Synthesis and biological evaluation of potential new inhibitors of the bacterial transferase MraY with a β-ketophosphonate structure

Article abstract of DOI:10.1039/c1ob06124k

Stable analogs of bacterial transferase MraY substrate or product with a pyrophosphate surrogate in their structure are described. β- ketophosphonates were designed as pyrophosphate bioisosteres and were investigated as UDP-GlcNAc mimics. The developed st

Full text of DOI:10.1039/c1ob06124k

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PAPER
Synthesis and biological evaluation of potential new inhibitors of the bacterial
transferase MraY with a b-ketophosphonate structure†
Nicolas Auberger,a Rok Frlan,‡a Bayan Al-Dabbagh,§b Ahmed Bouhss,b Muriel Crouvoisier,b
Christine Gravier-Pelletier*a and Yves Le Merrera
Received 8th July 2011, Accepted 14th September 2011
DOI: 10.1039/c1ob06124k
Stable analogs of bacterial transferase MraY substrate or product with a pyrophosphate surrogate in
their structure are described. b-ketophosphonates were designed as pyrophosphate bioisosteres and
were investigated as UDP-GlcNAc mimics. The developed strategy allows introduction of structural
diversity at a late stage of the synthesis. The biological activity of the synthesized compounds was
evaluated on the MraY enzyme.
ceptor undecaprenyl phosphate (C55-P) yielding undecaprenyl-
pyrophosphoryl-MurNAc-pentapeptide (lipid I) and releasing
Introduction
uridine monophosphate (UMP). Thereafter, MurG6 catalyzes the
transfer of the GlcNAc moiety from UDP-GlcNAc to lipid I
yielding undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)-
GlcNAc (lipid II). Lipid II is then translocated through the
membrane by a specific flippase. This translocation activity was
shown for FtsW7 from E. coli. In the outside of the membrane
the lipid II is then used as a substrate for the polymerisation
reactions, transglycosylation and transpeptidation, catalyzed by
the penicillin-binding proteins (PBPs) and the monofunctional
transglycosylases (MGTs).8
The enzymes involved in this biosynthetic pathway have been
demonstrated as ubiquitous and essential to bacteria2,3,4a,8,9 and
any anomaly in this biosynthesis leads to cell lysis. Therefore,
enzymes implicated in this process are ideal targets in the search
for new antibacterials.10 Indeed, several families of widely used
antibiotics, such as b-lactams and lipoglycopeptides, are already
known to interfere with certain enzymes implicated in pepti-
doglycan biosynthesis. However, because of the ever-increasing
resistance11 to these antibacterial agents and in the context of a
program12 directed to the synthesis and biological evaluation of
new antibacterials, we are focusing on the development of MraY
inhibitors. Indeed, this enzyme has been little exploited so far as a
consequence of its trans-membrane localisation.13 Therefore, since
there is no drug in clinical use targeting this essential enzyme,
this can be expected to delay the emergence of resistance. Our
goal is to target compounds which are stable analogs of MraY
substrate or product and displaying a pyrophosphate surrogate in
their structure (Fig. 3). Furthermore, some of these compounds
can also be considered as potential inhibitors of the MurG
enzyme due to their structural analogy with the substrate of this
enzyme. Results concerning the synthesis of a first compound
have been published in a preliminary form14 and we are now
aiming at reporting our full results dealing with a series of related
compounds.
Enzymes involved in peptidoglycan biosynthesis are key targets for
the development of new antibiotics because there are no parallels
of this pathway in eukaryotes. Peptidoglycan is a heteropolymer
of bacterial cell walls consisting of long glycan chains made of b-
1→4 alternating units of N-acetylmuramoyl-peptides (MurNAc-
peptides) and N-acetylglucosamine (GlcNAc) which are cross-
linked together through the short peptide chains.1 This macro-
molecule protects the cell from internal osmotic pressure effects
and contributes to the cell shape maintenance. Peptidoglycan
biosynthesis is a complex process which takes place in various
cell compartments: cytoplasm, membrane and periplasm2 (Fig.
1).
UDP-N-acetylglucosamine (UDP-GlcNAc) is first transformed
into UDP-N-acetylmuramoyl-pentapeptide (UDP-MurNAc-
pentapeptide) by the Mur enzymes (MurA, MurB, MurC, MurD,
MurE and MurF).3 Then, at the plasma membrane, two distinctive
proteins (MraY and MurG) synthesize polyprenyl-linked precur-
sors (lipid I and lipid II) that carry one complete cell-wall subunit
(Fig. 2).4
Thus, MraY5 transfers the phospho-MurNAc-pentapeptide
moiety from the cytoplasmic precursor to the membrane ac-
aUniversite´ Paris Descartes, UMR 8601 CNRS, Laboratoire de Chimie et
Biochimie Pharmacologiques et Toxicologiques, 45 rue des Saints Pe`res,
75006 Paris, France. E-mail: christine.gravier-pelletier@parisdescartes.fr;
Fax: +33 142868387; Tel: +33 142862181
bUniv Paris-Sud 11, Laboratoire des Enveloppes Bacte´riennes et Antibio-
tiques, Institut de Biochimie et Biophysique Mole´culaire et Cellulaire, UMR
8619, Orsay F-91405, France
† Electronic supplementary information (ESI) available: 1. Numbering
system, 2. Comprehensive experimental section, 3. 1H, 13C and 31P NMR
spectra. See DOI: 10.1039/c1ob06124k
‡ Present address: University of Ljubljana, Faculty of Pharmacy,
Asˇkercˇeva, 1000 Ljubljana, Slovenia.
§ Present address: Department of Internal Medicine, Faculty of Medicine
and Health Sciences, United Arab Emirates University, PO Box 17666, Al
Ain, United Arab Emirates.
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Fig. 1 Peptidoglycan biosynthesis pathway.
Fig. 2 Enzymatic reactions catalyzed by MraY and MurG enzymes.
regards to hydrolysis. Furthermore, the carbonyl group is proposed
as an alternative electrophilic center to the one involved in the
enzymatic reaction. The b-ketophosphonate moiety displaying
both a phosphonate anion and a polarized carbonyl group should
be able to chelate the Mg2+ co-factor. Consequently, the targeted b-
ketophosphonate pyrophosphate bioisosteres should be pertinent
mimics of the substrate or the product, since they have the required
electronic properties to coordinate the metallic co-factor of the
reaction as well as being stable to hydrolysis.
Fig. 3 General structure of MraY and MurG substrates and products
and targeted b-ketophosphonate isosteres.
The pyrophosphate present in substrate and product of the
reaction catalyzed by MraY is on one hand linked to a GlcNAc
derivative and on the other hand to either uridine or undecaprenol.
This diphosphate moiety is supposed to interact with the divalent
cation Mg2+ which is a co-factor of the enzymatic reaction,
suggested to proceed in either one15 or two steps.16 Furthermore,
it is prone to hydrolysis due to its positioning in the anomeric
position of the glycosyl derivative. That is why our strategy has
been to replace this pyrophosphate, site of the enzymatic reaction,
by a bioisostere displaying a b-ketophosphonate structure. In the
resulting compounds, the replacement of the sugar anomeric oxy-
gen atom is expected to enhance the stability of the inhibitors with
Results and discussion
The design and synthesis of pyrophosphate analogs of glycosyl
nucleotides have already been described (Fig. 4). Thus, methy-
lene diphosphonate17 and 1-C-phosphonophosphate18 analogs of
UDP-Glc or UDP-GlcNAc, respectively, have been reported.
Different ways of diphosphate-type connections between GlcNAc
and uridine moieties have also been investigated.19 In some cases,
the pyrophosphate has been replaced by a propenyl phosphonate,20
a hydroxypropyl phosphonate21 or a propyl phosphate.22
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Fig. 5 Retrosynthetic analysis.
has been chosen to mimic either the substrate or the product of
the reaction catalyzed by the transferase MraY. Accordingly, the
uridine moiety present in the substrate has been introduced as it is
or as a C5-alkyl uracil analog and the undecaprenyl chain, part of
lipid I, has been replaced by a citronellyl or a simple hexadecanyl
moiety.
The ester building block 3 (Scheme 1) was synthesized from
orthogonally protected a-1-C-allyl-N-acetylglucosamine 1 readily
obtained in four steps (50% overall yield as an a/b mixture of
anomers) from commercial GlcNAc.31 Protection of the secondary
alcohol function as its tert-butyldimethylsilyl ether derivative 2
allowed isolation of pure a anomer from the 24/1 a/b mixture.
Finally, ozonolysis of the alkene under basic conditions in the
presence of methanol gave the ester 3.32
Fig. 4 Structures of pyrophosphate analogs of glycosyl nucleotides
previously described.
Other mimics have no phosphorus atoms in their structure,
the pyrophosphate moiety being changed to a malonic linkage,23
a,b-ethylenic ester or amide with or without dihydroxylation of
the double bond.24 In addition, an oxycarbonyl aminosulfonyl
linker25 and a carbonyl linked to O- or N-sulfamoyl uridine26
have also been reported as well as allophanates and biuret
derivatives.27 Galactose-linked uridine derivatives without charge
or dipole contributions in the linker have been synthesized via
cross metathesis.28 Finally, replacement of the pyrophosphate by
a triazole moiety has also been recently described.29 However,
none of these pyrophosphate analogs display a glycosyl b-
ketophosphonate skeleton. Nevertheless, it should be mentioned
that a uridine-containing phosphonate ester analog has previously
been synthesized and tested as inhibitor of solubilised E. coli
transferase MraY (IC50 3.7 mM).30
Scheme 1 Synthesis of C-glycosyl ester 3. Reagents and conditions: a)
TBSCl, imidazole, DMF, 97%; b) O3, CH2Cl2, MeOH, NaOH, 65%.
According to path a, we next turned to the synthesis of
unsymmetrical methylphosphonates 7 and 8 substituted by an
uridinyl or a citronellyl moiety. Their preparation (Scheme 2)
involved NaH treatment, in the presence of methyl iodide,
of commercially available dibenzyl H-phosphonate, leading to
dibenzyl methylphosphonate 4. It was followed by benzyl mono-
deprotection in the presence of DABCO in refluxing toluene33 to
afford monobenzyl methylphosphonate 5. Esterification with iso-
propylidene N-Boc uridine34 or citronellol was performed under
Mitsunobu conditions in the presence of triphenyl phosphine and
diisopropyl azodicarboxylate affording the phosphonate 7 or 8 as
a diastereomeric mixture.
Chemical synthesis
Two complementary strategies to obtain the targeted com-
pounds have been investigated (Fig. 5). On one hand, the
retrosynthesis relies on the condensation of variously substituted
lithiomethylenephosphonates on the ester function of a conve-
niently protected methyl N-acetylglucosaminyl acetate (path a)
and on the other hand it involves the esterification of a b-
ketophosphonate derivative using different alcohol derivatives
(path b). The residue R3 to be introduced on the phosphonate
Access to the targeted b-ketophosphonates 9 and 10 was then
tentatively carried out, by condensation on the ester 3 of lithio
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ence of n-butyllithium, on the ester 3 giving the dibenzyl b-
ketophosphonate 11. Then, mono-benzyl deprotection in the
presence of DABCO led to 12 and was followed by the ester-
ification with isopropylidene N-Boc uridine under Mitsunobu
conditions in the presence of triphenylphosphine and diisopropyl
azodicarboxylate affording the targeted compound 9. It has to
be mentioned that N-Boc protection of uridine is necessary to
achieve this reaction since the esterification of 12 with isopropy-
lidene uridine under the same conditions was unsuccessful. In
order to compare biological activity, compound 11 was also
submitted to simultaneous acidic hydrolysis of benzylidene acetal
and silyl ether to afford 13 which was then mono-deprotected
with DABCO followed by DOWEX H+ treatment to provide
14. The complete deprotection of the phosphonate 9 was then
undertaken. Acidic hydrolysis was first performed and allowed
the simultaneous removal of benzylidene, isopropylidene, tert-
butyldimethylsilyl and Boc groups. Finally, cleavage of the benzyl
ester was achieved by hydrogenolysis in the presence of palladium
on charcoal in methanol leading to 16 as a first analog of the MraY
substrate.
In a complementary manner, access to other substrate or
product analogs of the reaction catalyzed by MraY were targeted
(Scheme 5). Therefore, on the one hand, a simplified structure of
the uridine moiety has been chosen in which the ribose of uridine
has been replaced by a C5 acyclic alkyl chain,35 retaining the same
five bond linkage between the alcohol function and the uracil
residue as in uridine. The imide function present in this 1-(5¢-
hydroxypentyl)uracil was protected as its tert-butyl carbamate 17
to avoid side reactions. On the other hand, the undecaprenyl chain
present within the product of the enzymatic reaction was replaced
by either citronellyl or hexadecanyl. The citronellyl moiety has
been preferred to a terpenic alcohol due to the better stability of
homo-allylic phosphonate in acidic conditions compared to allylic
phosphonate. Accordingly, phosphonate 12 has been reacted with
1-(5¢-hydroxypentyl)-N-Boc uracil 17, citronellol and hexade-
canol, under Mitsunobu conditions, affording the protected b-
ketophosphonates 18, 10 and 19, respectively, as diastereomeric
mixtures. Acidic hydrolysis of acid labile protective groups was
then carried out in the presence of 1/1 TFA/H2O which led
to 20, 21 and 23, respectively. Compound 21 resulted from the
addition of water on the citronellyl double bond, catalyzed by
the acidic conditions that were used. To limit this side reaction,
the acidic hydrolysis of citronellyl b-ketophosphonate 10 was
also performed in smoother conditions in the presence of 1/1/1
TFA/H2O/THF and afforded 22. Final benzyl ester cleavage of
compounds 20, 21 and 23 was achieved by hydrogenolysis in the
presence of palladium on charcoal 10% leading to the final b-
ketophosphonates 24, 25 and 26, respectively.
Scheme 2 Synthesis of unsymmetrical methyl phosphonates 7 and 8.
Reagents and conditions: a) i. NaH, THF, -15 ◦C to r.t.; ii. MeI, -40 ◦C to
r.t., 80%; b) i. DABCO, toluene, reflux; ii. H+, H2O, 82%; c) DIAD, PPh3,
THF, 83% for 7 and 95% for 8.
derivatives of phosphonates 7 and 8, generated in the presence
of n-butyllithium (Scheme 3). However, even if these conditions
proved successful for the synthesis of the b-ketophosphonate 10,
they did not allow that of the uridinyl related compound 9. It
has to be noted that at least 3 equivalents of the nucleophilic
entity are required to achieve the reaction due to both the acidity
of the acetamide and that of the methylene within the resulting
b-ketophosphonate. However, this excess of reagent renders its
separation from the formed compound 10 difficult and precludes
its use in higher excess in order to improve the yield.
Biological evaluation
Scheme 3 Towards targeted b-ketophosphonates 9 and 10 according to
path a. Reagents and conditions: a) i. nBuLi, THF, -78 ◦C, 1 h; ii. 3, THF,
-78 ◦C to r.t., 57% for 10.
The in vitro biological evaluation (Table 1) of compounds 13–16
and 20–26, on MraY purified from Bacillus subtilis, was carried
out as described in the experimental section. The residual activity
of the enzyme was measured in the presence of 1 mM of the tested
compounds. For the most active inhibitors, the IC50 value was
determined.
In an alternative manner, the synthesis of the b-
ketophosphonate 9 was envisaged according to the path b
(Scheme 4). It first involved the condensation of the dibenzyl
methylenephosphonate anion, generated from 4 in the pres-
Commercially available tunicamycin from Streptomyces sp. was
used as a positive control in the tests and resulted in an IC50 value
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Scheme 4 Synthesis of substrate analog 16 according to path b. Reagents and conditions: a) i. nBuLi, -78 ◦C, 1 h; ii. 3, -78 ◦C to r.t., 83%; b) i. DABCO,
toluene, reflux; ii. Dowex H+, MeOH, 90% for 12 and 88% for 14; c) TFA/H2O: 1/1, 97% for 13 from 11 and 94% for 15; d) 6, DIAD, PPh3, THF, 46%
or PyBOP, DIEA, DMF, 20%; e) H2, Pd/C 10%, MeOH, 85%.
Scheme 5 Generalisation to the synthesis of b-ketophosphonates 24, 25 and 26. Reagents and conditions: a) DIAD, PPh3, THF, 27% for 18, 58% for
10, 42% for 19; b) TFA/H2O: 1/1, 93% for 20, 94% for 21, 90% for 23; c) TFA/H2O/THF: 1/1/1, 84% for 22; d) H2, Pd/C 10%, MeOH, 94% for 24,
100% for 25, 87% for 26.
Table 1 Inhibitory activity of synthesized compounds on the MraY enzyme
Compound
13
14
15
20
21
22
23
Bn
H
IC50 (mM)
Compound
NI
NI
NI
0.94
0.19
NI
0.82
16
24
25
26
IC50 (mM)
NI
NI
1.3
0.85
NI: no inhibition at 1 mM concentration.
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equal to 0.012 mM. Furthermore, the inhibitors were also tested
on the MurG enzyme from E. coli which catalyzes the second
membrane step of peptidoglycan biosynthesis (Fig. 1), leading to
lipid II formation.
monitored by thin-layer chromatography with Merck 60F-254
precoated silica (0.2 mm) on glass. Flash chromatography was
performed with Merck Kieselgel 60 (200–500 mm); the solvent
systems were given v/v. Spectroscopic 1H, 13C and 31P NMR, MS
and/or analytical data were obtained using chromatographically
homogeneous samples.
The detected inhibitory activities were proven to be rather weak
concerning MraY inhibition since compounds 20, 23, 25 and
26 resulted in IC50 values in the mM range while compounds
13, 14, 15, 16, 22 and 24 proved not to be inhibitors at 1 mM
concentration. Interestingly, compound 21 exhibited significant
inhibitory activity with an IC50 value equal to 0.19 mM. Finally,
no inhibition of MurG activity was observed for the tested
compounds at 1 mM concentration. Therefore, the replacement
of the phosphorus atom within the pyrophosphate moiety by
a carbonyl group leads to low inhibitory activity which could
indicate the requirement of a tetrahedral geometry instead of a
trigonal one, in the related position, to ensure accurate interaction
of the inhibitor with the enzyme. Furthermore, by comparison
with compound 22, the activity observed for compound 21 is
encouraging and suggests a possible interaction with the enzyme
through a hydrogen bond involving the hydroxyl group present in
the inhibitor.
Benzyl (3-N-tert-butyloxycarbonyl-2¢,3¢-O-isopropylidene)-uridin-
5¢-yl 3-(2-acetamido-4,6-O-(R)-benzylidene-3-O-tert-butyl-
dimethylsilyl-2-deoxy-a-D-glucopyranosyl)-2-oxopropyl-
phosphonate 9
At 0 ◦C, DIAD (95 mL, 0.47 mmol, 1.5 eq.) was added dropwise to
a solution of 12 (200 mg, 0.32 mmol, 1 eq.), 6 (121 mg, 0.32 mmol,
1 eq.) and PPh3 (124 mg, 0.47 mmol, 1.5 eq.) in THF (5 mL).
The reaction mixture was stirred at r.t. for 16 h then concentrated.
Purification by flash chromatography (CH2Cl2/acetone, 8 : 2 to
7 : 3) afforded 9 (146 mg, 46%, white solid) as a mixture of epimers
(d.r. = 2/1): Rf 0.46 (CH2Cl2/acetone = 7 : 3); 1H NMR d 7.50–7.34
(m, 10H, Har), 7.28, 7.27 (2d, 1H, JH6u–H5u = 8.1 Hz, H6u◦, H6u*),
6.28 (d, 1H, JNH–H2¢ = 8.2 Hz, NH), 5.71, 5.66 (2d, 1H, JH5u–H6u = 8.1
Hz, H5u◦, H5u*), 5.66, 5.58 (2d, 1H, JH1¢¢–H2¢¢ = 2.1 Hz, H1¢¢*, H1¢¢◦),
5.50 (s, 1H, H7¢), 5.18–5.08 (m, 2H, CH2Ph), 4.98, 4.96 (2dd, 1H,
JH2¢¢–H3¢¢ = 6.4 Hz, JH2¢¢–H1¢¢ = 2.1 Hz, H2¢¢*, H2¢¢◦), 4.81 (dd, 0.7H,
JH3¢¢*–H2¢¢* = 6.4 Hz, JH3¢¢*–H4¢¢* = 3.7 Hz, H3¢¢*), 4.79–4.73 (m, 1.3H,
H1¢, H3¢¢◦), 4.37–4.33, 4.33–4.29 (2 m, 1H, H4¢¢◦, H4¢¢*), 4.29–4.19
(m, 2H, H2¢, H5¢¢a), 4.17–4.09 (m, 2H, H5¢¢b, H6¢eq), 3.85–3.77 (m, 1H,
H3¢), 3.67–3.61 (m, 1H, H6¢ax), 3.54–3.46 (m, 2H, H4¢, H5¢), 3.14, 3.08
(2dd, 1H, JH1a–P = 22.3 Hz, JH1a–H1b = 13.9 Hz, H1a◦, H1a*), 3.11,
2.99 (2dd, 1H, JH3a–H3b = 16.6 Hz, JH3a–H1¢ = 7.0 Hz, H3a◦, H3a*◦),
Conclusion
In summary, we have developed a simple and efficient method for
the synthesis of new pyrophosphate analogs of glycosyl nucleotides
from N-acetylglucosamine. The strategy offers the advantage of
allowing introduction of structural diversity at a late stage of the
synthesis rendering easy access to a series of compounds. The
method has been illustrated by the synthesis of several analogs
of MraY substrate or product. The activity of the synthesized
compounds has been evaluated on purified MraY and MurG.
Unexpectedly, the inhibitory activities were low, except for one
compound and the reason for this weak activity could lie in
the change in the geometry of the phosphorus atom, site of the
enzymatic reaction, probably preventing efficient recognition of
the compounds by the enzyme. This study led to reaching some
insight into the requirements for inhibition of the trans-membrane
protein MraY. Taking into account these results, our current work
is now focusing on new pyrophosphate mimics with a tetrahedral
geometry in the b position of the pyrophosphate. Special attention
will also be paid to the introduction of the pentapeptide side chain
in C3 position of GlcNAc moiety since it can be important for
binding to the enzyme.
3.07, 3.04 (2dd, 1H, JH1b–P = 22.9 Hz, JH1b–H1a = 13.9 Hz, H1b
,
H1b*), 2.75, 2.70 (2dd, 1H, JH3b–H3a = 16.6 Hz, JH3b–H1¢ = 6.6 Hz,
H3b◦, H3b*), 1.93, 1.91 (2 s, 3H, CH3CO*, CH3CO◦), 1.59, 1.58
(2 s, 9H, CO2tBu*, CO2tBu◦), 1.55, 1.36, 1.34 (3 s, 6H, CMe2),
0.83 (s, 9H, SitBu), 0.08, -0.01 (2 s, 4H, SiMe*), 0.07, -0.01 (2
s, 2H, SiMe◦); 13C NMR d 197.8 (d, JC2–P = 6.5 Hz, C2), 170.7,
170.7 (CH3CO◦, CH3CO*), 160.4 (C4u), 148.3 (C2u), 147.6, 147.6
(COBoc◦, COBoc*), 141.7, 141.3 (C6u◦, C6u*), 137.3 (Cqar), 135.3 (d,
JCq–P = 5.0 Hz, Cqar), 129.3, 129.1, 129.0, 128.5, 128.3, 128.2, 126.4,
◦
126.4 (CHar), 114.7, 114.7 (CMe2*, CMe2 ), 102.1, 10◦2.1, 102.0,
101.9 (C5u, C7¢), 95.7, 95.0 (C1¢¢◦, C1¢¢*), 87.4, 87.3 (CMe3 , CMe3*),
86.0, 85.8 (2d, JC4¢¢–P = 7.0 Hz, C4¢¢◦, C4¢¢*), 84.5, 84.5 (C2¢¢*, C2¢¢◦),
83.2, 83.2 (C4¢◦, C4¢*), 80.5 (C3¢¢), 70.7, 70.6 (C1¢◦, C1¢*), 70.1, 70.1
(C3¢*, C3¢◦), 69.2 (C6¢), 69.0, 68.9 (2d, JCH2Ph–P = 6.5 Hz, CH2Ph*,
CH2Ph◦), 65.7, 65.6 (2d, JC5¢¢–P = 6.5 Hz, C5¢¢◦, C5¢¢*), 65.3, 65.3
◦
(C5¢*, C5¢◦), 54.1, 54.1 (C2¢◦, C2¢*), 43.8, 43.7 (C3*, C3 ), 42.0, 41.8
Experimental
◦
◦
(2d, JC1–P = 127.0 Hz, C1*, C1 ), 27.6 (CMe3), 27.2, 25.4 (CMe2 ),
27.1, 25.3 (CMe2*), 25.8 (SitBu), 23.2, 23.1 (CH3CO*, CH3CO◦),
18.2 (SitBu), -3.9, -4.7 (SiMe); 31P NMR d 21.9 (s, 0.66P, P*), 21.7
(s, 0.33P, P◦); MS (ESI): m/z = 1022 [M + Na]+ 100%; HRMS calcd
for C48H66N3NaO16PSi+ 1022.3848, found 1022.3858.
Chemical synthesis
1H NMR (500 MHz), 13C NMR (125 MHz) and 31P NMR
(202 MHz) spectra were recorded in CDCl3 unless indicated
on a Bruker Avance or Avance II. Chemical shifts (d) are
reported in ppm and coupling constants are given in Hz. Optical
rotations were measured on a Perkin-Elmer 341 polarimeter with
a sodium (589 nm) lamp at 20 ◦C. Mass spectra, electrospray
(ESI) and high resolution (HRMS) were recorded by the Service
de Spectrome´trie de Masse, ICSN Gif sur Yvette. Tunicamycin
Benzyl (R)-citronellyl 3-(2-acetamido-4,6-O-(R)-benzylidene-3-
O-tert-butyldimethylsilyl-2-deoxy-a-D-glucopyranosyl)-2-
oxopropylphosphonate 10
Condensation of 8 on 3. The reaction was performed as
described for 11 with a solution of 8 (446 mg, 1.37 mmol, 3 eq.) in
THF (10 mL), nBuLi (630 mL, 1.42 mmol, 3.1 eq.) and a solution
R
from Streptomyces sp. was purchased from Sigma-Aldrichꢀ. All
reactions were carried out under a nitrogen atmosphere, and were
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of 3 (220 mg, 0.46 mmol, 1 eq.) in THF (5 mL). Two successive
purifications by flash chromatography (CH2Cl2/acetone, 9 : 1 and
cyclohexane/acetone, 2 : 1) afforded 10 (205 mg, 57%, pale yellow
oil) as a mixture of epimers (d.r. = 52/48).
organic layers were dried (MgSO4) and concentrated. Purification
by flash chromatography (CH2Cl2/acetone, 8 : 2) afforded 11 (600
mg, 83%, white solid): Rf 0.47 (CH2Cl2/acetone = 8 : 2); [a]2D0 +26
(c 1.0, CH2Cl2); IR (cm-1) 1718 (CO), 1250 (PO); 1H NMR d 7.51–
7.47 (m, 2H, Har), 7.42–7.30 (m, 13H, Har), 6.62 (d, 1H, JNH–H2¢
8.7 Hz, NH), 5.50 (s, 1H, H7¢), 5.12, 5.02 (AB from ABX, 2H, JAB
11.6 Hz, JA–P = 9.5 Hz, JB–P = 10.0 Hz, CH2Ph), 5.01 (d, 2H, JH–P
=
=
=
Mitsunobu with 12 and (R)-citronellol. At 0 ◦C, DIAD (70
mL, 0.36 mmol, 1.5 eq.) was added dropwise to a solution of 12
(150 mg, 0.24 mmol, 1 eq.), (R)-citronellol (44 mL, 0.24 mmol, 1
eq.) and PPh3 (93 mg, 0.36 mmol, 1.5 eq.) in THF (4 mL). The
reaction mixture was stirred at r.t. for 16 h then concentrated.
Two successive purifications by flash chromatography (cyclohex-
ane/acetone, 2 : 1 and CH2Cl2/acetone, 9 : 1) afforded 10 (107 mg,
58%, pale yellow oil) as a mixture of epimers (d.r. = 1/1): Rf 0.39
(cyclohexane/acetone = 2 : 1); IR (cm-1) 1719 (CO), 1250 (PO);
8.4 Hz, CH2Ph), 4.78 (ddd, 1H, JH1¢–H3a = 8.0 Hz, JH1¢–H2¢ = 5.7 Hz,
JH1¢–H3b = 5.1 Hz, H1¢), 4.31 (ddd, 1H, JH2¢–H3¢ = 9.8 Hz, JH2¢–NH
8.7 Hz, JH2¢–H1¢ = 5.7 Hz, H2¢), 4.15 (dd, 1H, JH6¢eq–H6¢ax = 10.3 Hz,
H6¢eq–H5¢ = 4.3 Hz, H6¢eq), 3.83 (dd, 1H, JH3¢–H2¢ = 9.8 Hz, JH3¢–H4¢ = 8.3
=
J
Hz, H3¢), 3.65 (dd, 1H, JH6¢ax–H6¢eq = 10.3 Hz, JH6¢ax–H5¢ = 9.2 Hz, H6¢ax),
3.51 (dd, 1H, JH4¢–H5¢ = 9.6 Hz, JH4¢–H3¢ = 8.3 Hz, H4¢), 3.47 (ddd, 1H,
J
H5¢–H4¢ = 9.6 Hz, JH5¢–H6¢ax = 9.2 Hz, JH5¢–H6¢eq = 4.3 Hz, H5¢), 3.19 (dd,
1H NMR d 7.51–7.33 (m, 10H, Har), 6.68, 6.54 (2d, 1H, JNH–H2¢
=
1H, JH3a–H3b = 17.1 Hz, JH3a–H1¢ = 8.0 Hz, H3a), 3.10, 3.05 (AB from
ABX, 2H, JAB = 13.5 Hz, JA–P = 23.2 Hz, JB–P = 21.9 Hz, H1a, H1b),
2.63 (dd, 1H, JH3b–H3a = 17.1 Hz, JH3b–H1¢ = 5.1 Hz, H3b), 1.85 (s, 3H,
CH3CO), 0.83 (s, 9H, SitBu), 0.10, 0.01 (2 s, 6H, SiMe); 13C NMR
d 197.4 (d, JC2–P = 4.7 Hz, C2), 170.7 (CH3CO), 137.4 (Cqar), 135.4,
135.3 (2d, JCq–P = 6.0 Hz, Cqar), 129.2, 129.1, 129.0, 129.0, 128.9,
128.5, 128.2, 128.2, 126.4 (CHar), 102.0 (C7¢), 83.4 (C4¢), 70.5 (C1¢),
70.1 (C3¢), 69.3 (C6¢), 68.6, 68.5 (2d, JCH2Ph–P = 6.5 Hz, CH2Ph),
65.7 (C5¢), 54.0 (C2¢), 43.9 (C3), 42.9 (d, JC1–P = 124.8 Hz, C1), 25.8
(SitBu), 23.0 (CH3CO), 18.2 (SitBu), -3.9, -4.7 (SiMe); 31P NMR
d 21.4 (s); MS (ESI): m/z = 1469 [2M + Na]+ 100%; HRMS calcd
for C38H50NNaO9PSi+ 746.2890, found 746.2898.
8.6 Hz, NH*, NH◦), 5.51, 5.50 (2 s, 1H, H7¢*, H7¢◦), 5.19–5.02 (m,
◦
◦
◦
◦
=
–H2¢
3H, CH2Ph, H6¢¢), 4.81 (ddd, 0.5H, JH1¢
= 7.9 Hz, JH1¢
–H3a
= 4.9 Hz, H1¢◦), 4.77 (ddd, 0.5H, JH1¢*–H3a* = 8.3
◦
◦
5.9 Hz, JH1¢
–H3b
Hz, JH1¢*–H2¢* = 5.9 Hz, JH1¢*–H3b* = 4.4 Hz, H1¢*), 4.36–4.28 (m, 1H,
H2¢), 4.20–4.13 (m, 1H, H6¢eq), 4.11–3.97 (m, 2H, H1¢¢), 3.87–3.80
(m, 1H, H3¢), 3.69–3.62 (m, 1H, H6¢ax), 3.57–3.43 (m, 2H, H4¢, H5¢),
◦
◦
◦
3.30 (dd, 0.5H, JH3a
= 17.0 Hz, JH3a
= 7.9 Hz, H3a◦), 3.22
◦
–H1¢
–H3b
(dd, 0.5H, JH3a*–H3b* = 17.2 Hz, JH3a*–H1¢* = 8.3 Hz, H3a*), 3.18 (dd,
◦
◦
◦
0.5H, JH1a
= 23.1 Hz, JH1a
= 13.5 Hz, H1a◦), 3.06, 3.06
◦
–H1b
–P
(AB from ABX, 1H, JAB = 13.3 Hz, JAX = 23.3 Hz, JBX = 21.6
◦
◦
◦
◦
◦
◦
Hz, H1a*, H1b*), 3.01 (dd, 0.5H, JH1b
= 21.8 Hz, JH1b
=
=
–P
–H1a
13.5 Hz, H1b◦), 2.74 (dd, 0.5H, JH3b
◦
◦
= 17.0 Hz, JH3b
–H1¢
–H3a
4.9 Hz, H3b◦), 2.59 (dd, 0.5H, JH3b*–H3a* = 17.2 Hz, JH3b*–H1¢* = 4.4
Hz, H3b*), 2.03–1.85 (m, 2H, H5¢¢), 1.89, 1.87 (2 s, 3H, CH3CO*,
CH3CO◦), 1.74–1.62 (m, 1H, H2¢¢a), 1.67 (s, 3H, H8¢¢), 1.59 (s, 3H,
H9¢¢), 1.59–1.38 (m, 2H, H3¢¢, H2¢¢b), 1.35–1.25, 1.21–1.11 (2 m, 2H,
H4¢¢), 0.89, 0.87 (2d, 3H, JH10¢¢–H3¢¢ = 6.5 Hz, H10¢¢◦, H10¢¢*), 0.83, 0.82
(2 s, 9H, SitBu◦, SitBu*), 0.09, 0.01 (2 s, 3H, SiMe*), 0.06, -0.01
(2 ◦s, 3H, SiMe◦); 13C NMR d 197.6, 197.4 (2d, JC2–P = 5.0 Hz,
C2 , C2*), 170.7, 170.6 (CH3CO*, CH3CO◦), 137.5, 137.4 (Cqar*,
Cqar◦), 135.6, 135.5 (2d, JCq–P = 5.5 Hz, Cqar*, Cqar◦), 131.6 (C7¢¢),
129.2, 129.1, 129.1, 129.0, 128.9, 128.5, 128.3, 128.2, 126.4 (CHar),
124.5 (C6¢¢), 102.0 (C7¢), 83.4, 83.4 (C4¢*, C4¢◦), 70.6, 70.5 (C1¢*,
Benzyl 3-(2-acetamido-4,6-O-(R)-benzylidene-3-O-tert-
butyldimethylsilyl-2-deoxy-a-D-glucopyranosyl)-2-
oxopropylphosphonate 12
To a solution of 11 (600 mg, 0.83 mmol) in toluene (8 mL) was
added DABCO (110 mg, 0.99 mmol, 1.2 eq.). The reaction mixture
was then refluxed for 7 h and concentrated. The residue was
taken up in methanol, acidified with DOWEX H+ (50WX8-100)
ion exchange resin. Methanol was removed in vacuo to afford 12
(475 mg, 90%, white solid): [a]2D0 +18 (c 1.0, CH2Cl2); H NMR
(acetone-d6) d 7.54–7.32 (m, 11H, NH, Har), 5.62 (s, 1H, H7¢), 5.18–
5.08 (m, 2H, CH2Ph), 4.66 (ddd, 1H, JH1¢–H3a = 6.8 Hz, JH1¢–H3b
6.3 Hz, JH1¢–H2¢ = 5.9 Hz, H1¢), 4.28 (ddd, 1H, JH2¢–H3¢ = 10.0 Hz,
JH2¢–NH = 9.4 Hz, JH2¢–H1¢ = 5.9 Hz, H2¢), 4.09 (dd, 1H, JH6¢eq–H6¢ax
9.7 Hz, JH6¢eq–H5¢ = 4.5 Hz, H6¢eq), 3.99 (dd, 1H, JH3¢–H2¢ = 10.0 Hz,
JH3¢–H4¢ = 8.9 Hz, H3¢), 3.68 (dd, 1H, JH6¢ax–H5¢ = JH6¢ax–H6¢eq = 9.7 Hz,
H6¢ax), 3.62 (ddd, 1H, JH5¢–H6¢ax = 9.7 Hz, JH5¢–H4¢ = 9.2 Hz, JH5¢–H6¢eq
4.5 Hz, H5¢), 3.52 (dd, 1H, JH4¢–H5¢ = 9.2 Hz, JH4¢–H3¢ = 8.9 Hz, H4¢),
3.37 (dd, 1H, JH1a–P = 22.8 Hz, JH1a–H1b = 13.4 Hz, H1a), 3.35 (dd,
1
C1¢◦), 70.2, 70.2 (C3¢*, C3¢◦), 69.4 (C6¢), 68.6, 68.6 (2d, JCH2Ph–P
=
6.0 Hz, CH2Ph), 65.8, 65.8 (C5¢◦, C5¢*), 65.7, 65.5 (2d, JC1¢¢–P = 7.0
=
◦
Hz, C1¢¢*, C1¢¢◦), 54.1, 54.0 (C2¢◦, C2¢*), 44.2, 44.0 (C3 , C3*), 42.8,
◦
42.5 (2d, JC1–P = 124.0 Hz, C1), 37.4, 37.3 (2d, JC2¢¢–P = 6.0 Hz, C2¢¢
,
=
C2¢¢*), 37.1, 37.0 (C4¢¢*, C4¢¢◦), 29.2, 29.1 (C3¢¢◦, C3¢¢*), 25.9 (SitBu,
C8¢¢)◦, 25.5 (C5¢¢), 23.2, 23.1 (CH3CO◦, CH3CO*), 19.4, 19.3 (C10¢¢*,
C10¢¢ ), 18.3 (SitBu), 17.8 (C9¢¢), -3.9, -4.7, -4.7 (SiMe); 31P NMR
d 20.9 (s, 0.52P, P*), 20.6 (s, 0.48P, P*); MS (ESI): m/z = 772 [M
+ H]+ 100%; HRMS calcd for C41H62NNaO9PSi+ 794.3829, found
794.3831.
=
1H, JH3a–H3b = 17.1 Hz, JH3a–H1¢ = 6.8 Hz, H3a), 3.24 (dd, 1H, JH1b–P
=
21.9 Hz, JH1b–H1a = 13.4 Hz, H1b), 3.00 (dd, 1H, JH3b–H3a = 17.1 Hz,
JH3b–H1¢ = 6.3 Hz, H3b), 1.84 (s, 3H, CH3CO), 0.82 (s, 9H, SitBu),
Dibenzyl 3-(2-acetamido-4,6-O-(R)-benzylidene-3-O-tert-
butyldimethylsilyl-2-deoxy-a-D-glucopyranosyl)-2-
oxopropylphosphonate 11
0.08, 0.00 (2 s, 6H, SiMe); 13C NMR (acetone-d6) d 199.9 (d, JC2–P
=
5.0 Hz, C2), 170.5 (CH3CO), 139.0 (Cqar), 137.6 (d, JCq–P = 6.0 Hz,
To a solution of 17 (890 mg, 3.2 mmol, 3.25 eq.) in THF
(15 mL) was added dropwise nBuLi (1.5 mL, 3.43 mmol, 3.5
eq.) at -78 ◦C. After 1 h stirring at -78 ◦C, the mixture was
added to a cold solution of 3 (475 mg, 0.99 mmol, 1 eq.) in
THF (3 mL). The reaction mixture was slowly warmed to r.t.
overnight and quenched with saturated aqueous NH4Cl solution.
The aqueous phase was extracted with EtOAc and the combined
Cqar), 129.6, 129.4, 129.3, 129.1, 128.7, 128.7, 127.3 (CHar), 102.5
(C7¢), 84.4 (C4¢), 72.0 (C1¢), 71.0 (C3¢), 69.7 (C6¢), 68.0 (d, JCH2Ph–P
=
5.0 Hz, CH2Ph), 65.9 (C5¢), 54.6 (C2¢), 43.8 (d, JC1–P = 124.0 Hz,
C1), 43.5 (C3), 26.3 (SitBu), 23.1 (CH3CO), 18.8 (SitBu), -3.8, -4.5
(SiMe); 31P NMR (acetone-d6) d 18.8 (s); MS (ESI): m/z = 632
[M - H]- 100%; HRMS calcd for C31H43NO9PSi- 632.2445, found
632.2460.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 8301–8312 | 8307
©

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Dibenzyl 3-(2-acetamido-2-deoxy-a-D-glucopyranosyl)-2-
Benzyl uridin-5¢-yl 3-(2-acetamido-2-deoxy-a-D-glucopyranosyl)-
oxopropylphosphonate 13
2-oxopropylphosphonate 15
To a suspension of 11 (100 mg, 0.14 mmol) in water (5 mL)
was added trifluoroacetic acid (5 mL) at 0 ◦C. The reaction
mixture was stirred at r.t. for 1 h and concentrated. Purification by
flash chromatography (CH2Cl2/MeOH, 85 : 15) and lyophilization
afforded 13 (70 mg, 97%, white solid): Rf 0.48 (CH2Cl2/MeOH =
85 : 15); [a]2D0 +38 (c 1.0, CH2Cl2); 1H NMR (DMSO-d6) d 7.67 (d,
1H, JNH–H2¢ = 7.8 Hz, NH), 7.41–7.31 (m, 10H, Har), 5.07–4.98 (m,
5H, OH4¢, CH2Ph), 4.88 (d, 1H, JOH3¢–H3¢ = 5.3 Hz, OH3¢), 4.44 (dd,
1H, JOH6¢–H6¢b = 6.0 Hz, JOH6¢–H6¢a = 5.8 Hz, OH6¢), 4.40 (ddd, 1H,
JH1¢–H3a = 9.1 Hz, JH1¢–H2¢ = 5.6 Hz, JH1¢–H3b = 4.6 Hz, H1¢), 3.71 (ddd,
1H, JH2¢–H3¢ = 10.0 Hz, JH2¢–NH = 7.8 Hz, JH2¢–H1¢ = 5.6 Hz, H2¢), 3.56
(ddd, 1H, JH6¢a–H6¢b = 11.6 Hz, JH6¢a–OH6¢ = 5.8 Hz, JH6¢a–H5¢ = 2.5 Hz,
H6¢a), 3.50 (dd, 1H, JH1a–P = 21.8 Hz, JH1a–H1b = 14.4 Hz, H1a), 3.47
To a suspension of 9 (146 mg, 0.15 mmol) in water (5 mL)
was added trifluoroacetic acid (5 mL) at 0 ◦C. The reaction
mixture was stirred at r.t. for 2 h and concentrated. Purification by
flash chromatography (CH2Cl2/MeOH, 3 : 1) and lyophilization
afforded 15 (91 mg, 94%, white solid) as a mixture of epimers
(d.r. = 2/1): Rf 0.10 (CH2Cl2/MeOH = 3 : 1); 1H NMR (DMSO-
d6) d 11.33 (s, 1H, H3u), 7.70 (d, 1H, JNH–H2¢ = 7.7 Hz, NH), 7.62,
7.58 (2d, 1H, JH6u–H5u = 8.0 Hz, H6u◦, H6u*), 7.42–7.31 (m, 5H, Har),
5.78 (d, 1H, JH1¢¢–H2¢¢ = 5.5 Hz, H1¢¢), 5.61, 5.57 (2d, 1H, JH5u–H6u
=
,
◦
8.0 Hz, H5u◦, H5u*), 5.48, 5.47 (2d, 1H, JOH2¢¢–H2¢¢ = 5.5 Hz, OH2¢¢
OH2¢¢*), 5.29 (d, 1H, JOH3¢¢–H3¢¢ = 4.8 Hz, OH3¢¢), 5.10–5.01 (m, 3H,
CH2Ph, OH4¢), 4.91 (d, 1H, JOH3¢–H3¢ = 5.1 Hz, OH3¢), 4.47–4.40 (m,
1H, OH6¢), 4.40 (ddd, 1H, JH1¢–H3a = 9.0 Hz, JH1¢–H2¢ = 5.5 Hz, JH1¢–H3b
=
(dd, 1H, (ddd, 1H, JH6¢b–H6¢a = 11.6 Hz, JH6¢b–OH6¢ = 6.0 Hz, JH6¢b–H5¢
5.7 Hz, H6¢b), 3.41 (dd, 1H, JH1b–P = 21.5 Hz, JH1b–H1a = 14.4 Hz,
H1b), 3.40 (ddd, 1H, JH3¢–H2¢ = 10.0 Hz, JH3¢–H4¢ = 8.0 Hz, JH3¢–OH3¢
5.3 Hz, H3¢), 3.35 (ddd, 1H, JH5¢–H4¢ = 8.8 Hz, JH5¢–H6¢b = 5.7 Hz,
JH5¢–H6¢a = 2.5 Hz, H5¢), 3.15 (ddd, 1H, JH4¢–H5¢ = 8.8 Hz, JH4¢–H3¢
8.0 Hz, JH4¢–OH4¢ = 5.3 Hz, H4¢), 2.90 (dd, 1H, JH3a–H3b = 16.1 Hz,
JH3a–H1¢ = 9.1 Hz, H3a), 2.66 (dd, 1H, JH3b–H3a = 16.1 Hz, JH3b–H1¢
=
4.2 Hz, H1¢), 4.21–4.10 (m, 2H, H5¢¢), 4.04 (ddd, 1H, JH2¢¢–H1¢¢ = 5.5
Hz, JH2¢¢–OH2¢¢ = 5.5 Hz, JH2¢¢–H3¢¢ = 5.5 Hz, H2¢¢), 4.02–3.91 (m, 2H,
H3¢¢, H4¢¢), 3.71 (ddd, 1H, JH2¢–H3¢ = 9.9 Hz, JH2¢–NH = 7.7 Hz, JH2¢–H1¢ =
=
5.5 Hz, H2¢), 3.60–3.28 (m, 6H, H1, H3¢, H5¢, H6¢), 3.14 (ddd, 1H,
JH4¢–H3¢ = 8.4 Hz, JH4¢–H5¢ = 8.4 Hz, JH4¢–OH4¢ = 5.4 Hz, H4¢), 2.89 (dd,
1H, JH3a–H3b = 16.0 Hz, JH3a–H1¢ = 9.0 Hz, H3a), 2.65, 2.65 (2dd, 1H,
JH3b–H3a = 16.0 Hz, JH3b–H1¢ = 4.2 Hz, H3b◦, H3b*), 1.78, 1.78 (2 s, 3H,
CH3CO*, CH3CO◦); 13C NMR (DMSO-d6) d 200.7, 200.7 (2d,
JC2–P = 6.0 Hz, C2), 169.4 (CH3CO), 163.0 (C4u), 150.6 (C2u), 140.7,
140.6 (C6u◦, C6u*), 136.2 (d, JCq–P = 6.5 Hz, Cqar), 128.5, 128.3,
=
=
4.6 Hz, H3b), 1.77 (s, 3H, CH3CO); 13C NMR (DMSO-d6) d 200.5
(d, JC2–P = 6.0 Hz, C2), 169.3 (CH3CO), 136.2 (d, JCq–P = 6.0 Hz,
Cqar), 128.4, 128.2, 127.7 (CHar), 74.9 (C5¢), 70.6 (C4¢), 70.3 (C3¢),
69.3 (C1¢), 67.0, 66.9 (2d, JCH2Ph–P = 6.0 Hz, CH2Ph), 60.9 (C6¢),
52.6 (C2¢), 41.8 (d, JC1–P = 126.0 Hz, C1), 41.4 (C3), 22.6 (CH3CO);
31P NMR (DMSO-d6) d 21.8 (s); MS (ESI): m/z = 544 [M +
Na]+ 100%; HRMS calcd for C25H32NNaO9P+ 544.1712, found
544.1707.
127.7 (CHar), 102.0 (C5u), 88.2, 88.2 (C1¢¢◦, C1¢¢*), 82.1 (d, JC4¢¢–P
6.0 Hz, C4¢¢), 74.9 (C5¢), 72.6 (C2¢¢), 70.6 (C4¢), 70.2 (C3¢), 69.5 (C3¢¢),
=
69.3 (C1¢), 67.1 (d, JCH2Ph–P = 5.5 Hz, CH2Ph), 65.5, 65.3 (2d, JC5¢¢–P
=
5.5 Hz, C5¢¢◦, C5¢¢*), ◦60.9 (C6¢), 52.7 (C2¢), 41.5 (C3), 41.4, 41.4 (2d,
JC1–P = 127.0 Hz, C1 , C1*), 22.6 (CH3CO); 31P NMR (DMSO-d6)
d 22.2 (s, 0.66P, P*), 22.0 (s, 0.33P, P◦); MS (ESI): m/z = 1337
[2M + Na]+ 100%; HRMS calcd for C27H36N3NaO14P + 680.1833,
found 680.1832.
Benzyl 3-(2-acetamido-2-deoxy-a-D-glucopyranosyl)-2-
oxopropylphosphonate 14
To a solution of 13 (34 mg, 65 mmol) in toluene (1 mL) was
added DABCO (9 mg, 78 mmol, 1.2 eq.). The reaction mixture was
then refluxed for 4 h and concentrated. The residue was taken up
in water, acidified with DOWEX H+ (50WX8-100) ion exchange
resin. Water was removed in vacuo to afford 14 (25 mg, 88%, white
solid): 1H NMR (DMSO-d6) d 7.73 (d, 1H, JNH–H2¢ = 8.0 Hz, NH),
7.42–7.28 (m, 5H, Har), 4.94 (d, 2H, JCH2Ph–P = 7.5 Hz, CH2Ph),
4.66 (ddd, 1H, JH1¢–H3a = 8.3 Hz, JH1¢–H3b = JH1¢–H2¢ = 5.2 Hz, H1¢),
3.72 (ddd, 1H, JH2¢–H3¢ = 10.3 Hz, JH2¢–NH = 8.0 Hz, JH2¢–H1¢ = 5.2 Hz,
H2¢), 3.56 (dd, 1H, JH6¢a–H6¢b = 11.5 Hz, JH6¢a–H5¢ = 2.2 Hz, H6¢a), 3.46
(dd, 1H, JH6¢b–H6¢a = 11.5 Hz, JH6¢b–H5¢ = 5.7 Hz, H6¢b), 3.41 (dd, 1H,
Uridin-5¢-yl 3-(2-acetamido-2-deoxy-a-D-glucopyranosyl)-2-
oxopropylphosphonate 16
A suspension of 15 (22 mg, 33 mmol) and Pd/C 10% (22 mg) in
methanol (2 mL) was purged from air and filled with H2. The
reaction was stirred at r.t. for 3 h then diluted with methanol,
filtered through a Celite pad and concentrated. Lyophilization
afforded 16 (16 mg, 84%, white solid): [a]2D0 +34 (c 1.0, H2O); 1H
NMR (DMSO-d6) d 11.27 (s, 1H, H3u), 8.22 (d, 1H, JNH–H2¢ = 6.0
Hz, NH), 7.85 (d, 1H, JH6u–H5u = 8.0 Hz, H6u), 5.80 (d, 1H, JH1¢¢–H2¢¢
=
5.5 Hz, H1¢¢), 5.62 (2d, 1H, JH5u–H6u = 8.0 Hz, H5u), 4.40–4.32 (m,
1H, H1¢), 4.11–4.05 (m, 1H, H2¢¢), 4.05–3.99 (m, 1H, H3¢¢), 3.97–
3.91 (m, 1H, H4¢¢), 3.91–3.83 (m, 2H, H5¢¢), 3.77–3.70 (m, 1H, H2¢),
3.61–3.54 (m, 1H, H6¢a), 3.47–3.25 (m, 3H, H3¢, H5¢, H6¢b), 3.09–2.96
(m, 2H, H4¢, H3a), 2.90 (dd, 1H, JH1a–P = 21.5 Hz, JH1a–H1b = 11.5
Hz, H1a), 2.81–2.67 (m, 2H, H1b, H3b), 1.76 (s, 3H, CH3CO); 13C
NMR (DMSO-d6) d 203.7 (d, JC2–P = 5.0 Hz, C2), 169.6 (CH3CO),
163.1 (C4u), 150.8 (C2u), 140.9 (C6u), 101.9 (C5u), 87.4 (C1¢¢), 83.5
(d, JC4¢¢–P = 6.5 Hz, C4¢¢), 74.8 (C5¢), 73.2 (C2¢¢), 71.3 (C4¢), 70.7 (C3¢),
70.3 (C3¢¢), 69.5 (C1¢), 63.5 (d, JC5¢¢–P = 5.0 Hz, C5¢¢), 61.4 (C6¢), 52.7
JH3¢–H2¢ = 10.3 Hz, JH3¢–H4¢ = 8.1 Hz, H3¢), 3.35 (ddd, 1H, JH5¢–H4¢
=
8.8 Hz, JH5¢–H6¢b = 5.7 Hz, JH5¢–H6¢a = 2.2 Hz, H5¢), 3.17, 3.12 (AB
from ABX, 2H, JAB = 13.3 Hz, JA–P = 21.7 Hz, JB–P = 22.0 Hz, H1),
3.12 (dd, 1H, JH4¢–H5¢ = 8.8 Hz, JH4¢–H3¢ = 8.1 Hz, H4¢), 2.88 (dd, 1H,
JH3a–H3b = 16.2 Hz, JH3a–H1¢ = 8.3 Hz, H3a), 2.75 (dd, 1H, JH3b–H3a
=
16.2 Hz, JH3b–H1¢ = 5.2 Hz, H3b), 1.76 (s, 3H, CH3CO); 13C NMR
(DMSO-d6) d 201.5 (d, JC2–P = 5.5 Hz, C2), 169.3 (CH3CO), 137.2
(d, JCq–P = 7.0 Hz, Cqar), 128.3, 127.8, 127.4 (CHar), 74.8 (C5¢),
70.8 (C4¢), 70.4 (C3¢), 69.5 (C1¢), 66.1 (d, JCH2Ph–P = 5.0 Hz, CH2Ph),
61.0 (C6¢), 52.7 (C2¢), 43.9 (d, JC1–P = 120.0 Hz, C1), 41.1 (C3), 22.6
(CH3CO); 31P NMR (DMSO-d6) d 16.2 (s); MS (ESI): m/z = 430
[M - H]- 100%.
(C2¢), 45.9 (d, JC1–P = 107.0 Hz, C1), 41.3 (C3), 22.7 (CH3CO); 31
P
NMR (DMSO-d6) d 8.9 (s); MS (ESI): m/z = 566 [M - H]- 100%;
HRMS calcd for C20H29N3O14P- 566.1387, found 566.1384.
8308 | Org. Biomol. Chem., 2011, 9, 8301–8312
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The Royal Society of Chemistry 2011
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Benzyl 5-(3-N-tert-butyloxycarbonyl-uracil-1-yl)pentyl
3-(2-acetamido-4,6-O-(R)-benzylidene-3-O-tert-
butyldimethylsilyl-2-deoxy-a-D-glucopyranosyl)-2-oxopropyl-
phosphonate 18
NH◦), 5.51, 5.50 (2 s, 1H, H7¢*, H7¢◦), 5.19–5.02 (m, 2H, CH2Ph),
4.81 (ddd, 0.45H, JH1¢–H3a = 7.6 Hz, JH1¢–H2¢ = 5.7 Hz, JH1¢–H3b = 5.0 Hz,
H1¢◦), 4.77 (ddd, 0.55H, JH1¢–H3a = 8.3 Hz, JH1¢–H2¢ = 5.5 Hz, JH1¢–H3b
=
4.4 Hz, H1¢*), 4.36–4.28 (m, 1H, H2¢), 4.20–4.13 (m, 1H, H6¢eq),
4.09–3.94 (m, 2H, H1¢¢), 3.84 (dd, 1H, J = 9.6 Hz, J = 8.1 Hz, H3¢),
3.65 (dd, 1H, JH6¢ax–H5¢ = JH6¢ax–H6¢eq = 9.6 Hz, H6¢ax), 3.56–3.43 (m, 2H,
H4¢, H5¢), 3.29 (dd, 0.45H, JH3a–H3b = 17.0 Hz, JH3a–H1¢ = 7.6 Hz, H3a◦),
3.22 (dd, 0.55H, JH3a–H3b = 17.2 Hz, JH3a–H1¢ = 8.3 Hz, H3a*), 3.18
(dd, 0.45H, JH1a–P = 23.0 Hz, JH1a–H1b = 13.4 Hz, H1a◦), 3.06, 3.04
(AB from ABX, 1.1H, JAB = 12.7 Hz, JA–P = 22.3 Hz, JB–P = 22.7 Hz,
H1a*, H1b*), 3.01 (dd, 0.45H, JH1b–P = 22.0 Hz, JH1b–H1a = 13.4 Hz,
H1b◦), 2.75 (dd, 0.45H, JH3b–H3a = 17.0 Hz, JH3b–H1¢ = 5.0 Hz, H3b◦),
2.59 (dd, 0.55H, JH3b–H3a = 17.2 Hz, JH3b–H1¢ = 4.4 Hz, H3b*), 1.89,
1.87 (2 s, 3H, CH3CO*, CH3CO◦), 1.69–1.57 (m, 2H, H2¢¢), 1.35–
1.22 (m, 26H, H3¢¢ to H15¢¢), 0.88 (t, 3H, JH16¢¢–H15¢¢ = 6.7 Hz, H16¢¢),
0.83, 0.82 (2 s, 9H, SitBu*,◦SitBu◦), 0.09, 0.01 (2 s, 3.3H, SiMe*),
At 0 ◦C, DIAD (47 mL, 0.24 mmol, 1.5 eq.) was added dropwise
to a solution of 12 (100 mg, 0.16 mmol, 1 eq.), 17 (47 mg,
0.16 mmol, 1 eq.) and PPh3 (62 mg, 0.24 mmol, 1.5 eq.) in
THF (2.5 mL). The reaction mixture was stirred at r.t. for 16
h then concentrated. Purification by preparative chromatography
(CHCl3/MeOH, 95 : 5) afforded 18 (39 mg, 27%, white solid) as a
mixture of epimers (d.r. = 65/35): Rf 0.35 (cyclohexane/acetone =
1
1 : 1); H NMR d 7.49–7.32 (m, 10H, Har), 7.15, 6.99 (2d, 1H,
JH6u–H5u = 8.0 Hz, H6u*, H6u◦), 6.57 (2d, 1H, JNH–H2¢ = 8.5 Hz, NH*,
NH◦), 5.69, 5.64 (2d, 1H, JH5u–H6u = 8.0 Hz, H5u*, H5u◦), 5.50,
5.49 (s, 1H, H7¢), 5.16–5.02 (m, 2H, CH2Ph), 4.79 (ddd, 0.35H,
◦
◦
◦
◦
◦
JH1¢
= 7.6 Hz, JH1¢
= 6.2 Hz, JH1¢
= 4.8 Hz, H1¢◦),
◦
–H2¢
0.06, -0.01 (2 s, 2.7H, SiMe ); 13C NMR d 197.7, 197.4 (2d, JC2–P
=
–H3a
–H3b
4.76 (ddd, 0.65H, JH1¢*–H3a* = 7.3 Hz, JH1¢*–H2¢* = 5.7 Hz, JH1¢*–H3b*
5.4 Hz, H1¢*), 4.32–4.24 (m, 1H, H2¢), 4.17–4.12 (m, 1H, H6¢eq),
4.08–3.90 (m, 2H, H1¢¢), 3.85–3.78 (m, 1H, H3¢), 3.73–3.61 (m, 3H,
=
◦
5.0 Hz, C2 , C2*), 170.7, 170.6 (CH3CO*, CH3CO◦), 137.4, 137.4
(Cqar*, Cqar◦), 135.6, 135.5 (2d, JCq–P = 6.0 Hz, Cqar*, Cqar◦), 129.1,
129.1, 129.0, 128.9, 128.9, 128.5, 128.2, 128.1, 126.4 (CHar), 102.0
(C7¢), 83.4, 83.4 (C4¢*, C4¢◦), 70.6, 70.5 (C1¢◦, C1¢*), 70.2 (C3¢), 69.4
◦
◦
=
H5¢¢, H6¢ax), 3.54–3.45 (m, 2H, H4¢, H5¢), 3.17 (dd, 0.35H, JH3a
–H3b
= 7.6 Hz, H3a◦), 3.17 (dd, 0.35H, JH1a
◦
◦
◦
◦
= 22.6
–P
16.8 Hz, JH3a
–H1¢
(C6¢), 68.6 (d, JCH2Ph–P = 6.5 Hz, CH2Ph), 67.3, 67.1 (2d, JC1¢¢–P
=
Hz, JH1a
= 14.6 Hz, H1a◦), 3.14 (dd, 0.65H, JH3a*–H3b* = 17.2
◦
◦
6.5 Hz, C1¢¢*, C1¢¢◦),◦ 65.7, 65.7 (C5¢*, C5¢◦), 54.0, 53.9 (C2¢◦, C2¢*),
–H1b
◦
◦
= 21.7 Hz,
Hz, JH3a*–H1¢* = 7.3 Hz, H3a*), 3.07 (dd, 0.65H, JH1b
◦
–P
44.1, 43.9 (C3*, C3 ), 42.7, 42.5 (2d, JC1–P = 124.0 Hz, C1*, C1 ),
JH1b
= 14.6 Hz, H1b◦), 3.06, 3.02 (AB from ABX, 1.3H, JAB
=
◦
◦
–H1a
32.0, 29.8, 29.8, 29.7, 29.6, 29.5, 29.2, 25.6, 25.5, 23.1, 23.1 ( C3¢¢
to C15¢¢), 30.5, 30.3 (2d, JC2¢¢–P = 6.0 Hz, C2¢¢◦, C2¢¢*), 25.8 (SitBu),
22.8 (CH3CO), 18.2 (SitBu), 14.2 (C16¢¢), -3.9, -4.7, -4.7 (SiMe);
31P NMR d 20.9 (s, 0.55P, P*), 20.6 (s, 0.45P, P◦); MS (ESI): m/z =
880 [M + Na]+ 100%; HRMS calcd for C47H75NO9PSi- 856.4949,
found 856.4951.
13.5 Hz, JAX = 23.0 Hz, JBX = 22.3 Hz, H1a*, H1b*), 2.81 (dd,
= 6.2 Hz, H3b◦), 2.66 (dd,
◦
–H1¢
◦
◦
◦
0.35H, JH3b
= 16.8 Hz, JH3b
–H3a
0.65H, JH3b*–H3a* = 17.2 Hz, JH3b*–H1¢* = 5.4 Hz, H3b*), 1.89 (2 s,
3H, CH3CO*, CH3CO◦), 1.72–1.58 (m, 4H, H2¢¢, H4¢¢), 1.59 (s, 9H,
CO2tBu), 1.41–1.30 (m, 2H, H3¢¢), 0.82 (s, 9H, SitBu), 0.08, 0.00
(2 s, 1.3H, SiMe*), 0.06, -0.01 (2◦s, 0.7H, SiMe◦); 13C NMR d
197.9, 197.8 (2d, JC2–P = 5.5 Hz, C2 , C2*), 170.7, 170.6 (CH3CO◦,
CH3CO*), 160.9, 160.9 (C4u*, C4u◦), 149.1 (C2u), 148.0 (COBoc),
143.9, 143.7 (C6u*, C6u◦), 137.4 (Cqar), 135.5 (d, JCq–P = 5.0 Hz,
Cqar), 129.2, 129.1, 129.0, 128.9, 128.5, 128.2, 126◦.4, 126.3 (CHar),
102.0, 101.9, 101.9 (C5u, C7¢), 86.9, 86.9 (CMe3 , CMe3*), 83.3
Benzyl 5-uracil-1-yl-pentyl 3-(2-acetamido-2-deoxy-a-D-
glucopyranosyl)-2-oxopropylphosphonate 20
To a suspension of 18 (37 mg, 40 mmol) in water (2 mL) was
(C4¢), 70.7, 70.5 (C1¢◦, C1¢*), 70.2 (C3¢), 69.3 (C6¢), 68.7 (d, JCH2Ph–P
=
◦
added trifluoroacetic acid (2 mL) at 0 C. The reaction mixture
6.5 Hz, CH2Ph), 66.4, 66.2 (2d, JC1¢¢–P = 7.0 Hz, C1¢¢*, C1¢¢◦), 65.6,
was stirred at r.t. for 3 h and concentrated. Purification by flash
chromatography (CH2Cl2/MeOH, 8 : 2) afforded 20 (23 mg, 93%,
white solid) as a mixture of epimers: Rf 0.50 (CH2Cl2/MeOH =
65.5 (C5¢*, C5¢◦), 54.1, 54.0 (C2¢◦, C2¢*), 49.2, 49.1 (C5¢¢*, C5¢¢◦◦), 43.8,
◦
43.3 (C3*, C3 ), 42.5, 42.4 (2d, JC1–P = 125.0 Hz, C1*, C1 ), 29.7,
29.6 (2d, JC2¢¢–P = 6.5 Hz, C2¢¢◦, C2¢¢*), 28.4, 28.3 (C4¢¢◦, C4¢¢*), 27.6
(CMe3), 25.8 (SitBu), 23.1 (CH3CO), 22.5, 22.5 (C3¢¢◦, C3¢¢*), 18.2
(SitBu), -3.9, -4.7 (SiMe); 31P NMR d 21.1 (s, 0.35P, P◦), 20.9 (s,
0.65P, P*); MS (ESI): m/z = 936 [M + Na]+ 100%; HRMS calcd
for C45H63N3O13PSi- 912.3868, found 912.3856.
1
8 : 2); H NMR (DMSO-d6) d 11.19 (s, 1H, H3u), 7.72–7.67 (m,
1H, NH), 7.65, 7.62 (2d, 1H, JH6u–H5u = 7.8 Hz, H6u◦, H6u*), 7.43–
◦
7.30 (m, 5H, Har), 5.54, 5.53 (2d, 1H, JH6u–H5u = 7.8 Hz, H5u
,
H5u*), 5.10–4.98 (m, 2H, CH2Ph), 4.39 (ddd, 1H, JH1¢–H3a = 9.2 Hz,
H1¢–H2¢ = 5.3 Hz, JH1¢–H3b = 4.5 Hz, H1¢), 3.99–3.88 (m, 2H, H1¢¢), 3.71
J
(ddd, 1H, JH2¢–H3¢ = 9.4 Hz, JH2¢–NH = 7.3 Hz, JH2¢–H1¢ = 5.3 Hz, H2¢),
3.62 (t, 2H, JH5¢¢–H4¢¢ = 7.2 Hz, H5¢¢), 3.56 (ddd, 1H, JH6¢a–H6¢b = 11.6
Hz, J = 5.8 Hz, J = 1.9 Hz, H6¢a), 3.47 (ddd, 1H, JH6¢b–H6¢a = 11.6
Hz, J = 5.8 Hz, J = 5.5 Hz H6¢b), 3.43–3.26 (m, 4H, H1, H3¢, H5¢),
Benzyl hexadecanyl 3-(2-acetamido-4,6-O-(R)-benzylidene-3-
O-tert-butyldimethylsilyl-2-deoxy-a-D-glucopyranosyl)-2-
oxopropylphosphonate 19
3.18–3.10 (m, 1H, H4¢), 2.90 (dd, 1H, JH3a–H3b = 16.2 Hz, JH3a–H1¢
=
At 0 ◦C, DIAD (54 mL, 0.27 mmol, 1.5 eq.) was added dropwise
to a solution of 12 (114 mg, 0.18 mmol, 1 eq.), hexadecan-1-
ol (44 mg, 0.18 mmol, 1 eq.) and PPh3 (71 mg, 0.27 mmol, 1.5
eq.) in THF (3 mL). The reaction mixture was stirred at r.t.
for 16 h then concentrated. Two successive purifications by flash
chromatography (cyclohexane/acetone, 2 : 1 and CH2Cl2/acetone,
9 : 1) afforded 19 (66 mg, 42%, colorless oil) as a mixture of epimers
(d.r. = 55/45): Rf 0.35, 0.25 (CH2Cl2/acetone = 9 : 1); 1H NMR d
7.52–7.31 (m, 10H, Har), 6.68, 6.53 (2d, 1H, JNH–H2¢ = 8.6 Hz, NH*,
9.2 Hz, H3a), 2.64 (dd, 1H, JH3b–H3a = 16.2 Hz, JH3b–H1¢ = 4.7 Hz,
H3b), 1.78, 1.76 (2 s, 3H, CH3CO*, CH3CO◦), 1.62–1.50 (m, 4H,
H2¢¢, H4¢¢), 1.33–1.21 (m, 2H, H3¢¢); 13C NMR (DMSO-d6) d 200.6
(C2), 169.3 (CH3CO), 163.7 (C4u), 150.9 (C2u), 145.6 (C6u), 136.3 (d,
JCq–P = 6.3 Hz, Cqar), 128.4, 128.2, 127.7 (CHar), 100.8 (C5u), 74.9
(C3¢), 70.6 (C4¢), 70.3 (C5¢), 69.4 (C1¢), 66.9 (2d, JCH2Ph–P = 5.8 Hz,
CH2Ph*, CH2Ph◦), 65.6 (2d, JC1¢¢–P = 5.5 Hz, C1¢¢*, C1¢¢◦), 60.9 (C6¢),
52.6 (C2¢), 47.2 (C5¢¢), 42.3 (C1), 41.3 (C3), 29.3 (d, JC2¢¢–P = 5.5 Hz,
C2¢¢), 27.9 (C4¢¢), 22.6 (CH3CO), 21.8 (C3¢¢); 31P NMR (DMSO-d6)
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 8301–8312 | 8309
©

View Online
d 21.16 (m); MS (ESI): m/z = 634 [M + Na]+ 100%; HRMS calcd
72.1 (C3¢, C4¢), 70.2 (C1¢), 69.7 (d, JCH2Ph–P = 6.7 Hz, CH2Ph), 66.7
(C1¢¢), 62.7 (C6¢), 54.4 (C2¢), 45.0 (C1), 42.8 (C3), 38.4 (2d, JC2¢¢–P = 6.8
Hz, C2¢¢), 38.1 (C4¢¢), 30.2 (C3¢¢), 26.5, 26.0 (C8¢¢, C5¢¢), 22.9 (CH3CO),
19.7 (C10¢¢◦, C10¢¢*), 17.9 (C9¢¢); 31P NMR (CD3OD) d 22.3 (m); MS
(ESI): m/z = 570 [M + H]+ 50, 592 [M + Na]+ 100%; HRMS calcd
for C28H45NO9P+ 570.2832, found 570.2806.
for C27H37N3O11P- 610.2166, found 610.2194.
Benzyl (R)-7-hydroxy-3,7-dimethyloctyl 3-(2-acetamido-2-
deoxy-a-D-glucopyranosyl)-2-oxopropylphosphonate 21
To a suspension of 10 (205 mg, 0.27 mmol) in water (5 mL) was
◦
added trifluoroacetic acid (5 mL) at 0 C. The reaction mixture
Benzyl hexadecanyl 3-(2-acetamido-2-deoxy-a-D-glucopyranosyl)-
2-oxopropylphosphonate 23
was stirred at r.t. for 2 h and concentrated. Purification by flash
chromatography (CH2Cl2/MeOH, 85 : 15) afforded 21 (147 mg,
94%, white solid) as a mixture of epimers (d.r. = 52/48): Rf 0.36
To a suspension of 19 (65 mg, 76 mmol) in water (2 mL) was
◦
1
(CH2Cl2/MeOH = 85 : 15); H NMR (DMSO-d6) d 7.69 (d, 1H,
added trifluoroacetic acid (2 mL) at 0 C. The reaction mixture
JNH–H2¢ = 7.7 Hz, NH), 7.41–7.32 (m, 5H, Har), 5.07–4.99 (m, 3H,
CH2Ph, OH4¢), 4.42 (ddd, 1H, J = 6.0 Hz, JOH6¢–H6¢a = 5.8 Hz, J =
3.1 Hz, OH6¢), 4.39 (ddd, 1H, JH1¢–H3a = 9.1 Hz, JH1¢–H2¢ = 5.6 Hz,
JH1¢–H3b = 4.6 Hz, H1¢), 4.01–3.90 (m, 2H, H1¢¢), 3.71 (ddd, 1H,
JH2¢–H3¢ = 9.9 Hz, JH2¢–NH = 7.7 Hz, JH2¢–H1¢ = 5.6 Hz, H2¢), 3.56 (ddd,
1H, JH6¢a–H6¢b = 11.6 Hz, JH6¢a–OH6¢ = 5.8 Hz, JH6¢a–H5¢ = 2.5 Hz, H6¢a),
3.50–3.44 (m, 1H, H6¢b), 3.43–3.27 (m, 4H, H1, H3¢, H5¢), 3.17–3.11
(m, 1H, H4¢), 2.90, 2.89 (2dd, 1H, JH3a–H3b = 16.1 Hz, JH3a–H1¢ = 9.1
Hz, H3a), 2.65, 2.64 (2dd, 1H, JH3b–H3a = 16.1 Hz, JH3b–H1¢ = 4.6 Hz,
H3b), 1.78, 1.78 (2 s, 3H, CH3CO*, CH3CO◦), 1.62–1.54 (m, 1H,
H2¢¢a), 1.52–1.43 (m, 1H, H3¢¢), 1.39–1.16 (m, 6H, H2¢¢b, H4¢¢a, H5¢¢,
H6¢¢), 1.10–1.01 (m, 1H, H4¢¢b), 1.05, 1.05 (2 s, 6H, H8¢¢, H9¢¢), 0.82,
0.83 (2d, 3H, JH10¢¢–H3¢¢ = 6.6 Hz, H10¢¢); 13C NMR (DMSO-d6) d
was stirred at r.t. for 3 h and concentrated. Purification by flash
chromatography (CH2Cl2/MeOH, 9 : 1) afforded 23 (45 mg, 90%,
white solid) as a mixture of epimers (d.r. = 55/45): Rf 0.32
1
(CH2Cl2/MeOH = 9 : 1); H NMR d 7.45–7.39 (m, 1H, NH),
7.38–7.28 (m, 5H, Har), 5.12–5.00 (m, 2H, CH2Ph), 4.76–469 (m,
1H, H1¢), 4.09–4.01 (m, 1H, H2¢), 3.97–3.88 (m, 2H, H1¢¢), 3.90–3.82
(m, 1H, H6¢a), 3.77–3.66 (m, 2H, H3¢, H6¢b), 3.63–3.52 (m, 2H, H4¢,
H5¢), 3.26–3.12 (m, 2H, H1), 3.04–2.82 (m, 2H, H3), 1.94, 1.93 (2
s, 3H, CH3CO*, CH3CO◦), 1.58–1.50 (m, 2H, H2¢¢), 1.33–1.18 (m,
26H, H3¢¢ to H15¢¢), 0.87 (t, 3H, JH16¢¢–H15¢¢ = 7.0 Hz, H16¢¢); 13C NMR d
◦
200.1, 200.0 (2d, JC2–P = 5.5 Hz, C2 , C2*), 172.2 (CH3CO), 135.9
(d, JCq–P = 5.5 Hz, Cqar), 128.8, 128.8, 128.8, 128.2, 128.1 (CHar),
75.1 (C5¢), 71.2 (C3¢), 70.7 (C4¢), 68.6 (C1¢), 68.5, 68.4 (2d, JCH2Ph–P
=
200.6 (d, JC2–P = 5.0 Hz, C2), 169.2 (CH3CO), 136.4 (d, JCq–P
=
6.5 Hz, CH2Ph*, CH2Ph◦), 67.3 (d, JC1¢¢–P = 6.5 Hz, C1¢¢), 61.6 (C6¢),
◦
6.0 Hz, Cqar), 128.4, 128.2, 127.7 (CHar), 74.8 (C5¢), 70.6 (C4¢), 70.3
(C3¢), 69.4 (C1¢), 68.7 (C7¢¢), 66.9, 66.8 (2d, JCH2Ph–P = 6.0 Hz, CH2Ph),
64.2 (d, JC1¢¢–P = 6.5 Hz, C1¢¢), 60.9 (C6¢), 52.6 (C2¢), 43.8 (C6¢¢), 41.8
(d, JC1–P = 120.0 Hz, C1), 41.3 (C3), 37.0, 36.9 (C4¢¢*, C4¢¢◦), 36.8,
36.7 (2d, JC2¢¢–P = 5.0 Hz, C2¢¢*, C2¢¢◦) 29.3, 29.2 (C8¢¢, C9¢¢), 28.7
(C3¢¢), 22.6 (CH3CO), 21.1 (C5¢¢), 19.1, 19.1 (C10¢¢◦, C10¢¢*); 31P NMR
(DMSO-d6) d 21.2 (s, 0.48P, P◦), 21.1 (s, 0.52P, P*); MS (ESI):
m/z = 610 [M + Na]+ 100%; HRMS calcd for C28H46NNaO10P+
610.2757, found 610.2779.
52.9 (C2¢), 42.9 (C3), 42.2, 42.1 (2d, JC1–P = 127.0 Hz, C1*, C1 ),
32.1, 29.9, 29.8, 29.8, 29.7, 29.5, 29.3, 25.5, 23.1 ( C3¢¢ to C15¢¢), 30.5,
30.4 (2d, JC2¢¢–P = 5.0 Hz, C2¢¢◦, C2¢¢*), 22.8 (CH3CO), 14.2 (C16¢¢); 31
P
NMR d 21.1 (s); MS (ESI): m/z = 1333 [2M + Na]+ 100%; HRMS
calcd for C34H58NNaO9P+ 678.3747, found 678.3765.
5-Uracil-1-yl-pentyl 3-(2-acetamido-2-deoxy-a-D-glucopyranosyl)-
2-oxopropylphosphonate 24
A suspension of 20 (15 mg, 24 mmol) and Pd/C 10% (15 mg) in
methanol (2 mL) was purged from air and filled with H2. The
reaction was stirred at r.t. for 4 h then diluted with methanol,
filtered through a Celite pad and concentrated. Lyophilization
afforded 24 (12 mg, 94%, white solid); [a]2D0 +26 (c 1.0, DMSO);
1H NMR (DMSO-d6) d 11.19 (s, 0.8H, H3u), 9.99 (s, 0.2H, H3u),
7.65 (d, 1H, JH6u–H5u = 7.6 Hz, H6u), 7.65 (d, 1H, JH5u–H6u = 7.6 Hz,
H5u), 7.28 (bs, 1H, NH), 5.01 (s, 1H, OH4¢), 4.80 (s, 1H, OH3¢),
Benzyl (R)-citronellyl 3-(2-acetamido-2-deoxy-a-D-
glucopyranosyl)-2-oxopropylphosphonate 22
To a solution of 10 (80 mg, 0.10 mmol) in THF (2 mL) was
added water (2 mL) and trifluoroacetic acid (2 mL) at 0 ◦C.
The reaction mixture was stirred at 0 ◦C for 2 h and at r.t. for
4 h. The mixture was then neutralized with aqueous NH4OH
solution. The aqueous phase was extracted with Et2O and the
combined organic layers were dried (MgSO4) and concentrated.
Purification by flash chromatography (CH2Cl2/MeOH, 85 : 15)
and lyophilization afforded 22 (50 mg, 84%, white solid): Rf 0.40
4.50 (m, 1H, OH6¢), 4.36 (ddd, 1H, JH1¢–H3a = 7.3 Hz, JH1¢–H3b
=
6.0 Hz, JH1¢–H2¢ = 5.7 Hz, H1), 3.75 (ddd, 1H, JH2¢–H3¢ = 9.8 Hz,
JH2¢–NH = 7.8 Hz, JH2¢–H1¢ = 5.7 Hz, H2¢), 3.73–3.67 (m, 2H, H1¢¢),
3.65 (t, 2H, JH5¢¢–H4¢¢ = 7.0 Hz, H5¢¢), 3.58 (ddd, 1H, JH6¢a–H6¢b = 11.4
Hz, JH6¢a–OH6¢ = 5.7 Hz, JH6¢a–H5¢ = 1.5 Hz, H6¢a), 3.47–3.35 (m, 2H,
H6¢b, H5¢), 3.34–3.25 (m, 1H, H3¢), 3.16–3.00 (m, 2H, H4¢, H3a), 2.86
(dd, 1H, JH1a–P = 19.7 Hz, JH1a–H1b = 11.6 Hz, H1a), 2.71 (dd, 1H,
1
(CH2Cl2/MeOH = 85 : 15); H NMR (CD3OD) d 7.38–7.20 (m,
5H, Har), 5.10–4.95 (m, 3H, H6¢¢, CH2Ph), 4.59–4.49 (m, 1H, H1¢),
4.03–3.88 (m, 2H, H1¢¢), 3.86 (dd, 1H, JH2¢–H3¢ = 9.5 Hz, JH2¢–H1¢
=
5.4 Hz, H2¢), 3.68–3.58 (m, 2H, H6¢), 3.49 (dd, 1H, JH3¢–H2¢ = 9.5
Hz, JH3¢–H4¢ = 8.3 Hz, H3¢), 3.45–3.38 (m, 1H, H5¢), 3.30–3.18 (m,
3H, H4¢, H1), 3.00–2.90 (m, 0.6H, H3a), 2.78–2.72 (m, 0.4H, H3b),
1.95–1.78 (m, 2H, H5¢¢), 1.85 (s, 3H, CH3CO), 1.62–1.50 (m, 1H,
JH1b–P = 19.7 Hz, JH1b–H1a = 11.6 Hz, H1b), 2.61 (dd, 1H, JH3b–H3a =
16.3 Hz, JH3b–H1¢ = 6.0 Hz, H3b), 1.76 (s, 3H, CH3CO), 1.65–1.45
(m, 4H, H2¢¢, H4¢¢), 1.36–1.20 (m, 2H, H3¢¢); 13C NMR (DMSO-d6)
d 203.5 (C2), 169.8 (CH3CO), 163.8(C4u), 151.0 (C2u), 145.8 (C6u),
H
2a¢¢), 1.56 (s, 3H, H8¢¢), 1.50 (s, 3H, H9¢¢), 1.47–1.38 (m, 1H, H3¢¢),
100.8 (C5u), 75.0 (C3¢), 71.3 (C4¢), 70.7(C5¢), 69.4 (C1¢), 63.4 (d, JC–P =
1.37–1.27 (m, 1H, H2b¢¢), 1.26–1.15 (m, 1H, H4b¢¢), 1.09–0.98 (m,
1H, H4a¢¢), 0.78, 0.77 (2d, JH10¢¢–H3¢¢ = 6.4 Hz, 3H, H10¢¢); 13C NMR
(CD3OD) d 201.8 (C2), 174.0 (CH3CO), 137.5 (d, JCq–P = 5.5 Hz,
Cqar), 132.3 (C7¢¢), 129.9, 129.4 (CHar), 125.8 (C6¢¢), 76.5 (C5¢), 72.1,
4.1 Hz, C1¢¢), 61.4 (C6¢), 52.7 (C2¢), 47.5 (C5¢¢), 45.7 (C1), 41.4 (C3),
29.3 (d, C2¢¢, JC–P = 6.6 Hz), 28.2 (C4¢¢), 22.7 (CH3CO), 21.4 (C3¢¢);
31P NMR (DMSO-d6) d 7.91 (m); MS (ESI): m/z = 520 [M - H]-
100%; HRMS calcd for C20H31N3O11P- 520.1696, found 520.1722.
8310 | Org. Biomol. Chem., 2011, 9, 8301–8312
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(R)-7-Hydroxy-3,7-dimethyloctyl 3-(2-acetamido-2-deoxy-a-D-
glucopyranosyl)-2-oxopropylphosphonate 25
heating at 100 ◦C for 1 min. The compounds were also tested
against MurG purified from E. coli as previously described.36,37
Reaction mixtures contained, in a final volume of 12.5 mL, 200 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 16 mM UDP-[14C]GlcNAc (1.7
kBq), 16 mM lipid I analogue, 30% (v/v) dimethyl sulfoxide and
MurG (7 ng). After 30 min at 37 ◦C, it was stopped by boiling for
3 min. Then, the mixture was lyophilised and taken up in 10 mL of
2-propanol/ammonium hydroxide/water (6 : 3 : 1; v/v/v). In both
cases, the radiolabeled substrate (UDP-MurNAc-pentapeptide in
the case of MraY, UDP-GlcNAc in the case of MurG) and reaction
product (lipid I, product of MraY, and lipid II, product of MurG)
were separated by TLC on silica gel plates LK6D (Whatman) using
2-propanol/ammonium hydroxide/water (6 : 3 : 1; v/v/v) as the
mobile phase. The radioactive spots were located and quantified
with a radioactivity scanner (model Multi-Tracemaster LB285;
EG&G Wallac/Berthold). Residual activities were calculated with
respect to a control assay without inhibitors. IC50 values were
determined with 7 inhibitor concentrations. Data represent the
mean of independent triplicate determinations, and the standard
deviations were less than 10%.
A suspension of 21 (75 mg, 0.13 mmol) and Pd/C 10% (75 mg)
in methanol (4 mL) was purged from air and filled with H2. The
reaction was stirred at r.t. for 3 h then diluted with methanol,
filtered through a Celite pad and concentrated. Lyophilization
afforded 25 (63 mg, 100%, white solid): [a]2D0 +51 (c 1.0, MeOH);1H
NMR (DMSO-d6) d 7.88 (d, 1H, JNH–H2¢ = 8.0 Hz, NH), 4.35 (ddd,
1H, JH1¢–H3b = 7.3 Hz, JH1¢–H2¢ = 6.0 Hz, JH1¢–H3a = 5.5 Hz, H1¢), 3.91–
3.78 (m, 2H, H1¢¢), 3.71 (ddd, 1H, JH2¢–H3¢ = 10.0 Hz, JH2¢–NH = 8.0
Hz, JH2¢–H1¢ = 6.0 Hz, H2¢), 3.56 (bd, 1H, JH6¢a–H6¢b = 11.6 Hz, H6¢a),
3.48–3.37 (m, 2H, H3¢, H6¢b), 3.36–3.29 (m, 1H, H5¢), 3.09 (dd, 1H,
J = 8.9 Hz, J = 8.3 Hz, H4¢), 3.01, 2.97 (AB from ABX, 1H, JAB
=
13.1 Hz, JAX = 21.6 Hz, JBX = 20.5 Hz, H1a, H1b), 2.84, 2.78 (AB
from ABX, 1H, JAB = 16.3 Hz, JAX = 5.5 Hz, JBX = 7.3 Hz, H3a,
H3b), 1.77 (s, 3H, CH3CO), 1.64–1.47 (m, 2H, H2¢¢a, H3¢¢), 1.40–
1.15 (m, 6H, H2¢¢b, H4¢¢a, H5¢¢, H6¢¢), 1.12–1.04 (m, 1H, H4¢¢b), 1.06
(s, 6H, H8¢¢, H9¢¢), 0.85 (d, 3H, JH10¢¢–H3¢¢ = 6.4 Hz, H10¢¢); 13C NMR
(DMSO-d6) d 201.9 (d, JC2–P = 5.0 Hz, C2), 169.4 (CH3CO), 74.8
(C5¢), 71.0 (C4¢), 70.4 (C3¢), 69.5 (C1¢), 68.7 (C7¢¢), 62.7 (d, JC1¢¢–P
=
6.0 Hz, C1¢¢), 61.1 (C6¢), 52.7 (C2¢), 44.3 (d, JC1–P = 121.0 Hz, C1),
43.9 (C6¢¢), 41.3 (C3), 37.2, 37.1 (C2¢¢, C4¢¢), 29.3, 29.2 (C8¢¢, C9¢¢), 28.9
(C3¢¢), 22.6 (CH3CO), 21.1 (C5¢¢), 19.2 (C10¢¢); 31P NMR (DMSO-d6)
d 14.1 (s); MS (ESI): m/z = 993 [2M - H]- 100%; HRMS calcd for
C21H39NO10P- 496.2312, found 496.2314.
Acknowledgements
We gratefully acknowledge the European Community for the
financial support of the Eur-INTAFAR integrated project within
the 6th PCRDT framework (contract No. LSHM-CT-2004-
512138) and for a post-doctoral fellowship granted to B. A. We
thank the “Re´gion Ile de France” for a doctoral grant to N.A. and
the “Ville de Paris” for a post-doctoral fellowship to R. F.
Hexadecanyl 3-(2-acetamido-2-deoxy-a-D-glucopyranosyl)-2-
oxopropylphosphonate 26
A suspension of 23 (40 mg, 61 mmol) and Pd/C 10% (40 mg)
in ethanol (10 mL) was purged from air and filled with H2. The
reaction was stirred at r.t. for 3 h then diluted with methanol.
Filtration through a Celite pad and concentration afforded 26 (30
mg, 86%, white solid): [a]2D0 +41 (c 1.0, MeOH); 1H NMR (DMSO-
d6) d 8.64 (d, 1H, JNH–H2¢ = 9.0 Hz, NH), 4.34–4.26 (m, 1H, H1¢),
3.81–3.70 (m, 1H, H2¢), 3.68–3.52 (m, 2H, H1¢¢), 3.50–3.38 (m, 1H,
H3¢), 3.38–3.22 (m, 4H, H3a, H5¢, H6¢), 3.04–2.96 (m, 1H, H4¢), 2.81
(dd, 1H, JH1a–P = 21.5 Hz, JH1a–H1b = 11.5 Hz, H1a), 2.60–2.35 (m,
2H, H1b, H3b), 1.73 (s, 3H, CH3CO), 1.50–1.40 (m, 2H, H2¢¢), 1.32–
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©