Iminosugar C-glycoside analogues of α-D-GlcNAc-1-phosphate: Synthesis and bacterial transglycosylase inhibition

Article abstract of DOI:10.1021/jo501340s

We herein describe the first synthesis of iminosugar C-glycosides of α-D-GlcNAc-1-phosphate in 10 steps starting from unprotected D-GlcNAc. A diastereoselective intramolecular iodoamination.cyclization as the key step was employed to construct the central piperidine ring of the iminosugar and the C-glycosidic structure of α-D-GlcNAc. Finally, the iminosugar phosphonate and its elongated phosphate analogue were accessed. These phosphorus-containing iminosugars were coupled efficiently with lipophilic monophosphates to give lipid-linked pyrophosphate derivatives, which are lipid II mimetics endowed with potent inhibitory properties toward bacterial transglycosylases (TGase).

Full text of DOI:10.1021/jo501340s

Article
pubs.acs.org/joc
Open Access on 08/19/2015
Iminosugar C‑Glycoside Analogues of α‑D‑GlcNAc-1-Phosphate:
Synthesis and Bacterial Transglycosylase Inhibition
†
,§,∥
‡,§,⊥
‡,#
†
†
Che-Hsiung Hsu,
Mathias Schelwies,†
Sebastian Enck, Lin-Ya Huang, Shih-Hsien Huang,
†
†
,†,‡
Yi-Fan Chang, Ting-Jen Rachel Cheng, Wei-Chieh Cheng, and Chi-Huey Wong*
†
Genomics Research Center, Academica Sinica, Taipei 115, Taiwan
‡
Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States
*
S Supporting Information
ABSTRACT: We herein describe the first synthesis of iminosugar C-glycosides of α-D-GlcNAc-1-phosphate in 10 steps starting
from unprotected D-GlcNAc. A diastereoselective intramolecular iodoamination−cyclization as the key step was employed to
construct the central piperidine ring of the iminosugar and the C-glycosidic structure of α-D-GlcNAc. Finally, the iminosugar
phosphonate and its elongated phosphate analogue were accessed. These phosphorus-containing iminosugars were coupled
efficiently with lipophilic monophosphates to give lipid-linked pyrophosphate derivatives, which are lipid II mimetics endowed
with potent inhibitory properties toward bacterial transglycosylases (TGase).
INTRODUCTION
potent substrate-based inhibitors that mimic the transition state
10
■
of TGase transglycosylation (Scheme 1). However, the idea
has been tested only with pyrrolidine mimics that were
Iminosugars are central structural motifs of small molecules that
act by inhibition of glycosidases or glycosyltransferases. Their
1
11
discovered by a combinatorial approach. Six-membered
strong inhibitory activity is attributed to electrostatic binding
interactions of the protonated nitrogen under physiological
conditions and to their “oxocarbenium-ion-like” structure that
mimics the transition state of the formation/cleavage of the
iminosugars would better represent the natural N-acetylglucos-
amine hydroxyl configurations, but they have yet to be studied.
Herein, we report structural iminosugar C-glycosidic lipid II
analogues such as compound 1 (Scheme 1), which contains a
simplified structure (e.g., shorter lipid chain and no peptide
moiety) when compared to that of lipid II but still possesses a
core structure with the potential to inhibit TGase. Also, a
molecule with a C-glycosidic linkage is expected to be stable
toward enzymatic hydrolysis. The synthesis of compound 1 is
challenging because the iminosugar α-C-glycoside of phospho-
nate analogue 2 has to be realized. Only a few examples have
been found for the synthesis of iminosugar analogues of
2
glycosidic linkage. Iminosugars have shown potential as
therapeutics toward cancer as well as viral and bacterial
infections and are used for the treatment of type 2 diabetes.
Because of the rise of infections from antibiotic-resistant
bacteria such as methicillin-resistant Staphylococcus aureus
MRSA) and vancomycin-resistant Enterococci (VRE), it is of
great interest to develop new drugs targeting the conserved
region of the bacterial cell wall. Peptidoglycan transglycosylase
TGase) is the enzyme responsible for catalyzing the
polymerization of β1,4-linked N-acetylglucosamine-N-acetyl-
muramic acid (GlcNAc-MurNAc) disaccharide of lipid II to
form the bacterial cell wall. TGases are attractive targets for
antibacterial agents because they are essential enzymes located
on the external cell surface and because no eukaryotic
counterpart is known. The only well-characterized TGase
inhibitor, moenomycin (Moe), however, is not effective in
humans because of its poor pharmacokinetic properties. From
recent structural studies, the transglycosylation mechanism
1
,3
(
4
12
(
GlcNAc. Herein, we present the synthesis of phosphonate 2
and its phosphate analogue. These compounds can be used to
efficiently construct lipid-linked pyrophosphate analogues such
as 1 that have the potential to display potent bacterial TGase
inhibition.
n
5
6
RESULTS AND DISCUSSION
■
7
To construct the iminosugar C-glycoside core of GlcNAc, we
utilized a ring-opening/cyclization strategy. Key steps are a
13
8
employed by TGase is that of a S 2-like reaction, which is
N
9
similar to other inverting glycosyltransferases (Figure 1).
Received: June 16, 2014
Published: August 19, 2014
Therefore, we expect iminosugar-based lipid II mimetics to be
©
2014 American Chemical Society
8629
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Figure 1. Transglycosylation by TGase to synthesize peptidoglycans.
Scheme 1. Iminosugar C-Glycosidic Lipid II Analogue
substituent in the 3-position leads to the formation of a
pyrrolidine when the hydroxyl group of 4 is transformed into a
good leaving group. Converting the hydroxyl group to an amine
was best completed by an oxidation/reductive amination
16
sequence. The ketone obtained by Swern oxidation was
found to be in equilibrium with N-acyl hemiaminal 5 (typical
1
3
C NMR appearance of the C-2 quaternary carbon at δ = 91
17
ppm), which was stable and could be subjected to reductive
amination directly without purification. Optimization of the
reaction conditions led to the use of excess NaBH CN/
3
NH OAc (10 equiv) at 50 °C in methanol. Under the optimal
4
reaction conditions, diaminoheptenitols 6a and 6b were
synthesized in 64% over two steps, with the (6R) diastereomer
being the main product in a ratio 6a/6b (6R/6S) = 67:33. The
ratio was determined by NMR analysis of the NH protons of 6a
and 6b acetamides in the crude product. It is noteworthy that
the use of other amine sources such as benzyl amine gave
higher yield and selectivity of the (6R) diastereomer, but the
subsequent cyclization reaction was not successful.
Wittig reaction followed by oxidation and reductive amination
leading to a diaminohepenitol, followed by an NIS-mediated
cyclization. Similar synthetic strategies were known before,
but have never been applied successfully to GlcNAc. The
synthesis of diaminoheptenitols is depicted in Scheme 2.
13
In the next step toward the synthesis of 7a, the cyclization of
a by internal iodoamination proceeds with a very high degree
Scheme 2. Synthesis of Diaminoheptenitols 6a and 6b
6
of stereoselectivity to form exclusively the α-epimer (Scheme
13b
3
).
The configuration of the stable compound 7a was
Scheme 3. Oxazine 9 for Structural Determination
14
Starting from GlcNAc, compound 3 was prepared and
subjected to optimized Wittig conditions. The obtained
determined by NMR analysis. Early attempts to determine the
two newly formed stereocenters at C-2 and C-6 on the basis of
NMR parameters of 7a failed. However, small coupling
constants and the absence of 1,3-diaxial NOE contacts
indicated that the conformation of 7a was not a chair, but it
was difficult to obtain unambiguous proof of its exact
1
5
aminoheptenitol 4
had to be transformed into the
corresponding amine to construct the piperidine ring.
Epimerization at C-6 of 4 followed by reinversion with a
nitrogen nucleophile was attempted but was found not to be
applicable to GlcNAc derivatives because the nitrogen
8
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configuration (see Figure S1). The structure of 7a was finally
determined by analyzing bicyclic oxazine 9, a side product in
the synthesis of 8 (a C2-acetamido analogue of homonojir-
imycin, HNJ), that was obtained by treating iodo derivative 7a
We are also interested in the synthesis of an analogue with an
extended phosphate group that has been found in several
2
0
glycosyltransferase inhibitors. The endocyclic secondary
amine function of 8 was initially required to be protected
because the amine group would engage in the phosphorylation
reaction, causing low yields. As a result, carboxybenzyl (Cbz)-
protected iminoheptitol 13 was prepared and transformed into
phosphate 14 in good yield by using the phosphoramidite
method (Scheme 6). The nitrogen was Cbz-protected because
with silver acetate in THF/H O (Scheme 3). The bicyclic
2
4
18
system locks the sugar ring in the chair conformation ( C ),
1
in which the C-2 substituent is in an axial position. NOEs
indicate spatial proximity of H3 and H5 on one side of the ring
and of H4 and H6 on the other side, respectively (Figure 2). In
addition, larger coupling constants throughout the molecule are
observed, which indicates larger vicinal diaxial coupling
Scheme 6. Synthesis of Phosphate Analogue 15
19
constants associated with the D-gluco conformation.
alkyl or aryl protecting groups gave significant amounts of N-
oxide after treatment with mCPBA. Deprotection of 14 by Pd/
Figure 2. Coupling constants and NOEs of bicyclic iminosugar 9.
Abbreviations of the NOE intensities are as follows: s, strong; m,
medium; and w, weak.
H2 in THF/H O/AcOH afforded the desired glycosyl
2
phosphate mimetic 15 in 90%.
Because L-iminosugars are being increasingly appreciated for
2
1
The synthesis of the first GlcNAc-derived iminosugar
phosphonate was achieved for the D-gluco series of compounds,
but initial attempts were not successful. The intramolecular
formation of aziridine intermediate 11 dominated in reactions
employing bases such as Cs CO (Scheme 4). The problem
pharmaceutical purposes, the procedure also offers access to
L-ido imino-C-glycosides of GlcNAc-1-phosphate 17 (Scheme
7), which allows biological studies to be extended to the
Scheme 7. Synthesis of L-ido Analogue 17
2
3
Scheme 4. Synthesis of Phosphonate Analogue 2
unnatural (L-ido) derivatives. The structure of 17 was
1
determined as the L-ido-isomer in a C -configuration by
NMR analysis (see Figure S2).
4
was solved by using a bulkier base such as NaHDMS.
Substitution of the iodine of 7a by deprotonated dibenzyl-
phosphite using NaHDMS led to the isolation of protected
phosphonate 10 in moderate yield (Scheme 4). Debenzylation
of 10 was achieved to afford 2 in high yield with Pd/C under a
H atmosphere in a solvent mixture of THF/H O/AcOH. In
addition to the phosphonate group, the versatile iodo
intermediate 7a enabled the introduction of structural diversity,
such as the introduction of the azido group (Scheme 5).
To synthesize lipid II analogues, phosphonate 2 and
phosphate 15 were transformed into the corresponding
pyrophosphates (Scheme 8). C15- (18, farnesyl) and C20-
phosphates (19, geranylgeranyl) were attached using the
2
2
22
carbonyldiimidazole (CDI) method. Accordingly, we treated
the ammonium salt of lipid monophosphates 18 and 19 with
CDI to generate a highly reactive monophosphate imidazolide
in situ, which was subsequently reacted with iminosugars 2 and
1
5. Surprisingly, pyrophosphate products 20, 21, and 1 were
Scheme 5. Synthesis of Azido Derivative 12
obtained in good yields (56−70%) with only a small amount of
self-coupled product of lipid monophosphate.
Clostridium difficile is a Gram-positive spore-forming
bacterium that can cause severe infectious diarrhea. The
frequency C. difficile infection has significantly increased in
North America and worldwide in recent years and is becoming
8
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Scheme 8. Synthesis of Lipid-Linked Pyrophosphate
Analogues
design. The C20 derivative 1 exhibited the best inhibitory
potency against C. difficile TGase, with a K value in the low
i
micromolar range (Table 1), although the inhibition is still
weak compared with that of moenomycin. The K value is
i
determined by using our recent reported FRET-based TGase
27
assay.
CONCLUSIONS
■
A variety of imino-C-glycosides of α-D-GlcNAc-1-phosphate, 2,
and 15, their L-ido analogue 17, and lipid-linked analogues 20,
2
1, and 1 were synthesized and evaluated for C. difficile TGase
inhibition. The synthetic procedure provides easy access to
diaminoheptenitol 6a and 6b, versatile iodo intermediates 7a
and 7b, and aminoheptenitol 4 which has been reported via a
15
less direct route (eight steps starting from D-arabinofuranose).
Our result demonstrated that the connection of iminosugar
phosphonate 2 with a lipid chain via a pyrophosphate group
results in a new design for generating active TGase inhibitors.
Because the lipid part greatly contributes to TGase inhibition,
optimization may offer even better TGase inhibitors. In
addition, such a glycolipid could be further elaborated by
enzymes such as N-acetylglucosamine transferase to increase
the glycan length to better fit the donor site of TGase, which
8
can accommodate a large substrate. Further investigations
based on these results are in progress. The efficient
pyrophosphate coupling could also be used for the synthesis
of analogues of UDP-GlcNAc, which could result in potential
23
28
one of the major causes of morbidity in healthcare facilities.
inhibitors of GlcNAc transferases such as OGT.
Because targeting C. difficile cell wall synthesis has proved to be
2
4
successful, we are interested in developing new C. difficile
TGase inhibitors by our newly synthesized iminosugars. The
inhibitory activity of compounds 2, 15, and 17 against C. difficile
EXPERIMENTAL SECTION
■
General. All chemicals were obtained from commercial sources and
used without further purification. Anhydrous solvents were purchased
and used without additional purification. TLC was performed on glass
plates precoated with Silica Gel 60 F254 (0.25 mm, E. Merck);
detection was executed by spraying with a solution of Ce-
25
TGase was then evaluated by the HPLC-based TGase assay.
Unfortunately, none of these derivatives (lacking a lipophilic
moiety) displayed a measurable inhibition at 500 and 100 μM.
It is noted that the TGase inhibition results of the three lipid-
linked compounds, 20, 21, and 1, showed increased inhibition
against TGase (Table 1). It is clear that the presence of a lipid
(NH ) (NO ) and (NH ) Mo O24 and subsequent heating on a
4 2 3 6 4 6 7
1
13
hot plate. H and C NMR spectra were recorded on 500 and 600
1
MHz spectrometers. In H NMR spectra, chemical shifts were
referenced to internal tetramethylsilane (δ 0.00 ppm in CDCl3),
C D H (δ 7.16 ppm in C D ), or HOD (δ 4.79 ppm in D O).
6
5
6
6
2
Table 1. Inhibition of Clostridium difficile TGase by
Synthesized Iminosugars
Chemical shifts are in ppm and are calibrated using the resonances of
the carbon and the residual proton of the deuterated solvent. Proton
1
1
a
peaks were assigned with the aid of 2D NMR techniques ( H− H
COSY, HSQC, DEPT135, DEPT90, and HMBC). Mass spectra (MS)
were recorded on an ESI-TOF mass spectrometer.
inhibition (%) (500 inhibition (%) (100
compd
μM)
μM)
Ki (μM)
b
2




0
Measurement of the Percent Inhibition of TGase Inhibitors.
HPLC analysis was performed on an anion-exchange column (4.6 mm
1
1
2
2
1
5
7
0
1


×
25 mm, 5 μm, LC-SAZ1, Supelco Co.). A linear gradient of
50
NH OAc (from 20 mM to 1 M in MeOH) was eluted at a flow rate of
4
100
100
100
30
1.0 mL/min over 30 min. Inhibition assays were composed of buffer (1
c
100
100
6.3 ± 1.4
μL, 0.85% decyl-PEG, 500 mM HEBES, 100 mM CaCl , pH 8.0),
2
d
DMSO/MeOH (1:1, 2.5 μL), NBD-lipid II (200 μM)/internal
standard (4 μM) (2:3, 1 μL), inhibitors (1 μL, different concentrations
ranging from 10 to 100 mM), TGase (CD) (1 μL, 25 μg/mL), and
Moe
0.0015 ± 0.0001
a
Inhibition percent calculated by comparing the reaction rate in the
presence of the compound to the rate in the absence of any inhibitory
H O (4.5 μL). After incubation at 37 °C for 2 h, reactions were
b
c
2
compound.  refers to no inhibition. Shown are the mean values of
stopped by addition of moenomycin A (1 μL, 100 μM). The reaction
mixture was analyzed by HPLC analysis. The percent inhibition was
calculated by the relative peak area of the remaining NDB-lipid II and
NBD internal standard via integration.
d
the data generated from three independent experiments. The data
was adopted from ref 27.
Determination of K Values of Transglycosylase Inhibitors.
i
chain provides increased potency, possibly due to increased
26
The K values of 1 were determined using our recently reported FRET-
i
27
lipophilicity and increased binding to TGases. By comparison
of 20 and 21, the elongation of the bond length between the
lipid and the sugar was found to decrease the inhibition activity
from 100 to 50% (500 μM) and 30 to 0% (100 μM), suggesting
that the phosphonate moiety is a better precursor for this
based assays. The reaction was performed and monitored as
described above with various concentrations of FRET-based lipid II
analogue (0−15 μM) in the presence of 0−40 μM 1. Initial velocity for
each progression curve was calculated and used for K determination.
i
The K value for 1 was determined with the competitive inhibition
i
8
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Article
For 6a, [α]20 ≅ −36.3 (c 1.0, CH Cl ). H NMR (600 MHz,
1
model using GraphPad Prism 5 (GraphPad Software Inc.). The data
shown are the mean value from three independent experiments.
N-((3R,4R,5R)-4,5,7-Tris(benzyloxy)-6-hydroxyhept-1-en-3-yl)-
acetamide (4). Under an atmosphere of argon in a three-necked flask
equipped with a dropping funnel, dry THF (100 mL) was added to
methyltriphenylphosphonium bromide (dried by heating at 150 °C at
D
2
2
CDCl ) δ 7.39−7.26 (m, 15H), 6.31 (d, J = 8.7 Hz, 1H, NHAc), 5.82
(ddd, J = 17.2, 10.4, 5.1 Hz, 1H, 2-H), 5.19−5.13 (m, 2H, 1-H), 4.81−
4.76 (m, 2H, CH Ph, 3-H), 4.72 (d, J = 10.8 Hz, 1H, CH Ph), 4.60
3
2
2
(dd, J = 10.6 Hz, 2H, CH Ph), 4.54 (s, 2H, CH Ph), 3.88 (dd, J = 7.1,
2
2
1.7 Hz, 1H, 4-H), 3.66 (dd, J = 9.2, 5.2 Hz, 1H, 7-H ), 3.60 (dd, J =
a
0
.01 mbar), forming a suspension that was cooled to 0 °C. nBuLi (2.2
7.0, 5.1 Hz, 1H, 5-H), 3.50 (dd, J = 9.2, 6.1 Hz, 1H, 7-H ), 3.24 (ddd, J
= 5.6, 5.6, 5.3 Hz, 1H, 6-H), 1.99 (s, 3H, NHAc). C NMR (151
b
1
3
M in hexanes, 103 mmol, 46 mL) was added dropwise over 15 min
using the droping funnel, forming a red−orange solution. This was
stirred for 10 min at 0 °C and 40 min at rt, forming a dark red
solution. Then a suspension of 3 (20.3 mmol, 10.0 g) in dry THF (150
mL) was added over 10 min via dropping funnel at 0 °C. The mixture
was stirred for 1 h at 0 °C, 12 h at rt, and 8 h under reflux when TLC
monitoring [CH Cl /MeOH 20:1, R (3) = 0.27, R (4) = 0.39, cerium
molybdate] showed full consumption of the starting material. Then,
saturated aqueous solutions of ammonium chloride (250 mL) and
diethyl ether (250 mL) were added followed by separation of the
phases and extraction of the aqueous phase with diethyl ether (3 × 250
mL), separating large amounts of unreacted phosphonium salts. The
combined organic extracts were dried over Na SO , and the solvent
MHz, CDCl ) δ 169.5 (CO), 138.5, 138.0, 137.9, 136.8 (C-2),
3
128.4, 128.3, 128.2, 127.9, 127.8, 127.7, 127.6, 127.4, 115.2 (C-1), 82.7
(C-5), 81.4 (C-4), 74.9, 73.2, 72.1 (C-7), 52.2 (C-6), 52.1 (C-3), 23.3
+
(CH
489.2753; found, 489.2748.
). HRMS (ESI-TOF) calcd for C30
H
N
O
(M + H) ,
3
37
2
4
For 6b, [α]2
0
≅ −43.8 (c 1.0, CH
Cl
2
). H NMR (CDCl
1
, 500
2
2
f
f
D
2
3
MHz) δ 2.07 (s, 3H, CH
), 3.23 (dd, J = 6.8, 6.8 Hz, 1H), 3.43 (dd, J
3
= 7.6, 7.6 Hz, 1H), 3.54 (dd, J = 8.4, 6.5 Hz, 1H), 3.71 (d, J = 8.4 Hz,
1H), 4.06 (d, J = 8.4 Hz, 1H), 4.5−4.6 (m, 3H), 4.74 (d, J = 10.6 Hz,
1H, CH Ph), 4.78 (d, J = 10.6 Hz, 1 H, CH Ph), 4.86−4.91 (m, 2H),
2 2
5.27−5.37 (m, 2H, 1-H), 6.04 (ddd, J = 15.1, 10.5, 3.8 Hz, 1H, 2-H),
1
3
7
(
.05 (d, J = 8.3 Hz, 1 H, NH), 7.30−7.43 (m, 15 H, Ar-H). C NMR
2
4
CDCl , 125 MHz), δ 23.6 (CH ), 51.2, 51.8 (2 CNHR), 73.1, 73.5,
was removed in vacuo. Separation from side products SP1 and SP2
(R (3) = 0.20, R (SP1) = 0.15, cerium molybdate, R (SP2) = 0.15,
3
3
7
4.9, 75.4 (4 t, OCH Ph, C-7), 80.4, 83.1, 115.6 (C-1), 127.7, 127.7,
[
2
f
f
f
1
27.8, 127.8, 128.0, 128.3, 128.4, 128.5, 128.54, 128.56 (9 d, Ar), 135.8
UV, toluene/MeOH 9:1] by column chromatography on silica gel
separation from SP2: 50 cm column, 1.500 g silica, toluene/MeOH
0:1; separation from SP1: 50 cm column, 500 g silica, hexanes/
(
C-2), 138.2, 138.3, 138.7 (3 s, Ar), 169.8 (CO). HRMS (ESI-
(
4
+
TOF) calcd for C H N O (M + H) , 489.2753; found, 489.2758.
30 37
2
4
N-((2S,3S,4R,5R,6R)-4,5-Bis(benzyloxy)-6-(benzyloxymethyl)-2-
(iodomethyl)piperidin-3-yl)acetamide (7a). Under an atmosphere of
argon in the dark, N-iodosuccinimide (0.65 mmol, 147 mg) was added
in one portion at rt to a solution of 6a (0.65 mmol, 320 mg) in dry
CH Cl (20 mL). The resulting solution was stirred at rt for 2 h (TLC
EtOAc 1:1) led to the isolation of 4 (5.97 g, 60%) as a colorless oil.
Analytical data was in full accordance with the reported data. H NMR
1
(
CDCl , 500 MHz) δ 1.89 (s, 3H, CH ), 2.96 (d, J = 5.6 Hz, 1 H),
3
3
3
1
.60−3.57 (m, 3H), 3.82 (dd, J = 6.0, 2.0 Hz, 1H), 3.94−3.99 (m,
2
2
H), 4.5−4.7 (m, 6 H, 3CH Ph), 4.78−4.82 (m, 1H), 5.12 (d, J = 10.3
2
monitoring, CH Cl /MeOH 20:1, R (6a) = 0.25, R (7a) = 0.60, cerium
2
2
f
f
Hz, 1H, 1-H ), 5.16 (d, J = 17.2 Hz, 1H, 1-H ), 5.82 (ddd, J = 17.2,
Z
E
molybdate]. Then, a saturated aqueous solution of sodium thiosulfate
(10 mL) was added followed by extraction with CH Cl (3 × 20 mL).
2
1
1
7
0.4, 5.2 Hz, 1 H, 2-H), 6.15 (d, J = 8.6 Hz, 1H, NH), 7.12−7.34 (m,
5 H, Ar-H). 13C NMR (CDCl , 125 MHz), δ 23.3 (CH ), 52.7 (C-3),
2
3
3
The combined organic extracts were dried over MgSO , and the
4
0.9 (t, C-7), 71.0, 73.4, 74.5, 75.1 (3 t, OCH Ph), 80.4, 81.0, 115.5
2
solvent was removed in vacuo. Purification by silica gel chromatog-
(
C-1), 125.3, 127.7, 128.8, 127.9, 128.0, 128.3, 128.4, 128.4, 128.5 (9
raphy (CH Cl /MeOH 100:1) gave 7a (168 mg, 50%) as a white
2
2
d, Ar), 129.1, 136.9 (C-2), 138.0, 138.3 (3 s, Ar), 169.7 (CO).
solid. [α]2
1
0
D
≅ +20.5 (c 0.4, CH
.82 (s, 3H, CH ), 2.99 (t, J = 10.2 Hz, 1H, 1-H ), 3.20 (dd, J = 10.2,
Cl
2
). H NMR (CDCl
1
, 600 MHz) δ
+
2
3
HRMS (ESI-TOF) calcd for C H NO (M + H) , 490.2593; found,
30
36
5
3
a
4
90.2588.
N-((3R,4R,5R,6S)-6-Amino-4,5,7-tris(benzyloxy)hept-1-en-3-yl)-
acetamide (6a) and N-((3R,4S,5R,6R)-6-Amino-4,5,7-tris-
benzyloxy)hept-1-en-3-yl)acetamide (6b). Oxalyl chloride (12
3
.3 Hz, 1H, 1-H ), 3.36−3.40 (m, 2H, 2-H and 7-Ha), 3.43 (m, 1H, 5-
b
H), 3.47 (m, 1H, 6-H), 3.56 (m, 1H, 4-H), 3.82 (dd, J = 9.4, 4.9 Hz,
H, 7-H ), 4.33 (m, 1H, 3-H), 4.41−4.45 (t, 2H, CH Ph), 4.60−4.49
1
b
2
(
(
m, 4H, CH Ph), 6.61 (d, J = 9.1 Hz, 1 H, NH), 7.19−7.33 (m, 15 H,
2
mmol, 1.02 mL) was added at −78 °C to a solution of DMSO (16
13
Ar-H). C NMR (CDCl , 150 MHz) 9.8 (CH I), 23.3 (CH ), 48.9
3
2
3
mmol, 1.13 mL) in dry CH Cl (20 mL) under an atmosphere of
2
2
(
C-3), 49.8 (C-2), 56.0 (C-6), 68.0 (C-7), 72.0, 72.1, 73.4 (OCH Ph),
2
argon in a Schlenk flask. After 30 min of stirring, a solution of 4 (4.0
7
4.5 (C-5), 75.4 (C-4), 138.0, 137.5, 137.3, 128.5, 128.5, 128.4, 128.0,
mmol, 1.96 g) in dry CH Cl (8 mL) was added carefully via syringe
2
2
1
27.9, 127.8, 127.7, 127.7, 127.5 (Ar), 137.3, 137.5, 138.0 (Ar), 170.0
over 10 min (note that precooling the cooled wall of the flask is
+
(
6
CO). HRMS (ESI-TOF) calcd for C H IN O (M + H) ,
30
36
2
4
necessary!). After 1 h of stirring at −78 °C, NEt (32 mmol, 4.44 mL)
3
15.1714; found, 615.1686.
N-((2S,3S,4R,5R,6R)-4,5-Bis(benzyloxy)-6-((benzyloxy)methyl)-2-
was added followed by 40 min of stirring at −78 °C. The solution was
warmed to 0 °C within 10 min with an ice bath, and CH Cl (20 mL)
2
2
(
hydroxymethyl)piperidin-3-yl)acetamide(8) and (4aS,6R,7R,8-
and a saturated aqueous solution of NaHCO (50 mL) were added
3
R,8aS)-7,8-Bis(benzyloxy)-6-((benzyloxy)methyl)-2-methyl-
followed by extraction of the aqueous phase with CH Cl (3 × 40
2
2
4a,5,6,7,8,8a-hexahydro-4H-pyrido[3,2-d][1,3]oxazine (9). A solu-
mL). The combined organic extracts were dried over Na SO , and the
2
4
tion of 7a (0.33 mmol, 200 mg) in THF/H O 1:1 (44 mL) was
2
solvent was removed in vacuo. A crude, instable intermediate was
obtained as a yellow oil [CH Cl /MeOH 20:1, R (4) = 0.39,
treated with Ag(OAc) (0.9 mmol, 150 mg) and stirred at rt for 30 min
followed by the addition of AcOH (200 μL). After 3 h (TLC
monitoring, CH Cl /MeOH 20:1, R (8) = 0.38, R (9) = 0.5, cerium
2
2
f
Rf(ketone) = 0.49, cerium molybdate]. Without purification, dry
methanol (40 mL) was added to the reaction crude under an
atmosphere of argon, and the resulting solution was treated with
2
2
f
f
molybdate], the mixture was filtered, and the solvent was removed in
vacuo. Dichloromethane (100 mL) was added to the residue, which
ammonium acetate (100%, 40 mmol, 3.08 g) and NaBH CN (20
was washed with saturated aqueous NaHCO . The collected organic
3
3
mmol, 1.30 g). After the resulting solution was stirred for 18 h at 50
phase was dried by MgSO and concentrated in vacuo. Purification by
4
°
C, TLC monitoring [2 × CH Cl /MeOH 20:1, R (ketone) = 0.66,
column chromatography on silica gel (CH Cl /MeOH/Et N 30:1:0.3)
2
2
f
2
2
3
R (alcohol) = 0.58, R (6b) = 0.41, R (6a) = 0.34, cerium molybdate],
led to the isolation of 8 (96.5 mg, 58%) and 9 (41.7 mg, 26%) as white
f
f
f
saturated NaHCO3 (40 mL) was added to the reaction mixture
followed by the removal of MeOH. The residual aqueous solution was
extracted by CH Cl (120 mL) twice. The collected organic phase was
solid and colorless oil, respectively.
1
Compound 8. H NMR (600 MHz, CDCl ) δ 7.38−7.14 (m,
3
2
2
15H), 6.98 (d, J = 7.3 Hz, 1H, NHAc), 4.60 (m, 2H, CH Ph), 4.51−
2
dried over MgSO and concentrated. NMR of the crude product
4.40 (m, 4H, CH Ph), 4.21 (m, 1H, 3-H), 3.95 (d, J = 6.2 Hz, 1H,
4
2
showed a ratio of 6a/6b = 67:33. Purification by column
OH), 3.81 (t, J = 8.9 Hz, 1H, 7-H ), 3.63 (m, 1H, 4-H), 3.58 (m, 1H,
a
chromatography on silica gel (196 g silica, CH Cl /MeOH 25:1) led
to the isolation of 6a (836 mg, 42%) and 6b (411 mg, 22%) as
colorless oils.
5-H), 3.48 (dd, J = 9.4, 5.9 Hz, 1H, 7-H ), 3.42−3.35 (m, 2H, 6-H, 1-
2
2
b
H ), 3.28−3.24 (m, 1H, 2-H), 3.23−3.18 (m, 1H, 1-H ), 1.86 (s, 3H).
b
a
13
C NMR (151 MHz, CDCl ) δ 171.4, 138.1, 137.5, 137.2, 128.7,
3
8
633
dx.doi.org/10.1021/jo501340s | J. Org. Chem. 2014, 79, 8629−8637

The Journal of Organic Chemistry
Article
1
7
28.5, 128.4, 128.2, 127.9, 127.9, 127.7, 127.4, 74.6 (C-4), 74.5 (C-5),
mL), dried with MgSO , and concentrated. The crude residue was
4
3.3, 72.2, 71.9 (OCH Ph), 68.4 (C-7), 62.2 (C-1), 55.0 (C-6), 48.9
purified by flash silica gel column chromatography (toluene/EtOAc
2
1
(
C-2), 46.2 (C-3), 23.2 (CH ). HRMS (ESI-TOF) calcd for
7:3) to afford compound 12 (32.3 mg, 65%) as colorless oil. H NMR
3
+
C H N O (M + H) , 505.2697; found, 505.2698.
(600 MHz, CDCl ) δ 7.40−7.31 (m, 11H), 7.29 (m, 2H), 7.25−7.22
3
0
37
2
5
3
1
Corrected H NMR Values for Compound 9 (Lorentz-to-Gauss
(m, 2H), 6.68 (d, J = 9.1 Hz, 1H), 4.61 (t, J = 12.0 Hz, 2H), 4.55 (q, J
= 12.0 Hz, 2H), 4.48 (t, J = 11.3 Hz, 2H), 4.22 (m, 1H, 3-H), 3.88 (t, J
= 8.6 Hz, 1H), 3.63 (t, J = 3.0 Hz, 1H), 3.54 (td, J = 8.9, 3.3 Hz, 1H),
3.50 (dd, J = 12.2, 3.8 Hz, 1H), 3.48−3.42 (m, 2H), 3.34−3.31 (m,
1
Transformed Spectra). Compound 9. H NMR (600 MHz, CDCl ) δ
3
7
4
1
.41−7.07 (m, 15H, Ar-H), 4.87 (dd, J = 10.7, 6.4 Hz, 2H, CH Ph),
2
.73 (d, J = 10.7 Hz, 1H, CH Ph), 4.53 (ddd, J = 10.9, 10.4, 5.1 Hz,
2
1
3
H, 2-H), 4.48−4.41 (m, 2H, CH Ph), 4.36 (d, J = 10.8 Hz, 1H), 4.29
1H), 3.17 (dd, J = 12.1, 9.5 Hz, 1H), 1.86 (s, 3H). C NMR (151
2
(
ddd, J = 10.4, 9.5, 1.2 Hz, 1H, 3-H), 3.79 (dd, J = 9.0, 3.0 Hz, 1H, 7-
MHz, CDCl ) δ 169.9, 138.1, 137.5, 137.3, 128.5, 128.5, 128.4, 128.0,
3
H ), 3.61 (dd, J = 9.5, 9.3 Hz, 1H, 4-H), 3.38 (dd, J = 9.0, 8.1 Hz, 1H,
127.8, 127.8, 127.7, 127.5, 75.0, 74.4, 73.3, 72.0, 67.9, 55.4, 53.6, 47.2,
a
+
7
1
1
-H ), 3.25 (dd, J = 13.6, 5.1 Hz, 1H, 1-H ), 3.19 (dd, J = 9.3, 9.2 Hz,
H, 5-H), 2.94 (ddd, J = 9.2, 8.1, 3.0 Hz, 1H, 6-H), 2.58 (dd, J = 13.6,
0.9 Hz, 1H, 1-H ), 1.94 (d, J = 1.2 Hz, 3H). C NMR (CDCl , 150
23.3. HRMS (ESI-TOF) calcd for C H N O (M + H) , 530.2762;
b
a
30
35
5
4
found, 530.2776.
13
b
3
Benzyl(2S,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6-
MHz) δ 164.31(CO), 138.98, 138.25, 137.86, 128.46, 128.29,
((benzyloxy)methyl)-2-(hydroxymethyl)piperidine-1-carboxylate
1
8
7
28.13, 127.97, 127.96, 127.83, 127.58, 127.28 (Ar), 84.53 (C-4),
(13). A solution of compound 8 (220 mg, 0.44 mmol) in dioxane/H
(3:2, 25 mL) was treated with NaHCO (361 mg, 4.40 mmol) and
3
2
O
2.52 (C-5), 79.84 (C-2), 75.88, 75.50, 73.38 (OCH Ph), 72.18 (C-3),
2
1.50 (C-7), 63.30 (C-6), 47.90 (CH O), 14.02 (CH ). HRMS (ESI-
CbzCl (503 μL, 3.52 mmol) at 0 °C. After stirring at rt for 4 h, the
2
3
+
TOF) calcd for C H N O (M + H) , 487.2591; found, 487.2616.
reaction mixture was diluted with CH Cl (30 mL) and washed with
30
35
2
4
2
2
Dibenzyl(((2S,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6-
(benzyloxy)methyl)piperidin-2-yl)methyl)phosphonate (10). To a
water (15 mL) and brine (15 mL) sequentially. The organic layer was
(
dried with MgSO and concentrated, and the residue was purified by
4
stirring solution of dibenzyl phosphate (111 μL, 0.48 mmol) in dry
DMF (2.5 mL) was added molecular sieve MS 4Å (150 mg), and the
mixture was stirred for 30 min at rt. NaHMDS (2 M solution in THF,
flash silica gel column chromatography (EtOAc/hexane 55:45 to
70:30) to afford 13 (347 mg, 92%) as a viscous oil. H NMR (600
1
MHz, C D , 45 °C) δ 7.38−7.07 (m, 20H, Ar-H), 6.61 (d, J = 9.1 Hz,
6
6
230 μL, 0.46 mmol) was then added. After 1 h, compound 7a (51 mg,
1H, NH), 5.26 (m, 1H, 6-H), 5.21 (d, J = 11.9 Hz, 1H, 7-H ), 5.05 (d,
a
0.08 mmol) in dry DMF (1.5 mL) was added to the reaction mixture
J = 9.8 Hz, 1H, 7-H ), 4.92 (ddd, J = 9.2, 2.3, 2.3 Hz, 1H, 3-H), 4.57
b
at rt, and the mixture was stirred for another 24 h (R (10) = 0.38, UV,
(m, 1H, 1-H ), 4.5−4.49 (m, 2H, 1-H , CH Ph), 4.46 (d, J = 11.9 Hz,
f
a
b
2
EtOAc/Hex 4:1). The reaction mixture was concentrated to remove
1H, CH Ph), 4.43−4.37 (m, 2H, CH Ph), 4.31 (d, J = 11.9 Hz, 1H,
2
2
DMF and purified by silica gel chromatography (CH Cl /MeOH
CH Ph), 4.17 (d, J = 11.3 Hz, 1H, CH Ph), 4.14−4.11 (ddd, J = 8.0,
2
2
2
2
1
00:1 to 10:2, 5.0 g silica) to afford 10 (24.8 mg, 40%) as a white solid.
6.0, 2.3 Hz, 1H, 1-H), 4.04 (t, J = 7.1 Hz, 1H, OH), 3.84 (p, J = 9.5
1
13
H NMR (600 MHz, CDCl ) δ 7.33−7.18 (m, 25H), 6.60 (d, J = 8.2
Hz, 3H, 5-H, CH Ph), 3.74 (m, 1H, 4-H), 1.62 (s, 3H, CH ).
C
3
2
3
Hz, 1H, NHAc), 4.97 (m, CH Ph, 2H), 4.92−4.87 (m, CH Ph, 2H),
NMR (151 MHz, C D , 45 °C) δ 169.9 (CO), 156.6 (CO),
2
2
6
6
4
.56 (d, J = 11.8 Hz, CH Ph, 1H), 4.52 (d, J = 11.5 Hz, CH Ph, 1H),
138.6, 137.8, 137.7, 137.0, 128.4, 128.4, 128.4, 128.3, 128.1, 128.0,
127.9, 127.7, 127.6 (Ar), 76.4 (C-4), 74.6 (C-5), 73.0, 71.6, 71.3, 67.9
2
2
4
.45−4.40 (m, CH Ph, 2H), 4.34 (d, J = 11.9 Hz, CH Ph 1H), 4.10
2
2
(
(
1
brd, J = 9.4 Hz, 1H, 3-H), 3.71−3.63 (m, 2H, 7-Ha and 2-H), 3.57
(OCH Ph), 67.0 (C-7), 61.4 (C-1), 55.7 (C-6), 55.0 (C-2), 46.8 (C-
2
+
m, 1H, 4-H), 3.53−3.47 (m, 2H, 7-Hb and 5-H), 3.32 (m, 6-H),
3), 22.3 (CH ). HRMS (ESI-TOF) calcd for C H N O (M + H) ,
3
38 42
2
7
13
.94−1.87 (m, 1H, 1-H ), 1.84−1.75 (m, 4H, COCH and 1-H ).
C
639.3065; found, 639.3084.
a
3
b
NMR (151 MHz, CDCl ) δ 169.95 (CO), 138.3, 137.7, 137.5,
Benzyl(2S,3S,4R,5R,6R)-3-acetamido-4,5-bis(benzyloxy)-6-
((benzyloxy)methyl)-2-(((bis(benzyloxy)phosphoryl)oxy)methyl)-
piperidine-1-carboxylate (14). To a stirred solution of 13 (16 mg,
0.03 mmol) and tetrazole (9.7 mg, 0.14 mmol) in MeCN (2 mL) at 0
°C was slowly added bis(benzyloxy)(diisopropylamino)phosphane (40
μL, 0.125 mmol) followed by removal of the ice bath. When the TLC
analysis showed consumption of the starting material, the reaction
mixture was cooled to 0 °C followed by the addition of mCPBA (75%,
9.3 mg, 0.032 mmol). The ice bath was removed, and the reaction
mixture was heated to rt. After 1 h, the reaction was quenched by
3
1
1
7
4
36.2, 136.2, 136.2, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 127.9,
27.8, 127.8, 127.6, 127.5, 75.4 (C-4), 74.1 (C-5), 73.2, 72.1, 68.8 (C-
), 67.2, 67.2, 67.2, 67.2 (dd, Jc,p = 4.2 Hz, 2C, POCH Ph), 55.4 (C-6),
2
9.8, 49.7 (d, Jc,p = 16.2 Hz, C-3), 43.6 (C-2), 29.3, 28.4 (d, J = 140.4
c,p
3
1
Hz, C-1), 23.3 (CH3). P NMR (CDCl , 202 MHz) δ 31.9. HRMS
3
+
(
ESI-TOF) calcd for C H N O P (M + H) , 749.3350; found,
44 49 2 7
7
49.3360.
((2S, 3S, 4R, 5R, 6R)-3-Acetamido-4, 5-dihydroxy-6-
hydroxymethyl)piperidin-2yl)methyl) phosphonic acid (2). To a
(
(
round-bottomed flask contained starting material 10 (20 mg, 0.027
saturated NaHCO (1.5 mL). The organic phase was collected. The
3
mmol) were added THF (2.5 mL), H O (2.5 mL), and AcOH (50
aqueous phase was extracted with CH Cl (10 mL) three times. The
2
2
2
μL). The solution was treated with Pd/C (10%, Degussa type, 26.0
mg) under a hydrogen atmosphere and stirred at rt for 18 h. The
mixture was filtered, and the solvent was removed in vacuo.
Purification by biogel P2 chromatography (eluted by water) led to
combined organic phase was dried over MgSO , filtered, and
concentrated. The reside was purified by silica gel chromatography
4
(EtOAc/hexanes 55:45) to yield compound 14 (21.3 mg, 79% over
1
two steps) as a colorless liquid. H NMR (600 MHz, C D , 45 °C) δ
6
6
the isolation of 2 (7.2 mg, 90%) as a white solid after lyophilization.
7.37−7.06 (m, 35H), 6.91 (brs, 1H, NHAc), 5.08−5.04 (m, 10H),
1
H NMR (600 MHz, D O) δ 4.10 (brd, J = 6.8 Hz, 1H, 3-H), 3.91−
4.55 (brs, 2H), 4.43 (m, 4H), 4.22 (d, J = 11.5 Hz, 1H), 4.02−3.85 (m,
2
13
3
.75 (m, 3H, 7-Ha, 7-Hb, 2-H), 3.68 (t, J = 9.5 Hz, 1H, 4-H), 3.56 (t, J
4H), 1.79 (brs, 3H, CH
(NHCOCH ), 156.4 (NHCOOBn), 138.0, 137.5, 137.3, 136.2, 135.8,
3
3
). C NMR (151 MHz, CDCl ) δ 170.0
3
=
(
9.0 Hz, 1H, 5-H), 3.22 (d, J = 8.2 Hz, 1H, 6-H), 2.01 (s, 3H), 1.79
m, 1H, 1-Ha), 1.66 (m, 1H, 1-Hb). 13C NMR (151 MHz, D O) δ
135.7, 135.7, 128.5, 128.4, 128.3, 128.1, 127.9, 127.9, 127.9, 127.8,
2
1
76.9 (CO), 71.9 (C-4), 71.6 (C-5), 60.4 (C-7), 56.3 (C-6), 53.9
127.6, 127.5, 74.3, 73.0, 71.8, 71.2, 69.3, 67.9, 67.5, 52.3, 47.7, 23.0
31
31
(
C-2), 53.7−53.6 (C-3, Jc,p = 28.9 Hz), 24.3. P NMR (D O, 202
(NHCOCH
3
). P NMR (CDCl
3
, 202 MHz) δ 0.32. HRMS (ESI-
2
+
MHz) δ 19.7 (brs). HRMS (ESI-TOF) calcd for C H N O P (M −
H) , 297.0846; found, 297.0860.
TOF) calcd for C52H N O10P (M + H) , 899.3667; found, 899.3692.
55 2
9
19
2
7
−
((2S,3S,4R,5R,6R)-3-Acetamido-4,5-dihydroxy-6-(hydroxymethyl)-
piperidin-2-yl)methyl Dihydrogen Phosphate (15). To a round-
bottomed flask containing starting material 14 (20 mg) were added
N-((2R,3S,4R,5R,6R)-2-(Azidomethyl)-4,5-bis(benzyloxy)-6-
(
(benzyloxy)methyl)piperidin-3-yl)acetamide (12). To a stirred
solution of iodo compound 7a (58 mg, 0.094 mmol) in dry DMF
6 mL) was added NaN3 (12.2 mg, 0.188 mmol). The reaction
mixture was heated at 80 °C and reacted for 8 h. After the
disappearance of 7a (toluene/EtOAc 3:2, Rf = 0.25, cerium
THF (2.5 mL), H O (2.5 mL), and AcOH (50 μL). The solution was
2
(
treated with Pd/C (10%, Degussa type, 26.0 mg) under a hydrogen
atmosphere and stirred at rt for 18 h. The mixture was filtered through
a pad of Celite, and the solvent was removed in vacuo. Purification by
1
molybdate) on TLC, the reaction mixture was cooled to rt. CH Cl2
P2 led to the isolation of 15 (6.5 mg, 91%) as a colorless solid. H
2
(10 mL) was added, and the mixture was washed twice with water (5
NMR (600 MHz, D O) δ 4.07 (m, 2H, 3-H and 1-H ), 3.97 (m, 1H,
2
a
8
634
dx.doi.org/10.1021/jo501340s | J. Org. Chem. 2014, 79, 8629−8637

The Journal of Organic Chemistry
Article
1
3
-H ), 3.88 (m, 2H, 4-H and 7-H ), 3.76 (d, J = 12.3 Hz, 1H, 7-H ),
1H), 1.35 (d, J = 13.7 Hz, 1H). 13C NMR (151 MHz, D O) δ 173.9
b
a
b
2
1
3
.61 (m, 1H, 2-H), 3.42 (m, 2H, 5-H and 6-H), 1.98 (s, 3H, CH3).
C
(CO), 67.2(C4), 65.9 (C5), 59.0 (C7), 57.0 (C6), 51.6 (C2, J
=
c,p
NMR (151 MHz, D O) δ 174.6 (CO), 71.0 (C-4), 70.1 (C-5), 60.1
2.6 Hz), 51.0, 50.9 (C-3, J = 13.6 Hz), 27.4, 26.5 (C-1, Jc,p = 129.1
2
c,p
31
31
(
C-1), 58.8 (C-7), 55.7 (C-6), 53.9 (C-2), 50.9 (C-3), 21.8 (CH ).
P
Hz), 24.6, 22.2 (CH3). P NMR (202 MHz, D O) δ −19.01. HRMS
3
2
+
NMR (D O, 202 MHz) δ 3.3. HRMS (ESI-TOF) calcd for
C H N O P (M − H) , 313.0795; found, 313.0808.
(ESI-TOF) calcd for C H N O P (M + H) , 299.1003; found,
2
9
20
2
7
−
299.1006.
9
19
2
8
N-((2S,3S,4R,5R,6S)-4,5-Bis(benzyloxy)-6-(benzyloxymethyl)-2-
(((2S, 3S, 4R, 5R, 6R)-3-Ace tamido-4, 5-dihydroxy-6-
(hydroxymethyl)piperidin-2-yl)methyl)phosphoric ((2Z,6E)-3,7,11-
trimethyldodeca-2,6,10-trien-1-yl phosphoric) Anhydride (20). A
round-bottomed flask containing lipid phosphate 18 (17 mg, 0.051
mmol) was dried in vacuo for 1 h. Anhydrous DMF (1 mL) was then
added to the flask followed by the addition of CDI (32.8 mg, 0.203
mmol). The resulting solution was kept stirring for 3 h. MeOH (30
μL) was added to quench the extra CDI for 1.5 h. The reaction residue
was then transferred to a vial containing compound 15 (5.3 mg, 0.016
mmol) followed by the addition of tetrazole solution in dry DMF (10
mg/mL, 0.041 mmol). The reaction was kept stirring for 48 h. The
mixture was transferred to a round-bottomed flask, and the vial was
washed with MeOH (1 mL) three times. The solvent was removed in
(iodomethyl)piperidinyl) Acetamide (7b). Under an atmosphere of
argon in the dark, N-iodosuccinimide (0.38 mmol, 85.5 mg) was added
in one portion at 0 °C to a solution of 6b (0.3 mmol, 147 mg) in dry
CH Cl (10 mL). The resulting solution was stirred at 0 °C for 2 h
until TLC monitoring [CH Cl /MeOH 20:1, R (6b) = 0.38, R (7b) =
0
material. Then, a saturated aqueous solution of sodium thiosulfate (15
mL) and a saturated aqueous solution of Na CO (15 mL) were added
followed by extraction with CH Cl (3 × 20 mL). The combined
organic extracts were dried over Na SO , and the solvent was removed
2
2
2
2
f
f
.67, cerium molybdate] showed full consumption of the starting
2
3
2
2
2
4
in vacuo. Purification by column chromatography on silica gel (20 g
silica, CH Cl /MeOH 99:1) led to the isolation of 7b (132 mg, 72%)
as a colorless oil. [α]D ≅ +25.9 (c 1.0, CH Cl ). H NMR (CDCl ,
2
2
20
1
vacuo and purified by silica gel chromatography (CHCl
NH OH 7:3:0.5 to MeOH/H O 1:1). The collected fractions
containing product with 90% purity were concentrated and further
purified by HPLC (20 mM NH HCO /MeCN) to afford compound
20 (6.7 mg, 70%) as a white solid after lyophilization. H NMR (600
MHz, D O, 1H),
O) δ 5.47 (td, J = 7.1, 1.0 Hz, lipid-CCHCH
O, 2H),
/MeOH/
2
2
3
3
5
1
2
3
00 MHz) δ 1.79 (s, 3H, CH ), 3.04 (dd, J = 10.9 Hz, J = 10.9 Hz, 1H,
4
2
3
-H ), 3.31 (dd, J = 10.7 Hz, J = 3.5 Hz, 1H, 1-H ), 3.29−3.33 (m, 1H,
-H), 3.37 (ddd, J = 8.5 Hz, J = 4.7 Hz, 2.1 Hz, 1H, 6-H), 3.43 (dd, J =
.2 Hz, J = 2.1 Hz, 1H, 5-H), 3.53 (dd, J = 9.0 Hz, J = 4.7 Hz, 1H, 7-
a
b
4
3
1
H ), 3.60 (dd, J = 3.1 Hz, J = 3.1 Hz, 1H, H-4), 3.63 (dd, J = 9.0 Hz, J
=
2
2
a
9.0 Hz, 1H, 7-H ), 4.37 (d, J = 11.2 Hz, 1H, CH Ph), 4.42 (d, J =
5.25−5.15 (m, 2 × CCH, 2H), 4.50 (m, lipid-CCHCH
2
b
2
11.2 Hz, 1H, CH Ph), 4.46 (ddd, J = 9.7 Hz, J = 3.2 Hz, 2.7 Hz, 1H, 3-
4.25−4.20 (m, 1H, 1-Ha), 4.08 (dt, J = 10.9, 3.8 Hz, 1H, 1-Hb), 4.01
(dd, J = 10.9, 6.1 Hz, 1H, 3-H), 3.90 (dd, J = 11.6, 2.9 Hz, 1H, 7-Ha),
3.85 (dd, J = 10.8, 9.0 Hz, 1H, 4-H), 3.60 (dd, J = 11.7, 6.6 Hz, 1H, 7-
Hb), 3.38 (m, 1H, 2-H), 3.27 (t, J = 9.5 Hz, 1H, 5-H), 3.12−3.07 (m,
2
H), 4.49 (d, J = 11.9 Hz, 2H, CH Ph), 4.59 (d, J = 11.8 Hz, 1H,
CH Ph), 4.68 (d, J = 11.8 Hz, 1H, CH Ph), 6.68 (d, J = 9.7 Hz, 1H,
2
2
2
13
NH), 7.17−7.40 (m, 15H, Ar-H). C NMR (CDCl , 125 MHz) δ 9.6
3
(
t, CH I), 23.4 (CH ), 48.8 (C-3), 55.5 (C-6), 55.9 (C-2), 71.0 (C-7),
1H, 6-H), 2.20−2.09 (m, 3 × lipid-CH
2.04 (m, lipid-CH , 2H), 1.73 (s, lipid-CH
3H), 1.64 (s, 2 × lipid-CH
, 6H). 13C NMR (151 MHz, D
(CO), 160.2 (NH HCO ), 143.0, 136.6, 133.4 (lipid CH
2
, 6H), 2.07 (s, NHAc, 3H),
, 3H), 1.70 (s, lipid-CH
O) δ 174.5
CH
C
O),
2
3
7
1
1
1.8, 73.06, 73.4 (3 t, OCH Ph), 73.5 (C-5), 75.1 (C-4), 127.7, 127.8,
2
3
,
3
2
27.8, 128.0, 128.4, 128.4, 128.5, 128.6, 128.7 (9 d, Ar), 137.2, 137.7,
38.1 (3 s, Ar), 170.0 (CO). HRMS (ESI-TOF) calcd for
3
2
4
3
3
2
+
C H IN O (M + H) , 615.1714; found, 615.1716.
CH), 124.4, 124.2 (2 X CCH), 119.4, 119.3 (lipid-CCHCH
2
30
36
2
4
Dibenzyl((2S,3S,4R,5R,6S)-3-acetamido-4,5-bis(benzyloxy)-6-
benzyloxymethyl) piperidin-2-yl)methylphosphonate (16). Under
an atmosphere of argon and strictly dry conditions, Cs CO (0.6
72.6 (C-5), 72.0 (C-4), 64.0, 63.9 (C-1), 63.0, 63.03 (lipid-C
(
CHCH O), 61.8 (C-7), 55.1 (C-6), 53.2, 53.1 (C-2), 52.8 (C-3), 38.7,
2
2
3
38.7, 25.7, 25.5 (4C, lipid-CH ), 24.8 (lipid-CH ), 21.9 (NHCOCH ),
2
3
3
31
mmol, 195 mg) was added to a solution of freshly distilled dibenzyl
phosphite (0.9 mmol, 237 mg) in dry DMF (1.5 mL). After 1 h of
stirring at rt, 7b (0.147 mmol, 90 mg) was added, and the resulting
solution was stirred for 12 h in the dark until TLC monitoring
16.9 (lipid-CH ), 15.6 (lipid-CH ), 15.2 (lipid-CH ). P NMR (202
3 3 3
MHz, D O) δ −10.91 (d, J = 21.7 Hz), −11.51 (d, J = 21.8 Hz).
2
−
HRMS (ESI-TOF) calcd for C H N O P (M − H) , 597.2337;
found, 597.2337.
24
44
2
11 2
[
CH Cl /MeOH 20:1, R (7b) = 0.64, R (16) = 0.39, cerium
(((2S, 3S, 4R, 5R, 6R)-3-Ace tamido-4, 5-dihydroxy-6-
(hydroxymethyl)piperidin-2-yl)methyl)phosphonic((2Z,6E)-3,7,11-
trimethyldodeca-2,6,10-trien-1-yl phosphoric) Anhydride (21). A
round-bottomed flask containing lipid phosphate 18 (13.6 mg, 0.041
mmol) was dried in vacuo for 1 h. Anhydrous DMF (1 mL) was then
added to the flask followed by the addition of CDI (26.2 mg, 0.163
mmol). The resulting solution was kept stirring for 3 h. MeOH (25
μL) was added to quench the extra CDI for 1.5 h. The reaction residue
was then transferred to a vial containing compound 2 (4.1 mg, 0.014
mmol). The reaction was completed when the reaction mixture turned
from cloudy to clear after 48 h of stirring. The mixture was transferred
to a round-bottomed flask, and the vial was washed with MeOH (1
mL) three times. The solvent was removed in vacuo and purified by
2
2
f
f
molybdate] showed full consumption of the starting material. Then,
the solvent was removed in vacuo. Purification by column
chromatography on silica gel (20 g silica, CH Cl /MeOH 98:2) led
2
2
1
to the isolation of 16 (49 mg, 45%) as a colorless oil. H NMR
(
3
4
CDCl , 500 MHz) δ 1.77 (s, 3H, CH ), 3.29−3.34 (m, 1H), 3.39−
3
3
.53 (m, 4H), 3.60 (dd, J = 4.9, 2.7 Hz, 1H), 4.19 (d, J = 9.7 Hz, 1H),
.34 (d, J = 11.2 Hz, 1H), 4.38 (d, J = 12.8 Hz, 1H), 4.42 (d, J = 12.4
Hz, 1H), 4.51 (d, J = 11.9 Hz, 1H), 4.62 (d, J = 11.9 Hz, 1H), 4.88−
5
.02 (m, 4 H), 6.71 (d, J = 10.0 Hz, 1H, NH), 7.15−7.35 (m, 25H, Ar-
13
H). C NMR (CDCl , 100 MHz) δ 23.4 (CH ), 49.2, 49.4, 49.6 (3
3
3
CNHR), 55.0, 67.2, 67.3, 67.43, 67.49, 70.5, 71.6, 73.0, 73.2, 73.4, 74.4,
127.6, 127.7, 127.8, 127.9, 128.0, 128.0, 128.3, 128.3, 128.4, 128.4,
128.4, 128.5, 128.62, 128.65, 128.65 (15 d, Ar), 136.2, 136.3, 137.4,
silica gel chromatography (CHCl
MeOH/H O 1:1). The collected fractions containing product with
90% purity were concentrated and further purified by HPLC (20 mM
NH HCO /MeCN) to afford compound 20 (5.0 mg, 62%) as a white
solid after lyophilization. H NMR (600 MHz, D O) δ 5.47 (brt, J =
2
3 4
/MeOH/NH OH 7:3:0.5 to
31
1
3
37.8, 138.1 (5 s, Ar), 169.9 (CO). P NMR (CDCl , 400 MHz) δ
2
3
+
2.4. HRMS (ESI-TOF) calcd for C H N O P (M + H) , 749.3350;
44
50
2
7
found, 749.3337.
4
3
1
((2S,3S,4R,5R,6S)-3-Acetamido-4,5-dihydroxy-6-(hydroxymethyl)-
piperidin-2-yl)methylphosphonic Acid (17). A solution of 16 (0.059
mmol, 37 mg) in MeOH (2 mL) was treated with Pd/C (10%,
Degussa type, 5.3 mg) and stirred at rt for 3 d under a hydrogen
atmosphere. The mixture was filtered, and the solvent was removed in
vacuo. Purification by column chromatography on RP silica gel (mini
6.3 Hz, lipid-CCHCH O,1H), 5.26−5.17 (m, 2 × CCH, 2H),
2
4.48 (t, J = 6.8 Hz, lipid-CCHCH O, 2H), 3.99 (brd, J = 7.1 Hz,
2
1H, 3-H), 3.93 (dd, J = 12.0, 2.8 Hz, 1H, 7-H ), 3.63 (m, 3H, 7-H , 2-
a
b
H, 4-H), 3.32 (t, J = 9.1 Hz, 1H, 5-H), 3.04 (brs, 1H, 6-H), 2.21−1.92
(m, lipid-CH , NHAc, 1-H, 13H), 1.74 (s, lipid-CH , 3H), 1.70 (s,
2
3
13
column, 2 g RP silica, eluent: H O) led to the isolation of L-ido-17 (6.8
mg, 47%) as a colorless solid. H NMR (600 MHz, D O) δ 4.31 (m,
lipid-CH , 3H), 1.64 (s, lipid-CH , 6H). C NMR (151 MHz, D O) δ
2
3
3
2
1
174.3 (CO), 160.2 (NH HCO ), 143.1, 136.6, 133.5 (lipid-CH C
2
4
3
3
1
H, 3-H), 4.04−4.02 (m, 2H, 4-H and 5-H), 3.96−3.90 (m, 1H, 2-H),
CH), 124.4, 124.2 (2 × CCH), 119.4, 119.3 (lipid-CCHCH O),
2
3.88 (dd, J = 12.1, 4.5 Hz, 1H, 7-H ), 3.85−3.79 (m, 1H, 7-H ), 3.69
72.4 (C-5), 71.1 (C-4), 62.9, 62.8 (lipid-CCHCH O), 61.7 (C-7),
a
b
2
(
dd, J = 9.8, 3.3 Hz, 1H, 6-H), 2.06 (s, 3H, NHAc), 1.91−1.83 (m, 2H,
54.1 (C-6), 53.3 (C-3), 50.3, 50.3 (C-2), 38.7, 38.7, 25.7, 25.5 (4C,
8
635
dx.doi.org/10.1021/jo501340s | J. Org. Chem. 2014, 79, 8629−8637

The Journal of Organic Chemistry
Article
lipid-CH ), 24.8 (lipid-CH ), 24.5, 23.6 (C-1), 22.0 (NHCOCH ),
AI072155, and by the Alexander von Humboldt Foundation
(Feodor Lynen Research Fellowships for M.S. and S.E.).
2
3
3
31
1
6.9 (lipid-CH ), 15.6 (lipid-CH ), 15.2 (lipid-CH ). P NMR (202
3 3 3
MHz, D O) δ 14.27 (d, J = 23.7 Hz), −10.71 (d, J = 26.3 Hz). HRMS
2
−
(
ESI-TOF) calcd for C H N O P (M − H) , 581.2387; found,
24
44
2
10
2
REFERENCES
■
5
81.2373.
((2S, 3S, 4R, 5R, 6R)-3-Acetamido-4, 5-dihydroxy-6-
hydroxymethyl)piperidin-2-yl)methyl)phosphonic((2Z,6E,10E)-
,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-yl phosphoric)
Anhydride (1). A round-bottomed flask containing lipid phosphate
9 (13.3 mg, 0.036 mmol) was dried in vacuo for 1 h. Anhydrous
(
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1
DMF (1 mL) was then added to the flask followed by the addition of
CDI (23.3 mg, 0.144 mmol). The resulting solution was kept stirring
for 3 h. MeOH (25 μL) was added to quench the extra CDI for 1.5 h.
The reaction residue was then transferred to a vial containing
compound 2 (3.6 mg, 0.012 mmol). The reaction was completed
when the reaction mixture turned from cloudy to clear after 48 h of
stirring. The mixture was transferred to a round-bottomed flask, and
the vial was washed with MeOH (1 mL) three times. The solvent was
removed in vacuo and purified by silica gel chromatography (CHCl3/
MeOH/NH OH 7:3:0.5 to MeOH/H O 1:1). The collected fractions
4
2
containing product with 90% purity were concentrated and further
purified by HPLC (20 mM NH HCO /MeCN) to afford compound 1
4
3
1
(
4.3 mg, 56%) as a white solid after lyophilization. H NMR (600
MHz, D O) δ 5.38 (m, lipid-CCHCH O, 1H), 5.13 (m, 2 × C
2
2
CH, 3H), 4.39 (t, J = 6.6 Hz, lipid-CCHCH O, 2H), 3.88−3.83 (m,
2
2
9
H, 3-H and 7-H ), 3.52−3.47 (m, 3H, 7-H , 2-H, 4-H), 3.19 (t, J =
a
b
.3 Hz, 1H, 5-H), 2.90 (m, 1H, 6-H), 2.12−1.80 (m, 17H, lipid-CH ,
2
NHAc, 1-H), 1.66 (s, 3H, lipid-CH ), 1.62 (s, 3H, lipid-CH ), 1.55
3
3
13
(
m, 9H, lipid-CH3). C NMR (151 MHz, D O) δ 174.3 (CO),
2
1
1
7
5
60.3 NH HCO ), 143.1, 136.6, 136.4, 133.4 (lipid-CH CCH),
4
3
3
24.5, 124.4, 124.3 (3 × CCH), 119.4, 119.3 (lipid-CCHCH O),
2
(11) Shih, H. W.; Chen, K. T.; Chen, S. K.; Huang, C. Y.; Cheng, T.
J. R.; Ma, C.; Wong, C. H.; Cheng, W. C. Org. Biomol. Chem. 2010, 8,
2.9 (C-5), 71.3 (C-4), 62.8, 62.8 (lipid-CCHCH O), 62.2 (C-7),
2
4.0 (C-6), 53.8, 53.7 (C-3), 50.3 (C-2), 38.8, 38.7, 25.6, 25.6, 25.5
2
586.
(
(
1
6C, lipid-CH ), 24.8 (lipid-CH ), 24.7, 23.8 (C-1), 22.0
2
3
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NHCOCH ), 16.9 (lipid-CH ), 15.6 (lipid-CH ), 15.2 (lipid-CH ),
3
3
3
3
31
5.2 (lipid-CH3). P NMR (202 MHz, D O) δ 14.66 (brs), −10.74
2
(
d, J = 26.5 Hz). HRMS (ESI-TOF) calcd for C H N O P (M −
29
52
2
10
2
−
H) , 649.3013; found, 649.3040.
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ASSOCIATED CONTENT
■
(
*
S
Supporting Information
1
H NMR, C NMR, and 31P NMR data, NOEs of compound
13
(
7
a and 17, and substrate−velocity curve of 1. This material is
1
available free of charge via the Internet at http://pubs.acs.org.
(
Smits, E.; Weber, M.; van Wiltenburg, J.; Wolberg, M.; Aerts, J. M. F.
AUTHOR INFORMATION
G.; Overkleeft, H. S. Org. Process Res. Dev 2008, 12, 414.
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Corresponding Author
E-mail: chwong@gate.sinica.edu.tw.
*
Present Addresses
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1998, 37, 2893. (b) Mitchell, M. L.; Tian, F.; Lee, L. V.; Wong, C. H.
Angew. Chem., Int. Ed. 2002, 41, 3041.
∥
(
C.-H.H) Department of Molecular and Experimental
Medicine, The Scripps Research Institute, La Jolla, California
2037, United States.
(
2
(
1
21) D’Alonzo, D.; Guaragna, A.; Palumbo, G. Curr. Med. Chem.
009, 16, 473.
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(24) Gerding, D. N.; Muto, C. A.; Owens, R. C. Clin. Infect. Dis.
008, 46, S32.
9
⊥
(
M.S.) Synthesis and Homogeneous Catalysis, BASF SE, Carl-
Bosch Str. 38, 67056 Ludwigshafen, Germany.
#
(
S.E.) Advanced Materials and Systems Research, BASF SE,
(
Carl-Bosch-Str. 38, 67056 Ludwigshafen, Germany.
2
Author Contributions
§
(25) Liu, C. Y.; Guo, C. W.; Chang, Y. F.; Wang, J. T.; Shih, H. W.;
Hsu, Y. F.; Chen, C. W.; Chen, S. K.; Wang, Y. C.; Cheng, T. J. R.; Ma,
C.; Wong, C. H.; Fang, J. M.; Cheng, W. C. Org. Lett. 2010, 12, 1608.
(26) (a) Perlstein, D. L.; Wang, T. S. A.; Doud, E. H.; Kahne, D.;
Walker, S. J. Am. Chem. Soc. 2010, 132, 48. (b) Ye, X. Y.; Lo, M. C.;
Brunner, L.; Walker, D.; Kahne, D.; Walker, S. J. Am. Chem. Soc. 2001,
Che-Hsiung Hsu and Mathias Schelwies contributed equally to
this work.
Notes
The authors declare no competing financial interest.
1
(
23, 3155.
27) Huang, S. H.; Wu, W. S.; Huang, L. Y.; Huang, W. F.; Fu, W. C.;
ACKNOWLEDGMENTS
■
This work was supported by the National Science Council and
Academia Sinica, by National Institute of Health grant
Chen, P. T.; Fang, J. M.; Cheng, W. C.; Cheng, T. J. R.; Wong, C. H. J.
Am. Chem. Soc. 2013, 135, 17078.
8
636
dx.doi.org/10.1021/jo501340s | J. Org. Chem. 2014, 79, 8629−8637

The Journal of Organic Chemistry
Article
(
28) Gloster, T. M.; Zandberg, W. F.; Heinonen, J. E.; Shen, D. L.;
Deng, L. H.; Vocadlo, D. J. Nat. Chem. Biol. 2011, 7, 174.
8
637
dx.doi.org/10.1021/jo501340s | J. Org. Chem. 2014, 79, 8629−8637