC–P = 5.8 Hz), 67.89, 68.79, 69.17, 69.23, 72.25, 72.93, 74.68,
75.36, 78.67, 80.39, 96.99, 127.64, 127.71, 127.77, 127.78, 127.89,
127.93, 127.99, 128.07, 128.42, 128.44, 128.46, 128.56, 135.81,
135.96, 137.21, 138.19, 138.44, 138.47; ESI HRMS: m/z calcd for
Supplementary data
49H52O9P [M+H]+ 815.3344, found 815.3328.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.019.
C
4.2.23. 7-O-Phosphono-6-deoxy-glycero-D-manno-
References and notes
heptopyranosyl phosphate (6)
To a solution of 28 (100 mg, 0.123 mmol) in MeOH (4.0 mL) was
added Pd(OH)2 on carbon (100 mg). The reaction was stirred in H2
atmosphere (50 psi) for 4 h. The reaction mixture was filtered and
washed with MeOH. The filtrate was concentrated to give a syrup 6
(31 mg, 92%). 1H NMR (D2O, 400 MHz): 1.68–1.74 (m, 1H), 2.13–
2.20 (m, 1H), 3.31–3.38 (m, 1H), 3.42–3.47 (m, 1H), 3.72–3.86
(m, 2H), 3.99–4.04 (m, 2H), 5.05 (s, 1H); 13C NMR (D2O,
100 MHz): d 31.60 (d, JC–P = 7.4 Hz), 62.96 (d, JC–P = 5.2 Hz), 68.12,
70.36, 71.09, 72.93, 93.95; HRMS: m/z calcd for C7H14O9P [MꢁH]ꢁ
273.0381, found 273.0380.
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