Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase

Article abstract of DOI:10.1039/b504330a

Thymidine analogues bearing a variety of functional groups at the C5-position via an amino-linker arm were prepared and the substrate activity for PCR using thermophilic KOD Dash DNA polymerase was examined. The enzyme accepted the thymidine analogues bea

Full text of DOI:10.1039/b504330a

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A R T I C L E
Expansion of repertoire of modified DNAs prepared by PCR using
KOD Dash DNA polymerase†
Tsutomu Ohbayashi, Masayasu Kuwahara, Masatoshi Hasegawa, Toshiyuki Kasamatsu,
Takehiro Tamura and Hiroaki Sawai*
Department of Applied Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan.
E-mail: sawai@chem.gunma-u.ac.jp
Received 29th March 2005, Accepted 12th May 2005
First published as an Advance Article on the web 9th June 2005
Thymidine analogues bearing a variety of functional groups at the C5-position via an amino-linker arm were
prepared and the substrate activity for PCR using thermophilic KOD Dash DNA polymerase was examined. The
enzyme accepted the thymidine analogues bearing pyridine, imidazole, biotin, a cationic-charged guanidinium, a
cationic-charged amino, mercaptopyridyl and phenanthrolne groups at the C5-position, forming the corresponding
PCR product. However, a thymidine analogue bearing a carboxyl group at the C5-position was a poor substrate and
the corresponding PCR products could not be obtained. The thymidine analogue bearing a mercapto group was also
a poor substrate for the enzyme, because it dimerized by disulfide linkage under PCR conditions. The enzyme hardly
accepts the thymidine analogues with a negatively-charged carboxyl group or a bulky group as a substrate. KOD
Dash DNA polymerase, having a broader substrate specificity than any other DNA polymerase, will expand the
variety of modified DNAs that can be prepared by PCR.
a large side-group and/or to the ionic effect of a substituting
Introduction
group, such as a cationic group or an anionic group. The
DNAs that have been chemically modified with a variety of func-
ability of the modified nucleotides to act as a substrate also
tional groups are important tools for biochemical and biological
depends on the kind of DNA polymerase.23–29 Furthermore,
studies.1–3 They are also useful for diagnostic and therapeutic
some DNA polymerases can accept two or four modified
purposes. In general, the applications rely on differences in
nucleotides simultaneously in the primer-extension reaction or
the chemical reactivity, electronic and stereochemical properties
polymerase chain reacion (PCR).23–26 Previously, we have shown
of the additional functional groups compared to the naturally
that KOD Dash DNA polymerase can readily use some new
occurring DNA. Among the many possible sites of the DNA-
thymidine analogue nucleotides as a substrate and can read a
modification, the C5-position of the thymidine is an ideal site
template DNA containing the modified thymidine, but no other
for the attachment of functional groups, because it is located
DNA polymerase, including Taq DNA polymerase, can accept
in the major groove of double-stranded DNA and does not
the thymidine analogues as a substrate.27 The new thymidine
inhibit A–T base pairing. Previously, we reported the synthesis of
analogues that have an sp3 hybridized carbon at the C5-position
thymidine analogues, C5-substituted 2ꢀ-deoxyuridines bearing
with methyl ester or an amino-linker arm are a good substrate
an amino-linker arm at the C5-position,4 and their introduc-
for the enzyme and the corresponding modified DNAs can be
tion into oligodeoxyribonucleotides chemically using a DNA
obtained by PCR using KOD Dash DNA polymerase.28
synthesizer.5
To expand the structural and functional repertoire of modified
DNAs, we have attached a variety of functional groups at the
C5-position of the thymidine analogue via the amino-linker
arm and examined whether they work as a substrate for KOD
If a DNA polymerase can accept modified nucleotides as
substrates and modified DNA can be prepared enzymatically
by PCR, the resulting modified DNA could be applied in the
production of a DNA aptamer or a catalytic DNA by in vitro
Dash DNA polymerase in forming the corresponding modified
selection. A variety of DNA aptamers and DNA catalysts have
DNA by PCR. The functional groups include pyridine, imida-
been produced from the natural-type DNAs.6–10 However, their
zole, biotinyl, cationic-charged guanidinium, cationic-charged
activity is, in general, not very strong when compared with the
amino, anionic-charged carboxyl, mercapto, mercaptopyridyl
corresponding functional proteins, antibodies and enzymes. One
and phenanthroline groups. Among the thymidine analogues
reason for the weak activity of the DNA aptamer or catalyst
examined, all analogues except the ones with a carboxyl or a
would be a lack of the functional groups which are present
sterically bulky group could be accepted as a substrate for the
within proteins. The modified DNA with a functional group
enzyme, yielding the modified DNA by PCR. The finding that
will improve the weak activity of natural-type DNA. Some
the thymidine analogues with an anionic-charged carboxyl and
improved DNA aptamers or catalysts have been prepared from
bulky groups are a poor substrate for the enzyme may be useful
the modified DNAs.11–14 However, more active DNA aptamers or
for study of the recognition process of the enzyme and substrate.
catalysts are still desired for wide applications. Thus, nucleotide
KOD Dash DNA polymerase has broader substrate specificity
analogues with a variety of functional groups, that are substrates
which will expand the variety of modified DNA that can be
for DNA polymerase, will be desirable for the production of a
prepared by PCR.
high-performance functional DNA. Some DNA polymerases
can use 5ꢀ-triphosphates of modified 2ꢀ-deoxyuridines with
a C5-side chain carrying an (E)-propenyl15–20 or a propynyl
group.18–24 The differences in reactivity of C5-substituted 2ꢀ-
deoxyuridines as a substrate may be due to the steric effect of
Results and discussion
Synthesis of modified substrate
The modified thymidine analogue, 5-N-(6-aminohexyl)carb-
amoylmethyl-2ꢀ-deoxyuridine 5ꢀ-triphosphate (1) was prepared
according to the method described previously.27 This analogue
is a good substrate for the thermostable KOD Dash DNA
† Electronic supplementary information (ESI) available: synthetic pro-
cedure for the 5’-triphosphates of thymidine analogues, 4–14 and 16. See
http://www.rsc.org/suppdata/ob/b5/b504330a/
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 6 3 – 2 4 6 8
2 4 6 3
©

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polymerase, which belongs to the B-family DNA polymerase
derived from thermophilic archae.30 The nucleotide 1 bears a
primary amino-group at the C5-position of thymidine via a
linker arm. Many kinds of functional groups can be introduced
to the terminal amino-group by amide bond formation or by
other reactions with the amino group, as shown in Scheme 1. The
hydroxysuccinimide activated esters of urocanic acid, imidazole
acetic acid, nicotinic acid, biotin, 3-(2-pyridyldithio)propionic
acid, trifluoroacetylaminohexanoic acid and 2-phenanthroline
carboxylic acid were reacted easily with 1 in aqueous DMF
solution. The corresponding thymidine analogues bearing func-
tional groups, 3–10, 12 and 16, were obtained in moderate
to high yields. The reaction of succinic anhydride with the
amino group of 1 gave a thymidine analogue with an anionic-
charged carboxyl group at the C5-position of 11. Isothiocyanate
of 2,9-dimethylphenanthroline or fluorescence reacts with the
amino group of 1 to give the 2,9-dimethylphenanthroline- or
fluorescence-substituted thymidine analogue, 13 or 14, respec-
tively. Similarly, guanidinium-substituted thymidine analogue15
was obtained by reaction of S-ethylthiopseudourea hydrobro-
mide with 1. Thus, thymidine analogues with catalytic function,
an aromatic compound hydrophobic moiety, a hydrophilic
moiety, an anionic-charged group, a cationic charged group,
a chelating group and fluorescent groups were obtained from
the nucleotide 1.
Synthesis of modified DNA by incorporation of the thymidine
analogues by PCR using KOD Dash DNA polymerase
We studied the ability of the thymidine analogues 1–16 to act as
a substrate in place of deoxythymidine triphosphate (TTP) for
KOD Dash DNA polymerase under typical PCR conditions,
using pUC18 plasmid DNA as a template, with oligonucleotide
A, 5ꢀ-GGAAACAGCTATGACCATGATTAC-3ꢀ, and oligonu-
cleotide B, 5ꢀ-CGACGTTGTAAAACGACGGCCAGT-3ꢀ, as
primers. Fig. 1 shows the results of PCR with the substrates. We
used ten times the concentration of the enzyme for the PCR with
the modified thymidine analogues compared to that with natural
substrate, TTP, to increase the yield of the modified DNA. Non-
specific amplification of DNA took place when the ten times
concentration of the enzyme was used for PCR with natural TTP
(lane 4 of Fig. 1B). We confirmed the previous report25 that KOD
Dash DNA polymerase can accept 1 or 2 as a substrate, giving
a 108 base-pair DNA product (2) (lane 4 and 5 of Fig. 1A). The
formation of the corresponding natural-type DNA from the nat-
ural substrate TTP was 1.4 times larger compared to that from 1
under the optimum conditions for the formation of natural type
DNA, where the enzyme quantity was reduced to suppress the
non-specific amplification. The 108 base-pair DNA containing
the primers and the template region has 40 modified thymidines
with a single-stretch of four successive thymidine residues. The
Scheme 1 Synthesis of 5ꢀ-triphosphates of thymidine analogues bearing various functional groups.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 6 3 – 2 4 6 8
2 4 6 4

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Scheme 2 Structures of DNA polymerase-tolerated modified nucleotides reported by other workers.
enzyme could accept thymidine analogues 3–7, 9–12 and 15,
forming the corresponding 108 bp PCR product. The mobility
shift of the modified DNA on the gel was observed depending on
the mass increase and the charge associated with modification.
However, the enzyme could not tolerate thymidine analogues, 8,
11, 13, 14 and 16 as a substrate for the PCR. Table 1 shows the
relative incorporation rate of the modified substrate, estimated
from the band intensity of the resulting modified DNA on the
gel after staining with ethidium bromide and quantification by
a molecular imager. The incorporation rate is expressed as the
ratio of the modified DNA from each substrate analogue to the
modified DNA from 1. Thus, modified DNAs bearing imidazole
acetic acid, urocanic acid nicotinic acid, phenanthroline 9-
carboxylic acid or biotin via an amide linkage were obtained
by PCR. The thymidine analogue with imidazole acetic acid or
urocanic acid via hexamethylenediamine linker, 4 or 5, works as
a substrate for KOD Dash DNA polymerase, giving the PCR
product (lane 7 or 8 of Fig. 1A). However, the enzyme could
not tolerate a similar analogue bearing an imidazole group
without a hexamenthylenediamine linker.28 Aminohexanoic acid
was added to the hexamethylenediamine linker of the thymidine
analogue 1 to increase the length of the linker, forming 10. The
yield of the modified DNA from 10 decreased slightly compared
to that from 1 (lane 13). The thymidine analogue containing
a cationic-charged guanidinium group at the C5-position was
also a good substrate for the enzyme. The modified DNA with
a guanidinium group was formed efficiently by PCR from the
modified substrate 15 (lane 6 of Fig. 1B). This is the first example
that a guanidinium-substituted thymidine analogue works as a
substrate during PCR, although a thymidine analogue bearing
a guanidinium group via a propynyl linker 17 has been reported
to be accepted by Tth or Pwo DNA polymerase in the template-
directed primer extension reactions using a natural or modified
DNA template (Scheme 2).25,26 The oligonucleotides with a
guanidinium group have been prepared by a DNA synthesizer
using phosphoramidite chemistry and are shown to have high
hybridization ability.31,32 The PCR with the modified substrate
15 using KOD Dash DNA polymerase will provide a simple
synthetic method for the modified DNA with a guanidinium ion,
which is expected to have high hybridization and cell permeation
ability and thus may be useful as an antisense agent. The
analogue with pydridyldithio group 7 was slightly incorporated
into the DNA by PCR. On the other hand, thymidine analogues
bearing the anionic-charged carboxyl group 11 (lane 14),
mercapto group 8 (lane 11) and 2,9-dimethylphenanthroline
via thiourea linkage 13 (lane 16) were a poor substrate for the
enzyme. The analogue with phenanthroline 9-carboxylic acid via
amide linkage 12 works as a substrate and could be incorporated
into the modified DNA (lane 15), whereas the analogue with 2,9-
dimethylphenanthroline via 5-thiourea linkage was a very poor
substrate for the enzyme. The analogue bearing fluorecsein via
thiourea linkage 14 was also a poor substrate and no fluorescein-
labelled DNA could be obtained when 14 was used in place of
TTP (lane 17). However, combination use of TTP and 14 in a
molar ratio of 5 : 5 gave the fluorescein-labelled DNA by PCR.33
The result indicates that the enzyme could tolerate a single
incorporation of 14, but not successive multiple incorporation.
The corresponding analogue with fluorescein carboxylic acid
via amide linkage 16 also could not work as a substrate for
the PCR, although the mixture of 16 and 1 in ratio of 9 : 1
yielded the resulting fluorescent-modified DNA. The enzyme
could not accept the substrate with a mercapto group at the C5-
position of 8, because the mercapto group of 8 was converted
to the disulfide-bridged dimer under the PCR conditions and
the resulting dimer could not work as a substrate. We confirmed
that the disulfide bridged dimer of 8 was formed easily by air
oxidation when 8 was kept under the PCR conditions, as the
buffer for the PCR did not contain a reducing agent such as
DTT. Held and Benner reported that the thymidine analogue
with a C5-position side chain carrying a thiol group protected
as the disulfide with t-butyl group 18 can be incorporated as
a substrate for PCR with Pwo DNA polymerase, although
the corresponding analogue with a free thiol group was not
a substrate for the enzyme.22 These results suggest that steric
Table 1 Relative yield of the modified DNA from thymidine analogues by PCR with KOD Dash DNA polymerase
Nucleotide
Relative yielda
Nucleotide
Relative yielda
Nucleotide
Relative yielda
1
2
3
4
5
6
1
7
0.04
0
0.43
0.76
0
13
14
15
16
0
0.
1.09
0
0.90
0.67
0.80
0.60
0.41
8
9
10
11
12
0.32
a Relative yield is expressed as the ratio of the resulting modified DNA from each thymidine analogue to that from 1 under the same PCR conditions,
estimated from the quantification of the modified DNA on the gel by a molecular imager.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 6 3 – 2 4 6 8
2 4 6 5

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Accurate incorporation of 5ꢀ-triphosphate of the modified
thymidine in place of TTP during PCR was checked by
sequencing of the resulting modified DNA. The modified
DNA was enzymatically converted into natural-type DNA,
inserted into plasmid DNA and then sequenced with an ABI
genetic analyzer. The sequencing of the modified DNA from 5ꢀ-
triphosphate of the thymidine analogue bearing the guanidinium
group is shown in Fig. 2. The result demonstrates that the
modified DNA obtained by PCR has an identical sequence to
that of the original one. Several other modified DNAs from other
thymidine analogues have also the same sequence.28,29 Thus, the
modified thymidine analogue was correctly incorporated into
the modified DNA and the resulting modified DNA can serve
as a template for sequence-specific DNA amplification.
Fig. 2 Sequencing of the PCR product from the 5ꢀ-triphosphate of
modified thymidine 15 in place of TTP. The figure shows DNA sequence
excluding the primer region.
Fig. 1 Modified DNA synthesis by PCR from dTTP and analogues
bearing various functional groups using KOD Dash DNA polymerase.
Total reaction mixture (20 ll) contained 0.5 ng ll−1 of DNA template
(pUC18 2686 bp), 0.2 lM of each primer, natural dNTPs or modified
dNTP mix (0.2 mM of each nucleotide) and 0.5 unit per 10 ll of DNA
polymerase (except lane 3, where natural TTP mix and 0.05 unit per
10 ll of DNA polymerase was used) in the buffer supplied by the
maker for the DNA polymerase reaction. PCR assays were carried out
at 94 ◦C for 1 min, 30 cycles of 94 ◦C for 30 sec, 52 ◦C for 30 sec,
74 ◦C for 1 min and 74 ◦C for 5 min. The PCR reaction mixture was
quenched by the addition of a formamide-dye solution and aliquots
of PCR products were analyzed by 2% agarose gel electrophoresis and
visualized by staining with ethidium bromide. A) lane 1: marker DNA
(100–1200 bp); lane 2: negative control without TTP (dATP + dCTP +
dGTP); lane 3: positive control: TTP + dATP + dCTP + dGTP; lane 4:
1 + dATP + dCTP + dGTP; lane 5: 2 + dATP + dCTP + dGTP: lane
6: 3 + dATP+ dCTP + dGTP; lane 7: 4 + dATP + dCTP + dGTP; lane
8: 5 + dATP + dCTP + dGTP; lane 9: 6 + dATP + dCTP + dGTP;
lane 10: 7 + dATP + dCTP + dGTP; lane 11: 8 + dATP + dCTP
+dGTP; lane 12: 9 + dATP + dCTP + dGTP: lane 13: 10 + dATP +
dCTP + dGTP; lane 14: 11 + dATP + dCTP + dGTP; lane 15: 12 +
dATP + dCTP + dGTP; lane 16: 13 + dATP + dCTP + dGTP; lane 17:
14 + dATP + dCTP + dGTP. B) lane 1: marker DNA (100–1200 bp);
lane 2: negative control without TTP (dATP + dCTP + dGTP); lane 3:
positive control: TTP + dATP + dCTP + dGTP; lane 4: TTP + dATP +
dCTP + dGTP (0.5 unit per 10 ll of DNA polymerase was used); lane
5: 1 + dATP + dCTP + dGTP: lane 6: 15 + dATP + dCTP + dGTP:
lane 7: 16 + dATP + dCTP + dGTP.
Conclusions
We have shown that triphosphates of thymidine analogues
bearing various functional groups except an anionic, mercapto
or bulky group can be incorporated into DNA by PCR
using KOD Dash DNA polymerase, yielding the corresponding
modified DNA. The modified DNAs with imidazole, pyridine
and phenanthroline derivatives obtained by PCR in this method
will be useful for the production of catalytic DNA by applying
in vitro selection. The in vitro selection using the modified DNA
with a cationic-charged guanidinium group or amino group may
help in the production of highly active DNA aptamers against
the anionic-charged target molecule, that cannot be obtained
from the natural-type DNA.
Experimental
Analytical methods and gel electrophoresis
ESI-Mass spectra were recorded on a PE-Sciex API-100 mass
spectrometer. UV spectra were obtained with a Shimazu 1200
1
spectrometer. H- and 31P-NMR spectra were obtained with a
JEOL a-500 or a JEOL AL-300 spectrometer. Triphosphates of
2ꢀ-deoxyuridine derivatives for NMR measurement were passed
through a Dowex 50-WX-8 (Na+ form) column to convert the
triethylammonium salt to the sodium salt. Tetramethylsilane
(TMS) and 85% phosphoric acid were used as the internal
standards for 1H- and 31P-NMR, respectively. High-pressure
liquid chromatography (HPLC) on an ODS-silica gel column
(4 mm × 250 mm) was carried out with a linear gradient
elution of acetonitrile in 50 mM triethylammonium acetate at
a flow rate of 1.0 ml min−1. Gel electrophoresis was carried out
using 2% agarose gel electrophoresis at 100 V for 45 min and
was visualized by staining with ethidium bromide. The band
intensities of the DNA on the gel were quantified with a Bio-Rad
Molecular Imager FX Pro. The sequencing of the PCR product
was carried out using a BigDye Terminator Cycle Sequencing
Kit (Applied Biosystems) and an ABI Prism 310NT Genetic
Analyzer.
effects of the substituent group and the length of a linker arm
at C5 of thymidine may be important for the acceptance of
the substrate by the enzyme. The enzyme also hardly accepted
the thymidine analogue with anionic-charged carboxyl group
11 (lane 11). The analogue bearing a carboxyl group without
a linker arm was also a poor substrate for the KOD DNA
polumerase.28 On the other hand, Pwo DNA polymerase accept
the 7-deazaadenosine nucleotide bearing a carboxyl group with
a propynyl linker 19 in place of dATP, forming the corresponding
modified DNA.25,26 KOD DNA polymerase has been reported
to have two aspartic acid residues in the active site and the
carboxyl group of the active site of the enzyme is crucial for
the DNA polymerization ability.34 The electrostatic repulsion
between the carboxylic acids at the active site of the enzyme and
the carboxylic acid of the substrate might be responsible for the
poor substrate activity of the substrate with carboxylic acid. A
cationic group at the C5-position of the thymidine analogues
does not impede the interaction of the substrate with the active
site of the enzyme, hence maintaining their substrate activity.
Materials
2ꢀ-Deoxynucleoside 5ꢀ-triphosphates, dATP, dGTP, dCTP and
TTP, were purchased from Roche Diagnostics Co. KOD Dash
2 4 6 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 6 3 – 2 4 6 8

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2H), 3.08 (m, 4H), 2.30 (m, 2H), 1.43 (m, 4H), 1.22 (m, 4H); 31
DNA polymerases were obtained from Toyobo Co. 5ꢀ-Triphos-
phates of 5-N-(6-aminohexyl)carbamoylmethyl-2ꢀ-deoxyuridine
5ꢀ-triphosphate (1) and 5-N-(6-trifluoroacetylaminohexyl)carb-
amoylmethyl-2ꢀ-deoxyuridine (2) were prepared as described
previously.27 Other C5-substituted deoxyuridine derivatives
were prepared from 1. The pUC18 plasmid DNA was from
Toyobo Co. Oligonucleotides used as primers, DNA A, 5ꢀ-
GGAAACAGCTATGACCATGATTAC-3ꢀ and DNA B, 5ꢀ-
CGACGTTGTAAAACGACGGCCAGT-3ꢀ, were from Sawa-
day Technology Co.
P
NMR (D2O) d −5.6 (d), −10.6 (d), −21.4 (t).
Synthetic procedure of 5ꢀ-triphosphates of thymidine ana-
logues, 4–14 and 16 was similar to that described above for
the synthesis of 3 and 15 and a detailed procedure is described
in the supporting information.† The yield and ESI-mass data of
the nucleotides, 4–14 and 16, are listed in the following.
5-N -(6-[4-Imidazoleacetyl]aminohexyl)carbamoylmethyl-2ꢀ -
deoxyuridine 5ꢀ-triphospahte (4). The yield of 4 was 43%
from 1. ESI-Mass (negative mode) m/z: found 731.3; calcd for
[M–H]− 731.1.
DNA ligation kit was purchased from Takara Co. GenElute
plasmid purification kit and X-Gal were from Sigma. All other
chemicals were reagent grade and were used without further
purification.
5-N -(6-[Urocanyl]aminohexyl)carbamoylmethyl-2ꢀ -deoxy-
uridine 5ꢀ-triphospahte (5). The nucleotide 5 was obtained in
96% yield from 1. ESI-Mass (negative mode) m/z: found 723.1;
calcd for [M–H]− 723.1.
Synthesis of 5ꢀ-triphosphates of 2ꢀ-deoxyuridine derivatives
5-N-(6-[Biotinyl]aminohexyl)carbamoylmethyl-2ꢀ-deoxyuridine
5ꢀ-triphospahte (6). The yield of 6 was 19% from 1. ESI-Mass
(negative mode) m/z: found 849.3; calcd for [M–H]− 849.2.
5-N -(6-[Nicotinyl]aminohexyl)carbamoylmethyl-2ꢀ -deoxy-
uridine 5ꢀ-triphospahte (3). A solution of dicyclohexylcarbodi-
imide (309 mg, 1.50 mmol) in dry DMF (5 ml) was added
gradually to a solution of nicotinic acid (123 mg, 1.00 mmol)
and N-hydroxysuccinimide (138 mg, 1.20 mmol) in dry DMF
(5 ml), under nitrogen, in an ice bath, with stirring. The mixture
was stirred for 1 h at 0 ◦C, then overnight at rt. The solution was
evaporated to dryness and the residue was dissolved in ethyl
acetate. The solution was filtered to remove the precipitate and
concentrated by evaporation to give the N-hydroxysuccinimide
ester of biotin as a white powder, 307 mg. The activated
ester was used without further purification and was generated
immediately prior to use.
5-N-(6-[3-(2-Pyridyldithio)propionyl]aminohexyl)carbamoyl-
methyl-2ꢀ-deoxyuridine 5ꢀ-triphospahte (7). The yield of the
nucleotide 7 was 59% from 1. ESI-Mass (negative mode) m/z:
found 820.0; calcd for [M–H]− 820.1.
5-N-(6-[3-Mercaptopropinonyl]aminohexyl)carbamoylmethyl-
2ꢀ-deoxyuridine 5ꢀ-triphospahte (8). The yield of the nucleotide
8 was 66%. ESI-Mass (negative mode) m/z: found 711.0; calcd
for [M–H]− 711.1.
5-N -(6-[e-Trifulouroacetylamidohexyl]aminohexynyl)carb-
amoylmethyl-2ꢀ-deoxyuridine 5ꢀ-triphospahte (9). The yield of
the nucleotide 9 was 88% from 1. ESI-Mass (negative mode)
m/z: found 832.2; calcd for [M–H]− 832.2.
5-N-(6-[Aminohexynyl]aminohexyl)carbamoylmethyl-2ꢀ-deoxy-
uridine 5ꢀ-triphospahte (10). The nucleotide 10 was obtained
in 93% yield from 1. ESI-Mass (negative mode) m/z: found
736.4; calcd for [M–H]− 736.2.
5-N-(6-Aminohexyl)carbamoylmethyl-2ꢀ-deoxyuridine
5ꢀ-
triphosphate (1) (60 OD260 nm, 6.5 lmol) was dissolved in a
solution containing distilled water (300 ll) and 1.0 M sodium
hydrogen carbonate buffer (pH 9.5) (200 ll) at rt. The solution
of the active ester (40 mg, 180 lmol) in dry DMF (500 ll)
was added and stirred for 20 h at 37 ◦C. The mixture was
concentrated and purified by HPLC on an ODS-silica gel
column (10 mm × 250 mm), with a linear gradient elution
of acetonitrile (2.1–30.1%) in 50 mM triethylammonium
acetate (pH 7.2) for 28 min at a flow rate of 3.0 ml min−1.
5-N-(6-Succinylaminohexyl)carbamoylmethyl-2ꢀ-deoxyuridine
5ꢀ-triphospahte (11). The nucleotide 11 was obtained in 70%
yield from 1. ESI-Mass (negative mode) m/z: found 723.1;
calcd for [M–H]− 723.1.
The purified 3 was obtained in 46% yield (42 OD260
,
nm
3.0 lmol). The yield was calculated from the estimation that the
molar absorption coefficient of the product is the sum of that
of 5-N-(6-aminohexyl)carbamoylmethyl-2ꢀ-deoxyuridine and
nicotinic acid. ESI-Mass (negative mode) m/z: found 728.0;
calcd for [M–H]− 728.1. 1H NMR (D2O) d: 8.86 (1H, s, Py–H2),
8.69 (1H, d, Py–H6), 8.17 (1H, d, J = 7.9 Hz, Py–H4), 7.91
(1H, s, H6), 7.58 (1H, dd, Py–H5), 6.29 (1H, dd, H1ꢀ), 4.69
(1H, m, H3ꢀ), 4.24–4.13 (3H, m, H4ꢀ and H5ꢀ), 3.40 (2H, t, J =
6.9 Hz, NHCH2), 3.35 (2H, s, C5–CH2), 3.20 (2H, m, CH2NH),
2.40 (2H, m, H2ꢀ), 1.63 (2H, m, NHCH2CH2), 1.53 (2H, m,
CH2CH2NH), 1.37 (4H, m, CH2CH2). 31P NMR d: −5.7 (d,
J = 60 Hz, c-P), −10.3 (d, J = 60 Hz, a-P), −21.3 (t, J = 60 Hz,
b-P).
5-N-(6-[9-Phenanthrylcarbonyl]aminohexyl)carbamoylmethyl-
2ꢀ-deoxyuridine 5ꢀ-triphosphate (12). The yield of 12 was 45%
yield from 1. ESI-Mass (negative mode) m/z: found 829.1;
calcd for [M–H]− 8291.
5-N-(6-[5-(2,9-Dimethylphenanthroline)thiouryl]aminohexyl)-
carbamoyl-methyl-2ꢀ-deoxyuridine 5ꢀ-triphosphate (13). The
yield of 13 was 54% from 1. ESI-Mass (negative mode) m/z:
found 888.1; calcd for [M–H]− 888.2.
5-N-(4-[Fluoresceinthiouryl]aminohexyl)carbamoyl-methyl-2ꢀ-
deoxyuridine 5ꢀ-triphosphate (14). The yield of 14 was 27%
yield from 1. ESI-Mass (negative mode) m/z: found 1012.3;
calcd for [M–H]− 1012.3.
5-N -(6-Guanidinumhexyl)carbamoylmethyl-2ꢀ -deoxyuridine
5ꢀ-triphospahte (15). The guanidylation reagent, S-ethylthio-
pseudourea hydrobromide was prepared according to the
published procedure.35 Triethylamine (659 ll, 4.75 lmol)
was added to a solution of 1 (100 OD260 nm, 10.8 lmol) and
S-ethylthiopseudourea hydrobromide (440 mg, 2.38 lmol) in
DMF (2.38 ll) with stirring at rt. The reaction mixture was
stirred for 10 h at rt. The mixture was concentrated and purified
by HPLC on an ODS-silica gel column (20 mm × 250 mm) with
a linear gradient elution of acetonitrile (3.5–14%) in 50 mM
triethylammonium acetate (pH 7.2) for 45 min at a flow rate of
8.0 ml min−1. The purified nucleotide 15 was obtained in 70%
yield (70 OD260 nm, 7.6 lmol). ESI-Mass (negative mode) m/z:
5-N-(4-[Fluoresceinylcarbonyl]aminohexyl)carbamoyl-methyl-
2ꢀ-deoxyuridine 5ꢀ-triphosphate (16). The yield of 16 was 6%
yield from 1. ESI-Mass (negative mode) m/z: found 981.0;
calcd for [M–H]− 981.1.
Synthesis of modified DNAs by PCR with modified TTP using
KOD Dash DNA polymerase. The reaction mixture (20 ll)
contained 0.5 ng ll−1 DNA template (pUC18 2686 bp), 0.2 lM
of each primer, 0.2 mM of natural dNTPs (dATP + dGTP +
dCTP + TTP) or modified dNTP mix (dATP + dGTP +
dCTP + modified TTP) and 0.05 unit per 10 ll or 0.5 unit per
10 ll of DNA polymerase, respectively, in the buffer supplied by
the maker for the DNA polymerase reaction. PCR assays were
carried out at 94 ◦C for 1 min, 30 cycles of 94 ◦C for 30 sec,
1
found 665.1; calcd for [M–H]− 665.1. H NMR (D2O) d 7.82
(s, 1H), 6.23 (t, 1H), 4.59 (m, 1H), 4.18–3.99 (m, 3H), 3.26 (s,
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 6 3 – 2 4 6 8
2 4 6 7

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52 ◦C for 30 sec, 74 ◦C for 1 min and 74 ◦C for 5 min. The
reaction mixture was quenched by the addition of a formamide-
dye solution (90% formamide : 1% bromophenolblue : 1%
xylenencyanol) and the PCR products were analyzed by 2%
agarose gel electrophoresis after staining with ethidium bromide.
The band intensity of the resulting modified DNA on the gel was
quantified with a Bio-Rad Molecular Imager. Relative yield of
the modified DNA from each thymidine analogue was estimated
from the band intensity.
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This work is partially supported by a Grant-in-Aid for Scientific
Research from The Ministry of Education, Science and Tech-
nology, Japan.
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2 4 6 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 6 3 – 2 4 6 8