Dipeptides as leaving group in the enzyme-catalyzed DNA synthesis

Article abstract of DOI:10.1002/cbdv.201200261

Conjugates of 2′-deoxyadenosine monophosphate with dipeptides have been synthesized and tested as substrates for several polymerases. Although the incorporation efficiency is not very high, it demonstrates that some of these dipeptides can be accommodated in the active site of polymerases and function as leaving groups in the enzymatic synthesis of DNA. Copyright

Full text of DOI:10.1002/cbdv.201200261

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Dipeptides as Leaving Group in the Enzyme-Catalyzed DNA Synthesis
by Xiao-Ping Song, Camille Bouillon, Eveline Lescrinier, and Piet Herdewijn*
Laboratory of Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven,
Minderbroedersstraat 10, B-3000 Leuven
(phone: þ3216337387; fax: þ3216337340; e-mail: Piet.Herdewijn@rega.kuleuven.be)
Conjugates of 2’-deoxyadenosine monophosphate with dipeptides have been synthesized and tested
as substrates for several polymerases. Although the incorporation efficiency is not very high, it
demonstrates that some of these dipeptides can be accommodated in the active site of polymerases and
function as leaving groups in the enzymatic synthesis of DNA.
Introduction. – Nucleic acids are synthesized in a cell making use of nucleoside
triphosphates as substrate and polymerases as enzymes. Pyrophosphate functions as
leaving group in this process. Diversifying this leaving group could serve the aim to
develop a new biochemistry for the enzymatic synthesis of nucleic acids. Besides the
potential application in drug design (developing modified nucleoside triphosphate
mimics as direct substrates for polymerases), these molecules can also be applied in
synthetic biology (developing an XNA information system) [1], and as tools to better
understand the chemical origin of nucleic acid replication in life.
The constructions of an XNA system could rely on a systematic diversification of
the sugar moiety, the base moiety, and the leaving group, as well as enzyme evolution.
Generally, native polymerases have limited ability to recognize modified nucleotide
building blocks. However, some engineered polymerase mutants are able to efficiently
incorporate unnatural substrates. For example, Pinheiro et al. reported that some
engineered polymerases are able to generate XNA sequences (HNA, CeNA, ANA,
FANA, TNA, and LNA) up to 72 nt, while still retaining canonical base-pairing
selectivity [2]. Previously, we have demonstrated that some natural amino acids (e.g., l-
aspartic acid) or unnatural amino acids (e.g., l-b-imidazole lactic acid, iminodiacetate
(IDA), iminodipropionate (IDP), and 3-phosphono-l-alanine) are able to act as
pyrophosphate mimics in enzymatic DNA polymerization [3–7]. We also observed
that d4TMP adducts of 3-phosphono-l-alanine (3-phosphono-l-alanine-d4TMP) fails
to deliver d4TMP into CEM/TKꢀ cells, suggesting the metabolic instability of this
nucleotide adduct [8].
In the present study, we have evaluated if dipeptides can function as leaving groups
in the DNA polymerization reaction. Dipeptides could have an additional advantage,
as they might contribute to the cell-penetration ability of the DNA precursors bearing
an alternative leaving group. Dipeptides might facilitate the membrane delivery of
these nucleotide adducts with the help of membrane transporters [9]. Dipeptides
consisting of two amino acid molecules joined by a peptide bond are metabolically
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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degradable by hydrolase enzymes in vivo [10]. Therefore, the dipeptide that would be
generated in the cell after nucleotide incorporation can be degraded into single amino
acids, making the polymerization reaction irreversible. Dipeptides bearing two
additional carboxylic acid residues could retain the chelating properties necessary for
binding the nucleotide substrates in the active site of polymerases. Compared to a
single amino acid, a dipeptide is a larger molecule, allowing us to probe the geometry of
the enzyme-active-site pocket.
Results and Discussion. – Synthesis of Compounds 1a–1k. The eleven compounds
synthesized are compiled in Table 1. The synthetic routes used to obtain compounds
1a–1k are outlined in Schemes 1–4.
Table 1. Synthesized Compounds 1a–1k
Compound No.
Structure
Compound No.
Structure
1a
1b
1c
1d
1e
1f
Asp-Asp-dAMP
Gly-Asp-dAMP
His-Asp-dAMP
Asp-His-dAMP
Asp-Gly-dAMP
His-IDA-dAMP
1g
1h
1i
1j
1k
Gly-IDA-dAMP
Asp-IDA-dAMP
Gly-IDP-dAMP
Asp-IDP-dAMP
Gly-Gly-dAMP
Scheme 1 depicts the synthesis of the phosphoramidate nucleoside dipeptide
analogs 1a–1e, obtained in four steps. First, the protected dipeptides 2a–2e were
obtained in good yield starting from commercially available amino acids, using the
conventional coupling reagent N,N’-dicyclohexylcarbodiimide (DCC)/N-hydroxysuc-
cinimide (HOSu) in CH2Cl2. The Boc protecting group was removed by treating 2a–2e
with CF3COOH (TFA) to give the corresponding amines 3a–3e, respectively, in
excellent yields. The synthesis of compounds 1a–1e was carried out according to the
procedure described by Wagner and co-workers [11], starting from 2’-deoxyadenosine-
5’-monophosphate (dAMP) and amines 3a–3e with DCC as coupling reagent. Finally,
deprotection of the carboxylic acid ester function with NaOH in MeOH/H2O solution
(for compounds 1a, 1b, and 1e) or by hydrogenation with 10% Pd/C (for compounds 1c
and 1d) provided the target compounds 1a–1e.
The synthesis of compound 1f–1h was accomplished as outlined in Scheme 2. N-
Alkylation of N-benzylglycine ethyl ester with tert-butyl 2-bromoacetate in the
presence of EtNiPr2 gave compound 4, which was further treated with TFA to remove
t
selectively the Bu group to give the corresponding acid 5 as TFA salt. Then, the
obtained compound 5 was coupled to commercially available amino acids, using the
coupling reagent DCC/HOSu or N-[3-(dimethylamino)propyl]-N’-ethylcarbodiimide
hydrochloride (EDCI)/1-hydroxybenzotriazole (HOBt) to afford compounds 6a–6c in
good yields. Compounds 7a–7c were obtained after removal of the PhCH2 (Bn)
protecting groups, and the resulting secondary amines were coupled with dAMP in the
presence of DCC to provide the corresponding protected nucleoside analogs. The
deprotection of the carboxylic acid functions with NaOH in MeOH/H2O solution gave
compounds 1f–1h.

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Scheme 1. Synthesis of Compound 1a–1e
i) N,N’-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (HOSu), N-methylmorpholine,
CH2Cl2, r.t. ii) CF3COOH (TFA), CH2Cl2, r.t. iii) 2’-Deoxyadenosine-5’-monophosphate (dAMP),
DCC, 1,4-dioxane/H2O, Et3N, 808. iv) 10% Pd/C, H2, r.t. for 1a, 1b, and 1e; 0.4m NaOH (MeOH/H2O),
r.t. for 1c and 1d. Im¼1H-Imidazol-4-yl.
The synthesis of compound 1i and 1j is depicted in Scheme 3. Two Michael addition
reactions of BnNH2 on methyl acrylate and tert-butyl acrylate successively gave
compounds 8 and 9, respectively. Treatment of the latter with TFA provided compound
10. The TFA salt 10 was coupled with glycine methyl ester or l-aspartic acid dimethyl
ester in the presence of EDCI/HOBt to furnish 11a and 11b respectively. The
hydrogenolyses gave the free amines 12a and 12b. Finally, as described previously, a
coupling reaction of 12a and 12b with dAMP, followed by a saponification reaction,
gave the final compounds 1i and 1j, respectively.
Scheme 4 displays the synthesis of compound 1k obtained in four steps, starting with
a coupling reaction between diethyl aminomalonate and N-(benzyloxy)carbonyl
(Cbz)-glycine. Then, compound 13 was engaged in a hydrogenation reaction in the
presence of HCl to avoid intermolecular and intramolecular side-reactions. The HCl
salt 14 was coupled with dAMP, and deprotection of the carboxylate function provided
compound 1k, after a decarboxylation reaction.
Single-Nucleotide-Incorporation Experiments. Polymerases play a central role
during the enzyme-catalyzed DNA synthesis. They promote DNA synthesis by
catalyzing the nucleophilic attack of the 3’-terminal OH group of a primer to the a-
phosphate of the incoming nucleoside triphosphate, leading to formation of a 5’-3’
phosphodiester bond [12]. To investigate the substrate property of compounds 1a–1k,
polymerases from different families were tested (HIV-1 RT, Taq, Vent (exoꢀ)

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Scheme 2. Synthesis of Compound 1f–1h
i) EtNiPr2, MeCN, r.t. – reflux. ii) TFA, CH2Cl2, r.t. iii) 1-Ethyl-3-[3-(dimethylamino)propyl]carbodi-
imide (EDCI), 1-hydroxybenzotriazole (HOBt), CH2Cl2, Et3N, r.t. for 6a; DCC, HOSu, N-
methylmorpholine, CH2Cl2, r.t. for 6b–6c. iv) H2, 10% Pd/C, MeOH, r.t. v) dAMP, DCC, 1,4-
t
dioxane/H2O, Et3N, 808 for 1g and 1h; dAMP, DCC, BuOH/H2O, Et3N, 808 for 1f. vi) 0.4m NaOH
(MeOH/H2O), r.t.
Scheme 3. Synthesis of Phosphoramidate Analogs 1i and 1j
i) Methyl acrylate, MeOH, r.t. ii) tert-Butyl acrylate, MeOH, r.t. iii) TFA, CH2Cl2, r.t. iv) EDCI, HOBt,
CH2Cl2, Et3N, r.t. v) H2, 10% Pd/C, MeOH, r.t. vi) dAMP, DCC, 1,4-dioxane/H2O, Et3N, 808. vii) 0.4m
NaOH (MeOH/H2O), r.t.

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Scheme 4. Synthesis of Phosphoramidate Analog 1k
t
i) EDCI, HOBt, CH2Cl2, Et3N, r.t. ii) H2, 10% Pd/C, MeOH, HCl, r.t. iii) dAMP, DCC, BuOH/H2O,
Et3N, 808. iv) 0.4m NaOH (MeOH/H2O), r.t.
polymerase, and Therminator DNA polymerase). Primer P1 and template T1 were used
as duplex (Table 2), 2’-deoxyadenosine triphosphate (dATP) was used as positive
control. Incorporation efficiency was evaluated by the polyacrylamide gel-based single-
nucleotide-incorporation assay [13][14].
Table 2. PrimerꢀTemplate Complex Used in the Incorporation Assay
Oligonucleotide
Sequence
P1
T1
5’-CAGGAAACAGCTATGAC-3’
3’-GTCCTTTGTCGATACTGTCCC-5’
Single-Nucleotide Incorporation by HIV-1 RT. HIV-1 RT is an error-prone
polymerase and therefore, has high mutation rate [15]. Its flexibility and high tolerance
towards chemically modified nucleotide substrates made it the primary choice as
enzyme catalyst for our pyrophosphate-mimic screening. The single-nucleotide-
incorporation results of compound 1a–1k by HIV-1 RT are compiled in Table 3.
Unfortunately, all eleven compounds were not good substrates for HIV-1 RT. Most of
the compounds only showed 10–35% yield of primer extension. Compound 1c, 1g, and
Table 3. Results of Incorporation of Compounds 1a–1k by HIV-1 RTa)
Compound No.
Incorporation [%]
Compound No.
Incorporation [%]
1a
1b
1c
1d
1e
1f
33
0
20
15
14
20
1g
1h
1i
1j
1k
0
0
18
5
21
a) Conditions: primer, P1; template; T1, [HIV-1 RT], 0.025 U/ml.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
1h were not able to extend the primer after 2 h reaction time. The best single
incorporation result was obtained by using compound 1a as substrate, with 33% yield of
Pþ1 strand. It is interesting to note that compounds 1a, 1b, and 1c, all of which contain
an l-aspartic acid moiety in the leaving group, showed quite different incorporation
results, with 30, 0, and 20% primer conversion, respectively. This is much less than the
90% conversion obtained when using l-Asp-dAMP as substrate.
Single-Nucleotide Incorporation by Therminator DNA Polymerase. Therminator
DNA polymerase is a 98N variant, recognized for its ability to incorporate a broad
spectrum of modified nucleotides [16][17]. As shown in Fig. 1, compounds 1a–1k
exhibited good substrate properties using Therminator as catalyst. Except for 1d, all of
the tested analogs were accepted as substrate by Therminator DNA polymerase quite
efficiently, some moderate-to-high yields of primer conversion were obtained.
However, using Therminator polymerase as catalyst, a higher ratio of misincorporation
was observed. For example, using compound 1g as substrate in the single-nucleotide-
incorporation assay over 90% primer conversion was obtained with Therminator
polymerase (Fig. 2), but only 22% of the primer was converted to the Pþ1 level, 49%
converted to the Pþ2 level, and 19% primer converted to the Pþ3 and Pþ4 level.
Fig. 1. Single-nucleotide incorporation of compounds 1a–1k into P1T1 by Therminator DNA polymerase.
[Therminator pol]¼0.05 U/ml.
Single-Nucleotide Incorporation by Other DNA Polymerases. Furthermore, the
substrate properties of compound 1a–1k were investigated by Taq and Vent (exoꢀ)
polymerases (Fig. 3). The incorporation efficiency was significantly less appealing than
using Therminator DNA polymerase as catalyst. When using Taq as polymerase, only
compounds 1d (30% primer conversion) and 1g (27% primer conversion) gave primer
extension. Most of the analogs were poor substrates for Taq polymerase. In contrast,
Vent (exoꢀ) polymerase showed a slightly better catalytic ability. Compound 1f with
iminodiacetic acid joint by l-histidine as leaving group demonstrates 75% primer
extension at a concentration of 500 mm (Fig. 4). Compound 1g containing an
iminodiacetic acid moiety and a glycine in the leaving group gave 60% Pþ1 product.
On the contrary, compound 1h composed of iminodiacetic acid and l-aspartic acid in

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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Fig. 2. Single incorporation of compound 1c into P1T1 by Therminator DNA polymerase. [Therminator
pol]¼0.05 U/ml. Time points: 10, 20, 30, 60, and 120 min.
Fig. 3. Single-nucleotide incorporation of compounds 1a–1k by Taq and Vent (exoꢀ) DNA polymerase.
[Vent (exoꢀ) pol]¼0.1 U/ml, [Taq pol]¼0.15 U/ml.
Fig. 4. Single incorporation of compound 1f into P1T1 by Vent (exoꢀ) DNA polymerase. [Vent (exoꢀ)
pol]¼0.1 U/ml. Time points: 10, 20, 30, 60, and 120 min.
the leaving group was a poorer substrate than 1f and 1g, and only 15% yield of Pþ1
product was obtained. The different incorporation results obtained for compounds 1f

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and 1g indicated that it is still challenging to balance binding properties and potential
substrate properties for polymerases of modified nucleoside triphosphates.
It is worth noting that compound 1k is the smallest-sized dipeptide among the tested
analogs. It contains two glycine molecules and only one carboxylic acid group in the
leaving group, and it still can be accepted as substrate by all four enzymes tested. It
showed better substrate properties than some of the analogs with two or three
carboxylic acid groups, especially when using Therminator polymerase as catalyst
(Fig. 1). Enzyme-catalyzed nucleotide incorporation is a very complicated procedure.
Several factors might be involved in this process. Based on the results obtained for
compound 1k and for other analogs that have been studied here, it is clear that the size
of the dipeptide leaving group is a very important factor influencing the incorporation
reactions. Co-crystallization experiments and molecular modeling to analyze the
possible interactions between the substrate and the enzyme active site will be very
helpful for the future rational design of new pyrophosphate mimics.
Conclusions. – A series of phosphoramidate analogs bearing dipeptides as leaving
groups were synthesized and tested as substrates by HIV-1 RT, Taq, Vent (exoꢀ), and
Therminator DNA polymerases. Therminator polymerase exhibited the best catalytic
efficiency of all the four enzymes used, with moderate-to-high yield of primer
conversions. A higher level of misincorporation was also observed with Therminator
polymerase. Compound 1k bearing only one carboxylic acid residue in the leaving
group is still well-accepted by the four polymerases. This suggests that the spacial size of
the dipeptide leaving group might play an important role in the recognition by the
enzyme active site. Although these dipeptide leaving groups show inferior incorpo-
ration efficiencies when compared with, for example, iminodipropionate, the obtained
results are encouraging. We will further explore the potential of using such protected or
unprotected dipeptideꢀdNMP analogs as potential prodrugs for modified nucleotides.
Another interesting question is, if such conjugates would be able to freely penetrate the
membrane of vesicles and/or function as substrate in a nonenzymatic nucleic acid
polymerization reaction.
Experimental Part
General. For all reactions, chemicals of anal. or synthetic grade were obtained from commercial
sources and used without purification. Technical-grade solvents were obtained from Brenntag (B-
Deerlijk). Flash chromatography (FC): Davisilꢂ silica gel 60, 0.040–0.063 mm (Grace Davison). Anal.
TLC: Alugramꢂ silica gel UV254 mesh 60, 0.20 mm (MachereyꢀNagel). NMR Spectra: Bruker Avance II
300- or 500-MHz NMR spectrometer; chemical shifts, d, expressed in ppm downfield from TMS (Me4Si)
for 1H- and 13C-NMR; J in Hz. 31P-NMR: chemical shifts are referenced to an external 85% H3PO4
standard (d 0.00 ppm). MS: a quadrupole/orthogonal-acceleration time-of-flight tandem mass spec-
trometer (Q-Tof 2, Micromass) equipped with a standard electrospray ionization (ESI) source; in m/z.
Dibenzyl (2S)-2-({(2S)-4-(Benzyloxy)-2-[(tert-butoxycarbonyl)amino]-4-oxobutanoyl}amino)buta-
nedioate (2a). General Procedure I (GP I). To a soln. of Boc-l-aspartic acid benzyl ester (646 mg,
2.0 mmol) and l-aspartic acid dibenzyl ester (971 mg, 2.0 mmol) in CH2Cl2 (15 ml), N-methylmorpholine
(NMM; 205 ml, 2.0 mmol), DCC (619 mg, 3.0 mmol) and HOSu (232 mg, 2.0 mmol) were added. The
mixture was stirred for 14 h under Ar at r.t., white precipitate was removed by filtration, and the resulting
crude material was purified by FC (hexane/AcOEt 5 :1). Yield: 92%. White solid. 1H-NMR (300 MHz,
CDCl3): 7.45–7.30 (m, 3 Ph); 5.14–5.07 (m, 3 PhCH2); 4.92–4.86 (m, HꢀC(a)(Aspa)); 4.60–4.55 (m,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2693
t
HꢀC(a)(Aspb)); 3.11–3.03 (m, CH2(b)(Aspa)); 2.93–2.70 (m, CH2(b)(Aspb)); 1.47 (s, Bu). 13C-NMR
(75 MHz, CDCl3): 171.2; 170.3; 170.1; 169.7; 155.2; 135.1; 134.8; 127.9–128.3 (15 C); 81.2; 67.2; 66.5
(2 C); 50.3; 48.6; 36.0; 35.8; 27.9. HR-ESI-MS: 641.2450 ([MþNa]þ , C34H38N2NaO9þ ; calc. 641.2470).
Benzyl (3S)-4-{[2-(Benzyloxy)-2-oxoethyl]amino}-3-[(tert-butoxycarbonyl)amino]-4-oxobutanoate
(2b). GP I was applied using glycine benzyl ester (404 mg, 2.0 mmol), Boc-l-aspartic acid benzyl ester
(646 mg, 2.0 mmol), NMM (205 ml, 2.0 mmol), DCC (619 mg, 3.0 mmol), and HOSu (232 mg, 2.0 mmol).
1
Purification was carried out according to the method used for 2a. Yield: 82%. White solid. H-NMR
(300 MHz, CDCl3): 7.37–7.35 (m, 2 Ph); 5.19 (s, PhCH2); 5.15 (s, PhCH2); 4.61–4.60 (m, NHCHCH2);
4.06 (d, J¼5.0, NHCH2CO2Bn); 3.12–3.05 (m, 1 H, NHCHCH2); 2.80–2.72 (m, 1 H, NHCHCH2); 1.47
t
(s, Bu). 13C-NMR (75 MHz, CDCl3): 171.7; 171.1; 169.4; 155.6; 135.5; 135.3; 128.4–128.7 (10 C); 80.7;
67.3; 66.9; 50.6; 36.2; 34.0; 28.4. HR-ESI-MS: 471.2112 ([MþH]þ , C25H32N2O7þ ; calc. 471.2126).
Methyl (3S)-3-[(tert-Butoxycarbonyl)amino]-4-{[(2S)-3-(1H-imidazol-4-yl)-1-methoxy-1-oxopro-
pan-2-yl]amino}-4-oxobutanoate (2c). GP I was applied using l-histidine methyl ester dihydrochloride
(484 mg, 2.0 mmol), Boc-l-aspartic acid methyl ester (495 mg, 2.0 mmol), NMM (205 ml, 2.0 mmol),
DCC (619 mg, 3.0 mmol), and HOSu (232 mg, 2.0 mmol). Purification was carried out according to the
method used for 2a. Yield: 58%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 7.72 (s, NHCH¼N); 6.79 (s,
NHCH¼C); 4.72–4.70 (m, NHCHCH2CO2Me); 4.51–4.50 (m, NHCHCH2Im); 3.65 (s, MeO); 3.63 (s,
t
MeO); 3.12–3.10 (m, ImCH2); 2.88–2.73 (m, NHCHCH2CO2Me); 1.39 (s, Bu). 13C-NMR (75 MHz,
CDCl3): 171.7; 170.9; 170.6; 155.3; 135.0; 131.1; 118.7; 80.1; 52.6; 52.1; 51.7; 50.6; 36.0; 29.3; 27.9. HR-ESI-
MS: 399.1873 ([MþH]þ , C17H27N4O7þ ; calc. 399.1880).
Dimethyl N-(tert-Butoxycarbonyl)-l-histidyl-l-aspartate (2d). GP I was applied using Boc-l-
histidine (510 mg, 2.0 mmol), Boc-l-aspartic acid dimethyl ester hydrochloride (395 mg, 2.0 mmol),
NMM (205 ml, 2.0 mmol), DCC (619 mg, 3.0 mmol), and HOSu (232 mg, 2.0 mmol). Purification was
carried out according to the method used for 2a. Yield: 74%. Colorless oil. 1H-NMR (300 MHz, CDCl3):
7.77 (s, NHCH¼N); 6.79 (s, NHCH¼C); 4.76–4.74 (m, NHCHCO2Me); 4.37–4.35 (m, NHCHCH2Im);
3.63 (s, MeO); 3.57 (s, MeO); 2.99–2.82 (m, ImCH2); 2.81–2.60 (m, NHCHCH2CO2Me); 1.31 (s, tBu).
13C-NMR (75 MHz, CDCl3): 174.7; 171.8; 170.9; 155.5; 135.0; 132.1; 118.0; 79.9; 54.3; 52.6; 51.9; 48.5;
35.8; 29.2; 28.1. HR-ESI-MS: 399.1871 ([MþH]þ , C17H27N4O7þ ; calc. 399.1880).
Dibenzyl N-(tert-Butoxycarbonyl)glycyl-l-aspartate (2e). GP I was applied using Boc-l-glycine
(350 mg, 2.0 mmol), l-aspartic acid dibenzyl ester (971 mg, 2.0 mmol), NMM (205 ml, 2.0 mmol), DCC
(619 mg, 3.0 mmol), and HOSu (232 mg, 2.0 mmol). Purification was carried out according to the
method used for 2a. Yield: 95%. White solid. 1H-NMR (300 MHz, CDCl3): 7.35–7.11 (m, 2 Ph); 5.19 (s,
PhCH2); 5.06 (s, PhCH2); 4.95–4.92 (m, NHCHCO2Bn); 3.82 (s, NHCH2CO); 3.13–2.82 (m,
t
NHCHCH2CO2Bn); 1.46 (s, Bu). 13C-NMR (75 MHz, CDCl3): 170.3; 169.9; 169.1; 155.6; 135.0; 134.8;
127.9–128.3 (10 C); 79.9; 67.3; 66.5; 48.3; 43.8; 36.0; 28.0. HR-ESI-MS: 493.1957 ([MþNa]þ
,
C25H31N2NaOþ7 ; calc. 493.1945).
Dibenzyl (2S)-2-{[(2S)-2-Amino-4-(benzyloxy)-4-oxobutanoyl]amino}butanedioate Trifluoroace-
tate (3a). General Procedure II (GP II). Compound 2a was dissolved in CH2Cl2 (10 ml), and TFA
(5 ml) was added to the soln. The soln. was stirred for 24 h at r.t. After removal of solvent and excess of
1
TFA, 3a was obtained as colorless oil. Yield: 98%. H-NMR (300 MHz, CDCl3): 7.30–7.22 (m, 3 Ph);
5.06–4.99 (m, 3 PhCH2); 4.56–4.50 (m, HꢀC(a)(Aspa)); 4.09–4.05 (m, HꢀC(a)(Aspb)); 3.01–2.88 (m,
CH2(b)(Aspa), CH2(b)(Aspb)). 13C-NMR (75 MHz, CDCl3): 170.4; 170.4; 169.4; 135.0; 134.7; 134.6;
127.9–128.2 (15C); 67.2; 66.6; 64.0; 49.3; 48.7; 35.2; 34.6. HR-ESI-MS: 519.2131 ([MþH]þ , C29H32N2O7þ ;
calc. 519.2126).
Benzyl (3S)-3-Amino-4-{[2-(benzyloxy)-2-oxoethyl]amino}-4-oxobutanoate Trifluoroacetate (3b).
GP II was applied using 2b (1.4 g, 3 mmol) and TFA (5 ml) to give 3b as TFA salt. Yield: 98%. Colorless
oil. 1H-NMR (300 MHz, CDCl3): 7.31–7.30 (m, 2 Ph); 5.11–5.09 (m, 2 PhCH2); 4.66–4.65 (m,
NH2CHCH2); 4.00–3.99 (m, NHCH2CO2Bn); 3.04–3.03 (m, NH2CHCH2). 13C-NMR (75 MHz, CDCl3):
170.9; 169.5; 168.6; 134.9; 134.6; 128.5–128.8 (10 C); 68.1; 67.8; 50.2; 34.7; 33.2. HR-ESI-MS: 371.1592
([MþH]þ , C20H24N2Oþ5 ; calc. 371.1601).
Methyl (3S)-3-Amino-4-{[(2S)-3-(1H-imidazol-4-yl)-1-methoxy-1-oxopropan-2-yl]amino}-4-oxobu-
tanoate Trifluoroacetate (3c). GP II was applied using 2c (1.2 g, 3 mmol) and TFA (5 ml) to give 3c as
TFA salt. Yield: 95%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 7.60 (s, NHCH¼N); 6.77 (s,

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NHCH¼C); 4.08–4.03 (m, NHCHCH2CO2Me); 3.87–3.83 (m, NHCHCH2Im); 3.66 (s, MeO); 3.61 (s,
MeO); 3.53–3.30 (m, NHCHCH2CO2Me); 2.56–2.43 (m, NHCHCH2Im). 13C-NMR (75 MHz, CDCl3):
172.9; 171.6; 169.9; 134.4; 133.2; 117.6; 55.3; 54.3; 52.2; 51.5; 36.6; 28.9. HR-ESI-MS: 299.1346 ([Mþ
H]þ , C12H19N4Oþ5 ; calc. 299.1355).
Dimethyl l-Histidyl-l-aspartate Trifluoroacetate (3d). GP II was applied using 2d (1.2 g, 3 mmol)
and TFA (5 ml) to furnish 3d as TFA salt. Yield: 90%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 7.64 (s,
NHCH¼N); 6.90 (s, NHCH¼C); 4.79–4.76 (m, NHCHCH2CO2Me); 4.30–4.26 (m, NHCHCH2Im); 3.65
(s, MeO); 3.60 (s, MeO); 3.15–3.06 (m, NHCHCH2CO2Me); 2.85–2.80 (m, NHCHCH2Im). 13C-NMR
(75 MHz, CDCl3): 174.9; 172.4; 169.3; 136.5; 134.2; 119.5; 56.3; 53.2; 52.7; 52.6; 38.7; 32.6. HR-ESI-MS:
299.1350 ([MþH]þ , C12H19N4Oþ5 ; calc. 299.1355).
Dibenzyl Glycyl-l-aspartate Trifluoroacetate (3e). GP II was applied using 2e (1.5 g, 3 mmol) and
TFA (5 ml) to afford 3e as TFA salt. Yield: 90%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 7.28–7.21
(m, 2 Ph); 5.00–4.97 (m, 2 PhCH2 , NHCHCH2); 3.95–3.86 (m, NHCH2CO); 3.02–2.85 (m,
NHCHCH2). 13C-NMR (75 MHz, CDCl3): 170.5; 170.2; 134.9; 134.6; 127.9–128.2 (10 C); 67.5; 66.7;
48.7; 40.7; 35.5. HR-ESI-MS: 371.1609 ([MþH]þ , C20H24N2O5þ ; calc. 371.1601).
5’-O-{[((1S)-1-(Carboxymethyl)-2-{[(1S)-1,2-dicarboxyethyl]amino}-2-oxoethyl)amino]hydroxy-
phosphinyl}-2’-deoxyadenosine (1a). General Procedure III (GP III). 2’-Deoxyadenosine-5’-monophos-
phate (dAMP; 100 mg, 0.30 mmol) and 3a (777 mg, 1.5 mmol) were dissolved in 1,4-dioxane (8 ml)/H2O
(2ml) in the presence of one drop of Et3N. A soln. of DCC (433 mg, 2.10 mmol) in 1,4-dioxane (2 ml) was
added, and the mixture was heated at 858 for 3 h under Ar. After completion, the mixture was cooled,
and the solvent was removed in vacuo. The resulting residue was purified by FC (CHCl3/MeOH/H2O
5 :2 :0.5) to provide the intermediate compound, 5’-O-[({(1S)-3-(benzyloxy)-1-[({(1S)-3-(benzyloxy)-1-
[(benzyloxy)carbonyl]-3-oxopropyl}amino)carbonyl]-3-oxopropyl}amino)hydroxyphosphinyl]-2’-deox-
yadenosine, as a white solid. Yield: 43%. 1H-NMR (300 MHz, D2O): 8.47 (s, HꢀC(8)); 8.17 (s, HꢀC(2));
7.29–7.24 (m, 3 Ph); 6.47 (t, J¼6.5, HꢀC(1’)); 5.04–4.99 (m, 3 PhCH2); 4.64–4.60 (m, HꢀC(3’)); 4.12–
4.10 (m, HꢀC(4’)); 4.02–3.95 (m, CH2(5’)); 3.47–3.44 (m, HꢀC(a)(Aspa), HꢀC(a)(Aspb)); 2.87–2.75
(m, HaꢀC(2’), CH2(b)(Aspa), CH2(b)(Aspb)); 2.50–2.45 (m, HbꢀC(2’)). 13C-NMR (125 MHz, D2O):
173.8; 171.2; 170.2; 170.0; 158.1; 155.5; 152.1; 148.7; 139.3; 135.6; 135.5; 135.2; 127.4–127.8 (15C); 118.6;
86.2; 83.6; 71.2; 66.6; 66.0; 65.6; 64.0; 52.0; 48.6; 39.6; 37.2; 35.2. 31P-NMR (121 MHz, D2O): 5.70. HR-
ESI-MS: 830.2591 ([MꢀH]ꢀ , C39H4ON7O12Pꢀ ; calc. 830.2556).
This intermediate was dissolved in MeOH (5 ml) and reduced under H2 for 24 h, in the presence of
10% Pd/C (20 mg). The mixture was filtered over Celite and concentrated in vacuo, and the resulting
crude material was purified by FC (CHCl3/MeOH/H2O 5 :2 :0.5, 5 :2 :1, 5 :3 :0.5) to provide 1a (64 mg,
85%). White solid. 1H-NMR (600 MHz, D2O): 8.45 (s, HꢀC(8)); 8.22 (s, HꢀC(2)); 6.49 (t, J¼6.6,
HꢀC(1’)); 4.68–4.64 (m, HꢀC(3’)); 4.34–4.31 (m, HꢀC(4’)); 4.24–4.22 (m, HꢀC(a)(Aspa)); 3.99–3.90
(m, CH2(5’)); 3.81–3.77 (m, HꢀC(a)(Aspb)); 2.83–2.78 (m, 1 HꢀC(2’)); 2.63–2.44 (m, 1 HꢀC(2’),
CH2(b)(Aspa), CH2(b)(Aspb)). 13C-NMR (150 MHz, D2O): 178.5; 178.3; 178.1; 175.7; 155.5; 152.6;
148.7; 139.9; 118.6; 86.1; 83.6; 71.4; 64.2; 53.1; 52.7; 40.3; 39.4; 38.9. 31P-NMR (121 MHz, D2O): 6.28. HR-
ESI-MS: 560.1154 ([MꢀH]ꢀ , C18H22N7O12Pꢀ ; calc. 560.1148).
5’-O-[({(1S)-1-(Carboxymethyl)-2-[(carboxymethyl)amino]-2-oxoethyl}amino)hydroxyphosphin-
yl]-2’-deoxyadenosine (1b). GP III was applied using dAMP (100 mg, 0.30 mmol), 3b (555 mg,
1.5 mmol), and DCC (433 mg, 2.10 mmol). After purification, the desired intermediate was obtained
as white solid. Yield: 76%. 1H-NMR (600 MHz, D2O): 8.33 (s, HꢀC(8)); 8.09 (s, HꢀC(2)); 7.33–7.12 (m,
2 Ph); 6.35 (t, J¼7.2, HꢀC(1’)); 5.08 (s, PhCH2); 4.88 (s, PhCH2); 4.64–4.63 (m, HꢀC(3’)); 4.18–4.17 (m,
HꢀC(4’), NHCHCH2CO2Bn); 3.95–3.94 (m, CH2(5’)); 3.88 (d, J¼2.4, NHCH2CO2Bn); 2.67–2.64 (m,
1 HꢀC(2’), NHCHCH2CO2Bn); 2.63–2.61 (m, 1 HꢀC(2’)). 13C-NMR (150 MHz, D2O): 175.3; 172.2;
170.9; 154.8; 151.8; 148.3; 139.7; 135.1; 135.1; 127.5–128.7 (10C); 118.5; 85.8; 83.7; 70.9; 67.4; 63.9; 52.1;
46.7; 41.4; 39.1; 37.9. 31P-NMR (121 MHz, D2O): 7.81. HR-ESI-MS: 682.2038 ([MꢀH]ꢀ , C30H29N7O9Pꢀ
;
calc. 682.2032).
The intermediate was dissolved in MeOH (5 ml) and reduced under H2 for 24 h, in the presence of
10% Pd/C (22 mg). The mixture was filtered on Celite, and concentrated in vacuo, the resulting crude
material was purified by FC (CHCl3/MeOH/H2O 5 :2 :0.5, 5 :2 :1, 5 :3 :0.5) to provide 1b. Yield: 88%.
1
White solid. H-NMR (600 MHz, D2O): 8.45 (s, HꢀC(8)); 8.24 (s, HꢀC(2)); 6.49 (t, J¼8.4, HꢀC(1’));

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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4.70–4.68 (m, HꢀC(3’)); 4.27–4.24 (m, HꢀC(4’)); 4.02–3.91 (m, CH2(5’)); 3.80–3.75 (m,
NHCHCH2CO2H); 2.85–2.79 (m, 1 HꢀC(2’)); 2.61–2.45 (m, 1 HꢀC(2’), NHCHCH2CO2H).
13C-NMR (150 MHz, D2O): 178.6; 176.4; 175.9; 155.6; 152.8; 148.8; 139.9; 118.7; 86.1; 83.8; 71.5; 64.2;
53.2; 43.4; 40.6; 39.0. 31P-NMR (121 MHz, D2O): 6.21. HR-ESI-MS: 502.1074 ([MꢀH]ꢀ , C16H21N7O10Pꢀ
;
calc. 502.1093).
5’-O-{[((1S)-2-{[(1S)-1-Carboxy-2-(1H-imidazol-4-yl)ethyl]amino}-1-(carboxymethyl)-2-oxoethyl)-
amino]hydroxyphosphinyl}-2’-deoxyadenosine (1c). GP III was applied using dAMP (100 mg,
0.30 mmol), 3c (447 mg, 1.5 mmol), and DCC (433 mg, 2.10 mmol). After purification, the desired
intermediate was obtained as white solid. The resulting crude product was treated with 0.4m NaOH soln.
in MeOH/H2O 1:4 (5 ml) for 3 h, the solvent was removed in vacuo, and the crude product was purified
by FC (CHCl3/MeOH/H2O 5 :2 :0.5, 5 :2 :1, 5 :3 :0.5) to provide 1c. Yield: 22% (over two steps). White
solid. 1H-NMR (600 MHz, D2O): 8.41 (s, HꢀC(8)); 8.14 (s, HꢀC(2)); 7.51 (s, NHCH¼N); 6.73 (s,
NHCH¼C); 6.41 (t, J¼6.7, HꢀC(1’)); 4.60–4.58 (m, HꢀC(3’)); 4.31–4.27 (m, HꢀC(a)(His)); 4.17–4.15
(m, HꢀC(4’)); 3.90–3.82 (m, CH2(5’)); 3.75–3.67 (m, HꢀC(a)(Asp)); 2.97–2.86 (m, CH2(b)(His));
2.83–2.68 (m, HaꢀC(2’)); 2.52–2.42 (m, CH2(b)(Asp), HbꢀC(2’)). 13C-NMR (150 MHz, D2O): 179.3;
172.6; 170.7; 155.2; 153.5; 146.8; 139.4; 136.5; 134.9; 119.2; 118.6; 89.4; 84.3; 72.6; 65.2; 56.1; 43.2; 40.9;
39.8; 32.4. 31P-NMR (121 MHz, D2O): 6.61. HR-ESI-MS: 582.1471 ([MꢀH]ꢀ , C19H25N9O10Pꢀ ; calc.
582.1467).
2’-Deoxy-5’-O-{[((1S)-2-{[(1S)-1,2-dicarboxyethyl]amino}-1-(1H-imidazol-4-ylmethyl)-2-oxoeth-
yl)amino]hydroxyphosphinyl}adenosine (1d). GP III was applied using dAMP (100 mg, 0.30 mmol),
3d (447 mg, 1.5 mmol), and DCC (433 mg, 2.10 mmol). After purification, the desired intermediate was
obtained as white solid, and the crude product was then treated with 0.4m NaOH soln. in MeOH/H2O 1:4
(5 ml) for 3 h. The solvent was removed in vacuo, and the resulting crude product was purified by FC
(CHCl3/MeOH/H2O 5 :2 :0.5, 5 :2 :1, 5 :3 :0.5) to provide 1d. Yield: 30% (over two steps). White solid.
1H-NMR (600 MHz, D2O): 8.43 (s, HꢀC(8)); 8.13 (s, HꢀC(2)); 7.45 (s, NHCH¼N); 6.70 (s, NHCH¼C);
6.41 (t, J¼7.1, HꢀC(1’)); 4.58–4.56 (m, HꢀC(3’)); 4.28–4.26 (m, NHCHCO2H); 4.15–4.13 (m,
HꢀC(4’)); 3.77–3.71 (m, CH2(5’), NHCHCH2Im); 2.50–2.46 (m, 1 HꢀC(2’), NHCHCH2CO2H); 2.61–
2.59 (m, 1 HꢀC(2’), NHCHCH2Im). 13C-NMR (150 MHz, D2O): 177.4; 176.9; 170.6; 155.3; 152.1; 148.9;
140.3; 139.6; 132.0; 118.8; 116.7; 89.8; 87.4; 70.2; 63.0; 52.2; 51.8; 38.4; 34.9; 32.1. 31P-NMR (121 MHz,
D2O): 6.02. HR-ESI-MS: 582.1475 ([MꢀH]ꢀ , C20H25N9O10Pꢀ ; calc. 582.1467).
2’-Deoxy-5’-O-{[(2-{[(1S)-1,2-dicarboxyethyl]amino}-2-oxoethyl)amino]hydroxyphosphinyl}adeno-
sine (1e). GP III was applied using dAMP (100 mg, 0.30 mmol), 3e (555 mg, 1.5 mmol), and DCC
(433 mg, 2.10 mmol). After purification, the desired intermediate was dissolved in MeOH (10 ml) and
reduced under H2 for 24 h in the presence of 10% Pd/C (22 mg). The mixture was filtered on Celite, and
concentrated in vacuo, and the resulting crude material was purified by FC (CHCl3/MeOH/H2O 5 :2 :0.5,
5 :2 :1, 5 :3 :0.5) to provide 1e. Yield: 49% (over two steps). White solid. 1H-NMR (600 MHz, D2O): 8.43
(s, HꢀC(8)); 8.22 (s, HꢀC(2)); 6.47 (t, J¼6.6, HꢀC(1’)); 4.67–4.66 (m, HꢀC(3’)); 4.33–4.31 (m,
NHCHCO2H); 4.23–4.22 (m, HꢀC(4’)); 3.97–3.95 (m, CH2(5’)); 3.42–3.39 (m, NHCH2CONH); 2.83–
2.78 (m, 1 HꢀC(2’)); 2.64–2.53 (m, 1 HꢀC(2’), NHCHCH2CO2H). 13C-NMR (150 MHz, D2O): 178.8;
178.2; 173.4; 155.6; 152.7; 148.8; 139.8; 118.6; 85.9; 83.6; 71.2; 64.2; 52.5; 44.4; 39.5; 38.9. 31P-NMR
(121 MHz, D2O): 7.81. HR-ESI-MS: 502.1090 ([MꢀH]ꢀ , C16H20N7O10Pꢀ ; calc. 502.1093).
N-Benzyl-N-[2-(tert-butoxy)-2-oxyethyl]glycine Ethyl Ester (4). To a soln. of N-benzylglycine ethyl
ester (2.06 g, 10.7 mmol) and EtNiPr2 (3.5 ml, 21.3 mmol) in anh. MeCN (10 ml), tert-butyl 2-
bromoacetate (1.55 ml, 10.7 mmol) was added dropwise. The mixture was stirred for 30 min at r.t. and
refluxed for 4 h, the solvent was then removed in vacuo. The residue was diluted with AcOEt (10 ml),
washed with 10% NaHCO3 soln. (2ꢁ10 ml), and the org. layer was dried (Na2SO4), and concentrated in
vacuo. The resulting crude material was purified by FC (hexane/AcOEt 5 :1) to provide 4. Yield: 87%.
Yellow oil. 1H-NMR (300 MHz, CDCl3): 7.42–7.27 (m, Ph); 4.18 (q, J¼7.2, 6 H, OCH2Me); 3.93 (s,
t
t
NCH2Ph); 3.55 (s, NCH2CO2Et); 3.45 (s, NCH2CO2 Bu); 1.49 (s, Bu); 1.29 (t, J¼7.2, OCH2Me).
13C-NMR (125 MHz, CDCl3): 171.3; 170.5; 138.4; 129.0; 128.3; 127.2; 81.0; 60.3; 57.7; 55.1; 54.3; 28.2; 14.2.
HR-ESI-MS: 308.1837 ([MþH]þ , C17H26NOþ4 ; calc. 308.3926).
2-[Benzyl(2-ethoxy-2-oxoethyl)amino]acetic Acid Trifluoroacetate (5). Ester 4 (2.84 g, 9.2 mmol)
was treated by TFA (6.8 ml, 92.3 mmol) in CH2Cl2 (60 ml) for 2 d at r.t. After removal of solvent and

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
excess of TFA, the oily crude residue was triturated with Et2O and filtered to afford 5 as a TFA salt.
Yield: 78%. White solid. 1H-NMR (300 MHz, MeOD): 7.52–7.44 (m, Ph); 4.42 (s, NCH2Ph); 4.23 (q, J¼
7.1, OCH2Me); 4.07 (s, NCH2CO2Et); 4.02 (s, NCH2CO2H); 1.27 (t, J¼7.1, OCH2Me). 13C-NMR
(75 MHz, MeOD): 170.6; 168.5; 139.8; 132.3; 131.0; 130.3; 63.4; 60.3; 54.9; 54.6; 14.3. HR-ESI-MS:
252.1229 ([MþH]þ , C13H18NOþ4 ; calc. 252.1236).
Methyl N-Benzyl-N-(2-ethoxy-2-oxoethyl)glycyl-l-histidinate (6a). General Procedure IV (GP IV).
A soln. of 5 (TFA salt; 500.0 mg, 1.4 mmol), l-histidine methyl ester dihydrochloride (331.4 mg,
1.4 mmol), and Et3N (630 ml, 4.5 mmol) in CH2Cl2 (5 ml) was stirred at r.t. for 30 min. The mixture was
cooled to 08, and HOBt (278.5 mg, 2.1 mmol) and EDCI (393.6 mg, 2.1 mmol) were added. The soln. was
stirred for 24 h at r.t. After removal of the solvent, the residue was diluted with AcOEt (20 ml), washed
with sat. NaHCO3 soln. (2 ꢁ 10 ml) and brine (10 ml), dried (MgSO4), and concentrated in vacuo. The
resulting crude material was purified by FC (CH2Cl2/MeOH 10 :1) to provide 6a (431.0 mg, 75%). White
1
solid. H-NMR (300 MHz, CDCl3): 9.77 (br. s, NH); 8.39 (d, NH); 7.49 (s, NHCH¼N); 7.26–7.18 (m,
Ph); 6.78 (s, NHCH¼C); 4.85–4.78 (m, NHCHCO2Me); 4.10 (q, J¼7.1, OCH2Me); 3.82–3.69 (m,
NCH2CONH); 3.61 (s, CO2Me); 3.31 (s, NCH2Ph, NCH2CO2Et); 3.14–3.11 (m, ImCH2); 1.19 (t,
OCH2Me). 13C-NMR (75 MHz, CDCl3): 171.5; 170.9; 170.8; 137.1; 135.1; 132.9; 128.9; 128.2; 127.4; 116.7;
60.4; 58.6; 57.5; 54.1; 52.0; 51.9; 29.2; 13.9. HR-ESI-MS: 403.1981 ([MþH]þ , C20H19N4O5þ ; calc.
403.1981).
Methyl N-Benzyl-N-(2-ethoxy-2-oxoethyl)glycylglycinate (6b). GP I was applied using glycine
methyl ester (279 mg, 2.0 mmol), 5 (502 mg, 2.0 mmol), NMM (205 ml, 2.0 mmol), DCC (619 mg,
3.0 mmol), and HOSu (232 mg, 2.0 mmol). Purification was carried out according to the method used for
2a. Yield: 45%. White solid. 1H-NMR (300 MHz, CDCl3): 7.25–7.40 (m, Ph); 4.17–4.12 (m, 2 H,
OCH2Me); 3.90 (d, J ¼12.4, CH2(Gly)); 3.72 (s, MeO); 3.32–3.49 (m, 2 NCH2CO); 1.29–1.22 (t, J ¼7.4,
OCH2Me). 13C-NMR (75 MHz, CDCl3): 170.5; 169.0; 166.2; 137.4; 129.0; 128.3; 127.5; 60.4; 58.9; 57.4;
53.8; 51.7; 40.6; 14.0. HR-ESI-MS: 323.1601 ([MþH]þ , C16H24N2O5þ ; calc. 323.1601).
Dimethyl N-Benzyl-N-(2-ethoxy-2-oxoethyl)glycyl-l-aspartate (6c). GP I was applied using l-
aspartic acid dimethyl ester hydrochloride (395 mg, 2.0 mmol), 5 (502 mg, 2.0 mmol), NMM (205 ml,
2.0 mmol), DCC (619 mg, 3.0 mmol), and HOSu (232 mg, 2 mmol). Purification was carried out
according to the method used for 2a. Yield: 75%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 7.36–7.27
(m, Ph); 4.91–4.87 (m, NHCHCO2Me); 4.20–4.15 (m, 2 H, OCH2Me); 3.85–3.77 (m, PhCH2); 3.74 (s,
MeO); 3.69 (s, MeO); 3.39–3.35 (m, 2 NCH2CON); 3.06–3.02 (m, 1 H, NHCHCH2CO2Me); 2.84–2.79
(m, 1 H, NHCHCH2CO2Me); 1.28 (t, J¼8.4, OCH2Me). 13C-NMR (75 MHz, CDCl3): 171.2; 171.1; 170.8
(2C); 137.4; 129.1; 128.6; 127.7; 60.7; 58.8; 57.6; 54.6; 52.7; 52.0; 48.0; 36.2; 14.2. HR-ESI-MS: 395.1810
([MþH]þ , C19H27N2Oþ7 ; calc. 395.1818).
Methyl N-(2-Ethoxy-2-oxoethyl)glycylglycinate (7b). General Procedure V (GP V). Compound 6b
(322.0 mg, 1.0 mmol) was dissolved in MeOH (5 ml) and reduced under H2 for 24 h in the presence of
10% Pd/C (37 mg). The mixture was filtered on Celite and concentrated in vacuo to give 7b. Yield:
1
280.0 mg, 100%. Colorless oil. H-NMR (300 MHz, CDCl3): 4.23–4.16 (m, OCH2Me); 4.07 (d, J¼5.7,
NHCH2CO2Me); 3.76 (s, MeO); 3.47 (s, CH2CO2Et); 3.42 (s, NHCH2CON); 1.28 (d, J¼7.1, OCH2Me).
13C-NMR (75 MHz, CDCl3): 171.8; 171.6; 170.3; 61.2; 52.4; 51.9; 50.6; 40.8; 14.2. HR-ESI-MS: 233.1138
([MþH]þ , C9H18N2Oþ5 ; calc. 233.1132).
Dimethyl N-(2-Ethoxy-2-oxoethyl)glycyl-l-aspartate (7c). GP V was applied using 6c (788.0 mg,
2.0 mmol) and 10% Pd/C (37 mg) under H2 to afford 7c. Yield: 95%. Colorless oil. 1H-NMR (300 MHz,
CDCl3): 4.93–4.87 (m, NHCHCO2Me); 4.23–4.16 (m, PhCH2); 3.77 (s, MeO); 3.70 (s, MeO); 3.42 (s,
CH2CO2Et); 3.36 (s, NHCH2CON); 3.08–2.82 (m, NHCHCH2CO2Me); 1.28 (t, J¼7.1, OCH2Me).
13C-NMR (75 MHz, CDCl3): 172.1; 171.3; 171.2; 171.2; 61.1; 52.8; 52.2; 52.1; 50.7; 48.1; 36.3; 14.1. HR-
ESI-MS: 305.1455 ([MþH]þ , C12H21N2O7þ ; calc. 305.1349).
5’-O-{[(2-{[(1S)-1-Carboxy-2-(1H-imidazol-4-yl)ethyl]amino}-2-oxoethyl)(carboxymethyl)amino]-
hydroxyphosphinyl}-2’-deoxyadenosine (1f). GP V was applied using 6a (431.0 mg, 1.1 mmol) and 10%
Pd/C (37 mg) under H2 to furnish 7a (358 mg) as colorless oil. dAMP (50 mg, 0.15 mmol), 7a (200 mg,
0.75 mmol), and DCC (156 mg, 0.75 mmol) were dissolved in a mixture tBuOH (3 ml)/H2O (1 ml). The
mixture was heated at 858 for 6 h under Ar. After completion, the mixture was cooled, and solvent was
removed in vacuo. The resulting crude material was purified by FC (iPrOH/H2O/NH3 7:0.5 :0.5, 7:1:1,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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7:1:2), the obtained product was treated with 0.4m NaOH soln. in MeOH/H2O 1:4 (3 ml) for 4 h, the
solvent was removed in vacuo, and the resulting crude material was purified by HPLC to give 1f. Yield:
22% (over two steps). White solid. 1H-NMR (500 MHz, D2O): 8.40 (s, HꢀC(8)); 8.11 (s, HꢀC(2)); 7.50 (s,
NHCH¼C); 6.66 (s, NHCH¼N); 6.41 (t, J¼5.8, HꢀC(1’)); 4.58–4.56 (m, HꢀC(3’)); 4.28–4.26 (m,
CONHCH); 4.15–4.14 (m, HꢀC(4’)); 4.01–3.94 (m, CH2(5’)); 3.70–3.56 (m, NCH2CO2H); 3.41–3.13
(m, NCH2CONH); 2.97–2.82 (m, CHCH2Im) 2.55–2.48 (m, CH2(2’)). 13C-NMR (75 MHz, D2O): 178.2;
171.6; 173.1; 155.2; 152.2; 148.4; 139.5; 135.3; 135.2; 118.2; 118.1; 85.8; 83.2; 71.1; 64.3; 54.8; 51.7; 51.1;
38.3; 29.9. 31P-NMR (121 MHz, D2O): 7.98. HR-ESI-MS: 582.1469 ([MꢀH]ꢀ , C20H25N9O10Pꢀ ; calc.
582.1467).
5’-O-[((Carboxymethyl){2-[(carboxymethyl)amino]-2-oxoethyl}amino)hydroxyphosphinyl]-2’-de-
oxyadenosine (1g). GP III was applied using dAMP (100 mg, 0.30 mmol), 7b (348 mg, 1.5 mmol), and
DCC (433 mg, 2.10 mmol). Purification gave the desired intermediate as a white solid. Yield: 33%.
1H-NMR (600 MHz, D2O): 8.40 (s, HꢀC(8)); 8.17 (s, HꢀC(2)); 6.42 (t, J¼7.8, HꢀC(1’)); 4.63–4.62 (m,
HꢀC(3’)); 4.24–4.23 (m, HꢀC(4’)); 4.09–3.99 (m, CH2(5’), OCH2Me); 3.93 (s, NHCH2CO2H); 3.69 (s,
MeO); 3.68–3.67 (m, NCH2CO2Et, NCH2CON); 2.88–2.82 (m, 1 HꢀC(2’)); 2.64–2.59 (m, 1 HꢀC(2’));
1.14 (t, J¼9.0, OCH2Me). 13C-NMR (150 MHz, D2O): 174.3; 173.4; 172.8; 172.2; 155.5; 152.6; 148.7;
139.8; 118.5; 85.9; 83.5; 71.2; 64.4; 62.0; 52.7; 50.2; 48.8; 38.7; 35.2; 13.2. 31P-NMR (121 MHz, D2O): 6.89.
HR-ESI-MS: 544.1545 ([MꢀH]ꢀ , C19H26N7O10Pꢀ ; calc. 544.1562).
This intermediate was then treated with 0.4m NaOH soln. in MeOH/H2O 1:4 (5 ml) for 3 h, the
solvent was removed in vacuo, and the resulting residue was purified by FC (iPrOH/H2O/NH3 7:0.5 :0.5,
7:1:1, 7:1:2) to provide 1g. Yield: 44%. White solid. 1H-NMR (600 MHz, D2O): 8.49 (s, HꢀC(8)); 8.24
(s, HꢀC(2)); 6.49 (t, J¼7.8, HꢀC(1’)); 4.70–4.67 (m, HꢀC(3’)); 4.24–4.22 (m, HꢀC(4’)); 4.10–4.02 (m,
CH2(5’)); 3.75 (d, J¼9.6, NCH2CO2H); 3.73 (s, NHCH2CO2H); 3.59 (d, J¼13.2, NCH2CON); 2.85–2.80
(m, 1 HꢀC(2’)); 2.61–2.59 (m, 1 HꢀC(2’)). 13C-NMR (150 MHz, D2O): 178.8; 176.5; 174.2; 155.6; 152.7;
148.8; 134.0; 118.7; 86.1; 83.7; 71.3; 64.3; 52.4; 52.2; 43.1; 39.0. 31P-NMR (121 MHz, D2O): 8.12. HR-ESI-
MS: 502.1079 ([MꢀH]ꢀ , C16H21N7O10Pꢀ ; calc. 502.1088).
5’-O-{[(Carboxymethyl)(2-{[(1S)-1,2-dicarboxyethyl]amino}-2-oxoethyl)amino]hydroxyphosphin-
yl}-2’-deoxyadenosine (1h). GP III was applied using dAMP (100 mg, 0.30 mmol), 7c (456 mg,
1.5 mmol), and DCC (433 mg, 2.10 mmol). After purification, the desired intermediate as a obtained
as a white solid. Yield: 59%. 1H-NMR (600 MHz, D2O): 8.40 (s, HꢀC(8)); 8.19 (s, HꢀC(2)); 6.43 (t, J¼
6.6, HꢀC(1’)); 4.69–4.67 (m, HꢀC(3’), NHCHCO2Me); 4.19–4.18 (m, HꢀC(4’)); 4.06–3.97 (m, CH2(5’),
OCH2Me); 3.68 (s, MeO); 3.64 (s, MeO); 2.87–2.82 (m, NHCH2CO2Me, 1 HꢀC(2’)); 2.60–2.56 (m,
1 HꢀC(2’)); 1.14 (t, J¼7.2, OCH2Me). 13C-NMR (150 MHz, D2O): 173.4; 173.4; 172.8; 172.2; 155.5;
152.6; 148.7; 139.8; 118.6; 85.9; 83.5; 71.2; 64.4; 62.0; 53.1; 52.5; 52.0; 50.3; 48.8; 38.7; 35.2; 13.2. 31P-NMR
(121 MHz, D2O): 7.14. HR-ESI-MS: 544.1545 ([MꢀH]ꢀ , C19H26N7O10Pꢀ ; calc. 544.1562).
This intermediate was then treated with 0.4m NaOH soln. in MeOH/H2O 1:4 (5 ml) for 3 h, the
solvent was removed in vacuo, and the resulting residue was purified by FC (iPrOH/H2O/NH3 7:0.5 :0.5,
7:1:1, 7:1:2) to provide 1h. Yield: 38%. White solid. 1H-NMR (600 MHz, D2O): 8.47 (s, HꢀC(8)); 8.17
(s, HꢀC(2)); 6.44 (t, J¼7.8, HꢀC(1’)); 4.67–4.65 (m, HꢀC(3’)); 4.43–4.40 (m, HꢀC(a)(Asp)); 4.23–4.22
(m, HꢀC(4’)); 4.08–3.97 (m, CH2(5’)); 3.82–3.72 (m, NCH2CO2H); 3.70–3.47 (dd, J¼11.5, J¼17.9,
NCH2CONH); 2.81–2.76 (m, 1 HꢀC(2’)); 2.69–2.65 (m, 1 H, CH2(b)(Aspa)); 2.57–2.50 (m, 1 HꢀC(2’),
CH2(b)(Aspb), 1 H of CH2(b)(Aspa)). 13C-NMR (150 MHz, D2O): 178.2; 178.1; 178.0; 173.1; 155.1;
152.3; 148.3; 139.6; 118.1; 85.8; 83.2; 71.0; 64.1; 52.9; 51.5; 51.0; 39.4; 38.5. 31P-NMR (121 MHz, D2O):
8.05. HR-ESI-MS: 560.1153 ([MꢀH]ꢀ , C19H24N7O10Pꢀ ; calc. 560.1142).
Methyl 3-(benzylamino)propanoate (8). A soln. of BnNH2 (1.0 g, 9.33 mmol) in MeOH (1 ml) was
stirred at r.t. for 1 d, in the presence of methyl acrylate (756 ml, 8.40 mmol). The mixture was then
concentrated in vacuo, the resulting crude material was purified by FC (hexane/AcOEt 5 :1) to furnish 8.
Yield: 93%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 7.34–7.26 (m, Ph); 3.82 (s, NHCH2Ph); 3.70 (s,
MeO); 3.92 (t, J¼6.5, NHCH2CH2CO2Me); 2.55 (t, J¼6.5, NHCH2CH2CO2Me). 13C-NMR (75 MHz,
CDCl3): 173.1; 140.0; 128.4; 128.1; 127.0; 53.7; 51.6; 44.4; 34.5. HR-ESI-MS: 194.1190 ([MþH]þ
,
C11H16NOþ2 ; calc. 194.1181).
tert-Butyl Methyl 3,3’-(Benzylimino)dipropanoate (9). A soln. of 8 (1.67 g, 8.65 mmol) in MeOH
(1 ml) was stirred at r.t. for 1 d in the presence of tert-butyl acrylate (1.40 ml, 9.52 mmol). The mixture

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
was then concentrated in vacuo, and the resulting crude material was purified by FC (hexane/AcOEt
5 :1) to give 9. Yield: 66%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 7.27–7.22 (m, Ph); 3.61 (s, MeO);
t
3.56 (s, NCH2Ph); 2.79–2.73 (m, NCH2CH2CO2Me, NCH2CH2CO2 Bu); 2.44 (t, J¼7.2, NCH2CH2-
t
t
CO2Me); 2.36 (t, J¼7.2, NCH2CH2CO2 Bu); 1.41 (s, Bu). 13C-NMR (75 MHz, CDCl3): 172.9; 171.8;
139.1; 128.7; 128.1; 126.9; 80.2; 58.3; 51.4; 49.4; 49.1; 33.8; 32.5; 28.0. HR-ESI-MS: 322.2016 ([MþH]þ
,
C18H28NOþ4 ; calc. 322.4192).
N-Benzyl-N-(3-methoxy-3-oxopropyl)-b-alanine Trifluoroacetate (10). Diester 9 (1.83 g, 5.69 mmol)
was treated with TFA (4.23 ml, 56.9 mmol) in CH2Cl2 (40ml) for 2 d at r.t. After removal of the solvent
and excess of TFA, the crude residue was triturated with Et2O and filtered to afford 10 as TFA salt. Yield:
97%. White solid. 1H-NMR (300 MHz, (D6)DMSO): 7.55–7.46 (m, Ph); 4.40 (s, NCH2Ph); 3.62 (s,
MeO); 3.35–3.27 (m, NCH2CH2CO2Me, NCH2CH2CO2H); 2.89 (t, J¼7.4, NCH2CH2CO2Me); 2.79 (t,
J¼7.4, NCH2CH2CO2H). 13C-NMR (75 MHz, (D6)DMSO): 171.5; 170.4; 131.2; 129.8; 129.7; 129.0; 56.6;
51.9; 47.7; 47.4; 28.2; 28.0. HR-ESI-MS: 266.1386 ([MþH]þ , C14H20NO4þ ; calc. 266.3129).
Methyl N-Benzyl-N-(3-methoxy-3-oxopropyl)-b-alanylglycinate (11a). GP IV was applied using 10
(TFA salt) (500.0 mg, 1.32 mmol), glycine methyl ester hydrochloride (166.0 mg, 1.32 mmol), Et3N
(550 ml, 3.95 mmol), HOBt (267.1 mg, 1.97 mmol), and EDCI (379.0 mg, 1.97 mmol). After purification,
11a was obtained. Yield: 57%. White solid. 1H-NMR (300 MHz, CDCl3): 8.08 (br. s, NH); 7.38–7.30 (m,
Ph); 3.95 (d, J¼5.4, NHCH2CO2Me); 3.70 (s, NCH2CH2CO2Me); 3.57 (s, NHCH2CO2Me); 3.55 (s,
NCH2Ph); 2.80 (t, J¼6.9, NCH2CH2CONH); 2.73 (t, J¼6.2, NCH2CH2CO2Me); 2.50 (t, J¼6.9,
NCH2CH2CONH); 2.40 (t, J¼6.2, NCH2CH2CO2Me). 13C-NMR (75 MHz, CDCl3): 172.6; 172.5; 170.2;
137.6; 129.0; 128.2; 127.1; 66.0; 51.8; 51.4; 49.8; 48.9; 40.7; 32.8; 31.8. HR-ESI-MS: 337.1763 ([MþH]þ
,
C17H25N2Oþ5 ; calc. 337.3908).
Dimethyl N-Benzyl-N-(3-methoxy-3-oxopropyl)-b-alanyl-l-aspartate (11b). GP IV was applied
using 10 (TFA salt) (500.0 mg, 1.32 mmol), l-aspartic acid dimethyl ester hydrochloride (260.8 mg,
1.32 mmol), Et3N (550 ml, 3.95 mmol), HOBt (267.1 mg, 1.97 mmol) and EDCI (379.0 mg, 1.97 mmol).
After purification, 11b was obtained. Yield: 57.5%. White solid. 1H-NMR (300 MHz, CDCl3): 8.20 (br. s,
NH); 7.27–7.21 (m, Ph); 4.89–4.83 (m, NHCHCO2Me); 3.70 (s, NHCHCO2Me); 3.62 (s,
NHCHCH2CO2Me); 3.60 (s, NCH2CH2CO2MeþNCH2Ph); 2.97–2.83 (m, NHCHCH2CO2Me); 2.81–
2.71 (m, NCH2CH2CONH, NCH2CH2CO2Me); 2.49 (t, J¼7.2, NCH2CH2CO2Me); 2.38 (t, J¼6.0,
NCH2CH2CONH). 13C-NMR (75 MHz, CDCl3): 172.5; 171.9; 171.0; 17O.5; 137.6; 128.8; 128.1; 127.0;
57.8; 52.3; 51.6; 51.3; 49.8; 48.5; 48.1; 36.0; 33.0; 31.3. HR-ESI-MS: 409.1969 ([MþH]þ , C20H29N2O7þ ;
calc. 409.1975).
Methyl N-(3-Methoxy-3-oxopropyl)-b-alanylglycinate (12a). GP V was applied using 11a (250.0 mg,
0.74 mmol) and 10% Pd/C (26 mg) under H2. After filtration and concentration, 12a was obtained. Yield:
96%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 8.23 (br. s, NH); 3.98 (s, NHCH2CO2Me); 3.70 (s,
NHCH2CO2Me, NHCH2CH2CO2Me); 3.35–3.23 (m, NHCH2CH2CO2Me, NHCH2CH2CONH); 3.01–
2.94 (m, NHCH2CH2CO2Me, NHCH2CH2CONH). 13C-NMR (75 MHz, CDCl3): 171.0; 170.9; 170.5;
52.3; 52.2; 44.5; 43.6; 40.9; 31.1; 30.2. HR-ESI-MS: 247.1285 ([MþH]þ , C10H19N2O5þ ; calc. 247.2683).
Dimethyl N-(3-Methoxy-3-oxopropyl)-b-alanyl-l-aspartate (12b). GP V was applied using 11b
(310.0 mg, 0.76 mmol) and 10% Pd/C (26 mg) under H2. After filtration and concentration, 12b was
obtained. Yield: 71%. Colorless oil. 1H-NMR (300 MHz, CDCl3): 8.24 (d, NH); 4.82–4.76 (m,
NHCHCO2Me); 4.66 (br. s, NH); 3.67 (s, NHCHCO2Me); 3.63 (s, NHCHCH2CO2Me); 3.61 (s,
NHCH2CH2CO2Me); 3.00–2.93 (m, NHCH2CH2CONH, NHCH2CH2CO2Me); 2.92–2.74 (m, NHCH-
CH2CO2Me); 2.64 (t, J¼6.2, NHCH2CH2CO2Me); 2.56–2.52 (m, NHCH2CH2NCONH);13C-NMR
(75 MHz, CDCl3): 172.2; 171.6; 171.1; 171.0; 52.5; 51.8; 51.7; 48.3; 44.7; 44.0; 35.9; 33.7; 32.6. HR-ESI-
MS: 319.1501 ([MþH]þ , C13H23N2O7þ ; calc. 319.1505).
5’-O-[((2-Carboxyethyl){3-[(carboxymethyl)amino]-3-oxopropyl}amino)hydroxyphosphinyl]-2’-de-
oxyadenosine (1i). GP III was applied using dAMP (100 mg, 0.30 mmol), 12a (369 mg, 1.5 mmol), and
DCC (433 mg, 2.10 mmol). After purification, the obtained crude material was treated with 0.4m NaOH
soln. in MeOH/H2O (1:4) (5 ml) for 3 h. Solvent was removed in vacuo, and the resulting crude product
was purified by FC (iPrOH/H2O/NH3 7:0.5 :0.5, 7:1:1, 7:1:2) to furnish 1i. Yield: 28% (over two steps).
1
White solid. H-NMR (600 MHz, D2O): 8.42 (s, HꢀC(8)); 8.18 (s, HꢀC(2)); 6.45 (t, J¼6.8, HꢀC(1’));
4.70–4.69 (m, 2 H, HꢀC(3’)); 4.21–4.20 (m, HꢀC(4’)); 3.89–3.85 (m, CH2(5’)); 4.64–3.58 (m,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2699
NHCH2COOH); 3.14–3.12 (m, NCH2CH2CONH); 3.05–3.02 (m, NCH2CH2COOH); 2.88–2.79 (m,
2 H, CH2CH2COOH, 1 HꢀC(2’)); 2.59–2.52 (m, 1 HꢀC(2’)); 2.34–2.56 (m, CH2CH2COOH); 2.24–2.17
(m, 1 H, NCH2CH2CONH). 13C-NMR (150 MHz, D2O): 178.6; 176.0; 173.7; 156.9; 152.8; 149.7; 141.3;
119.9; 85.8; 85.1; 70.6; 63.4; 42.7 (3C); 38.0; 35.5; 35.3. 31P-NMR (121 MHz, D2O): 8.48. HR-ESI-MS:
530.1409 ([MꢀH]ꢀ , C18H25N7O10Pꢀ ; calc. 530.1401).
5’-O-{[(2-Carboxyethyl)(3-{[(1S)-1,2-dicarboxyethyl]amino}-3-oxopropyl)amino]hydroxyphos-
phinyl}-2’-deoxyadenosine (1j). GP III was applied using dAMP (100 mg, 0.30 mmol), 12b (477 mg,
1.5 mmol), and DCC (433 mg, 2.10 mmol). After purification, the resulting crude product was treated
with 0.4m NaOH soln. in MeOH/H2O (1:4) (5 ml) for 3 h. Solvent was removed in vacuo, and the
resulting crude product was purified by FC (iPrOH/H2O/NH3 7:0.5 :0.5, 7:1:1, 7:1:2) to furnish 1j.
White solid. Yield: 32%. 1H-NMR (600 MHz, D2O): 8.43 (s, HꢀC(8)); 8.19 (s, HꢀC(2)); 6.43 (t, J¼6.6,
HꢀC(1’)); 4.70–4.68 (m, HꢀC(3’)); 4.39–4.37 (m, HꢀC(a)(Asp)); 4.19–4.17 (m, HꢀC(4’)); 3.87–3.83
(m, CH2(5’)); 3.06–3.00 (m, 4 H, NCH2CH2CO); 2.86–2.81 (m, 1 HꢀC(2’)); 2.68–2.65 (m, 1 H,
CH2(b)(Aspa)); 2.58–2.53 (m, 2 H, CH2(b)(Aspb), 1 HꢀC(2’)); 2.31–2.29 (m, 4 H, NCH2CH2CO).
13C-NMR (150 MHz, D2O): 179.7; 178.6; 178.0; 175.0; 155.6; 150.4; 149.7; 139.9; 119.9; 85.8; 85.6; 83.6;
70.6; 63.3; 51.3; 42.8; 42.4; 38.5; 35.6; 34.8. 31P-NMR (121 MHz, D2O): 8.48. HR-ESI-MS: 588.1462
([MꢀH]ꢀ , C20H27N7O12Pꢀ ; calc. 588.1455).
Ethyl N-[(Benzyloxy)carbonyl]glycyl-O-ethyl-3-oxoserinate (13). GP IV was applied using diethyl
aminomalonate hydrochloride (1.0 g, 4.72 mmol), Et3N (722 ml, 5.20 mmol), N-[(benzyloxy)carbonyl]-
glycine (988.4 mg, 4.72 mmol), HOBt (957.5 mg, 7.809 mmol), and EDCI (1.36 g, 7.09 mmol). After
purification, 13 was obtained. Yield: 87%. White solid. 1H-NMR (300 MHz, CDCl3): 7.38–7.29 (m, Ph);
6.91 (br. s, NH); 5.36, (s, NH); 5.16–5.14 (m, PhCH2, CHCO2Et); 4.33–4.23 (m, 2 CO2CH2Me); 4.00–
3.98 (m, NHCH2); 1.30 (t, J¼7.0, 2 CO2CH2Me). 13C-NMR (75 MHz, CDCl3): 168.7; 167.3; 166.0; 139.9;
128.5; 128.2; 128.1; 67.3; 62.8; 55.1; 44.3; 14.0 . HR-ESI-MS: 367.1508 ([MþH]þ , C17H23N2O7þ ; calc.
367.1500).
Ethyl Glycyl-O-ethyl-3-oxoserinate Hydrochloride (14). Compound 13 (300 mg, 0.82 mmol) was
dissolved in EtOH (5 ml) and reduced under H2 for 6 h, in the presence of 10% Pd/C (29 mg) and HCl
(50 ml). The mixture was then filtered on Celite and concentrated in vacuo. Compound 14 was obtained as
a colorless oil without additional purification. Yield: 88%. 1H-NMR (300 MHz, CDCl3): 9.12 (br. s, NH);
5.20, (s, CHCO2Et); 4.27–4.20 (m, 2 CO2CH2Me); 3.78 (s, CH2NH2); 1.29 (t, J¼7.0, 2 CO2CH2Me).
13C-NMR (75 MHz, CDCl3): 167.4; 167.3; 63.7; 57.9; 41.5; 14.3. HR-ESI-MS: 233.1135 ([MþH]þ
,
C9H17N2Oþ5 ; calc. 233.1132).
5’-O-[({2-[(Carboxymethyl)amino]-2-oxoethyl}amino)hydroxyphosphinyl]-2’-deoxyadenosine (1k).
dAMP (50 mg, 0.15 mmol), 14 (194 mg, 0.76 mmol), and DCC (156 mg, 0.76 mmol) were dissolved in a
mixture tBuOH (3 ml)/H2O (1 ml). The mixture was heated at 858 for 6 h under Ar. After completion,
the mixture was cooled, and the solvent was removed in vacuo. The resulting crude material was purified
by FC (iPrOH/H2O/NH3 7:0.5 :0.5, 7:1:1, 7:1:2), the obtained product was treated with 0.4m NaOH
soln. in MeOH/H2O 1:4 for 4 h, solvent was removed in vacuo, the resulting crude material was purified
by HPLC to give 1k. Yield: 32% (over two steps). White solid. 1H-NMR (500 MHz, D2O): 8.39 (s,
HꢀC(8)); 8.20 (s, HꢀC(2)); 6.45 (t, J¼5.8, HꢀC(1’)); 4.70–4.67 (m, HꢀC(3’)); 4.17–4.14 (m, HꢀC(4’));
4.03–3.92 (m, CH2(5’)); 3.64 (s, NHCH2CO2H); 3.54 (d, NHCH2CONH); 2.84–2.52 (m, CH2(2’)).
13C-NMR (75 MHz, D2O): 178.2; 171.6; 155.5; 152.6; 148.6; 139.7; 118.6; 85.6; 83.6; 71.2; 64.1; 44.3; 42.8;
38.6. 31P-NMR (121 MHz, D2O): 7.73. HR-ESI-MS: 444.1042 ([MꢀH]ꢀ , C14H19N7O8Pꢀ ; calc. 444.1033).
Enzymatic Reactions. DNA Duplexes. Oligonucleotides P1 and T1 were purchased from Sigma
Genosys. The concentrations were determined with a Varian Cary-300-Bio UV spectrophotometer. The
primer P1 was 5’-33P-labeled with 5’-[g33P]-ATP (Perkin-Elmer) using T4 polynucleotide kinase (New
England Biolabs) according to the procedure provided by the supplier. The labeled oligonucleotide was
further purified using IllustraTM Microspin TM G-25 columns (GE Healthcare).
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 758 for 10 min, followed by slowly cooling
to r.t. over a period of 1.5 h. For the incorporation of 1a–1k, a series of 20-ml-batch reactions were
performed with the enzyme (HIV-1 RT, Taq, Vent (exoꢀ), Therminator DNA polymerases). The final
mixture contained 125 nm primerꢀtemplate complex, reaction buffer (50 mm Tris·HCl, 50 mm KCl,

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
10 mm MgCl2, 0.5 mm spermidine, 10 mm dithiothreitol (DTT); pH 8.3 for HIV-1 RT; 20 mm Tris·HCl,
10 mm KCl, 2 mm MgSO4, 0.1% Triton X-100, pH 8.8 for thermostable polymerases), appropriate
concentration of enzyme, and different concentrations of phosphoramidate building blocks (1 mm,
500 mm, 200 mm, and 100 mm). In the control reaction, a 10-mm dATP was used as reference. The mixture
was incubated at 378 or 758, resp., aliquots were quenched after 10, 20, 30, 60, and 120 min.
Polyacrylamide Gel Electrophoresis. All polymerase-reaction aliquots (2.5 ml) were quenched by
addition of 10 ml of loading buffer (90% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol, and
50 mm ethylenediaminetetraacetic acid (EDTA)). Samples were heated at 858 for 3 min prior to analysis
by electrophoresis for 2.5 h at 2000 V on a 30 cmꢁ40 cmꢁ0.4 mm 20% (mono/bis 19 :1) 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 imaging device Cycloneꢂ and the Optiquant image analysis
software (Perkin-Elmer).
The authors thank FWO and K.U. Leuven (IDO, GOA) for financial support, Jef Rozenski for
providing HR-MS, Luc Baudemprez for recoding the NMR spectra and Chantal Biernaux for editorial
help.
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Received July 26, 2012