Synthesis of nucleobase-caged peptide nucleic acids having improved photochemical properties

Article abstract of DOI:10.1039/c4ob00418c

A nucleobase-caged peptide nucleic acid (PNA) having a (6-bromo-7- methoxycoumarin)-4-ylmethoxycarbonyl (Bmcmoc) caging group was newly synthesized. The Bmcmoc-caged PNAs were photolyzed to produce parent PNAs with a high photochemical efficiency. Introduction of a single Bmcmoc group was sufficient to suppress polymerase chain reaction (PCR) clamping activity and triplex invasion complex formation. Photo-mediated restoration of the PCR clamping activity was also demonstrated.

Full text of DOI:10.1039/c4ob00418c

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Synthesis of nucleobase-caged peptide nucleic
acids having improved photochemical properties†
Cite this: Org. Biomol. Chem., 2014,
12, 5089
Takayoshi Watanabe,‡ Tomoko Hoshida, Jun Sakyo, Mariko Kishi, Satoshi Tanabe,
Junichi Matsuura, Shingo Akiyama, Makiko Nakata, Yasuaki Tanabe,
Akinobu Z. Suzuki, Soichiro Watanabe and Toshiaki Furuta*
Received 24th February 2014,
Accepted 30th May 2014
DOI: 10.1039/c4ob00418c
www.rsc.org/obc
A
nucleobase-caged peptide nucleic acid (PNA) having
a
molecules to induce translation inhibition in live zebrafish
(6-bromo-7-methoxycoumarin)-4-ylmethoxycarbonyl (Bmcmoc) embryos.9 Very recently, nucleobase-caged PNAs were reported
caging group was newly synthesized. The Bmcmoc-caged PNAs and used in light-triggered PNA/PNA recognition by Diederichsen’s
were photolyzed to produce parent PNAs with a high photo- group.10 Introduction of two or three 2-(2-nitrophenyl)propyl
chemical efficiency. Introduction of a single Bmcmoc group was (NPP) caging groups into each strand of complementary
sufficient to suppress polymerase chain reaction (PCR) clamping PNAs was necessary to inhibit duplex formation. Modification
activity and triplex invasion complex formation. Photo-mediated of a single nucleobase in one PNA strand partially prevented
restoration of the PCR clamping activity was also demonstrated.
PNA/PNA duplex formation, which is consistent with obser-
vations of nucleobase-caged DNAs.11
Caged compounds are molecules designed such that their bio-
logical functions are temporarily masked and are reactivated
by the application of external triggers such as ultraviolet light.1
N-(2-Aminoethyl)glycine peptide nucleic acid (aeg-PNA) is a
DNA mimetic in which the sugar–phosphate backbone has
been replaced by a 2-aminoethylglycine linkage.2 Because aeg-
PNA molecules can bind to their complementary oligonucleo-
tides with high affinity and improved specificity, PNAs can be
potential antisense and antigene agents.3 PNAs have also been
used to create biochemical tools4 used in transcription acti-
vation,5 polymerase chain reaction (PCR) clamping,6 target
detection,7 and vector tagging.8 If produced correctly, caged
PNA molecules can be used as photochemically controllable
chemical tools for the manipulation of oligonucleotide func-
tions both in cells and in vitro. To prepare caged PNAs, a
caging group should be introduced into a functional group of
nucleobases as a steric mask or should be used as a photo-
cleavable linker incorporated into a hairpin-shaped PNA/oligo-
nucleotide duplex. The latter approach was demonstrated by
Dmochowski’s group, who reported the synthesis of hairpin-
shaped caged negatively charged PNAs (ncPNAs) as photo-
chemically controllable antisense molecules and used the
We have been studying the preparation, photochemical pro-
perties and biological use of caged nucleotides.12 We reported
the synthesis and photochemistry of the (6-bromo-7-methoxy-
coumarin-4-yl)methoxy carbonyl (Bmcmoc) group as a new
photo-removable protecting group for nucleobases.12d
Bmcmoc-caged nucleosides have 10–100 times better photo-
lytic efficiency than those of other caging groups, including
the NPP group. Therefore the Bmcmoc group is a good candi-
date for preparing nucleobase-caged PNAs. In this study, we
elucidated whether the oxycarbonyl linkage of the Bmcmoc
group can tolerate through coupling, deprotection, and clea-
vage conditions of a standard solid-phase PNA synthesis. In
addition, we tested whether the high photochemical efficiency
of the Bmcmoc group is maintained when the group is incorpo-
rated into a rather hydrophobic oligo PNA strand. Further-
more, we examined whether more than two caging groups are
always necessary to suppress the original function of PNA.
Finally, we examined the introduction of a single Bmcmoc
group to ascertain whether it can suppress a strand-invasion
type (PNA)2/DNA triplex formation between a 16-mer PNA and
a dsDNA.13
We synthesized two caged PNA monomers having
a
Bmcmoc protecting group on nucleo-bases (Fig. 1). A Bmcmoc
group was introduced into the amino moiety of tert-butyl
cytosine acetate via the corresponding chloroformate inter-
mediate (Bmcmoc-Cl12d). Modification of the adenine base was
achieved by the reaction of BmcCH2OH12d with an isocyanate
generated from 8 14 and CDI.15 The Bmcmoc protected cytosin-
1-yl acetic acid (3) and adenin-9-yl acetic acid (10) were
coupled to a (2-aminoethyl)glycine unit (4), followed by depro-
Department of Biomolecular Science, Faculty of Science, Toho University,
2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
E-mail: furuta@biomol.sci.toho-u.ac.jp; Fax: +81-47-472-1169; Tel: +81-47-472-1169
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4ob00418c
‡Present address: School of Materials Science, Japan Advanced Institute of
Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292.
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Fig. 2 Chemical structures of the caged compounds used in this study.
were almost identical at 350 nm (ε350 = 5100 M−1 cm−1 for
3 and 5200 M−1 cm−1 for 13). Photolysis was performed at
350 nm (two RPR 350 nm lamps, 4 mJ s−1). The photolytic con-
sumption of the starting compounds followed single exponen-
tial decay, from which we obtained a photolysis quantum yield
of 0.24 for 3 and 0.0014 for 13 (Fig. S3†). Consequently, the
photolytic efficiency, εΦ, value of the disappearance of 3 was
calculated to be 1220 M−1 cm−1, which is comparable to that
of the Bmcmoc-caged cytosine12d and is more than 170 times
larger than that of 13. The Bmcmoc-caged PNA monomer 7
and the ABmcmoc-AcOH (10) have similar photophysical and
photochemical properties to those of 3 (Table 1). When incor-
porated into PNA oligomers, the CBmcmoc group showed
slightly decreased photolytic quantum yields. In the PNA oligo-
mers, one would expect the Bmcmoc to experience a more
hydrophobic environment; this could explain the decrease in
the photolysis quantum yields.19 The observed εΦ values of
1090 M−1 cm−1 for 10-mer and 760 M−1 cm−1 for 16-mer are
still two orders of magnitude larger than that of CNVOC-AcOH
(Table 1). Moreover, almost quantitative production of the
Fig. 1 Synthesis of Bmcmoc-caged PNA monomers.
t
tection of the Bu groups to yield Fmoc-protected caged PNA
monomers, Fmoc-CBmcmoc-aeg-OH (6) and Fmoc-ABmcmoc-aeg-
OH (12). The Fmoc protecting group of 6 was deprotected by
20% piperidine to give CBmcmoc-aeg-OH (7), which was used in
a photolysis reaction for comparison. Similarly, CNVOC-AcOH
(13) was synthesized by the reaction of compound 1 16 with
NVOC-Cl (82% yield), followed by deprotection of the tBu
group (87% yield).
Caged PNA oligomers were synthesized on a 0.02 mmol
scale by following an Fmoc SPPS protocol using HATU as a
condensation agent. We synthesized 5-mer, 10-mer, and
16-mer caged PNAs having a single Bmcmoc-caged cytosine
(CBmcmoc) (Fig. 2). The 10-mer sequence (TAGCTGTTTC) was
chosen to target the start codon region of E. coli lac Z gene.17
The 16-mer homopyrimidine sequence (TTCTCTTCCTTCTCTT)
was designed to produce a (PNA)2/DNA triplex using a strand
invasion mechanism.18 The C-terminus glycine was introduced
as a spacer for the improvement of the yields of the coupling
reactions. After cleavage from the Alko-PEG resin, the desired
products were purified using reversed-phase HPLC and were
characterized using ESI-MS. The results show that the
Bmcmoc group tolerates through coupling, deprotection, and
cleavage conditions of the Fmoc SPPS.
Table 1 Selected photophysical and chemical properties of nucleo-
base-caged peptide nucleic acids
b
c
d
e
λmaxa (nm) Φdis
εΦdis
Φapp
εΦapp
CBmcmoc-AcOH (3)
CNVOC-AcOH (13)
294, 328
295, 349
270, 329
0.24
0.0014 7.3
0.18
0.22
0.18
0.14
0.13
1220
A
C
Bmcmoc-AcOH (10)
1140
Bmcmoc-aeg-OH (7) 291, 329
1250
1020
1090
760
5-mer cPNA (14)
10-mer cPNA (15)
16-mer cPNA (16)
268, 329
260, 331
268, 331
0.16
920
460
0.075
a Absorption maximum (nm) measured in K-MOPS (pH 7.2).
b Quantum yields for disappearance of starting materials upon
irradiation (350 nm). Samples (10 μM) in K-MOPS (pH 7.2) were
The photophysical and chemical properties of the Bmcmoc-
caged PNAs were measured in a simulated physiological saline photolyzed with two RPR 350 nm lamps. c Product of the photolysis
quantum yield for disappearance and molar absorptivity at 350 nm
solution. The photolytic efficiency of the Bmcmoc caging
group was compared with that of the NVOC group. The molar
(M−1 cm−1). d Quantum yields for the appearance of the PNAs upon
irradiation (350 nm). e Product of the photolysis quantum yield for
absorptivities of the caged CBmcmoc-AcOH (3) and CNVOC-AcOH (13) appearance and molar absorptivity at 350 nm.
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Fig. 4 PCR clamping and triplex invasion study. Agarose gel electro-
phoresis of the PCR products (a, b). PCRs were performed in the pres-
ence of 10-mer PNA or 10-mer cPNA (15): (a) lanes 1 and 7, no PNAs;
lane 8, 12 μM; lanes 2 and 9, 18 μM; lanes 3 and 10, 24 μM; lanes 4
and 11, 30 μM; lanes 5 and 12, 36 μM; lane 6, 42 μM; (b) PCR mixtures
containing 15 were exposed to UV light (350 nm, 2 mJ s−1, 120 s). Lane 1,
no cPNA; lane 2, 24 μM; lane 3, 48 μM; (c) gel-shift analysis of triplex
invasion complexes between the 50-mer dsDNA and the 16-mer PNA or
16-mer cPNA (16). Lanes 1 and 5, no PNAs; lanes 2 and 6, 0.2 μM; lanes
3 and 7, 1 μM; lanes 4 and 8, 2 μM; I, triplex invasion complex; T, triplex;
D, duplex.
Fig. 3 Time course for photolysis of the caged PNAs. Samples (10−5 M)
were irradiated at 350 nm (4 mJ s−1) under simulated physiological con-
ditions (10 mM K-MOPS buffer at pH 7.2). Closed circles show the con-
sumption of 14. Open circles show the yield of 5-mer PNA. Closed
squares show the consumption of 16. Open squares show the yield of
16-mer PNA. Solid lines show the least-squares curve fit to a simple
decaying exponential for 14 and 16. The dotted line shows the rising
exponential for 5-mer PNA.
parent PNA was observed from photolysis of the 5-mer cPNA
(14) with a quantum yield of 0.16 (Fig. 3). The molar absorptiv-
ities of the Bmcmoc-caged PNAs (14–16) are approximately
10 000 M−1 cm−1 at 330 nm and 200 M−1 cm−1 at 385 nm
(Fig. S2†). The compounds can be photolyzed at wavelengths
of up to 385 nm.
activity of a caged PNA on DNA polymerization would be
detected efficiently using PCR clamping experiments. We used
PCR as a simple model of DNA polymerization in cells: DNA
replication and DNA repair.
The effects of the non-caged PNA and the cPNA were
measured in standard PCRs containing Blend-Taq DNA poly-
merase, pUC18 as a template, and a set of primers (398 bp
product). Either the 10-mer PNA or the 10-mer cPNA (15) was
added to the PCR mixture. After 20 cycles of amplification, the
amplicons were analyzed using agarose gel electrophoresis
(Fig. 4a and b). The amount of the 398 bp product was
decreased by 80% in the presence of the non-caged PNA (120
times molar excess to the primer). The affinity of the primer
(15-mer, Tm = 36 °C) is almost identical to that of the competi-
tive 10-mer PNA (Tm = 36.7 °C). Therefore, a partial clamping
activity was observed. However, the amount of the amplified
product (398 bp) was restored to 90% when compound 15 was
added to the PCR mixtures. The same amount of reduction
was observed when a different primer that is not competitive
with the 10-mer PNA was used. Therefore, the 10% reduction
in the product amount is expected to be attributable to the
sequence-independent suppression of the amplification in the
presence of PNA molecules. Exposure to 350 nm UV light
restored the clamping activity of the 10-mer cPNA (15) by a
level comparable to that of the non-caged PNA (Fig. 4b). The
results indicate that the introduction of a single nucleobase-
caged cytosine enabled the suppression of sequence selective
inhibitory activity of the 10-mer PNA on a DNA polymerization
reaction.
To determine the extent of destabilization of a PNA/DNA
duplex from the introduction of a single Bmcmoc group,
the melting temperature (Tm) difference of 10-mer PNA/DNA
between the non-caged and the caged PNAs was measured. The
Tm value of the non-caged 10-mer PNA (H2N-TAGCTGTTTC-
COOH) and its complementary DNA was 36.7 °C. The stability
of the duplex was decreased by 7.7 °C for the 10-mer cPNA (15)/
DNA (Tm = 29.0 °C). This decrease is consistent with the fact
that introduction of a single mismatch caused a decrease in Tm
value by 8–15 °C in PNA/DNA duplexes.20 The results show that
the nucleobase-caged Bmcmoc-cytosine corresponded to a mis-
matched base. Therefore complete suppression of PNA/DNA
duplex formation was not achieved by the introduction of
a single Bmcmoc group on a nucleobase. After exposure to
350 nm light (4 mJ s−1, 120 s), an increase of Tm value (33.2 °C)
observed in a 15–DNA mixture confirmed photo-deprotection of
the Bmcmoc group to give the parent 10-mer PNA.
We hypothesized that nucleobase-caged PNAs having a
single Bmcmoc caging group are useful as caged PNAs in
applications in which a smaller affinity difference in PNA/DNA
duplexes between an intact PNA and a cPNA would be ampli-
fied or integrated. To test this hypothesis, we conducted a
study using PCR clamping. PNA-mediated PCR clamping is
achieved by annealing of a PNA to its complementary template
DNA and inhibition of primer extension reactions. The
method is based on the higher selectivity and specificity of
duplex formation between complementary PNA and DNA than
those of a DNA/DNA duplex. Therefore, the residual inhibitory
Oligopyrimidinyl PNAs are known to interact with dsDNAs
having homopurine target sequences to form triple helixes,
conventional PNA–dsDNA Hoogsteen type triplexes, or triplex
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invasion (PNA)2DNA complexes involving an unbound DNA
strand in the P-loop structure.18a Wang et al. reported that
homopyrimidine PNAs longer than 12-mer formed triplex inva-
sion complexes with dsDNAs and induced transcription
initiation both in vitro and in human cultured cells.18b The
triplex invasion complex includes combined Hoogsteen and
Watson–Crick type base pairings. Therefore, the affinity differ-
ence between the intact PNAs and the cPNAs to their comp-
lementary dsDNA targets would be integrated and enhanced.
To test this hypothesis, the effects of the single Bmcmoc group
were investigated in the reported triplex invasion complex
between the 16-mer PNA and the 50-mer dsDNA.18b The 16-
mer PNA (H2N-TTCTCTTCCTTCTCTT-CO2H) or the 16-mer
cPNA (16) was mixed with the 50-mer dsDNA having a PNA
binding sequence (AAGAGAAGGAAGAGAA). Results were ana-
lyzed using polyacrylamide gel electrophoresis. Two slower-
migrating bands appeared at the expense of the dsDNA (D in
Fig. 4c) when more than five-fold molar excess of the 16-mer
PNA (1 μM) was used (lanes 3 and 4 in Fig. 4c). Incubation was
carried out in low ionic strength solutions. Therefore, the
intense band should be assigned as the triplex invasion
complex (I) containing two PNA molecules.21 Another band
must be the conventional PNA–dsDNA triplex (T). However, for-
mation of the triplex invasion complex was markedly sup-
pressed when the dsDNA was incubated with 1 μM of the
16-mer cPNA (lane 7 in Fig. 4c). The use of ten-fold excess of the
cPNA (2 μM) causes the formation of a substantial amount of
the triplex and the invasion complex. These results indicate that
the 16-mer cPNA having a single Bmcmoc-caged cytosine base
can be used as a caged compound of triplex invasion forming
PNA when an appropriate amount of the compound is used.
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