Direct PCR amplification of various modified DNAs having amino acids: Convenient preparation of DNA libraries with high-potential activities for in vitro selection

Article abstract of DOI:10.1016/j.bmc.2005.11.030

We synthesized modified 2′-deoxyuridine triphosphates bearing amino acids at the C5 position and investigated their substrate properties for KOD Dash DNA polymerase during polymerase chain reaction (PCR). PCR using C5-modified dUTP having an amino acyl group (arginyl, histidyl, lysyl, phenylalanyl, tryptophanyl, leucyl, prolyl, glutaminyl, seryl, O-benzyl seryl or threonyl group) gave the corresponding full-length PCR products in good yield. Although dUTP analogues bearing aspartyl, glutamyl or cysteinyl were found to be poor substrates for PCR catalyzed by KOD Dash DNA polymerase, optimization of the reaction conditions resulted in substantial generation of full-length product. In the case of reaction using dUTP analogue having a cysteinyl group, addition of a reducing agent improved the reaction yield. Thus, PCRs using KOD Dash DNA polymerase together with amino acyl dUTP provide convenient and efficient preparation of various modified DNA libraries with potential protein-like activities.

Full text of DOI:10.1016/j.bmc.2005.11.030

Bioorganic & Medicinal Chemistry 14 (2006) 2518–2526
Direct PCR amplification of various modified DNAs having
amino acids: Convenient preparation of DNA libraries with
high-potential activities for in vitro selection
Masayasu Kuwahara,a,b Kazuo Hanawa,a Kazuomi Ohsawa,a Rina Kitagata,a
Hiroaki Ozakia and Hiroaki Sawaia,*
aDepartment of Applied Chemistry, Faculty of Engineering, Gunma University, Gunma 376-8515, Japan
bPRESTO, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan
Received 9 October 2005; revised 12 November 2005; accepted 15 November 2005
Available online 15 December 2005
Abstract—We synthesized modified 20-deoxyuridine triphosphates bearing amino acids at the C5 position and investigated their sub-
strate properties for KOD Dash DNA polymerase during polymerase chain reaction (PCR). PCR using C5-modified dUTP having
an amino acyl group (arginyl, histidyl, lysyl, phenylalanyl, tryptophanyl, leucyl, prolyl, glutaminyl, seryl, O-benzyl seryl or threonyl
group) gave the corresponding full-length PCR products in good yield. Although dUTP analogues bearing aspartyl, glutamyl or
cysteinyl were found to be poor substrates for PCR catalyzed by KOD Dash DNA polymerase, optimization of the reaction con-
ditions resulted in substantial generation of full-length product. In the case of reaction using dUTP analogue having a cysteinyl
group, addition of a reducing agent improved the reaction yield. Thus, PCRs using KOD Dash DNA polymerase together with ami-
no acyl dUTP provide convenient and efficient preparation of various modified DNA libraries with potential protein-like activities.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
engineered variant of T7 DNA polymerase (Sequenase)
and Moloney murine leukemia virus (M-MLV) reverse
transcriptase (SuperScript II), respectively. Thus, primer
extension16–18 or one-primer polymerase chain reaction
(PCR) is used widely instead of symmetric PCR, which
can amplify DNA exponentially, to prepare modified
DNA for in vitro selection. It is vital in symmetric
PCR that the modified nucleoside triphosphates act as
good substrates for thermostable DNA polymerase and
that the modified DNAs work as templates during the
amplification process. Therefore, the combinations of
modified nucleoside triphosphates and thermostable
DNA polymerases that result in efficient amplification
by PCR are limited.
In vitro selection techniques have been used to select
DNA catalysts and DNA aptamers that have activities
similar to those of protein–enzymes and antibodies.1–6
To date, various functionally modified nucleic acids have
been developed to enhance activity or to diversify func-
tion.7–15 In particular, Perrin et al.13,14 and Sidorov
et al.15 invented modified DNAzymes with two function-
alities, imidazolyl and a primary amino group, that can
catalyze RNA hydrolysis in the absence of divalent metal
ions. These examples demonstrated that a DNA mole-
cule modified with plural functionalities found in the ac-
tive sites of protein–enzymes can exhibit protein-like
activity. However, preparation of modified DNAs hav-
ing plural functionalities is difficult using polymerase
reactions. These modified DNA libraries were prepared
by primer extension reaction using C8- or C7-modified
dATP and C5-modified dUTP together with a genetically
Among commercially available thermostable DNA
polymerases, it is known that KOD Dash DNA poly-
merase from Pyrococcus kodakaraensis shows high poly-
merase activity and excellent fidelity.19–22 For instance,
polymerization reaction rate with KOD Dash DNA
polymerase is twofold faster than that with Taq DNA
polymerase and about sixfold faster than that with Pfu
DNA polymerase. Moreover, the fidelity of KOD Dash
DNA polymerase is about 3.4-fold higher than that
of Taq DNA polymerase. Thus, KOD Dash DNA
Keywords: Modified DNA; Modified thymidine triphosphate; Poly-
merase chain reaction; Amino acid.
*
Corresponding author. Tel.: +81 27 730 1220; fax: +81 27 730
1224; e-mail: sawai@chem.gunma-u.ac.jp
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.030

M. Kuwahara et al. / Bioorg. Med. Chem. 14 (2006) 2518–2526
2519
polymerase is the one of the best catalysts for PCR. In a
previous study, KOD Dash DNA polymerase was found
to accept 5-(2-(6-aminohexylamino)-2-oxoethyl)-dUTP
(TC6) as an alternative to TTP during PCR, efficiently
yielding the corresponding modified DNA.23 We report
here the convenient and efficient preparation of modified
DNAs having two different functionalities (primary ami-
no group and a group such as imidazolyl, guanidinyl,
hydroxyl, etc.) by PCR using a series of C5-modified
dUTPs carrying amino acids.
We first investigated the effects of linker structure on
amplification efficiency of PCR. Under condition 3,
the analogue TC6 with a 2-(6-aminohexylamino)-2-oxo-
ethyl linker was found to be the best substrate for
PCR among several known C5-modified dUTPs used
in this study (Fig. 1, Table 1). The formation of the cor-
responding natural-type DNA from the natural sub-
strate TTP was 1.4 times larger compared with that
from TC6 under optimized conditions for the formation
of natural-type DNA, where the enzyme quantity was
reduced to suppress the non-specific amplification. With
regard to relative yields of PCR using KOD Dash DNA
polymerase, that of TC2 with 2-(2-aminoethylamino)-2-
oxoethyl linker four methylene units shorter than that
in TC6 decreased to 0.48. While use of TAL provided a
product in a relative yield of 0.77, use of TPR, which is
known to be a substrate for PCR using Taq DNA poly-
merase,32 did not provide any product. Moreover, use of
TDH was found to decrease the relative yield to 0.34,
which was lower than when TC2 or TAL was used. These
results indicated that the distance between amino groups
with positive charge and nucleobase, the chemical struc-
ture of the linker as well as a type of DNA polymerase
affects generation of the PCR product. Interestingly
however, these five modified dUTPs were found to be
good substrates for template-directed primer extension
reactions (see Supplementary material). Therefore, when
preparing modified DNAs by PCR, template activities
of the PCR product as well as substrate properties of
the modified nucleoside triphosphates for thermostable
DNA polymerases are important factors in amplifying
modified DNAs.
2. Results
All aminoacyl dUTPs, except for sulfur-containing ana-
0
logues, such as TC6C and TC6C , were synthesized from
amino acids with hydrogenation-sensitive protecting
groups such as benzyloxycarbonyl and benzyl groups
(Scheme 1). Reaction of the amino acid with N-hydroxy-
succinimide in the presence of N,N0-dicyclohexylcarbo-
diimide gave the corresponding N-succinimide ester.
Then, coupling of TC6 with the ester followed by
hydrogenation to remove the protecting groups gave
T
T
C6H, TC6K, TC6R, TC6F, TC6W, TC6L, TC6P, TC6Q
,
0
C6S, TC6S , TC6T, TC6D, and TC6E. The sulfur-contain-
0
ing analogues (TC6C and TC6C ) were synthesized from
N-a-(9-fluorenylmethoxycarbonyl)-S-tert-butylthio-L-
cysteine because sulfur inhibits catalysis of the Pd/C
used for the hydrogenation reaction. After the coupling
reaction, the Fmoc and S-t-butyl groups were success-
fully removed by treatment with diethylamine and
dithiothreitol (DTT), respectively.
CbzHN
NHCbz
CbzHN
NHCbz
NHCbz
N
O
O
N
A
H
N
N
N
O
CbzHN
N
H
NH
O
HOSu
TC6
CbzHN
O
O
P
O
P
O
P
O
N
DCC
DMF
1M NaHCO3 aq
DMF
HO
O
O
O
O
OH
OH
CbzHN
CbzHN
OH OH OH
O
O
O
NH
O
O
N
H
N
H2N
N
H
N
H
NH
O
H2, Pd/C
H2N
O
O
P
O
P
O
P
Water/MeOH (1/1)
HO
O
O
O
O
OH
OH OH OH
TC6R
O
O
H
B
tBuS
N
StBu
S
StBu
S
S
N
NH
H
O
HOSu
O
TC6
FmocHN
N
O
O
P
O
P
O
P
OH
O
FmocHN
FmocHN
N
DCC
DMF
1M NaHCO3 aq
DMF
HO
O
O
O
O
O
O
OH OH OH
O
OH
O
O
O
O
N
H
H
tBuS
N
N
S
N
NH
HS
N
H
NH
H
O
O
Diethylamine
DTT
H2N
H2N
O
N
O
O
O
P
O
P
O
P
O
O
P
HO
O
O
O
HO
P
O
P
O
O
O
O
OH OH OH
OH OH OH
OH
OH
TC6C'
TC6C
Scheme 1. Synthetic routes of modified dUTPs bearing (A) argininyl group and (B) cysteinyl group via 2-(6-aminohexylamino)-2-oxoethyl linker at
the C5 position of uracil, respectively.

2520
M. Kuwahara et al. / Bioorg. Med. Chem. 14 (2006) 2518–2526
R1 =
O
H
N
R1
TAL
HN
NHR2
NH2
NH2
O
O
N
O
P
O
P
O
P
HO
O
O
O
H
N
O
OH OH OH
TC2
TPR
NH2
OH
O
O
O
TDH
NH2
TC6
NH2
N
R2 =
H
O
O
O
O
O
NH2
NH2
NH2
NH2
NH
NH
TC6H
TC6K
TC6R
TC6F
TC6W
NH
NH2
NH2
HN
O
O
O
O
O
H
N
NH2
NH2
NH2
OH
NH2
O
TC6L
TC6P
TC6Q
TC6S
TC6S'
O
NH2
O
O
O
O
O
NH2
OH
NH2
SH
NH2
NH2
O
NH2
S
S
TC6T
TC6C
TC6C'
TC6D
TC6E
OH
O
OH
Figure 1. Chemical structures and abbreviations of C5-modified dUTP analogs used in this study.
Table 1. Relative yield of the modified DNAs from dUTP analogs by
PCR
dUTP analog
residue < cationic residue <aromatic residue 6 aliphatic
residue 0ꢀ neutral hydrophilic residue, except for TC6C
or TC6C . For TC6C, PCR under conditions 1 and 2 pro-
vided no product, presumably due to dimerization of
TC6C by disulfide bond formation,24–26 because addition
of the reducing agent dithiothreitol (DTT) to the reac-
tion mixture improved generation of the PCR product
(Fig. 4). Actually, in our previous study, a dUTP ana-
Relative yield
TC6
TC2
TDH
TAL
TPR
1
0.48
0.34
0.77
0
logue with a thiol group forms a disulfide bond, which
Relative yield is expressed by the ratio of the resulting modified DNA
from each dUTP analogue to that from TC6 under the PCR condition
3 (20 cycles, 0.5 min at 94 ꢁC for denaturing, 0.5 min at 52 ꢁC for
annealing, and 1 min at 74 ꢁC for extension). All experiments were run
five times and the averaged data are shown.
0
was found to be reduced by DTT.26 For TC6C , the
PCR product obtained in the absence of DTT formed
smears (Fig. 3, lane 15). Addition of DTT to the reac-
tion mixture led to a sharp band of gel mobility equal
to that derived from TC6C in the presence of DTT,
although long-chain DNA which could be formed by
non-specific amplification was present in a substantial
amount (Fig. 4). The sharp band was identified by
sequencing involving a cloning method as described in
experimental section.
We then investigated the effects of tethered amino acids
on amplification efficiency of PCR. Fifteen amino acyl
dUTPs were synthesized and their substrate properties
were evaluated by PCR assays (Figs. 2A–C, Fig. 3,
and Table 2). In all cases, relative yields of PCR were
similar or somewhat improved by changing the reaction
conditions. However, it is still necessary to optimize
PCR parameters, such as temperature and time for each
process as well as number of cycles. For example, with
To confirm the accurate incorporation of amino acyl
dUTPs instead of natural TTP during PCR, modified
DNAs derived from TC6H, TC6R, TC6F, TC6W, TC6L
,
TC6C (with 4 mM DTT) or TC6E were sequenced (Figs.
2C–F). Five samples were chosen at random from
among single colonies derived from each modified
DNA, and their sequences were confirmed. Misincorpo-
ration was not observed in any of the samples analyzed,
indicating that the modified dUTPs were accurately
incorporated into the corresponding modified DNAs,
in which the original sequence was conserved (Fig. 5).
T
C6D or TC6E, a small amount of the full-length product
of modified DNA was provided under condition 2, while
no product was yielded under condition 1. The results of
PCR assays indicated that the chemical structure of the
side chain residue on the tethered amino acid consider-
ably affects the amplification efficiency of PCR, and
relative yields were found to be as follows: anionic

M. Kuwahara et al. / Bioorg. Med. Chem. 14 (2006) 2518–2526
2521
Figure 2. Schematic illustration of PCR assays (A–C) and sequence analyses of PCR products (C–F).
3. Discussion
Sequence analyses of the PCR products showed that the
original sequences were conserved in the products derived
from modified dUTPs, indicating that the modified
DNAs obtained in this method are applicable to in vitro
selection. Modification of DNA with residues found in
the catalytic centers of enzymes (histidyl, lysyl, seryl, cys-
teinyl, aspartyl or glutamyl) would enhance the potential
of libraries for catalytic DNA. Modification with residues
such as phenylalanyl, tryptophanyl, leucyl or threonyl
may cause local distortion of conformation that induces
formation of unique hydrophobic cavities for catalytic-
or binding sites via hydrogen bonding or p-p stacking
interactions between strands of DNA. In addition, highly
active DNA aptamers against anionic target molecules,
which cannot be obtained from natural DNA, may
emerge from modified DNA libraries having cationic
guanidinyl (Arg) or amino groups (Lys). A combinatorial
modified DNA library containing arginyl uracil bases was
prepared using TC6R, and screening for glutamic acid-
binding aptamers from this library is currently underway.
Moreover, apart from applications to in vitro selection,
enzymatic preparation of modified DNAs could be ap-
plied to conferring new abilities to DNA. For instance,
serial sequences of uracil with guanidinyl groups, which
were enzymatically attached to the 30-end of oligodeoxy-
nucleotide (ODN) using a TC6 derivative, performed as a
carrier for cellular uptake of ODN34. Thus, expansion of
modified DNA repertories with unique functionalities,
We synthesized C5-modified dUTPs bearing amino
acids by coupling of TC6 with commercially available
protected amino acids and subsequent removal of the
protecting groups by conventional hydrogenation,
which is applicable to TC6 derivatives because the linker
arm is not sensitive to catalytic reduction. This synthetic
method would also be useful for tethering a variety of
short peptides to the amino terminus of TC6. Prepara-
tions of modified DNA with 2–4 functionalities by sym-
metric PCR simultaneously using 2–4 different modified
nucleoside triphosphates have been reported27,28; how-
ever, multiple functionalities could be conveniently
incorporated into DNA by using only a single type of
dUTP analogue bearing amino acids or peptides.
Using a series of amino acyl dUTPs instead of natural
TTP, a variety of modified DNAs with amino acids were
prepared by PCR catalyzed with KOD Dash DNA poly-
merase. The relative yields of PCR products were found
to depend on the structure of substituents in the ana-
logue, as reported in previous studies.23,25–33 The sub-
strate specificity of the modified nucleotides is likely
varied with their chemical structures, amino acid
sequences, and ternary structure of DNA polymerase;
however, direct experimental evidence to explain this
phenomenon has not yet been reported.

2522
M. Kuwahara et al. / Bioorg. Med. Chem. 14 (2006) 2518–2526
M
1
2
3
4
5
M
1
2
5
6
7
8
9
3
4
200 nt
200 nt
100 nt
100 nt
Figure 4. PAGE gel image of the ethidium bromide stained and UV
visualized 108 nt PCR product derived from0 pUC18 and the
C5-modified dUTP analogs (TC6, TC6C or TC6C ) with or without
M
10 11 12 13 14 15 16
17
dithiothreitol (DTT) under condition 2. The reaction mixtures
0
containing dATP, dGTP, dCTP, and TC6 (lane 1), TC6C with 2 mM
DTT (lane 2), TC6C with 2 mM DTT (lane 3), TC6C with 4 mM DTT
(lane 4) or TC6C with 10 mM DTT (lane 5) were electrophoresed on
denaturing PAGE, respectively. All reaction mixtures, except for
positive control, do not contain natural TTP. A reaction mixture
containing all four natural dNTPs with 2 mM DTT for positive
control provided the PCR product, and a reaction mixture containing
dATP, dGTP, and dCTP with 2 mM DTT for negative control did not
result in formation of any product (data omitted).
200 nt
100 nt
Figure 3. PAGE gel image of the ethidium bromide stained and UV
visualized 108 nt PCR product derived from pUC18 and the
C5-modified dUTP analogs under condition 2. The full experimental
conditions are described in Materials and methods. The reaction
mixtures containing dATP, dGTP, dCTP and TC6 (lane 1, 10), TC6H
(lane 2), TC6K (lane 3), TC6R (lane 4), TC6F (lane 5), TC6W (la0ne 6), TC6L
(lane 7), TC6P (lane 8), TC6Q (lane 9), TC6S (lane 11), TC6S (lane 12),
C6C0
TC6T (lane 13), TC6C (lane 14), T
(lane 15), TC6D (lane 16) or TC6E
(lane 17) were electrophoresed on denaturing PAGE, respectively. All
reaction mixtures, except for positive control, do not contain natural
TTP. A reaction mixture containing all four natural dNTPs for
positive control provided the PCR product, and a reaction mixture
containing dATP, dGTP, and dCTP for negative control did not result
in formation of any product (data omitted).
Figure 5. Sequence analysis of the PCR product derived from pUC18
and phenylalanyl dUTP (TC6F) instead of natural TTP. The figure
shows DNA sequences (25–108) excluding the primer region.
such as amino acyl or peptidyl groups, would enhance the
potential of DNA as a functional molecule.
under atmospheric pressure ionization conditions. UV
analyses were performed on a Shimadzu UV-1200 spec-
1
trometer. H and 31P NMR spectra were recorded on a
4. Experimental
JEOL JNM-AL300 or JNM-LA500 FT-NMR spec-
trometer. Tetramethylsilane (TMS) and 85% phosphoric
4.1. General methods
1
acid were used as the internal standards for H and 31P
NMR, respectively. Sodium salt of the dUTP analogue,
into which the corresponding triethylammonium salt
Mass spectral analysis for nucleoside analogues was per-
formed on an ABI MDS-Sciex API-100 spectrometer
Table 2. Relative yield of the modified DNAs with amino acids by PCR
dUTP analog
Relative yield
dUTP analog
Relative yield
Condition 1
Condition 2
Condition 1
Condition 2
TC6
1
1
TC6Q
1.22
1.14
0.96
1.21
0
1.29
1.29
1.19
1.28
0
TC6H
TC6K
TC6R
TC6F
TC6W
TC6L
TC6P
0.70
0.63
0.56
1.08
0.82
1.18
1.16
0.79
0.61
0.59
1.24
0.94
1.28
1.13
TC6S
0
TC6S
TC6T
TC6C
0
TC6C
TC6D
TC6E
ND
0
ND
0.08
0.10
0
Relative yield is expressed by the ratio of the resulting modified DNA from each dUTP analogue to that from TC6 under the PCR condition 1 or 2;
condition 1 (30 cycles, 0.5 min at 94 ꢁC for denaturing, 0.5 min at 52 ꢁC for annealing, and 1 min at 74 ꢁC for extension) and condition 2 (30 cycles,
0.5 min at 94 ꢁC for denaturing, 1 min at 50 ꢁC for annealing, and 2 min at 74 ꢁC for extension). All experiments were run five times and the averaged
data are shown.
ND, not determined because smear band appeared.

M. Kuwahara et al. / Bioorg. Med. Chem. 14 (2006) 2518–2526
2523
was converted with Dowex 50WX8 (Na+ form), was
used for NMR measurement. Reversed-phase HPLC
was performed using a JASCO Gulliver system with
UV detection at 260 nm and a packed column (C18,
4.6 · 250 mm).
4.3.2. 5-((2-(6-(Arginamido)hexylamino)-2-oxoethyl))-20-
deoxyuridine-50-triphosphate (TC6R). The analogue TC6
(20 OD260nm, 2.14 lmol) was dissolved in distilled water
(100 lL), and a mixture of sodium bicarbonate buffer
(1.0 M, 80 lL) and DMF (215 lL) was then added. To
the above mixture, N-a,N-x1,N-x2-tricarbobenzoxy-L-
arginineN-succinimidyl ester in dry DMF (200 mM,
215 lL, 20 equiv) was added and the reaction mixture
was stirred at room temperature for 3 h. After evapora-
tion, the residue was suspended with water and
centrifuged to remove insoluble matter, and the super-
natant was evaporated to dryness. The residue was puri-
fied by reversed-phase HPLC with a 3.5–70% gradient of
acetonitrile in 50 mM triethylammonium acetate buffer
(pH 7.2) over 50 min at a flow rate of 1.0 mL/min to give
4.2. Materials
All amino acids with protecting groups were purchased
from WATANABE Chemical Industries. The following
dUTP analogues were used as reference samples: 5-(2-(6-
aminohexylamino)-2-oxoethyl)-20-deoxyuridine-50-tri-
phosphate (TC6); 5-(2-(2-aminoethylamino)-2-oxoethyl)-
20-deoxyuridine-50-triphosphate (TC2); 5-((2-(2-amino-
ethoxy)ethoxy)methyl)-20-deoxyuridine-50-triphosphate
(TDH); 5-((E)-3-aminoprop-1-enyl)-20-deoxyuridine-50-
triphosphate (TAL); and 5-(3-aminoprop-1-ynyl)-20-de-
oxyuridine-50-triphosphate (TPR). Among these, the
analogues TC6, TC2, and TDHwere synthesized according
to procedures reported previously,23,30 while the ana-
logues TAL and TPR were purchased from TriLink
BioTechnologies and from JENA Bioscience, respective-
5-((2-(6-(N-a,N-x1,N-x2-tricarbobenzoxy-arginami-
0
do)hexylamino)-2-oxoethyl))-dUTP (TC6R , 1.26 lmol)
in 59% yield; ESI-MS (negative ion mode) m/z: found
1181.5; calcd for [(MꢁH)ꢁ] 1181.3. A suspension con-
taining 5% Pd/C (ca. 10 mg) in 1 mL water/methanol
(1:1) was stirred under an H2 atmosphere. After activa-
tion of Pd/C, 10 drops of the suspension were added to
0
ly.
Natural
20-deoxynucleoside-50-triphosphates
the analogue TC6R (5.78 lmol) in 1 mL water/methanol
(1:1), and this was stirred under an H2 atmosphere at
room temperature for 3 h. The reaction mixture was fil-
tered with a membrane filter (Millex-LH, Millipore) to
remove the catalyst and then the filtrate was evaporated
to dryness. The residue was purified by reversed-phase
HPLC with a 0–56% gradient of acetonitrile in triethy-
lammonium acetate buffer (50 mM, pH 7.2) over
50 min at a flow rate of 1.0 mL/min to give the analogue
TC6R (2.08 lmol) in 36% yield. 1H NMR (300 MHz,
D2O): d = 7.77 (s, 1H), 6.21 (t, 1H), 4.07 (m, 3H), 3.86
(t, 1H), 3.23 (s, 2H), 3.20 (m, 1H), 3.06 (m, 6H), 2.28
(m, 2H), 1.78 (m, 2H), 1.53 (m, 2H), 1.37 (m, 4H),
1.17 (m, 4H); 31P NMR (500 MHz, D2O): d = ꢁ7.79
(d), ꢁ10.94 (d), ꢁ22.04 (t); ESI-MS (negative ion mode)
m/z: found 779.5; calcd for [(MꢁH)ꢁ] 779.2.
(dATP, dGTP, dCTP, and TTP) were obtained from
Roche Diagnostics. KOD Dash DNA polymerase was
obtained from Toyobo. Primer oligonucleotides (#1:
50-GGAA ACAGCTATGACCATGATTAC-30, #1F:
50-FAM-labeled-GGAAACAGCTATGACCATGATT
AC-30, #2: 50-CGACGTTGTAAAACGACGGCCAG
T-30, and #3: 50-AAGTTGGGTAACGCCAGGGTT
TTC-30) were purchased from JBioS and pUC18 tem-
plate DNA was obtained from TaKaRa Biomedicals.
4.3. Synthesis of aminoacyl dUTP analogues
All aminoacyl dUTPs, except 0 for sulfur-containing
analogs, such as TC6C and TC6C , were prepared using
protocol similar to that for arginyl dUTP (TC6R), as
follows. Aminoacyl dUTPs were synthesized by cou-
pling TC6 with commercially available protected
amino acids followed by hydrogenation to remove
4.3.3. 5-((2-(6-(Asparatamido)hexylamino)-2-oxoethyl))-
20-deoxyuridine-50-triphosphate (TC6D). This product
was prepared by coupling of TC6 (0.35 lmol) with
N-a-carbobenzoxy-L-aspartic acid a-N-succinimidyl b-
benzyl diester followed by hydrogenation to give the
the protecting groups (Scheme 1). For synthesis of
0
TC6C and TC6C , cysteine protected with an Fmoc
group was used. The yields were calculated from
the estimation that the molar absorption coefficient
of the product is the sum of that of 5-(2-(6-amin-
ohexylamino)-2-oxoethyl)-20-deoxyuridine and a teth-
ered amino acid.
analogue TC6D (0.14 lmol) in 40% yield from TC6
.
ESI-MS (negative ion mode) m/z: found 738.1; calcd
for [(MꢁH)ꢁ] 738.1.
4.3.4. 5-((2-(6-(S-tert-Butylthio-cysteinamido)hexylami-
C6C0
4.3.1. N-a, N-x1,N-x2-tricarbobenzoxy-arginine N-hy-
droxysuccinimide ester. N,N0-Dicyclohexylcarbodiimide
(64 mg, 1.5 equiv) in dry DMF (1.0 mL) was added
dropwise to a stirred solution of N-a,x1,x2-tricarbo-
benzoxy-arginine (120 mg), and hydroxysuccinimide
(29 mg, 1.2 equiv) in dry DMF (1.0 mL) at 0 ꢁC. The
reaction mixture was stirred at 0 ꢁC for 1 h and at room
temperature for 18 h, and was then filtered to remove
DCUrea. The filtrate was evaporated and the resulting
residue was dried well and used for the next reaction
without further purification. Assuming that the succini-
mide ester was obtained in quantitative yield, the crude
ester was dissolved in dry DMF such that the final con-
centration would be 200 mM.
no)-2-oxoethyl))-20-deoxyuridine-50-triphosphate ðT Þ.
The analogue TC6 (10 OD260nm, 1.07 lmol) was dis-
solved in distilled water (120 lL) and sodium bicarbon-
ate buffer (1.0 M, 20 lL) was then added. To the above
mixture, N-a-(9-fluorenylmethoxycarbonyl)-S-tert-butyl-
thio-L-cysteine N-succinimidyl ester in dry DMF
(100 mM, 260 lL, 25 equiv) was added and the reaction
mixture was stirred at room temperature for 3 h. After
evaporation, the residue was suspended in water and
centrifuged to remove insoluble materials, and the
supernatant was evaporated to dryness. The residue
was purified by reversed-phase HPLC with a 3.5–56%
gradient of acetonitrile in 50 mM triethylammonium
acetate buffer (pH 7.2) over 35 min at a flow rate of

2524
M. Kuwahara et al. / Bioorg. Med. Chem. 14 (2006) 2518–2526
1.0 mL/min to give 5-((2-(6-(N-a-(9-fluorenylmethoxy-
carbonyl)-S-tert-butylthio-cysteinamido)hexylamino)-2-
4.3.10. 5-((2-(6-(Lysinamido)hexylamino)-2-oxoethyl))-
20-deoxyuridine-50-triphosphate (TC6K). This product
was prepared by coupling of TC6 (0.86 lmol) with N-
a-N-e-di-carbobenzoxy-L-lysine a-N-succinimidyl ester
followed by hydrogenation to give the analogue TC6K
(0.72 lmol) in 84% yield from TC6. ESI-MS (negative
ion mode) m/z: found 753.3; calcd for [(MꢁH)ꢁ] 753.2.
0
oxoethyl))-dUTP (TC6C -Fmoc, 0.50 lmol) in 47% yield
from TC6; ESI-MS (negative ion mode) m/z: found
1036.2; calcd for [(MꢁH)ꢁ] 1036.2. The analogue
0
T
C6C -Fmoc (0.50 lmol) was dissolved with water
(1 mL) and a drop of diethylamine was added at 0 ꢁC.
The reaction mixture was stirred for 2 h at 0 ꢁC and
was then evaporated to dryness. The residue was puri-
fied by reversed-phase HPLC with a 3.5–56% gradient
of acetonitrile in triethylammonium acetate buffer
(50 mM, pH 7.2) over 35 min at0 a flow rate of 1.0
4.3.11. 5-((2-(6-(Tryptophanamido)hexylamino)-2-oxo-
ethyl))-20-deoxyuridine-50-triphosphate (TC6W). This
product was prepared by coupling of TC6 (0.63 lmol) with
N-a-carbobenzoxy-L-tryptophan a-N-succinimidyl ester
followed by hydrogenation to give the analogue TC6W
(0.24 lmol) in 38% yield from TC6. ESI-MS (negative
ion mode) m/z: found 809.0; calcd for [(MꢁH)ꢁ] 809.2.
mL/min to give the analogue TC6C (0.34 lmol) in 68%
0
yield from TC6C -Fmoc. ESI-MS (negative ion mode)
m/z: found 814.1; calcd for [(MꢁH)ꢁ] 814.1.
4.3.5. 5-((2-(6-(Cysteinamido)hexylamino)-2-oxoethyl))-
20-deoxyuridine-50-triphosphate (TC6C). The tert-butyl-
thio-protecting group was removed by addition of
Tris–HCl buffer (100mM, pH 8.0) and DTT (10 mM)
to the analogue TC6C (0.66 lmol). The reaction mixture
was stirred for 10 min at room temperature and then
evaporated to dryness. The residue was purified by re-
versed-phase HPLC with a 3.5–56% gradient of acetoni-
trile in triethylammonium acetate buffer (50 mM, pH
7.2) over 35 min at a flow rate of 1.0 mL/min to give
4.3.12. 5-((2-(6-(Phenylalaninamido)hexylamino)-2-oxo-
ethyl))-20-deoxyuridine-50-triphosphate
(TC6F).
This
product was prepared by coupling of TC6 (0.66 lmol)
with N-a-carbobenzoxy-L-phenylalanine a-N-succinim-
idyl ester followed by hydrogenation to give the ana-
logue TC6F (0.18 lmol) in 27% yield from TC6. ESI-
MS (negative ion mode) m/z: found 770.2; calcd for
[(MꢁH)ꢁ] 770.2.
4.3.13. 5-((2-(6-(Prolinamido)hexylamino)-2-oxoethyl))-
20-deoxyuridine-50-triphosphate (TC6P). This product
was prepared by coupling of TC6 (0.41 lmol) with
N-a-carbobenzoxy-L-proline a-N-succinimidyl ester fol-
lowed by hydrogenation to give the analogue TC6P
(0.12 lmol) in 29% yield from TC6. ESI-MS (negative
ion mode) m/z: found 720.1; calcd for [(MꢁH)ꢁ] 720.2.
the analogue TC6C (0.053 lmol) in 8% yield from
0
T
C6C . ESI-MS (negative ion mode) m/z: found 726.1;
calcd for [(MꢁH)ꢁ] 726.1.
4.3.6.
5-((2-(6-(Glutaminamido)hexylamino)-2-oxoeth-
yl))-20-deoxyuridine-50-triphosphate (TC6Q). This product
was prepared by coupling of TC6 (0.66 lmol) with N-a-
carbobenzoxy-L-glutamine a-N-succinimidyl ester fol-
lowed by hydrogenation to give the analogue TC6Q
(0.51 lmol) in 77% yield from TC6. ESI-MS (negative
ion mode) m/z: found 751.4; calcd for [(MꢁH)ꢁ] 751.2.
4.3.14.
5-((2-(6-(O-Benzyl-serinamido)hexyla0mino)-2-
oxoethyl))-20-deoxyuridine-50-triphosphate ðTC6S Þ. Cou-
pling of the analogue TC6 with N-a-carbobenzoxy-O-
benzyl-L-serine a-N-succinimidyl ester was performed
similarly to the procedure described for TC6R. The ana-
00
4.3.7.
5-((2-(6-(Glutaminamido)hexylamino)-2-oxoeth-
logue TC6S , 5-((2-(6-(N-a-carbobenzoxy-O-benzyl-seri-
namido)hexylamino)-2-oxoethyl))-20-dUTP (0.49 lmol)
was obtained in 54% yield from TC6 (0.90 lmol). A sus-
pension containing 5% Pd/C (ca. 10 mg) in 1 mL of
water/methanol (1:1) was stirred under an H2 atmo-
yl))-20-deoxyuridine-50-triphosphate (TC6E). This product
was prepared by coupling of TC6 (0.89 lmol) with N-a-
carbobenzoxy-L-glutamic acid a-N-succinimidyl c-ben-
zyl diester followed by hydrogenation to give the
analogue TC6E (0.73 lmol) in 82% yield from TC6
;
ESI-MS (negative ion mode) m/z: found 752.2; calcd
sphere. After activation of Pd/C, ten drops of the sus-
00
pension were added to TC6S (0.35 lmol) in 1 mL of
water/methanol (1:1), and this was stirred under an H2
atmosphere at room temperature for 3 h. The reaction
mixture was filtered with a membrane filter (Millex-
LH, Millipore) to remove the catalyst, and the filtrate
was evaporated to dryness. The residue was purified
by reversed-phase HPLC with a 3.5-70% gradient of ace-
tonitrile in triethylammonium acetate buffer (50 mM,
pH 7.2) over 35 min at a flow rate of 1.0 mL/min to give
for [(MꢁH)ꢁ] 752.1.
4.3.8. 5-((2-(6-(Histidinamido)hexylamino)-2-oxoethyl))-
20-deoxyuridine-50-triphosphate (TC6H). This product
was prepared by coupling of TC6 (0.65 lmol) with
N-a, N-p-dicarbobenzoxy-L-histidine a-N-succinimidyl
ester followed by hydrogenation to give the analogue
TC6H (0.15 lmol) in 23% yield from TC6. ESI-MS (neg-
ative ion mode) m/z: found 760.1; calcd for [(MꢁH)ꢁ]
760.2.
0
00
the analogue TC6S (0.077 lmol) in 22% yield from TC6S
.
ESI-MS (negative ion mode) m/z: found 800.2; calcd for
[(MꢁH)ꢁ] 800.2.
4.3.9. 5-((2-(6-(Leucinamido)hexylamino)-2-oxoethyl))-
20-deoxyuridine-50-triphosphate (TC6L). This product
was prepared by coupling of TC6 (0.35 lmol) with N-
a-carbobenzoxy-L-leucine a-N-succinimidyl ester fol-
lowed by hydrogenation to give the analogue TC6L
(0.15 lmol) in 43% yield from TC6. ESI-MS (negative
ion mode) m/z: found 736.0; calcd for [(MꢁH)ꢁ] 736.2.
4.3.15. 5-((2-(6-(Serinamido)hexylamino)-2-oxoethyl))-
20-deoxyuridine-50-triphosphate (TC6S). A suspension
containing 5% Pd/C (ca. 10 mg) in 1 mL water/methanol
(1:1) was stirred under H2 atmosphere. After activation
of Pd/C, ten drops of the suspension were added to TC6S
(0.25 lmol) in 1 mL water/methanol (1:1), and this was
00

M. Kuwahara et al. / Bioorg. Med. Chem. 14 (2006) 2518–2526
2525
then stirred under an H2 atmosphere at room tempera-
ture overnight. The reaction mixture was filtered with
a membrane filter (Millex-LH, Millipore) to remove
the catalyst, and the filtrate was evaporated to dryness.
The residue was purified by reversed-phase HPLC with
a 3.5–70% gradient of acetonitrile in triethylammonium
acetate buffer (50 mM, pH 7.2) over 35 min at a flow
subjected to the TA Cloningꢂ method: Taq DNA poly-
merase has a non-template-dependent activity that adds
a single 20-deoxyadenosine at the 30-end of PCR prod-
uct. Cloning was performed using a TA Cloningꢂ kit
(Invitrogen) according to the instruction manual and
five samples were chosen at random from among single
colonies derived from modified DNA for sequencing.
Plasmids extracted from the single colonies were se-
quenced with a Genetic Analyzer 310 (ABI) according
to standard protocol using a BigDye Terminatorꢂ v3.0
Sequencing Kit together with primer #3.
rate of 1.0 mL/min to give the analogue TC6S
00
(0.038 lmol) in 15% yield from TC6S . ESI-MS (negative
ion mode) m/z: found 710.0; calcd for [(MꢁH)ꢁ] 710.1.
4.3.16. 5-((2-(6-(Threoninamido)hexylamino)-2-oxoeth-
yl))-20-deoxyuridine-50-triphosphate (TC6T). This product
was prepared by coupling of TC6 (0.58 lmol) with
N-a-carbobenzoxy-O-benzyl-L-threonine a-N-succinim-
idyl ester followed by hydrogenation to give the
Acknowledgments
This work was supported by a Grant-in-Aid for Scientif-
ic Research on Priority Areas (B) and (C) from the Min-
istry of Education, Culture, Sports, Science and
Technology of Japan, and PRESTO from the Japan Sci-
ence and Technology Agency.
analogue TC6T (0.070 lmol) in 12% yield from TC6
.
ESI-MS (negative ion mode) m/z: found 724.0; calcd
for [(MꢁH)ꢁ] 724.1.
4.4. PCR amplification
PCR experiments were performed with a 20 lL reaction
volume containing 10 ng pUC 18 template DNA, prim-
ers #1 and #2 at 0.4 lM each, 1.0 U KOD Dash DNA
polymerase, reaction buffer supplied with enzyme (at
1times concentration), and triphosphates at 200 lM
each; PCR with natural triphosphates dATP, dGTP,
dCTP, and TTP was used as a positive control; PCR
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.11.030.
with one of the dUTP analogues (TC6, TC2, TDH, TAL
,
,
References and notes
T
T
PR, TC6H, TC6K, TC6R, TC6F,0 TC6W, TC6L, TC6P,TC6Q
0
C6S, TC6S , TC6T, TC6C, TC6C , TC6D or TC6E) instead
1. Gold, L.; Polisky, B.; Uhlenbeck, O.; Yarus, M. Annu.
Rev. Biochem. 1995, 64, 763.
of TTP was used for the actual assays (Fig. 1); PCR with
dATP, dGTP, and dCTP (water was added instead of
TTP) was used as a negative control. For all amplifica-
tions, a hot start (1 min at 94 ꢁC) was used, followed
by amplification and final incubation (5 min at 74 ꢁC).
Conditions for the amplification process (denaturing,
annealing, and extension) were as follows; condition 1
(30 cycles, 0.5 min at 94 ꢁC, 0.5 min at 52 ꢁC, and
1 min at 74 ꢁC); condition 2 (30 cycles, 0.5 min at
94 ꢁC, 1 min at 50 ꢁC, and 2 min at 74 ꢁC); condition 3
(20 cycles, 0.5 min at 94 ꢁC, 0.5 min at 52 ꢁC, and
1 min at 74 ꢁC). PCR products were resolved by dena-
turing PAGE, visualized by ethidium bromide staining,
and gel images were recorded on a Molecular Imagerꢂ
FX (Bio-Rad). The intensity of each band correspond-
ing to full-length DNA product was quantified using
Quantity Oneꢂ software. The amount of full-length
PCR product formed during reaction with TC6 was set
at 1. Here, calculated relative yields are averages of five
independent experiments.
2. Santoro, S. W.; Joyce, G. F. Proc. Natl. Acad. Sci. U. S. A.
1997, 94, 4262.
3. Breaker, R. R. Chem. Rev. 1997, 97, 371.
4. Wilson, D. S.; Szostak, J. W. Annu. Rev. Biochem. 1999,
68, 611.
5. Osborne, S. E.; Ellington, A. D. Chem. Rev. 1997, 97, 349.
6. Famulok, M.; Mayer, G.; Blind, M. Chem. Res. 2000, 33,
591.
7. Latham, J. A.; Johnson, R.; Toole, J. J. Nucleic Acids Res.
1994, 22, 2817.
8. Battersby, T. R.; Ang, D. N.; Burgstaller, P.; Jurczyk, S.
C.; Bowser, M. T.; Buchanan, D. D.; Kennedy, R. T.;
Benner, S. A. J. Am. Chem. Soc. 1999, 121, 9781.
9. Santoro, S. W.; Joyce, G. F.; Sakthivel, K.; Gramatikova,
S., ; Barbas, C. F., III J. Am. Chem. Soc. 2000, 122, 2433.
10. Kusser, W. Rev. Mol. Biotechnol. 2000, 74, 27.
11. Verma, S.; Jager, S.; Thum, O.; Famulok, M. Chem. Rec.
2003, 3, 51.
12. Masud, M. M.; Kuwahara, M.; Ozaki, H.; Sawai, H.
Bioorg. Med. Chem. 2004, 12, 1111.
´ `
13. Perrin, D. M.; Garestier, T.; Helene, C. J. Am. Chem. Soc.
2001, 123, 1556.
14. May, J. P.; Ting, R.; Lermer, L.; Thomas, J. M.; Roupioz,
Y.; Perrin, D. M. J. Am. Chem. Soc. 2004, 126, 4145.
15. Sidorov, A. V.; Grasby, J. A.; Williams, D. M. Nucleic
Acids Res. 2004, 32, 1591.
´ `
16. Perrin, D. M.; Garestier, T.; Helene, C. Nucleosides
Nucleotides 1999, 18, 377.
17. Thum, O.; Ja¨ger, S.; Famulok, M. Angew. Chem. Int. Ed.
2001, 40, 3990.
18. Takagi, M.; Nishioka, M.; Kakihara, H.; Kitabayashi, M.;
Inoue, H.; Kawakami, B.; Oka, M.; Imanaka, T. Appl.
Environ. Microbiol. 1997, 63, 4504.
4.5. Cloning and sequencing of PCR products
Modified DNA was purified by agarose gel electropho-
resis and eluted from the gel into TBE buffer (45 mM
Tris-borate, 1 mM EDTA). The eluted sample was con-
verted into natural DNA by a second PCR using KOD
Dash DNA polymerase under condition 1. After purifi-
cation by agarose gel electrophoresis and elution, the
dsDNA product was re-amplified by a third PCR using
Taq DNA polymerase under condition 3 and was

2526
M. Kuwahara et al. / Bioorg. Med. Chem. 14 (2006) 2518–2526
19. Mizuguchi, H.; Nakatsuji, M.; Fujiwara, S.; Takagi, M.;
Imanaka, T. J. Biochem. 1999, 126, 762.
27. Ja¨ger, S.; Famulok, M. Angew. Chem. Int. Ed. 2004, 43,
3337.
20. Nishioka, M.; Mizuguchi, H.; Fujiwara, S.; Komatsubara,
S.; Kitabayashi, M.; Uemura, H.; Takagi, M.; Imanaka,
T. J. Biotechnol. 2001, 88, 141.
28. Ohbayashi, T.; Masud, M. M.; Ozaki, A. N.; Ozaki, H.;
Kuwahara, M.; Sawai, H. Bioorg. Med. Chem. Lett. 2002,
12, 1167.
21. Hashimoto, H.; Nishioka, M.; Fujiwara, S.; Takagi, M.;
Imanaka, T.; Inoue, T.; Kai, Y. J. Mol. Biol. 2001, 306, 469.
22. Sawai, H.; Ozaki, A. N.; Satoh, F.; Ohbayashi, T.; Masud,
M. M.; Ozaki, H. Chem. Commun. 2001, 24, 2604.
23. Vaish, N. K.; Fraley, A. W.; Szostak, J. W.; McLaughlin,
L. W. Nucleic Acids Res. 2000, 28, 3316.
24. Held, H. A.; Benner, S. A. Nucleic Acids Res. 2002, 30,
3857.
25. Obayashi, T.; Kuwahara, M.; Hasegawa, M.; Kasamatsu,
T.; Tamura, T.; Sawai, H. Org. Biomol. Chem. 2005, 3,
2463.
29. Kuwahara, M.; Takahata, Y.; Shoji, A.; Ozaki, A. N.;
Ozaki, H.; Sawai, H. Bioorg. Med. Chem. Lett. 2003, 13,
3735.
30. Sakthivel, K.; Barbas, C. F., III Angew. Chem. Int. Ed.
1998, 37, 2872.
31. Lee, S. E.; Sidorov, A.; Gourlain, T.; Mignet, N.; Thorpe,
S. J.; Brazier, J. A.; Dickman, M. J.; Hornby, D. P.;
Grasby, J. A.; Williams, D. M. Nucleic Acids Res. 2001,
29, 1565.
32. Okamoto, A.; Tanaka, K.; Nishiza, K.; Saito, I. Bioorg.
Med. Chem. 2004, 12, 5875.
26. Gourlain, T.; Sidorov, A.; Mignet, N.; Thorpe, S. J.; Lee,
S. E.; Grasby, J. A.; Williams, D. M. Nucleic Acids Res.
2001, 29, 1898.
33. Ohmichi, T.; Kuwahara, M.; Sasaki, N.; Hasegawa, M.;
Nishikata, T.; Sawai, H.; Sugimoto, N. Angew. Chem. Int.
Ed. 2005, 44, 6682.