Iminodipropionic Acid as the Leaving Group for DNA Polymerization by HIV-1 Reverse Transcriptase

Article abstract of DOI:10.1002/cbic.201100160

Previous studies have demonstrated that some selected amino monoacids and amino diacids can function as leaving groups in the polymerase-catalyzed incorporation of deoxynucleotides into DNA. Among these, the iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) represents an interesting example, as it could overcome some of the problems observed when using L-aspartic acid as the leaving group, that is, poor chain elongation. We have now synthesized and evaluated a series of IDA-dAMP analogues that bear either an extended aliphatic chain in the amino acid function, or a phosphonic acid moiety (substituting for the carboxylic acid function). Among these compounds, the nucleotide with an iminodipropionic acid leaving group (IDP-dAMP) was identified as the best substrate; the excellent single incorporation (91% conversion to a P+1 strand at 50 μM) was at a substrate concentration ten times lower than that used for IDA-dAMP). This nucleotide also presented improved kinetics and elongation capability compared to IDA-dAMP. The analogues with T, G, and C base moieties were also investigated for their incorporation ability with HIV-1 RT. The incorporation efficiency was found to decrease in the order A>T>G>C. The properties of the iminodipropionic acid as the leaving group surpass those of previously evaluated leaving groups; this acid will be a prime candidate for in vivo testing.

Full text of DOI:10.1002/cbic.201100160

DOI: 10.1002/cbic.201100160
Iminodipropionic Acid as the Leaving Group for DNA
Polymerization by HIV-1 Reverse Transcriptase
Xiao-Ping Song, Camille Bouillon, Eveline Lescrinier, and Piet Herdewijn*[a]
Previous studies have demonstrated that some selected amino
monoacids and amino diacids can function as leaving groups
in the polymerase-catalyzed incorporation of deoxynucleotides
into DNA. Among these, the iminodiacetic acid phosphorami-
date of deoxyadenosine monophosphate (IDA-dAMP) repre-
sents an interesting example, as it could overcome some of
the problems observed when using l-aspartic acid as the leav-
ing group, that is, poor chain elongation. We have now synthe-
sized and evaluated a series of IDA-dAMP analogues that bear
either an extended aliphatic chain in the amino acid function,
or a phosphonic acid moiety (substituting for the carboxylic
acid function). Among these compounds, the nucleotide with
an iminodipropionic acid leaving group (IDP-dAMP) was identi-
fied as the best substrate; the excellent single incorporation
(91% conversion to a P+1 strand at 50 mm) was at a substrate
concentration ten times lower than that used for IDA-dAMP).
This nucleotide also presented improved kinetics and elonga-
tion capability compared to IDA-dAMP. The analogues with T,
G, and C base moieties were also investigated for their incor-
poration ability with HIV-1 RT. The incorporation efficiency was
found to decrease in the order A>T>G>C. The properties of
the iminodipropionic acid as the leaving group surpass those
of previously evaluated leaving groups; this acid will be a
prime candidate for in vivo testing.
Introduction
During the polymerase-catalyzed synthesis of DNA, the 3’-hy-
droxyl group of the primer attacks the a-phosphate group of
the incoming nucleoside triphosphate, and the resulting pyro-
phosphate acts as the leaving group. We have investigated the
possibility of using alternative leaving groups for this reaction,
in order to establish if the pyrophosphate moiety of the tri-
phosphate nucleoside could be replaced by other functionali-
ties for the enzymatic synthesis of DNA. Such investigations
also allow a better understanding of the real role of the pyro-
phosphate moiety in the mechanism of the polymerization re-
action itself. We initially observed that some phosphoramidate
compounds (in which an amino acid moiety is linked to 2’-de-
oxyadenosine 5’-monophosphate through a PÀN bond) can be
employed as the substrate in enzyme-catalyzed nucleic acid
polymerization.[1–3] Among these phosphoramidate com-
pounds, l-Aspartic acid-dAMP showed the best results in a
single nucleotide incorporation experiment when using HIV-1
reverse transcriptase (RT) as the catalyst (90% conversion to a
P+1 strand in 60 min at 500 mm substrate concentration). How-
ever, stalling occurs after the addition of two nucleobase resi-
dues in a chain elongation experiment. The reason for this
stalling is not clear. We have demonstrated that a phosphoest-
er bond provides better leaving group properties than a phos-
phoramidate bond, but the stalling issue remains.[4] It was also
shown that the chemistry of the leaving group need not be
limited to that of an a-amino acid.[5] Iminodiacetic acid dAMP
(IDA-dAMP; compound 1, Scheme 1) has shown incorporation
ability similar to that of l-Asp-dAMP in a single-nucleotide in-
corporation assay, but with enhanced elongation capacity and
improved kinetics.[5]
Scheme 1. Structure of phosphoramidate compounds 1–7.
In order to broaden the chemical space and to obtain fur-
ther insight in the structure–activity relationship, we synthe-
sized and evaluated a series of IDA-dAMP analogues (2–7,
Scheme 1) as potential substrates for HIV-1 RT. In these com-
pounds either the carboxylic acid function was replaced by a
phosphonate group, or the alkyl chain in the leaving group
was extended. It has already been demonstrated that the
introduction of a phosphonate group could have a beneficial
[a] X.-P. Song, Dr. C. Bouillon, Prof. Dr. E. Lescrinier, Prof. Dr. P. Herdewijn
Rega Institute, Laboratory of Medicinal Chemistry
Katholieke Universiteit Leuven
Minderbroederstraat 10, 3000 Leuven (Belgium)
E-mail: piet.herdewijn@rega.kuleuven.be
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DNA Polymerization by HIV-1 Reverse Transcriptase
effect on the substrate properties of the nucleotide,[6] and that
longer alkyl groups are acceptable as leaving groups.[7]
The ability to incorporate 2–7 into a growing DNA chain cat-
alyzed by HIV-1 RT was evaluated by using a primer-template
assay. Among these compounds, iminodipropionic acid-dAMP
(IDP-dAMP) 2 showed better properties than those of IDA-
dAMP, with 91% conversion to a P+1 strand in 60 min at
50 mm concentration (a substrate concentration ten times
lower than that observed for IDA-dAMP) in the single incorpo-
ration assay. Full elongation was observed in the chain elonga-
tion experiment, and less stalling occurred than in the case of
IDA-dAMP, thus making this nucleotide analogue a prime can-
didate for evaluation in a cellular system.
Results and Discussion
Scheme 3. Synthesis of phosphoramidate analogue 4. Reagents and condi-
tions: A) diethyl phosphite, 37% formaldehyde, 0–1008C; B) 10% Pd/C, am-
monium formate, ethanol/H2O, 1008C; C) dAMP, DCC, 1,4-dioxane/DMF,
808C; D) TMSI, Et3N, CH2Cl2, 08C.
Synthesis of phosphoramidates
The synthesis of 2–7 is shown in Schemes 2–6. First, the syn-
thesis of 2 and 3 was carried out according to the procedure
described by Wagner and colleagues,[8] by starting from com-
mercially available deoxyadenosine monophosphate (dAMP)
and an amino acid diester, with dicyclohexylcarbodiimide
(DCC) as the coupling reagent. Deprotection was accomplished
with 0.4m sodium hydroxide in a methanol-water solution
(Scheme 2).
this aza-Michael addition, we used a 2:1 ratio of vinylphospho-
nate/amine, and water as the solvent. After hydrogenolysis of
the benzyl group, 14 was isolated in 90% yield, and succes-
sively coupled with dAMP affording 15. Deprotection of the
phosphonate ester functions with TMSI at 08C provided 5
(Scheme 4).
Intermediate 10 was prepared by starting from commercially
available reagents, according to the method described by
Aime et al.[9] Compound 11 was obtained after the removal of
the benzyl protecting group and the resulting secondary
amine was coupled with dAMP in the presence of DCC to pro-
vide 12. Finally the biphosphonate 4 was isolated in good
yield after deprotection with iodotrimethylsilane (TMSI) at 08C
(Scheme 3).
Condensation of 1,3,5-tribenzylhexahydro-1,3,5-triazine with
three equivalents of diethyl phosphite at 1008C for 3 h afford-
ed 16 in good yield.[10] N-alkylation of this compound with
ethyl bromoacetate in the presence of diisopropylethylamine
(DIEA) gave 17, and this was deprotected by hydrogenation to
afford the secondary amine 18. Compound 18 was then cou-
pled with dAMP in the presence of DCC to obtain 19. Depro-
tection of 19 was carried out in two steps: first, hydrolysis of
Compound 5 was prepared by a four-step synthesis, by
starting from benzylamine and diethylvinylphosphonate. For
Scheme 2. Synthesis of phosphoramidate analogues 2 and 3. Reagents and
conditions: A) DCC, 1,4-dioxane/DMF, 808C; B) 0.4m NaOH (MeOH/H2O),
room temperature.
Scheme 4. Synthesis of phosphoramidate analogue 5. Reagents and condi-
tions: A) H2O, reflux; B) 10% Pd/C, ammonium formate, ethanol/H2O, 1008C;
C) dAMP, DCC, 1,4-dioxane/DMF, 808C; D) TMSI, Et3N, CH2Cl2, 08C.
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P. Herdewijn et al.
the phosphonate ester with TMSI at 08C, then removal of the
carboxylic ester function with sodium hydroxide in a metha-
nol-water solution to give 6 (Scheme 5).
Scheme 6. Synthesis of phosphoramidate analogue 7. Reagents and condi-
tions: A) methyl acrylate, MeOH, room temperature; B) diethyl vinylphosph-
onate, H2O, room temperature; C) H2, 10% Pd/C, MeOH; room temperature;
D) dAMP, DCC, tBuOH/H2O, 808C; E) TMSBr, Et3N, CH2Cl2, 08C; F) 0.4m NaOH
(MeOH/H2O), room temperature.
Scheme 5. Synthesis of phosphoramidate analogue 6. Reagents and condi-
tions: A) diethyl phosphonate, 1008C; B) ethylbromoacetate, DiEA, anhy-
drous CH3CN, 25–1008C; C) 10% Pd/C, ammonium formate, ethanol/H2O,
1008C; D) dAMP, DCC, 1,4-dioxane/DMF, 808C; E) TMSI, Et3N, CH2Cl2, 08C;
F) 0.4m NaOH (MeOH/H2O), room temperature.
Table 1. Overview of primer–template complexes used in the DNA poly-
merase reactions.[a]
Compound 22 was obtained after two Michael additions by
starting from benzylamine in the presence of methylacrylate
and diethyl vinyl phosphonate. After hydrogenation of the
benzyl group, the secondary amine 23 was coupled with
dAMP to provide 24. Finally, deprotection of the carboxylic
acid and phosphonate functions gave phosphoramidate 7
(Scheme 6).
Primer P1
primer P2
5’-CAGGAAACAGCTATGAC-3’
5’-CAGGAAACAGCTATGACTGA-3’
3’-GTCCTTTGTCGATACTGTCCC-5’
3’-GTCCTTTGTCGATACTGTTTTTTTGGAC-5’
3’-GTCCTTTGTCGATACTGACTGC-5’
3’-GTCCTTTGTCGATACTGCTTTT-5’
3’-GTCCTTTGTCGATACTGACTGAAAAA-5’
template T1
template T2
template T3
template T4
template T5
[a] Boldface letters indicate template overhang in the hybridized primer–
template duplex.
Single nucleotide incorporation experiment
Human immunodeficiency virus type-1 (HIV-1) is a pathogen
responsible for the acquired immunodeficiency syndrome; it
encodes a polymerase that is critical for viral replication. This
HIV-1 reverse transcriptase is an important target for anti-HIV
therapies.[11] It is an error-prone polymerase with a high muta-
tion rate; therefore it exhibits flexibility and high tolerance to-
wards chemically modified nucleotide substrates.[12,13] The abili-
ty of HIV-1 reverse transcriptase to incorporate phosphorami-
date analogues 2–7 into a growing DNA strand was analyzed
by a gel-based single-nucleotide incorporation assay[14,15] by
using primer-template complex P1T1 (Table 1) and the natural
nucleotide triphosphate (dATP) as reference compound.
the primer to a P+1 strand, with 90% yield in 60 min (Fig-
ure 1B); a concentration ten times lower than that needed for
IDA-dAMP. Efficient incorporation was still detected when the
substrate concentration was decreased to 5 mm (data not
shown) and 10 mm (18% and 33% incorporation, respectively).
In contrast, the corresponding analogue 3 was less well recog-
nized under the same conditions, and only 63% conversion
was observed at 500 mm (Figure 1C).
A phosphonic acid group has an additional ionizable func-
tion when compared to a carboxylic acid, and thus its presence
might be beneficial as electrostatic interactions are important
during the polymerization process, as recently observed in a
study of phosphono alanine as a leaving group.[6] Analogues 4,
5, 6, and 7 (related to IDA-dAMP and IDP-dAMP) possess either
one or two phosphonic acid groups substituted for the carbox-
Among 2–7, iminodipropionic acid-dAMP (IDP-dAMP, 2)
showed the best single incorporation result. Maximal primer
extension was obtained with a substrate concentration above
100 mm (Figure 1A). However, at 50 mm, 2 was able to convert
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DNA Polymerization by HIV-1 Reverse Transcriptase
Figure 3. Incorporation efficiency (% elongated primer) of compound 6 by
HIV-1 RT (0.025 UmLÀ1), measured after 10 ( ) and 120 ( ) min.
~
*
at 100 mm after 10 min incubation (Figure 4A and B). This
means that 6 is a potential inhibitor at high concentration
while it is a substrate at low concentration. The corresponding
Figure 1. Single incorporation of phosphoramidate analogues into P1T1
(125 nm) by HIV-1 RT (0.025 UmLÀ1). Time points: 10, 20, 30, 60, and
120 min. A) and B) Incorporation of compound 2; C) Incorporation of com-
pound 3.
ylic acid functions. When tested under the same conditions, 4
and 5 were incorporated with 90% and 83% conversion to the
P+1 strand in 60 min at 500 mm, respectively (Figure 2A and
Figure 4. Single incorporation of phosphoramidate analogues into P1T1
(125 nm) by HIV-1 reverse transcriptase (HIV-1 RT; 0.025 UmLÀ1). Time points:
10, 20, 30, 60, and 120 min. A) and B) Incorporation of compound 6; C) in-
corporation of compound 7.
Figure 2. Single incorporation of phosphoramidate analogues into P1T1
(125 nm) by HIV-1 reverse transcriptase (0.025 UmLÀ1). Time points: 10, 20,
30, 60, and 120 min. A) Incorporation of compound 4; B) Incorporation of
compound 5.
analogue 7 was less efficiently recognized, with only 14% in-
corporation at 500 mm, and 26% at 1 mm (Figure 4C). In sum-
mary, 3, 4, 5, 6 and 7 all showed inferior incorporation ability
when compared to 2.
B); these are inferior to the result obtained for IDP-dAMP. The
incorporation results for 1–5 suggest that small changes in the
chemical structures of the leaving group might lead to signifi-
cant differences in biochemical properties. Based on the above
and previously published results,[6] it can be deduced that the
beneficial effect of a phosphonate group is highly dependent
on its position within the leaving group moiety.
In an attempt to assemble more information about the
structure of the nucleotide mimic that can be accepted as sub-
strate by HIV-1 RT, two additional nucleotide phosphorami-
dates, 32 and 34 (containing a primary amine in the leaving
group), were synthesized and evaluated (Scheme 7). Com-
pound 32 was prepared according to a four-step synthesis by
starting from benzylamine and dimethylgluconate. After Mi-
chael addition, the benzyl group was removed by hydrogenol-
ysis to afford 28, which was then coupled with dAMP to pro-
vide 30. Deprotection of the carboxylic ester functions gave
32. Compound 34 was prepared by using the same procedure,
followed by deprotection of the phosphonate function.
Incorporation experiments demonstrated that there is a
large difference between compounds that bear a primary
Compound 6, which bears a carboxylic acid and a phospho-
nate group as the leaving group moiety, was also accepted as
substrate by HIV-1 RT. It is interesting to note that in this case
decreased incorporation efficiency was observed with sub-
strate concentrations above 100 mm. At the highest concentra-
tion (1 mm) only 16% incorporation was obtained (Figure 3).
At lower concentrations, 6 showed very good incorporation
ability, with 24% conversion at 10 mm, 72% at 50 mm, and 82%
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P. Herdewijn et al.
Figure 5. Elongation into P1T2 (125 nm) by HIV-1 reverse transcriptase
(0.025 UmLÀ1). Time points: 15, 30, 60, 90, and 120 min.
conditions)[5] and to 3-phosphono-l-Ala-dAMP (16% P+7 prod-
uct at 500 mm, 24% P+7 at 1 mm).[6]
Compounds 3, 4, 5 and 6 showed less efficient chain elonga-
tion properties than 2 (Table 2), while stalling occurred after
primer extensions with two or three nucleobases. Likewise,
Table 2. Elongation results for compounds 2, 3, 4, 5, and 6. Conditions:
primer P1, template T2, HIV-1 RT (0.025 UmLÀ1).
Product
2[a]
28.6
5.1
7.9
traces
21.5
30.2
3.5
3[a]
4[a]
5[a]
6[a]
6.8
2.5
3.2
2.2
14.9
42
Scheme 7. Synthesis of phosphoramidate analogues 32 and 34. Reagents
and conditions: A) MeOH, room temperature; B) H2, 10% Pd/C, MeOH (for
28) or EtOH (for 29), room temperature; C) dAMP, DCC, tBuOH/H2O, 808C;
D) 0.4m NaOH (MeOH/H2O), room temperature; E) TMSBr, Et3N, CH2Cl2, 08C.
P+7
P+6
P+5
P+4
P+3
P+2
P+1
0
0
0
0
0
0
0
0
0
traces
9.4
53.7
9.6
traces
10.8
53.1
8.6
traces
4.9
35.4
15.2
amine and those that bear a secondary amine group, even
when these possess the same acidic functions. Compound 32
shows very poor incorporation (only 16% incorporation at
1 mm), and 34 was totally unreactive (as a diastereoisomer
mixture) under the same incorporation conditions (data not
shown).
7.7
[a] Percentage of total radio-emitting oligonucleotides in the mixture.
with 6 complete chain extension to P+7 product was obtained
but only in limited amount; the elongation reaction mainly
stopped at the P+1 strand.
Elongation experiments
Kinetic experiments
In order to investigate the strand elongation capability of
these phosphoramidates (2–6), a template-dependent incorpo-
ration assay of more than one nucleotide was carried out. Pre-
viously we observed that templates with long overhangs
might benefit the chain elongation result, as this increases the
affinity of the primer–template duplex for the enzyme.[5] In this
experiment a template with seven thymidines and flanked by
four non-thymidine nucleotides at the 3’-end was used. HIV-1
RT was used as the polymerase. A range of concentrations was
tested, reactions were quenched after 15, 30, 60, 90, and
120 min, and the samples were analyzed by 20% polyacryl-
amide gel electrophoresis.
The kinetic parameters for the incorporation of modified sub-
strates (IDA-dAMP and IDP-dAMP) and the natural substrate
(dATP) were investigated in parallel, on the basis of the Single
Completed Hit model.[15,16] IDA-dAMP, IDP-dAMP, and dATP
were processed by HIV-1 RT, and displayed a Michaelis–Menten
kinetic profile, when P1 and T1 were used as primer and tem-
plate. The steady-state kinetic values, KM and Vmax, and the de-
rived value Vmax/KM are given in Table 3.
As can be seen from Table 3, the Vmax value for IDP-dAMP is
similar to that for the natural substrate, thus suggesting that
the IDP-dAMP is accepted as efficiently as dATP. However, an
increase in KM for the modified substrate was observed, com-
pared to dATP. This implies that the binding affinity of the sub-
strate to the enzyme is reduced when the iminodipropionic
acid substitutes the pyrophosphate (PPi) leaving group. There-
fore, the catalytic efficiency of IDP-dAMP towards HIV-1 RT was
reduced 83-fold compared to dAMP. In comparison, the ratio
of Vmax/KM for IDA-dAMP was 940-fold lower than the natural
Full-length elongation was observed for IDP-dAMP
(Figure 5), with 29% P+7 product obtained at 500 mm sub-
strate concentration, and 100% P+7 at 1 mm, over 2 h. Al-
though during the elongation process of IDP-dAMP a stalling
effect was still present at P+2 and P+3, improvements were
observed when compared both to IDA-dAMP (where a non-
negligible amount of primer was still present under the same
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DNA Polymerization by HIV-1 Reverse Transcriptase
Table 3. Steady-state kinetics for the incorporation of phosphoramidates
IDP-dAMP, IDA-dAMP and dATP by HIV-1 RT.
Kinetics of
IDP-dAMP and dATP[a]
Kinetics of
IDA-dAMP and dATP[b]
dATP
IDP-dAMP
dATP
IDA-dAMP
Vmax [nmminÀ1
KM [mm]
]
12.37Æ0.45 11.78Æ0.48 24.53Æ1.10
13.44Æ1.36
0.93Æ0.13 73.66Æ9.87
0.44Æ0.09 226.1Æ61.48
Vmax/KM [minÀ1
]
13.30ꢁ10À3
0.16ꢁ10À3 55.75ꢁ10À3
0.06ꢁ10À3
[a] 0.0125 UmLÀ1 HIV-1 RT, [b] 0.025 UmLÀ1 HIV-1 RT.
substrate, therefore IDP-dAMP also showed enhanced kinetics
over IDA-dAMP.
Control experiments
Scheme 8. Structures of the base counterparts of IDA-dAMP and IDP-dAMP.
To confirm Watson–Crick base pair fidelity for incorporation of
2, we carried out control experiments with a mismatched se-
quence (A against A, G, and C). No misincorporation was ob-
served (Figure 6) at 500 mm for 2.
Furthermore, we compared the incorporation efficiency of
the four different bases when using iminodiacetic acid and imi-
nodipropionic acid as leaving groups. Compounds 35–40 (the
base counterparts of IDA-dAMP and IDP-dAMP) were synthe-
sized using previously described procedures (Scheme 8).[8]
A
series of gel-based single nucleotide incorporation assays was
carried out, by using primer P1 and template T3 for thymine, P1
and T4 for guanine, and P2 and T5 for cytosine (Table 1).
All of these six compounds (35–40) were recognized by HIV-
1 RT and incorporated into the corresponding DNA strand, but
the incorporation efficiency of the T, G and C nucleotides was
less efficient than that of the related IDA-dAMP and IDP-dAMP.
We observed 89% conversion to the P+1 strand for IDP-dTMP
(Figure 7A), 85% conversion for IDP-dGMP (Figure 7B), and
35% conversion for IDP-dCMP (Figure 7C) at 1 mm substrate
concentration. (IDP-dAMP showed 91% conversion at 50 mm in
60 min.) In the case of IDA-dNMP phosphoramidate analogues
(N=A, T, G, or C), the incorporation efficiency was lower than
that of IDA-dAMP, with 35% (IDA-dTMP), 27% (IDA-dGMP), and
25% (IDA-dCMP) conversion to the P+1 strand at 1 mm, re-
spectively (data not shown). The above results show that when
using either iminodiacetic acid or iminodipropionic acid as
leaving group, the incorporation efficiency decrease in the
order A>T>G>C. The reasons for this base-specific phenom-
enon are not clear. However, when looking at the results of
Figure 7. Single incorporation with IDP-dNMP (N=T, G, C) by HIV-1 RT. Re-
action conditions: primer/template (P1/T3, P1/T4, P2/T5:125 nm), HIV-1 RT
(0.025 UmLÀ1). Time points: 10, 20, 30, 60, and 120 min. A) Incorporation of
38 with P1/T3; B) Incorporation of 39 with P1/T4; C) Incorporation of 40 with
P2/T5.
IDA-dNMP and IDP-dNMP, iminodipropionic acid clearly ap-
pears as a better leaving group than iminodiacetic acid.
Conclusions
In conclusion, a series of analogues of IDA-dAMP have been
synthesized and evaluated as substrates for HIV-1 RT. IDP-
dAMP is clearly a better substrate than IDA-dAMP in poly-
merase-catalyzed DNA synthesis
when using HIV-1 RT as the
enzyme, with excellent single
incorporation and improved
elongation ability. Kinetic pa-
rameters showed that the Vmax
/
KM of IDP-dAMP is 83 times
lower than that for the natural
substrate. Structure–activity re-
Figure 6. Control experiments. Reaction conditions: primer/template (P1/T3, P1/T4, P2/T5:125 nm), HIV-1 RT
(0.025 UmLÀ1), IDP-dAMP (500 mm). Time points: 10, 20, 30, 60, and 120 min. A) IDP-dAMP with P1/T3; B) IDP-dAMP
with P1/T4; C) IDP-dAMP with P2/T5.
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P. Herdewijn et al.
2’-Deoxyadenosine-5’-(iminodipropionic acid)phosphoramidate
(2), (General procedure II): A solution of 8 (80 mg, 0.16 mmol) in
MeOH/H2O (4:1 v/v, 3 mL, containing NaOH (0.4m)) was stirred at
room temperature under nitrogen for 2 h, and the solvent was re-
moved under reduced pressure. The resulting crude material was
purified by chromatography (gradient: CHCl3/CH3OH/H2O 5:1:0,
5:2:0.25, v/v/v; 5:3:0.5, v/v/v; and 5:4:1, v/v/v), to provide 2 as
white solid (64 mg, yield 85%). 1H NMR (300 MHz, D2O): d=8.37
(s, 1H; H8), 8.15 (s, 1H; H2), 6.41 (t, J=6.6 Hz, 1H; H1’), 4.78–4.70
(m, 1H; H3’), 4.19–4.17 (m, 1H; H4’), 3.87–3.84 (m, 2H; H5’), 3.13–
3.02 (m, 4H; CH2CH2CO2H), 2.81–2.72 (m, 1H; H2’a), 2.57–2.49 (m,
1H; H2’b), 2.30–2.25 (m, 4H; CH2CH2CO2H); 13C NMR (75 MHz, D2O):
d=180.7, 155.2, 152.3, 148.4, 139.6, 118.4, 85.7, 83.4, 70.8, 63.5,
43.1, 37.1, 32.1; 31P NMR (121 MHz, D2O): d=9.09; HRMS (ESI) m/z
calcd for C16H22N6O9PÀ: 473.1191, found: 473.1202.
lationship studies for 1–7, 32, and 34 further support the hy-
pothesis that recognition and incorporation of the incoming
nucleotide is very specific and controlled by structural and
electrostatic features.[1] Investigation of the influence of the
base moiety of IDA-dNMP and IDP-dNMP on the inhibition
properties revealed that the incorporation efficiency is base-
dependent following the order A>T>G>C. In control experi-
ments, the incorporation of IDP-dAMP proved to follow
Watson–Crick rules for incorporation of adenosine nucleotide.
In summary, the iminodipropionic acid moiety has been identi-
fied as the best leaving group when using HIV-1 RT as a poly-
merase, and its potential as a constituent of an antiviral nu-
cleoside triphosphate mimic will be evaluated.
O-(2’-Deoxyadenosin-5’-yl)-N-(methoxycarbonylethyl)-N’-(meth-
oxycarbonylmethyl)phosphoramidate (9): General procedure I
was applied with dAMP (100 mg, 0.30 mmol), methyl 3-(N-methox-
ycarbonylmethyl)amino propanoate (286 mg, 1.50 mmol), and DCC
(436 mg, 2.11 mmol). Purification was carried out according to the
method used for 8. Compound 9 was obtained as white solid
Experimental Section
General: For all reactions, chemicals of analytical or synthetic
grade were obtained from commercial sources and were used
without purification. Technical solvents were obtained from Brenn-
tag (Deerlijk, Belgium). All DNA oligonucleotides were purchased
from Sigma Genosys. The primer oligonucleotides were 5’-labeled
with [g-33P]ATP (PerkinElmer) by using T4 polynucleotide kinase
(New England Biolabs) according to standard procedures. Labeled
oligonucleotides were further purified on Illustra Microspin G-25
columns (GE Healthcare). Flash silica chromatography was per-
formed on Davisil silica gel 60 (0.040–0.063 mm; Grace Davison,
Lokeren, Belgium). Analytical thin layer chromatography was per-
formed on Alugram silica gel UV254 mesh 60 (0.20 mm; Macher-
ey–Nagel). NMR Spectra were recorded on a Bruker Avance II NMR
spectrometer (300 MHz or 500 MHz). Chemical shifts are ex-
1
(103 mg, yield 68%). H NMR (500 MHz, D2O): d=8.36 (s, 1H; H8),
8.15 (s, 1H; H2), 6.39 (t, 1H; J=6.5 Hz, H1’), 4.66–4.64(m, 1H; H3’),
4.21–4.16 (m, 1H; H4’), 3.88–3.86 (m, 2H; H5’), 3.60 (d, 2H; J=
11.0 Hz, CH2CO2Me), 3.51 (s, 3H; CH2CO2CH3), 3.45 (s, 3H;
CH2CH2CO2CH3), 3.11–3.01 (m, 2H; CH2aCH2CO2Me), 2.97–2.77 (m,
1H; H2’a), 2.56–2.49 (m, 1H; H2’b), 2.37–2.23 (m, 2H;
CH2bCH2CO2Me); 13C NMR (125 MHz, D2O): d=174.8, 174.2, 155.0,
152.2, 148.5, 139.7, 118.3, 85.8, 83.3, 71.0, 63.8, 51.9, 51.7, 48.8,
43.3, 38.4, 33.3; 31P NMR (121 MHz, D2O): d=7.58; HRMS (ESI) m/z
calcd for C17H24N6O9PÀ: 487.1348, found: 487.1324.
1
pressed as d units (ppm) downfield from tetramethyl silane for H
O-(2’-Deoxyadenosin-5’-yl)-N-(carboxyethyl)-N’-(carboxymethyl)-
phosphoramidate (3): General procedure II was applied by using 9
(80 mg, 0.16 mmol) in MeOH/H2O (4:1 v/v, 3 mL, containing NaOH
(0.4m)). Purification was carried out according to the method used
for 2. Compound 3 was obtained as white solid (54 mg, yield
and 13C. 31P NMR chemical shifts are referenced to an external 85%
H3PO4 standard (d=0.00 ppm). Exact mass spectra were obtained
with a quadrupole/orthogonal-acceleration time-of-flight tandem
mass spectrometer (Micromass Q-TOF 2; Waters, Milford, MA)
equipped with a standard electrospray ionization (ESI) source. Pu-
rification by HPLC was performed on a reverse phase C18 column
by gradient elution (acetontitrile and 50 mm triethylammonium bi-
carbonate (TEAB) buffer).
1
72%). H NMR (300 MHz, D2O): d=8.38 (s, 1H; H8), 8.12 (s, 1H; H2),
6.39 (t, 1H; J=6.6 Hz, H1’), 4.64–4.63 (m, 1H; H3’), 4.18–4.17 (m, 1H;
H4’), 3.90–3.92 (m, 2H; H5’), 3.49–3.46 (m, 2H; CH2CO2H), 3.17–3.15
(m, 2H; CH2CH2CO2H), 2.79–2.71 (m, 1H; H2’a), 2.55–2.50 (m, 1H;
H2’b), 2.30–2.26 (m, 2H; CH2CH2CO2H); 13C NMR (75 MHz, D2O): d=
174.8, 174.2, 155.0, 152.2, 148.4, 139.7, 118.3, 85.8, 83.3, 71.0, 63.7,
49.8, 44.6, 38.7, 32.2; 31P NMR (121 MHz, D2O): d=8.88; HRMS (ESI)
m/z calcd for C15H20N6O9PÀ: 459.1035, found: 459.1043.
O-(2’-Deoxyadenosin-5’-yl)-N,N’-[di(methoxycarbonylethyl)]-
phosphoramidate (8), (General procedure I): In a two-neck flask,
2’-deoxyadenosine-5’-monophosphoric acid (dAMP, 100 mg,
0.30 mmol) and dimethyl N,N’-iminodipropanoate (284 mg,
1.50 mmol) were suspended in a mixture of 1,4-dioxane (8 mL) and
N,N’-dimethylformamide (DMF, 2 mL). One drop of triethylamine
(Et3N) was added to the solution to facilitate dissolution. Then, a
solution of DCC (433 mg, 2.10 mmol) in 1,4-dioxane (2 mL) was
added, and the reaction mixture was heated at 858C for 3 h under
argon. After completion, the reaction mixture was cooled, and the
solvent was removed under reduced pressure. The resulting crude
material was purified by chromatography on silica gel (gradient:
CHCl3/CH3OH/H2O 5:1:0, v/v/v; 5:2:0.25, v/v/v; 5:3:0.25, v/v/v;
5:3:0.5, v/v/v) to provide 8 as white solid (103 mg, yield 68%).
1H NMR (300 MHz, D2O): d=8.34 (s, 1H; H8), 8.14 (s, 1H; H2), 6.38
(t, J=6.4 Hz, 1H; H1’), 4.71–4.67 (m, 1H; H3’), 4.20–4.17 (m, 1H; H4’),
3.87–3.82 (m, 2H; H5’), 3.50 (s, 6H; OCH3), 3.06–2.97 (m, 4H;
NCH2CH2), 2.85–2.78 (m, 1H; H2’a), 2.60–2.52 (m, 1H; H2’b), 2.35–2.28
(m, 4H; CH2CH2); 13C NMR (125 MHz, D2O) 174.9, 155.1, 152.3,
148.4, 139.6, 118.3, 85.7, 83.3, 70.8, 63.5, 51.7, 41.9, 38.4, 33.4;
31P NMR (121 MHz, D2O): d=8.36; HRMS (ESI) m/z calcd for
C18H26N6O9PÀ: 501.1504, found: 501.1505.
Tetraethyl N,N’-(benzylamino)di(methyl phosphonate) (10): In a
50 mL round-bottomed flask benzylamine (980 mg, 9.33 mmol)
and diethyl phosphate (2.57 g, 18.70 mmol) were mixed and
cooled to 0–58C. 37% Aqueous formaldehyde (2.10 mL,
28.0 mmol) was added at a rate to maintain the reaction tempera-
ture under 108C (over ~15 min). The resulting emulsion was stirred
at room temperature for 30 min, then heated to 1008C for 1 h. Re-
moval of the water and excess formaldehyde in vacuo gave 10 as
1
a colorless oily compound (3.27 g, yield 86%). H NMR (300 MHz,
CDCl3): d=7.39–7.24 (m, 5H; Ph), 4.13–4.08 (m, 8H; CH2CH3), 3.96
(s, 2H; CH2Ph), 3.14 (d, J=9.1 Hz, 4H; NCH2), 1.30 (t, J=7.1 Hz,
12H; CH2CH3); 13C NMR (75 MHz, CDCl3): d=137.7, 128.9, 127.9,
127.1, 61.5, 60.6, 49.2, 16.1; 31P NMR (121 MHz, CDCl3): d=24.74;
HRMS (ESI) m/z calcd for C16H27NO5P+: 344.1621, found: 344.1630.
Tetraethyl iminodi(methyl phosphonate) (11): In a 50 mL flask 10
(3.20 g, 7.86 mmol) was dissolved in ethanol (20 mL). Ammonium
formate (2.47 g, 39.30 mmol) was dissolved in distilled water (3 mL)
1874
www.chembiochem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1868 – 1880

DNA Polymerization by HIV-1 Reverse Transcriptase
and added to the mixture, followed by 10% Pd/C (0.5 g). The mix-
ture was stirred to reflux for 1 h, then ammonium formate (2.47 g,
39.30 mmol) in water (3 mL) was again added, and reflux was
maintained for a further 12 h. The reaction mixture was then fil-
tered on Celite, and concentrated under reduced pressure. The res-
idue was diluted with CH2Cl2 (10 mL), washed with 10% Na2CO3 so-
lution (2ꢁ10 mL), the organic layer was dried over anhydrous
Na2SO4, and concentrated. Compound 11 was obtained as colorless
(2 mL) and added to the mixture, followed by 10% Pd/C (57 mg).
The mixture was stirred to reflux for 1 h, then ammonium formate
(217 mg, 3.45 mmol in 2 mL water) was again added, and reflux
maintained for a further 2 h. The reaction mixture was then filtered
on Celite and concentrated under reduced pressure. The residue
was diluted with CH2Cl2 (10 mL), washed with 10% Na2CO3 (2ꢁ
10 mL), dried over anhydrous Na2SO4, filtered, and concentrated.
Compound 14 was obtained as colorless oil (214 mg, yield 90%).
1H NMR (300 MHz, CDCl3): d=4.10–4.03 (m, 8H; CH2CH3), 2.90–2.81
(m, 4H; NCH2), 1.98–1.87 (m, 4H; NCH2CH2), 1.29 (t, 12H; J=7.1 Hz,
CH2CH3); 13C NMR (75 MHz, CDCl3): d=61.3, 42.6, 27.0, 16.1;
31P NMR (121 MHz, CDCl3): d=30.22; HRMS (ESI) m/z calcd for
C12H30NO6P2+: 346.1543, found: 346.1559.
1
oil (2.37 g, yield 95%). H NMR (300 MHz, CDCl3): d=4.18–4.11 (m,
8H; CH2CH3), 3.13 (d, J=10.8 Hz, 4H; NCH2), 1.34 (t, J=7.0 Hz, 12H;
CH2CH3); 13C NMR (75 MHz, CDCl3): d=61.8, 46.7, 16.1; 31P NMR
(121 MHz, CDCl3): d=24.89; HRMS (ESI) m/z calcd for
C10H26NO6P2+: 318.1229, found: 318.1220.
O-(2’-Deoxyadenosin-5’-yl)-N,N’-[di((diethoxyphosphoryl)meth-
yl]-phosphoramidate (12): General procedure I was applied by
using dAMP (100 mg, 0.30 mmol), 11 (666 mg, 2.10 mmol), and
DCC (436 mg, 2.11 mmol). Purification was carried out according to
the method used for 8, and 12 was obtained as white solid
O-(2’-Deoxyadenosin-5’-yl)-N,N’-[di((diethoxyphosphoryl)ethyl)]-
phosphoramidate (15): General procedure I was applied with
dAMP (100 mg, 0.30 mmol), 14 (725 mg, 2.10 mmol), and DCC
(436 mg, 2.11 mmol). Purification was carried out according to the
method used for 8, and 15 was obtained as white solid (105 mg,
yield 53%). 1H NMR (500 MHz, D2O): d=8.42 (s, 1H; H8), 8.23 (s,
1H; H2), 6.46 (t, J=6.5 Hz, 1H; H1’), 4.73–4.70 (m, 1H; H3’), 4.23–
4.17 (m, 1H; H4’), 4.05–4.02 (m, 8H; OCH2CH3’), 3.99–3.96 (m, 2H;
H5’), 3.17–3.10 (m, 4H; NCH2CH2), 2.88–2.83 (m, 1H; H2’a), 2.65–2.62
(m, 1H; H2’b), 2.02–1.92 (m, 4H; NCH2CH2), 1.27–1.23 (m, 12H;
CH2CH3); 13C NMR (125 MHz, D2O): d=155.1, 152.2, 148.2, 139.6,
118.3, 85.5, 83.4, 70.5, 63.4, 62.8, 39.5, 24.2, 23.4, 15.2; 31P NMR
(121 MHz, D2O): d=32.61, 7.64; HRMS (ESI) m/z calcd for
C20H40N6O11P3À: 657.1973, found: 657.1978.
1
(138 mg, yield 73%). H NMR (500 MHz, D2O): d=8.45 (s, 1H; H8),
8.24 (s, 1H; H2), 6.48 (t, J=6.5 Hz, 1H; H1’), 4.74–4.72 (m, 1H; H3’),
4.31–4.25 (m, 1H; H4’), 4.12–4.06 (m, 8H; CH2CH3’), 3.98–3.96 (m,
2H; H5’), 3.63–3.61 (m, 4H; NCH2), 2.94–2.88 (m, CH2CH3), 2.94–2.88
(1H; H2’a), 2.65–2.60 (m, 1H; H2’b), 1.29–1.23 (m, 12H; OCH2CH3);
13C NMR (125 MHz, D2O): d=155.1, 152.2, 148.5, 139.6, 118.3, 85.7,
83.4, 71.2, 63.9, 63.2, 41.8, 38.2, 15.2; 31P NMR (121 MHz, D2O): d=
26.98, 5.60; HRMS (ESI) m/z calcd for C20H36N6O11P3À: 629.1660,
found: 629.1663.
O-(2’-Deoxyadenosin-5’-yl)-N,N’-[di(phosphonomethyl)]-phos-
phoramidate (4), (General procedure III): Iodotrimethylsilane
(348 mL, 2.53 mmol) was added to a solution of 12 (100 mg,
0.16 mmol), Et3N (444 mL, 3.16 mmol), and dry CH2Cl2 (10 mL) at
08C under argon. The reaction mixture was stirred at room temper-
ature for 6 h, quenched with TEAB (1m), and then the mixture was
concentrated in vacuo. The resulting crude material was purified
by chromatography on silica gel (gradient CHCl3/MeOH/H2O 5:1:0,
v/v/v; 5:2:0.25, v/v/v; 5:3:0.25, v/v/v; 5:3:0.5, v/v/v; 5:3:0.5, v/v/v;
5:4:1, v/v/v) to give crude 4. Further purification by HPLC on a C18
column running a gradient of acetonitrile and TEAB (50 mm) buffer
O-(2’-Deoxyadenosin-5’-yl)-N,N’-[di(phosphonoethyl)]-phosphor-
amidate (5): General procedure III was applied with 15 (80 mg,
0.12 mmol), Et3N (337 mL, 2.40 mmol), dry CH2Cl2 (10 mL), and iodo-
trimethylsilane (262 mL, 1.92 mmol). Purification was carried out
according to the method used for 4, and 5 was obtained as white
1
solid (42 mg, yield 64%). H NMR (300 MHz, D2O): d=8.40 (s, 1H;
H8), 8.19 (s, 1H; H2), 6.43 (t, J=6.5 Hz, 1H; H1’), 4.67–4.60 (m, 1H;
H3’), 4.20–4.19 (m, 1H; H4’), 3.91–3.85 (m, 2H; H5’), 3.12–3.05 (m,
4H; NCH2CH2), 2.78–2.72 (m, 1H; H2’a), 2.57–2.52 (m, 1H; H2’b),
1.82–1.69 (m, 4H; NCH2CH2); 13C NMR (125 MHz, D2O): d=154.7,
151.8, 148.2, 139.7, 118.3, 85.6, 83.5, 70.9, 60.1, 42.9, 38.9, 26.7;
31P NMR (121 MHz, D2O): d=22.58, 8.86; HRMS (ESI) m/z calcd for
C14H24N6O11P3À: 545.0716, found: 545.0712.
1
yielded a white solid (70 mg, yield 84%). H NMR (300 MHz, D2O):
d=8.37 (s, 1H; H8), 8.14 (s, 1H; H2), 6.38 (t, 1H; J=6.1 Hz, H1’),
4.78–4.70 (m, 1H; H3’), 4.23–4.17 (m, 1H; H4’), 4.05–3.98 (m, 2H;
H5’), 3.26–3.16 (m, 4H; CH2P(O)(OH)2), 2.74–2.65 (m, 1H; H2’a), 2.52–
2.48 (m, 1H; H2’b); 13C NMR (75 MHz, D2O): d=154.1, 150.8, 148.1,
140.0, 118.2, 85.6, 83.5, 70.8, 64.1, 45.2, 38.8; 31P NMR (121 MHÀz,
Diethyl N-(benzylamino)methylphosphate (16):
A mixture of
1,3,5-tribenzyl hexahydro-1,3,5-trizaine (1.00 g, 2.82 mmol) and di-
ethyl phosphonate (1.16 g, 8.46 mmol) was heated at 1008C over-
night, then the product was dried at high vacuum and used in the
D2O): d=19.50, 10.09; HRMS (ESI) m/z calcd for C12H20N6O11P3
:
1
next step without further purification (2.16 g). H NMR (300 MHz,
517.0408, found: 517.0417.
CDCl3): d=7.35–7.28 (m, 5H; Ph), 4.19–4.14 (m, 4H; CH2CH3), 3.89
(s, 2H; CH2Ph), 2.99 (m, J=12.6 Hz, 4H; NCH2), 1.37–1.29 (t, J=
7.05 Hz, 12H; OCH2CH3); 13C NMR (125 MHz, CDCl3): d=138.8,
128.1, 127.9, 126.9, 61.8, 54.5, 44.8, 16.2; 31P NMR (121 MHz,
CDCl3): d=26.23; HRMS (ESI) m/z calcd for C12H21NO3P+: 258.1253,
found: 258.1262.
Tetraethyl N,N’-(benzylamino)di(ethyl phosphonate) (13): Diethyl
vinylphosphonate (328 mg, 2 mmol) was added to a solution of
benzylamine (107 mg, 1.0 mmol) in water (2 mL), at room tempera-
ture. The reaction was refluxed for 4 h, then the solvent was re-
moved under reduced pressure, and 13 was obtained as white
solid (318 mg, yield 73%) without further purification. 1H NMR
(300 MHz, CDCl3): d=7.30–7.21 (m, 5H; Ph), 4.08–3.99 (m, 8H;
CH2CH3), 3.77 (s, 2H; CH2Ph), 2.94–2.87 (m, 4H; NCH2), 2.84–2.72 (m,
4H; NCH2), 2.05–1.86 (m, 4H; NCH2CH2), 1.30–1.22 (m, 12H;
CH2CH3); 13C NMR (75 MHz, CDCl3): d=139.0, 128.1, 127.8, 126.8,
61.2, 57.1, 46.0, 24.9, 16.1; 31P NMR (121 MHz, CDCl3): d=30.36;
HRMS (ESI) m/z calcd for C19H36NO6P2+: 436.2012, found: 436.2025.
N-Benzyl-N’-(diethoxyphosphorylmethyl)glycine ethyl ester (17):
Ethyl bromoacetate was added dropwise to a solution of 16 and
diisopropylethylamine (2.16 g, 16.78 mmol) in anhydrous acetoni-
trile (10 mL). The reaction mixture was stirred for 30 min at room
temperature and refluxed for 4 h. The solvent was then removed
in vacuo. The residue was diluted with CH2Cl2 (10 mL), washed
with 10% NaHCO3 (2ꢁ10 mL), the organic layer dried over anhy-
drous Na2SO4, and concentrated under reduced pressure. The re-
sulting crude material was purified by flash chromatography (SiO2,
CHCl3/AcOEt), to provide 17 as a yellow oil (2.15 g, yield 74%).
Tetraethyl iminodi(ethylphosphonate) (14): Compound 13
(300 mg, 0.69 mmol) was dissolved in ethanol (10 mL). Ammonium
formate (217 mg, 3.45 mmol) was dissolved in distilled water
ChemBioChem 2011, 12, 1868 – 1880
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www.chembiochem.org
1875

P. Herdewijn et al.
1H NMR (300 MHz, CDCl3): d=7.39–7.26 (m, 5H; Ph), 4.22–4.07 (m,
6H; CH2CH3), 3.94 (s, 2H; CH2Ph), 3.60 (s, 2H; NCH2C(O)), 3.19 (d,
J=10.3 Hz, 2H; NCH2P(O)), 1.36–1.25 (m, 9H; OCH2CH3); 13C NMR
(125 MHz, CDCl3): d=170.7, 137.9, 128.8, 128.0, 127.1, 61.7, 59.9,
59.3, 54.2, 49.2, 16.2, 13.9; 31P NMR (121 MHz, CDCl3): d=24.82;
HRMS (ESI) m/z calcd for C16H27NO5P+: 344.1621, found: 344.1630.
CH2P(O)(OH)2), 2.73–2.66 (m, 1H; H2’a), 2.52–2.44 (m, 1H; H2’b);
13C NMR (75 MHz, D2O): d=179.9, 155.3, 152.4, 148.4, 139.8, 118.3,
85.7, 83.1, 70.2, 63.3, 49.8, 45.2, 38.6; 31P NMR (121 MHz, D2O): d=
16.90, 9.70; HRMS (ESI) m/z calcd for C13H19N6O10P2À: 481.0643,
found: 481.0640.
Methyl 3-(benzylamino)propanoate (21): A solution of benzyla-
mine (1.0 g, 9.33 mmol) in methanol (1 mL) was stirred at room
temperature for three days, in the presence of methylacrylate
(756 mL, 8.40 mmol). The reaction mixture was then concentrated
under reduced pressure and the resulting crude material was puri-
fied by chromatography on silica gel (EtOAc/hexane) to afford 21
as a colorless oil (1.53 g, yield 85%). Rf =0.37 (EtOAc). 1H NMR
(300 MHz, CDCl3): d=7.34–7.26 (m, 5H; Ph), 3.82 (s, 2H; NHCH2Ph),
3.70 (s, 3H; OCH3), 3.92 (t, J=6.5 Hz, 2H; NHCH2CH2CO2Me), 2.55 (t,
J=6.5 Hz, 2H; NHCH2CH2CO2Me); 13C NMR (75 MHz, CDCl3): 173.1,
140.0, 128.4, 128.1, 127.0, 53.7, 51.6, 44.4, 34.5; HRMS (ESI): m/z
calcd for C11H16NO2+: 194.1181, found: 194.1190.
Ethyl 2-[N-(diethoxyphosphoryl)methylamino] acetate (18): In a
50 mL flask 17 (2.15 g, 6.26 mmol) was dissolved in ethanol
(20 mL). Ammonium formate (1.98 g, 31.30 mmol) dissolved in dis-
tilled water was added, followed by 10% Pd/C (0.60 g). The mix-
ture was stirred and heated to reflux; after 1.5 h ammonium for-
mate (1.98 g, 31.30 mmol) was again added, and reflux was main-
tained for a further 1.5 h. The reaction mixture was then filtrated
on Celite and concentrated under reduced pressure. The residue
was diluted with CH2Cl2 (20 mL), washed with 10% NaHCO3 (2ꢁ
15 mL); the organic layer was dried over anhydrous Na2SO4, and
concentrated. Compound 18 was afforded as a colorless oil (1.17 g,
yield 74%). 1H NMR (300 MHz, CDCl3): d=4.18–4.13 (m, 6H;
CH2CH3), 3.48 (s, 2H; NCH2C(O)), 3.04 (d, J=12.5 Hz, 2H; NCH2P(O)),
1.36–1.24 (m, 9H; OCH2CH3); 13C NMR (75 MHz, CDCl3): d=171.5,
61.9, 60.5, 51.2, 45.3, 16.2, 13.9; 31P NMR (121 MHz, CDCl3): d=
25.20; HRMS (ESI) m/z calcd for C9H21NO5P+: 254.1152, found:
254.1161.
Methyl 3-[N-benzyl(N’-2-(diethoxyphosphoryl)ethyl)amino]pro-
panoate (22): A solution of 21 (125 mg, 0.65 mmol) in water
(1 mL) was stirred at room temperature for 24 h in the presence of
diethyl vinylphosphonate (300 mL, 1.94 mmol). The reaction mix-
ture was then concentrated under reduced pressure, and the re-
sulting crude material was purified by chromatography on silica
gel (EtOAc/hexane) to afford 22 as a colorless oil (149 mg, yield
64%). Rf 0.18 (EtOAc). 1H NMR (300 MHz, CDCl3): d 7.31–7.21 (m,
5H; Ph), 4.02 (q, 4H; POCH2CH3), 3.66 (s, 3H; OCH3), 3.57 (s, 2H;
NHCH2Ph), 2.82–273 (m, 4H; NHCH2CH2CO2Me and NHCH2CH2P),
2.47 (t, J=6.6 Hz, 2H; NHCH2CH2CO2Me), 1.98–1.86 (m, 2H;
NHCH2CH2P), 1.25 (t, J=7.2 Hz, 6H; POCH2CH3); 13C NMR (75 MHz,
CDCl3): 172.7, 138.6, 128.5, 128.1, 126.6, 61.3, 57.8, 51.4, 48.7, 46.5,
32.5, 23.0, 16.20; 31P NMR (121 MHz, CDCl3): 30.50; HRMS (ESI): m/z
calcd for C17H29NO5P+: 358.1783, found: 358.1775.
O-(2’-Deoxyadenosin-5’-yl)-N-(diethoxyphosphorylmethyl)-N’-
(ethoxycarbonylmethyl)phosphoramidate (19): General proce-
dure I was applied with dAMP (100 mg, 0.30 mmol), 18 (532 mg,
2.10 mmol), and DCC (436 mg, 2.11 mmol). Purification was carried
out according to the method used for 8, and 19 was obtained as
1
white solid (144 mg, yield 88%). H NMR (300 MHz, D2O): d=8.36
(s, 1H; H8), 8.13 (s, 1H; H2), 6.38 (t, J=6.8 Hz, 1H; H1’), 4.65–4.61
(m, 1H; H3’), 4.18–4.14 (m, 1H; H4’), 3.97–3.92 (m, 9H; CH2CH3 and
H5’), 3.69 (d, J=11.3 Hz, 2H; NCH2CO2Et), 3.44–3.36 (m, 2H;
NCH2P(O)(OEt)2), 2.87–2.78 (m, 1H; H2’a), 2.57–2.48 (m, 1H; H2’b),
1.16–1.07 (m, 12H; CH2CH3); 13C NMR (75 MHz, D2O): d=172.6,
155.0, 152.1, 148.6, 139.9, 118.4, 86.0, 83.6, 71.5, 64.3, 63.5, 61.7,
Methyl 3-[2-(diethoxyphosphoryl)ethylamino]propanoate (23):
Compound 22 (145 mg, 0.41 mmol) was dissolved in methanol
(3 mL) and reduced under H2 atmosphere for 6 h in the presence
of 10% Pd/C (14 mg). The reaction mixture was then filtered on
Celite, concentrated under reduced pressure, and the resulting
crude material was purified by chromatography on silica gel
(EtOAc/MeOH) to afford 23 as a colorless oil (53 mg, yield 49%).
1H NMR (300 MHz, CDCl3): d=4.10–4.01 (m, 4H; P(O)OCH2CH3),
3.66 (s, 3H; OCH3), 2.91–2.83 (m, 4H; NHCH2CH2CO2Me and
NHCH2CH2P), 2.51 (t, J=6.6 Hz, 2H; CH2CH2CO2Me), 2.02–1.90 (m,
2H; CH2CH2P), 1.30 (t, J=7.1 Hz, 6H; POCH2CH3); 13C NMR (75 MHz,
CDCl3): 172.9, 61.6, 51.6, 44.5, 43.1, 34.1, 26.2, 16.4; 31P NMR
(121 MHz, CDCl3): 30.17; HRMS (ESI): m/z calcd for C10H23NO5P+:
268.1314, found: 268.1287.
48.8, 42.6, 38.6, 15.5, 13.1; 31P NMR (121 MHz, D2O): d=26.85, 6.68;
À
HRMS (ESI) m/z calcd for C19H31N6O10P2
: 565.1582, found:
565.1586.
O-(2’-Deoxyadenosin-5’-yl)-N-(phosphonomethyl)-N’-(ethoxycar-
bonylmethyl)phosphoramidate (20): General procedure III was
applied with 19 (100 mg, 0.18 mmol), Et3N (249 mL, 1.77 mmol),
and iodotrimethylsilane (193 mL, 1.42 mmol). Purification was car-
ried out according to the method used for 4, and 20 was obtained
1
as white solid (69 mg, yield 77%). H NMR (300 MHz, D2O): d=8.40
(s, 1H; H8), 8.15 (s, 1H; H2), 6.41 (t, J=6.4 Hz, 1H; H1’), 4.67 (m, 1H;
H3’), 4.17 (m, 1H; H4’), 3.99 (m, 5H; CH2CH3 and H5’), 3.49 (m, 2H;
NCH2CO2Et), 3.35 (m, 2H; NCH2P(O)(OEt)2), 2.76 (m, 1H; H2’a), 2.52
(m, 1H; H2’b), 1.06 (m, 3H; CH2CH3); 13C NMR (125 MHz, D2O): d=
173.2, 155.2, 152.2, 148.4, 139.7, 118.3, 85.8, 83.4, 71.2, 64.0, 61.3,
O-(2’-Deoxyadenosin-5’-yl)-N-(diethoxyphosphorylethyl)-N’-(me-
thoxycarbonylethyl)phosphoramidate (24): 2’-Deoxyadenosine-5’-
monophosphoric acid (50 mg, 0.15 mmol), 23 (200 mg, 0.75 mmol),
and DCC (156 mg, 0.75 mmol) were dissolved in a mixture of tert-
butanol (3 mL) and water (1 mL). The reaction was heated for 6 h
under argon atmosphere. After completion, the reaction mixture
was cooled, and the solvent was removed under reduced pressure.
The resulting crude material was purified by chromatography on
silica gel (iPrOH/H2O/NH3 7:0.5:0.5, v/v/v) to provide 24 as a color-
less oil (74 mg, yield 85%). Rf 0.51 (iPrOH/H2O/NH3 7:2:1, v/v/v).
1H NMR (300 MHz, D2O): d=8.36 (s, 1H; H8), 8.16 (s, 1H; H2), 6.39
(t, 1H; J=6.6 Hz, H1’), 4.64–4.68 (m, 1H; H3’), 4.15–4.04 (m, 5H; H4’
and P(O)OCH2CH3), 3.95–3.86 (m, 2H; NHCH2CH2P), 3.64 (s, 1H; H5’),
3.27 (s, 3H; OCH3), 3.20–3.16 (m, 2H; NCH2CH2CO2Me), 2.81–2.63
(m, 2H; H2’), 2.53–2.50 (m, 2H; NCH2CH2CO2Me), 2.34–2.26 (m, 2H;
45.0, 43.0, 38.7, 12.9; 31P NMR (121 MHz, D2O): d=18.50, 8.12;
À
HRMS (ESI) m/z calcd for C15H23N6O10P2
: 509.0956, found:
509.0962.
O-(2’-Deoxyadenosin-5’-yl)-N-(phosphonomethyl)-N’-(carboxyme-
thyl)phosphoramidate (6): General procedure II was applied with
20 (60 mg, 0.12 mmol) and a solution of NaOH (0.4m) in MeOH/
H2O (4:1 v/v, 2 mL). Purification was carried out according to the
method used for 2, and 6 was obtained as a white solid (45 mg,
yield 80%). 1H NMR (300 MHz, D2O): d=8.43 (s, 1H; H8), 8.14 (s,
1H; H2), 6.39 (t, J=6.8 Hz, 1H; H1’), 4.71–4.70 (m, 1H; H3’), 4.13–
4.10 (m, 1H; H4’), 4.06–4.02 (m, 1H; H5’a), 3.94–3.90 (m, 1H; H5’b),
3.76 (d, J=12.5 Hz, 2H; CH2CO2H), 3.12 (t, J=9 Hz, 2H;
1876
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1868 – 1880

DNA Polymerization by HIV-1 Reverse Transcriptase
NHCH2CH2P), 1.27–1.23 (m, 6H; P(O)OCH2CH3); 13C NMR (75 MHz,
D2O): d=173.1, 154.8, 152.2, 148.4, 139.7, 118.2, 85.5, 83.4, 70.6,
64.1, 63.7, 54.3, 44.3, 41.0, 38.6, 32.6, 24.4, 15.4; 31P NMR (121 MHz,
D2O): 28.21 (P-C), 7.98 (P-N); HRMS (ESI): m/z calcd for
C20H33N6O10P2À: 579.1739, found: 579.1749.
61%). Rf 0.18 (EtOAc). 1H NMR (300 MHz, CDCl3): d 7.34–7.22 (m,
5H; Ph), 4.16–4.01 (m, 6H; CO2CH2CH3 and P(O)OCH2CH3), 3.80 (s,
2H; NHCH2Ph), 3.38 (q, J=6.6 Hz, 1H; NHCHCH2), 2.65 (d, J=
5.7 Hz, 2H; CHCH2CO2Et), 2.28 (s, 1H; NH), 2.12–2.02 (m, 2H;
CHCH2P(O)(OEt2)), 1.31–1.22 (m, 9H; CO2CH2CH3 and P(O)OCH2CH3);
13C NMR (75 MHz, CDCl3): d=171.7, 139.8, 128.3, 128.1, 126.9, 61.5,
60.3, 50.8, 49.5, 39.5, 30.2, 16.3, 14.1; 31P NMR (121 MHz, CDCl3):
d=29.26; HRMS (ESI): m/z calcd for C17H29NO5P+: 358.1783, found:
358.1798.
O-(2’-Deoxyadenosin-5’-yl)-N-(phosphonoethyl)-N’-(methoxycar-
bonylethyl)phosphoramidate (25): Bromotrimethylsilane (37 mL,
0.3 mmol) was added dropwise to a solution of 24 (20 mg,
0.03 mmol) and Et3N (40 mL, 0.3 mmol) in dry CH2Cl2 (2 mL) at 08C
under argon. The reaction mixture was stirred at room temperature
for 24 h, and then quenched with triethylammonium bicarbonate
(1.5 mL, 1m). The mixture was concentrated in vacuo, and the re-
sulting crude material was purified by chromatography on silica
gel (gradient : CHCl3/MeOH/H2O 6:4:0.5, v/v/v; 5:4:1, v/v/v; 5:5:1,
Dimethyl 3-aminopentanedioate (28): Compound 26 (200 mg,
0.75 mmol) was dissolved in methanol (4 mL) and reduced under
H2 atmosphere for 6 h in the presence of 10% Pd/C (26 mg). The
reaction mixture was then filtered on Celite and concentrated
under reduced pressure. Compound 28 was obtained as a colorless
oil without additional purification (130 mg, yield 98%). Rf 0.25
(EtOAc). 1H NMR (300 MHz, CDCl3): d=3.68 (s, 6H; OCH3), 3.65–
3.60 (m, 1H; NH2CHCH2), 2.54–2.35 (m, 4H; NHCHCH2), 1.66 (s, 2H;
NH2); 13C NMR (75 MHz, CDCl3): d=172.2, 51.6, 45.3, 41.5; HRMS
(ESI): m/z calcd for C7H14NO4+: 176.0933, found: 176.0926.
1
v/v/v) to provide 25 as a colorless oil (17 mg, yield 90%). H NMR
(500 MHz, D2O): d 8.42 (s, 1H; H8), 8.17 (s, 1H; H2), 6.44 (t, 1H; J=
5.5 Hz, H1’), 4.70 (s, 1H; H3’), 4.19 (s, 1H; H4’), 3.85 (s, 2H; H5’), 3.43
(s, 3H; OCH3), 3.11–3.04 (m, 2H; NHCH2CH2P), 3.01–2.97 (m, 2H;
NHCH2CH2CO2Me), 2.87–2.83 (m, 1H; H2’a), 2.58–2.52 (m, 1H; H2’b),
2.32–2.18 (m, 2H; NHCH2CH2CO2Me), 1.77–1.73 (m, 2H;
NHCH2CH2P(O)(OH)2); 13C NMR (125 MHz, D2O): d=174.6, 155.7,
152.3, 148.4, 139.5, 118.2, 85.7, 83.2, 70.7, 63.4, 51.6, 41.5, 40.7,
38.3, 32.7, 27.7; 31P NMR (201.7 MHz, D2O): d=22.23 (P-C), 8.73 (P-
N); HRMS (ESI): m/z calcd for C16H25N6O10P2À: 523.1107, found:
523.1115.
Ethyl-3-amino-4-(diethoxyphosphoryl)butanoate (29): Compound
27 (213 mg, 0.59 mmol) was dissolved in ethanol (4 mL) and re-
duced under H2 atmosphere for 6 h in the presence of 10% Pd/C
(21 mg). The reaction mixture was then filtered on Celite and con-
centrated under reduced pressure. Compound 29 was obtained as
a colorless oil without additional purification (148 mg, yield 93%).
Rf 0.23 (EtOAc). 1H NMR (300 MHz, CDCl3): d=4.11–4.00 (m, 6H;
CO2CH2CH3 and P(O)OCH2CH3), 3.58–3.50 (m, 1H; NHCHCH2), 2.53–
2.31 (m, 2H; CHCH2CO2Et), 1.96–1.72 (m, 4H; NH2 and CHCH2P(O)-
(OEt2)), 1.28–1.16 (m, 9H; CO2CH2CH3 and P(O)OCH2CH3); 13C NMR
(75 MHz, CDCl3): d=171.5, 61.6, 60.3, 43.8, 42.9, 33.5, 16.3, 14.0;
31P NMR (121 MHz, CDCl3): d=29.16; HRMS (ESI): m/z calcd for
C10H23NO5P+: 268.1314, found: 268.1315.
O-(2’-Deoxyadenosin-5’-yl)-N-(phosphonoethyl)-N’-(carboxyeth-
yl)phosphoramidate (7): A solution of 25 (82 mg, 0.16 mmol) in
MeOH/H2O (4:1 v/v, 2 mL, containing NaOH (0.4m)) was stirred at
room temperature for one day. After completion the solvent was
removed under pressure. The resulting crude material was purified
by HPLC by using a C18 column running a gradient of acetonitrile
and TEAB (50 mm) buffer to provide 7 as a colorless oil (67 mg,
yield 82%). 1H NMR (600 MHz, D2O): d=8.43 (s, 1H; H8), 8.16 (s,
1H; H2), 6.44 (t, J=6.6 Hz, 1H; H1’), 4.68 (s, 1H; H3’), 4.21 (s, 1H;
H4’), 3.89 (s, 2H; H5’), 3.11–3.05 (m, 4H; NHCH2CH2P and
NHCH2CH2CO2H), 2.82–2.76 (m, 1H; H2’a), 2.57–2.52 (m, 1H; H2’b),
2.29–2.24 (m, 2H; NHCH2CH2CO2H), 1.83–1.76 (m, 2H;
NHCH2CH2P(O)(OH)2); 13C NMR (150 MHz, D2O): d=180.8, 155.1,
152.1, 148.3, 139.4, 118.2, 85.9, 83.3, 70.7, 63.3, 42.4, 40.9, 38.6,
O-(2’-Deoxyadenosin-5’-yl)-N-[(1-methoxycarbonylmethyl)-2-me-
thoxycarbonylethyl]phosphoramidate (30): 2’-Deoxyadenosine-5’-
monophosphoric acid (50 mg, 0.15 mmol), 28 (132 mg, 0.76 mmol),
and DCC (187 mg, 0.91 mmol) were dissolved in a mixture of tert-
butanol (3 mL) and water (1 mL). The reaction was heated for 6 h
under argon atmosphere, cooled to room temperature, and the
solvent was removed under reduced pressure. The resulting crude
material was purified by chromatography on silica gel (iPrOH/H2O/
NH3 7:0.5:0.5, v/v/v) to provide 30 as a colorless oil (71 mg, yield
36.9, 27.7; 31P NMR (201.7 MHz, D2O): d=22.30 (P-C), 8.87 (P-N);
À
HRMS (ESI): m/z calcd for C15H23N6O10P2
: 509.0656, found:
509.0948.
1
96%). Rf 0.74 (iPrOH/H2O/NH3 7:2:1, v/v/v). H NMR (500 MHz, D2O):
Dimethyl 3-(benzylamino)pentanedioate (26): A solution of ben-
zylamine (1.0 g, 9.33 mmol) in methanol (1 mL) was stirred at room
temperature for three days in the presence of dimethyl gluconate
(1.18 mL, 8.40 mmol). The reaction mixture was then concentrated
under reduced pressure and the resulting crude material was puri-
fied by chromatography on silica gel (EtOAc/hexane) to afford 26
as a colorless oil (1.66 g, yield 66%). Rf =0.67 (EtOAc). 1H NMR
(300 MHz, CDCl3): d=7.32–7.21 (m, 5H; Ph), 3.80 (s, 2H; NHCH2Ph),
3.66 (s, 6H; OCH3), 3.44 (t, J=6.3 Hz, 1H; NHCHCH2), 2.57 (d, J=
6.3 Hz, 4H; NHCHCH2), 1.92 (s, 1H; NH); 13C NMR (75 MHz, CDCl3):
d=172.0, 139.9, 128.2, 127.9, 126.8, 51.4, 51.0, 50.7, 38.4; HRMS
(ESI): m/z calcd for C14H20NO4+: 266.1392, found: 266.1372.
d=8.41 (s, 1H; H8), 8.15 (s, 1H; H2), 6.41 (t, J=6.7 Hz, 1H; H1’),
4.69–4.67 (m, 1H; H3’), 4.19–4.18 (m, 1H; H4’), 3.86–3.85 (m, 2H;
H5’), 3.64 (s, 6H; OCH3), 3.60–3.53 (m, 1H; NHCHCH2), 2.85–2.81 (m,
1H; H2’a), 2.60–2.51 (m, 1H; H2’b), 2.41–2.31 (m, 4H; NHCHCH2);
13C NMR (125 MHz, D2O): d=175.0, 155.7, 152.5, 149.6, 141.3,
119.5, 87.1, 84.7, 72.3, 65.4, 53.2, 46.9, 41.5, 39.9; 31P NMR
(201.7 MHz, D2O): d=5.79; HRMS (ESI): m/z calcd for C17H24N6O9PÀ:
487.1342, found: 487.1351.
O-(2’-Deoxyadenosin-5’-yl)-N-[1-((diethoxyphosphoryl)methyl)-2-
ethoxycarbonylethyl]-phosphoramidate (31): 2’-Deoxyadenosine-
5’-monophosphoric acid (30 mg, 0.09 mmol), 29 (121 mg,
0.45 mmol), and DCC (94 mg, 0.45 mmol) were dissolved in a mix-
ture of tert-butanol (3 mL) and water (1 mL). The reaction was
heated for 6 h under argon atmosphere. After completion, the re-
action mixture was cooled, and the solvent was removed under re-
duced pressure. The resulting crude material was purified by chro-
matography on silica gel (iPrOH/H2O/NH3 7:0.5:0.5, v/v/v) to pro-
vide 31 as a colorless oil (43 mg, yield 84%). Rf =0.70 (iPrOH/H2O/
Ethyl 3-(benzylamino)-4-(diethoxyphosphoryl)butanoate (27): A
solution of benzylamine (500 mg, 4.66 mmol) in methanol (3 mL)
was stirred at room temperature for three days in the presence of
triethyl-4-phosphonocrotonate (930 mL, 4.19 mmol). The reaction
mixture was then concentrated under reduced pressure, and the
resulting crude material was purified by chromatography on silica
gel (EtOAc/hexane) to afford 27 as a colorless oil (1.02 g, yield
1
NH3 7:2:1, v/v/v). H NMR (300 MHz, D2O): d=8.38 (s, 1H; H8), 8.14
ChemBioChem 2011, 12, 1868 – 1880
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
1877

P. Herdewijn et al.
(s, 1H; H2), 6.38 (t, J=6.6 Hz, 1H; H1’), 4.69–4.67 (m, 1H; H3’), 4.34–
4.32 (m, 1H; H4’), 4.16–4.06 (m, 8H; H5’ and CO2CH2CH3 and
P(O)OCH2CH3), 3.93–3.88 (m, 1H; NHCHCH2), 3.33–3.32 (m, 2H;
CHCH2CO2Et), 2.85–2.71 (m, 2H; H2’a and H2’b), 2.44–2.33 (m, 2H;
CHCH2P(O)(OEt)2), 1.26–1.14 (m, 9H; CO2CH2CH3 and P(O)OCH2CH3);
13C NMR (75 MHz, D2O): d=173.0, 154.8, 152.0, 148.4, 139.8, 118.2,
85.6, 83.3, 70.8, 64.0, 62.8, 61.3, 50.8, 44.0, 38.7, 32.0, 15.2, 13.0;
31P NMR (121 MHz, D2O): d=31.15 and 30À.90 (P-C), 5.62 (P-N);
d=18.16 and 16.15 (P-C), 7.70 and 7.39 (P-N); HRMS (ESI): m/z
calcd for C14H21N6O10P2À: 495.0794, found: 495.0797.
O-(2’-Deoxythymin-5’-yl)-(iminodiacetic acid)phosphoramidate
(35), (General procedure IV): In a two-neck flask, 2’-deoxythymi-
dine-5’-monophosphate (100 mg, 0.31 mmol) and dimethyl imino-
diacetate hydrochloride (306 mg, 1.55 mmol) were suspended in a
mixture of 1,4-dioxane (8 mL) and DMF (2 mL). One drop of Et3N
was added to the solution to facilitate dissolution. Then a solution
of DCC (447 mg, 2.17 mmol) in 1,4-dioxane (2 mL) was added and
the reaction mixture was heated at 858C for 3 h under argon at-
mosphere. After completion, the reaction mixture was cooled, and
the solvent was removed under reduced pressure. The resulting
crude material was purified by chromatography on silica gel (gradi-
ent: CHCl3/CH3OH/H2O 5:1:0, v/v/v; 5:2:0.25, v/v/v; 5:3:0.25, v/v/v;
5:3:0.5, v/v/v) to provide compound O-(2’-deoxythymin-5’-yl)-N,N’-
[di(methoxycarbonylethyl)]-phosphoramidate. This compound was
then treated with NaOH (0.4m) in MeOH/H2O (v/v, 1:4, 3 mL) for
1.5 h, the solvent was removed under reduced pressure, the result-
ing crude material was purified by chromatography on silica gel
(gradient: CHCl3/CH3OH/H2O 5:1:0, 5:2:0.25, v/v/v; 5:3:0.5, v/v/v;
and 5:4:1, v/v/v) to give 35 as a white solid (29 mg, yield 22% after
HRMS (ESI): m/z calcd for C20H33N6O10P2
: 579.1733, found:
579.1718.
O-(2’-Deoxyadenosin-5’-yl)-N-[(1-carboxymethyl)-2-carboxyeth-
yl]-phosphoramidate (32): A solution of 30 (70 mg, 0.14 mmol) in
MeOH/H2O (4:1 v/v, 2 mL, containing NaOH (0.4m)) was stirred at
room temperature for 2 h. After completion the solvent was re-
moved under reduced pressure. The resulting crude material was
purified by chromatography on silica gel (gradient: iPrOH/H2O/NH3
7:0.5:0.5, v/v/v; 7:1:1, v/v/v; 7:2:1, v/v/v) to provide 32 as colorless
1
oil (48 mg, yield 64%). Rf 0.71 (iPrOH/H2O/NH3 7:2:1, v/v/v). H NMR
(500 MHz, D2O): d=8.46 (s, 1H; H8), 8.17 (s, 1H; H2), 6.46 (t, J=
6.6 Hz, 1H; H1’), 4.73 (s, 1H; H3’), 4.25 (s, 1H; H4’), 3.95 (s, 2H; H5’),
3.62–3.60 (m, 1H; NHCH), 2.85–2.80 (m, 1H; H2’a), 2.60–2.57 (m, 1H;
1
2’b), 2.43–2.34 (m, 4H; CH2CO2H); 13C NMR (125 MHz, D2O): d=
two steps). H NMR (500 MHz, D2O): d=7.72 (s, 1H; H6), 6.34 (t, J=
H
6.8 Hz, 1H; H1’), 4.56–4.54 (m, 1H; H3’), 4.15–4.14 (m, 1H; H4’), 4.04–
4.02 (m, 2H; H5’), 3.63–3.61 (m, 4H; CH2CO2H), 2.41–2.31 (m, 2H;
H2’), 1.91 (s, 3H; CH3); 13C (75 MHz, D2O): d=179.5, 167.7, 152.6,
137.3, 111.8, 85.5, 84.9, 71.1, 64.2, 52.1, 38.5, 11.8; 31P NMR
(121 MHz, D2O): d=8.71; HRMS (ESI) m/z calcd for C14H19N3O11PÀ:
436.0763, found: 436.0737.
178.3, 154.2, 151.0, 147.8, 139.4, 118.9, 85.5, 83.0, 70.5, 63.3, 47.0,
42.8, 38.4; 31P NMR (201.7 MHz, D2O): d=6.68; HRMS (ESI): m/z
calcd for C15H20N6O9PÀ: 459.1029, found: 459.1035.
O-(2’-Deoxyadenosin-5’-yl)-N-[1-((ethoxy(hydroxy)phosphoryl)-
methyl)-2-carboxyethyl]phosphoramidate (33): A solution of 31
(97 mg, 0.17 mmol) in MeOH/H2O (4:1 v/v, 2 mL, containing
NaOH(0.4m)) was stirred at room temperature for one day. After
completion the solvent was removed under pressure. The resulting
crude material was purified by chromatography on silica gel (gradi-
ent: iPrOH/H2O/NH3 7:0.5:0.5, v/v/v; 7:1:1, v/v/v; 7:2:1, v/v/v) to pro-
vide 33 as a colorless oil (71 mg, yield 76%). Rf 0.58 (iPrOH/H2O/
O-(2’-Deoxyguanosin-5’-yl)-(iminodiacetic acid)phosphoramidate
(36): General procedure IV was applied with 2’-deoxyguanosine-5’-
monophosphate (100 mg, 0.29 mmol), dimethyl iminodiacetate hy-
drochloride (381 mg, 2.02 mmol), and DCC (415 mg, 2.02 mmol).
After reaction the obtained compound was treated with NaOH
(0.4m) in MeOH/H2O (v/v, 1:4) (3 mL); purification was carried out
according to the method used for 35 to give 36 as a white solid.
(30 mg, yield 21% after two steps). 1H NMR (300 MHz, D2O): d=
8.01 (s, 1H; H8), 6.20 (t, 1H; J=7.0 Hz, H1’), 4.58–4.56 (m, 1H; H3’),
4.12–4.11 (m, 1H; H4’), 3.93–3.92 (m, 2H; H5’), 3.60 (d, 4H; J=
10.7 Hz, NCH2), 2.71–2.68 (m, 1H; H2’a), 2.44–2.41 (m, 1H; H2’b);
13C NMR (75 MHz, D2O): d=179.2, 158.6, 153.6, 151.3, 137.3, 115.9,
85.7, 83.2, 71.2, 63.8, 51.3, 38.2; 31P NMR (121 MHz, D2O): d=7.91;
HRMS (ESI) m/z calcd for C14H18N6O10PÀ: 461.0827, found: 461.0812.
1
NH3 7:2:1, v/v/v). H NMR (500 MHz, D2O): d=8.43 (s, 1H; H8), 8.12
(s, 1H; H2), 6.40 (t, 1H; J=6.6 Hz, H1’), 4.70 (s, 1H; H3’), 4.22 (s, 1H;
H4’), 3.94 (s, 2H; H5’), 3.81–3.75 (m, 2H; P(O)OCH2CH3), 3.55–3.50 (m,
1H; NHCHCH2), 2.81–2.76 (m, 1H; H2’a), 2.58–2.54 (m, 2H;
CHCH2CO2H), 2.44–2.37 (m, 1H; H2’b), 1.95–1.76 (m, 2H;
CHCH2P(O)(OH)OEt), 1.16–1.11 (m, 3H; P(O)(OH)OCH2CH3); 13C NMR
(125 MHz, D2O): d=177.4, 153.7, 150.3, 147.8, 139.8, 117.9, 85.7,
83.3, 70.9, 63.6, 60.6, 45.6, 42.2, 38.7, 32.9, 15.5; 31P NMR
(201.7 MHz, D2O): d=23.44 and 23.34 (P-C),À6.43 and 6.39 (P-N);
HRMS (ESI): m/z calcd for C16H25N6O10P2
: 523.1107, found:
O-(2’-Deoxycytidin-5’-yl)-(iminodiacetic
acid)phosphoramidate
523.1108.
(37): General procedure IV was applied with 2’-deoxycytidine-5’-
monophosphate (100 mg, 0.31 mmol), dimethyl iminodiacetate hy-
drochloride (407 mg, 2.15 mmol), and DCC (443 mg, 2.15 mmol).
After reaction, the obtained molecular was treated with NaOH
(0.4m) in MeOH/H2O (1:4, v/v, 3 mL); purification was carried out
according to the method used for 35 to give 37 as a white solid
(44 mg, yield 32% after two steps). 1H NMR (300 MHz, D2O): d=
7.88 (d, J=7.4 Hz, 1H; H6), 6.22 (t, J=6.7 Hz, 1H; H1’), 6.02 (d, J=
7.5 Hz, 1H; H5), 4.43–4.42 (m, 1H; H3’), 4.07–4.05 (m, 1H; H4’), 3.95–
3.91 (m, 2H; H5’), 3.63 (d, J=10.7 Hz, 4H; NCH2), 2.29–2.23 (m, 1H;
H2’a), 2.21–2.19 (m, 1H; H2’b); 13C NMR (75 MHz, D2O): d=179.2,
165.6, 156.9, 141.4, 96.1, 85.6, 85.5, 70.7, 63.7, 51.6, 39.2; 31P NMR
(121 MHz, D2O): d=7.79; HRMS (ESI) m/z calcd for C13H18N4O10PÀ:
421.0766, found: 421.0786.
O-(2’-Deoxyadenosin-5’-yl)-N-[1-((phosphono)methyl)-2-carboxy-
ethyl]phosphoramidate (34): Bromotrimethylsilane (53 mL,
0.4 mmol) was added dropwise to a solution of 33 (30 mg,
0.05 mmol) and Et3N (56 mL, 0.4 mmol) in dry CH2Cl2 (2 mL) at 08C
under argon. The reaction mixture was stirred at room temperature
for 24 h, and then quenched with triethylammonium bicarbonate
(1.5 mL, 1m). The mixture was concentrated in vacuo, and the
resulting crude material was purified by chromatography on silica
gel (gradient: CHCl3/MeOH/H2O 5:4:1, v/v/v; 6:4:0.5, v/v/v; 5:5:1,
v/v/v) to provide 34 as a colorless oil (26 mg, yield 83%). Rf 0.41
(iPrOH/H2O/NH3 7:2:1, v/v/v). 1H NMR (600 MHz, D2O): d=8.44 (s,
1H; H8), 8.14 (s, 1H; H2), 6.43 (t, J=5.7 Hz, 1H; H1’), 4.73–468 (m,
1H; H3’), 4.21–4.19 (m, 1H; H4’), 3.97–3.89 (m, 2H; H5’), 3.56–3.51
(m, 1H; NHCHCH2), 2.80–2.42 (m, 3H; CHCHCO2H and H2’a), 2.36–
2.25 (m, 1H; H2’b), 1.81–1.54 (m, 2H; CHCH2P(O)(OH)2); 13C NMR
(150 MHz, D2O): d=177.9, 155.2, 152.2, 148.3, 139.5, 118.2, 85.7,
83.1, 70.3, 63.4, 47.5, 45.3, 38.6, 37.0; 31P NMR (201.7 MHz, D2O):
O-(2’-Deoxythymin-5’-yl)-(iminodipropionicacid)phosphorami-
date (38): General procedure IV was applied with thymidine-5’-
monophosphate (100 mg, 0.31 mmol), dimethyl N,N’-iminodipropa-
noate (411 mg, 2.17 mmol), and DCC (448 mg, 2.17 mmol). After re-
1878
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ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2011, 12, 1868 – 1880

DNA Polymerization by HIV-1 Reverse Transcriptase
action, the obtained compound was treated with NaOH (0.4m) in
MeOH/H2O (1:4, v/v, 3 mL); purification was carried out according
to the method used for 35 to give 38 as a white solid (51 mg,
yield 35% after two steps). 1H NMR (500 MHz, D2O): d=7.76 (s,
1H; H6), 6.35 (m, J=6.5 Hz, 1H; H1’), 4.59–4.56 (m, 1H; H3’), 4.17–
4.16 (m, 1H; H4’), 3.99–3.92 (m, 2H; H5’), 3.26–3.20 (m, 4H;
NCH2CH2), 2.44–2.41 (m, 4H; NCH2CH2), 2.38–2.35 (m, 2H; H2’), 1.93
(s, 3H; CH3); 13C NMR (150 MHz, D2O): d=181.0, 166.3, 151.5,
Steady-state kinetics of single nucleotide incorporation: The
steady-state kinetics of single nucleotide incorporation of 1, 2, and
dATP (the natural nucleoside triphosphate) were determined by a
gel-based polymerase assay. Template T1 and primer P1 were used
in all experiments. Primer and template in a 1:2 molar ratio were
hybridized in a buffer (Tris·HCl (20 mm pH 8.3), KCl (10 mm), MgSO4
(2 mm), and 0.1% Triton X-100) and used in an amount to provide
125 nm primer in each 20 mL reaction. A range of “building block”
concentrations was used: phosphoramidate (10 mm to 1 mm), natu-
ral substrate (0.1 mm to 10 mm). The final concentrations of primer-
137.0, 111.4, 85.3, 84.6, 70.8, 63.6, 43.0, 38.3, 37.2, 11.3; 31P NMR
À
(121 MHz, D2O): d=8.84; HRMS (ESI) m/z calcd for C16H23N3O11P3
:
464.1076, found: 464.1074.
template complex and HIV-1 RT were 125 nm, and 0.0125 UmLÀ1
,
respectively. Reaction mixtures were incubated at 378C and ali-
quots were drawn at six time intervals. The reactions were
quenched by addition of a buffer (80% formamide, EDTA (2 mm),
and 1ꢁTBE). The analysis of the polymerase reaction was per-
formed by polyacrylamide gel electrophoresis (see below). The in-
corporation rates (V) were calculated based on the percentage of
the extended oligonucleotide in the mixture (P+1 band). The ki-
netics parameters (Vmax and KM) were determined by plotting V
(nmminÀ1) versus substrate concentration (mm), and fitting the
data to a non-linear Michaelis–Menten regression by using Graph-
Pad Prism Software version 5.0.
O-(2’-Deoxyguanosin-5’-yl)-(iminodipropionicacid)phosphorami-
date (39): General procedure IV was applied with 2’-deoxyguano-
sine-5’-monophosphate (100 mg, 0.29 mmol), dimethyl N, N’-imino-
dipropanoate (381 mg, 2.02 mmol), and DCC (415 mg, 2.02 mmol).
After reaction the obtained molecular was treated with NaOH
(0.4m) in MeOH/H2O (1:4, v/v, 3 mL); purification was carried out
according to the method used for 35 to give 39 as a white solid
(30 mg, yield 21% after two steps). 1H NMR (600 MHz, D2O): d=
7.99 (s, 1H; H8), 6.24 (t, J=6.6 Hz, 1H; H1’), 4.64–4.63 (m, 1H; H3’),
4.14–4.13 (m, 1H; H4’), 3.89–3.84 (m, 2H; H5’), 3.11–3.07 (m, 4H;
NCH2CH2), 2.79–2.74 (m, 1H; H2’a), 2.46–2.44 (m, 1H; H2’b), 2.31–2.28
(m, 4H; NCH2CH2); 13C NMR (150 MHz, D2O): d=179.0, 159.1, 153.2,
151.3, 137.5, 116.2, 85.8, 83.5, 71.2, 63.9, 43.3, 38.3, 37.5; 31P NMR
(121 MHz, D2O): d=9.14; HRMS (ESI) m/z calcd for C16H22N6O10PÀ:
489.1140, found: 489.1123.
Electrophoresis: All polymerase reaction aliquots (2.5 mL) were
quenched by the addition of 10 mL of loading buffer (90% forma-
mide, 0.05% bromophenol blue, 0.05% xylene cyanol and EDTA
(50 mm)). Samples were heated at 858C for 3 min, then subjected
to electrophoresis (2.5 h, 2000 V) on a 30 cmꢁ40 cmꢁ0.4 mm 20%
(19:1 acrylamide:bis-acrylamide) denaturing gel in the presence of
Tris-borate (100 mm) and EDTA (2.5 mm, pH 8.3). Products were vi-
sualized by phosphorimaging. The amount of radioactivity in the
bands corresponding to the products of enzymatic reactions was
determined by using a Cyclone imaging device with Optiquant
image analysis software (Perkin–Elmer).
O-(2’-Deoxytcytidin-5’-yl)-(iminodipropionicacid)phosphorami-
date (40): General procedure IV was applied with 2’-deoxycytidine-
5’-monophosphate (100 mg, 0.31 mmol), dimethyl N,N’-iminodipro-
panoate (407 mg, 2.15 mmol), and DCC (443 mg, 2.15 mmol). After
reaction the obtained compound was treated with NaOH (0.4m) in
MeOH/H2O (1:4, v/v, 3 mL); purification was carried out according
to the method used for 35 to give 40 as a white solid (44 mg,
yield 32% after two steps). 1H NMR (500 MHz, D2O): d=7.97 (d,
J=7.5 Hz, 1H; H6), 6.32 (t, J=6.5 Hz, 1H; H1’), 6.11 (d, J=7.5 Hz,
1H; H5), 4.57–4.55 (m, 1H; H3’), 4.19–4.18 (m, 1H; H4’), 4.02–3.93
(m, 2H; H5’), 3.22–3.16 (m, 4H; NCH2CH2), 2.45–2.40 (m, 5H; NCH2
and H2’a), 2.31–2.26 (m, 1H; H2’b); 13C NMR (150 MHz, D2O): d=
181.3, 166.0, 157.5, 141.3, 96.4, 85.8, 85.6, 70.7, 63.5, 43.3, 39.6,
37.5; 31P NMR (121 MHz, D2O): d=9.03; HRMS (ESI): m/z calcd. for
C15H22N4O10PÀ: 449.1079, found: 449.1067.
Acknowledgements
The research was financially supported by K.U. Leuven, Bijzonder
Onderzoeksfonds, Geconcerteerde Onderzoeksacties. We would
like to thank Prof. Jef Rozenski for providing HRMS, Luc Baudem-
prez for recording the NMR spectra, Dr. Elisabetta Groaz for care-
ful reading of the manuscript, and Chantal Biernaux for editorial
help.
DNA polymerase reactions: End-labeled primer was annealed to
its template by combining primer and template in a molar ratio of
1:2, and heating the mixture to 708C for 10 min followed by slow
cooling to room temperature over 2 h. For incorporation of the
phosphoramidates (compounds 2–7, 32, and 34), a series of 20 mL
batch reactions was incubated with HIV-1 RT (10 UmLÀ1 stock solu-
tion, specific activity 8.095 UmgÀ1; Ambion). The final mixture con-
tained primer/template complex (125 nm), RT buffer (Tris·HCl
(250 mm, pH 8.3), KCl (250 mm), MgCl2 (50 mm), spermidine
(2.5 mm), dithiothreitol (DTT, 50 mm)), HIV-1 RT (0.025 UmLÀ1), and
different concentrations of phosphoramidate building blocks
(1 mm, 500 mm, 200 mm, and 100 mm). In the control reaction (natu-
ral nucleotides), dATP, dGTP, dTTP and dCTP (10 mm each) were
used. The mixture was incubated at 378C, and aliquots were
quenched after 10, 20, 30, 60, and 120 min. For elongation experi-
ments, the same mixture with appropriate primer/template hybrid
was incubated at 378C, and aliquots were quenched after 15, 30,
60, 90, and 120 min. In the reaction with the natural nucleotide,
dATP (50 mm) was used.
Keywords: DNA
·
HIV-1
reverse
transcriptase
·
iminodipropionic acid · nucleotides · phosphoramidates
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