Synthesis and evaluation of glucosamine-6-phosphate analogues as activators of glmS riboswitch

Article abstract of DOI:10.1016/j.tet.2012.09.015

The glmS riboswitch is a ribozyme found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). Given the importance of GlcN6P for cell wall biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics. Herein, we describe the efficient synthesis of three GlcN6P analogues and their evaluation on inducing self-cleavage of the glmS riboswitch from Bacillus subtilis. Our results provide valuable information for further elucidation of the structure-activity relationships and drug design for glmS riboswitch antibiotics.

Full text of DOI:10.1016/j.tet.2012.09.015

Tetrahedron 68 (2012) 9405e9412
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and evaluation of glucosamine-6-phosphate analogues as activators
of glmS riboswitch
,
,
,
*
Guan-Nan Wang a, Pui Sai Lau b c, Yingfu Li b c, Xin-Shan Ye a
a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Rd No. 38, Beijing 100191, China
b Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada
c Department of Chemistry and Chemical Biology, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2012
Accepted 4 September 2012
Available online 10 September 2012
The glmS riboswitch is a ribozyme found in numerous Gram-positive bacteria and responds to the cel-
lular concentrations of glucosamine 6-phosphate (GlcN6P). Given the importance of GlcN6P for cell wall
biosynthesis, the glmS riboswitch has become a new drug target for the development of antibiotics.
Herein, we describe the efficient synthesis of three GlcN6P analogues and their evaluation on inducing
self-cleavage of the glmS riboswitch from Bacillus subtilis. Our results provide valuable information for
further elucidation of the structureeactivity relationships and drug design for glmS riboswitch
antibiotics.
Keywords:
glmS Riboswitch
GlcN6P analogues
Antibiotics
Ó 2012 Elsevier Ltd. All rights reserved.
Synthesis
Carbohydrate
1. Introduction
few small molecule agonists and antagonists have been de-
veloped.16,17 Activators that have comparable or superior activity
Riboswitches are structural mRNA domains that regulate gene
expression in response to the intracellular concentration of specific
metabolites. They control essential genes in many pathogenic
bacteria, thus representing an intriguing class of RNA target for the
development of antibiotics and chemicalebiological tools.1e3 No-
tably, the glmS riboswitch, which is located upstream of the gene
encoding glucosamine-6-phosphate synthase (GlmS) in numerous
Gram-positive bacteria, is unique in that it is the first example of
a natural ribozyme that is also a riboswitch.4 As part of gene reg-
ulation, it binds to its small molecule metabolite, glucosamine-6-
phosphate (GlcN6P), which triggers self-cleavage targeting its
own mRNA.5,6 Functionally, the glmS ribozyme controls the amount
of GlmS to regulate cellular production of GlcN6P,7,4 which is an
essential metabolite for the biosynthesis of bacterial cell walls and
fungal cell wall chitin.8e11 Given the importance of GlcN6P, in-
hibition of the metabolite is lethal to microorganisms. Conse-
quently, the glmS riboswitch is emerging as a new drug target, and
small molecules that mimic GlcN6P to trigger riboswitch activity
have the potential to become antibiotics.
than natural GlcN6P are in great demand for a better understanding
of the riboswitch and paving the way to the discovery of effective
antibiotics. In this research, three GlcN6P analogues have been
designed and synthesized (Fig. 1). Carba-sugar 1 was designed to
address the role of the oxygen atom in pyranose ring for riboswitch
activation. Compounds 2 and 3 were designed to add an extra hy-
droxyl group at the C-6 position with the consideration of providing
extra binding sites around the pocket. Instead of 6-phosphate, the
6-phosphonate was introduced to the 6-position. It is known that
the PeC bond makes compounds resistant to enzymatic hydroly-
sis18,19 and has conformational preferences different from those in
phosphates.20 These analogues were subsequently investigated for
their ability to induce self-cleavage of the glmS riboswitch from
Bacillus subtilis.
Although substantial structural and mechanistic information of
the glmS riboswitch have been disclosed over recent years,5,6,12e15
* Corresponding author. Tel.: þ86 10 82805736; fax: þ86 10 82802724; e-mail
Fig. 1. The structures of three GlcN6P analogues.
address: xinshan@bjmu.edu.cn (X.-S. Ye).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.09.015

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G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
2. Results and discussion
Catalytic hydrogenolysis of 7 provided the phosphated carba-sugar
1 smoothly.
The synthesis of compounds 2 and 3 started from glucosamine
The synthesis of carba-sugar 1 is shown in Scheme 1. Carbocyclic
compound
5
was prepared from methyl glucopyranoside
4
8. As shown in Scheme 2, perbenzylation of 8 provided compound
9, and selective acetolysis of 9 afforded the 6-acetylated compound
10 in good yield. It was reported that the anomeric position is
usually acetylated prior to the 6-position in substrates, such as
perbenzylated glucose, galactose and mannose.23,24 However, in
the case of compound 9, the dibenzylated amino group protected
the anomeric position from acetolysis. Removal of the acetyl group
in 10 led to compound 11, which was oxidized by DesseMartin
periodinane followed by the Pudovik reaction25,26 with dibenzyl
according to the procedures described by Barton et al.21 Compound
5 was treated with dibenzyl N,N-diisopropylphosphoramidite, fol-
lowed by oxidation with meta-chloroperoxybenzoic acid (m-CPBA)
to give the fully protected phosphate 6 in 86% isolated yield. The
chemical shift of compound 6 in 31P NMR spectroscopy is at
d 3.14,
indicating the formation of phosphate.22 Subsequently, basic
opening of the oxazolidinone in 6 also resulted in the removal of
one of the benzyl groups on phosphate, yielding compound 7.
Scheme 1. Synthesis of carba-sugar 1.
Scheme 2. Synthesis of GlcN6P analogues 2 and 3.

G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
9407
phosphonate and triethylamine to yield the
a
-hydrox-
H-5 proton of the major diastereomer 14a was downfield
yphosphonate 12 in 93% isolated yield as an inseparable mixture of
diastereoisomers (3:1). The separation of two diastereoisomers was
achieved after benzoylation of the nascent hydroxyl groups.
Deprotection of the benzoyl groups with methyl amine solution,
which was followed by deprotection of the benzyl groups using
catalytic hydrogenolysis, afforded the target compounds 2 and 3 in
good yields, respectively.
(
d
¼3.93 ppm) relative to that of the minor diastereomer 14b
(d
¼3.56 ppm). These confirmed that the configuration of major
product 12a is S and the configuration of minor product 12b is R.
The Pudovik reaction, one of the most versatile methods for
construction of CeP bonds, involves the addition of organophos-
phorus compounds to unsaturated bonds. With the potential to
synthesize biologically important
a-hydroxyphosphonic acids
The absolute configurations of diastereoisomers 12a and 12b
were assigned by the Mosher method (also known as Mosher ester
analysis).27e29 As displayed in Scheme 3, each of the diastereomeric
S- and R-MTPA esters of major diastereomer 12a was prepared from
(R)-MTPAeCl and (S)-MTPAeCl, respectively (S-Mosher acid chlo-
ride gives rise to the R-Mosher ester due to relative priority: CF3 is
lower than COCl but higher than COOR). The H-5 proton signal of
each diastereomeric Mosher ester was assigned by 1He1H COSY
experiments. The chemical shift of H-5 in S-MTPA ester is dS¼3.93,
while the chemical shift of H-5 in R-MTPA ester is dR¼3.85. That is,
derivatives,31e33 this reaction has received more and more atten-
tion to make the process highly enantioselective.34,35 The examples
presented herein showed the versatility of the Mosher method as
a convenient and alternative approach to the assignment of ste-
reochemistry of Pudovik products especially when the crystalliza-
tion of products is impossible. It is applicable to either a single
enantiomer or a pair of enantiomers.
The synthetic compounds 1, 2 and 3 were subsequently in-
vestigated for their ability to induce self-cleavage of 50-32P-la-
belled glmS riboswitch from B. subtilis. As shown in Table 1 and
Fig. 4, carba-sugar 1 showed moderate activation of self-cleavage
of glmS riboswitch from B. subtilis. The percentage of riboswitch
self-cleavage induced by compound 1 is around half to that by
natural metabolite GlcN6P. At the same time during the prepa-
ration of this manuscript, Wittmann and Mayer et al. reported
the DdSR
¼dSꢀdR¼3.93ꢀ3.85¼0.08 was positive. According to the
Mosher empirical rule, the absolute configuration of the 6-position
in compound 12a was S-. This could be explained by the MTPA
plane figures (Fig. 2). The H-5 of R-MTPA ester, in which H-5 and
phenyl group reside are on the same side of the MTPA plane, is
relatively more shielded (upfield in its spectrum) than H-5 in the S-
MTPA ester due to the magnetic shielding effect of phenyl group.
Correspondingly, the minor diastereomer was assigned as R-
configuration.
their research of carba-sugar
1
on glmS riboswitch of
vancomycin-resistant Staphylococcus aureus.36 In their study, it
was found that the potency of compound 1 for glmS riboswitch of
S. aureus is similar to that of GlcN6P. They also mentioned the
Scheme 3. Synthesis of R- and S-Mosher esters from compound 12a.
Fig. 2. The MTPA plane of S- and R-MTPA esters.
To further confirm the assignment of the absolute configura-
activation by compound 1 in the glmS riboswitch of B. subtilis.
The activation achieved by compound 1 in our experiments
compared favourably with the data reported by them. It seems
that different sources of glmS ribozyme might have different
tions of 12a and 12b, the S-MTPA esters (14a and 14b) generated
from both diastereoisomers 12a and 12b were compared
(Fig. 3).27,30 The 1H NMR analysis of both products showed that the
Fig. 3. The MTPA plane of S-MTPA esters 14a and 14b made from 12a and 12b.

9408
G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
Table 1
moderate self-cleavage of the glmS riboswitch from B. subtilis. The
displacement of phosphate in GlcN6P by -hydroxyphosphonate led
Effect of synthetic compounds on glmS riboswitch
a
to massive loss of activation. These results highlight the role of
phosphate moiety in contributing to the binding of the glmS ribos-
witch. These insights are useful for further development of chemical
agonists or antagonists for the glmS riboswitch. It seems that glmS
riboswitch exhibits high level of discrimination against many closely
Sample
% Cleavage
1 min
2 min
No reaction (NR)
d
d
No magnesium (-Mg2þ
)
d
d
GlcN6P
67.50
29.08
0
65.12
38.68
0.37
0.29
1
2
3
related GlcN6P analogues. Our results provide
a deeper un-
derstanding of the glmS riboswitch and further elucidation on the
structureeactivity relationships of metabolite derivatives.
0
Fig. 4. Cleavage of glmS RNA with and without compounds. (a) Cleavage of glmS RNA after 1 min reaction time; (b) Cleavage of glmS RNA after 2 min reaction time. A 200 mM
concentration of GlcN6P or a test compound was added to 0.5 pmol of 50-32P labelled glmS RNA. Samples were incubated in 50 mM of HEPES (pH 7.5), 10 mM of MgCl2, 200 mM of
KCl at room temperature for a duration of 1 or 2 min. Negative control samples included: i) addition of double-distilled water instead of compound, or ii) addition of GlcN6P, without
Mg2þ in the reaction buffer. RNA products were separated by 10% denaturing polyacrylamide gel electrophoresis (PAGE).
sensitivities to GlcN6P analogues. On the other hand, the
6-phosphonate derivatives 2 and 3 did not exhibit activity under
the tested assay conditions.
4. Experimental section
4.1. General procedures
It was determined that the oxygen ring and phosphate group
are required for stabilization of the interaction of the riboswitch
with GlcN6P rather than being involved in the catalytic
reaction.37,38 The results herein show that the destabilization of
the interaction can tolerate carba-sugar to some extent, whereas
the 6-phosphate group is critical for interaction.16,38 The phos-
phate moiety forms the fixed network of Mg2þ-coordinated
interactions with the various functional groups of the ligand
Air- and/or moisture-sensitive reactions were carried out under
an atmosphere of argon using flame-dried glassware and standard
syringe/septa techniques. All chemicals were purchased as reagent
grade and used without further purification, unless otherwise noted.
Dichloromethane (CH2Cl2) and pyridine were distilled over calcium
hydride (CaH2). Methanol was distilled from magnesium. DMF was
stirred with CaH2 and distilled under reduced pressure. Tetrahy-
drofuran (THF) was distilled over sodium/benzophenone. Reactions
were monitored by analytical thin-layer chromatography on silica
gel 60 F254 precoated on aluminium plates (E. Merck). Spots were
detected under UV (254 nm) and/or by staining with acidic ceric
ammonium molybdate. Column chromatography was performed on
silica gel (200e300 mesh). 1H NMR, 13C NMR, and 31P NMR spectra
were recorded on a JEOL AL-300, or Varian INOVA-500 spectrome-
ters at 25 ꢁC. Chemical shifts (in parts per million) were referenced to
binding pocket.15 The electrical properties of
a-hydroxyl-
phosphonate are slightly different from 6-phosphate. The change
in electrical properties of 6-phosphonate might change the co-
ordination with cations and further change the interaction with
the binding pocket.13 The other factor may rely on the unfilled
space within the ligand binding pocket in the absence of the
bridging oxygen.
3. Conclusions
tetramethylsilane (
d
¼0 ppm) in deuterated chloroform. 13C NMR
spectra were obtained by using the same NMR spectrometers and
Three GlcN6P analogues have been prepared to investigate their
effect on the glmS riboswitch. The Pudovik reaction was applied to
were calibrated with CDCl3
(
d
¼77.00 ppm) or CD3OD
(d
¼49.00 ppm). 31P NMR spectra were reported parts per million (
d)
synthesize the
rations of two diastereomeric products were assigned by the Mosher
method. Among the three analogues, carba-sugar induced
a-hydroxyphosphonate substrates and the configu-
relative to H3PO4 (0.00 ppm) as an internal reference. Mass spectra
were recorded using a PE SCIEX QSTAR spectrometer. Elemental
analysis data were recorded on a Vario EL-Z elemental analyser.
1

G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
9409
4.2. Dibenzyl((3aS,4R,5R,6R,7aS)-3-benzyl-4,5-bis(benzyloxy)-
2-oxooctahydrobenzo[d]oxazol-6-yl)methyl phosphate (6)
(121.5 MHz, D2O)
258.0737; found, 258.0733.
d
2.84; HRMS: calcd for C7H16NO7P Na [MþNa]þ,
To a solution of dibenzyl N,N-diisopropylphosphoramidite (90%,
4.5. (3R,4R,5S,6R)-N,N-Dibenzyl-2,4,5-tris(benzyloxy)-6-(ben-
zyloxymethyl)tetrahydro-2H-pyran-3-amine (9)
111
(42.5 mg, 607
for 15 min under argon atmosphere, and then the CH2Cl2 solution
(2 mL) of compound 5 (57.0 mg, 120 mol) was added. The mixture
mL, 300
mmol) in CH2Cl2 (10 mL) was added 1H-tetrazole
m
mol). The mixture was stirred at room temperature
To a suspension of D-glucosamine (5.0 g, 27.9 mmol) in anhy-
m
drous DMF (70 mL) was added NaH (3.7 g, 60% dispersion in min-
eral oil, 92.5 mmol) at 0 ꢁC. Benzyl bromide (8.1 mL, 66.6 mmol)
was added dropwise over 5 min. After stirring at room temperature
for 1 h, same quantities of NaH and benzyl bromide were added at
was stirred for another 4 h at room temperature under argon at-
mosphere. The mixture was cooled to 0 ꢁC and m-CPBA (70%,
89.0 mg, 361 mmol) was added. After stirring for 45 min, the mix-
ture was diluted with EtOAc (50 mL). The solution was washed with
aqueous 10% Na2SO3 (2ꢂ20 mL), 1 M HCl (2ꢂ20 mL), saturated
NaHCO3 (2ꢂ20 mL), and brine (3ꢂ20 mL). The organic layer was
dried (MgSO4) and concentrated. Purification by column chroma-
tography (petroleum ethereethyl acetate, 1:1) gave the phosphate
triester 6 as an amorphous solid (76.0 mg, 86% yield). 1H NMR
0
ꢁC. After stirring for another 1 h, the third portion of NaH (3.8 g,
95.0 mmol) and benzyl bromide (8.2 mL, 67.6 mmol) were added
consecutively at 0 ꢁC and then the mixture was warmed to room
temperature. The reaction mixture was stirred overnight, and then
MeOH was added slowly to quench the excess of the NaH. DMF was
removed under reduced pressure. The residue was dissolved in
CH2Cl2 (150 mL) and washed with water (20 mL) and brine (20 mL).
The organic layer was dried (Na2SO4), filtered, and evaporated to
give a yellow oil. The residue was purified by column chromatog-
raphy on silica gel (petroleum ethereethyl acetate, 20:1 to 6:1) to
give 9 as a colourless oil (17.8 g, 89% yield) as a mixture of anomers
(300 MHz, CDCl3)
d
1.65e1.73 (m, 1H), 1.89e1.94 (d, J¼14.4 Hz, 1H),
2.04 (brs, 1H), 3.41e3.43 (m, 1H), 3.56e3.59 (m, 1H), 3.67 (brs, 1H),
3.84e3.95 (m, 2H), 4.14 (brs, 1H), 4.40 (d, J¼11.7 Hz, 1H), 4.49 (s,
2H), 4.54 (brs, 1H), 4.66 (d, J¼12.3 Hz, 1H), 4.83 (d, J¼15.3 Hz, 1H),
5.02 (brs, 4H), 7.07e7.32 (m, 25H); 13C NMR (75 MHz, CDCl3)
d 26.8,
35.7 (d, JPC¼8.0 Hz), 46.3, 55.9, 68.1 (d, JPC¼5.6 Hz), 69.5 (d,
JPC¼3.8 Hz), 72.6, 72.9, 73.3, 77.2 (d, JPC¼4.4 Hz), 77.8, 127.6, 127.7,
127.9, 128.0, 128.2, 128.5, 128.6, 128.8, 135.5, 135.6, 137.4, 137.6,
(
a
/
b
¼1:8). 1H NMR (300 MHz, CDCl3)
d
3.03 (t, J¼8.4 Hz, 1H), 3.45
(brs, 1H), 3.54e3.86 (m, 8H), 3.92 (d, J¼14.1 Hz, 2H), 4.04e4.73 (m,
7H), 4.84 (d, J¼10.8 Hz,1H), 4.98 (t, J¼10.8 Hz, 2H), 5.19 (d, J¼2.7 Hz,
158.0; 31P NMR (121.5 MHz, CDCl3)
d
3.14. HRMS: calcd for
0.1H, H-1
), 55.2 (
70.8 ( ), 71.4 (
), 79.2 ( ), 79.9 (
a
), 7.08e7.48 (m, 35H); 13C NMR (75 MHz, CDCl3)
d
54.8
), 70.4 ( ),
), 74.7 ( ), 74.8
), 103.6 ( ), 126.7,
C43H44NO8P Na [MþNa]þ, 756.2702; found, 756.2704.
(b
a
), 55.9 (
), 72.2 (
), 80.0 (
a
), 63.4 (
), 73.3 (
), 81.3 (
b
), 69.2 (
), 73.4 (
), 100.5 (
b
), 69.6 (
a
), 70.3 (
b
a
a
a
a
a
b
), 74.3 (
b
b
(b
b
a
a
b
b
a
4.3. Sodium benzyl((1R,2R,3R,4S,5S)-4-(benzylamino)-2,3-
bis(benzyloxy)-5-hydroxy cyclohexyl)methyl phosphate (7)
127.0, 127.2, 127.6, 127.8, 128.0, 128.2, 128.3, 128.4, 128.6, 128.8,
137.5, 138.1, 139.0, 139.2, 139.7; Anal. Calcd for C48H49NO5: C, 80.08;
H, 6.86; N, 1.95; found: C, 79.83; H, 6.80; N, 1.84; HRMS: calcd for
C48H50NO5 [MþH]þ, 720.3684; found, 720.3673.
A solution of compound 6 (100.0 mg, 0.14 mmol) in ethanolic-
sodium hydroxide solution (10%) (v/v¼1:1, 5 mL) was heated un-
der reflux overnight. The solvents were removed and the residue
was purified by C-18 reversed-phase column chromatography (el-
uent: H2O to CH3OH/H2O, 1:1) to give 7 (80.0 mg, 92% yield) as an
4.6. ((2R,3S,4R,5R,6R)-3,4,6-Tris(benzyloxy)-5-(dibenzyl-
amino)tetrahydro-2H-pyran-2-yl)methyl acetate (10)
amorphous solid. 1H NMR (300 MHz, CD3OD)
d
1.54 (t, J¼12.6 Hz,
To a solution of compound 9 (3.0 g, 4.2 mmol) in acetic anhy-
dride and acetic acid (20 mL, v:v¼1:1), the 1.5% H2SO4 solution in
acetic anhydride (1.0 mL) was added dropwise. The mixture was
stirred at 60 ꢁC for 5 h under argon. Then the mixture was cooled
down to room temperature and poured to ice water (80 mL). So-
dium bicarbonate was added to the stirred solution in portions until
no bubble occurred. The solution was extracted with CH2Cl2
(100 mLꢂ3) and the combined organic phase was dried over
Na2SO4. The solvent was evaporated under reduced pressure and
the residue was purified by column chromatography on silica gel
(petroleum ethereethyl acetate, 6:1 to 4:1) to give 10 as a colour-
1H), 1.87e1.91 (m, 1H), 2.11 (brs, 1H), 2.51 (dd, J¼2.4, 9.9 Hz, 1H),
3.46 (t, J¼10.2 Hz, 1H), 3.63 (t, J¼9.6 Hz, 1H), 3.65 (d, J¼13.2 Hz, 1H),
3.85 (d, J¼13.2 Hz, 1H), 3.92e3.96 (m, 1H), 4.06e4.07 (m, 1H),
4.12e4.18 (m, 1H), 4.63 (d, J¼11.4 Hz, 1H), 4.73e4.90 (m, 5H),
7.18e7.35 (m, 20H); 13C NMR (75 MHz, CD3OD)
d 32.9, 39.2 (d,
JPC¼8.6 Hz), 51.9, 63.9, 65.8, 66.4 (d, JPC¼5.6 Hz), 68.1 (d,
JPC¼5.6 Hz), 75.8, 75.9, 83.1, 83.7, 128.15, 128.2, 128.4, 128.5, 128.6,
128.8, 128.9, 129.3, 129.4, 139.8, 139.9, 140.1 (d, JPC¼3.2 Hz), 140.2,
141.2; 31P NMR (121.5 MHz, CDCl3)
d 2.69; HRMS: calcd for
C35H40NO7PNa[MþNa]þ, 640.2440; found, 640.2440.
less oil (2.66 g, 95% yield) as major
CDCl3)
b
-anomer. 1H NMR (300 MHz,
2.03 (s, 3H), 3.03 (t, J¼8.4 Hz, 1H), 3.51e3.57 (m, 2H),
4.4. ((1R,2R,3R,4S,5S)-4-Amino-2,3,5-trihydroxycyclohexyl)-
methyl dihydrogen phosphate (1)
d
3.75e3.79 (m, 3H), 3.90e3.94 (m, 2H), 4.22 (dd, J¼4.2, 11.7 Hz, 1H),
4.35 (d, J¼11.7 Hz, 1H), 4.48 (d, J¼10.8 Hz, 1H), 4.58e4.67 (m, 2H),
4.76 (d, J¼10.8 Hz, 1H), 4.84 (d, J¼11.1 Hz, 1H), 4.93 (d, J¼11.4 Hz,
1H), 5.03 (d, J¼11.1 Hz, 1H), 7.12e7.48 (m, 25H); 13C NMR (75 MHz,
A methanolic solution (8 mL) of compound 7 (50.0 mg,
0.078 mmol) was stirred at room temperature in the presence of
10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 48 h. The
catalyst was then removed by filtration through Celite, and the
filtrate was concentrated. In most cases, direct lyophilization gave
the pure compound 1 (19.5 mg, 97% yield) as white amorphous
solids. In the cases when further purification was needed, the water
solution of residue was adjusted to pH¼2 by 1 N HCl, and purified
by Dowex 50Wꢂ8 (Hþ form, 200e400 mesh, eluent: H2O) to give 1
as white amorphous solids after lyophilization. 1H NMR (300 MHz,
CDCl3) d 20.8, 54.6, 63.2, 63.4, 70.6, 72.9, 74.4, 74.7, 78.8, 81.1, 100.2,
126.7, 127.2, 127.3, 127.8, 127.9, 128.0, 128.2, 128.3, 128.5, 128.7, 137.1,
137.7, 138.7, 139.5, 170.7; Anal. Calcd for C43H45NO6: C, 76.87; H,
6.75; N, 2.08; found: C, 76.60; H, 6.78; N, 1.87; HRMS: calcd for
C43H46NO6 [MþH]þ, 672.3320; found, 672.3331.
4.7. ((2R,3S,4R,5R,6R)-3,4,6-Tris(benzyloxy)-5-(dibenzyl-
amino)tetrahydro-2H-pyran-2-yl)methanol (11)
D2O)
d
1.43 (t, J¼13.5 Hz, 1H), 1.71e1.80 (m, 2H), 2.96 (d, J¼11.1 Hz,
1H), 3.17 (t, J¼9.9 Hz, 1H), 3.48 (t, J¼10.5 Hz, 1H), 3.77e3.82 (m, 1H),
Compound 10 (2.5 g, 3.7 mmol) was dissolved in methanol
(20 mL), to which NaOMe (1 M solution in methanol, 1.0 mL,
1.0 mmol) was added and the resulting solution was stirred for 1 h
3.89e3.95 (m, 1H), 4.03 (s, 1H); 13C NMR (75 MHz, D2O)
d 31.7, 37.4
(d, JPC¼7.4 Hz), 56.9, 65.6, 66.9 (d, JPC¼5.0 Hz), 71.7, 72.6; 31P NMR

9410
G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
at room temperature. Dowex 50w Hþ resin was added to neutralize
the solution (pH¼7), after which the resin was removed by filtra-
tion and washed with ethyl acetate. The solvent was removed to
afford 11 (2.3 g, quantitative) as an amorphous solid without fur-
JPC¼6.2 Hz), 136.9, 138.2, 138.9, 139.9, 164.8 (d, JPC¼6.2 Hz); 31P
NMR (121.5 MHz, CDCl3)
d 20.70; HRMS: calcd for C62H61NO9P
[MþH]þ, 994.4078; found, 994.4079.
ther purification. 1H NMR (300 MHz, CDCl3)
d
1.87 (t, J¼6.6 Hz, 1H),
4.9. Dibenzyl (S)-hydroxy((2S,3S,4R,5R,6R)-3,4,6-
tris(benzyloxy)-5-(dibenzylamino) tetrahydro-2H-pyran-2-yl)-
methylphosphonate (12a)
2.98 (t, J¼9.6 Hz, 1H), 3.30e3.34 (m, 1H), 3.54 (t, J¼9.3 Hz, 1H),
3.64e3.71 (m, 1H), 3.77e3.82 (m, 4H), 3.93 (d, J¼13.8 Hz, 2H),
4.55e4.69 (m, 3H), 4.76 (d, J¼10.8 Hz, 1H), 4.86e4.93 (ABq,
J¼11.4 Hz, 2H), 5.01 (d, J¼11.1 Hz, 1H), 7.18e7.48 (m, 25H); 13C NMR
Compound 13a (100.0 mg, 0.1 mmol) was treated with 33%
methyl amine solution in ethanol (10 mL) at 0 ꢁC for 20 min. Then
the mixture was diluted with dichloromethane. The mixture was
concentrated and the residue was purified by column chroma-
tography on silica gel (petroleum ethereethyl acetate, 5:1) to af-
ford compound 12a (82.0 mg, 92% yield) as a colourless oil. 1H
(75 MHz, CDCl3)
d 54.9, 62.3, 63.3, 70.9, 74.3, 74.8, 75.1, 79.2, 81.0,
101.0, 126.8, 127.3, 127.8, 127.9, 128.1, 128.3, 128.4, 128.8, 137.3, 137.9,
138.9, 139.7; Anal. Calcd for C41H43NO5: C, 78.19; H, 6.88; N, 2.22;
found: C, 77.89; H, 6.91; N, 2.05; HRMS: calcd for C41H44NO5
[MþH]þ, 630.3219; found, 630.3223.
NMR (300 MHz, CDCl3)
d 2.87e2.98 (m, 2H), 3.68e3.75 (m, 4H),
4.8. (Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)-3,4,6-
tris(benzyloxy)-5(dibenzylamino) tetrahydro-2H-pyran-2-yl)-
methyl benzoate (12)
3.86 (d, J¼14.1 Hz, 2H), 4.21 (t, J¼11.1 Hz, 1H), 4.46 (d, J¼11.7 Hz,
1H), 4.58e4.66 (m, 1H), 4.65 (d, J¼8.4 Hz, 1H), 4.75e4.86 (m, 3H),
4.98 (d, J¼11.1 Hz, 1H), 5.05e5.09 (m, 4H), 7.16e7.38 (m, 35H); 13C
NMR (75 MHz, CDCl3)
d
54.6, 63.0, 66.6 (d, JPC¼159.5 Hz), 67.9 (d.
A solution of 11 (60.0 mg, 0.10 mmol) in dichloromethane
(5 mL) was added DesseMartin periodinane (65.0 mg, 0.15 mmol).
After stirring at room temperature for 2 h, the reaction was
quenched by adding saturated aqueous solutions of NaHCO3
(5 mL) and Na2S2O3 (5 mL). The mixture was diluted with diethyl
ether (30 mL) and stirred at room temperature for 30 min. The
aqueous layer was extracted with diethyl ether (20 mLꢂ3); the
organic layer was dried over Na2SO4 and concentrated under re-
duced pressure. The residue was dissolved in dichloromethane
JPC¼6.8 Hz), 68.1 (d, JPC¼6.8 Hz), 70.3, 73.6, 74.4, 74.7, 77.7 (d,
JPC¼9.9 Hz), 80.9, 100.6, 126.7, 127.2, 127.7, 127.9, 128.0, 128.2,
128.3, 128.4, 128.6, 128.7, 136.0 (d, JPC¼5.0 Hz), 136.1 (d,
JPC¼5.0 Hz), 137.1, 138.1, 138.8, 139.6; 31P NMR (121.5 MHz, CDCl3)
d
25.57; HRMS: calcd for C55H57NO8P [MþH]þ, 890.3816; found,
890.3834.
4.10. Dibenzyl (R)-hydroxy((2S,3S,4R,5R,6R)-3,4,6-
tris(benzyloxy)-5-(dibenzylamino) tetrahydro-2H-pyran-2-yl)-
methylphosphonate (12b)
(3 mL), triethylamine (0.1 mL) and dibenzyl phosphonate (25.0 mL,
0.15 mmol) were added. After stirring for 24 h at room tempera-
ture under argon, the reaction mixture was concentrated under
reduced pressure. Purification by column chromatography on
silica gel (petroleum ethereethyl acetate, 5:1 to 4:1) followed by
Sephedex (LH20) size-exclusion chromatography afforded com-
pound 12 (79.0 mg, 93% yield) as a colourless oil, which is a mix-
ture of diastereomers (3:1).
Compound 13b (50.0 mg, 0.05 mmol) was treated with 33%
methyl amine solution in ethanol (5 mL) at 0 ꢁC for 2 h. Then the
mixture was diluted with dichloromethane. The mixture was
concentrated and the residue was purified by column chroma-
tography on silica gel (petroleum ethereethyl acetate, 5:1) to af-
ford compound 12b (39.0 mg, 88% yield) as a colourless oil. 1H NMR
Benzoyl chloride (65.0
a solution of 12 (100.0 mg, 0.11 mmol) and Et3N (235.0
m
L, 0.55 mmol) was added dropwise to
L,
(300 MHz, CDCl3)
d
3.05 (t, J¼8.7 Hz, 1H), 3.38 (brs, 1H), 3.73e3.81
m
(m, 4H), 3.90 (d, J¼13.5 Hz, 2H), 4.02 (t, J¼8.1 Hz,1H), 4.26 (brs,1H),
4.56e4.73 (m, 5H), 4.87 (t, J¼11.1 Hz, 2H), 5.00e5.13 (m, 4H),
1.65 mmol) in dichloromethane (15 mL) under argon. The reaction
was stirred at room temperature for 15 h, then diluted with
dichloromethane (50 mL). The organic phase was washed by so-
dium bicarbonate solution, brine, dried over Na2SO4, and con-
centrated. The residue was purified by column chromatography on
silica gel (petroleum ethereethyl acetate, 6:1 to 3:1) to afford 13a
(79.0 mg) and 13b (24.0 mg) as colourless oil, in total yield of 93%.
7.13e7.39 (m, 35H); 13C NMR (75 MHz, CDCl3)
d 54.8, 63.0, 67.7 (d,
JPC¼6.8 Hz), 68.4 (d, JPC¼6.8 Hz), 69.0 (d, JPC¼158.3 Hz), 70.6, 73.5,
74.2, 76.2, 79.7 (d, JPC¼3.1 Hz), 81.3, 100.7, 126.7, 127.1, 127.3, 127.7,
127.8, 128.0, 128.1, 128.2, 128.4, 128.5, 128.8, 136.0 (d, JPC¼5.6 Hz),
136.4 (d, JPC¼6.2 Hz),137.2,137.8,138.5,139.6; 31P NMR (121.5 MHz,
24.38; HRMS: calcd for C55H57NO8P [MþH]þ, 890.3816;
Data for compound 13a: 1H NMR (300 MHz, CDCl3)
d 3.06 (t,
CDCl3)
found, 890.3823.
d
J¼8.4 Hz, 1H), 3.51 (t, J¼9.0 Hz, 1H), 3.74e3.83 (m, 3H), 3.88e4.00
(m, 3H), 4.30 (d, J¼9.9 Hz, 1H), 4.66 (d, J¼10.8 Hz, 2H), 4.77e4.82
(m, 2H), 4.91 (d, J¼12.0 Hz, 1H), 5.06e5.16 (m, 5H), 6.18 (d,
J¼11.4 Hz, 1H), 7.18e7.59 (m, 38H), 8.06 (d, J¼8.1 Hz, 2H); 13C NMR
4.11. (1S)-((2S,3S,4R,5R)-5-Amino-3,4,6-trihydroxytetrahydro-
2H-pyran-2-yl)(hydroxy) methylphosphonic acid (2)
(75 MHz, CDCl3)
d
54.5, 63.5, 66.4 (d, JPC¼165.1 Hz), 68.1 (d,
JPC¼6.2 Hz), 68.5 (d, JPC¼6.8 Hz), 70.4, 73.1, 74.7 (d, JPC¼5.0 Hz),
77.4, 77.6, 82.0, 100.9, 126.6, 127.1, 127.3, 127.7, 128.0, 128.1, 128.2,
128.3, 128.4, 128.5, 128.9, 130.0, 130.2, 133.6, 135.8 (d, JPC¼6.2 Hz),
137.3, 137.8, 138.6, 139.6, 165.1; 31P NMR (121.5 MHz, CDCl3)
A methanolic solution (3 mL) of compound 12a (50.0 mg,
0.056 mmol) was stirred at room temperature in the presence of
10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 2 days. The
catalyst was then removed by filtration through Celite, and the
filtrate was concentrated. The residue was purified by C-18
reversed-phase column chromatography (eluent: H2O) to give
d
21.14; HRMS: calcd for C62H61NO9P [MþH]þ, 994.4078; found,
994.4093. Data for compound 13b: 1H NMR (300 MHz, CDCl3)
d
3.08 (dd, J¼8.1, 9.6 Hz, 1H), 3.66e3.79 (m, 2H), 3.76 (d, J¼13.8 Hz,
compound 2 (14.0 mg,
a
/
b
¼2:1, 96% yield) as white amorphous
2H), 3.89 (d, J¼13.8 Hz, 2H), 4.16 (t, J¼8.4 Hz, 1H), 4.56e4.65 (m,
2H), 4.76e4.81 (m, 3H), 4.85e5.05 (m, 4H), 5.05e5.22 (m, 2H),
6.06 (dd, J¼2.7, 11.1 Hz, 1H), 7.14e7.59 (m, 38H), 8.03e8.06 (m,
solids after lyophilization. 1H NMR (300 MHz, D2O)
d
2.85 (t,
J¼9.3 Hz, 0.5H), 3.11 (dd, J¼3.3, 10.5 Hz, 1H), 3.45e3.60 (m, 2.2H),
3.79 (t, J¼9.6 Hz, 1H), 3.88e4.00 (m, 2.8H), 4.79 (d, J¼8.7 Hz, 0.5H),
2H); 13C NMR (75 MHz, CDCl3)
d
54.7, 63.0, 67.6 (d, JPC¼6.2 Hz),
5.29 (d, J¼3.3 Hz, 1H); 13C NMR (75 MHz, D2O) data for both
a and
68.0 (d, JPC¼165.0 Hz), 68.5 (d, JPC¼5.6 Hz), 70.1, 73.9, 74.5, 75.9,
77.2 (d, JPC¼5.0 Hz), 79.5, 81.4, 100.5, 126.7, 127.0, 127.1, 127.4,
127.6, 127.7, 127.8, 128.08, 128.15, 128.26, 128.35, 128.5, 128.7,
128.8, 129.4, 129.9, 133.4, 135.9 (d, JPC¼6.2 Hz), 136.5 (d,
b
anomers:
d
54.8, 57.2, 66.5 (d, JPC¼155.8 Hz), 66.7 (d,
JPC¼155.7 Hz), 69.4, 69.6 (d, JPC¼9.2 Hz), 70.4, 71.5, 72.6, 76.0, 89.8,
93.6; 31P NMR (121.5 MHz, CDCl3)
d 19.25 (a), 19.62 (b); HRMS:
calcd for C6H15NO8P [MþH]þ, 260.0530; found, 260.0533.

G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
9411
4.12. (1R)-((2S,3S,4R,5R)-5-Amino-3,4,6-
4.16. Polymerase chain reaction
trihydroxytetrahydro-2H-pyran-2-yl)(hydroxy) methyl-
phosphonic acid (3)
The glmS DNA template has a sequence of 50-GAATT CTAAT
ACGAC TCACT ATAGG TCTTG TTCTT ATTTT CTCAA TAGGA AAAGA
AGACG GGATT ATTGC TTTAC CTATA ATTAT AGCGC CCGAA CTAAG
CGCCC GGAAA AAGGC TTAGT TGACG AGGAT GGAGG TTATC GAATT
TTCGG CGGAT GCCTC CCGGC TGAGT GTGCA GATCA CAGCC GTAAG
GATTT CTTCA AACCA AGGGG GTGAC TCCTT GAACA AAGAG AAATC
ACATG ATCTT CCAAA AAACA TGTAG GAGGG GAC-30, which was
obtained through PCR amplification of a lab plasmid, originally
derived from B. subtilis chromosomal DNA (1A40). DNA primers
were obtained through automated synthesis using standard phos-
phoramidite chemistry (Integrated DNA Technologies, Iowa, USA).
Before usage, the oligonucleotides were purified by 10% denaturing
PAGE and quantified by absorbance at 260 nm. Primers used in-
clude the forward sequence 50-GAATT CTAAT ACGAC TCACT ATAGG
TCTTG TTCTT ATT-30 (T7 RNA polymerase promoter sequence is
shown in italicized letters; the þ1 transcription initiation site and
critical nucleotides for in vitro transcription are shown in bold
letters), and the reverse sequence 50-GTCCC CTCCT ACATG TTTTT
TGG-30. PCR was conducted in 75 mM TriseHCl (pH 9.0), 2 mM
A methanolic solution (3 mL) of compound 12b (50.0 mg,
0.056 mmol) was stirred at room temperature in the presence of
10% Pd/C (5.0 mg) under 4 atm hydrogen pressure for 2 days. The
catalyst was then removed by filtration through Celite, and the
filtrate was concentrated. The residue was purified by C-18
reversed-phase column chromatography (eluent: H2O) to give
compound 3 (13.5 mg,
a/
b
¼2:1, 92% yield) as white amorphous
solids after lyophilization. 1H NMR (300 MHz, D2O)
d
2.86 (t,
J¼8.7 Hz, 0.5H), 3.15 (dd, J¼3.0, 10.2 Hz, 1H), 3.49e4.00 (m, 6H),
4.76 (d, J¼8.4 Hz, 0.5H), 5.27 (d, J¼2.7 Hz, 1H); 13C NMR (75 MHz,
D2O) data for both
a and b anomers: d 56.6, 59.1, 70.1, 71.9 (d,
JPC¼152.6 Hz), 71.8 (d, JPC¼151.4 Hz), 72.1, 72.2, 74.7, 75.1 (d,
JPC¼7.2 Hz), 79.5 (d, JPC¼8.0 Hz), 91.7, 95.4; 31P NMR (121.5 MHz,
CDCl3)
d
18.50 (
a
), 18.10 (
b
); HRMS: calcd for C6H15NO8P [MþH]þ,
260.0530; found, 260.0531.
4.13. (S)-((S)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)-3,4,6-
tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-yl)-
methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (14a)
MgCl2, 50 mM KCl, 20 mM (NH4)2SO4, each primer at 0.5 mM, DNA
plasmid at w3 nM, each dNTP at 0.5 mM, and 5 units of Taq DNA
polymerase (Biotools, Madrid, Spain). Thermal cycling steps were:
94 ꢁC for 1 min, 20 cycles of 94 ꢁC to 50 ꢁC to 72 ꢁC (30 s for each
temperature), and finally 72 ꢁC for 8 min.
(R)-(ꢀ)-
a
-Methoxy-
mol) was added dropwise to a solution of 12a (5.0 mg,
mol), Et3N (6.4 L, 45.0 mol) and 4-dimethylaminopyridine
mol) in dichloromethane (2 mL) under argon. The
a
-trifluoromethylphenylacetyl
chloride
(4.3
5.6
mL, 22.4 m
m
m
m
(1.0 mg, 8.4
m
reaction mixture was stirred at room temperature overnight. The
mixture was concentrated. The residue was purified by column
chromatography on silica gel (petroleum ethereethyl acetate, 5:1)
to afford 14a as colourless oil (5.0 mg, 80% yield). 1H NMR
4.17. RNA preparation
In vitro transcription was conducted at 37 ꢁC for 3 h in 150
mL of
40 mM TriseHCl, pH7.9, 6 mM MgCl2, 10 mM DTT, 10 mM NaCl,
2 mM spermidine, 25 pmol PCR amplified DNA, 2.5 mM each of GTP,
(300 MHz, CDCl3)
d
2.90 (dd, J¼8.1, 10.2 Hz, 1H), 3.22 (t, J¼9.0 Hz,
1H), 3.49 (s, 3H), 3.70e3.72 (m, 1H), 3.73 (d, J¼14.0 Hz, 2H), 3.88 (d,
J¼14.0 Hz, 2H), 3.93 (dd, J¼4.2, 9.3 Hz, 1H, H-5), 4.32 (d, J¼10.5 Hz,
1H), 4.47 (d, J¼11.7 Hz, 1H), 4.64e4.75 (m, 4H), 4.81 (dd, J¼7.8,
12.0 Hz, 1H), 4.91e5.05 (m, 4H), 6.02 (d, J¼11.1 Hz, 1H), 7.06e7.40
(m, 38H), 7.57 (d, J¼7.8 Hz, 2H).
CTP, UTP, ATP, 1.07 units/
mL RiboLock Ribonuclease Inhibitor and
1.33 units/ L T7 RNA polymerase (Fermentas, Canada). The tran-
m
scription mixture was then treated with DNase I (3 units) in the
presence of 0.2 mM CaCl2 at 37 ꢁC for 15 min (Fermentas, Canada).
The transcribed RNA was subsequently concentrated through
standard ethanol precipitation, purified by 10% denaturing PAGE
and then quantified by absorbance at 260 nm. Afterwards, the 50-
triphosphate of the RNA was dephosphorylated with calf intestine
alkaline phosphatase following manufacturer’s protocol (Fermen-
tas, Canada), and extracted with phenol/chloroform (1:1) (BioShop,
Canada). RNA samples were concentrated through standard etha-
4.14. (R)-((S)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)-
3,4,6-tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-
yl)methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (15a)
Compound 15a was prepared from compound 12a and (S)-
(þ)-
scribed in the preparation of compound 14a, yielding 15a (84%
yield) as a colourless oil. 1H NMR (300 MHz, CDCl3)
2.81 (t,
a
-methoxy-
a
-trifluoromethylphenylacetyl chloride as de-
nol precipitation, before labelling the 50-terminus with [ 32P]ATP
g-
(Perkin Elmer, Massachusetts, USA) using T4 polynucleotide kinase
according to manufacturer’s protocol (Fermentas, Canada). 50-32P
labelled RNA was purified again by 10% denaturing PAGE before
usage.
d
J¼9.0 Hz, 1H), 3.05 (t, J¼9.0 Hz, 1H), 3.43 (s, 3H), 3.65e3.73 (m,
3H), 3.83e3.87 (m, 3H, H-5 and NeCH2), 4.26 (d, J¼10.2 Hz, 1H),
4.44 (d, J¼12.0 Hz, 1H), 4.58e4.68 (m, 4H), 4.93e5.10 (m, 5H),
5.95 (d, J¼11.4 Hz, 1H), 7.09e7.41 (m, 38H), 7.57 (d,
J¼7.5 Hz, 2H).
4.18. Ribozyme cleavage assays
4.15. (S)-((R)-(Bis(benzyloxy)phosphoryl)((2S,3S,4R,5R,6R)-
3,4,6-tris(benzyloxy)-5-(dibenzylamino)tetrahydro-2H-pyran-2-
yl)methyl)3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (14b)
A 200 mM concentration of glucosamine-6-phosphate (GlcN6P)
(Sigma, Oakville, Canada), or a test compound (1, 2 or 3) was added
to 0.5 pmol 50-32P labelled glmS RNA. Samples were incubated in
50 mM HEPES (pH7.5), 10 mM MgCl2, 200 mM KCl at room tem-
perature for a duration of 1 or 2 min. Negative control samples
included: i) addition of double-distilled water instead of com-
pound, or ii) addition of GlcN6P, without Mg2þ in the reaction
buffer. After the testing period, reactions were terminated by the
addition of loading dye solution containing 100 mM EDTA and 7 M
urea (Sigma, Oakville, Canada). RNA products were separated by
10% denaturing PAGE and visualized using a PhosphorImager and
ImageQuant software. Percent cleavage for each reaction was cal-
culated using the following equation:
Compound 14b was prepared from compound 12b and (R)-
(ꢀ)-
scribed in the preparation of compound 14a, yielding 14b (85%
yield) as a colourless oil. 1H NMR (300 MHz, CDCl3)
2.99 (t,
a-methoxy-a-trifluoromethylphenylacetyl chloride as de-
d
J¼8.7 Hz, 1H), 3.46 (s, 3H), 3.56 (d, J¼10.8 Hz, 1H, H-5), 3.70e3.76
(m, 3H), 3.88 (d, J¼13.5 Hz, 2H), 4.12 (t, J¼9.3 Hz, 1H), 4.27 (d,
J¼11.4 Hz, 1H), 4.40 (d, J¼8.1 Hz, 1H), 4.56 (d, J¼12.0 Hz, 1H),
4.71e5.09 (m, 8H), 6.00 (d, J¼11.7 Hz, 1H), 7.18e7.39 (m, 38H), 7.54
(d, J¼7.8 Hz, 2H).

9412
G.-N. Wang et al. / Tetrahedron 68 (2012) 9405e9412
ꢀ
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ꢁ
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This work was financially supported by the National Natural
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Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2012.09.015.
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