Deoxynucleoside Triphosphate Containing Pyridazin-3-one Aglycon as a Thymidine Triphosphate Substitute for Primer Extension and Chain Elongation by Klenow Fragments

Article abstract of DOI:10.1021/acs.joc.8b00918

Deoxynucleoside 5′-triphosphate was synthesized with 3-oxo-2H-pyridazin-6-yl (Pz^O) - a uracil analogue lacking a 2-keto group - as the nucleobase. Theoretical analyses and hybridization experiments indicated that Pz^O recognizes adeni

Full text of DOI:10.1021/acs.joc.8b00918

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Article
Deoxynucleoside Triphosphate Containing Pyridazin-3-
one Aglycon as a Thymidine Triphosphate Substitute for
Primer Extension and Chain Elongation by Klenow fragments
Takahito Tomori, Kento Nagaoka, Leo Takeshita, Takashi Shiozawa,
Yuya Miyatake, Yoshiaki Masaki, Mitsuo Sekine, and Kohji Seio
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00918 • Publication Date (Web): 28 Jun 2018
Downloaded from http://pubs.acs.org on June 29, 2018
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Deoxynucleoside Triphosphate Containing Pyridazinꢀ3ꢀone
Aglycon as a Thymidine Triphosphate Substitute for Primer
Extension and Chain Elongation by Klenow Fragments
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Takahito Tomori, Kento Nagaoka, Leo Takeshita, Takashi Shiozawa, Yuya Miyatake, Yoshiaki
Masaki, Mitsuo Sekine, and Kohji Seio*
School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku,
Yokohama, 226-8501, Japan
kseio@bio.titech.ac.jp
Abstract
Deoxynucleoside 5'ꢀtriphosphate was synthesized with 3ꢀoxoꢀ2Hꢀpyridazinꢀ6ꢀyl (PzO)—a uracil
analog lacking a 2ꢀketo group—as the nucleobase. Theoretical analyses and hybridization
experiments indicated that PzO recognizes adenine (A) for formation of a WatsonꢀCrick base pair.
Primer extension reactions using nucleoside 5'ꢀtriphosphate and Klenow fragment revealed that the
synthetic nucleoside 5'ꢀtriphosphate was incorporated into the 3' end of the primer through
recognition of A in the template strand. Moreover, the 3'ꢀnucleotide residue harboring PzO as the
base was resistant to the 3'ꢀexonuclease activity of Klenow fragment exo+. The primer bearing the
PzO base at the 3' end could function in subsequent chain elongation. These properties of PzO were
attributed to the presence of an endocyclic nitrogen atom at the position ortho to the glycosidic bond,
which was presumed to form an Hꢀbond with the amino acid residue of DNA polymerase for
effective recognition of the 3' end of the primer for primer extension. These results provide a basis
for designing new nucleobases by combining a nitrogen atom at the position ortho to the glycosidic
bond and baseꢀpairing sites for WatsonꢀCrick hydrogen bonding.
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Introduction
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The development of deoxynucleotides bearing unnatural nucleobases that can be introduced into
genomic DNA by DNA polymerases1,2,3 has been widely studied in the field of synthetic biology. To
this end, many types of unnatural base pairs including NaMꢀ5SICS,4,5 DsꢀPn(x),6,7 and PꢀZ8 have
been synthesized. Artificial nucleobases must have several features for incorporation into genomic
DNA. Firstly, the artificial deoxynucleoside 5'ꢀtriphosphate must be capable of incorporation into the
3' terminal hydroxyl group of the primer recognizing the counter nucleobase on the template.
Secondly, the primer bearing the artificial nucleoside residue at the 3' end must be amenable to
extension by DNA polymerases. Thirdly, the artificial nucleotide at the 3' end of the primer must be
resistant to the proofreading 3'→5' exonuclease activity DNA polymerases.
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To meet the first criterion, artificial base pairs recognizing counter bases either through
hydrogen bonding8 or shape fitting4,5,6,7,9 have been developed. For the second criterion, an Hꢀbond
acceptor on the minor groove side of nucleobases—referred to as the minor groove Hꢀbond
acceptor—is required. In the case of the natural nucleobases, the nitrogen at position 3 (N3) and
2ꢀketo group of purine and pyrimidine nucleobases, respectively, function as minor groove Hꢀbond
acceptors. Their importance has been supported by Xꢀray crystallographic studies10ꢀ12 of binary
complexes consisting of DNA polymerase and a primerꢀtemplate duplex, or of ternary complexes
containing an additional nucleoside triphosphate. For example, in the binary complex consisting of
Thermus aquaticus (Taq) DNA polymerase I—a DNA polymerase belonging to the A family—and a
primerꢀtemplate duplex, Arg573 of the polymerase recognized nucleobases at the 3' end of the
primer, at the N3 of guanine (G) or 2ꢀketo of cytosine (C).11 The same crystallographic structures
also demonstrated the recognition of the 2ꢀketo of C or the N3 of G in the corresponding position in
the template by Gln754. In fact, successful artificial base pairs have minor groove Hꢀbond acceptors.
For example, the NaMꢀ5SICS pair has a methoxy group in NaM and a thioketo group in 5SICS; the
DsꢀPn pair has an endocyclic nitrogen atom in Ds and a nitro group in Pn; and the PꢀZ pair has an
endocyclic nitrogen in P and keto group in Z. Xꢀray crystallographic studies of these artificial base
pairs have revealed the expected Hꢀbonding in the minor groove.10, 13
Various heteroaromatic groups harboring an endocylic nitrogen or oxygen atom at the position
ortho to the glycosidic bond have been reported as a potential scaffold for new artificial nucleobase
analogs bearing a minor groove Hꢀbond acceptor, including benzofuranꢀ2ꢀyl or benzotriazolꢀ2ꢀyl14
and pyridineꢀ2ꢀyl (2Py).15 An investigation of the DNA polymerase reaction using a primerꢀtemplate
duplex containing a 2Py residue at both 3' ends of the primer and at the counter position of the
template as a 2Pyꢀ2Py selfꢀpair15 found that strand extension catalyzed by Klenow fragment exoꢀ
(Kf−) proceeded more rapidly from the primer harboring 2Py than from the primerꢀtemplate duplex
containing other pairs of sixꢀmembered rings without a nitrogen atom at the position ortho to the
glycosidic bond. These results suggest that this nitrogen atom acts as a minor groove Hꢀbond
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acceptor. However, 2Py itself has not been used for artificial selfꢀpairing because it can
accommodate dATP or dGTP more efficiently when incorporated into the template strand. Therefore,
if the 2Py structure is used as the scaffold for artificial nucleobases, some functional groups must be
introduced that recognize specific nucleobase analogs via appropriate noncovalent interactions such
as hydrogen bonds.
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Based on these considerations, we designed pyridazinꢀ3ꢀone (PzO; Figure 1a) as a new
artificial nucleobase. PzO was designed with Hꢀbonding sites to form WatsonꢀCrick base pairs with A,
including a carbonyl group at position 3 and an imino group at position 2. It also has a nitrogen atom
at position 2 as the minor groove Hꢀbond acceptor in the case of 2Py (Figure 1a). Since PzO was
designed to recognize the natural nucleobase A, it cannot itself be a component of new artificial pairs.
The aim of this study was to demonstrate that the combination of nitrogen at the ortho position and
hydrogen sites for base pairing is an effective design strategy for artificial bases.
We synthesized a deoxynucleoside triphosphate (1) with PzO as an aglycon (Figure 1b), and
characterized its properties including baseꢀpairing stability and selectivity by preparing DNA
duplexes in which PzO was incorporated using phosphoramidite (2) (Figure 1b). Additionally, we
examined reactions catalyzed by Kf− and Klenow fragment exo+ (Kf+) using 1 instead of thymidine
5'ꢀtriphosphate (TTP). Our results show that PzO can selectively recognize A during DNA duplex
formation and enzymatic base incorporation. In addition, the primer harboring PzO at the 3' end
could be elongated by either Kf− or Kf+, despite the absence of the 2ꢀketo carbonyl group.
Figure 1. a) Design and structure of a pyridazineꢀtype nucleobase (PzO) with sites for hydrogen bonding with A and
an enzyme recognition site. b) Structure of 1 and 2.
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Results and Discussion
Hydrogen bond properties of PzO
The hydrogen bond energies between PzO or thymine (T) and A or G were calculated according to
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the
density
functional
theory
of
electronic
structure
at
the
M06ꢀ2X/6ꢀ311+G(2df,p)//M06ꢀ2X/6ꢀ31+G(d,p) level (Figure 2).16 In this calculation, we used
6ꢀmethylpyridazinꢀ3ꢀone, 1ꢀmethylthymine, 9ꢀmethyladenine, and 9ꢀmethylguanine as model
structures for PzO, T, A, and G, respectively. Baseꢀpairing energies were calculated as energy
differences between XꢀY complexes such as AꢀT, GꢀT, AꢀPzO, and GꢀPzO, and the sum of each base.
The basis set superposition error was estimated using the counterpoise method.17 Base pair structures
and baseꢀpairing energies are shown in Figure 2. PzO formed base pairs with A in the WatsonꢀCrick
geometry (Figure 2a). The baseꢀpairing energy for the AꢀPzO pair was −13.8 kcal/mol, which was
almost identical to that of the AꢀT base pair (−13.7 kcal/mol) (Figure 2b). These results suggest that
the AꢀPzO base pairing is as stable as AꢀT.
We also calculated the baseꢀpairing energy of GꢀPzO (Figure 2c) and GꢀT (Figure 2d) pairs
starting from wobble geometry. The value of the GꢀPzO pair was −10.9 kcal/mol, which was larger
than that of GꢀT base pair (−14.6 kcal). The lower stability of the GꢀPzO base pair can be explained
by two factors. Firstly, although two hydrogen bonds were formed in both base pairs, the absence of
a 2ꢀketo group in PzO shortened the distance between the 2ꢀamino group of G and the methyl group
corresponding to the C1' position of PzO. Consequently, the planes of PzO and G were twisted by
14.3° while T and G were coꢀplanar (Figure S1), which prevented steric clashing between the methyl
and amino groups. Secondly, as indicated by the electrostatic potential map of PzO (Figure 3a) and T
(Figure 3b), the N1 of PzO was less negative than the 2ꢀketo group of T. Thus, hydrogen bonding
between the N1 of PzO and the amino group of G may be intrinsically weaker than that between the
2ꢀketo of T and the amino group of G.
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Figure 2. Baseꢀpair structures and energies of a) AꢀPzO, b) AꢀT, c) GꢀPzO, and d) GꢀT.
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Figure 3. Electrostatic potential of electron density from total selfꢀconsistent field density (isovalue = 0.001) of a) T
and b) PzO. The color bar indicates electrostatic potential from −7.0e−2 to 7.0e−2. Images were generated with
GaussView v.6.0.16. Chemical structures were overlaid to show the approximate positions of atoms.
Synthesis of ribonucleoside, deoxynucleoside, triphosphate, and phosphoramidite
harboring PzO as a nucleobase
We synthesized the nucleoside triphosphate 1 and phosphoramidite 2. Although methods for
synthesizing a Cꢀnucleosides from a glycal and a heteroaromatic compound using transition metal
catalyst has been reported so far, the pyridazin deravatives have never been introduced by these
methods.18,19 Indeed, we applied these method using 3,6ꢀdibromopyiridazon, but the Cꢀglycosidic
bond did not form (data not shown). Therefore, we carried out the synthesis of ribonucleoside 1020,21
and deoxynucleoside 13 (dPzO)22 with PzO as the base moiety by optimizing previously reported
procedure. The compounds were synthesized via known intermediates 4,23 5,20,21,22 6,21 and 9,21 but
the conditions and routes of synthesis were modified for maximal yield.
The new synthetic route for synthesis of dPzOTP (1) and phosphoramidite derivatives (2) is
outlines in Scheme 1.
We first synthesized compound 423 from 3 by FriedelꢀCrafts Cꢀglycosilation catalyzed by
SnCl4. Under the previously reported conditions,23 the major product was a compound with two
furan rings at position 1 of the ringꢀopened ribose skeleton. This perꢀfuranylation was suppressed
when the amount of SnCl4 was reduced from 1.1 to 0.1 equiv. The acetyl groups of 4 were removed
by sodium methoxide; reꢀprotection with benzoyl groups yielded 5, which had increased lipophilicity.
Oxidation of compound 5 produced 6, which was converted to compound 7 by hydrogenation. We
used 4ꢀ(4,6ꢀdimethoxyꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ4ꢀmethylmorpholinium chloride as the condensing agent
in the cyclization of 7 to 8 using hydrazine, and 8 was then converted to 10 via oxidization by
2,3ꢀdichloroꢀ5,6ꢀdicyanobenzoquinone22 and deprotection by treatment with 28% aqueous NH3.
We next synthesized deoxynucleoside derivatives from ribonucleoside 10. The 3' and 5'
hydroxyl groups were protected with 1,1,3,3ꢀtetraisopropyldisiloxaneꢀ1,3ꢀdiyl to obtain 11, which
was deoxygenated by treatment with Oꢀphenyl chlorothionoformate followed by reduction using
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Tris(trimethylsilyl)silane and azobisisobutyronitrile as reducing agent and radical initiator,
respectively. The resultant compound 12 was deprotected using triethylamineꢀtris(hydrogen fluoride)
to obtain the deoxynucleoside 13 (73% yield). Compound 13 was converted to the triphosphate
derivative 1 in three steps (27% yield) using phosphoryl chloride and pyrophosphate,24,25 and then
separated on a diethylꢀaminoethyl Sephadex Aꢀ25 column (Figures S2 and S3). The phosphoramidite
derivative 2 was also synthesized. The 5'ꢀhydroxyl group was protected by 4, 4' dimethoxytrityl to
obtain 14 (53% yield), which was phosphitylated to obtain 2 (50% yield). The structures of the
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1
synthesized compounds were characterized by Hꢀ and other types of nuclear magnetic resonance
spectroscopy (see the Supporting Information) and by electrospray ionization (ESI) timeꢀofꢀflight
(TOF) mass spectrometry (MS).
Scheme 1. Synthesis of dPzO triphosphate (1) and phosphoramidite derivatives (2).
Oligodeoxynucleotide (ODN) synthesis and ultraviolet (UV) melting curve analysis
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To investigate the baseꢀpairing capacity of PzO, the hybridization properties of ODNs containing PzO
were examined by measuring the melting temperature (Tm) of ODN duplexes modified with PzO and
their complementary and nonꢀcomplementary ODNs. The ODNs were synthesized according to the
standard phosphoramidite method on an automated DNA synthesizer without modifications. The
sequences of the synthesized and purchased ODNs are shown in Figure 4a. ODN1 had PzO
incorporated at the position indicated by [X], and was synthesized from 2 and commercially
available deoxynucleoside phosphoramidites. The ODNs were purified by anion exchange
highꢀperformance liquid chromatography and characterized by matrixꢀassisted laser
desorption/ionizationꢀTOF MS (Figures S4 and S5).
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ODN2 was synthesized as an unmodified ODN containing T instead of PzO. The sequence of
cODN1 was complementary to that of ODN2, with an A at the position indicated by [Y]. cODN2–4
were mismatched sequences with G, C, and T, respectively, at the [Y] position.
Figure 4. a) Sequences of ODNs used in hybridization studies. b) Sequences of primers and 25ꢀnt templates used in
single nucleotide insertion and fullꢀlength primer extension studies. c) Sequences of 30ꢀnt templates used in the chain
elongation reaction. d) Sequences of size markers used in PAGE analyses.
The Tm values of duplexes formed between ODN1 or ODN2 and cODN1–4 were determined
from UV melting curves (Figures S6 to S9) and are summarized in Table 1. The Tm of ODN1 and
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cODN1 was 54.3°C, which was 3.5°C lower than that of the ODN2/cODN1 duplex (Tm = 57.8°C).
The higher Tm of the latter may reflect the stacking effects of the methyl and keto groups at positions
5 and 2, respectively, of the T at position [X], which stabilized the duplex. On the other hand, the Tm
values of mismatched duplexes containing ODN1 and ODN2 were 27.4°C and 31.3°C, respectively,
for the duplex with cODN2; 40.2°C and 41.2°C, respectively, for the duplex with cODN3; and
45.2°C and 48.3°C, respectively, for the duplex with cODN4. Base recognition by PzO and T were
compared according to differences in Tm values between matched duplexes (e.g., ODN1/cODN1 and
ODN2/cODN1) and the most stable mismatched duplexes (e.g., ODN1/cODN4 and ODN2/cODN4).
The base recognition capacities of ODN1 and ODN2 were nearly identical (−9.1°C vs. –9.5°C).
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Table 1. Base recognition capacity of PzO
ODN
cODN
Tma/°C
ꢁ
−
Tmb/°C
(X)
(Y)
ODN1
cODN1
54.3
(X = PzO) (Y = A)
cODN2
(0.25)
27.4
−26.9
−14.1
−9.1
−
(0.98)
(Y = G)
cODN3
40.2
(0.25)
(Y = C)
cODN4
45.2
(0.11)
(Y = T )
ODN2
cODN1
(Y = A)
cODN2
(Y = G)
cODN3
(Y = C)
cODN4
(Y = T )
57.8
(0.49)
(X = T)
31.3
−26.5
−16.6
−9.5
(0.25)
41.2
(0.25)
48.3
(0.24)
aTm values (and standard deviations in parentheses) were measured in 10 mM sodium phosphate (pH 7.0) containing
0.1 M NaCl, 0.1 mM EDTA, and 2.0 µM each ODN.
bꢁTm = Tm −Tm (Y = A)
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Single nucleotide insertion by Kf−
To investigate the templateꢀspecific incorporation of 1 by DNA polymerase, we examined single
nucleotide insertion using the Kf− of Escherichia coli DNA polymerase lacking 3' to 5' exonuclease
activity. Template and primer sequences are shown in Figure 4b. The templates T1, T2, T3, and T4
were single stranded 25ꢀnt ODNs containing A, G, C, and T, respectively, at position [Z]. Primer P1
was a singleꢀstranded 18ꢀnt DNA whose 5' end was labeled with a carboxyfluorescein residue (FAM
in Figure 4b). One of T1 to T4 along with P1, and 10 µM of 1 or 10 µM TTP was incubated in the
presence of Kf− for single nucleotide incorporation (Figure 5a). The reaction products were analyzed
by 20% polyacrylamide gel electrophoresis (PAGE) containing 7 M urea (Figure 5b). Lane 1 shows
the band corresponding to P1; lanes 2–5 are for experiments using 1 and T1–T4, in which A, G, C,
and T, respectively, occupied position [Z] of the template; lanes 6–9 are for experiments using TTP
and T1–T4, respectively. Shown in lane 10 is the synthetic 19ꢀnt control (P2 in Figure 4d), whose
sequence was identical to P1 but with an additional T residue at the 3' end. Compound 1 was
incorporated when [Z] was A (lane 2) but not when it was G, C, or T (lanes 3–5). Under these
conditions, TTP was incorporated when [Z] = A (lane 6), but minor band corresponding to the 19ꢀnt
product was observed even when [Z] = G, C, and T (lanes 7–9), because the TTP concentration was
higher than the Km of the Kf−ꢀcatalyzed TTP incorporation reaction (discussed below). Thus, 10 µM
1 was predominantly incorporated into the site opposite to A without giving misꢀincorporated
products.
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Figure 5. PAGE analysis of single nucleotide incorporation of 1 or TTP into P1 in the presence of 25ꢀnt T1–T4.
Lanes 1 and 10 correspond to the synthetic 18ꢀnt primer P1 and 19ꢀnt synthetic control P2, respectively. Conditions:
0.1 µM template (T1–T4); 0.1 µM P1; 10 µM each TTP or 1; 0.01 U/µl Kf−; and reaction time = 10 min.
To determine the incorporation efficiency of 1, we evaluated the steadyꢀstate kinetic
parameters for the single nucleotide insertion reaction using Kf− (Table 2). The Km, Vmax, and
Vmax/Km values were determined using T1 as the template. The Km values for 1 and TTP were 9 and
0.07 µM, respectively. On the other hand, 1 and TTP had nearly identical Vmax values (21%·and
25%·min−1, respectively). The large Km of 1 indicated that the affinity of 1 for the binary complex
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consisting of Kf− and the P1/T1 duplex was roughly two orders of magnitude lower than that of TTP.
On the other hand, the similar Vmax values of 1 and TTP indicated that the kinetics of phosphodiester
bond formation in the ternary complex consisting of Kf−, 1, and the P1/T1 duplex were as rapid as
those in the complex with TTP instead of 1. Accordingly, the gross reaction efficiency as evaluated
by Vmax/Km was 2.9 × 108 and 2.8 × 106 for TTP and 1, respectively, indicating that 1 was
incorporated about 100 times less efficiently than TTP.
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Table 2. Steadyꢀstate kinetic parameters for the insertion of a single dNTP into a P1/T1 duplex by Kf−
Vmax/Km
N
Km (µM)
Vmax (%·min−1)
(%·min−1·M−1)
2.9 x 108
T
0.07 (0.02)
21 (4.0)
25 (4.0)
PzO 9.0 (0.9)
2.8 x 106
Fullꢀlength primer extension by Kf−
We performed the fullꢀlength extension of P1 using T1, Kf−, and a mixture of dATP, dGTP, and
dCTP in the presence or absence of 1 or TTP (Figure 6). The band in lane 1 corresponds to P1, and
lane 5 shows the synthetic 25ꢀnt P3 (Figure 4c) with a chain length identical to that of the expected
fullꢀlength product. Lane 2, which is the reaction product in the absence of 1 or TTP, had a thin band
corresponding to the 19ꢀnt product generated by incorporation of dGTP, dCTP, or dATP mismatched
with A.
Figure 6. Fullꢀlength primer extension by Kf− using P1 and T1 by. Lane 1, P1: lane 2, reaction in the presence of
dATP, dGTP, and dCTP; lane 3, reaction in the presence of 1, dATP, dGTP, and dCTP; lane 4, reaction in the
presence of TTP, dATP, dGTP, and dCTP; lane 5, 25ꢀnt control P3. Conditions: 0.1 µM T1, 0.1 µM P1, 10 µM each
dNTP, 0.01 U/µl Kf−, and reaction time = 10 min.
The reaction in the presence of 1, dGTP, dCTP, and dATP (lane 3) yielded a products that
appeared at the same position as the synthetic 25ꢀnt control P3 (lane 5) and 26ꢀnt product. The
mobility of these bands were also identical to those of the band in lane 4, in which TTP was used
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instead of 1. These results indicate that 1 was incorporated into the position opposite A without
disrupting subsequent chain elongation, resulting in a fullꢀlength product.
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Incorporation of 1 by Kf− at two positions
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To further evaluate the properties of 1, we used a duplex consisting of P1 and the 30ꢀnt template T9
(Figure 4c) containing two A residues flanking the 5'ꢀCGTꢀ3' trinucleotide sequence in the
singleꢀstranded region of the primerꢀtemplate duplex (Figure 7). Shown in lane 1 is the band
corresponding to P1; lanes 7 and 14 show the synthetic control P4 (Figure 4d), whose chain length
was 30ꢀnt. When 10 µM 1 was used (left panel), an intermediate product (*) presumed to be P5
(Figure 4f) was observed at 5–60 min. On the other hand, the expected fullꢀlength products at the
position close to that of the synthetic control P4 (lanes 7 and 14) were not observed in lanes 2 and 3
at 5 and 10 min. The amount of fullꢀlength product increased over time (lanes 4–6), becoming the
main band after 60 min (lane 6). It is worth noting that in these experiments, the fullꢀlength product
comprised 31ꢀnt ODNs that were longer than the synthetic control P4 by one nucleotide, as
determined based on gel mobility. The sequence of the fullꢀlength product was likely 31 nt with one
of the four nucleotide residues at the 3'́ end resulting from the templateꢀindependent incorporation
activity of Kf−.26
The same experiments carried out at 100 µM 1 (right panel) showed that the fullꢀlength
product was obtained as the major product within 30 min (lane 10).
Figure 7. Time course of fullꢀlength primer extension with incorporation of two dPzO residues. Lane 1, P1; lanes 2–
6, time course of reaction in the presence of 10 µM 1; lane 7, 30ꢀnt synthetic control P4; lane 8, P1; lanes 9–13, time
course of reaction in the presence of 100 µM 1; lane 14, 30ꢀnt synthetic control P4. Conditions: 0.1 µM T9; 0.1 µM
P1; 10 µM dATP, dGTP, and dCTP; 0.01 U/µl Kf−.
To further evaluate the properties of 1 in the chain elongation reactions, we used templates T5
and T6–T10 (Figure 4c), incorporating two consecutive A residues or two A residues flanking one to
four nucleotides. The chain elongation reactions were carried out in the presence of 100 µM 1
(Figure 8). Lane 1 corresponds to P1; lane 2 represents the reaction with P1, T5, dGTP, dATP, and
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dCTP; lanes 3–8 corresponded to the reaction of P1 and T5–T10 in the presence of dGTP, dATP,
dCTP, and 1; and lane 10 shows the reaction of P1 and T5 in the presence of the four canonical
deoxynucleoside triphosphates. As shown by PAGE, chain elongation was terminated only in the
reaction shown in lane 4 that used template T6 containing two consecutive A residues. The position
of the band was the same as that observed in lane 2, in which neither TTP nor 1 was included and
chain elongation was halted after incorporation of a mismatched nucleotide. In contrast, the
fullꢀlength products were observed in lanes 5–8. Thus, once 1 was incorporated at the first position,
a second 1 could also be incorporated by Kf− as long as the template was designed so that two A
residues were not arranged consecutively. The reason for the failure of consecutive incorporation is
unclear. However, the Xꢀray crystallographic studies revealed that the nucleobases of dNTP and the
3' ends of the primer were well stacked in the ternary complex consisting of DNA polymerase,
primerꢀtemplate duplex, and dNTP,27 thus, the second 1 may not be properly fixed at the 3' end of the
primer when there is a weak stacking interaction between two PzO residues which lack 5ꢀmethyl and
2ꢀketo groups.
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Figure 8. Incorporation of one (lane 3) or two (lanes 4–8) dPzO residues at various positions. Lane 1, P1; lane 2,
reaction in the presence of P1, T5, dATP, dGTP, and dCTP; lane 3, conditions for lane 2 plus 1; lanes 4–8, reaction
using T6–T10; lane 9, conditions for lane 2 plus TTP; lane 10, P4. Conditions: 0.1 µM template; 0.1 M P1; 10 µM
dATP, dGTP, TTP, and dCTP; 100 µM 1; 0.01 U/µl KF−; and reaction time = 60 min.
Single nucleotide and fullꢀlength primer extension by Kf+
We evaluated the properties of 1 as a substrate of Kf+—which possesses 3' to 5' exonuclease
activity—by performing the single nucleotide extension reaction using primer P1 and the 25ꢀnt
template T5 (Figure 9). As seen in lane 2, 1 was incorporated into the 3' end of P1, yielding a 19ꢀnt
product and a thin band (**) corresponding to the primer shortened by the 3' to 5' exonuclease
activity. In other reactions with templates T2, T3, and T4 containing G, C, and T, respectively (lanes
3–5), there was no 19ꢀnt product, and denser bands were observed for the shortened primer. These
results suggest that the dPzO residue incorporated by Kf+ was resistant to 3' to 5' exonuclease
activity. Thus, base pairing between PzO and A is recognized as legitimate by Kf+.
It was previously reported that 3ꢀdeazaꢀdeoxyadenosine—which lacks a minor groove Hꢀbond
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acceptor—incorporated at the 3' end of a primer was excised by the exonuclease activity of Kf+.28
This result indicated that Kf+ recognized the minor groove Hꢀbond acceptor and distinguished
between complementary and misincorporated nucleotides. The fact that dPzO was not excised by
Kf+ suggests that the N1 nitrogen of PzO acted as the minor groove Hꢀbond acceptor, as in the case
of the N3 of purine or 2ꢀketo of pyrimidine nucleobases.
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We carried out fullꢀlength chain elongation using primer P1, template T1, and the mixture of
dATP, dGTP, and dCTP in the presence or absence of 1 or TTP and found that the fullꢀlength product
was generated when 1 was used instead of TTP (Figure 10).
Figure 9. PAGE analysis of single nucleotide insertion using 1 to P1 in the presence of 18ꢀnt T1–T4. Lanes 1 and 6
correspond to synthetic 18ꢀnt primer P1 and 19ꢀnt synthetic control P2, respectively. Conditions: 0.1 µM T1–T4; 0.1
µM P1; 10 µM each dNTP; 0.01 U/µl Kf−; reaction time = 10 min.
Figure 10. PAGE analysis of single nucleotide insertion using 1 to P1 in the presence of 18ꢀnt T1. Lanes 1 and 5
correspond to 18ꢀnt primer P1 and 25ꢀnt synthetic control P3, respectively. Conditions: 0.1 µM T1; 0.1 µM P1; 10
µM each dNTP; 0.01 U/µl Kf−, and reaction time = 10 min.
Conclusion
In this study, we synthesized dPzO (13) as well as its triphosphate 1 and phosphoramidite derivative
2, and determined that PzO in DNA recognizes and selectively forms WatsonꢀCrick base pairs with A.
In addition, we demonstrated that 1 is incorporated into DNA by Kf− and Kf+, and was not excised
by the 3' to 5' exonuclease activity. Finally, we found that 1 could be introduced at multiple sites as
long as the template was designed so as to avoid consecutive A bases.
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Compound 13 is a new thymidine analog lacking 2ꢀketo and 5ꢀmethyl groups while
maintaining WatsonꢀCrick baseꢀparing properties. Although similar pyrimidine deoxynucleoside
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analogs
such
as
5ꢀ(βꢀ
Dꢀ2ꢀdeoxyribofuranosyl)ꢀ3ꢀmethylꢀ2ꢀpyridone
and
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5ꢀ(βꢀ ꢀ2ꢀdeoxyribofuranosyl)ꢀ2ꢀaminopyridine—Cꢀnucleosides with a methine instead of the 2ꢀketo
D
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group—have been reported,29 their properties as substrates of DNA polymerases are not known since
they inhibit DNA polymerase reactions. We also demonstrated for the first time that the 2ꢀketo group
of pyrimidines is not essential for the DNA polymerase reaction, since 1—which has a nitrogen atom
that can form a hydrogen bond instead of the 2ꢀketo group—was a substrate of Kf− and Kf+. Our
results suggest that combining a baseꢀpairing site with an endocyclic nitrogen atom ortho to the
glycosidic bond is a promising design for novel artificial nucleoside triphosphates that can be
incorporated into DNA by DNA polymerases. In the case of PzO, we combined a Tꢀlike hydrogen
and endocyclic nitrogen atom, but other base pair motifs such as metalꢀmediated30 or halogen
bonding31ꢀ33 interactions may be equally effective.
In its present form, compound 1 cannot be incorporated by Kf− in a consecutive manner.
Although this property may be useful if 1 is used as a chain terminator at the consecutive position in
antiviral or anticancer reagents,34 it is nonetheless a limitation that must be overcome for broad
application of this compound in synthetic biology. A possible reason for the failure of consecutive
incorporation is the low stacking potential of PzO; the incorporation efficiency can in theory be
improved by chemically modifying dPzO so as to enhance the stacking interaction. Another reason is
the lower hydrogen bonding capacity of the N1 of PzO with the amino acid residues of Kf+ and Kf−.
As shown by the quantum chemical calculation of the GꢀPzO base pair, the hydrogen bonding
capacity of the N1 of PzO was weaker than that of the 2ꢀketo of T. Thus, DNA polymerases that can
form strong hydrogen bonds with the N1 of PzO or that do not recognize the minor groove hydrogen
bond acceptor with high stringency would be expected to efficiently incorporate 1.
In this study, we used Kf− and Kf+ as DNA polymerases. These belong to family A, whose
members directly recognize minor groove Hꢀbond acceptors at the 3' end of primer and the counter
nucleobase in the template via amino acid residues such as Arg and Gln.35,36 On the other hand,
RB69 polymerase—which belongs to family B—was found to recognize the N3 of A via Tyr567,
which formed a waterꢀmediated hydrogen bond.37,12 This suggests that recognition of the minor
groove Hꢀbond site is more flexible in family B than in family A polymerases. Therefore, the
potential for 1 to serve as a substrate for polymerases of family B as well as families C, D, X, and
Y38 merits further investigation.
We are currently developing new PzO derivatives and are screening polymerases to achieve
more efficient incorporation of 1 and its derivatives. Moreover, a new artificial nucleobase bearing a
nitrogen atom at the ortho position is being synthesized and will be reported elsewhere.
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Experimental Section
General Methods
1H, 13C, 31Pꢀ nuclear magnetic resonance (NMR) spectra were recorded at 500, 126, 202 MHz for
1H, 13C and 31PꢀNMR, respectively. The chemical shifts were measured from the residual
nonꢀdeuterated solvents of CDCl3 (7.26 ppm), DMSOꢀd (2.49 ppm), CD3ODꢀd4 (3.31 ppm) and D2O
(4.79 ppm) for 1HꢀNMR spectra, and solvents’ signal of CDCl3 (77.16 ppm), DMSOꢀd6 (39.52 ppm)
and CD3ODꢀd4 (49.00 ppm) for 13CꢀNMR spectra and 85% phosphoric acid (0.00 ppm) for 31PꢀNMR
spectra. Highꢀperformanceꢀliquidꢀchromatography (HPLC) analysis was run using reverseꢀphase
(RP) C18 column (5 µm, 4.6 × 150 mm) or anionꢀexchange column (4 × 250 mm).
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Measurements of UVꢀmelting temperature
ODNs were dissolved in deionized distilled water and their concentration were determined from
absorbance measurements at 260 nm. The absorption coefficients of ODNs were calculated
according to the nearest–neighbor method39 using the Oligo Analyzer 3.1
(http://sg.idtdna.com/calc/analyzer). The absorption coefficients of ODN1 were calculated by
assuming that they are identical to those of ODNs incorporating T instead of PzO.
The ODNs (1.2 nmol each) were dissolved in 10 mM sodium phosphate buffer (pH 7.0, 100 mM
NaCl, 0.1 mM EDTA, 600 µL). The final concentration of the duplexes was 2 µM. The mixture was
placed in quartz cells (10 mm) and incubated at 90 °C. After 10 min, the mixture was cooled to 5 °C
then heated to 90 °C at a rate of 0.5 °C/min. The absorption at 260 nm was recorded and used to
draw the UVꢀmelting curves. The UVꢀmelting curve was smoothed by the Savitzky–Golay method.
Melting temperature was calculated as the temperature that gave the maximum of the first deviation
of each UV melting curve.
Synthetic procedures
2ꢀ(2,3,5ꢀTriꢀOꢀacetylꢀβꢀDꢀribofuranosyl)furan (4)
1.0 M solution of SnCl4 in CH2Cl2 (630 µL, 0.63 mmol) was added to a solution of tetraꢀOꢀacetylꢀ
βꢀDꢀribose (2.0 g, 6.28 mmol) and furan (960 µL, 12.6 mmol) in CH2Cl2 (32 mL) at 0 °C.
The reaction mixture was stirred and warmed to ambient temperature for 2 h. The mixture was
quenched with sat. NaHCO3 aq. (300 mL). The mixture was filtered by celite® pad, and extracted
twice with CH2Cl2 (100 mL each). The organic layers were combined and was dried over MgSO4,
filtered and evaporated under reduced pressure. The residue was purified by chromatography on a
column of silica gel (60N, 50 g) using nꢀhexane–EtOAc (90:10–70:30, v/v) to give the fractions
containing 4. The fractions were collected and evaporated under reduced pressure to give 4 (625 mg,
31%).
1HꢀNMR (500 MHz, DMSOꢀd6): δ 2.01 (s, 3H, 2´ꢀ, 3´ꢀ or 5´ꢀAc), 2.03 (s, 3H, 2´ꢀ, 3´ꢀ or 5´ꢀAc),
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2.07 (s, 3H, 2´ꢀ, 3´ꢀ or 5´ꢀAc), 4.08 (dd, 1H, J = 4.7 Hz, 12.0 Hz, Hꢀ5´), 4.21 (m, 1H, Hꢀ4´), 4.31 (dd,
1H, J = 3.4 Hz, 12.0 Hz, Hꢀ5´´), 4.95 (d, 1H, J = 6.5 Hz, Hꢀ1´), 5.25 (t, 1H, J = 5.2 Hz, Hꢀ3´), 5.35 (t,
1H, J = 6.1 Hz, Hꢀ2´), 6.46 (dd, 1H, J = 1.8 Hz, 3.2 Hz, aromaticꢀH), 6.54 (d, 1H, J = 3.2 Hz,
aromaticꢀH), 7.71 (dd, 1H, J = 0.9 Hz, 1.8 Hz, aromaticꢀH); 13CꢀNMR (126 MHz, DMSOꢀd6): δ 20.3,
20.4, 20.6, 63.1, 71.2, 72.5, 74.8, 79.0, 109.9, 110.7, 143.9, 150.0, 169.4, 169.6, 170.0; ESIꢀTOF
mass: calcd. for C15H18NaO8 [M+Na]+ 349.0894, found 349.0885.
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2ꢀ(2,3,5ꢀTriꢀOꢀbenzoylꢀβꢀDꢀribofuranosyl)furan (5)
Sodium methoxide (45 mg, 0.81 mmol) was added to a solution of 4 (2.56 g, 7.83 mmol) in
MeOH (40 mL). The reaction mixture was stirred at ambient temperature for 1 h. The reaction
mixture was evaporated under reduced pressure. The residue was dissolved in H2O (40 mL), then the
solution was passed through a DOWEX 50W X 8 (100–200 mesh, H+ form, 14 mL). The fractions
were collected and evaporated under reduced pressure to give a crude material. The residue was
dissolved in pyridine (80 mL), and benzoyl chloride (3.7 mL, 31.3 mmol) was added. After 1 h, the
reaction mixture was quenched with EtOH (5 mL). The reaction mixture was diluted with EtOAc
(200 mL), washed twice with H2O (150 mL each), twice with brine (150 mL each). The organic layer
was dried over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by
chromatography on a column of silica gel (C200, 100 g) using nꢀhexane–EtOAc (90:10–70:30, v/v)
to give the fractions containing 5. The fractions were collected and evaporated under reduced
pressure to give 5 (3.75 g, 93%, 2 steps)
1HꢀNMR (500 MHz, CDCl3): δ 4.57–4.60 (dd, 1H, J = 4.2 Hz, 11.9 Hz, Hꢀ5´ or Hꢀ5´´), 4.69 (m,
1H, Hꢀ4´), 4.77 (dd, 1H, J = 3.4 Hz, 12.0 Hz, Hꢀ5´ or Hꢀ5´´), 5.32 (d, 1H, J = 4.9 Hz, Hꢀ1´), 5.90–
5.95 (m, 1H, Hꢀ2´ and Hꢀ3´), 6.33–6.34 (m, 1H, aromaticꢀH), 6.45 (d, 1H, J = 3.0 Hz, aromaticꢀH),
7.34–7.44 (m, 8H, BzꢀH), 7.52–7.57 (m, 4H, BzꢀH and aromaticꢀH), 7.93–7.98 (m, 4H, BzꢀH);
13CꢀNMR (126 MHz, DMSOꢀd6): δ 64.6, 72.9, 74.2, 75.8, 79.8, 110.7, 111.4, 129.2, 129.4, 129.5,
134.2, 134.5, 134.6, 144.6, 150.7, 165.2, 165.5, 166.1; ESIꢀTOF mass: calcd. for C30H24NaO8
[M+Na]+ 535.1363, found 535.1354.
4ꢀ(2,3,5ꢀTriꢀOꢀbenzoylꢀβꢀDꢀribofuranosyl)ꢀ4ꢀoxobutylic acid (7)
Concentrated sulfuric acid (6 mL) was added to a solution of chromium trioxide (5.0 g, 58.5
mmol) in water (18 mL). The mixture was stirred at 0 °C for 30 min. The 2.4 M Jones reagent (24
mL) was added to a solution of 5 (6.0 g, 11.7 mmol) in acetone (65 mL). The reaction mixture was
stirred at 0 °C for 1 h. The reaction mixture was carefully poured into a twoꢀlayered mixture of
CH2Cl2 (200 mL), and H2O (200 mL) containing ice (50 g). The mixture was diluted with CH2Cl2
(100 mL), and extracted four times with CH2Cl2 (100 mL each). The organic layer was dried over
MgSO4, filtered and evaporated under reduced pressure. The residue was dissolved in EtOH (87 mL),
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and to a suspention of 5% palladiumꢀcarbon (470 mg) in EtOH (7 mL) was added. The reaction
mixture was stirred at ambient temperature for 1 h. The mixture was filtered on celite® pad, and the
solvent was evaporated under reduced pressure. The residue was purified by chromatography on a
column of silica gel (C300, 80 g) using nꢀhexane–EtOAc (50:50–10:90, v/v) to give the fractions
containing 7. The fractions were collected and evaporated under reduced pressure to give 7 (4.42 g,
93%, 2 steps).
1HꢀNMR (500 MHz, DMSOꢀd6): δ 2.46 (m, 2H, ꢀC(O)CH2CH2ꢀ), 2.81–2.98 (m, 2H,
ꢀC(O)CH2CH2ꢀ), 4.58–4.61 (m, 1H, Hꢀ5´ or Hꢀ5´´), 4.65–4.68 (m, 1H, Hꢀ5´ or Hꢀ5´´), 4.73 (m, 1H,
Hꢀ4´), 4.92 (m, 1H, Hꢀ1´), 5.66 (t, 1H, J = 5.9 Hz, Hꢀ3´), 5.89 (t, 1H, J = 4.9 Hz, Hꢀ2´), 7.39 (t, 2H,
J = 7.7 Hz, BzꢀH), 7.47 (t, 4H, J = 7.7 Hz, BzꢀH) 7.58–7.66 (m, 3H, BzꢀH), 7.82–7.99 (m, 6H,
BzꢀH), 12.4 (br, 1H, ꢀC(O)OH); 13CꢀNMR (126 MHz, DMSOꢀd6): δ 28.0, 34.1, 64.6, 72.5, 73.6,
79.7, 85.5, 129.2, 129.3, 129.4, 129.5, 129.8, 129.9, 130.0, 134.2, 134.4, 134.5, 165.2, 165.3, 166.2,
174.4, 207.2; ESIꢀTOF mass: calcd. for C30H26NaO10 [M+Na]+ 569.1418, found 569.1414.
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6ꢀ(2,3,5ꢀTriꢀOꢀbenzoylꢀβꢀDꢀribofuranosyl)ꢀ4,5ꢀdihydropyridazinꢀ3ꢀone (8)
Hydrazine monohydrate (140 µL, 4.25 mmol) and 4ꢀ(4,6ꢀdimethoxyꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ
4ꢀmethylmorpholinium chloride (1.18 g, 4.25 mmol) was added to a solution of 7 (1.53 g, 2.83
mmol) in dry EtOH (84 mL). The reaction mixture was stirred at ambient temperature for 2 h.
The mixture was diluted with nꢀhexane–EtOAc (200 mL, 1:1, v/v), washed twice with H2O (150 mL
each), and twice with brine (150 mL each). The organic layer was dried over MgSO4, filtered and
evaporated under reduced pressure. The residue was purified by chromatography on a column of
silica gel (60N, 100 g) using nꢀhexane–EtOAc (70:30–50:50, v/v) to give the fractions containing 8.
The fractions were collected and evaporated under reduced pressure to give 8 (1.07 g, 70%).
1HꢀNMR (500 MHz, CDCl3): δ 2.38–2.48 (m, 2H, Hꢀ5), 2.57–2.69 (m, 2H, Hꢀ4), 4.53 (dd, 1H, J =
3.6 Hz, 12.2 Hz, Hꢀ5´ or Hꢀ5´´), 4.66–4.68 (m, 1H, Hꢀ4´), 4.80–4.83 (m, 2H, Hꢀ1´ and Hꢀ5´ or
Hꢀ5´´), 5.82 (t, 1H, J = 4.4 Hz, Hꢀ3´), 5.85 (t, 1H, J = 6.0 Hz, Hꢀ2´), 7.35–7.49 (m, 6H, BzꢀH), 7.53–
7.61 (m, 3H, BzꢀH), 7.93–8.09 (m, 6H, BzꢀH), 8.27 (br, 1H, NꢀH); 13CꢀNMR (126 MHz, CDCl3): δ
21.1, 26.1, 64.0, 72.7, 72.9, 80.9, 82.4, 128.7, 128.8, 128.9, 129.0, 129.2, 129.6, 129.6, 130.0, 133.7,
133.8, 133.9, 151.2, 165.6, 165.7, 166.3, 167.4; ESIꢀTOF mass: calcd. for C30H26N2NaO8 [M+Na]+
565.1581, found 565.1566.
6ꢀ(2,3,5ꢀTriꢀOꢀbenzoylꢀβꢀDꢀribofuranosyl)pyridazinꢀ3ꢀone (9)
2,3ꢀdichloroꢀ5,6ꢀdicyanobenzoquinone (840 mg, 3.68 mmol) and pꢀtoluenesulfonic acid
monohydrate (350 mg, 1.84 mmol) was added to a solution of 8 (1.0 g, 1.84 mmol) in dry toluene
(45 mL). The reaction mixture was stirred at ambient temperature for 2 h. The reaction mixture was
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diluted with EtOAc (300 mL), washed twice with sat. NaHCO3 aq. (300 mL, each), once brine (300
mL). The organic layer was dried over MgSO4, filtered and evaporated under reduced pressure. The
residue was purified by chromatography on a column of silica gel (NꢀH, 20 g) using nꢀhexane–
EtOAc (70:30–30:70, v/v) to give the fractions containing 9. The fractions were collected and
evaporated under reduced pressure to give 9 (680 mg, 68%).
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1HꢀNMR (500 MHz, DMSOꢀd6): δ 4.59 (dd, 1H, J = 4.5 Hz, 12.1 Hz, Hꢀ5´ or Hꢀ5´´), 4.65 (1H, dd,
J = 3.6 Hz, 12.2 Hz, Hꢀ5´ or Hꢀ5´´), 4.74–4.77 (m, 1H, Hꢀ4´), 5.19 (d, 1H, J = 5.9 Hz, Hꢀ1´), 5.79 (t,
1H, J = 5.4 Hz, Hꢀ3´), 5.91 (t, 1H, J = 5.8 Hz, Hꢀ2´), 6.89 (d, 1H, J = 9.8 Hz, Hꢀ4), 7.42–7.52 (m,
6H, BzꢀH), 7.57 (d, 1H, J = 9.8 Hz, Hꢀ5), 7.62–7.68 (m, 3H, BzꢀH), 7.85–7.93 (m, 6H, BzꢀH), 13.1
(br, 1H, NꢀH); 13CꢀNMR (126 MHz, DMSOꢀd6): δ 64.0, 72.3, 73.9, 79.9, 80.6, 128.7, 128.8, 129.0,
129.1, 129.1, 129.4, 129.5, 129.5, 130.7, 132.3, 133.9, 134.1, 134.2, 144.3, 160.8, 164.9, 165.0,
165.8; ESIꢀTOF mass: calcd. for C30H24N2O8Na [M+Na]+ 563.1425, found 563.1418.
6ꢀ(βꢀDꢀribofuranosyl)pyridazinꢀ3ꢀone (10)
28% NH3 aq. (19 mL) was added to a solution of 9 (1.3 g, 2.41 mmol) in MeOH (12 mL). The
reaction mixture was sealed and heated at 80 °C for 12 h. After being cooled to the ambient
temperature, the solvent was evaporated under reduced pressure. The residue was purified by
chromatography on a column of silica gel (C200, 10 g) using CH2Cl2–MeOH (100:0–80:20, v/v) to
give the fractions containing 10. The fractions were collected and evaporated under reduced pressure
to give 10 (468 mg, 85%).
1HꢀNMR (500 MHz, CD3ODꢀd4): δ 3.65–3.68 (dd, 1H, J = 4.2 Hz, 12.1 Hz, Hꢀ5´ or Hꢀ5´´), 3.73–
3.77 (dd, 1H, J = 3.4 Hz, 12.1 Hz, Hꢀ5´ or Hꢀ5´´), 3.97–3.99 (m, 1H, Hꢀ4´), 4.07–4.12 (m, 2H, Hꢀ2´
and Hꢀ3´), 4.63 (d, 1H, J = 6.5 Hz, Hꢀ1´), 6.97 (d, 1H, J = 9.8 Hz, Hꢀ4), 7.71 (d, 1H, J = 9.8 Hz,
Hꢀ5); 13CꢀNMR (126 MHz, CD3ODꢀd4): δ 63.3, 73.0, 76.9, 83.9, 87.0, 130.8, 133.9, 149.4, 163.8;
ESIꢀTOF mass: calcd. for C9H11N2O5 [M–H]– 227.0673, found 227.0668.
UVꢀvis spectrum (in 10 mM sodium phosphate buffer pH 7.0), λmax = 284 nm (ε = 2.43 × 103)
6ꢀ[3,5ꢀOꢀ(1,1,3,3ꢀTetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀ βꢀDꢀribofuranosyl]pyridazinꢀ3ꢀone (11)
Compound 10 (350 mg, 1.43 mmol) was coꢀevaporated four times with pyridine, and dissolved in
dry pyridine (15 mL) under an argon atmosphere. To the solution of 10 were added 1,3ꢀdichloroꢀ
1,1,3,3ꢀtetraisopropyldisiloxane (540 µL, 1.68 mmol). The mixture was stirred at ambient
temperature for 15 h. The reaction mixture was quenched with EtOH (5 mL). The reaction mixture
was diluted with EtOAc (100 mL) washed twice with H2O (100 mL), and twice with brine (100 mL
each). The organic layer was dried over MgSO4, filtered and evaporated under reduced pressure. The
residue was purified by chromatography on a column of silica gel (60N, 20 g) using nꢀhexane–
EtOAc (90:10–30:70, v/v) to give the fractions containing 11. The fractions were collected and
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evaporated under reduced pressure to give 11 (504 mg, 70%).
1HꢀNMR (500 MHz, DMSOꢀd6): δ 0.97–1.03 (m, 28H, TIPDSꢀH), 3.85–3.90 (m, 2H, Hꢀ4, and
Hꢀ5´ or 5´´), 3.99 (dd, 1H, J = 3.2 Hz, 12.4 Hz, Hꢀ5´ or 5´´), 4.09 (dd, 1H, J = 5.1 Hz, 8.0 Hz, Hꢀ3´),
4.14–4.16 (m, 1H, Hꢀ2´), 4.56 (d, 1H, J = 1.7 Hz, Hꢀ1´), 5.28 (d, 1H, J = 5.0 Hz, 2´ꢀOH), 6.85 (d, 1H,
J = 9.8 Hz, Hꢀ4), 7.44 (d, 1H, J = 9.8 Hz, Hꢀ5), 12.9 (s, 1H, NꢀH); 13CꢀNMR (126 MHz, DMSOꢀd6):
δ 12.0, 12.2, 12.5, 12.8, 16.8, 16.9, 17.0, 17.1, 17.2, 17.4, 60.9, 70.8, 74.2, 81.1, 83.8, 130.0, 131.4,
146.1, 160.4; ESIꢀTOF mass: calcd. for C21H37N2O6Si2 [M–H]– 469.2196, found 469.2195.
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6ꢀ[3,5ꢀOꢀ(1,1,3,3ꢀTetraisopropyldisiloxaneꢀ1,3ꢀdiyl)ꢀ2ꢀdeoxyꢀβꢀDꢀribofuranosyl]pyridazinꢀ3ꢀon
e (12)
N,Nꢀdimethylꢀ4ꢀaminopyridine (640 mg, 5.1 mmol) and Oꢀphenylchlorothionoformate (350 µL,
2.55 mmol) was added to a solution of 11 (800 mg, 1.70 mmol) in dry MeCN (34 mL). The reaction
mixture was stirred at ambient temperature for 1 h. The reaction mixture was evaporated under
reduced pressure to give a crude material. This material was dissolved in dry toluene (36 mL), and
2,2´ꢀazodiisobutyronitrile (26 mg, 0.14 mmol), tris(trimethylsilyl)silane (665 µL, 2.16 mmol) were
added. The reaction mixture was stirred at 90 °C for 15 h. The reaction mixture was diluted with
EtOAc (100 mL), and washed twice with 5% NaClO aq. (150 mL each). The organic layer was dried
over MgSO4, filtered and evaporated under reduced pressure. The residue was purified by
chromatography on a column of silica gel (60N, 15 g) using nꢀhexane–EtOAc (80:20–50:50, v/v) to
give the fractions containing 12. The fractions were collected and evaporated under reduced pressure
to give 12 (498 mg, 76%).
1HꢀNMR (500 MHz, CDCl3): δ 1.01–1.08 (m, 28H, TIPDSꢀH), 2.26–2.32 (m, 1H, Hꢀ2´ or 2´´),
2.34–2.39 (m, 1H, Hꢀ2´ or 2´´), 3.82–3.87 (m, 2H, Hꢀ5´ and 5´´), 4.03–4.07 (m, 1H, Hꢀ4´), 4.44–
4.78 (m, 1H, Hꢀ3´), 4.98 (dd, 1H, J = 6.5 Hz, 7.5 Hz, Hꢀ1´), 6.80 (d, 1H, J = 9.8 Hz, Hꢀ4), 7.47 (d,
1H, J = 9.8 Hz, Hꢀ5), 11.8 (s, 1H, NꢀH); 13CꢀNMR (126 MHz, CDCl3): δ 12.7, 13.1, 13.4, 13.6, 17.1,
17.2, 17.2, 17.4, 17.5, 17.5, 17.5, 17.7, 39.6, 62.8, 72.1, 86.2, 130.6, 132.0, 148.9, 162.0.; ESIꢀTOF
mass: calcd. for C21H38N2NaO5Si2 [M+Na]+ 477.2211, found 477.2204.
6ꢀ(2ꢀDeoxyꢀβꢀDꢀribofuranosyl)pyridazinꢀ3ꢀone (13)
Triethylamine (455 µL, 3.24 mmol) and triethylamine trihydrofluoride (530 µL, 3.24 mmol) were
added to a solution of 12 (490 mg, 1.08 mmol) in dry THF (11 mL). The reaction mixture was stirred
at ambient temperature for 16 h. The reaction was quenched with trimethylethoxysilane (840 µL,
5.40 mmol). The mixture was evaporated under reduced pressure. The residue was purified by
chromatography on a column of silica gel (60N, 5 g) using CH2Cl2–MeOH (100:0–80:20, v/v) to
give the fractions containing 13. The fractions were collected and evaporated under reduced pressure
to give 13 (170 mg, 73%).
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1HꢀNMR (500 MHz, CD3ODꢀd4): δ 2.11–2.15 (m, 2H, Hꢀ2´ and Hꢀ2´´), 3.01–3.67 (m, 2H, Hꢀ5´
and Hꢀ5´´), 3.94 (m, 1H, Hꢀ4´), 4.34 (m, 1H, Hꢀ3´), 5.01 (dd, 1H, J = 7.1 Hz, 9.3 Hz, Hꢀ1´), 6.97 (d,
1H, J = 9.8 Hz, Hꢀ4), 7.71 (d, 1H, J = 9.8 Hz, Hꢀ5); 13CꢀNMR (126 MHz, CD3ODꢀd4): δ 42.0, 63.7,
74.1, 79.8, 89.6, 130.9, 133.9, 150.5, 163.8; ESIꢀTOF mass: calcd. for C9H12N2NaO4 [M+Na]+
235.0689, found 235.0691.
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1Rꢀ1,2ꢀdideoxyꢀ1ꢀ(3ꢀoxoꢀ2Hꢀpyridazinꢀ6ꢀyl)ribofuranose 5ꢀtriphosphate
tris(triethylammonium) salt (1)
To a solution of 12 (21 mg, 0.1 mmol) in trimethyl phosphate (500 µL), phosphoryl chloride (12
µL, 0.13 mmol) was added at 0 °C. The reaction mixture was stirred at 0 °C for 1 h.
Triꢀnꢀbutylamine (100 µL, 0.42 mmol) was added to the reaction mixture, and then 0.5 M
bis(tributylammonium) pyrophosphate in a DMF solution (500 µL) was added. After 2 h, the
reaction was quenched by the addition of 2.0 M triethylammonium bicarbonate (TEAB, 1.0 mL).
The resulting crude product was purified by 1.5 cm × 20 cm DEAE Sephadex Aꢀ25 column
chromatography (eluted by a linear gradient of 50 mM TEAB to 1.0 M TEAB), and then by
RPꢀHPLC (eluted by a linear gradient of MeCN, 0% to 15% in 0.1 M triethylammonium acetate
buffer) to give 1 (26.8 µmol, 27%).
1HꢀNMR (500 MHz, D2O): δ 2.26 (m, 2H, Hꢀ2´ and Hꢀ2´´), 4.09 (m, 2H, Hꢀ5´ and Hꢀ5´´), 4.23 (m,
1H, Hꢀ4´), 4.60 (m, 1H, Hꢀ3´), 5.11 (t, 1H, J = 8.3 Hz, Hꢀ1´), 7.15 (d, 1H, J = 9.8 Hz, Hꢀ4), 7.83 (d,
1H, J = 9.8 Hz, Hꢀ5); 31PꢀNMR (202 MHz, D2O): δ –22.99 (t, J = 19.8 Hz), –11.09 (d, J = 19.9 Hz),
–10.49 (d, J = 19.4 Hz); ESIꢀTOF mass: calcd. for C9H14N2O13P3 [M–H]– 450.9714, found 450.9714.
6ꢀ(5´ꢀOꢀDimethoxytritylꢀ2ꢀdeoxyꢀβꢀDꢀribofuranosyl)pyridazinꢀ3ꢀone (14)
Compound 13 (85 mg, 0.4 mmol) was coꢀevaporated four times with pyridine, and dissolved in
dry pyridine (4 mL) in under an argon atmosphere. To the solution of 13 was added
4,4´ꢀdimethoxytrityl chloride (169 mg, 0.48 mmol). The mixture was stirred at ambient temperature
for 4 h. The reaction was quenched with EtOH (1 mL). The reaction mixture was diluted with
CH2Cl2 (15 mL) washed twice with H2O (15 mL each), and twice with brine (15 mL each). The
organic layer was dried over MgSO4, filtered and evaporated under reduced pressure. The residue
was purified by chromatography on a column of silica gel (60N, 6 g) using CH2Cl2–MeOH (100:0–
90:10, v/v) to give the fractions containing 14. The fractions were collected and evaporated under
reduced pressure to give 14 (110 mg, 53%).
1HꢀNMR (500 MHz, DMSOꢀd6): δ 2.00–2.13 (m, 2H, Hꢀ2´ and Hꢀ2´´), 3.01 (dd, 1H, J = 5.3 Hz,
10.1 Hz, Hꢀ5´ or Hꢀ5´´), 3.07 (dd, 1H, J =3.8 Hz, 10.1 Hz, Hꢀ5´ or Hꢀ5´´), 3.72 (s, 6H,
DMTrꢀOCH3 ), 3.89–3.91 (m, 1H, Hꢀ4´), 4.16–4.17 (m, 1H, Hꢀ3´), 4.90 (dd, 1H, J = 6.1 Hz, 9.4 Hz,
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Hꢀ1´), 5.18 (br, 1H, Hꢀ4), 6.85–6.87 (m, 5H, DMTrꢀH), 7.19–7.23 (m, 5H, DMTrꢀH), 7.27–7.30 (m,
2H, DMTrꢀH), 7.34–7.36 (m, 2H, DMTrꢀH), 7.41–7.43 (m, 1H, Hꢀ5), 12.9 (br, 1H, NꢀH); 13CꢀNMR
(126 MHz, DMSOꢀd6): δ 55.0, 55.1, 64.2, 72.2,77.9, 78.0, 85.4, 86.3, 113.2, 126.7, 127.7, 127.8,
129.70, 129.73, 130.2, 131.8, 135.6, 144.9, 147.2, 158.0, 160.6; ESIꢀTOF mass: calcd. for
C30H30N2NaO6+ [M+Na]+ 537.1996, found 537.2001.
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1Rꢀ1,2ꢀdideoxyꢀ5ꢀOꢀ(4,4ʹꢀdimethoxytrityl)ꢀ1ꢀ(3ꢀoxoꢀ2Hꢀpyridazinꢀ6ꢀyl)ribofuranose
3ꢀ(2ꢀcyanoethyl N, Nꢀdiisopropylphosphoramidite) (2)
Compound 14 (100 mg, 0.19 mmol) was coꢀevaporated four times each with pyridine and toluene,
and dissolved in dry CH2Cl2 (2 mL) in under an argon atmosphere. N, Nꢀdiisopropylethylamine (86
µL, 0.49 mmol) and 2ꢀcyanoethyl N, Nꢀdiisopropylchlorophosphoramidite (55 µL, 0.25 mmol) was
added to a solution of 14. The mixture was stirred at ambient temperature for 30 min. The reaction
mixture was diluted with CH2Cl2 (10 mL), washed three times with NaHCO3 aq. (15 mL each), and
twice with brine (15 mL each). The organic layer was dried over MgSO4, filtered and evaporated
under reduced pressure. The residue was purified by chromatography on a column of silica gel (60N,
3 g) using CH2Cl2–MeOH (100:0–95:5, v/v) to give the fractions containing 2. The fractions were
collected and evaporated under reduced pressure to give 2 (68 mg, 50%).
1HꢀNMR (500 MHz, CDCl3): δ 1.09–1.19 (m, 12H, -CH(CH3)2), 2.16–2.42 (m, 2H, Hꢀ2´ and
Hꢀ2´´), 2.45 (t, 1H, J = 6.5 Hz), 2.61 (t, 1H, J = 6.4 Hz), 3.20–3.32 (m, 1H, Hꢀ5´ and Hꢀ5´´), 3.54–
3.62 (m, 2H, ꢀCH(CH3)2ꢀ), 3.54–3.85 (m, 10H, ꢀCH2CH2CN, ꢀCH2CH2CN, DMTrꢀOCH3), 4.21–4.24
(m, 1H, Hꢀ4´), 4.53–4.56 (m, 1H, Hꢀ3´), 5.02–5.25 (m, 1H, Hꢀ1´), 6.80–6.86 (m, 5H, DMTrꢀH),
7.19–7.25 (m, 5H, DMTrꢀH and Hꢀ4), 7.27–7.31 (m, 2H, DMTrꢀH), 7.40–7.42 (m, 2H, DMTrꢀH),
7.41–7.43 (m, 1H, Hꢀ5), 10.7 (br, 1H, NꢀH); 13CꢀNMR (126 MHz, CDCl3): δ 20.., 20.38, 20.39,
20.52, 20.58, 24.60, 24.67, 24.71, 24.73, 24.77, 24.79, 39.35, 40.26, 40.29, 40.38, 40.40, 43.27,
43.34, 43.38, 43.44, 55.36, 55.37, 55.39, 55.41, 58.25, 58.29, 58.30, 58.40, 58.43, 58.44, 63.91,
64.02, 75.40, 75.53, 75.72, 75.85, 78.61, 78.66, 86.24, 86.26, 86.59, 86.30, 86.44, 86.45, 86.51,
86.54, 113.25, 117.58, 117.67, 127.03, 127.04, 127.97, 128.28, 128.33, 130.20, 130.24, 130.73,
132.20, 135.87, 135.90, 144.74, 144.77, 148.48, 148.51, 158.65, 158.66, 161.16; 31PꢀNMR (202
MHz, CDCl3): δ 149.2, 149.5; ESIꢀTOF mass: calcd. for C39H47N4NaO7P+ [M+Na]+ 737.3075, found
737.3065.
Singleꢀnucleotide insertion by Kf– or Kf+
The equimolar amount of the template DNA and P1 were mixt in a 5 µL of buffer containing 100
mM NaCl, 20 mM TrisꢀHCl (pH 7.9), 20 mM MgCl2 and 2 mM DTT so that the final concentration
of the duplex became 0.2 ꢂM. The duplex was annealed by heating at 90 °C for 1 min and slow
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cooling to ambient temperature. The duplex mixture (5 ꢂL) was mixed with a solution containing
Kf– (0.1 Unit·ꢂLꢀ1, 1 ꢂL). Then, the mixture was incubated at 37 °C for 2 min. The reactions were
initiated by adding a solution containing 1 (25 ꢂM, 4 ꢂL) to the DNA–enzyme mixture at 37 °C. The
reaction mixture was incubated for 10 min at 37 °C and quenched by the addition of 20 ꢂL of stop
solution (10 M Urea, 50 mM EDTA, 0.1% BPB solution). After vigorous stirring, the 3 µL aliquot of
reaction mixtures were analyzed by electrophoresis with 20% denaturing polyacrylamide gel
containing 7 M urea (40 W, 55 °C, 60 min) and visualized in fluoroimage analyzer (FUJIFILM
FLA–9500).
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Steadyꢀstate kinetics of singleꢀnucleotide insertion by Kf–
Equal amount of T1 and P1 were dissolved in 3 µL of buffer containing 500 mM NaCl, 100 mM
Tris–HCl, 100 mM MgCl2 and 10 mM DTT so that the final concentration of the duplex to 0.5–2.5
µM. Then T1 was annealed with P1 by heating at 90 °C for 1 min and following slow cooling to
ambient temperature. The duplex mixture (3 µL) was mixed with a solution containing Kf–(0.025–
2.5 Unit·µL–1, 2 µL). Then, the mixture was incubated at 37 °C for 2 min. The reactions were
initiated by adding a solution containing 1 (2–10 µM, 5 µL) or TTP (0.04–240 µM, 5 µL) to the at
37 °C. The reaction mixture was incubated for 1 min at 37 °C and quenched by the addition of 20 µL
of stop solution (10 M urea, 50 mM EDTA, 0.1% BPB solution). After vigorous stirring, 3 µL
aliquots of the reaction mixtures were analyzed by electrophoresis with 20% denaturing
polyacrylamide gel containing 7 M urea (40 W, 55 °C, 60 min) and visualized on fluoroimage
analyzer (FUJIFILM FLA–9500). The kinetic parameters (Km and Vmax) were determined from
Hanes–Woolf plots.
Chain elongation reaction using Kf– or Kf+ in the presence of various dNTPs
The equimolar amount of the template DNA and P1 were mixed in a 5 µL of buffer containing 100
mM NaCl, 20 mM TrisꢀHCl (pH 7.9), 20 mM MgCl2 and 2 mM DTT so that the final concentration
of the duplex became 0.2 ꢂM. The duplex was annealed by heating at 90 °C for 1 min and slow
cooling to ambient temperature.
The duplex mixture (5 ꢂL) was mixed with a solution containing Kf– (0.1 Unit·ꢂLꢀ1, 1 ꢂL). Then,
the mixture was incubated at 37 °C for 2 min. The reactions were initiated by adding a 4 µL of the
solution containing 1 (25 ꢂM), dATP (25 ꢂM), dGTP (25 ꢂM) and dCTP (25 ꢂM) to the DNA–
enzyme mixture at 37 °C. The reaction mixture was incubated for 10 min at 37 °C and quenched by
the addition of 20 ꢂL of stop solution (10 M Urea, 50 mM EDTA, 0.1% BPB solution). After
vigorous stirring, the 3 µL aliquot of the reaction mixtures were analyzed by electrophoresis with
20% denaturing polyacrylamide gel containing 7 M urea (40 W, 55 °C, 60 min) and visualized in
fluoroimage analyzer (FUJIFILM FLA–9500).
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Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website. General
procedure, HPLC charts, MALDIꢀTOF mass charts, UVꢀmelting curves, and HPLC charts,
Coordinates used for the quantum chemical calculations, and the calculated energies and frequencies
are included.
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Acknowledgement
This study was financially supported by JSPS KAKENHI 17H05230, 26288075, 17H04886.
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