Inhibition of tRNA-dependent ligase MurM from Streptococcus pneumoniae by phosphonate and sulfonamide inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.03.028

Ligase MurM catalyses the addition of Ala from alanyl-tRNA^Ala, or Ser from seryl-tRNA^Ser, to lipid intermediate II in peptidoglycan biosynthesis in Streptococcus pneumoniae, and is a determinant of high-level penicillin resistance. Phosphorus-based transition state analogues were designed as inhibitors of the MurM-catalysed reaction. Phosphonamide analogues mimicking the attack of a lysine nucleophile upon Ala-tRNA^Ala showed no inhibition of MurM, but adenosine 3′-phosphonate analogues showed inhibition of MurM, the most active being a 2′-deoxyadenosine analogue (IC₅₀ 100 μM). Structure/function studies upon this analogue established that modification of the amino group of the aminoalkylphosphonate resulted in loss of potency, and modification of the adenosine 5′-hydroxyl group with either a t-butyl dimethyl silyl or a carbamate functional group resulted in loss of activity. A library of 48 aryl sulfonamides was also screened against MurM using a radiochemical assay, and two compounds showed sub-millimolar inhibition. These compounds are the first small molecule inhibitors of the Fem ligase family of peptidyltransferases found in Gram-positive bacteria.

Full text of DOI:10.1016/j.bmc.2009.03.028

Bioorganic & Medicinal Chemistry 17 (2009) 3443–3455
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of tRNA-dependent ligase MurM from Streptococcus pneumoniae
by phosphonate and sulfonamide inhibitors
Elena Cressina a, Adrian J. Lloyd b, Gianfranco De Pascale a,b, B. James Mok c, Stephen Caddick c,
David I. Roper b, Christopher G. Dowson b, Timothy D. H. Bugg a,
*
a Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
b Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
c Department of Chemistry, University College London, 20 Gower Street, London WC1H 0AJ, UK
a r t i c l e i n f o
a b s t r a c t
Ligase MurM catalyses the addition of Ala from alanyl-tRNAAla, or Ser from seryl-tRNASer, to lipid inter-
mediate II in peptidoglycan biosynthesis in Streptococcus pneumoniae, and is a determinant of high-level
penicillin resistance. Phosphorus-based transition state analogues were designed as inhibitors of the
MurM-catalysed reaction. Phosphonamide analogues mimicking the attack of a lysine nucleophile upon
Ala-tRNAAla showed no inhibition of MurM, but adenosine 30-phosphonate analogues showed inhibition
Article history:
Received 4 December 2008
Accepted 14 March 2009
Available online 21 March 2009
Keywords:
MurMN
FemABX
Ligase
Peptidoglycan biosynthesis
Streptococcus pneumoniae
Inhibition
of MurM, the most active being a 20-deoxyadenosine analogue (IC50 100
lM). Structure/function studies
upon this analogue established that modification of the amino group of the aminoalkylphosphonate
resulted in loss of potency, and modification of the adenosine 50-hydroxyl group with either a t-butyl
dimethyl silyl or a carbamate functional group resulted in loss of activity. A library of 48 aryl sulfona-
mides was also screened against MurM using a radiochemical assay, and two compounds showed sub-
millimolar inhibition. These compounds are the first small molecule inhibitors of the Fem ligase family
of peptidyltransferases found in Gram-positive bacteria.
Antibacterial
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
A
C
B
MurNAc
L-Ala
MurNAc
L-Ala
Infections caused by Streptococcus pneumoniae are responsible
for approximately 3 million deaths each year worldwide due to
pneumonia, meningitis and sepsis, and cause serious upper airway
infections such as sinusitis and otitis media.1 Penicillin resistance
in S. pneumoniae is a significant clinical problem in a number of
countries worldwide, with up to 39% of S. pneumoniae isolates
resistant to penicillin in some European countries in 2005.2 Given
the steady worldwide increase in resistance rates, and the lack of
effective new antibacterial agents appearing from pharmaceutical
development,3 there is a need to identify new targets for antibac-
terial action and strategies to combat antibiotic resistance.
The peptidoglycan layer of many highly penicillin resistant
strains of S. pneumoniae contain additional -Ala-Ala- and -Ser-
Ala- inter-strand cross-links,4 as shown in Figure 1. The presence
of these cross-links is associated with particular sequences of the
murM and murN genes,5 in the same gene family as the femABX
genes responsible for formation of the (Gly)5 cross-link in Staphy-
lococcus aureus.6 The encoded MurMN proteins catalyse the addi-
tion of Ala(Ser) and Ala, respectively, to lipid intermediate II in
MurNAc
L-Ala
MurNAc
L-Ala
D-Gln
L-Lys
D-Gln
D-Gln
L-Lys
D-Gln
L-Lys
D-Ala
L-Lys L-Ala(Ser) L-Ala D-Ala
D-Ala
D-Ala
D-Ala
D-Ala
C55-OPP
C55-OPP
MurNAc GlcNAc
L-Ala
MurNAc GlcNAc
L-Ala
MurM
γ-D-Glu
γ-D-Glu
Ala-tRNA
L-Lys NH2
L-Lys L-Ala NH2
D-Ala
D-Ala
D-Ala
D-Ala
Figure 1. Peptidoglycan cross-links found in the cell walls of (A) penicillin-sensitive
and (B) highly penicillin-resistant Streptococcus pneumoniae. (C) Reaction catalysed
by S. pneumoniae ligase MurM. C55-OPP, undecaprenyl diphosphate.
peptidoglycan biosynthesis,7 using Ala-tRNAAla or Ser-tRNASer
respectively (see Fig. 1C), analogous to the addition of five glycine
,
* Corresponding author. Tel.: +44 02476 573018; fax: +44 02476 524112.
E-mail address: T.D.Bugg@warwick.ac.uk (T.D.H. Bugg).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.028

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E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
units from Gly-tRNAGly by FemABX in S. aureus.8 Deletion of the
murM gene in S. pneumoniae leads to loss of the high-level resis-
tance phenotype,5 therefore MurM is a potential target for anti-
resistance agents that could be applied synergistically with penicil-
lin, against antibiotic-resistant bacteria.
R1
O
R1
a,b
O
CbzNH
P
OH
Ph
O
NH2
H
O
OR2
1a-e
We have recently reported the in vitro reconstitution of the
MurM-catalysed reaction for penicillin-resistant and -susceptible
MurM variants of S. pneumoniae,9 allowing the study of inhibitors
of MurM. The catalytic mechanism of the MurM-catalysed reaction
is likely to proceed via a tetrahedral transition state, shown in
Figure 2. This transition state could be effectively mimicked by a
phosphonate or phosphonamide group. We have previously re-
ported in a communication the identification of the first synthetic
inhibitor of MurM, an adenosine phosphonate transition state ana-
logue.10 In this paper we report the synthesis and evaluation of two
series of phosphorus-based transition state analogues for the
MurM-catalysed reaction, and the screening of a library of aryl sul-
fonamides against MurM. The first series of phosphonamides mi-
mic the attack of a lysine nucleophile upon Ala-tRNAAla; the
second series of adenosine phosphonates incorporate the adeno-
sine base found at the 30-terminus of tRNA, as shown in Figure 2.
c,d
R1
R1
O
e
O
H2N
P
NHR3
CbzNH
P
NHR3
OR2
OR2
3a-i
2a-i
O
P
NHBoc
O
P
OR2
NH3
OR2
NH3
N
H
MeO2C
N
H
R1
R1
R1 R2
R1 R2
Me -CH3
3a
3b
3c
3d
3e
2. Results
Me -CH3
3f
Me -CH2CH2OCH3
Me -CH2CH2OCH3
Me -n-Bu
3g
3h
3i
Me -n-Bu
2.1. Synthesis and assay of phosphonamide transition state
analogues
Et
-CH3
Ph -CH3
Ph -CH3
The first series of phosphonamide transition state analogues
(see Fig. 2) was synthesised using the synthetic route illus-
trated in Figure 3. Cbz-protected aminoalkylphosphonate mon-
Figure 3. Synthetic route for phosphonamide series. Reagents: (a) RCHO, PCl3,
CH2Cl2; (b) R1OH, CH2Cl2; (c) (COCl)2, DMF; (d) R2NH2, Et3N, CH2Cl2; (e) H2, Pd/C,
MeOH.
esters 1a–e were prepared in
a one-pot condensation of
aldehydes the phosphonate diester and diacid were also
observed, which were separated by chromatography. Three dif-
ferent alcohols were used for monoester synthesis: methanol,
benzylcarbamate, an aldehyde and PCl3, as reported by Yuan
et al.11 This reaction proceeded in 25–37% yield, to give the phos-
phonate monoester as the major product, though with aliphatic
NH2
A
NH2
N
N
N
N
N
tRNA
tRNA
C55-OPP
C55-OPP
MurNAc GlcNAc
N
O
O
N
N
MurNAc GlcNAc
L-Ala
O
O
L-Ala
O
MurM
O
γ-D-Glu
O
OH
O
OH
γ-D-Glu
L-Lys
N
H
L-Lys
NH2
D-Ala
D-Ala
NH3
NH3
D-Ala
D-Ala
NH2
B
N
N
HO
-O
N
N
O
NHBoc
O
P
O
R
OR
MeO2C
O
P
N
H
NH3
NH3
Phosphonamide analogues
R = -CH3, -CH2CH2OCH3
Adenosine phosphonates
R = OH, H
Figure 2. Transition state analogues designed for MurM. (A) The presumed oxyanion intermediate for the MurM-catalysed reaction. (B) Phosphonamide and adenosine
phosphonate transition state analogues.

E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
3445
2-methoxyethanol (mimicking the 20-hydroxyl group of the
adenosine nucleotide) and 1-butanol.
derivative were unsuccessful using a range of peptide coupling
agents (data not shown), indicating that the 30-position of this
nucleoside is sterically hindered. Zemlicka et al. have previously
reported the coupling of phosphonate diacid 4 to a similar pro-
tected adenosine derivative, to give a mixture of 20- and 30-
substituted phosphonate esters, using DCC as coupling agent.12
In our hands, phosphonate diacid 4 was coupled using EDC to
the known adenosine derivative 5,13 to give a 3:1 mixture of
30- and 20-substituted phosphonate esters 6a and 6b in 60% over-
all yield. Deprotection of the N6-benzoyl group of adenosine with
ammonia/methanol gave 7a/7b in 97% yield; desilylation with
Et3NꢀHF gave 8a/8b in 99% yield; and removal of the Cbz group
was achieved by hydrogenation in 87% yield, to give 9a/9b as a
3:1 mixture of regioisomers.
Phosphonate monoesters 1a–e were then converted to the cor-
responding phosphonochloridate using oxalyl chloride, and then
a
coupled with either 1-propylamine or N -Boc-lysine methyl ester
to give protected phosphonamides 2a–i, typically in 40–70% yield.
Deprotection of the N-Cbz group was achieved by hydrogenation,
typically in 80–100% yield.
Each phosphonamide was tested as an inhibitor of recombinant
MurM from the highly penicillin-resistant strain 159 of S. pneumo-
niae, using a radiochemical assay, but no inhibition was observed
at 1 mM concentration for any of the compounds. The stability of
the phosphonamides under the assay conditions (pH 6.8, 37 °C)
was verified, therefore this series of compounds do not act as
inhibitors for MurM.
Attempts were made to separate 9a and 9b, and, although chal-
lenging to separate, a small sample of a single regioisomer was ob-
tained after MonoQ anion exchange chromatography, which was
found to be the 20-regioisomer. Analysis by C18 reverse phase HPLC
confirmed that the two regioisomers 9a and 9b had retention times
of 15.5 and 16.1 min, and the purified sample of 9b showed a single
peak at 16.06 min.
2.2. Synthesis and assay of adenosine phosphonate transition
state analogues
The synthetic route employed for the adenosine 30-phospho-
nate analogue is illustrated in Figure 4. Earlier attempts to cou-
ple either a phosphonate monoester 1a or the corresponding
The corresponding derivative of 20-deoxyadenosine was syn-
thesised using a similar synthetic sequence, illustrated in
phosphonate diacid
4
to
a
20,50-TBDMS-protected adenosine
Figure 5. The N6-benzoyl,50-OTBDMS derivative 10 was prepared
using literature methods.13 Coupling to phosphonate diester 4
was found to proceed in low yield using EDC, but coupling with
phosphonate monoester 1a was successful using DCC as coupling
agent, giving the demethylated product 11 in 54% yield after
6 days reaction time. 11 was deprotected by treatment with
ammonia/methanol to give debenzoylated 12 in 85% yield, fol-
lowed by desilylation with Et3NꢀHF to give 13 in 99% yield,
and hydrogenation to give the target phosphonate 14 in 62%
yield.
NHBz
N
N
N
TBDMSO
N
O
OH
O
CbzNH
P
The sample of pure 20-phosphonate 9b showed weak inhibition
OH
HO OH
of MurM (IC50 780 lM), whereas the 3:1 mixture of 9a/9b showed
5
4
ꢁ20% inhibition of MurM at 1 mM concentration, therefore indi-
cating that the 20-regioisomer binds more tightly to MurM than
the 30-regioisomer (see Section 3). 20-Deoxyadenosine phospho-
a
NHR2
N
NHR2
N
N
N
N
NHBz
N
N
R1O
R1O
N
N
N
O
O
N
TBDMSO
N
O
O
O
O
CbzNH
P
OMe
a
O
OH
OH O
R1
R2
HO
P
P
OH
b
c
6a/6b TBDMS Bz
7a/7b TBDMS H
OH
OH
1a
10
NHCbz
8a/8b
H
H
NHCbz
d
NH2
N
NH2
N
NHR2
NH2
N
N
N
N
N
N
N
HO
N
N
HO
N
N
N
R1O
HO
N
HO
O
O
N
O
O
d
O
O
O
O
O
OH
OH O
O
O
R1
R2
-O
P
11 TBDMS Bz
-O
P
P
OH
P
b
c
9a 9b
3 : 1
14
12 TBDMS
13
H
H
H
NHCbz
NH3
NH3
NH3
Figure 5. Synthetic route for 20-deoxyadenosine phosphonate. Reagents and
conditions: (a) DCC, Dowex (pyr+), pyridine, 5–6 days; (b) NH3, MeOH; (c) Et3NꢀHF,
THF; (d) H2, Pd/C, MeOH/H2O.
Figure 4. Synthetic route for adenosine phosphonates. Reagents and conditions: (a)
EDC, Dowex (pyr+), pyridine, 5–6 days; (b) NH3, MeOH; (c) Et3NꢀHF, THF; (d) H2, Pd/
C, MeOH/H2O.

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E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
whereas the fully protected compound
6
and the N-Cbz,50-
100
90
80
70
60
50
40
30
20
10
0
OTBDMS protected derivative 7 showed no enzyme inhibition. In
the 20-deoxyphosphonate series, the N-Cbz protected derivative
13 showed no enzyme inhibition, neither did the protected deriv-
atives 11 and 12.
14
9b
20-Deoxyadenosine showed only 13% inhibition of MurM at
1 mM concentration, indicating the importance of the phosphonate
group. 20-Deoxyadenosine 30-phosphate was tested against MurM,
and showed 20% inhibition at 1 mM concentration, indicating that
the aminoalkyl substituent is required for efficient binding. A
methylphosphonate derivative 15 lacking the amino-substituent
was synthesised by coupling of commercially available methyl-
phosphonic acid to 10 using DCC, in 47% yield, followed by depro-
tection as before. Phosphonate 15 was assayed against MurM, but
showed only 7% inhibition at 1 mM concentration, less active than
the 30-phosphate derivative. Therefore, the amino-substituent does
appear to be required for efficient binding to the MurM active site.
In the Ala-tRNA substrate for MurM, the adenosine nucleotide is
at the 30-terminus of a large tRNA molecule, therefore, synthetic
modification at the 50 position of 14 seemed a logical approach to
higher affinity MurM inhibitors. The 50-OTBDMS derivative 16 was
prepared by hydrogenation of 11, in 99% yield. Compound 16 was as-
sayed against MurM, but showed no enzyme inhibition at 1 mM,
indicating that a bulky substituent is not tolerated in this position.
As a neutral mimic of the phosphodiester linkage present in alanyl
tRNAAla, the 50-carbamate derivative 17 was synthesised. Using the
method of Gotor et al.,14 20-deoxyadenosine was reacted with ace-
tone O-(phenyloxycarbonyl) oxime in the presence of Candida ant-
arctica lipase B, yielding the 50-phenyl carbonate derivative in 73%
yield, which was then reacted with methylamine, followed by
deprotection, to give 17. Assays versus MurM showed only 3% inhi-
bition by 17 at 1 mM concentration, therefore, modification to the 50
carbamate has reduced activity significantly, which is surprising, gi-
ven that a phosphodiester linkage is found at this position in alanyl
tRNA.
0
200
400
600
800
1000
Concentration (µM)
Figure 6. Inhibition of MurM by adenosine phosphonates 14 and 9b.
nate 14 showed much higher levels of inhibition, as shown in
Figure 6, giving an IC50 value of 100 lM.
20-Deoxyadenosine phosphonate 14 was tested for antibacterial
activity against S. pneumoniae strains pn16 (penicillin-sensitive)
and 159 (penicillin-resistant, MIC 16
was observed at 1 mM concentration (380
penicillin MIC was observed for strain 159. Compound 14 was also
tested against other strains containing femABX genes, namely
S. aureus, Enterococcus faecalis and Micrococcus flavus, but no anti-
microbial activity was observed in each case.
l
g/ml). No growth inhibition
g/ml), and no effect on
l
2.3. Structure/activity studies on adenosine phosphonate
inhibitors
In order to assess the role of different functional groups in the
inhibitor structure, several protected compounds were also tested
against MurM, as listed in Table 1. In the ribo-phosphonate series,
the N-Cbz protected derivative 8 showed 15% inhibition of MurM
at 1 mM concentration, similar to the deprotected compound,
NH2
N
NH2
N
N
N
Me
N
HO
N
N
Si
O
O
N
O
Me
Table 1
O
Structure/activitiy data for inhibition of S. pneumoniae 159 MurM by adenosine
derivatives
O
O
O
-O
P
-O
P
15
16
Compound Description
% Inhibition
at 1 mM
CH3
NH3
concentration
6a/6b
7a/7b
8a/8b
50-OTBDMS, N-Cbz, Bz-Ad ribofuranose phosphonate
0
0
15
50-OTBDMS, N-Cbz ribofuranose phosphonate
N-Cbz ribofuranose phosphonate (3:1 mixture of
20,30 isomers)
NH2
N
N
Ribofuranose phosphonate (3:1 mixture of 20,30
isomers)
19
N
N
N
N
9a/9b
N
N
NHMe
O
9b
—
—
Ribofuranose 20-phosphonate
20-Deoxyadenosine
55
13
15
0
N
O
O
O
HO
O
20-Deoxyadenosine 30-phosphate
50-OTBDMS, N-Cbz, Bz-Ad deoxyribofuranose
phosphonate
11
O
O
NH OH
NH3
12
13
14
15
16
17
50-OTBDMS, N-Cbz deoxyribofuranose phosphonate
N-Cbz deoxyribofuranose phosphonate
Deoxyribofuranose phosphonate
Deoxyribofuranose 30-methylphosphonate
50-OTBDMS deoxyribofuranose phosphonate
50-Methylcarbamate deoxyribofuranose
phosphonate
0
0
95
7
0
3
-O
P
17
18
NH3
18
Puromycin
0
OMe

E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
3447
Puromycin (18) is a 30-acylamino derivative of adenosine, which
inhibits the action of the bacterial ribosome by mimicking tyrosyl
tRNA.15 Puromycin was tested as an inhibitor of MurM, but showed
no inhibition at 1 mM concentration.
In view of the similarity of phosphonate 14 to the transition
state for the formation of acyl-tRNA by aminoacyl tRNA synthetase
enzymes, which are known antibacterial targets,16 14 was also as-
sayed as an inhibitor of S. pneumoniae alanyl tRNA synthetase
(which is used here for reconstitution of MurM9), and seryl-tRNA
synthetase, as described in Section 4. At 1 mM concentration, phos-
phonate 14 showed 22% inhibition of S. pneumoniae alanyl-tRNA
synthetase, and 28% inhibition of S. pneumoniae seryl tRNA synthe-
that functions as a transition state mimic, but no inhibition was ob-
served using phosphonamide dipeptide analogues 3a–i, and only
weak inhibition was observed using the corresponding ribo-phos-
phonate analogue 9b. The lack of activity of the phosphonamide
series could be due to a requirement for the adenosine nucleoside
structure for binding, or might indicate that a negatively charged
phosphonate group is needed, in order to mimic the transition
state structure.
One possible rationalisation of the higher activity of the 20-
deoxy analogue 14, compared with the ribofuranose analogue 9,
is that the conformation of the furanose ring is important for bind-
ing to MurM. Ribo-nucleoside derivatives are known to adopt pref-
erentially the 30-endo conformation, in which the 20-substituent is
axial and 30-substituent equatorial, whereas 20-deoxy derivatives
prefer the 20-endo conformation, in which the 30-substituent is
axial, as shown in Figure 8.19 The 1H–1H NMR coupling constant
between H-10 and H-20 is a reliable reporter of furanose conforma-
tase; no inhibition was observed at 100 lM concentration.
2.4. Screening of library of aryl sulfonamides
Sulfonamides represent an alternative transition state mimic to
phosphonates, and, being less polar and uncharged, offer improved
pharmacokinetic properties. Caddick et al. have recently reported
the use of microwave-assisted synthesis to prepare libraries of aryl
sulfonamides.17 Using this methodology, a library of 48 aryl sulfon-
amides having the general structure shown in Figure 6 was pre-
pared, and was screened for inhibition of MurM at 1 mM
concentration, using the radiochemical enzyme assay. Two com-
pounds were found to show inhibition of MurM, whose structures
are shown in Figure 7. They showed 50% (R = H) and 34% (R = Br)
inhibition of MurM at 1 mM concentration of inhibitor. These
structures represent possible lead structures for development of
non-nucleoside inhibitors of the MurMN/FemABX ligase family.
tion.19 In the case of the ribo-phosphonates 9a/9b, J1 2 values of
6.6 Hz and 6.0 Hz were measured, respectively, which are similar
to literatures values of 5.8 Hz for adenosine 30-phosphate, and
5.9 Hz for adenosine 20-phosphate,20 consistent with the adoption
of a 30-endo conformation in solution. In the case of 20-deoxyribo-
0
0
0
0
phosphonate 14, J1 2 values of 7.7 and 5.7 Hz were measured.
The calculated J1 2 values for a 20-endo conformation are 10.1 and
5.5 Hz, whereas the calculated values for a 30-endo conformation
are 7.4 and 0.1 Hz,21 with one very small coupling constant, due
to a 90° dihedral bond angle, that is not observed in 14. Therefore,
the NMR data for 20-deoxy analogue 14 are consistent with a 20-
endo conformation, bearing an axial 30-phosphonate group,
whereas the 30-phosphonate of ribo-analogue 9a is equatorial,
implying that an axial phosphonate group is required for efficient
binding to MurM. This is supported by the observation that 20-
phosphonate 7b, which contains an axial phosphonate at C-20, still
shows modest enzyme inhibition.
0
0
3. Discussion
The MurMN/FemABX family of aminoacyl-tRNA ligases repre-
sent an possible target for chemotherapeutic intervention against
a number of pathogenic Gram-positive bacteria that contain inter-
strand cross-links in their peptidoglycan structures, including S.
aureus, E. faecalis and penicillin-resistant S. pneumoniae. Many
Gram-positive bacteria contain inter-strand cross-links, and con-
siderable diversity in the nature of these cross-links exists,18 there-
fore it could be possible to target certain types of bacteria, if a
selective agent could be developed. One would expect that such
an agent would increase the susceptibility towards penicillin, and
might be bacteriocidal, since it is known for example that the femX
gene in S. aureus is essential.6
The lack of antibacterial activity for 14 against S. pneumoniae
strain 159, and the lack of effect on penicillin MIC, suggests that
14 is not effectively transported across the cell membrane. A
tRNA-based inhibitor for Weissella viridescens FemX has been re-
ported, which also shows no antibacterial activity.22 It would
therefore be of interest to prepare neutral, less polar MurM inhib-
itors that would be more likely to be taken up into the cell. It is
therefore of interest that two aryl sulfonamides obtained from li-
brary screening show weak inhibitory activity against MurM.
There is protein structural data available for S. aureus FemA23
and W. viridescens FemX,24 in the latter case co-crystallised with
UDPMurNAc-pentapeptide.24 At present there is no structure avail-
able containing the aminoacyl tRNA substrate, therefore the pre-
cise location of the aminoacyl-tRNA binding site is not known.
Active site residue Arg-211, located close to the diphosphate link-
age, has been implicated in substrate binding, using site-directed
mutagenesis.25 On the opposite side of the FemX active site, as
shown in Figure 9, there is a cluster of conserved amino acid resi-
dues, comprising Tyr-72, Asp-108, and Gln-143, of which Asp-108
has been suggested to act as a catalytic base.26
In this work, we have shown that S. pneumoniae MurM is inhib-
ited in vitro by a 20-deoxyadenosine 30-phosphonate analogue 14,
O
O
R1 = allyl, t-butyl,
2-hydroxyethyl,
4-methylbenzyl
S
R1NH
O
R2 = alkyl, aryl
N
R2
Me
These three amino acid residues are conserved in all MurM/
FemABX homologues, and seem a possible site for acyl transfer. Sit-
uated close to this conserved triad is the sidechain of Lys-75, which
might provide electrostatic stabilisation to the oxyanion transition
state. An electrostatic interaction of this type might rationalise
why enzyme inhibition is only observed using charged phospho-
nate analogues, whereas the phosphonamide series of analogues
was uncharged at the phosphorus centre.
Phosphonate analogue 14 was also tested as an inhibitor of
seryl and alanyl-tRNA synthetases from S. pneumoniae, since it
also mimics the transition state for acyl tRNA formation. Only
rather weak inhibition was observed, in the millimolar range,
O
O
S
N
H
O
N
Me
O
X = H
X = Br
X
Figure 7. Structures of aryl sulfonamide library, and the two compounds found to
inhibit MurM.

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E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
Ribofuranoses - 3'-endo conformation:
2'-deoxyfuranose- 2'-endo conformation:
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
HO
N
HO
O
HO
N
O
O
N
O
HO
O
O
-O
OH
P
O
O
O
-O
P
P
-O
NH3
NH3
NH3
14
IC50 100 µM
9a
INACTIVE
9b
IC50 780 µM
Figure 8. Expected lowest energy conformations for the ribofuranose ring of phosphonates 9a, 9b and 14.
Figure 9. Active site of Weissella viridescens FemX, complexed with UDPMurNAc-pentapeptide (in cyan, Lys-NH2 at position 3 and D-Ala-COOꢂ at position 5 are indicated),
showing active site residues Arg-211, Tyr-72, Asp-108, Gln-143, Lys-75 and Arg-106. Figure prepared using PYMOL software, from PDB file 1FYZ.
whereas binding constants for tRNA substrates by these en-
zymes are typically in the sub micromolar range. Therefore,
there is a prospect for development of a selective transition
state analogue for the MurMN/FemABX family of enzymes,
provided that transport across the cell membrane can be
achieved.
of Fernandez et al.27 Puromycin (18) was purchased from Sigma
Aldrich Chemical Co. Undecaprenyl phosphate was purchased
from Larodan Fine Chemicals AB. UDP-MurNAc-pentapeptide (AE-
KAA), M. flavus membranes, lipid intermediate II, recombinant S.
pneumoniae Pn16 alanyl-tRNA synthetase (AlaRS) and S. pneumo-
niae MurM, and M. flavus tRNA were prepared as previously
described.9
4. Experimental
4.1. Materials
4.2. General procedure for monoalkyl 1-(benzyloxycarbonyl-
amino) phosphonates (1a–e) (based upon Ref. 11)
N6-Benzoyl 20-deoxyadenosine was prepared from 20-deoxya-
denosine, using the procedure of Ti et al.13 N6-Benzoyl
50-(tert-butyldimethylsilyl) adenosine (5) and N6-benzoyl 50-
(tert-butyldimethylsilyl) 20-deoxyadenosine (10) were prepared
by silylation of N6-benzoyl adenosine and N6-benzoyl 20-deoxya-
denosine, respectively, using the procedure of Ogilvie et al.13 Ace-
tone O-((phenyloxy)carbonyl) oxime was prepared by the method
Benzylcarbamate (2 g, 13.2 mmol) was suspended in dry CH2Cl2
(50 mL) under N2 and cooled to approximately ꢂ15 °C in a ice/
methanol bath. The appropriate aldehyde (15.8 mmol) was added
to the suspension followed by phosphorus trichloride (1.38 mL,
15.8 mmol). The mixture was brought to 0 °C in an ice bath and left
stirring for 1 h 30 min. After this time, the solution was warmed to
rt and flushed with N2 to eliminate the residue aldehyde. Then dry

E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
3449
alcohol (25.0 mmol) was added dropwise and the mixture was stir-
red for 4 h. After this time, the solvent was removed under reduced
pressure and the remaining yellow oil was dissolved in ethyl ace-
tate (50 mL) and extracted with 5% NaHCO3 (3 ꢃ 100 mL). The
combined water phases were acidified to pH 1 with concentrated
HCl and extracted with CH2Cl2 (3 ꢃ 100 mL). The combined organic
phases were dried over MgSO4 and the solvent removed under re-
duced pressure. The residue was purified by flash chromatography
on silica gel (eluent CH2Cl2/MeOH from 100% CH2Cl2 to 98:2 at
0.5% gradient of methanol).
4.2.6. 1-(Benzyloxycarbonylamino) ethyl phosphonic acid (4)
The above procedure was followed, using acetaldehyde
(1.09 mL, 19.5 mmol), except that the reaction was quenched with
a chilled solution of NaOH (500 mg in 10 mL). The standard work-
up yielded acid 4 (1.336 g 5.15 mmol, 40% yield) as a white solid.
1H NMR (300 MHz; CDCl3/CD3OD): dH 7.32 (5H, m, Ph), 5.04 (2H,
m, CH2-Ph Cbz), 4.00 (1H, m, CH–P), 1.35 (3H, m). 13C NMR
(75.5 MHz; CDCl3/CD3OD): dC 158.6, 138.2, 130.0, 129.6, 129.5,
68.5, 47.1, 45.1 (d, JCP = 159), 17.0. 31P NMR (121.5 MHz; CDCl3/
CD3OD): dP 27.8. LSI MS: m/z 260.04 (M+H+), 282.01 (M+Na+). HR
MS (FAB): 260.0696 Da (calcd for C10H15NO5P (M+H+): 260.0687).
4.2.1. Methyl 1-(benzyloxycarbonylamino) ethyl phosphonate (1a)
Yield: 33% (white solid). Mp: 120–123 °C (lit.11 119–120 °C). Rf
(CH2Cl2/MeOH 8:2) = 0.1. 1H NMR (300 MHz; CDCl3/CD3OD): dH
7.30 (5H, m, Ph), 5.06 (2H, s, CH2-Ph), 4.03 (1H, m, CH–P), 3.67
(3H, d, J = 10.2, OCH3), 1.28 (3H, dd, JPH = 16.4, J3 = 7.3, CH3–CH–
P). 13C NMR (75.5 MHz; CDCl3/CD3OD): dC 155.5, 135.8, 128.3,
128.0, 127.9, 67.0, 52.5, 43.8, 41.7 (d, JCP = 159), 15.4. 31P NMR
(121.5 MHz; CDCl3): dP 22.9. ES-MS: m/z 318.1 (M+2Na+). HR MS
(FAB): 274.0845 Da (calcd for C11H17NO5P (M+H+): 274.0844). IR
4.3. General procedure for O-alkyl N-alkyl 1-(benzyloxycarbo-
nylamino) alkyl(aryl) phosphonamides (2a–i)
Phosphonate monoester 1 (1.48 mmol) was suspended in dry
CH2Cl2 (20 mL) and added dropwise to a solution of (COCl)2
(258 lL, 2.96 mmol) and DMF (10 lL, 0.132 mmol) in dry CH2Cl2
(20 mL) and the solution was stirred at room temperature for 1 h
under nitrogen. After this time, the volatile components were re-
moved under reduced pressure and the residue re-dissolved in
dry CH2Cl2 (30 mL) under N2 and cooled to 0 °C. Triethylamine
(neat):
m
(cmꢂ1) 3304, 2956, 1694, 1531, 1454.
4.2.2. 2-(Methoxy)ethyl 1-(benzyloxycarbonylamino) ethyl
phosphonate (1b)
(412
pylamine (182
l
L, 2.38 mmol) is added dropwise followed by either n-pro-
l
L, 2.22 mmol) or solution of Boc-Lys-OMe
a
Yield: 36% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.15. 1H NMR
(300 MHz; CDCl3/CD3OD): dH 7.32 (5H, m, Ph), 5.10 (2H, m, CH2-
Ph), 4.10 (3H, m, CH–P, CH2–O–P), 3.55 (2H, m, CH2–O–CH3), 3.31
(3H, m, OCH3 and CD3OD), 1.36 (3H, dd, J3 = 7.3, JPH = 16.5, CH3–
CH–P). 13C NMR (125 MHz; CDCl3): dC 156.4, 136.7, 128.9, 128.4,
74.0, 71.8, 67.5, 65.8, 62.0, 44.7, 43.4 (d, JCP = 161), 16.2. 31P NMR
(121.5 MHz; CDCl3): dP 28.16. ES-MS: m/z 362.3 (M+2Na+). IR
(613 mg, 2.35 mmol) in CH2Cl2. The solution was brought to room
temperature and stirred for 1 day. After this time, the solvent was
removed under reduced pressure and the residue was dissolved in
ethyl acetate (50 mL) and extracted with 5% NaHCO3 (3 ꢃ 50 mL)
then with water (20 mL) and brine (20 mL). The organic phase
was dried over MgSO4 and the solvent was removed under reduced
pressure. The crude product was purified by flash chromatography
on silica gel (eluent: CH2Cl2 100% to CH2Cl2/MeOH 98:2, 0.5% gra-
dient of methanol).
(neat):
m
(cmꢂ1) 3295, 2941, 1698, 1530, 1453.
4.2.3. n-Butyl 1-(benzyloxycarbonylamino) ethyl phosphonate (1c)
Yield: 31% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.15. 1H NMR
(300 MHz; CDCl3): dH 11.6 (1H, br, OH), 7.33 (5H, m, Ph), 5.57
(1H, br, NH), 5.10 (2H, s, CH2-Ph), 4.16 (1H, m, CH–P), 4.02 (2H,
dd, JPH = 13.4, J3 = 7.2, CH2O but), 1.60 (2H, qui, J3 = 7.2, CH2 but),
1.35 (5H, m, CH2 but, CH3–CH–P), 0.89 (3H, t, J3 = 7.2, CH3 but).
13C NMR (75.5 MHz; CDCl3): dC 156.2, 136.6, 128.9, 128.6, 128.5,
67.5, 66.6, 66.5 (t, JC–P = 6), 44.7, 42.6 (d, JC–P = 160), 32.7, 19.0,
16.1, 13.9. 31P NMR (121.5 MHz; CDCl3): dP 28.3. LSI MS: 316.09
(M+H+), 338.07 (M+Na+). HR MS: 316.1318 Da (calcd for
4.3.1. O-Methyl N-propyl 1-(benzyloxycarbonylamino) ethyl
phosphonamide (2a)
Yield: 79% (oil). Rf (AcOEt/MeOH 95:5): 0.5. 1H NMR (300 MHz;
CDCl3): dH 7.25 (5H, m, Ph), 6.19 and 5.65 (1H, d, J = 9.8, NH Cbz,
two diastereoisomers), 5.01 (2H, s, CH2-Ph), 3.98 (1H, m, CH–P),
3.55, 3.53 (3H, 2 ꢃ d, JPH = 10.5, OCH3, two diastereoisomers), 3.05
(1H, m, NH–P), 2.74 (2H, m, CH2–NH prop), 1.32 (5H, m, CH2–CH2–
NH, CH3–CH–P), 0.78 (3H, m, CH3 prop). 13C NMR (75.5 MHz; CDCl3):
dC 156.0, 155.6, 136.2, 128.2, 127.9, 127.8, 127.7, 66.5, 50.6, 50.5,
50.4, 45.2, 43.3 (d, JCP = 144), 44.1, 41.8 (d, JCP = 146), 42.6, 42.3,
25.0, 15.4, 15.1, 10.8. 31P NMR (121.5 MHz; CDCl3): dP 33.3, 32.0.
ES-MS: m/z 337.2 (M+Na+). Anal. Calcd for C14H23N2O4P: C, 53.50;
C14H23NO5P (M+H+): 316.1313). IR (neat):
2958, 2873, 1688, 1541, 1453.
m
(cmꢂ1) 3285, 3063,
4.2.4. Methyl 1-(benzyloxycarbonylamino) propyl phosphonate
(1d)
H, 7.38; N, 8.91. Found: C, 52.99; H, 7.41; N, 8.66. IR (neat):
m
(cmꢂ1) 3233, 3033, 2961, 2875, 1702, 1537, 1453.
Yield: 25% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.1. 1H NMR (300 MHz;
DMSO-d6): dH 8.31 (1H, s, OH), 7.35 (5H, s, Ph), 5.05 (2H, s, CH2-Ph),
3.58 (4H, m, CH–P, OCH3), 1.75–1.43 (2H, m, CH2–CH–P), 0.85 (3H,
m, CH2–CH3). 13C NMR (75.5 MHz; DMSO-d6): dC 156.5, 137.5,
128.6, 128.0, 128.7, 65.7, 55.2, 50.6, 48.6 (d, JCP = 154), 22.5, 11.3.
31P NMR (121.5 MHz; DMSO-d6): dP 24.7. LSI MS: m/z 288.10
4.3.2. O-(2-(Methoxy)ethyl) N-propyl 1-(benzyloxycarbonylami-
no) ethyl phosphonamide (2b)
Yield: 26% (oil). Rf (CH2Cl2/MeOH 97:3) = 0.5. 1H NMR
(300 MHz; CDCl3): dH 7.21 (5H, m, Ph), 5.67 and 5.33 (1H, d,
J = 9.7, NH Cbz, two diastereoisomers), 5.03 (2H, m, CH2-Ph),
4.08–3.96 (3H, m, CH–P, CH2–O–P), 3.45 (2H, m, CH2–O–CH3),
3.28 (3H, 2 ꢃ s, OCH3, diastereoisomers), 2.87–2.68 (3H, m, CH2–
NH prop, NH–P), 1.41–1.26 (5H, m, CH2–CH2–NH, CH3–CH–P),
0.78 (3H, t, CH3 prop). 13C NMR (75.5 MHz; CDCl3): dC 156.4,
136.8, 128.8, 128.5, 128.4, 128.3, 72.1, 67.2, 63.7, 63.6, 63.5, 63.4,
59.3, 46.3, 44.4 (d, JCP = 144), 45.3, 43.3 (d, JCP = 146), 42.9, 25.6,
16.2, 15.8, 11.4. 31P NMR (121.5 MHz; CDCl3): dP 32.6, 30.6. ES-
MS: m/z 358.1 (M+), 381.2 (M+Na+). LSI MS: m/z 359.10 (M+H+),
381.07 (M+Na+), 397.03 (M+K+). HR MS (FAB): 359.1731 Da (calcd
for C16H28N2O5P (M+H+): 359.1735). Anal. Calcd for C16H27N2O5P:
C, 53.62; H, 7.59; N, 7.82. Found: C, 52.95; H, 7.56; N, 7.11. IR
(M+H+), 310.13 (M+Na+). IR (neat):
1531, 1454.
m
(cmꢂ1) 3304, 2956, 1694,
4.2.5. Methyl 1-(benzyloxycarbonylamino) benzyl phosphonate
(1e)
Yield: 37% (white solid). Mp: 178–180 °C (lit.11 175 °C). 1H NMR
(300 MHz; DMSO-d6): dH 8.27 (1H, d, J = 8.8, NH), 7.32 (10H, m, Ph),
5.02 (3H, m, CH–P, CH2-Ph), 3.50 (3H, d, JPH = 10.3, OCH3). 13C NMR
(75.5 MHz; DMSO-d6): dC 155.3, 137.2, 128.6, 128.4, 128.3, 128.2,
128.0, 127.5, 66.1, 52.8, 53.8, 51.8 (d, JCP = 150). 31P NMR
(121.5 MHz; DMSO-d6): dP 20.5. ES-MS: m/z 336.3 (M+H+), 358.6
(M+Na+). IR (neat):
m
(cmꢂ1) 3294, 2936, 1714, 1543.
(neat): m
(cmꢂ1) 3323, 3179, 3067, 2953, 2872, 1691, 1528, 1452.

3450
E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
4.3.3. O-n-Butyl N-propyl 1-(benzyloxycarbonylamino) ethyl
phosphonamide (2c)
Boc, NH Cbz), 5.02 (2H, s, CH2-Ph), 4.23–4.00 (4H, m,
CH2–O–P, CH–P), 3.70 (3H, s, COOCH3), 3.51 (2H, m, CH2–O–CH3),
a-CH Lys,
Yield: 86% (white solid). Mp: 42–48 °C. Rf (CH2Cl2/MeOH 97:3):
0.4. 1H NMR (300 MHz; CDCl3): dH 7.33 (5H, m, Ph), 5.57 and 5.10
(1H, d, J = 9.8, NH Cbz, two diastereoisomers), 5.11 (2H, m, CH2-Ph),
4.03 (2H, m, CH2O), 3.90 (1H, m, CH–P), 2.92–2.58 (3H, m, CH2–NH,
NH–P), 1.60 (2H, m, CH2 but), 1.51–1.28 (7H, m, CH3–CH–P, CH2–
CH2–NH, CH2 but), 0.95–0.82 (6H, m, CH3 but, CH3 prop). 13C
NMR (75.5 MHz; CDCl3): dC 156.5, 136.8, 128.9, 128.8, 128.6,
128.5, 128.4, 67.3, 64.4, 64.1, 46.1, 44.9, 44.2, 43.3, 43.0, 32.9,
32.8, 25.7, 25.6, 19.1, 16.2, 15.9, 14.0, 11.5. 31P NMR (121.5 MHz;
CDCl3): dP 31.8, 30.6. LSI MS: m/z 357.12 (M+H+), 379.09
(M+Na+). HR MS (FAB): 357.1936 Da (calcd for C17H30N2O4P
3.33 (3H, s, OCH3), 2.96–2.86 (3H, m, e-CH2 Lys, NH–P), 1.73–
1.31 (18H, m). 13C NMR (100.5 MHz; CDCl3): dC 173.3, 155.4,
136.4, 128.4, 128.1, 128.0, 79.8, 71.7, 66.9, 63.3, 58.9, 53.3, 52.2,
44.3, 43.4, 40.4, 32.2, 31.6, 28.3, 22.2, 15.7. 31P NMR (121.5 MHz;
CDCl3): dP 32.4, 32.3, 32.0, 30.9. LSI MS: m/z 560.19 (M+H+),
582.19 (M+Na+). HR MS (FAB): 560.2724 Da (calcd for C25H43N3O9P
(M+H+): 560.2736). IR (neat):
m
(cmꢂ1) 3269, 2935, 1703, 1525.
a
4.3.8. O-n-Butyl N-( -amino N -tert-butoxycarbonylamino
e
lysyl methylester) 1-(benzyloxycarbonylamino) ethyl
phosphonamide (2h)
(M+H+): 357.1943). FT-IR (neat):
2957, 2932, 2872, 1688, 1529.
m
(cmꢂ1) 3319, 3183, 3035,
Yield: 38% (oil). Rf (CH2Cl2, MeOH 95:5) = 0.5. 1H NMR
(400 MHz; CDCl3): dH 7.28 (5H, m, Ph), 5.91–5.24 (2H, br, NH
Cbz, NH-Boc), 5.08 (2H, s, CH2-Ph), 4.20 (1H, m,
(2H, m, CH2O but), 3.82 (1H, m, CH–P), 3.66 (3H, s, OCH3), 3.09–
2.96 (1H, m, NH–P), 2.83 (2H, m, -CH2 Lys), 1.69–1.27 (22H, m),
a-CH Lys), 3.96
4.3.4. O-Methyl N-propyl 1-(benzyloxycarbonylamino) propyl
phosphonamide (2d)
e
Yield: 35% (oil). Rf (AcOEt) = 0.2. 1H NMR (300 MHz; CDCl3): dH
7.32 (5H, m, Ph), 6.03 and 5.46 (1H, d, J = 9.7, NH Cbz, two diaste-
reoisomers), 5.11 (2H, m, CH2-Ph), 3.89 (1H, m, CH–P), 3.62 (3H, m,
OCH3), 3.10–2.69 (3H, m, CH2–NH, NH–P), 1.75–1.43 (2H, m, CH2–
CH–P, CH2–CH2–NH), 0.90 (6H, m, CH3–CH2–CH–P, CH3 prop). 13C
NMR (75.5 MHz; CDCl3): dC 157.1, 136.9, 128.8, 128.4, 128.3,
128.2, 67.2, 51.8, 51.0, 50.9, 49.9, 48.9, 43.1, 25.6, 22.2, 11.5,
10.9. 31P NMR (121.5 MHz; CDCl3): dP 32.6, 31.1. LSI MS: m/z
0.85 (3H, m, CH3 but). 13C NMR (100.5 MHz; CDCl3): dC 173.3,
155.9, 155.4, 136.4, 128.4, 128.0, 127.9, 79.7, 66.7, 64.0, 53.3,
52.1, 45.3, 44.4, 43.9, 42.9, 40.6, 40.4, 40.1, 32.5, 32.0, 31.7, 28.2,
22.3, 18.8, 15.6, 13.6. 31P NMR (121.5 MHz; CDCl3): dP 32.0, 31.6,
30.7, 30.6. LSI MS: m/z 558.23 (M+H+), 580.2 (M+Na+). HR MS
(FAB): 558.2928 Da (calcd for C26H45N3O8P (M+H+): 558.2944). IR
(neat):
m
(cmꢂ1) 3266, 2956, 1703, 1524.
a
329.14 (M+H+), 657.29 (2M+H+). IR (neat):
2961, 1686, 1535.
m
(cmꢂ1) 3312, 3200,
4.3.9. O-Methyl N-(
e
-amino N -tert-butoxycarbonylamino lysyl
methylester) 1-(benzyloxycarbonylamino) benzyl
phosphonamide (2i)
4.3.5. O-Methyl N-propyl 1-(benzyloxycarbonylamino) benzyl
phosphonamide (2e)
Yield: 67% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.4. 1H NMR (400 MHz;
CDCl3): dH 7.33 (10H, m, Ph), 6.08 (1H, br, NH Cbz), 5.05 (4H, m,
Yield: 56% (white solid). Mp: 107–130 °C. Rf (AcOEt) = 0.4. 1H
NMR (300 MHz; CDCl3): dH 7.35 (10H, m, Ph), 6.24 and 6.07 (1H,
dd, J = 9.2, J = 5.5, NH Cbz, 2 ꢃ diastereoisomers), 5.08 (3H, m, CH–
P, CH2-Ph), 3.66 and 3.47 (3H, d, JPH = 10.7, OCH3, 2 ꢃ diastereoiso-
mers), 2.83 (2H, m, CH2–NH prop), 2.65 (1H, br m, NH–P), 1.42 and
1.29 (2H, 2 ꢃ qui, J = 7.3, CH2–CH2–NH, two diastereoisomers),
0.85 and 0.79 (3H, 2 ꢃ t, J = 7.3, CH3 prop, two diastereoisomers).
13C NMR (75.5 MHz; CDCl3): dC 155.7, 136.1, 128.4, 128.3, 128.2,
CH-Ph, CH2-Ph, NH-Boc), 4.22 (1H, br, a-CH Lys), 3.72 and 3.71
(3H, 2 ꢃ s, COOCH3, diastereoisomers), 3.67 and 3.46 (3H, 2 ꢃ d,
JPH = 10.9, POCH3, diastereoisomers), 2.88 (1H, m, NH–P), 2.72–
2.54 (2H, m,
e
-CH2 Lys), 1.68–1.18 (15H, m). 13C NMR
(100.5 MHz; CDCl3): dC 173.2, 155.4, 136.1, 128.7, 128.6, 128.5,
128.1, 127.9, 79.9, 67.2, 53.3, 52.2, 51.5, 40.6, 31.9, 28.3, 22.1. 31P
NMR (121.5 MHz; CDCl3): dP 29.2, 29.0, 28.9. ES-MS: m/z 600.3
(M+Na+), 616.6 (M+K+). LSI MS: m/z 578.21 (M+H+). HR MS (FAB):
578.2628 Da (calcd for C28H41N3O8P (M+H+): 578.2631). IR (neat):
127.8, 127.7, 67.0, 53.7, 51.9 (d, JCP = 147), 51.3, 42.8, 25.1, 11.0. 31
P
NMR (121.5 MHz; CDCl3): dP 29.3, 28.5. ES-MS: m/z 399.1 (M+Na+).
LSI MS: m/z 377.10 (M+H+). HR MS (FAB): 377.1634 Da (calcd for
C19H26N2O4P (M+H+): 377.1630). Anal. Calcd for C19H25N2O4P: C,
60.63; H, 6.69; N, 7.44. Found: C, 60.34; H, 6.72; N, 7.29. IR (neat):
m
(cmꢂ1) 3340, 2948, 1697, 1497.
4.4. General procedure for O-alkyl N-alkyl 1-amino alkyl(aryl)
phosphonamides (3a–i)
m
(cmꢂ1) 3326, 3211, 2931, 2873, 1688, 1533, 1454.
Phosphonamide 2 (100 mg) was dissolved in methanol (10 mL)
and Pd/C 10% (10 mg) was added. The solution was stirred under
H2 until the reaction went to completion (monitored by TLC). The
catalyst was then filtered on Celite and the remaining solution
was removed under reduced pressure. The crude product 3 re-
quired generally further purification which was performed by
flash chromatography on silica gel (eluents: CH2Cl2 100% to
CH2Cl2/MeOH 95:5 (1% gradient) or AcOEt 100% to AcOEt/MeOH
95:5 (1% gradient)).
a
4.3.6. O-Methyl N-(
e
-amino N -tert-butoxycarbonylamino lysyl
methylester) 1-(benzyloxycarbonylamino) ethyl
phosphonamide (2f)
Yield: 64% (oil). Rf (AcOEt/MeOH 95:5) = 0.5. 1H NMR (400 MHz;
CDCl3): dH 7.32 (5H, m, Ph), 6.10–5.76 (1H, br m, NH Cbz), 5.37 (1H,
br, NH Boc), 5.09 (2H, s, CH2-Ph), 4.25 (1H, m,
m, CH–P), 3.76–3.60 (6H, m, COOCH3, POCH3), 3.21 (1H, br, NH–P),
2.88 (2H, m,
-CH2 Lys), 1.62–1.33 (18H, m). 13C NMR (100.5 MHz;
a-CH Lys), 4.04 (1H,
e
CDCl3): dC 173.3, 156.1, 155.5, 136.4, 128.4, 128.0, 127.9, 79.7, 66.9,
53.4, 52.1, 50.9, 45.2, 44.2, 43.7, 43.5, 42.7, 40.5, 40.2, 40.1, 32.0,
31.9, 31.7, 31.4, 28.3, 22.3, 15.7, 15.3. 31P NMR (121.5 MHz; CDCl3):
dP 33.5, 33.2, 32.2, 32.0. LSI MS: m/z 516.15 (M+H+). HR MS (FAB):
516.2466 Da (calcd for C23H39N3O8P (M+H+): 516.2474). IR (neat):
4.4.1. O-Methyl N-propyl 1-amino ethyl phosphonamide (3a)
Yield: 77% (oil). Rf (AcOEt/MeOH 95:5) = 0.2. 1H NMR (300 MHz;
CDCl3): dH 3.61 and 3.60 (3H, 2 ꢃ d, JPH = 10.4, OCH3, diastereoiso-
mers), 3.07–2.80 (4H, m), 1.70(2H, br s, NH2), 1.47 and 1.46 (2H, sext,
J = 7.2, CH2–CH2–NH, two diastereoisomers), 1.26 and 1.25 (3H,
2 ꢃ dd, JPH = 16.9, J3 = 7.2, two diastereoisomers), 0.87 (3H, t,
J = 7.2, CH3 prop). 13C NMR (75.5 MHz; CDCl3): dC 50.9, 50.7, 46.2,
45.7, 44.3 and 43.6 (2 ꢃ d, JCP = 140, diastereoisomers), 43.3, 26.0,
17.7, 17.3, 11.5. 31P NMR (121.5 MHz; CDCl3): dP 36.6, 36.2. ES-MS:
m/z 180.103 (M+). HR MS (FAB): 180.1030 Da (calcd for C6H18N2O2P
m
(cmꢂ1) 3259, 2949, 1698, 1526, 1454.
a
4.3.7. O-(2-(Methoxy)ethyl) N-(
e
-amino N -tert-butoxycarbo-
nylamino lysyl methylester) 1-(benzyloxycarbonylamino)
ethyl phosphonamide (2g)
Yield: 48% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.75. 1H NMR
(400 MHz; CDCl3): dH 7.31 (5H, m, Ph), 5.64–5.05 (2H, br m, NH-
(M+): 180.1027). IR (neat):
m
(cmꢂ1) 3218, 2961, 2874, 1595, 1456.

E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
3451
4.4.2. O-2-((Methoxy)ethyl) N-propyl 1-amino ethyl
phosphonamide (3b)
36.0, 35.6. LSI MS: m/z 382.22 (M+H+), 404.21 (M+Na+). HR MS
(FAB): 382.2098 Da (calcd for C15H33N3O6P (M+H+): 382.2107). IR
Yield: 81% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.3. 1H NMR (300 MHz;
CDCl3): dH 4.07 (2H, m, CH2–O–P), 3.52 (2H, m, CH2–O–CH3), 3.32
(3H, s, OCH3), 3.07 (1H, m, CH–P), 2.88 (2H, m, CH2–NH), 2.77
(1H, m, NH–P), 1.75 (2H, br s, NH2), 1.46 and 1.45 (2H, 2 ꢃ qui,
J = 7.3, CH2–CH2–NH, two diastereoisomers), 1.26 and 1.25 (3H,
2 ꢃ dd, JC–H = 7.2, JP–H = 17.1, CH3–CH, diastereoisomers), 0.86
(3H, t, J = 7.3, CH3 prop). 13C NMR (75.5 MHz; CDCl3): dC 71.7,
62.5, 58.7, 45.9, 44.1 and 43.6 (2 ꢃ d, JCP = 140, diastereoisomers),
42.7, 25.5, 17.0, 16.7, 10.9. 31P NMR (121.5 MHz; CDCl3): dP 35.4,
35.1. LSI MS: m/z 225.13 (M+H+). HR MS (FAB): 225.1369 Da (calcd
(neat):
m
(cmꢂ1) 3258, 2948, 2867, 1742, 1704, 1525.
a
4.4.7. O-(2-(Methoxy)ethyl) N-(
e
-amino N -tert-butoxycarbonyl-
amino lysyl methylester) 1-amino ethyl phosphonamide (3g)
Yield: 99% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.3. 1H NMR (400 MHz;
CDCl3): dH 5.16 (1H, br, NH-Boc), 4.20 (1H, m,
a-CH Lys), 4.06 (2H,
m, CH2–O–P), 3.66 (3H, s, COOCH3), 3.52 (2H, m, CH2–O–CH3), 3.32
(3H, s, OCH3), 3.08–2.77 (4H, m, e-CH2 Lys, CH–P, NH–P), 1.96 (2H,
br s, NH2), 1.72–1.22 (18H, m). 13C NMR (100.5 MHz; CDCl3): dC
173.3, 155.4, 79.8, 71.9, 66.9, 62.9, 58.9, 53.3, 52.2, 45.5, 44.1,
40.6, 32.2, 31.9, 28.3, 22.3, 17.0. 31P NMR (121.5 MHz; CDCl3): dP
35.4, 35.0. LSI MS: m/z 426.23 (M+H+). HR MS (FAB): 426.2355
for C8H22N2O3P (M+H+): 225.1368). IR (neat):
2934, 2876, 1658, 1453.
m
(cmꢂ1) 3255, 2962,
(calcd for C17H37N3O7P (M+H+): 426.2369). IR (neat):
3272, 2932, 1741, 1705, 1526.
m )
(cmꢂ1
4.4.3. O-n-Butyl N-propyl 1-amino ethyl phosphonamide (3c)
Yield: 65% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.4. 1H NMR (300 MHz;
CDCl3): dH 3.96 and 3.85 (2H, 2 ꢃ m, CH2O, diastereoisomers), 3.02
(1H, m, CH–P), 2.90 (2H, m, CH2–NH), 2.63 (1H, m, NH–P), 1.58
(4H, m, NH2, CH2 but), 1.46 and 1.45 (2H, 2 ꢃ sext, J = 7.2, CH2–
CH2–NH, diastereoisomers), 1.33 (2H, sext, J = 7.2, CH2 but), 1.25
and 1.24 (3H, 2 ꢃ dd, JC–H = 7.3, JP–H = 16.9, CH3–CH, diastereoiso-
mers), 0.86 (6H, m). 13C NMR (75.5 MHz; CDCl3): dC 62.5, 45.0,
44.5, 43.2 and 42.6 (2 ꢃ d, JCP = 138, diastereoisomers), 42.0, 31.6,
24.7, 17.8, 16.4, 16.0, 12.6, 10.1. 31P NMR (121.5 MHz; CDCl3): dP
29.6, 29.4. LSI MS: m/z 223.15 (M+H+). HR MS (FAB): 223.1586 Da
a
4.4.8. O-n-Butyl N-(
e
-amino N -tert-butoxycarbonylamino lysyl
methylester) 1-amino ethyl phosphonamide (3h)
Yield: 80% (oil). Rf (AcOEt 100%): 0.4. 1H NMR (400 MHz;
CDCl3): dH 5.29 (1H, br, NH-Boc), 4.27 (1H, m, -CH Lys), 4.04
and 3.91 (2H, 2 ꢃ m, CH2–O–P), 3.73 (3H, s, OCH3), 3.09–2.93
(3H, m, CH–P, -CH2 Lys), 2.76 (1H, br, NH–P), 1.79–1.37 (22H,
a
e
m), 1.32 and 1.31 (3H, 2 ꢃ dd, JC–H = 7.2, JP–H = 16.7, CH–CH3, dia-
stereoisomers), 0.94 (3H, t, J = 7.2, CH3 but). 13C NMR
(100.5 MHz; CDCl3): dC 173.2, 155.4, 79.8, 63.6, 53.2, 52.2, 45.9,
45.3, 44.5, 43.9, 40.8, 32.6, 32.1, 31.9, 28.2, 22.3, 18.8, 17.5, 17.1,
13.6. 31P NMR (121.5 MHz; CDCl3): dP 34.5, 34.1. LSI MS: m/z
424.18 (M+H+), 446.08 (M+Na+). HR MS (FAB): 424.2562 Da (calcd
(calcd for C9H24N2O2P (M+H+): 223.1575). IR (neat):
2960, 2933, 2874, 1598.
m
(cmꢂ1) 3209,
4.4.4. O-Methyl N-propyl 1-amino propyl phosphonamide (3d)
Yield: 96% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.5. 1H NMR (300 MHz;
CDCl3): dH 3.68 (3H, d, JPH = 10.5, OCH3), 3.03–2.76 (4H, m, CH–P,
CH2–NH, NH–P), 1.85 (3H, m, CHAHB–CH–P, NH2), 1.52 (3H, m,
CHAHB–CH–P, CH2–CH2–NH), 1.06 (3H, t, J = 7.3, CH3–CH2–CH–P),
0.93 (3H, m, CH3 prop). 13C NMR (100.5 MHz; CDCl3): dC 51.9,
51.0, 50.5 and 49.6 (2 ꢃ d, JCP = 139, diastereoisomers), 50.3, 42.9,
25.6, 24.4, 24.1 (t, JCP = 36), 11.1, 10.9. 31P NMR (121.5 MHz;
CDCl3): dP 36.1, 35.5. LSI MS: m/z 195.08 (M+H+). HR MS (FAB):
for C23H41N3O6P (M+H+): 424.2576). IR (neat):
2959, 2871, 1743, 1704, 1527.
m
(cmꢂ1) 3237,
4.4.9. O-Methyl N-propyl 1-amino benzyl phosphonamide (3i)
Yield: 100% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.5. 1H NMR
(300 MHz; CDCl3): dH 7.30 (5H, m, Ph), 4.25 and 4.22 (1H, 2 ꢃ d,
JPH = 15.5, CH–P, two diastereoisomers), 3.67 and 3.62 (3H, 2 ꢃ d,
JPH = 10.7, OCH3), 2.80–2.69 (2.5H, m, CH2–NH prop, NH–P diaste-
reoisomer A), 2.50 (0.5H, m, NH–P, diastereoisomer B), 2.07 (2H,
br s, NH2), 1.40 (2H, m, CH2–CH2–NH), 0.84 (3H, m, CH3 prop).
13C NMR (75.5 MHz; CDCl3): dC 138.8, 138.5, 128.8, 128.2, 128.1,
128.0, 127.9, 71.7, 56.2, 54.4 (d, JCP = 137), 51.5, 43.5, 25.8, 11.5.
31P NMR (121.5 MHz; CDCl3): dP 32.0, 31.6. LSI MS: m/z 243.15
(M+H+), 485.27 (2M+H+), 727.35 (3M+H+). HR MS (FAB):
243.1274 Da (calcd for C11H20N2O2P (M+H+): 243.1262). IR (neat):
194.1176 Da (calcd for C7H19N2O2P (M+): 194.1184). IR (neat):
(cmꢂ1) 3220, 2960, 2874, 1457.
m
4.4.5. O-Methyl N-propyl 1-amino benzyl phosphonamide (3e)
Yield: 100% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.5. 1H NMR
(300 MHz; CDCl3): dH 7.30 (5H, m, Ph), 4.25 and 4.22 (1H, 2 ꢃ d,
JPH = 15.5, CH–P, two diastereoisomers), 3.67 and 3.62 (3H,
2 ꢃ d, JPH = 10.7, OCH3), 2.80–2.69 (2.5H, m, CH2–NH prop, NH–P
diastereoisomer A), 2.50 (0.5H, m, NH–P, diastereoisomer B),
2.07 (2H, br s, NH2), 1.40 (2H, m, CH2–CH2–NH), 0.84 (3H, m,
CH3 prop). 13C NMR (75.5 MHz; CDCl3): dC 138.8, 138.5, 128.8,
128.2, 128.1, 128.0, 127.9, 71.7, 56.2, 54.4 (d, JCP = 137), 51.5,
43.5, 25.8, 11.5. 31P NMR (121.5 MHz; CDCl3): dP 32.0, 31.6. LSI
MS: m/z 243.15 (M+H+), 485.27 (2M+H+), 727.35 (3M+H+). HR
MS (FAB): 243.1274 Da (calcd for C11H20N2O2P (M+H+):
m
(cmꢂ1) 3215, 2958, 2873, 1668, 1602, 1554, 1494, 1452.
4.4.10. N6-Benzoyl 50-(tert-butyldimethylsilyl) 30-(1-(benzylo-
xycarbonylamino) ethyl phosphonyl) adenosine (6a) and N6-
benzoyl 50-(tert-butyldimethylsilyl) 20-(1-(benzyloxycarbon-
ylamino) ethyl phosphonyl) adenosine (6b)
Phosphonic acid 4 (163 mg, 0.61 mmol) was coevaporated three
times with dry pyridine, then dissolved in the same solvent (2 mL).
N6-Benzoyl 50-(tert-butyldimethylsilyl) adenosine (5) (244 mg,
0.50 mmol) was coevaporated three times with dry pyridine, dis-
solved in the same solvent (5 mL) and added to the stirring suspen-
sionof 62. ThenDowex50W8Xresin(pyr+)(0.2 g), andEDC(477 mg,
2.5 mmol) were added, and the solution was stirred at 45 °C for
2 days. Water (10 mL) was added, and the solution was stirred for
10 min filtered, and removed under reduced pressure. The residue
was dissolved in CHCl3 (20 mL) and extracted with water
(2 ꢃ 20 mL) and brine (20 mL). The organic phase was dried over
MgSO4, and removed under reduced pressure. The residue was puri-
fied by column chromatography on silica gel (eluent CHCl3/MeOH/
Et3N: gradient from 68:2:0.1 to 5:2:0.1) to yield a 3:1 mixture of
6a and 6b as triethylammonium salts (219 mg, 0.35 mmol, 70%
243.1262). IR (neat):
1554, 1494, 1452.
m
(cmꢂ1) 3215, 2958, 2873, 1668, 1602,
a
4.4.6. O-Methyl N-(
e
-amino N -tert-butoxycarbonylamino lysyl
methylester) 1-amino ethyl phosphonamide (3f)
Yield: 100% (oil). Rf (CH2Cl2/MeOH 9:1) = 0.3. 1H NMR (400 MHz;
CDCl3): dH 5.28 (1H, br, NH-Boc), 4.20 (1H, m,
a-CH Lys), 3.72 and
3.60 (3H, 2 ꢃ d, JPH = 10, POCH3, diastereoisomers), 3.66 (3H, s,
COOCH3), 3.11–2.97 (4H, m, e-CH2 Lys, CH–P, NH–P), 1.89–1.18
(20H, m). 13C NMR (100.5 MHz; CDCl3): dC 173.2, 155.4, 79.7,
53.2, 52.2, 50.5, 45.7, 45.2, 44.6, 44.3, 43.8, 43.3, 43.1, 40.7, 32.1,
31.7, 28.2, 22.3, 17.4, 17.3, 17.0. 31P NMR (121.5 MHz; CDCl3): dP

3452
E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
yield). Rf (CHCl3/MeOH/Et3N 6:1:0.1): 0.3. 1H NMR (400 MHz;
CDCl3): dH 11.90 (1H, br s, OH), 9.28 (1H, br s, Ad NH), 8.68 (1H, s,
CH Ad), 8.30 and 8.27 (1H, 2 ꢃ s, CH Ad, 2 isomers), 7.96 (2H, d,
J = 7.3, 2,6 CH Bz), 7.51 (1H, m, Bz), 7.42 (2H, m, Bz), 7.27–7.08 (5H,
m, Cbz), 6.21 and 6.18 (0.2H, 2 ꢃ d, J = 3.6, 10 CH (6b), two diastere-
oisomers), 6.13 and 6.11 (0.8H, 2 ꢃ d, J = 5.5, 10 CH (6a), two diaste-
reoisomers), 5.69 and 5.62 (0.2H, 2 ꢃ d, J = 8.5, NH Cbz (6b), two
diastereoisomers), 5.44 and 5.33 (0.8H, 2 ꢃ d, J = 7.0 and 9.2, NH
Cbz (6a), two diastereoisomers), 5.07–4.93 (2.2H, m, CH2 Cbz and
20 CH (6b)), 4.83 and 4.74 (0.8H, 2 ꢃ m, 30 CH (6a), two diastereoiso-
mers), 4.61 and 4.55 (0.8H, 2 ꢃ m, 20 CH2 (6a), two diastereoiso-
mers), 4.45 (0.2H, m, 30 CH (6b)), 4.25–4.05 (1H, m, 40 CH), 3.96
(1H, m, CH–P), 3.84–3.64 (2H, m, 50 CH2), 2.97 (6H, q, J = 7.2,
(CH3CH2)3NH+ salt), 1.34 (3H, m, CH3–CH–P), 1.22 (9H, t, J = 7.2,
(CH3CH2)3NH+ salt), 1.19 and 0.85 and 0.82 (9H, 3 ꢃ s, (CH3)3C–Si,
mixture was stirred at room temperature overnight. The solvent
was then removed under reduced pressure, and the residue dis-
solved in ethyl acetate (10 mL) and extracted with water
(3 ꢃ 20 mL). The combined aqueous phases were evaporated under
reduced pressure and the residue was purified by column chroma-
tography on silica gel (eluent CHCl3/MeOH/NH3: gradient from
6:1:0.1 to 5:2:0.1) to yield a 3:1 mixture of 8a and 8b as ammo-
nium salts (100 mg, 0.19 mmol, 99%). Rf (CHCl3/MeOH/NH3
6:1:0.1): 0.2. 1H NMR (400 MHz; CD3OD): dH 8.27 and 8.23 (1H,
2 ꢃ s, CH Ad, two isomers), 8.16 and 8.15 (1H, 2 ꢃ s, CH Ad, two
isomers), 7.35–7.23 (5H, m, Cbz), 6.12 and 6.09 (0.2H, 2 ꢃ d,
J = 5.7, 10 CH (8b), two diastereoisomers), 5.98 and 5.95 (0.8H,
2 ꢃ d, J = 7.0 and 6.5, 10 CH (8a), two diastereoisomers), 5.31
(0.2H, m, 20 CH (8b)), 5.12–4.77 (3.6H, m, CH2 Cbz, 20 and 30 CH
(8a)), 4.56 and 4.52 (0.2H, 2 ꢃ dd, J = 3.2 and 5.0, 30 CH (8b), two
diastereoisomers), 4.33 and 4.29 (0.8H, 2 ꢃ m, 40 CH (8a), two dia-
stereoisomers), 4.18 (0.2H, m, 40 CH (8b)), 3.95 (1H, m, CH–P),
3.84–3.73 (2H, m, 50 CH2), 1.39 and 1.38 (2.4H, 2 ꢃ dd, JC–H = 7.2,
JP–H = 15.0, CH3–CH–P (8a), two diastereoisomers), 1.22 (0.6H, m,
CH3–CH–P (8b)). 13C NMR (100.5 MHz; CD3OD): dC 157.5, 153.5,
150.0, 142.0, 138.3, 129.5, 129.0, 128.8, 128.7, 121.0, 91.0, 90.9,
87.9, 87.8, 87.6, 87.5, 78.1, 78.0, 76.3, 75.1, 72.4, 67.7, 67.6, 63.2,
63.0, 47.1, 45.6 (d, JCP = 154, isomers), 46.7, 45.2 (d, JCP = 152, iso-
mers), 17.0. 31P NMR (121.5 MHz; CD3OD): dP 22.7, 22.2, 22.0,
21.9. ES-MS (ꢂve ion): m/z 507.1 (MꢂH+), (+ve ion): m/z 509.1
(M+H+), 531.1 (M+Na+). LSI MS: m/z 509.15 (M+H+), HR MS
(FAB): 509.1535 Da (calcd for C20H26N6O8P (M+H+) 509.1549). IR
isomers), 0.04 and 0.00 and ꢂ0.02 (6H, 3 ꢃ s, CH3–Si, isomers). 13
C
NMR (75.5 MHz; CDCl3): dC 165.4, 156.6, 156.4, 156.3, 152.9,
152.7, 152.6, 152.0, 149.9, 141.8, 141.7, 137.1, 136.9, 134.0, 133.0,
129.3, 129.0, 128.8, 128.6, 128.4, 128.2, 123.6, 123.5, 87.8, 87.6,
86.1, 85.7, 85.9, 76.0, 75.6, 67.0, 66.9, 63.5, 46.4, 44.3 (d, JCP = 153,
isomers), 45.9, 44.6, 42.6 (d, JCP = 150, isomers), 26.4, 26.3, 18.7,
17.5, 16.9 (q, JCP = 48), 8.8, ꢂ5.0, ꢂ5.1. 31P NMR (121.5 MHz; CDCl3):
dP 24.9, 22.4, 21.8. ES-MS (ꢂve ion): m/z 725.4 (MꢂH+), (+ve ion): m/z
727.1 (M+H+), 749.2 (M+Na+). LSI MS: m/z 727.26 (M+H+), HR MS
(FAB): 727.2683 Da (calcd for C33H44N6O9PSi (M+H+): 727.2676).
IR (neat):
m
(cmꢂ1) 3255, 2931, 2857, 1697, 1609, 1578.
4.4.11. 50-(tert-Butyldimethylsilyl) 30-(1-(benzyloxycarbonyla-
mino) ethyl phosphonyl) adenosine (7a) and 50-(tert-butyl di-
methyl silyl) 20-(1-(benzyloxycarbonylamino) ethyl
(neat): m
(cmꢂ1) 3180, 2935, 1693, 1645, 1577.
4.4.13. 30-(1-Amino ethyl phosphonyl) adenosine (9a) and 20-(1-
amino ethyl phosphonyl) adenosine (9b)
phosphonyl) adenosine (7b)
Compound 6a/6b (250 mg, 0.30 mmol) was dissolved in metha-
nol (5 mL), then NH3 aq 33% (15 mL) was added, and the mixture
was stirred at room temperature overnight. Then, the solvents were
removed under reduced pressure and the residue was purified by
column chromatography on silica gel (eluent CHCl3/MeOH/Et3N:
gradient from 65:5:0.1 to 5:2:0.1) to yield a 3:1 mixture of 7a and
7b as triethylammonium salts (210 mg, 0.29 mmol, 97%). Rf
(CHCl3/MeOH/Et3N 6:1:0.1): 0.3. 1H NMR (400 MHz; CDCl3): dH
11.7 (1H, br s, OH), 8.23 and 8.22 (1H, 2 ꢃ s, CH Ad, two isomers),
8.12 and 8.09 (1H, 2 ꢃ s, CH Ad, two isomers), 7.29 (5H, m, Cbz),
6.87 (2H, br s, Ad NH2), 6.46 and 6.39 (1H, 2 ꢃ d, J = 7.7, J = 9.0, NH
Cbz), 6.15 (0.2H, m, 10 CH (7b)), 6.06 (0.8H, m, 10 CH (7a)), 5.10–
4.98 (2.2H, m, CH2 Cbz and 20 CH (7b)), 4.88 and 4.79 (0.8H, 2 ꢃ m,
30 CH (7a), two diastereoisomers), 4.66 and 4.50 (0.8H, 2 ꢃ m, 20
CH2 (7a), two diastereoisomers), 4.58 (0.2H, m, 30 CH (7b)), 4.27–
3.92 (2H, m, 40 CH, CH–P), 3.84–3.68 (2H, m, 50 CH2), 2.95 (6H, q,
J = 7.3, (CH3CH2)3NH+ salt), 1.37 (3H, m, CH3–CH–P), 1.22 (9H, t,
J = 7.3, (CH3CH2)3NH+ salt), 0.87 and 0.83 (9H, 3 ꢃ s, (CH3)3C–Si, iso-
mers), 0.06 and 0.00, (6H, 2 ꢃ s, CH3–Si). 13C NMR (100.5 MHz;
CDCl3): dC 156.1, 156.0, 155.6, 152.6, 150.2, 150.0, 138.7 139.6,
136.9, 136.7, 128.9, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7,
119.5, 119.4, 87.3, 87.0, 85.6, 85.5, 85.2, 85.1, 75.5, 75.2, 74.8, 66.4,
66.3, 63.2, 45.7, 44.0 (d, JCP = 174, isomers), 45.7, 44.2, 42.5 (d,
JCP = 171, isomers), 25.9, 25.8, 18.4, 17.8, 16.4 (q, JCP = 137), 8.4,
ꢂ5.4, ꢂ5.5. 31P NMR (121.5 MHz; CDCl3): dP 24.3, 22.2, 21.8, 21.5.
ES-MS (ꢂve ion): m/z 621.3 (MꢂH+), (+ve ion): m/z 623.2 (M+H+),
645.2 (M+Na+). LSI MS: m/z 623.30 (M+H+), HR MS (FAB):
623.2427 Da (calcd for C26H40N6O8SiP (M+H+): 623.2414). IR (neat):
Compound 8a/8b (229 mg, 0.35 mmol), was dissolved in MeOH
(5 mL), then water (5 mL) and Pd/C 5% Degussa type (5 mg) were
added to the solution. The mixture was stirred under H2 atmo-
sphere for 18 h, then the solution was diluted with water
(10 mL) filtered on paper in a Hirsch funnel. The catalyst was
repeatedly washed with water (20 mL), then the filtrate was evap-
orated under reduced pressure and coevaporated several times
with ammonia to yield a 3:1 mixture of 9a and 9b in as ammonium
salts (121 mg, 0.31 mmol, 86%).
1H NMR (500 MHz; D2O): dH 8.42 and 8.41 (1H, 2 ꢃ s, CH Ad,
isomers), 8.31 and 8.29 (1H, 2 ꢃ s, CH Ad, isomers), 6.30 and 6.29
(0.25H, 2 ꢃ d, J = 5.8, 10 CH (9b)), 6.21 and 6.20 (0.75H, 2 ꢃ d,
J = 6.6, 10 CH (9a)), 5.36 (0.25H, m, 20 CH (9b)), 5.00 (1.5H, m, 20
and 30 CH (9a)), 4.66 (0.25H, m, 30 CH (9b)), 4.55 (0.75H, m, 40
CH (9a)), 4.40 (0.25H, m, 40 CH (9b)), 4.04–3.90 (2H, m, 50 CH2),
3.60 (0.75H, m, CH–P (9a)), 3.20 (0.25H, m, CH–P (9b)), 1.57
(2.25H, dd, JCH = 7.4, JPH = 16.8, CH3–CH–P (9a)), 1.41 and 1.36
(0.75H, 2 ꢃ dd, JCH = 7.3, JPH = 16.7, CH3–CH–P (9b), two diastereo-
isomers). 13C NMR (100.5 MHz; CDCl3): dC 154.9, 154.7, 152.0,
151.9, 147.8, 147.7, 140.3, 118.3, 87.8, 87.6, 87.5, 85.9, 85.8, 85.4,
76.2, 76.1, 74.9, 74.5, 72.8, 70.0, 69.9, 61.3, 61.2, 61.0, 45.2, 44.6,
43.7, 43.1, 13.3, 13.1. 31P NMR (121.5 MHz; D2O): dP 17.8, 17.1,
16.9, 16.6. ES-MS (ꢂve ion): m/z 373.1 (MꢂH+), 747.2 (2MꢂH+),
769.2 (2MꢂH++Na+); (+ve ion): m/z 375.1 (M+H+), 397.1 (M+Na+),
749.2 (2M+H+), 771.2 (2M+Na+). LSI MS: m/z 375.12 (M+H+). HR
MS (FAB): 375.1184 Da (calcd for C12H20N6O6P (M+H+) 375.1181).
RP-HPLC: tR, 9a, 15.50 min; 9b, 16.10 min.
A sample of 8b (40 mg) was purified by anion exchange chro-
matography using a MonoQ FPLC column, eluting with 0.25 M
NH4HCO3. The fractions were monitored at 260 nm and the first
two peaks eluted were collected and freeze dried. The residue
was then dissolved in MeOH (2 mL), then water (2 mL) and Pd/C
5% Degussa type (2 mg) was added to the solution. The mixture
was stirred under H2 atmosphere for 3 days, then it was diluted
with water (5 mL) and filtered. The catalyst was repeatedly washed
m
(cmꢂ1) 3182, 2930, 2858, 1710, 1642, 1600, 1573.
4.4.12. 30-(1-(Benzyloxycarbonylamino) ethyl phosphonyl)
adenosine (8a) and 20-(1-(benzyloxycarbonylamino) ethyl
phosphonyl) adenosine (8b)
Compound 7a/7b (140 mg, 0.193 mmol) was dissolved in THF
(5 mL), then Et3Nꢀ3HF (155
lL, 0.965 mmol) was added and the

E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
3453
with water (5 mL), then the filtrate was freeze-dried to yield a
sample of single isomer 9b (3.0 mg) in >95% purity. 1H NMR
(400 MHz; D2O): dH 8.42 (1H, s, CH Ad), 8.32 (1H, s, CH Ad), 6.30
and 6.29 (1H, 2 ꢃ d, J = 6.0, 10 CH, two diastereoisomers), 5.36
(1H, m, 20 CH), 4.65 (1H, m, 30 CH), 4.40 (1H, m, 40 CH), 3.00 (1H,
dd, J = 2.7,13.0, 50 CHAHB), 3.92 (1H, dd, J3 = 2.7,13.0, 50 CHAHB),
3.23 (1H, m, CH–P), 1.32 (3H, m, CH3–CH–P). 31P NMR
(121.5 MHz; D2O): dP 17.2, 16.8. ES-MS (+ve ion): m/z 375.1174
(M+H+); (ꢂve ion): m/z 373.1. (MꢂH+). HR MS (TOF):
375.1176 Da (calcd for C12H20N6O6P (M+H+) 375.1181). RP-HPLC:
tR, 16.06 min.
JCH = 7.3, JPH = 15.7, CH3–CH–P), 1.29 (9H, t, J = 7.0, (CH3–
CH2)3NH+), 0.85 (9H, s, (CH3)3C–Si), 0.05 (6H, s, CH3–Si). 13C NMR
(100.5 MHz; CD3OD): dC 158.1, 154.9, 150.2, 150.0, 141.7, 138.4,
129.9, 129.4, 129.0, 127.8, 127.9, 120.2, 88.9, 86.4, 76.7, 76.5,
67.6, 64.7, 47.7, 46.5, 45.0 (d, JCP = 149), 41.8, 41.6, 26.5, 19.2,
16.8, 9.2, ꢂ5.2. 31P NMR (121.5 MHz; CD3OD): dP 21.5. ES-MS
(ꢂve ion): m/z 605.3 (MꢂH+). LSI MS: m/z 607.24 (M+H+). HR MS
(FAB): 607.2460 Da (calcd for C26H40N6OSiP (M+H+): 607.2465).
IR (neat):
1601, 1573, 1535.
m
(cmꢂ1) 3205, 3034, 2953, 2930, 2857, 1709, 1644,
4.4.16. 30-(1-(Benzyloxycarbonylamino) ethyl phosphonyl) 20-
deoxyadenosine (13)
4.4.14. N6-Benzoyl 50-(tert-butyl dimethyl silyl) 30-(1-(benzyloxy-
carbonylamino) ethyl phosphonyl) 20-deoxyadenosine (11)
Phosphonic acid 1a (528 mg, 1.93 mmol) was coevaporated
three times with dry pyridine, then dissolved in the same solvent
(2 mL). N6-Benzoyl 50-(tert-butyldimethylsilyl) 20-deoxyadenosine
(10) (755 mg, 1.60 mmol) was coevaporated three times with dry
pyridine, dissolved in the same solvent (5 mL) and added to the
stirred solution of 1a. Dowex 50W 8X resin (pyr+) (0.2 g), and dicy-
clohexylcarbodiimide (1.648 g, 8 mmol) were added, and the solu-
tion was stirred at room temperature for 6 days. Water (10 mL)
was added, and the solution was stirred for 30 min. After this time,
the suspension was filtered on paper in a Hirsch funnel and the so-
lid was washed repeatedly with chloroform. The filtrate was then
evaporated under reduced pressure, the residue was dissolved in
CHCl3 (20 mL) and extracted twice with water (20 mL). The organic
phase was dried over MgSO4, and evaporated under reduced pres-
sure. The residue was purified by column chromatography on silica
gel (eluent CHCl3/MeOH/Et3N: gradient from 68:2:0.1 to 6:1:0.1) to
yield compound 11 as the triethylammonium salt (702 mg,
0.86 mmol, 54% yield). Rf: (CHCl3/MeOH/Et3N 6:1:0.1): 0.4. 1H
NMR (400 MHz; CDCl3): dH 12.36 (1H, br s, OH), 9.72 (1H, br s,
NH Ad), 8.73 (1H, s, CH Ad), 8.35 and 8.32 (1H, 2 ꢃ s, CH Ad, two
diastereoisomers), 8.04 (2H, d, J = 7.5, 2,6 CH Bz), 7.54 (1H, m,
Bz) 7.45 (2H, m, Bz), 7.27 (5H, m, Cbz), 6.53 (1H, m, 10 CH), 5.78
(1H, br m, NH Cbz), 5.11–4.99 (3H, m, CH2 Cbz, 30 CH), 4.31 and
4.20 (1H, 2 ꢃ s, 40 CH, two diastereoisomers), 3.98 (1H, m, CH–P),
3.79 (2H, m, 50 CH2), 2.96 (6H, q, J = 7.0, (CH3–CH2)3NH+), 2.50–
2.37 (2H, m, 20 CH2), 1.36 (3H, dd, J3 = 7.0, JPH = 14.8, CH3–CH–P),
1.35 (9H, t, J = 7.0, (CH3–CH2)3NH+), 0.85 (9H, s, (CH3)3C–Si), 0.07
and 0.05 (6H, 2 ꢃ s, CH3–Si). 13C NMR (100.5 MHz; CDCl3): dC
165.0, 156.1, 156.0, 152.3, 151.6, 141.3, 137.8, 136.7, 133.7,
132.5, 128.6, 128.3, 128.1, 128.0, 127.9, 124.4, 87.5, 84.5, 75.9,
75.7, 66.5, 63.6, 45.4, 45.2, 43.7 (d, JCP = 149), 45.0, 43.5 (d,
JCP = 150), 41.2, 40.8, 26.0, 18.3, 16.9, 8.5, ꢂ5.4. 31P NMR
(121.5 MHz; CDCl3): dP 20.8. ES-MS (ꢂve ion): m/z 709.3 (MꢂH+).
LSI MS: m/z 711.56 (M+H+), 733.45 (M+Na+), 749.53 (M+K+). HR
MS: 711.2722 Da (calcd for C33H44N6O8SiP (M+H+) 711.2727). IR
Compound 12 (120 mg, 0.13 mmol) was dissolved in THF
(5 mL), then Et3Nꢀ3HF (67
lL, 0.418 mmol) was added, and the
mixture was stirred at room temperature overnight. The solvent
was removed under reduced pressure, and the residue dissolved
in ethyl acetate and extracted three times with water. The com-
bined water phases were evaporated under reduced pressure and
the residue was purified by column chromatography on silica gel
(eluent CHCl3/MeOH/NH3: gradient from 65:5:0.1 to 6:1:0.1) to
yield compound 13 as the ammonium salt (66 mg, 0.13 mmol,
99%). Rf (CHCl3/MeOH/NH3 6:1:0.1): 0.2. 1H NMR (400 MHz;
CD3OD): dH 8.26 and 8.24 (1H, 2 ꢃ s, CH Ad, two diastereoisomers),
8.17 (1H, s, CH Ad), 7.35–7.03 (5H, m, Cbz), 6.65 (1H, d, J = 8.3, NH),
6.41 (1H, m, 10 CH), 5.15–4.91 (3H, m, 30 CH, CH2 Cbz), 4.23 and
4.14 (1H, 2 ꢃ m, 40 CH, two diastereoisomers), 3.89 (1H, m, CH–
P), 3.77 (2H, m, 50 CH2), 2.70 (1H, m, 20 CHAHB), 2.36 (1H, m, 20
CHAHB), 1.35 (3H, dd, JCH = 7.3, JPH = 15.6, CH3–CH–P). 13C NMR
(100.5 MHz; CD3OD): dC 165.7, 158.1, 157.7, 153.5, 150.3, 142.1,
138.7, 129.8, 129.7, 129.4, 129.3, 121.3, 89.7, 87.7, 77.5, 77.3,
67.8, 64.7, 41.2, 17.1. 31P NMR (121.5 MHz; CD3OD): dP 21.7. ES-
MS (ꢂve ion): m/z 491.1 (MꢂH+). LSI MS: m/z 493.15 (M+H+). HR
MS (FAB): 493.1578 Da (calcd for C20H26N6O7P (M+H+):
493.1600). IR (neat):
m
(cmꢂ1) 3205, 2933, 1694, 1646, 1618, 1577.
4.4.17. 30-(1-Amino ethyl phosphonyl) 20-deoxyadenosine (14)
Compound 13 (66 mg, 0.13 mmol), was dissolved in methanol
(5 mL), then water (10 mL) and Pd/C 5% Degussa type (5 mg) were
added to the solution. The mixture was stirred under H2 atmo-
sphere for 2 days, then diluted with water (10 mL) filtered on paper
in a Hirsch funnel. The catalyst was repeatedly washed with water
(20 mL), then the filtrate was evaporated under reduced pressure to
yield compound 14 (30 mg, 0.08 mmol, 62%) as a glassy solid.
1H NMR (400 MHz; D2O): dH 8.44 (1H, s, CH Ad), 8.35 (1H, s, CH
Ad), 6.62 (1H, dd, J = 5.7, 7.7, 10 CH), 5.17 (1H, m, 30 CH), 4.46 (1H,
m, 40 CH), 3.92 (2H, m, 50 CH2), 3.54 (1H, m, CH–P), 3.00 (1H, m, 20
CHAHB), 2.82 (1H, m, 20 CHAHB), 1.56 (3H, dd, JCH = 7.3, JPH = 15.8,
CH3–CH–P). 13C NMR (100.5 MHz; D2O): dC 154.9, 151.9, 147.9,
140.1, 118.6, 86.8, 84.6, 75.0, 61.5, 44.8, 43.4 (d, JCP = 146), 38.7,
13.4. 31P NMR (121.5 MHz; D2O): dP 17.1. ES-MS (ꢂve ion): m/z
357.5 (MꢂH+). LSI MS: m/z 359.12 (M+H+), 371.22 (M+Na+). HR
MS (FAB): 359.1223 Da (calcd for C12H20N6O5P (M+H+):
(neat):
m
(cmꢂ1) 3243, 2931, 2856, 1697, 1577.
4.4.15. 50-(tert-Butyl dimethyl silyl) 30-(1-(benzyloxycarbonyla-
mino) ethyl phosphonyl) 20-deoxyadenosine (12)
Compound 11 (485 mg, 0.598 mmol) was dissolved in methanol
(5 mL) then aq NH3 33% (15 mL) was added and the mixture was
stirred at room temperature overnight. Then, the solvents were re-
moved under reduced pressure and the residue was purified by
column chromatography on silica gel (eluent CHCl3/MeOH/Et3N:
gradient from 65:5:0.1 to 6:1:0.1) to yield compound 12 as the tri-
ethylammonium salt (362 mg, 0.512 mmol, 85%). Rf (CHCl3/MeOH/
Et3N 6:1:0.1): 0.3.
359.1232). IR (neat): m
(cmꢂ1) 3103, 2922, 2590, 1695, 1645,
1606, 1573. RP-HPLC: tR, 15.0 min.
4.4.18. 30-Methylphosphonyl 20-deoxyadenosine (15)
Methyl phosphonic acid (122 mg, 1.27 mmol) was coevaporated
three times with dry pyridine, then dissolved in the same solvent
(5 mL). N6-Benzoyl 50-TBDMS 20-deoxyadenosine 10 (500 mg,
1.06 mmol) was coevaporated three times with dry pyridine, dis-
solved in the same solvent (5 mL) and added. Then Dowex 50W
8X resin (pyr+) (0.2 g), and dicyclohexylcarbodiimide (1.09 g,
5.3 mmol) were added, and the solution was stirred at room
temperature for 2 days. Water (15 mL) was added and the solution
was stirred for 10 min. The solvent was removed under reduced
1H NMR (400 MHz; CD3OD): dH 8.38 (1H, s, CH Ad), 8.26 (1H, s,
CH Ad), 7.16 (5H, m, Cbz), 6.41 (1H, m, 10 CH), 5.12 (1H, m, 30 CH),
4.99 (2H, m, CH2 Cbz), 4.27 and 4.18 (1H, 2 ꢃ s, 40 CH, two diaste-
reoisomers), 3.98–3.15 (3H, m, CH–P, 50 CH2), 3.17 (6H, q, J = 7.0,
(CH3–CH2)3NH+), 2.67–2.55 (2H, m, 20 CH2), 1.37 (3H, dd,

3454
E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
pressure, and the residue was partitioned between CHCl3 (20 mL)
and water. The organic phase was then extracted with water
(2 ꢃ 20 mL), dried over MgSO4, and evaporated under reduced
pressure. The residue was purified by column chromatography
on silica gel (eluent CHCl3/MeOH/Et3N: gradient from 68:2:0.1 to
65:5:0.1) to yield N6-benzoyl 50-(tert-butyl dimethyl silyl) 30-
(methyl phosphonyl) 20-deoxyadenosine as the triethylammonium
salt (276 mg, 0.42 mmol, 42% yield). 1H NMR (400 MHz; CDCl3): dH
9.44 (1H, br, OH), 8.66 (1H, s, CH Ad), 8.30 (1H, s, CH Ad), 7.95 (2H,
d, J = 7.2, 2,6 CH Bz), 7.48 (1H, d, J = 7.2, 4 CH Bz), 7.40 (1H, t, J = 7.2,
3,5 CH Bz), 6.51 (1H, dd, J = 5.7, J = 7.5, 10 CH), 4.88 (1H, m, 30 CH),
4.25 (1H, m, 40 CH), 3.82 (2H, m, 50 CH2), 2.92 (6H, q, (CH3CH2)NH+),
2.67 (1H, m, 20 CHAHB), 2.57 (1H, m, 20 CHAHB), 1.22 (13H, m, CH3–P,
(CH3CH2)NH+), 0.80 (9H, s, (CH3)3C–Si), 0.00 (6H, s, CH3–Si). 31P
NMR (121.5 MHz; CDCl3): dP 21.7. ES-MS (ꢂve ion): m/z 546.1
(MꢂH+); (+ve ion): m/z 548.0 (M+H+); 570.1 (M+Na+). HR MS
(FAB): 548.2085 Da (calcd for C24H35N5O6SiP (M+H+) 548.2094).
The above compound (200 mg, 0.308 mmol) was dissolved in
methanol (2 mL), then aq NH3 33% (6 mL) were added and the mix-
ture was stirred at room temperature overnight. The solvents were
removed under reduced pressure and the residue was purified by
column chromatography on silica gel (eluent CHCl3/MeOH/Et3N:
gradient from 65:5:0.1 to 6:1:0.1) to yield the debenzylated com-
pound as the triethylammonium salt (128 mg). This material was
CD3OD): dP 14.9. ES-MS (+ve ion): m/z 473.2 (M+H+); (ꢂve ion):
m/z 471.2 (MꢂH+). HR MS (TOF): 473.2105 Da (calcd for
C18H34N6O5PSi (M+H+): 473.2092). IR (neat):
2929, 2852, 1615, 1574.
m ) 3182,
(cmꢂ1
4.6. 50-(Methylcarbamoyl) 20-deoxyadenosine (17)
Following the method of Fernandez et al. (ref), acetone O-((phe-
nyloxy)carbonyl) oxime (828 mg, 4.3 mmol) was added a solution
of 20-deoxyadenosine (270 mg, 1 mmol), 4 Å molecular sieves
(0.2 g), and freshly dried C. antarctica (CAL-B, 0.2 g, >10,000 U/g)
in anhydrous THF (20 mL) under N2, and the mixture was shaken
at 200 rpm, 37 °C in an incubator/shaker for 3 days. After this time,
the suspension was filtered on a synthered glass filter, the filter
was washed first with THF (10 mL) and then with methanol
(20 mL) and the filtrate was evaporated under reduced pressure.
The residue was purified by flash chromatography on silica gel
(eluent: gradient from AcOEt 100% to AcOEt/MeOH 9:1), affording
the 50-phenylcarbonate derivative (272 mg). 1H NMR (400 MHz;
CD3OD): dH 8.27 (1H, s, CH Ad), 8.21 (1H, s, CH Ad), 7.35 (2H, m),
7.21 (1H, m), 7.10 (2H, m), 6.47 (1H, t, J = 6.5, 10 CH), 4.67 (1H,
m, 30 CH), 4.53 (1H, dd, J = 11.8,3.9, 50 CHAHB), 4.45 (1H, dd,
J = 11.8, 4.7, 50 CHAHB), 4.23 (1H, dd, J = 4.0, 8.5, 40 CH), 2.83 (1H,
m, 20 CHACHB), 2.52 (1H, ddd, J = 13.8, 6.5, 4.2, 20 CHACHB). HR MS
(FAB): 372.1314 Da (calcd for C17H18N5O5 (M+H+): 372.1307).
The above material was dissolved in anhydrous THF (5 mL) un-
dissolved in THF (5 mL), then Et3Nꢀ3HF (220
lL, 1.38 mmol) was
added and the mixture was stirred at room temperature overnight.
The solvent was removed under reduced pressure and the residue
was partitioned between ethyl acetate (10 mL) and water (20 mL).
The organic phase was further extracted with water (2 ꢃ 10 mL)
and the combined water phases were evaporated under reduced
pressure. The residue was purified by flash chromatography on sil-
ica gel (eluent: CHCl3/MeOH/NH3: gradient from 6:1:0.1 to 5:2:0.1)
to yield compound 15 as ammonium salt (74 mg, 70% overall). 1H
NMR (400 MHz; CD3OD): dH 8.34 (1H, br s, CH Ad), 8.18 (1H, br s,
CH Ad), 6.45 (1H, dd, J = 5.7,8.0, 10 CH), 5.0 (1H, m, 30 CH), 4.26 (1H,
m, 40 CH), 3.87 (1H, dd, J = 2.8,12.4, 50 CHAHB), 3.83 (1H, dd,
J = 3.1,12.4, 50 CHAHB), 2.91 (1H, m, 20 CHACHB), 2.60 (1H, ddd,
der N2 and 2.0 M solution of methylamine in THF (336 lL,
0.673 mmol) was added. The solution was stirred at 50 °C until
the starting material is consumed (18 h). After this time, the sol-
vent was removed under reduced pressure and the result solid
was triturated diethyl ether, and filtered. The solid was washed
with enough Et2O to remove phenol completely. The product 17
(226 mg, 72% overall) was afforded as a white solid in 99% yield.
Rf (AcOEt/MeOH 9:1): 0.15. 1H NMR (400 MHz; D2O): dH 7.99
(1H, s, CH Ad), 7.90 (1H, s, CH Ad), 6.16 (1H, t, J = 6.0, 10 CH), 4.53
(1H, m, 30 CH), 4.27–3.99 (3H, m), 2.3 (1H, m, 20 CHACHB), 2.50
(1H, m, 20 CHACHB), 2.45 (3H, s, CH3). 13C NMR (100.5 MHz; D2O):
dC 158.2, 154.4, 152.3, 148.0, 139.0, 118.2, 84.6, 83.6, 70.5, 63.5,
38.6, 26.5. LSI MS: m/z 309.13 (M+H+). HRMS (FAB): 309.1318 Da
J = 2.6, 5.7,13.5, 20 CHACHB), 1.32 (3H, d, JPH = 16.5, CH3–P).13
NMR (100.5 MHz; D2O): dC 157.5, 153.5, 149.9, 141.6, 120.9, 89.1,
C
87.1, 75.5, 63.4, 40.7, 14.2, 12.8 (d, JCP = 137).31
P
NMR
(calcd for C12H17N6O4 (M+H+): 309.1311). FT-IR (neat):
3325, 2946, 1697, 1613, 1574.
m )
(cmꢂ1
(121.5 MHz; D2O): dP 24.4. ES-MS (ꢂve ion): m/z 328.0 (MꢂH+),
679.1 (2(MꢂH+)+Na+); (+ve ion): m/z 330.1 (M+H+), 352.1
(M+Na+). HR MS (TOF): 328.0827 Da (calcd for C11H15N5O5P
4.7. MurM Radiochemical enzyme assays
(MꢂH+) 328.0816). IR (neat):
RP-HPLC: tR, 8.60 min.
m
(cmꢂ1) 3042, 2861, 1652, 1606.
200
37.5
500 cpm/pmol) or 65.7
114 mCi/mmol, 0.1 mCi/mL), 4 units of inorganic pyrophospha-
tase (IPP), 0.16 mg Alanyl tRNA synthetase (AlaRSPn16), 10 mM
DTT, in 30 mM HEPES pH 7.6, 15 mM MgCl2, 25 mM KCl, 2 mM
l
M
L incubations containing 0.66 mg total M. flavus tRNA,
[3H]-Ala (1.45
Ci, ꢁ3,200,000 cpm, stock 0.5 mM,
M [14C]-Ala (1.45
l
l
4.5. 50-(tert-Butyl dimethyl silyl) 30-(1-amino ethyl phosphonyl)
l
l
Ci, ꢁ3,200,000 cpm,
20-deoxyadenosine (16)
Compound 11 (100 mg, 0.123 mmol) was dissolved in MeOH/
NH3 aq 1:3 (15 mL). Pd/C 5% Degussa type (5 mg) was added and
the reaction was stirred at room temperature under H2 atmosphere
for 18 h. After this time, the catalyst was filtered and the filtrate
was evaporated under reduced pressure. The residue was purified
by flash column chromatography on silica gel (eluent CHCl3/
MeOH/NH3: gradient from 6:1:0.1 to 5:2:0.1), affording compound
16 (60 mg, 0.123 mmol) in 99% yield. Rf (CHCl3/MeOH/Et3N
6:1:0.1): 0.1. 1H NMR (400 MHz; CDCl3/CD3OD/D2O 2:3:1): dH
8.21 (1H, s, CH Ad), 8.11 (1H, s, CH Ad), 6.39 (1H, dd, J = 5.7, 8.2,
10 CH), 4.97 (1H, m, 30 CH), 4.26 (1H, m, 40 CH), 3.82 (2H, m, 50
CH2), 3.19 (1H, m, CH–P), 3.00 (1H, m, 20 CH2), 2.67 (1H, ddd,
J3 = 2.0, 5.7, 13.8, 20 CHAHB), 2.55 (1H, m, 20 CHAHB), 1.37 (3H, dd,
J3 = 7.2, JPH = 14.8, CH3–CH–P), 0.79 (9H, s, (CH3)3C–Si), 0.00 (6H,
s, CH3–Si). 13C NMR (100.5 MHz; CDCl3/CD3OD/D2O 2:3:1): dC
157.1, 153.9, 150.1, 140.4, 120.4, 88.6, 86.0, 76.8, 64.8, 46.7, 45.3
(d, JCP = 149), 41.8, 27.0, 19.6, 15.2, ꢂ4.57. 31P NMR (121.5 MHz;
ATP were incubated at 37 °C for 2 h. Then, 20
lL of 3 M Na ace-
tate pH 4.5 were added, followed by 205 L of phenol equili-
l
brated with 10 mM Tris HCl, pH 8.0, 1 mM EDTA. The emulsion
was mixed and centrifuged for 3 min at 13,000 rpm. The aqueous
phase was pooled and added to 205
alcohol 24:1. After centrifugation, the aqueous phase was pooled
and supplemented with 520
lL of chloroform/isoamyl
l
L of absolute ethanol at ꢂ20 °C. The
precipitated aminoacyl-tRNA and was collected by centrifugation
at 13000 rpm. The pellet was washed with 1 mL of 70% ethanol,
dried under reduced pressure in a desiccator and resuspended
into 50
lL of 3 mM Na acetate pH 4.5. The specific activity for
each incubation (Cpm/pmol) was estimated by spotting 0.5
lL
of crude incubation mixture (before tRNA isolation) on filter pa-
per, which was dried and placed directly into scintillation liquid
for counting. The radiolabelled aminoacyl tRNA concentration
was estimated from precipitation of radioactivity from a 1 lL

E. Cressina et al. / Bioorg. Med. Chem. 17 (2009) 3443–3455
3455
sample of purified tRNA onto filter paper disks in ice cold 10% (w/
v) trichloroacetic acid for 15 min. This wash was repeated twice,
and the papers disks were finally washed in ice-cold ethanol,
dried and placed into scintillation liquid for counting.
and Escherichia coli in liquid culture, using the NCCLS protocol, at
1–5 mM final concentration.
Acknowledgements
To follow the generation of Lipid 2-[3H]-Ala or Lipid 2-[14C]-Ala
in the presence of an inhibitor, an assay mix typically contained in a
This work was supported by the EC Framework 6 COBRA net-
work., the Medical Research Council (grant G0400848), the Well-
40 lL total volume, 50 mM MOPS pH 6.8, 30 mM KCl, 10 mM MgCl2,
1.5% CHAPS, 1 mM DTT, 1 mM
L
-alanine, 50
l
M Lipid 2, 0.2
l
M
come Trust (grant 066443), a BBSRC studentship, and the
Medical Research Fund (University of Warwick).
[3H]-Ala-tRNA (5.5 ꢃ 10ꢂ3
l
Ci, 12,000 cpm total counts in each
incubation) or 0.5
l
M [14C]-Ala-tRNA (5.5 ꢃ 10ꢂ3
lCi, 12,000 cpm
total counts in each incubation), 50 nM (0.09
l
g) MurM159 and
References and notes
the inhibitor solution in water (1–0.1 mM). The incubations were
kept at 37 °C for 5 min and then terminated with the addition of
40 lL 6 M pyridinium acetate pH 4.5 and 80 lL of n-butanol. The
incubations were then mixed and centrifuged at 13,000 rpm for
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ing the solvent (4
l
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S. pneumoniae alanyl tRNA synthetase and seryl tRNA synthe-
tase were expressed and purified as previously described.9 Activity
was monitored by trichloroacetic acid precipitation and subse-
quent quantitation of [3H]-alanyl or seryl-tRNA formed by the en-
zymes in the presence of 37.5
pmolꢂ1), 2 mM ATP, 30 mM HEPES, 15 mM MgCl2 5 mM DTT,
25 mM KCl, 1 M alanyl or seryl
M tRNAAla or tRNASer and 0.17
tRNA synthetase. Enzyme inhibition assays were carried out in
the presence of 100 M and 1 mM inhibitor.
l
M [3H]-amino acid (204 cpm
l
l
l
4.9. Antibacterial testing
Inhibitors were tested for growth inhibition of S. pneumoniae
strains 159 and pn16, S. aureus (MRSA, MSSA), E. faecalis, M. flavus