δ-Di-carboxybutyl phosphoramidate of 2′-deoxycytidine-5′-monophosphate as substrate for DNA polymerization by HIV-1 reverse transcriptase

Article abstract of DOI:10.1016/j.bmc.2009.08.001

The replacement of the pyrophosphate moiety of 2′-deoxynucleoside triphosphates by non natural δ-dicarboxylic butyl amino acid allows incorporation of natural 2′-deoxycytidine into DNA using HIV-1 reverse transcriptase (RT) as enzyme. In contrast, the 3′-

Full text of DOI:10.1016/j.bmc.2009.08.001

Bioorganic & Medicinal Chemistry 17 (2009) 7008–7014
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
d-Di-carboxybutyl phosphoramidate of 20-deoxycytidine-50-monophosphate
as substrate for DNA polymerization by HIV-1 reverse transcriptase
b
a
Ivan Zlatev a, Anne Giraut b, François Morvan a, , Piet Herdewijn , Jean-Jacques Vasseur
*
a Institut des Biomolécules Max Mousseron, UMR 5247, Université Montpellier 2, CC1704, Place E. Bataillon, 34095 Montpellier Cedex 5, France
b Rega Institute for Medical Research, K.U.Leuven, Laboratory of Medicinal Chemistry, Minderbroedersstraat 10, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
The replacement of the pyrophosphate moiety of 20-deoxynucleoside triphosphates by non natural d-
dicarboxylic butyl amino acid allows incorporation of natural 20-deoxycytidine into DNA using HIV-1
reverse transcriptase (RT) as enzyme. In contrast, the 30-deoxycytidine analogue was not a substrate of
the HIV-1 RT.
Article history:
Received 18 June 2009
Revised 30 July 2009
Accepted 3 August 2009
Available online 8 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Polymerase
Modified nucleotide
Nucleotide analog
Phosphoramidate
HIV
1. Introduction
phate mimic and can efficiently act as a leaving group in a tem-
plate-directed, polymerase-catalyzed DNA oligomerization.4
For the past decades, nucleoside analogues have revealed an
outstanding potential in therapeutics as efficient antiviral or anti-
metabolic agents. These compounds act like prodrugs, as they need
intracellular phosphorylation by kinases before interacting with
cellular or viral polymerases. However, the intracellular metaboli-
sation to their nucleoside 50-triphosphate (NTP) entities is cumber-
some and thus a limiting factor for their therapeutic activity.
Modified nucleoside 50-monophosphate/phosphonate analogues
(phosphotriesters or phosphoramidates) have been synthesized1
and can be introduced in the cell, where the removal of the mask-
ing group(s) leads to the corresponding free nucleoside 50-mono-
phosphate/phosphonate (NMP) entities to be transformed by
kinases to the target NTPs. Yet, the two remaining steps of kinase
phosphorylation remain a problem due to substrate specificity of
cellular enzymes.2,3 Hence, the use of NTP analogues, that would
serve as direct substrates/inhibitors to polymerases, bypassing all
the kinase activation pathways, would be of great interest.
In particular, among various AA-dNMPs, only L-Asp-dNMPs (11,
Fig. 1) and L-His-dNMPs (12, Fig. 1) exhibited efficient incorpora-
tion by HIV-1 RT, suggesting that Asp and His amino acids posses
the required structural and electronic features for efficient coordi-
nation with the catalytic metal cations inside the polymerase ac-
tive site.5 L-Asp-dNMPs demonstrated by far the most efficient
incorporation and their kinetic analysis indicated Km values much
higher than those measured for natural dNTPs, and, however,
OH
O
P O
NH
Base
O
R2
R1
OH
R1
R2
-COOH
-COOH
-CH2COOH
11
N
Herdewijn et al. recently showed that amino acid phosphoram-
idates of 20-deoxynucleoside-50-monophosphates (AA-dNMPs) can
serve as substrates for polymerization by the HIV Reverse Trans-
criptase (RT) or other thermostable cellular polymerases.4–6 In fact,
-CH2-
12
NH
-COOH
-H
-CH2CH2COOH
-CH2COOH
13
14
1
interestingly, a natural
a-L-amino acid moiety, attached to the
-H
-(CH2)2CH(COOH)2
NMP by a 50-phosphoramidate linkage is a successful pyrophos-
Base = C for 1, A for 11-14
* Corresponding author. Tel.: +33 0 467 144 961; fax: +33 0 467 042 029.
E-mail address: morvan@univ-montp2.fr (F. Morvan).
Figure 1. Structures of amino acid phosphoramidates of nucleoside 50-monophos-
phates; used in previous studies (11, 12, 13 and 14), and in the present study (1).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.001

I. Zlatev et al. / Bioorg. Med. Chem. 17 (2009) 7008–7014
7009
importantly, Vmax values only 3.1-fold lower.6 This latter observa-
tion indicates efficient nucleophilic displacement of the amino acid
moiety, but poor selectivity/affinity of the AA-dNMP analogue for
the polymerase active site. Therefore, the design of novel phos-
phoramidate analogues with higher affinity for the polymerization
active site would be of significant interest towards the efficiency
improvement of this type of compounds.
tion of the 20-hydroxyl group, subsequent detritylation and then
introduction of the H-phosphonate monoester on the 50 position
of 3 using diphenyl phosphite9 (Scheme 1B). Compound 4 was then
oxidized by CCl4 in presence of a silylating agent, triethylamine
and amine 10, affording the fully protected diethylester phosphor-
amidate derivative, which was further deprotected firstly by treat-
ment with aqueous 0.4 M sodium hydroxide and secondly with
28% aqueous ammonia (Scheme 1B). Target dicarboxylic phosphor-
amidate 1 was obtained after a final purification by chromatogra-
phy and sodium ion exchange.
Along this line, we present herein the synthesis and the incor-
poration properties of a novel phosphoramidate conjugate of
20-deoxycytidine-50-monophosphate (dCMP), exhibiting a non-nat-
ural amino acid moiety, namely the d-di-carboxylic amino acid
moiety (compound 1, Fig. 1). This non-natural amino acid was de-
signed as a novel pyrophosphate mimic, featuring the closely situ-
ated double negative charge, as for aspartic residue in 11, of its
malonate moiety, attached via the relatively long and thus flexible
propyl chain. The structure concept was based on the previous
findings for AA-dNMP conjugates, assessing the poor HIV-1 RT sub-
strate nature for the Glu analogue5 (13, Fig. 1) and the b-Ala one11
(14, Fig. 1). Hence, the design of a non-natural amino acid side
chain taking into account these observations, in the aim to improve
polymerase affinity, seemed of particular interest (vide infra for
further discussion). On the other hand, the malonate dianion rep-
resents good bidentate chelating features, as it is well known for
forming six membered chelate rings with metal ions, and was
hence chosen in the aim to achieve coordination of a magnesium
cation within the polymerization site.
The 30-deoxy isomer of the title compound 1, compound 2
(Fig. 2), was synthesized in a similar way (see Supplementary
data), in order to be used as a negative control in the polymerase
assay, since 20-hydroxyl nucleotides are not substrates of HIV-1 RT.
3. Single nucleotide incorporation assay
The essential role of the HIV-1 RT in HIV replication renders this
viral polymerase the main target enzyme in treating HIV infection.
The HIV-1 RT is an error-prone polymerase with a high mutation
rate, exhibiting important flexibility and high tolerance towards
chemically modified nucleotide substrates.10 In previous stud-
ies,4–6 it was showed that HIV-1 RT was able to successfully incor-
porate AA-dNMPs instead of natural dNTPs, during polymerase-
catalyzed, template-directed DNA synthesis. Here, interestingly,
compound 1, bearing the newly introduced, non-natural d-di-car-
boxylic amino moiety, was smoothly recognized as a substrate by
HIV-1 RT, and was efficiently incorporated into the growing DNA
primer, resulting in a (n + 1) elongation of the primer P affording
2. Results and discussion
The target d-dicarboxylic-butyl phosphoramidate 20-deoxycyti-
dine derivative 1 was prepared using a strategy involving the ami-
dative oxidation of the di-30-O,N4-benzoylated 20-deoxycytidin-50-
yl-H-phosphonate monoester 4 by CCl4 in the presence of the
appropriate amino diethyl ester derivative 10 (Scheme 1).7
First, compound 10 was prepared in a three-step sequence,
starting from commercially available 3-bromopropylamine hydro-
bromide (Scheme 1A). After protection of the amino function with
a tert-butoxycarbonyl (Boc) group, the di-carboxylic moiety was
introduced as its diethyl ester, affording compound 9 after the
nucleophilic substitution of the bromine atom in 8 by the in situ
generated diethyl malonate carbanion.8 Deprotection of the Boc
group was accomplished by treating 9 with trifluoroacetic acid,
affording the desired di-carboxylic aminobutyric acid diethyl ester
10 as its trifluoroacetate salt (Scheme 1A). On the other hand, the
conveniently protected 20-deoxycytidine 50-H-phosphonate (4)
was prepared from commercially available N4-benzoyl-20-deoxy-
50-O-dimethoxytritylcytidine, in three steps, including benzoyla-
P+1 (Fig. 3, Panel I), up to 54% in 60 min at 500 lM nucleotide con-
centration. No significant improvement of the elongation was ob-
served when 1 mM concentration of the nucleotide was used
(Fig. 3). As expected, no incorporation was observed when the 30-
deoxyribocytidine phosphoramidate 2 was used in the experiment
(Fig. 3, Panel II) demonstrating that the incorporation of 1 was in-
deed performed by HIV-1 RT.
Compound 1 was also substrate for elongation of P complemen-
tary to a three-deoxyguanosine sequence. At 500 lM and 1 mM,
60% of the primer was elongated up to a P+3 product, while the rest
remained unreacted showing that three successive non natural
phosphoramidite 20-deoxycytidine 1 could be incorporated (Fig. 4).
When these results were compared to the ones obtained with
the incorporation of the L-Asp-dCMP analogue (Fig. 1, 8 11,
Base = Cytidinyl),6 a 1.5-fold decrease in the amount of the (n + 1)
product was observed (54% for 1 vs 82% for 11, for 60 min of reac-
tion at 500
lM concentration of nucleotide). This demonstrates
lesser efficiency for the non-natural AA-dCMP conjugate 1, sug-
COOEt
COOEt
COOEt
COOEt
b
a
Br
c
BocHN
H3N
Br
A
B
BocHN
HBr.H2N
CF3COO
9
10
8
NHBz
N
NHBz
N
NHBz
NH2
N
Na
N
O
O
O P O
DMTrO
HO
O
P O
NH
O
N
O
O
O
N
N
N
d, e
O
f
O
O
O
g, h, i
NHEt3
H
NaOOC
OH
OH
OBz
OBz
COONa
3
1
4
Scheme 1. (A) Synthesis of amino acid 10. Reagents and conditions: (a) Boc2O, CH2Cl2, NEt3, 90%; (b) diethyl malonate, sodium, EtOH, reflux, 77%; (c) CF3COOH, CH2Cl2, 98%;
(B) Synthesis of amino acid phosphoramidate 1. Reagents and conditions: (d) benzoyl chloride, pyridine; (e) 10% benzene sulfonic acid, CH2Cl2, MeOH, 93%; (f) diphenyl
phosphite, pyridine; then H2O/NEt3, 90%; (g) N,O-bis-trimethylsilylacetamide, pyridine, CCl4, NEt3, 10; (h) 0.4 M NaOH, CH3CN; then DOWEX-50WX8-pyridinium; (i) 28%
NH4OH; then DOWEX-50WX8-sodium, 38%.

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I. Zlatev et al. / Bioorg. Med. Chem. 17 (2009) 7008–7014
NH2
N
Na
O
O
P O
NH
O
N
O
NaOOC
R1 R2
COONa
R1
R2
1
2
-OH
-H
-H
Figure 4. Elongation of P primer hybridized to T2 template with 1 as substrate and
HIV-1 RT as enzyme. Conditions: primer P was 50-labeled with 33P, followed by
-OH
annealing to template T2. Reactions were carried out with 125 nM primerꢀtemplate
Figure 2. Chemical structures of the prepared 4,4-dicarboxybutyl phosphorami-
dates of 20-deoxy (1) and 30-deoxy (2) cytidine.
(PꢀT2), [HIV-1 RT] = 0.025 U l , time points: 30, 60, 90, 120 min. Blank: no
Lꢁ1
nucleotide analogue—500
lM: concentration of 1—1 mM: concentration of 1—
50 lM concentration of dCTP.
gesting a lower affinity of compound 1 for the polymerase active
site and/or less efficient displacement of the non-natural amino
acid, acting as leaving group. This could be due to a poorer pyro-
phosphate mimic character for this non-natural amino acid moiety,
compared to the Asp residue. The relatively important distance be-
tween the first negative charge borne by the 50-phosphorus group
and the charges of the malonate moiety might be pointed out as a
negative feature of this pyrophosphate mimic. However, interest-
ingly, compound 1 was more efficiently incorporated by HIV-1
RT than the Glu phosphoramidate conjugate (13, Fig. 1),5 suggest-
ing that the distance between the phosphoramidate monoester
group and the pending carboxylate group(s) is not the sole param-
eter encountered. Likewise, one might suggest that the flexible
propyl chain allows efficient positioning of the dicarboxylic moiety
inside the polymerization site and thus a smooth nucleophilic dis-
placement of the leaving group. However, the exact mechanism of
nucleotidyl transfer using AA-dNMP conjugates remains unknown.
The successful incorporation of compound 1, implying that the
non-natural amino acid moiety acts indeed as a pyrophosphate mi-
cating the importance of the carboxy group being fixed at the
-position of the amino acid.11 Interestingly, the compound 1 pre-
sented herein represents an alternative AA-dNMP conjugate lack-
ing the -carboxy group and successfully incorporated into the
a
a
DNA chain by the polymerase.
4. Conclusion
In conclusion, a newly designed phosphoramidate conjugate of
20-deoxycytidine-50-monophosphate 1, bearing a non-natural d-di-
carboxybutylamino moiety was prepared. As previously demon-
strated for the aspartyl phosphoramidate moiety, the d-dicarbox-
ylic butyl-amino moiety linked to the phosphorous atom acts as
a mimic of a pyrophosphate group and successfully behaves as a
leaving group in a nucleotidyl transfer reaction, allowing the ex-
tend of a DNA primer when the non-natural AA-dCMP phosphor-
amidate 1 was used as a substrate for template-directed DNA
synthesis, catalyzed by HIV-1 RT and allowed a partial elongation
of several non-natural AA-dCMP phosphoramidates 1. This result
should contribute to the development of this new type of NTP ana-
logues, which can find future applications in therapeutics and bio-
technology, serving as direct substrates/inhibitors to polymerases,
bypassing all the kinase activation pathways.
mic, replacing the b- and c-phosphate groups, remains interesting
in regards of the flexibility of HIV-1 RT, able to accept modified
nucleotide analogues as substrates, and pointing out the possibility
of designing new AA-dNMP conjugates, exhibiting a large variety of
natural or non-natural amino acid moieties. Another interesting
point in this study is the successful incorporation of a phosphor-
amidate conjugate containing d-carboxylic amino acid moiety. In-
deed, so far, all the phosphoramidates used in the previous
5. Experimental
5.1. General chemistry procedures
studies exhibit an
a-carboxy group adjacent to the amino group
linked with the 50 phosphorus atom (Fig. 1). Moreover, the b-Ala
dNMP conjugate (14, Fig. 1) was recently designed and evaluated
by us, without observing any incorporation by the HIV-1 RT, indi-
TLC was performed on Merck silica coated plates 60F254 (art.
5554). Compounds were revealed on UV light (254 nm) and after
Figure 3. Panel I: incorporation of phosphoramidate 1 by HIV-1 RT. Panel II: incorporation of phosphoramidate 2 by HIV-1 RT. Conditions: primer P was 50-labeled with 33P,
followed by annealing to template T1. Reactions were carried out with 125 nM primerꢀtemplate (PꢀT1), [HIV-1 RT] = 0.025 U
Lꢁ1, time points: 5, 10, 20, 30 and 60 min.
l

I. Zlatev et al. / Bioorg. Med. Chem. 17 (2009) 7008–7014
7011
spraying with 10% sulfuric acid ethanol solution and heating.
5.1.2. Synthesis of 2-[3-(tert-butoxycarbonylamino)propyl]-
diethyl malonate (9)
Phosphorus containing compounds were revealed after spraying
with the Hanes molybdate reagent12 and heating. Amines were
revealed after spraying with saturated ninhydrine solution in eth-
anol (7.0 g/L) and heating. Column chromatography was per-
formed on silica gel Merck 60 (art. 9385). Flash chromatography
was performed using a Biotage SP1 apparatus and Biotage silica
gel columns. Ion exchange purifications were carried using Phar-
macia Sephadex A-25 resin (art. 170), ISCO TRIS Pump and UA-6
detector (254 nm). Triethylammonium bicarbonate (TEAB) 1 M,
10 mL of freshly distilled ethanol were introduced into a two-
necked flask equipped with a water condenser. 270 mg of sodium
(12 mmol, 2 equiv) were added and the mixture was stirred for
5 min. 1.9 mL of diethyl malonate (12 mmol, 2 equiv) were then
introduced and the mixture was stirred at room temperature until
consumption of sodium. 1.5 g of compound 8 (6.2 mmol, 1 equiv),
dissolved in 10 mL of freshly distilled ethanol, were then introduced
to the reaction mixture over 5 min.8 The mixture was stirred under
reflux for 1 h, cooled to room temperature, diluted with 20 mL of
AcOEt and hydrolyzed with 10 mL of water. It was then washed
twice with water, and then the aqueous layers were back-extracted
with AcOEt. The combined organic layers were dried over Na2SO4,
filtered and evaporated to dryness. The crude product was purified
by column chromatography (gradient: 100% of cyclohexane to 15%
of AcOEt), affording pure 9 as a colorless oil (1.55 g, 77%). Rf: 0.34—
cyclohexane/AcOEt—7:3 (v/v). 1H NMR: (CDCl3, 200 MHz) d 4.60
(1H, pt, br, NHBoc), 4.22 (4H, q, OCH2CH3, J = 7.0 Hz), 3.36 (1H,
t, –CH(COOEt)2, J = 7.4 Hz), 3.16 (2H, q, CH2NHBoc, J = 6.4 Hz), 1.94
(2H, q, CH2CH(COOEt)2, J = 7.6 Hz), 1.50 (2H, m, –CH2–), 1.46 (9H,
s, CH3-Boc), 1.29 (6H, t, OCH2CH3, J = 7.0 Hz). 13C NMR: (CDCl3,
100 MHz) d 169.3 (COOEt), 155.9 (OC(O)NH), 79.2 (C(CH3)3), 61.4
(OCH2CH3), 51.6 (CH(COOEt)2), 40.0 (CH2-NHBoc), 28.4 (CH3-Boc),
27.8 (–CH2–), 25.9 (CH2CH(COOEt)2), 14.1 (OCH2CH3). ESI+ m/z 635
(2 M+H)+, 318 (M+H)+, 262 (MꢁtBu+H)+.
pH 7.5 was prepared from triethylamine, milliQ water (18.2 M
X cm)
and CO2 gas. Ion exchange reactions were performed using DOWEX
50WX8-200 H+ resin (Aldrich), different ion forms were obtained
after washing with corresponding 2 M aqueous solutions and then
water until neutrality. Analytical HPLC were performed using a
Nucleosil C18 column, (150 ꢂ 4.6 mm, 5
l
m) equipped with a pre-
m) on a Waters appara-
filter and a precolumn (Nucleosil, C18, 5
l
tus using a Waters-616 Pump and a Waters 966 Photodiode Array
Detector. Compounds were eluted using a linear gradient 0–80%
of acetonitrile in a 50 mM triethylammonium acetate buffer (pH
7), with a 1 mL/min flow rate. All moist-sensitive reactions were
carried under anhydrous conditions using dry glassware, anhy-
drous solvents and Argon atmosphere. Acetonitrile was distilled
from CaH2 and stored over activated 3 Å molecular sieves. Pyri-
dine and triethylamine were distilled from CaH2. Dichlorometh-
ane and carbon tetrachloride were distilled from P2O5. Ethanol
was distilled from sodium. All commercially available reagents
were used as received. 3 Å molecular sieves were dried in a
300 °C oven during 3 h prior to use. ESI and ESI-HRMS spectra
were recorded on a Q-Tof Micromass spectrometer. 1H and 13C
NMR spectra were recorded at room temperature on Brüker
spectrometers at 200 or 300 MHz (1H) and 75 or 100 MHz (13C).
Chemical shifts are given in ppm referenced to the solvent resid-
ual peak (CDCl3—7.27 and 77.0 ppm; DMSO-d6—2.49 and
39.5 ppm; D2O—4.79 ppm + drop of CH3OH at 49.5 ppm as inter-
nal reference).13 Coupling constants are given in hertz. Signal
splitting patterns are described as singlet (s), doublet (d), triplet
(t), quartet (q), broad signal (br), doublet of doublet (dd), doublet
of triplet (dt), doublet of quartet (dq), pseudo (p), multiplet (m).
Signal assignments were based on 2D homo-1H/1H, NOE or heter-
onuclear-1H/13C correlations and D2O exchange. 31P NMR spectra
were recorded at 121 MHz on proton-decoupled mode and on
proton-coupled mode for H-Phosphonates. Chemical shifts are gi-
ven in ppm referenced to external H3PO4-80%; coupling constants
are given in hertz. UV spectra are recorded on a Varian Cary 300
Bio UV/Vis spectrometer.
5.1.3. Synthesis of 2-(3-amino-propyl)-diethyl malonate, TFA
salt (10)
Compound 9 (1.4 g, 4.4 mmol, 1 equiv) was dissolved in 40 mL
of anhydrous DCM, 10 mL of TFA (30 equiv) were added and the
mixture was stirred at room temperature for 10 min. Solvents were
then removed under reduced pressure, the residue was coevapo-
rated three times with 95 ethanol and dried on an oil pump. Com-
pound 10 was a yellow oil (1.43 g, 98%). Rf: 0.44—DCM/MeOH/
AcOH—75:20:5 (v/v/v). 1H NMR: (CDCl3, 300 MHz) d 7.85 (3H, s,
br, NH3þ), 4.20 (4H, q, OCH2CH3, J = 7.1 Hz), 3.37 (1H, t, –CH(COO-
Et)2, J = 7.1 Hz), 3.05 (2H, q, CH2NH3), 1.97 (2H, q, systA2B2,
CH2CH(COOEt)2, J = 7.3 Hz), 1.76 (2H, m, systA2B2, –CH2–), 1.27
(6H, t, OCH2CH3, J = 7.1 Hz). 13C NMR: (CDCl3, 75 MHz) d 169.3
(COOEt), 61.9 (OCH2CH3), 51.1 (CH(COOEt)2), 39.5 (CH2NH3), 25.3
(CH2CH(COOEt)2), 24.9 (–CH2–), 13.8 (OCH2CH3). ESI+ m/z 435
(2(MꢁTFA)+H)+, 218 (MꢁTFA+H)+.
5.1.4. Synthesis of N4-acetyl-30-deoxy-50-O-(4,40-dimethoxytri-
tyl)cytidine (5)
To a solution of 30-deoxycytidine14 (0.54 g, 2.4 mmol, 1 equiv)
in anhydrous DMF (12 mL) was added acetic anhydride (0.25 mL,
2.6 mmol, 1.1 equiv). The mixture was stirred under Argon for
24 h.15 DMF was removed under reduced pressure (oil pump)
and the residue was dried by azeotropic distillation with anhy-
drous pyridine (three times). It was then dissolved in 24 mL of
dry pyridine and DMTrCl was added (1 g, 2.9 mmol, 1.2 equiv).
Mixture was stirred for 1 h at room temperature. 5 mL of methanol
were added; the mixture was reduced to half volume, diluted with
dichloromethane and washed with saturated bicarbonate solution.
The organic layers were combined, dried over Na2SO4 and evapo-
rated to dryness. Crude product was then purified by column chro-
matography (gradient: DCM 99%, NEt3 1–5% of MeOH) affording
pure 5 as white foam (1.1 g, 80%). Rf: 0.52—DCM/MeOH—9:1 (v/
v). 1H NMR: (CDCl3, 300 MHz) d 9.06 (1H, s, br, NHAc), 8.31 (1H,
d, H6, J = 7.4 Hz), 7.33–7.17 (9H, m, HDMTr), 7.12 (1H, d, H5,
J = 7.4 Hz), 6.79–6.76 (4H, m, HDMTr), 5.70 (1H, d, H-10, J = 0.9 Hz),
5.1.1. Synthesis of N-(tert-butoxycarbonyl)-3-
bromopropylamine (8)
To a solution of 3-bromopropylamine hydrobromide (1.1 mg,
5 mmol, 1 equiv) in 30 mL of anhydrous DCM, were added: 2 g of
Boc2O (10 mmol, 2 equiv), in solution in 20 mL of anhydrous
DCM, and 0.8 mL of anhydrous triethylamine. The mixture was
stirred at room temperature for 1 h, then diluted with 20 mL of
DCM, and washed twice with saturated bicarbonate solution, then
once with brine. The organic layers were dried over Na2SO4, fil-
tered and evaporated to dryness. The crude product was purified
by column chromatography (gradient: 100% of cyclohexane to
15% of AcOEt), affording pure 8 as a colorless oil (1.08 g, 90%). Rf:
0.35—cyclohexane/AcOEt—8:2 (v/v). 1H NMR: (CDCl3, 200 MHz) d
4.70 (1H, s, l, NHBoc), 3.46 (2H, t, CH2Br, J = 6.5 Hz), 3.31 (2H, q,
CH2NHBoc, J = 6.4 Hz), 2.07 (2H, qn, –CH2–, J = 6.5 Hz), 1.46 (9H,
s, CH3-Boc). 13C NMR: (CDCl3, 100 MHz) d 155.9 (OC(O)NH), 79.4
(C(CH3)3), 39.0 (CH2NHBoc), 32.7 (–CH2–), 30.8 (CH2Br), 28.4
(CH3-Boc). ESI+ m/z 238, 240 (M+H)+, 182, 184 (MꢁtBu+H)+.
0
0
0
4.63 (1H, m, H4 ), 4.38 (2H, m, br, OH2 , H2 ), 3.73 (6H, 2s, O-
0
00
CH3DMTr), 3.73–3.23 (2H, m, H5 ,5 ), 2.17 (3H, s, NHC(O)CH3),
2.10–1.90 (2H, m, H3 ,3 ). 13C NMR: (CDCl3, 75 MHz) d 170.1
0
00

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I. Zlatev et al. / Bioorg. Med. Chem. 17 (2009) 7008–7014
(C(O)CH3), 162.5 (C4), 158.7, 158.6 (COCH3DMTr), 156.1 (C2), 144.7
(C6), 144.3 (CDMTr), 135.5, 135.4, 130.1, 130.0, 128.1, 128.0, 127.1,
(BH2)+, 102 (TEAH)+; FAB- (GT) m/z 570 (MꢁTEAH)ꢁ, 152 (B)-. HRMS:
FAB+ (GT) m/z calcd for (C27H45N3O8P)+: 570.2944, found: 570.2953.
0
113.3 (CHDMTr, CDMTr), 96.3 (C5), 95.2 (C1 ), 86.8 (CAr3-DMTr), 81.3
(C4 ), 77.0 (C2 ), 63.7 (C5 ), 55.3 (O-CH3DMTr), 33.1 (C3 ), 25.0
(C(O)CH3). FAB+ (GT) m/z 572 (M+H)+, 330 (DMTr)+, 154 (BH2)+ ;
FAB- (GT) m/z 570 (M-H)-, 152 (B)-.
5.1.7. Synthesis of O-(30-deoxycytidin-50-yl)-N-[(3-malon-2-yl)-
propyl]-phosphoramidate, sodium salt (2)
0
0
0
0
5.1.7.1. Oxidation. The H-phosphonate monoester
7 (35 mg,
0.05 mmol, 1 equiv), was dried by azeotropic distillation with anhy-
drous pyridine (2ꢂ) and vacuum dried under P2O5. It was then dis-
solved in 0.1 mL of anhydrous pyridine and 0.15 mL of N,O-bis-
trimethylsilyl acetamide (BSA) and the solution was stirred
30 min at room temperature under Argon. 0.5 mL of anhydrous
CCl4 (50 equiv), 0.2 mL of anhydrous triethylamine (10 equiv) and
200 mg (0.6 mmol, 12 equiv) of 10, dissolved in 0.1 mL of anhy-
drous pyridine, were simultaneously added. The reaction mixture
was stirred at room temperature for 2 h, the solvents were removed
under vacuum, and the residue was coevaporated with acetonitrile.
5.1.5. Synthesis of N4-acetyl-30-deoxy-20-O-palmitoylcytidine
(6)
To a solution of 5 (1.3 g, 2.3 mmol, 1 equiv) dried by azeotropic
distillation with anhydrous pyridine, in 10 mL of dry pyridine, was
added palmitoyl chloride (3.3 mL, 11.5 mmol, 5 equiv). Mixture
was stirred for 2 h at room temperature. 5 mL of methanol were
added, and then the reaction mixture was concentrated to half vol-
ume, diluted with 30 mL of dichloromethane and washed with
concentrated bicarbonate solution. The organic layers were com-
bined, dried over Na2SO4 and evaporated to dryness. The resulting
residue was coevaporated with dry toluene to remove pyridine,
and was then dissolved in 35 mL of CH2Cl2/MeOH—7:3 (v/v). Solu-
tion was chilled at 0 °C and benzene sulfonic acid (1.1 g, 7 mmol,
3 equiv), dissolved in 11 mL of CH2Cl2/MeOH—7:3 (v/v), was added
dropwise. Mixture was stirred for 30 min at 0 °C, and then neutral-
ized with 30 mL of saturated bicarbonate solution. After extraction
with dichloromethane, the organic layers were washed with NaH-
CO3, dried over Na2SO4 and evaporated to dryness. Crude product
was then purified by column chromatography (gradient: CH2Cl2
100–5% of methanol) affording pure 6 as a yellow gel (0.77 g,
70%). Rf: 0.22—DCM/MeOH—95:5 (v/v). 1H NMR: (DMSO-d6,
300 MHz, 50 °C) d 10.77 (1H, s, br, NHAc), 8.38 (1H, d, H6,
5.1.7.2. Deprotection. The crude product was dissolved in 2 mL
of THF/ACN—1:1 (v/v) and 5 mL of 0.4 M aqueous NaOH were
added, then the mixture was stirred for 10 h at room temperature.
The mixture was quenched by adding some DOWEX-50WX8 pyrid-
inium resin, then the resin was filtered off and well rinsed. The fil-
trates were evaporated to dryness and coevaporated three times
with ethanol. The crude was then dissolved in 10ꢁ3 M TEAB and
purified by ion exchange DEAE-A25 Sephadex chromatography
(linear gradient of 10ꢁ3 M TEAB to 0.4 M). The fractions containing
the product were gathered and evaporated to dryness, then the
residue was further purified by RP-18 flash chromatography (linear
gradient of ACN: 0–5% in 20 mM TEAB). The appropriate fraction
were gathered and evaporated to dryness. The final target com-
pound was eluted on an ion exchange DOWEX-Na+ column and
after lyophilization from water compound 2 was a white powder
(8 mg, 32%). tR—HPLC: 5.9 min (>99%)—0 to 40% ACN in 30 min.
0
J = 7.5 Hz), 7.18 (1H, d, H5, J = 7.5 Hz), 5.82 (1H, d, H1 , J = 1.2 Hz),
0
0
5.27 (1H, pd, H2 , J = 5.5 Hz), 5.09 (1H, s, br, OH5 ), 4.35–4.30 (1H,
0
0
00
m, H4 ), 3.83–3.58 (2H, 2dd, H5 ,5
,
2J = 12.0 Hz), 2.36 (2H, t,
C(O)CH2–, J = 7.3), 2.22–2.14 (1H, ddd, H3 , 2J = 14.0 Hz,
0
3J = 10.3 Hz, 3J = 5.7 Hz), 2.12 (3H, s, NHC(O)CH3), 1.95–1.90 (1H,
UV: (H2O) kmax = 272 nm (
8.06 (1H, d, H6, JH6–H5 = 7.5 Hz), 6.06 (1H, d, H5, JH5–H6 = 7.5 Hz),
e
= 7 500). 1H NMR: (D2O, 300 MHz) d
3
3
ddd, H3
,
2J = 14.0 Hz, 3J = 5.5 Hz, 3J = 1.5 Hz), 1.56 (2H, qn,
00
0
0
0
C(O)CH2CH2–, J = 7.1), 1.26 (24H, s, br, 12 ꢂ –CH2–), 0.87 (3H, t,
5.82 (1H, s, H1 ), 4.65–4.60 (1H, m, H4 ), 4.44 (1H, pd, H2 ), 4.20–
2
–CH2CH3, J = 6.6 Hz). FAB+ (GT) m/z 508 (M+H)+.
4.14 (1H, ddd, systAB, H5 ,
JH5 -H5 = 11.7 Hz, 3J = 3.9 Hz,
0
0
00
3J = 2.1 Hz), 3.96–3.90 (1H, m, systAB, H5 ), 3.07 (1H, t, CH(COO)2,
00
5.1.6. Synthesis of N4-acetyl-30-deoxy-20-O-palmitoylcytidine-
50-yl hydrogenophosphonate, triethylammonium salt (7)
3J = 7.2 Hz), 2.80 (2H, pq, PNHCH2, 3J = 7.8 Hz), 2.20–2.10 (1H, m,
2
0
00
00
0
systAB, H3 ), 2.04–1.97 (1H, ddd, systAB, H3
,
JH3 –H3 = 14.7 Hz,
Nucleoside 6 (0.76 g, 1.5 mmol, 1 equiv) was dried by azeotropic
distillation with anhydrous pyridine and then dissolved in 7.5 mL of
dry pyridine. 2 mL of diphenylphosphite (10.5 mmol, 7 equiv) were
added and the mixture was stirred under Argon for 30 min.9 Reac-
tion was hydrolyzed by addition of 6 mL of water/triethylamine—
1:1 (v/v) and stirred for 10 more min. Solvents were removed under
reduced pressure, the resulting oil was dissolved in CH2Cl2 and
washed twice with saturated bicarbonate solution. The organic lay-
ers were dried over Na2SO4 and evaporated to dryness. Crude prod-
uct was then purified by column chromatography (gradient: CH2Cl2
95%, NEt3 5–6% of methanol) affording pure 7 as a yellow oil (0.84 g,
84%). Rf: 0.27—DCM/MeOH—9:1 (v/v). 1H NMR: (CDCl3, 300 MHz) d
12.32 (1H, s, br, NHTEAH), 9.86 (1H, s, br, NHAc), 8.48 (1H, d, H6,
J = 7.5 Hz), 7.31 (1H, d, H5, J = 7.5 Hz), 6.86 (1H, d, 1JH–P = 682.2 Hz),
3J = 6.0 Hz, 3J = 2.4 Hz), 1.76–1.38 (4H, m, 2 ꢂ –CH2–). 31P NMR:
(D2O, 121 MHz) d 9.5 (P–N). ESI- m/z 449 (Mꢁ3Na+2H)ꢁ. HRMS:
ESI- m/z calcd for (C15H22N4O10P)ꢁ: 449.1074, found: 449.1078.
5.1.8. Synthesis of N4,30-O-dibenzoyl-20-deoxycytidine (3)
To a solution of commercial N4-benzoyl-50-O-DMTr-20-deoxy-
cytidine (1.3 g, 2 mmol, 1 equiv), dried by azeotropic distillation
with anhydrous pyridine (3ꢂ), in 6.5 mL of anhydrous pyridine,
at 0 °C and under Argon atmosphere, were added 0.5 mL of benzoyl
chloride (4 mmol, 4 equiv). The ice bath was removed and the mix-
ture was stirred for 30 min. 5 mL of methanol were added and the
mixture was concentrated to half volume, diluted with 30 mL of
dichloromethane (DCM) and washed 3 ꢂ 20 mL with a saturated
bicarbonate solution. The organics were gathered, dried over
Na2SO4, filtered and evaporated to dryness. The crude residue
was then coevaporated three times with acetonitrile, and dissolved
in 40 mL of DCM/MeOH—7:3 (v/v) and cooled to 0 °C. Benzene sul-
fonic acid 10% solution was then added dropwise (1 g of BSA,
6 mmol, 3 equiv, dissolved in 10 mL of DCM/MeOH—7:3 (v/v))
and the mixture was stirred for 30 min at 0 °C. The reaction mix-
ture was finally neutralized with 20 mL of a saturated NaHCO3
solution, and stirred for 20 more min. The neutralized mixture
was pored into a separating funnel and extracted with dichloro-
methane. The organics were then washed three times with a satu-
rated bicarbonate solution, dried over Na2SO4, filtered and
evaporated to dryness. The residue was purified by silica gel chro-
0
0
0
5.91 (1H, s, H1 ), 5.25 (1H, pd, H2 , J = 4.8 Hz), 4.45 (1H, m, H4 ),
0
00
4.31–3.96 (2H, 2dd, H5 ,5 ), 3.03 (6H, q, CH2TEAH), 2.35–2.21 (3H, t,
0
00
C(O)CH2–, H3 ), 2.18 (3H, s, NHC(O)CH3), 1.98–1.90 (1H, m, H3 ),
1.57 (2H, qn, C(O)CH2CH2–, J = 7.5), 1.28 (9H, t, CH3TEAH, J = 7.5 Hz),
1.18 (24H, s, br, 12 ꢂ –CH2–), 0.81 (3H, t, –CH2CH3, J = 6.7 Hz).
13C NMR: (CDCl3, 75 MHz) d 172.4 (CO-Pal), 170.9 (C(O)CH3), 162.7
0
0
(C-4), 155.1 (C2), 145.3 (C6), 96.3 (C5), 91.2 (C1 ), 80.6 (C4 ,
3
0
JC–P = 8.0 Hz), 62.7 (C5 ), 53.4 (C(O)CH2–), 52.9 (C(O)CH2CH2–),
0
45.6 (CH2TEAH), 34.2 (C3 ), 31.9, 31.1, 29.7, 29.5, 29.3, 29.2, 29.1,
25.0, 24.8 (–CH2–), 22.7 (C(O)CH3), 14.1 (CH3CH2–), 8.6 (CH3-TEA).
1
3
31P NMR: (CDCl3, 121 MHz) d 4.7 (dt, JH–P = 621.4 Hz, JH–P
=
5.9 Hz). FAB+ (GT) m/z 673 (M+H)+, 594 (MꢁTEAH+2H)+, 154

I. Zlatev et al. / Bioorg. Med. Chem. 17 (2009) 7008–7014
7013
matography (linear gradient: 0–5% of MeOH in DCM). Compound 3
was a white foam (0.81 g, 93%). Rf: 0.52—DCM/MeOH—9:1 (v/v). 1H
NMR: (DMSO-d6, 300 MHz) d 11.29 (1H, s, br, NHBz), 8.43 (1H, d, H6,
Sephadex chromatography (linear gradient of 10ꢁ3 M TEAB to
0.4 M). The fractions containing the product were gathered and
evaporated to dryness, then the residue was further purified by
RP-18 flash chromatography (linear gradient of ACN: 0–5% in
20 mM TEAB). The appropriate fraction were gathered and evapo-
rated to dryness. The final target compound was eluted on an ion ex-
change DOWEX-Na+ column and after lyophilization from water
compound 1 was a white powder (5 mg, 38%). tR—HPLC: 6.2 min
3JH6–H5 = 7.5 Hz), 8.04–8.01 (4H, m, Hortho), 7.73–7.49 (6H, m, Hpara
,
=
3
Hmeta), 7.40 (1H, d, H5, 3JH5–H6 = 6.6 Hz), 6.30 (1H, pt, H1 , JH1 –H2
0
0
0
3
0
00
0
0
7.4 Hz, JH1 –H2 = 6.0 Hz), 5.51 (1H, m, H3 ), 5.30 (1H, pt, br, OH5 ),
0
0
00
4.32 (1H, m, H4 ), 3.75 (2H, m, H5 ,5 ), 2.71–2.64 (1H, dd, systAB,
H2 , JH2 –H2 = 14.8 Hz, 3JH2 –H1 = 5.7 Hz), 2.44–2.35 (1H, m, systAB,
2
0
0
00
0
0
H2 ). 13C NMR: (DMSO-d6, 75 MHz) d 165.2 (COBz), 163.1 (C4), 154.3
(>98%)—0 to 40% ACN in 30 min. UV: (H2O) kmax = 272 nm (e =
00
(C2), 144.8 (C6), 133.6, 133.1, 132.7, 129.3, 128.8, 128.4 (CHBz),
7300). 1H NMR: (D2O, 300 MHz) d 7.97 (1H, d, H6, 3JH6–H5 = 7.5 Hz),
3
128.3 (CBz), 96.3 (C5), 86.4 (C1 ), 85.5 (C4 ), 75.6 (C3 ), 61.8 (C5 ),
6.32 (1H, t, H1 , JH1 –H2 = 6.9 Hz), 6.10 (1H, d, H5,3JH5–H6 = 7.5 Hz),
0
0
0
0
0
0
0
38.3 (C2 ). ESI m/z 871 (2 M+H)+, 436 (M+H)+.
4.56–4.52 (1H, m, H4 ), 4.18 (1H, pt, H3 ), 4.02–3.93 (2H, m, H5 ,
00
+
0
0
0
0
H5 ), 3.04 (1H, t, CH(COO)2, 3J = 7.8 Hz), 2.79 (2H, pq, PNHCH2,
5.1.9. Synthesis of N4,30-O-dibenzoyl-20-deoxycytidin-50-yl
hydrogenophosphonate, triethylammonium salt (4)
J = 7.8 Hz), 2.46–2.38 (1H, ddd, systAB, H2 , JH2 –H2 = 14.1 Hz,
3
2
0
0
00
3J = 6.3 Hz, 3J = 3.9 Hz), 2.34–2.25 (1H, m, systAB, H2 ), 1.74–1.37
(4H, m, 2 ꢂ –CH2–). 31P NMR: (D2O, 121 MHz) d 9.4 (P–N). ESI-: m/z
449 (Mꢁ3Na+2H)-. HRMS: ESIꢁ m/z calcd for (C15H22N4O10P)ꢁ:
449,1074, found: 449,1056.
00
Toa solutionof 3 (0.37 g, 0.85 mmol, 1 equiv), dried by azeotropic
distillation with anhydrous pyridine (2ꢂ), in 5 mL of anhydrous pyr-
idine at room temperature and under Argon atmosphere, were
added 1.5 mL of diphenylphosphite (7 mmol, 7 equiv). The mixture
was stirred at room temperature for 1 h, and then hydrolyzed by
adding 4 mL of water/triethylamine—1:1(v/v) solution. The solvents
were removed under vacuum, and the residue was dissolved in
dichloromethane, then washed twice with a saturated bicarbonate
solution. The organics were dried over sodium sulfate, filtered and
evaporatedto dryness. Theresiduewaspurified bysilicagel chroma-
tography (linear gradient: 0 to 5% of MeOH in DCM/Net3—95:5).
Compound 4 was a white foam (0.46 g, 90%). Rf: 0.35—DCM/
MeOH—9:1 (v/v). 1H NMR: (CDCl3, 300 MHz) d 12.35 (1H, s, br,
5.2. Single nucleotide incorporation and elongation tests
Chemicals: Highly purified 2’-deoxycytidine triphosphate used
in DNA polymerase reactions was purchased from Pharmacia.
Oligodeoxyribonucleotides: Oligodeoxyribonucleotides P, T1 and
T2 were purchased from Sigma Genosys or Eurogentec. The concen-
trations were determined with a Varian Cary-300-Bio UV Spectro-
photometer.
The lyophilized oligonucleotides were dissolved in diethylpyro-
carbonate (DEPC)-treated water and stored at ꢁ20 °C. The primer
3
NHTEAH), 9.10 (1H, s, br, NHBz), 8.53 (1H, d, H6, JH6–H5 = 7.5 Hz),
8.04–7.88 (4H, m, Hortho), 7.60–7.42 (7H, m, Hpara, H5, Hmeta), 6.91
oligonucleotides were 50-33P-labeled with 50-[ 33P]-ATP (Perkin El-
c
1
3
0
0
0
(1H, d, H-P, JH–P = 621.3 Hz), 6.48 (1H, pt, H1 , JH1 –H2 = 5.1 Hz),
mer) using T4 polynucleotide kinase (NEB) according to standard
0
0
0
00
5.61 (1H, m, H3 ), 4.43 (1H, m, H4 ), 4.21 (2H, m, H5 ,5 ), 3.09 (6H, q,
procedures. The labeled oligonucleotide was further purified using
Illustra Microspin G-25 columns (GE Healthcare).
3
TM
TM
0
CH2-TEAH
,
J = 7.5 Hz), 2.86–2.80 (1H, m, H2 ), 2.41–2.31 (1H, m,
H2 ), 1.33 (9H, CH3-TEAH
165.9 (COBz), 162.3 (C4), 155.1 (C2), 145.2 (C6), 133.4, 133.2, 133.1,
129.7, 129.3, 128.9, 128.5, 127.7 (CHBz), 97.0 (C5), 87.2 (C1 ), 84.8
00
,
3J = 7.5 Hz). 13C NMR: (CDCl3, 75 MHz) d
DNA polymerase reactions: End-labeled primer was annealed to
its template by combining primer P and template (T1 for incorpo-
ration and T2 for elongation) in a molar ratio of 1:2 and heating the
mixture to 70 °C for 10 min followed by slow cooling to room tem-
perature over a period of 1.5 h. For the incorporation of compounds
0
3
2
0
0
0
(C4 , JC–P = 7.7 Hz), 76.3 (C3 ), 63.4 (C5 , JC–P = 4.1 Hz), 45.5 (CH2-
TEAH), 39.2 (C2 ), 8.6 (CH3-TEAH). 31P NMR: (CDCl3, 121 MHz) d 4.3
0
(dt, JH–P = 621.3 Hz, JH5 ,5 –P = 6.0 Hz). ESI-: m/z 498 (M-TEAH)-.
HRMS: ESI- m/z calcd for (C23H21N3O8P)+: 498,1066, found:
498,1089.
1 and 2, a series of 20
lL-batch reactions was performed with the
1
3
0
00
enzyme HIV-1 RT (Ambion, 10 U/
lL stock solution). The final mix-
ture contained 125 nM primerꢀtemplate complex, RT buffer
(250 mM TrisꢀHCl, 250 mM KCl, 50 mM MgCl2, 2.5 mM spermidine,
5.1.10. Synthesis of O-(20-deoxycytidin-50-yl)-N-[(3-malon-2-
yl)propyl]-phosphoramidate, sodium salt (1)
50 mM dithiothreitol (DTT); pH 8.3), 0.03 U/lL HIV-1 RT, and dif-
ferent concentrations of phosphoramidate building blocks (1 mM,
500 M, 200 M and 100 M). In the control reaction with the nat-
ural nucleotide, a 10 M dCTP concentration was used. The mix-
5.1.10.1. Oxidation. The H-phosphonate monoester 4 (15 mg,
0.025 mmol, 1 equiv), was dried by azeotropic distillation with
anhydrous pyridine (2ꢂ) and vacuum dried under P2O5. It was then
dissolved in 0.1 mL of anhydrous pyridine and 0.15 mL of N,O-bis-
trimethylsilyl acetamide (BSA) and the solution was stirred 30 min
at room temperature under Argon. 0.25 mL of anhydrous CCl4
(50 equiv), 0.2 mL of anhydrous triethylamine (20 equiv) and
100 mg (0.3 mmol, 12 equiv) of 10, dissolved in 0.1 mL of anhydrous
pyridine, were simultaneously added. The reaction mixture was stir-
red at room temperature for 2 h, the solvents were removed under
vacuum, and the residue was coevaporated with acetonitrile.
l
l
l
l
ture was incubated at 37 °C and aliquots were quenched after 5,
10, 20, 30 and 60 min. For the elongation with 1, the same mixture
containing the appropriate P:T2 hybrid was incubated at 37°C and
aliquots were quenched after 30, 60, 90 and 120 min.
Electrophoresis: All polymerase reaction aliquots (2.5
lL) were
quenched by the addition of 10 L of loading buffer (90% formam-
l
ide, 0.05% bromophenol blue, 0.05% xylene cyanol and 50 mM eth-
ylenediaminetetraacetic acid (EDTA)). Samples were heated at
85 °C for 3 min prior to resolution by electrophoresis for 3 h at
2000 V on a 0.4 mm 20% denaturing gel in the presence of a
100 mM Tris-borate, 2.5 mM EDTA buffer; pH 8.3. Products were
visualized by phosphorimaging. The amount of radioactivity in
the bands corresponding to the products of enzymatic reactions
was determined by using the Cyclone imaging apparatus and its
Optiquant image analysis software (both Perkin Elmer).
5.1.10.2. Deprotection. The crude product was dissolved in 1.5 mL
of ACN and 4 mL of 0.4 M aqueous NaOH were added, then the mix-
ture was stirred for 10 h at room temperature. The mixture was
quenched by adding some DOWEX-50WX8 pyridinium resin, then
the resin was filtered off and well rinsed. The filtrates were evapo-
rated to dryness and coevaporated three times with ethanol. The res-
idue was dissolved in 5 mL of 28% ammonia solution in a
hermetically closed flask and was stirred for 5 h at room tempera-
ture. Solvent was removed under vacuum and the crude was then
dissolved in 10ꢁ3 M TEAB and purified by ion exchange DEAE-A25
Acknowledgments
This work was supported by grant from the French ‘Agence
Nationale de Recherche Contre le Sida’ (ANRS). I.Z. thanks the ‘Min-

7014
I. Zlatev et al. / Bioorg. Med. Chem. 17 (2009) 7008–7014
4. Adelfinskaya, O.; Herdewijn, P. Angew. Chem., Int. Ed. 2007, 46, 4356.
5. Adelfinskaya, O.; Terrazas, M.; Froeyen, M.; Marliere, P.; Nauwelaerts, K.;
Herdewijn, P. Nucleic Acids Res. 2007, 35, 5060.
istère National de la Recherche et de la Technologie’ for the award
of a research studentship. F.M. is from Inserm.
6. Terrazas, M.; Marliere, P.; Herdewijn, P. Chem. Biodiversity 2008, 5, 31–39.
7. Atherton, F. R.; Openshaw, H. T.; Todd, A. R. J. Chem. Soc. 1945, 660.
8. Nelson, P. S.; Kent, M.; Muthini, S. Nucleic Acids Res. 1992, 20, 6253.
9. Jankowska, J.; Sobkowski, M.; Stawinski, J.; Kraszewski, A. Tetrahedron Lett.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.001.
10. Bebenek, K.; Abbotts, J.; Wilson, S. H.; Kunkel, T. A. J. Biol. Chem. 1993, 268,
10324.
11. Giraut, A.; Song X.-P.; Dyubankova, N.; Herdewijn, P. Chembiochem 2009 early
view (DOI: 10.1002/cbic.200900185).
References and notes
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