Peptide-nucleic acids (PNAs) with pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H, 6H,8H)-tetraone (PPT) as a universal base: Their synthesis and binding affinity for oligodeoxyribonucleotides

Article abstract of DOI:10.1039/b821193k

Peptide-nucleic acids (PNAs) including pyrimido[4,5-d]pyrimidine-2,4,5,7- (1H,3H,6H,8H)-tetraone (PPT) as a nucleobase were synthesized, and their binding affinity for the complementary oligodeoxyribonucleotides was investigated. We found that PNAs with o

Full text of DOI:10.1039/b821193k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Peptide-nucleic acids (PNAs) with pyrimido[4,5-d]pyrimidine-2,4,5,7-
(1H,3H,6H,8H)-tetraone (PPT) as a universal base: their synthesis and
binding affinity for oligodeoxyribonucleotides†
Taisuke Hirano,a Kenji Kuroda,a Masanori Kataokab and Yoshihiro Hayakawa*a
Received 27th November 2008, Accepted 6th April 2009
First published as an Advance Article on the web 27th May 2009
DOI: 10.1039/b821193k
Peptide-nucleic acids (PNAs) including pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H,6H,8H)-tetraone
(PPT) as a nucleobase were synthesized, and their binding affinity for the complementary
oligodeoxyribonucleotides was investigated. We found that PNAs with one or two PPT(s) and natural
nucleobases (i.e., adenine, cytosine, guanine, or thymine) have excellent binding affinity for
oligodeoxyribonucleotides with complementary bases at the positions facing the natural nucleobases,
and with adenine, cytosine, guanine, and thymine at the positions opposite PPT in PNAs. The binding
affinity of the PPT-containing PNA is higher than or comparable to that of a PNA consisting of all
complementary natural nucleobases, viz. a PNA with a suitable natural nucleobase in place of PPT in
the PPT-containing PNA. Consequently, it was concluded that PPT serves as a useful universal base
that can recognize all natural nucleobases.
quite good universality, recognizing and forming base pairs with
Introduction
all four natural nucleobases, but the stability of the resulting
In the field of nucleic acid research, much effort has been
base pairs is not always high. The reason for this low stability
expended to synthesize a universal base that is able to recognize
is considered to be because 3-nitropyrrole and 5-nitroindole are
natural nucleobases, i.e., adenine, cytosine, guanine, and thymine
hydrophobic bases and thus form base pairs through p–p stacking
(or uracil),1 in order to form stable pairs with these bases. Such a
interactions (space or intercalative interactions). However, such
universal base would be useful for various investigations dealing
interactions are not always strong enough to allow satisfactory
with nucleic acids that cannot be easily carried out using only
base-recognizing ability. Therefore, in order to create a base pair
the four kinds of natural nucleobases. Such investigations include
that possesses high binding affinity to natural nucleobases, it is
discrimination of single-nucleotide polymorphisms (SNPs)
in DNA hybridization by means of artificial mismatches,2,3
design of oligonucleotide primers or hybridization probes,
necessary to design a base that forms pairs with all the natural
nucleobases through not only the p–p stacking interactions but
also the hydrogen-bond interactions that are observed in natural
where the identity of one or more bases in the target sequence
base pairs.
is unknown,4–8 and sequence-specific recognition of duplex
As such a base, we recently designed pyrimido[4,5-d]pyrimidine-
DNA, thereby blocking gene expression.9,10 Various candidates
have been proposed as potential universal bases. These include
hypoxanthine (inosine),4 3-nitropyrrole,5 5-nitroindole,11 isocar-
bostyril derivatives,12 (3,4-dihydropyrimido)oxazine derivatives,13
9-deazaguanine,14 8-aza-7-deazapurine derivatives,15 pyridazine
2,4,5,7-(1H,3H,6H,8H)-tetraone (PPT).21 As we have reported,21
this artificial base indeed serves as a perfect universal base to form
a stable 1:1 complex with all the natural nucleosides. Initially, we
planned to prepare oligonucleotides containing a ribonucleoside
with PPT (PPT-nucleoside) (1). However, trials for preparation
of the targeted nucleotide resulted in failure, because the PPT-
nucleoside is too labile in the presence of acids to use as a building
unit for the synthesis of oligonucleotides. For example, the PPT-
nucleoside underwent cleavage of the glycoside bond by treatment
with dichloroacetic acid in dichloromethane, which is generally
used for removal of the 5¢-O-p,p¢-dimethoxytrityl protecting
group in oligonucleotide synthesis. In addition, conversion of the
PPT-nucleoside to the phosphoramidite could not be achieved,
because the PPT-nucleoside is poorly soluble in various organic
solvents, such as acetonitrile. Given the problems encountered
with the stability and solubility of the PPT-nucleoside chemistry,
it was decided to investigate the applicability of our system in
peptide/nucleic acids (PNAs) containing the PPT group as the
nucleobase.
derivatives,16
2-oxoimidazole-4-carboxamide.18
4-N-(N-quinon-5-yl)carbamoylcytosine,17
and
However, these bases are not useful in all situations. With the
exception of 3-nitropyrrole and 5-nitroindole, all of the above
bases form a stable pair with only some of the natural nucleobases.
For example, inosine, which was the first universal base and thus
has been most frequently used, discriminates cytosine and forms
a stable base with it, but does not do so with the other natural
nucleobases.19,20 In contrast, 3-nitropyrrole and 5-nitroindole have
aGraduate School of Information Science and CREST of JST, Nagoya
University, Chikusa, Nagoya, 464-8601, Japan. E-mail: yoshi@is.nagoya-
u.ac.jp; Fax: +81-52-789-5646; Tel: +81-52-789-4848
bResearch Center for Computational Science, Institutes for Natural Sciences,
Okazaki, 444-8787, Japan
As is well known, PNAs – oligonucleotide analogs in which
the phosphate backbone of the oligonucleotide is replaced
† Electronic supplementary information (ESI) available: NMR spectra.
See DOI: 10.1039/b821193k
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27
= =
by an uncharged polyamide backbone consisting of an N-(2-
aminoethyl)glycine unit22 – form duplexes with complementary
DNA. PNAs have numerous advantages over DNA for forming a
duplex with DNA, because the polyamide backbone of the PNA
is neutral. The advantages include (i) the higher thermodynamic
stability of the PNA–DNA duplex than a DNA–DNA duplex,
(ii) the higher sensitivity of PNA for recognizing mismatched
bases, and (iii) the relative independence of PNA from the salt
concentration of a solution.23–25 Therefore, PNAs are important
as biotechnological, diagnostic, and medicinal agents,23–25 and are
frequently employed in various biological investigations, such as
studies investigating antisense chemotherapy.23–25 Accordingly, the
PPT-containing PNAs are substances of great interest. In this
study, we prepared a PPT-containing PNA and investigated its
binding affinity for deoxyribonucleotides with complementary
natural nucleobases.
and then with ethyl isocyanatoformate (O C NCOOC2H5) in
◦
DMF (25 C, 24 h) gave 3 in 92% overall yield. Subsequently, 3
was reacted with sodium ethoxide in refluxing ethyl alcohol for
17 h to provide 4 as a mono-sodium salt28 in 92% yield. Subse-
quently, the O-TBDPS protecting group of 4 was removed using
triethylamine trihydrofluoride [(C2H5)3N·3HF] (25 ◦C, 24 h) to
give 5 in 82% yield. The alcohol 5 was converted to the carboxylic
acid 6 in 52% yield by oxidation using a mixture of 2,2,6,6-
tetramethyl-1-piperidinyloxy (TEMPO), NaBr, and NaClO
(25 ◦C, 1 h). The product 6 was condensed with tert-butyl N-[2-(N-
9-fluorenylmethoxycarbonyl)aminoethyl]glycinate29 [FmocNH-
(CH2)2NHCH2COOC(CH3)3] using 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDC) hydrochloride as a promoter in DMF
◦
(25 C, 24 h) to provide 7 in 74% yield. Finally, the O-tert-butyl
protecting group of 7 was removed by treatment with a 1:2 (v/v)
mixture of dichloromethane and trifluoroacetic acid (TFA) (25 ◦C,
24 h) to give 8 in 87% yield. The PPT-substituted amino acid
skeleton – the parent skeleton of 7 and 8 – was not decomposed
at all by this acid treatment. Removal of the N-Fmoc protecting
group of 8 was also achieved without decomposition of the parent
skeleton by using a 2:2:96 (v/v/v) mixture of 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU), piperidine and DMF (25 ◦C, 15 min).
These results indicated that the PPT-substituted amino acid is
stable under the usual acidic and basic conditions required for the
synthesis of PPT-containing PNAs.
Synthesis of PPT-containing PNA oligomers
Results and discussion
We next prepared two PPT-containing PNAs, H2N-Lys-
CCT(PPT)TCC-Gly-H (9) and H2N-Lys-CCTTTCC(PPT)(PPT)-
Gly-H (10), on a solid phase, starting from commercially available
Preparation and stability of the PPT-containing amino acid
R
We first prepared an amino acid with PPT (PPT-amino acid), to
be used as a building block for the PPT-containing PNA, and
checked its stability of the PPT-amino acid under acidic and basic
conditions.
The synthesis of the PPT-amino acid 8 was carried out according
to the procedure shown in Scheme 1. Treatment of 1-hydroxyethyl-
6-aminouracil (2)26 with tert-butyldiphenylsilyl chloride (TBDP-
SCl) in the presence of imidazole in DMF (60 ◦C, 1.5 h)
TentaGelꢀ S-RAM with lysine 11 (0.20 mmol/g) and using the
amino acids 8, 12,29 and 1330 as building units according to a
typical Fmoc solid-phase peptide synthesis. The resulting crude
product was purified by HPLC on a COSMOSIL 5C18-AR-300
column to give 9 and 10 in 40% and 41% yields, respectively.
The HPLC profile of 9 is shown in Fig. 1. In a similar manner,
H2N-Lys-CCTTTCC-Gly-H (14), which is required for the control
experiment, was separately synthesized.
Scheme 1 Reagents and conditions: (a) TBDPSCl, imidazole, DMF, 60 ◦C, 1.5 h; (b) OCNCH2COOC2H5, DMF, 25 ◦C, 24 h; (c) C2H5ONa, C2H5OH,
reflux, 17 h; (d) (C2H5)3N·3HF, 25 ◦C, 24 h; (e) (f) TEMPO, NaBr, NaClO, KOH, H2O, 25 ◦C, 2 h; (g) FmocNH(CH2)2NHCH2COOC(CH3)3, EDC·HCl,
DMF, 25 ◦C, 24 h; (h) TFA, CH2Cl2, 25 ◦C, 24 h.
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Fig. 2 Tm values of a 1:1 molar mixture of 5¢-GGAXAGG-3¢ (X = A,
G, C, or T) (15, 16, 17 or 18) and H2N-Lys-CCTYTCC-Gly-H (Y = T or
PPT) (14 or 9). N.D. = not detected.
Fig. 1 HPLC profile of the compound 9. Conditions: COSMOSIL
5C18-MS column (4.6 ¥ 250 mm); eluent A, 0.1% TFA/water; eluent B,
0.1% TFA/acetonitrile; gradient, linear 0–100% B over 35 min; detection
260 nm; flow rate, 1 mL/min; 55 ◦C.
Fig. 2 indicates that 9 and 14 have quite different binding
affinities compared to 15, 16, 17 and 18. Thus, as predicted, 14
makes a stable duplex only with 15, and does not form a stable
duplex with 16, 17 or 18. In contrast, 9 forms stable duplexes with
15, 16, 17 and 18, though the Tm value (i.e. the binding ability),
differs slightly according to the particular nucleobase. DG values
of the duplex estimated31,32 on the basis of the observed Tm values
are -35.9 kJ/mol for the duplex of 9 and 15, -34.2 kJ/mol for the
duplex of 9 and 16, -35.3 kJ/mol for the duplex of 9 and 17, and
-35.0 kJ/mol for the duplex of 9 and 18. These DG values are all
similar, indicating that, as expected, PPT serves as a universal base
that recognizes all natural nucleobases, regardless of whether they
are purine or pyrimidine bases.
Subsequently, the ability of the PNA 10, which in-
cludes two PPTs, to hybridize the complementary DNA,
5¢-GGAAAGGY1Y2-3¢ (type I) (Y1, Y2 = A, G, C or T) or
3¢-GGAAAGGY1Y2-5¢ (type II) (Y1, Y2 = A, G, C or T), was
examined in a manner similar to that described above. Thus, we
produced duplexes of 10 and complementary DNAs of type I
or type II and measured the Tm values of the resulting duplexes
(Fig. 3). We found that the PNA 10 forms duplexes with both
DNAs, and that these duplexes have similar Tm values. Thus,
the difference between the maximum and minimum Tm values
(Tm,max and Tm,min) is only 5.8 ◦C. This is smaller than the
difference between Tm,max and Tm,min in the duplexes between PNA
9 (which contains one PPT) and the complementary DNA 15.
This shows that, when two PPTs are incorporated in PNAs,
the resulting PNAs form duplexes that have closer stabilities
than those with one PPT, regardless of whether the nucleobase
facing the PPT unit is purine or pyrimidine. Consequently, we
consider that an increase in the number of PPTs enhances the
binding affinity for all natural nucleobases, and thus allows the
Binding affinity of the PPT-containing PNA for a
complementary DNA
First, we investigated the hybridizing ability of the PNA 9, which
contains one PPT, for complementary DNAs 15, 16, 17, and
18 (Table 1) as follows. We prepared a duplex of the DNA
and 9, by mixing 2.0 mmol of 15, 16, 17 or 18 and 9 in
10 mM phosphate buffer (pH 7) (1.0 mL), and estimated the
stability of each of the resulting duplexes by thermal denaturation
experiments using absorbance spectroscopy. In this experiment,
all absorbance–temperature curves were measured at 260 nm, and
melting temperatures (Tm) were determined on the basis of the
resulting melting curves by parallel melting data analysis software
(JASCO, version 1.01.01). In order to confirm the universality of
PPT as a nucleobase, we also carried out similar experiments using
14 as a PNA, where PPT in 9 is replaced by T. The Tm of a 1:1
molar mixture of the DNA (15, 16, 17 or 18) and 14 was measured
and compared with that of a 1:1 molar mixture of the DNA and
9. The results obtained by these experiments are shown in Fig. 2.
Table 1
DNA
15
5¢-GGAXAGG-3¢
X = A
16
X = G
17
18
X = C
X = T
PNA
9
14
H2N-Lys-CCTYTCC-Gly-H
(Y = PPT)
(Y = T)
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expected to be useful in various investigations dealing with nucleic
acids.
Experimental
General procedures, materials, and solvents
UV spectra were measured in DMSO on a JASCO V-550
spectrometer. IR spectra were obtained in a KBr tablet on a
JASCO FT/IR-5300 spectrometer. NMR spectra were taken in
DMSO-d6 on a JEOL JNM-a400 or ECA-500 instrument. The
1H- and 13C-NMR chemical shifts are described as d values in ppm
1
relative to H and 13C of (CH3)4Si. ESI-TOF mass spectra were
obtained on Applied Biosystems Mariner spectrometers. HPLC
analysis was carried out using a COSMOSIL 5C18-MS column
(ODS-5 mm, 4.6 ¥ 250 mm; Nacalai Tesque, Kyoto, Japan) on a
Waters 2695 Separations Module chromatograph with a Waters
2996 Photodiode Array detector. Column chromatography was
performed using Nacalai Tesque silica gel 60 (neutrality, 75 mm).
Unless otherwise noted, synthetic reactions were carried out at
ambient temperature. The reactions requiring anhydrous condi-
tions were carried out under an argon atmosphere in flasks dried
by heating at 400 ◦C under 133–400 Pa, or by washing with a 5%
solution of dichlorodimethylsilane in dichloromethane followed
by anhydrous dichloromethane, and then heating at 100 ◦C. DMF
and dichloromethane were distilled from calcium hydride. Other
organic reagents were used as commercially supplied without any
purification. Solid and amorphous organic substances were used
after drying over P2O5 at 50–60 ◦C for 8–12 h under 133–400 Pa.
The Fmoc protected C-monomer 13 was purchased from Applied
Biosystems (Foster City, USA). DNA oligomers were purchased
from Gene Design Inc, Japan.
Fig.
3
Tm values of a 1:1 molar mixture of H2N-Lys-CCTTT-
CC(PPT)(PPT)-Gly-H (10) and 5¢-GGAAAGGY1Y23¢ (type I) or
3¢-GGAAAGGY1Y25¢ (type II) (Y1, Y2 = A, G, C or T).
PNA to form more stable duplexes with the complementary
DNA.
Conclusions
We designed pyrimido[4,5-d]pyrimidine-2,4,5,7-(1H,3H,6H,8H)-
tetraone (PPT) as a new universal base and investigated its
universality using PPT-containing PNAs. The results revealed
that PPT serves as an excellent universal base to recognize all
four natural nucleobases, A, G, C, and T. It is important to
determine how PPT recognizes the these different kinds of natural
nucleobases – in other words, to clarify the configuration in which
PPT forms base pairs with the natural nucleobases. At present,
we have no direct observations that could answer this query.
However, based on the finding that the Tm values of the duplex
of the PPT-containing PNA and the complementary DNA are
comparable to those of duplexes of fully complementary PNAs
and DNAs, such as 9 and 15–18, we think that PPT forms the
base pairs by not only by p–p stacking interaction, but also by
hydrogen-bond formation through dynamic transformations such
as conformational change and keto–enol tautomerization. Here,
we think that the conformational change allows PPT to take
a pyrimidine base form when forming hydrogen bonds with a
purine base, i.e., A or G, but a purine base form when forming
hydrogen bonds with a pyrimidine base, i.e., C or T. At the same
time, the keto–enol tautomerization allows PPT to have structures
suitable for forming base pairs with natural nucleobases via
hydrogen-bonding interaction. In conclusion, we have developed
a PNA with PPT as a universal base and demonstrated that
this PPT-containing PNA has the ability to form a duplex
with a complementary DNA, in which all of the four natural
nucleobases face the PPT. Thus, these PPT-containing PNAs are
Ethyl {6-Amino-1-[2-(tert-butyldiphenylsilanyloxy)ethyl]-2,4-
dioxo-1,2,3,4-tetrahydro-pyrimidine-5-carbonyl}carbamate
(3).
To a suspension of 1-hydroxyethyl-6-aminouracil (2) (1.0 g,
5.8 mmol) in anhydrous DMF (6.0 mL) were added imidazole
(0.88 g, 13 mmol) and tert-butyldiphenylchlorosilane (1.8 mg,
6.4 mmol). The resulting mixture was stirred at 60 ◦C for 1.5 h and
then was added dropwise to water (1.0 L). The resulting precipitate
was collected by filtration and dried under reduced pressure to
give 1-(2-tert-butyldiphenylsilanyloxy)ethyl-6-aminouracil (2.2 g,
92% yield) as a colorless powder: mp 250 ◦C; 1H NMR (400 MHz)
0.96 (s, 9H), 3.76 (br, 2H), 4.04 (br, 2H), 4.60 (br, 1H), 6.70
(br, 2H), 7.41 (m, 6H), 7.58 (m, 4H), 10.32 (br, 1H); 13C NMR
(100 MHz) 19.1, 26.9, 42.1, 61.6, 76.2, 128.0, 128.3, 130.3, 130.1,
134.9, 135.5, 151.9, 158.8, 162.9. This product (14 g, 33 mmol)
was suspended in anhydrous DMF (40 mL) and to the resulting
suspension was added ethyl isocyanatoformate (3.9 g, 34 mmol)
dropwise over 30 min at 25 ◦C. The mixture was stirred at 25 ◦C
for 24 h and concentrated in vacuo. The resulting solid material
was washed with ethyl acetate, collected by filtration, and dried
under reduced pr◦essure to give 3 (7.0 g, 40% yield) as a colorless
powder: mp 275 C; 1H NMR (400 MHz) 0.94 (s, 9H), 1.22 (t,
J = 7.2 Hz, 3H), 3.81 (d, J = 4.8 Hz, 2H), 4.12 (q, J = 4.8 Hz,
2H), 4.18 (t, J = 4.8 Hz, 2H), 7.42 (m, 6H), 7.53 (M, 4H), 8.36
(br, 1H), 10.85 (br, 1H), 11.35 (br, 1H), 12.39 (br, 1H); 13C NMR
(100 MHz) 14.7, 19.1, 26.9, 43.1, 60.6, 61.1, 81.1, 128.3, 130.4,
132.9, 135.4, 148.8, 150.8, 159.9, 164.5, 166.2; IR 3389, 3111,
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1735, 1626, 1508, 1426, 1288, 1203, 1113, 1024 cm-1; UV lmax
272 nm (e 30,200).
(1.1 g, 2.8 mmol) in anhydrous DMF (10 mL) was added
6
(0.72 g, 2.8 mmol), and the mixture was stirred to
dissolve 6. To the resulting solution was added 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.1 g,
5.6 mmol). The mixture was stirred at 25 ◦C for 24 h and then
concentrated under reduced pressure. Water (30 mL) was ◦added
to the resulting viscous oil. The mixture was stirred at 25 C for
several minutes to produce a white solid. The solid was collected
by filtration and dried under reduced pressure. This crude product
was subjected to flash chromatography and eluted with a 19:80:1
(v/v/v) mixture of methanol and dichloromethane and acetic acid
to give 7 (1.3 g, 74%) as a colorless powder: mp 297 ◦C; 1H NMR
(400 MHz) 1.37–1.45 (m, 9H), 2.75 (br, 1H), 3.00–3.42 (m, 4H),
3.89–3.92 (br, 1H), 4.14–4.33 (m, 4H), 4.73–4.94 (m, 2H), 7.30–
7.41 (m, 5H), 7.66–7.68 (m, 2H), 7.86–7.91 (m, 2H), 9.87 (br, 1H),
10.6 (br, 1H); 13C NMR (100 MHz) 28.1, 42.3, 47.2, 47.5, 49.5,
65.4, 66.0, 81.4, 82.4, 83.6, 86.5, 120.6, 125.5, 125.6, 127.5, 128.1,
141.2, 144.3, 144.4, 150.5, 156.6, 156.8, 166.3, 167.4, 168.6, 168.9;
MS (ESI+) calcd for C31H32N6O9 (M + H+) 633.29, found 633.23;
IR 3386, 3216, 3071, 2975, 2822, 1719, 1547, 1508, 1449, 1255,
1204, 1146, 1039 cm-1; UV lmax 268 nm (e 36,000).
1-[2-(tert-Butyldiphenylsilanyloxy)ethyl]-1H,8H-pyrimido[4,5-
d]pyrimidine-2,4,5,7-tetraone (4). A solution of sodium ethoxide
in ethanol prepared using sodium (60 mg, 2.6 mmol) and dry
ethanol (20 mL) was added to 3 (0.60 g, 1.1 mmol). The mixture
was refluxed for 17 h and then cooled to 25 ◦C. The resulting
precipitate was collected by filtration. The collected solid was
washed with a 1.0 M aqueous HCl solution until the pH of the
filtrate became 7 and then dried under reduced pressure to give 4
(0.50 g, 92% yield) as a colorless powder: mp 300 C; H NMR
(400 MHz) 0.94 (s, 9H), 3.83 (t, J = 4.8 Hz, 2H), 4.23 (t, J =
4.8 Hz, 2H), 7.38 (m, 6H), 7.57 (m, 4H), 9.79 (br, 1H), 10.53 (br,
1H); 13C NMR (100 MHz) 19.2, 27.0, 42.3, 61.1, 86.0, 128.3, 130.1,
133.6, 135.4, 151.6, 157.9, 161.6, 162.6, 164.8; MS (ESI+) calcd for
C24H25N4O5Si (M + H+) 479.21, found 479.17; IR 3424, 1693,
1542, 1507, 1427, 1263, 1228, 1112, 1058 cm-1; UV lmax 279 nm
(e 20,000).
◦
1
1-(2-Hydroxyethyl)-1H,8H-pyrimido[4,5-d]pyrimidine-2,4,5,7-
tetraone (5). Compound 4 (1.7 g, 3.6 mmol) was mixed with
triethylamine trishydrofluoride (4.9 g, 31 mmol) in a polypropylene
flask and the mixture was stirred at 25 ◦C for 24 h. The resulting
mixture was poured into a 2 M aqueous KOH solution in a glass
flask, and then to this mixture was added a 2 M HCl solution
to adjust the pH of the mixture to 7. The precipitated solid
was collected by filtration and dried under reduced pressure to
give 5 (0.70 g, 82% yield) as a colorless powder: mp 295 ◦C;
1H NMR (400 MHz) 3.49 (br, 2H), 4.02 (br, 2H), 4.88 (br,
1H), 9.44 (br, 1H), 10.20 (br, 1H); 13C NMR (100 MHz) 43.0,
58.8, 86.1, 151.5, 157.9, 161.3, 162.7, 164.5; MS (ESI+) calcd
for C8H8N4O5 (M + H+) 241.05, found 241.06;; IR 2961, 2812,
1746, 1636, 1536, 1418, 1263, 1066 cm-1; UV lmax 278 nm
(e 32,200).
N-[2-(N-9-Fluorenylmethoxycarbonyl)aminoethyl]-N-[(2,4,5,7-
tetraoxo-3,4,5,6,7,8-hexahydro-2H-pyrimido[4,5-d]pyrimidin-1-
yl)acetyl]glycine (8). To a suspension of 7 (1.9 g, 3.0 mmol) in
anhydrous dichloromethane (10 mL) was added trifluoroacetic
acid (20 mL). This mixture was stirred at 25 ◦C for 24 h. The
resulting solution was concentrated to give a residual oil, to
which was added ether with stirring to afford a precipitate. This
was collected by filtration, dried under reduced pressure, and
recrystallized from a 4:1 (v/v) mixture of methanol and ether
◦
1
to provide 8 (1.5 g, 87%) as a colorless powder: mp 295 C; H
NMR (400 MHz) 2.75 (br, 1H), 3.10–3.64 (m, 4H), 3.99 (br, 1H),
4.23–4.37 (m, 4H), 4.79–4.97 (m, 2H), 7.28–7.46 (m, 5H), 7.65–
7.69 (m, 2H), 7.87–7.91 (m, 2H), 10.68 (br, 1H), 11.16 (br, 1H),
12.66 (br, 1H); 13C NMR (100 MHz) 47.2, 47.4, 48.3, 54.4, 65.4,
66.0, 87.0, 87.1, 120.6, 120.7, 125.4, 125.5, 125.6, 127.5, 128.1,
128.2, 141.2, 144.2, 144.3, 150.1, 156.6, 156.8, 159.2, 167.0, 169.9,
170.9, 171.2 ppm; MS (ESI+) calcd for C27H24N6O9 (M + H+)
577.19, found 577.17; IR 3420, 3252, 3068, 2979, 2818, 1698, 1550,
1504, 1451, 1369, 1240, 1154, 1124, 1036 cm-1; UV lmax 269 nm
(e 24,400).
(2,4,5,7-Tetraoxo-3,4,5,6,7,8-hexahydro-2H-pyrimido[4,5-d]-
pyrimidin-1-yl)acetic acid (6). Compound 5 (1.0 g, 4.2 mmol)
was suspended in water (70 mL) containing 2,2,6,6-tetramethyl-1-
piperidinyloxy (TEMPO) (0.70 g, 4.2 mmol) and sodium bromide
(0.92 g, 8.4 mmol). The suspension was adjusted to pH 10 by
adding a 0.4 M aqueous NaOH solution. To this suspension was
added an 11% aqueous sodium hypochlorite solution (5.4 mL),
and the pH of the resulting mixture was adjusted to 11 by adding
a 0.4 M aqueous NaOH solution. The resulting mixture was stirred
Solid-phase synthesis of PNA oligomers
◦
at 25 C for 1.5 h and then ethanol (400 mL) was added to the
2-(N-9-Fluorenylmethoxycarbonyl)amino-6-(tert-butoxycarbo-
nylamino)hexanoic acid-loaded Tentagel S-RAM resin (37 mg,
10 mmol of the amino group) was used as a solid support for the
solid-phase synthesis. A Teflon syringe with a frit at the bottom of
a 5 mL polypropylene cartridge was used as reactor.
mixture. The precipitated solid was collected by filtration. The
solid was dissolved in water (30 mL). To this solution was added a
2 M HCl solution. The precipitated solid was collected by filtration
and dried under reduced pr◦essure to give 6 (0.53 g, 50% yield) as
a colorless powder: mp 296 C; 1H NMR (400 MHz) 4.71 (s, 2H),
11.23 (br, 1H), 11.44 (br, 1H), 13.22 (br, 1H); 13C NMR (100 MHz)
44.6, 87.5, 149.6, 150.3, 155.4, 158.5, 159.7, 169.1; MS (ESI+) calcd
for C8H6N4O6 (M + H+) 255.04, found 255.03; IR 3385, 3218, 3071,
2931, 2857, 1697, 1653, 1599, 1490, 1428, 1395, 1285, 1181, 1112,
1007 cm-1; UV lmax 275 nm (e 30,800).
The chain elongation of PNA was carried out as follows: (1)
the resin in the cartridge was washed with anhydrous DMF
(0.5 mL) at 25 ◦C; (2) the resin was mixed with 0.5 mL of
a 2:2:96 (v/v/v) mixture of 1,8-diazabicyclo-[5.4.0]undec-7-ene
◦
(DBU) and piperidine and anhydrous DMF at 25 C for 7 min;
(3) steps (1) and (2) were repeated; (4) the resin was washed with
anhydrous DMF (0.5 mL) at 25 ◦C; (5) step (4) was repeated
three times; (6) the resin was washed with 0.5 mL of a 1:1 (v/v)
mixture of anhydrous N-methylpyrrolidone and anhydrous DMF
at 25 ◦C; (7) step (6) was repeated twice; (8) the resin was treated
tert-Butyl N-[2-(N-9-Fluorenylmethoxycarbonyl)amino ethyl]-
N-[(2,4,5,7-tetraoxo-3,4,5,6,7,8-hexahydro-2H-pyrimido[4,5-d]-
pyrimidin-1-yl)acetyl]glycinate (7). To
a solution of tert-
butyl N-[2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]glycinate
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with a homogeneous mixture of an Fmoc-protected monomer [8
(17 mg, 30 mmol) or 12 (15 mg, 30 mmol) or 13 (21 mg, 30 mmol)],
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluo-
rophosphate (10 mg, 27 mmol), a 0.4 M solution of N,N¢-
diisopropylethylamine in anhydrous DMF (83 mL), a 0.6 M
solution of 2,6-lutidine in anhydrous DMF (55 mL), and a 1:1
(v/v) mixture of anhydrous N-methylpyrrolidone and anhydrous
DMF (0.2 mL) at 25 ◦C for 1.5–2.0 h; (9) the resin was collected
by filtration and washed with anhydrous DMF (0.5 mL) at 25 ◦C;
(10) the resin was treated with a 5:6:89 (v/v/v) mixture of acetic
Measurement of Tm of a PNA–DNA duplex
A solution of a PNA oligomer (2.0 mmol) and a DNA oligomer
(2.0 mmol) in a 10 mM phosphate buffer solution of pH 7 (1.0 mL)
was prepared. The resulting solution was placed in a quartz cuvette
◦
of 1 cm path length and heated at 95 C for 5 min. The solution
was then cooled to 5 ◦C at a rate of 1 ◦C/min, and kept at
this temperature. The resulting solution was subjected to UV
measurement at 260 nm from 5 ◦C to 60 ◦C or 80 ◦C using a
JASCO V-550 spectrometer equipped with a JASCO ETC-505T
Peltier temperature controller by increasing the temperature at a
rate of 1 ◦C/min. The obtained UV absorbance was plotted to
prepare the melting curve of the duplex of the PNA oligomer and
the DNA oligomer. The Tm value was estimated on the basis of
the resulting melting curve using parallel melting data analysis
software (JASCO, version 1.01.01).
◦
anhydride, 2,6-lutidine, and anhydrous DMF (0.5 mL) at 25 C
for 5 min; (11) steps (9) and (10) were repeated. Steps (1) to
(11) were then repeated until a chain of the desired length was
obtained.
The resin in the cartridge was washed with anhydrous DMF
(0.5 mL) at 25 ◦C, and then treated with 0.5 mL of a 2:2:96
(v/v) mixture of DBU, piperidine, and anhydrous DMF at
25 ◦C for 7 min. These two procedures were repeated twice.
The resin was washed with anhydrous DMF (0.5 mL) at
25 ◦C three times and with 0.5 mL of a 1:1 (v/v) mixture
of anhydrous N-methylpyrrolidone and anhydrous DMF at
25 ◦C twice. The resin was treated with a mixture of (N-9-
fluorenylmethoxycarbonylamino)acetic acid (12 mg, 40 mmol),
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluo-
rophosphate (14 mg, 36 mmol), a 0.4 M solution of N,N¢-
diisopropylethylamine in anhydrous DMF (0.11 mL), a 0.6 M
solution of 2,6-lutidine in anhydrous DMF (73 mL), and a 1:1
(v/v) mixture of anhydrous N-methylpyrrolidone and anhydrous
DMF (0.2 mL) at 25 ◦C for 2.0 h. The resulting resin was separated
by filtration, washed successively with anhydrous DMF (0.5 mL),
anhydrous methanol (0.5 mL), and anhydrous dichloromethane
(0.5 mL), and then dried under reduced pressure. The resin was
mixed with a 1:9 (v/v) mixture of m-cresol and trifluoroacetic
acid (1.0 mL) at 25 ◦C for 2 h. The resin was removed by
filtration and the filtrate was concentrated. To the resulting oil
was added ether (10 mL). The precipitated solid was collected
by filtration. The solid product was dissolved in a 93:7 (v/v)
mixture of buffer A (a 0.1% trifluoroacetic acid/water solution)
and buffer B (a 0.1% trifluoroacetic acid/acetonitrile solution)
and subjected to HPLC purification using a C-18 reversed phase
column. Elution of products was carried out by the linear gradient
elution method starting from a 93:7 (v/v) mixture of buffer A
and buffer B to a 1:1 (v/v) mixture of buffer A and buffer B as
eluent (flow rate 10 ml/min), where a UV detector (254, 260, and
266 nm) was used for monitoring the elution course. The fractions
containing the desired product were combined and lyophilized.
The resulting material was treated with a 20% piperidine/water
solution at 0 ◦C for 45 min. The solution was again dried by
lyophilization. The residue was dissolved in a 0.1% trifluoroacetic
acid/water solution (8.0 mL). The resulting solution was subjected
to HPLC purification on a C-18 reversed phase column, eluted
with 100% of buffer A to a 1:1 (v/v) mixture of buffer A
and buffer B (linear gradient over 35 min). The combined
fractions containing the target product were concentrated by
lyophilization. This sequence of procedures gave 9 (8.3 mg) in a
40% isolated yield [MS (ESI+) calcd for C82H110N38O28 (M + 2H+)
1038.4, found 1038.5] and 10 (11 mg) in a 41% isolated yield
[MS (ESI+) calcd for C105H136N48O38 (M + 3 H+) 893.35, found
893.37].
Acknowledgements
This study was partly supported by CREST of JST (Japan Science
and Technology) and by a Grant-in-Aid for Scientific Research
(No. 16011223) from the Ministry of Education, Culture, Science,
Sports and Technology of Japan.
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