Synthesis of peptide nucleic acids containing pyridazine derivatives as cytosine and thymine analogs, and their duplexes with complementary oligodeoxynucleotides

Article abstract of DOI:10.1021/acs.orglett.5b00522

Synthesis of peptide nucleic acids (PNAs) is reported with new pyridazine-type nucleobases: 3-aminopyridazine (aPz) and 1-aminophthalazine (aPh) as cytosine analogs, and pyridazin-3-one (Pz^O) and phthalazin-1-one (Ph^O) as thymine ana

Full text of DOI:10.1021/acs.orglett.5b00522

Letter
pubs.acs.org/OrgLett
Synthesis of Peptide Nucleic Acids Containing Pyridazine Derivatives
As Cytosine and Thymine Analogs, and Their Duplexes with
Complementary Oligodeoxynucleotides
Takahito Tomori, Yuya Miyatake, Yuta Sato, Takashi Kanamori, Yoshiaki Masaki, Akihiro Ohkubo,
Mitsuo Sekine,* and Kohji Seio*
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan
S
* Supporting Information
ABSTRACT: Synthesis of peptide nucleic acids (PNAs) is reported with new
pyridazine-type nucleobases: 3-aminopyridazine (aPz) and 1-aminophthalazine (aPh)
as cytosine analogs, and pyridazin-3-one (PzO) and phthalazin-1-one (PhO) as
thymine analogs. The PNAs having an aPz or a PzO formed duplexes with each
complementary oligodeoxynucleotide forming a base pair with G or A, respectively,
as evaluated by using UV melting analyses and circular dichroism (CD) spectra.
NA and RNA contain pyrimidine nucleobases cytosine,
Dthymine, and uracil, which form base pairs in various base-
pairing motifs. Many pyrimidine base analogs have been reported
for the development of nucleoside derivatives and oligonucleo-
tide derivatives.1−5 These analogs were designed from various
six-membered heteroaromatic rings containing nitrogen atoms
such as pyridine,1 pyrimidine,2 pyrazine,3 and triazine4 rings. The
nucleotides and oligonucleotides incorporating these analogs
Figure 1. Structure of a PNA incorporating pyridazine-type
exhibit unique physicochemical and biochemical properties. For
example, a nucleoside analog incorporating 4-amino-2-oxo-1,3,5-
triazine as a base exhibits antiviral and antirumor activities.5 2-
Aminopyridine,1f pseudoisocytosine,6 and 6-amino-pyrazin-2-
one3 base analogs incorporated into triplex-forming oligonucleo-
tides (TFOs) have been used to recognize guanine residues of
DNA duplex forming hydrogen bonds under neutral pH. 2-
Aminopyrimidine7 has also been used in TFOs to recognize
cytosine residues within the DNA duplex. These studies suggest
that pyrimidine base analogs are useful for the development of
new artificial oligonucleotides and bioactive nucleoside or
nucleotide analogs.
Here, we report the synthesis of new pyrimidine base analogs.
Pyridazine-type nucleobases 3-aminopyridazine (aPz) and 1-
aminophthalazine (aPh) as cytosine analogs, and pyridazin-3-
one (PzO) and phthalazin-1-one (PhO) as thymine analogs
(Figure 1), were synthesized and incorporated into peptide
nucleic acids (PNAs) to study their base-pairing abilities. In
previous studies, only three pyridazine derivatives have been
described as pyrimidine analogs. For example, Furukawa et al.
have reported the synthesis of a ribonucleoside derivative having
a PzO residue as the base moiety.8,9 However, its properties,
including its base-pairing abilities, have not been reported.
Additionally, Rozners et al.10 and Nielsen et al.11 reported PNAs
harboring PzO as a thymine analog. These PNAs were designed
to bind to the DNA duplex as TFOs, forming hydrogen bonds
with the uracil of the U-A base pair. In these PNAs, the
nucleobases. The atoms of aPz and PzO are shown.
aminoethylglycine backbone and the PzO were connected via
linkers derived from β-alanine10 or 3-butenoic acid,11which are
two atoms longer than the 1-oxyethylene linker used in the
typical PNA unit shown in Figure 1. Therefore, the base-pairing
properties of PzO in canonical PNAs have not yet been
investigated. In contrast to these studies of PzO derivatives, the
incorporation of aPz derivatives into any kind of artificial nucleic
acid has not been reported. Here, we report the successful
synthesis of PNAs containing pyridazine-type nucleobases and
the evaluation of the base-pairing properties of these molecules.
First, the electrostatic potential around 3-aminopyridazines
was calculated and compared with that of cytosine. As shown in
Figure 2, the shapes of the electrostatic potential surfaces around
the amino groups and the endocyclic nitrogen atoms, N3 for
cytosine (Figure 2, left) and N1 for aPz (Figure 2, second left),
were similar. Although the exocyclic carbonyl oxygen of cytosine
makes the surface around its C2O2 position larger than that of
the endocyclic N1 position of aPz, these surfaces were
electrostatically negative. Romesberg and co-workers12 studied
the DNA polymerase reaction using DNA incorporating a
pyridine-2-yl (2Py) and demonstrated that the Klenow fragment
Received: February 19, 2015
Published: March 10, 2015
© 2015 American Chemical Society
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2 was converted to 3 by the reaction with hydrazine.13 The ethyl
ester of 3 was hydrolyzed to the carboxylic acid 4a. Compound
4a was condensed with PNA backbone 514 using hydroxybenzo-
triazolyltetramethyluronium hexafluorophosphate (HBTU) and
N-methylmorpholine (NMM) as a condensing agent and a base,
respectively, to give 6a. Subsequently, compound 6a was
hydrolyzed to the PNA unit 1a. Similarly, the PNA unit of PhO
(1b) was synthesized using the carboxylic acid 4b.15
Figure 2. Electrostatic potential maps of cytosine, aPz, thymine, and PzO
(from left to right) calculated at the HF/6-31G* level of theory.
Chemical structures are overlaid to show approximate atom positions.
The PNA unit having aPh (14b, Scheme 2) was also
synthesized by using 7.15 Compound 7 was converted to the
recognized the endocyclic nitrogen atom of 2Py instead of the 2-
keto group of canonical pyrimidine nucleobases and catalyzed
the strand extension from primer DNA having a 2Py at the 3′-
terminal. Because the N1 of aPz and the nitrogen atom of 2Py
were at the same endocyclic position, the α position to the
glycosidic bond, it was expected that the N1 atom of aPz could
also act as a mimic of the 2-keto group. We also calculated the
electrostatic potential of thymine (Figure 2, second right) and
PzO (Figure 2, right). Although the negative potential surface at
around N1 of PzO was very small, the positions of the hydrogen
donors and acceptors were similar.
Scheme 2. Synthesis of a PNA Unit Incorporating aPz (14a)
and aPh (14b)
The hydrogen bond energies between aPz and guanine were
calculated. The base-pairing energies (ΔE) of aPz and guanine
were −25 kcal/mol, which was less stable by 5 kcal/mol than the
cytosine−guanine base pair. This was because of the lack of a
hydrogen bond of the exocyclic carbonyl oxygen in the case of
aPz guanine base pair. However, the ΔE of the aPz-guanine base
pair was more stable than that of the thymine−adenine base pair
by 7 kcal/mol. This result suggested that although there were
only two apparent hydrogen bonds in aPz-guanine base pairs,
there was an additional attractive dipole−dipole interaction
between the amino group of the guanine and N1 of aPz. In
addition, the hydrogen bond energy between PzO and adenine
was calculated. The ΔE was −18.5 kcal/mol, which was slightly
more stable than the thymine−adenine base pair (ΔE = −18.0
kcal/mol).
To incorporate these molecules into PNAs, we first
synthesized a PNA unit having PzO (1a) (Scheme 1). Compound
Scheme 1. Synthesis of a PNA Unit Incorporating PzO (1a)
and PhO (1b)
chloride 8b using POCl3. Subsequently, compound 8b was
treated with NaN3, giving an azide compound that spontaneously
cyclized to the tetrazole derivative 9b as in the case of the
reaction of 2-chloropyridine and sodium azide.16 Then, the
tetrazole moiety was reduced by treatment with P(n-Bu)3, and
successive hydrolysis gave the aPh derivative 10b. After
protection of the amino group with a benzhydryloxycarbonyl
(Bhoc) group to give 11b, the ethyl ester was hydrolyzed by
using LiOH to give the lithium salt 12b as an intermediate.
Notably, if 12b was converted to the corresponding free acid, it
rapidly decomposed by decarboxylation, yielding benzhydryl (4-
methylphthalazin-1-yl)carbamate. Therefore, lithium salt 12b
was directly condensed with 5 to give 13b in 47% yield. Finally,
compound 13b was hydrolyzed to yield the PNA unit of aPh
(14b). Because 14b was not stable under purification on a silica
gel column and storage, 14b was immediately used for PNA
synthesis without further purification after the removal of the
residual 5 and other reagents by flash column chromatography.
According to a similar procedure detailed in the Supporting
Information (Scheme S1), the PNA unit of aPz (14a) was also
synthesized starting from 3. One of the differences between the
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routes to 14a and 14b was that, in 14a, the amino group was
protected with a tert-butyloxycarbonyl (tBoc) group instead of a
Bhoc group (Scheme 2 in the parentheses) because the Bhoc
group introduced to the amino group of aPz was too labile under
purification conditions. The other difference was that aPz
derivative 10a (Scheme 3) was directly synthesized from 8a via
Similarly, PNA-PzO formed a duplex with DNA-A, showing a
single transition of the UV-melting curve at 36 °C, which was
lower than that of the PNA-T/DNA-A duplex, for which the Tm
was 45 °C. This result was unexpected because the molecular
orbital calculation suggested that the PzO-A base pair was more
stable than the T-A pair. One of the reasons might be that the
stacking effects of the methyl group at position 5 and the keto
group at position 2 of the thymine residue contributed to the
higher stability of the duplex containing the T-A pair. For the
mixtures of PNA-PzO with DNA-G, DNA-T, and DNA-C and
the mixtures of PNA-PhO with DNA-A, DNA-G, DNA-T, and
DNA-C, the transitions of the melting curves occurred below 30
°C, and the Tm’s of the duplexes were not determined.
Scheme 3. Preparation of the Intermediate 10a
To study the structures of the complexes of PNAs and DNAs,
we measured the circular dichroism (CD) spectra of PNA-aPz
and PNA-aPh (Figure 4) and PNA-PzO and PNA-PhO (Figure
the intermediate 15 using a reaction in which sodium azide and
P(n-Bu)3 coexisted.17 Stepwise synthesis via 9a failed because 9a
was too stable toward the reduction by P(n-Bu)3.
By using the PNA units 1a, 1b, 14a, 14b and commercially
available PNA units and Fmoc-Lys-OH,18 PNAs (Figure 3) were
synthesized. After cleavage from the NovaSyn TGR resin,19 the
PNAs were purified using reversed-phase HPLC and charac-
terized by ESI-TOF mass spectrometry.
Figure 4. Circular dichroism (CD) spectra of PNA-aPz (red), PNA-aPh
(blue), and PNA-C (black) with DNA-G. The green lines indicate the
CD spectra of single stranded DNA-G. Conditions: 2.0 μM
oligonucleotides, 10 mM sodium phosphate buffer (pH 7.0), 100 mM
NaCl, 0.1 mM EDTA at 5 °C.
Figure 3. Sequences of PNAs and oligodeoxynucleotides used for
hybridization studies.
Figure 5. Circular dichroism (CD) spectra of PNA-PzO (red), PNA-
PhO (blue), and PNA-T (black) with DNA-A. The green lines indicate
the CD spectra of single-stranded DNA-A. Conditions: 2.0 μM
oligonucleotides, 10 mM sodium phosphate buffer (pH 7.0), 100 mM
NaCl, 0.1 mM EDTA at 5 °C.
Next, the hybridization of PNA-aPz and PNA-aPh with DNAs
(Figure 3) were studied using thermal denaturation studies
(Table S1).
The UV melting curve of PNA-aPz and DNA-G showed a
transition at 40 °C, which was lower by 12 °C than that of the
duplex of PNA-C and DNA-G, whose Tm was 52 °C. The lower
Tm of PNA-aPz was because of one less hydrogen bond in
comparison with the C−G pair. On the other hand, in the case of
the duplexes with DNA-A, DNA-C, and DNA-T, the UV melting
curves showed multiple transitions between 30 and 40 °C. PNA
has been reported to form preorganized structures that melt at
around 40 °C in the single-stranded state.19 Therefore, the
multiple transitions must be the overlap of the transitions of
PNA-DNA duplexes and the single-stranded PNAs.
In the case of PNA-aPh, the Tm’s of the duplexes were too low
to be determined because the multiple transitions of the melting
curves occurred at temperatures lower than 40 °C for any
combination of PNA-aPh and the DNAs. The lower affinity of
PNA-aPh and DNAs may be explained by the steric hindrance of
the fused benzene ring of aPh, which interfered with the
formation of stable duplexes.
5) at 5 °C in the presence of the complementary DNAs. As
shown in Figure 4, PNA-aPh/DNA-G and PNA-aPz/DNA-G
showed CD curves having positive bands at around 270 and 220
nm and a negative band at around 240 nm; these spectra were the
same as those of the PNA-C/DNA-G duplex and different from
those of single-stranded DNA-G. The single-stranded PNAs did
not show any strong CDs. These peaks were characteristic of
PNA-DNA duplexes19,20 and suggested the formation of the
PNA/DNA duplexes. In particular, the spectra of PNA-aPz/
DNA-G and PNA-C/DNA-G were quite similar at around 270
and 220 nm. When the spectra of the PNA-aPh/DNA-G duplex
and the PNA-C/DNA-G duplex were compared, we found that
the peak intensities at 270, 240, and 220 nm were smaller in the
case of PNA-aPh/DNA-G than in PNA-C/DNA-G. Because the
shape of the CD spectrum is sensitive to the relative geometry of
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In conclusion, we successfully synthesized peptide nucleic
acids incorporating aPz or aPh as a cytosine analog and PzO or
PhO as a thymine analog. The PNA having one of these bases
formed a duplex with complementary DNAs with lower affinity
than that of the canonical PNA having thymine or cytosine.
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endocyclic nitrogen at the α position relative to the glycosidic
linkage can form a hydrogen bond with the amino acid residues
of enzymes in DNA polymerase reactions. Therefore, nucleoside
triphosphates having aPz or PzO as base surrogates may be good
substrates for DNA polymerase reactions. If so, such molecules
may be useful for the development of new anticancer or antiviral
agents. We are now studying the synthesis of such nucleosides
having aPz or PzO; the results will be reported elsewhere.
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ASSOCIATED CONTENT
* Supporting Information
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The synthetic procedures for newly synthesized compounds and
their NMR data are provided. This material is available free of
charge via the Internet at http://pubs.acs.org.
(17) Kandalkar, R. S.; Kaduskar, D. R.; Ramaiah, A. P.; Barawkar, A. D.;
Bhyniya, D.; Deshpande, M. A. Tetrahedron Lett. 2013, 54, 414−418.
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AUTHOR INFORMATION
Corresponding Authors
■
(19) Tomac, S.; Sarkar, M.; Ratilainen, T.; Wittung, P.; Nielsen, P. E.;
Norden, B.; Graslund, A. J. Am. Chem. Soc. 1996, 118, 5544−5552.
́
̈
*E-mail: msekine@bio.titech.ac.jp.
*E-mail: kseio@bio.titech.ac.jp.
Notes
(20) Faccini, A.; Tortori, A.; Tedeschi, T.; Sforza, S.; Tonelli, R.;
Pession, A.; Corradini, R.; Marchelli, R. Chirality 2008, 20, 494−500.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by JSPS KAKENHI Grant No.
26288075.
■
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