Synthesis and characterization of flavin-tethered peptide nucleic acid

Article abstract of DOI:10.1016/S0040-4039(01)00228-3

We synthesized flavin-containing PNA monomer unit 5 from lumiflavin and prepared PNAs containing a flavin moiety (FPNA) by the standard tBoc chemistry. Each PNA oligomer was purified by reversed-phase HPLC and characterized by MALDI-TOF MS and UV spectra. Thermodynamic analyses indicated that the PNA oligomer containing a flavin moiety near the amino terminal considerably stabilized the PNA-DNA hybrids.

Full text of DOI:10.1016/S0040-4039(01)00228-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2529–2531
Synthesis and characterization of flavin-tethered peptide nucleic
acid
Hisafumi Ikeda,† Kohzo Yoshida, Makoto Ozeki and Isao Saito*
Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, CREST,
Japan Science and Technology Corporation, Yoshida, Sakyo, Kyoto 606-8501, Japan
Received 11 December 2000; revised 8 February 2001; accepted 9 February 2001
Abstract—We synthesized flavin-containing PNA monomer unit 5 from lumiflavin and prepared PNAs containing a flavin moiety
(FPNA) by the standard tBoc chemistry. Each PNA oligomer was purified by reversed-phase HPLC and characterized by
MALDI-TOF MS and UV spectra. Thermodynamic analyses indicated that the PNA oligomer containing a flavin moiety near
the amino terminal considerably stabilized the PNA–DNA hybrids. © 2001 Elsevier Science Ltd. All rights reserved.
PNA is an artificial biopolymer in which the DNA
sugar-phosphate backbone is replaced by a peptide
backbone.1 It has several advantages over natural
nucleic acids: (i) PNA oligomers can be easily synthe-
sized by solid-phase tBoc or Fmoc chemistry,2 (ii) are
extremely stable to cellular nucleases and proteases,3
and (iii) can hybridize with complementary DNA with
high affinity.4 However, it has been known that there is
a serious limitation to the uptake of PNA into cells.5
Modification of PNA backbones has been examined
directed toward the improvement of their binding to
DNA and/or RNA and of the membrane permeability.6
Simultaneously, several functionalized PNA oligomers,
which contain functional molecules in place of native
nucleobases, have been designed for the improvement
of water solubility and for the study on electron trans-
fer chemistry through a DNA duplex.7
DNA photolyase is well-known to photoconvert the
cyclobutane ring of a pyrimidine dimer directly to the
O
O
a
c
N
N
OR
N
N
NH
N
O
N
O
N
O
1: R = Et
2: R = H
Lumiflavin
b
F
N
N
O
O
H
N
F
F
F
N
N
O
O
N
H
OH
O
F
O
O
O
4
N
N
O
N
O
O
d
N
N
O
O
N
H
OH
3
5
Scheme 1. (a) Ethyl bromoacetate, potassium carbonate, DMF, rt, 2 days, 85%; (b) conc. HCl, reflux, 6 h, 99%; (c)
pentafluorophenol, EDCI, DMF, 0°C, 1 h then rt, 2 h, 85%; (d) 4, diisopropylethylamine, DMF, rt, 15 h, 96%.
* Corresponding author.
†
Current address: Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Science University of Tokyo,
2641 Yamazaki, Noda, Chiba 278–8510, Japan.
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00228-3

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H. Ikeda et al. / Tetrahedron Letters 42 (2001) 2529–2531
Figure 1. UV spectra of PNA1 and FPNA1. Concentration of single stranded PNA was 5 mM (strand concentration) in 50 mM
sodium cacodylate (pH 7.0). Bold F means flavin PNA unit.
two corresponding pyrimidines without the nucleotide
excision.8 Recently, flavins, which are chromophores of
DNA photolyases,8,9 have been investigated for the
mechanism of the DNA repair system.10 Also, an
remove water and TFA, and then the PNA oligomer
was further collected by ether precipitation. Each PNA
oligomer was characterized by MALDI-TOF MS
(Table 1)17 and UV spectra (Fig. 1). The yields of pure
PNA oligomers were moderate (40–60% from the
MBHA resin), but this synthetic procedure is suitable
for the preparation of a PNA oligomer in large
amounts.
attempt to synthesize
a
flavin-containing DNA
oligomer as an antisense molecule has been examined.11
However, flavins catalyze the conversion of phospho-
ramidite to phosphoramidate via intramolecular photo-
sensitized oxidation during the amidite synthesis,
implying that the synthesis of flavin-containing DNA
oligomers can be hardly synthesized by conventional
methods. We now wish to report herein the synthesis
and characterization of a flavin-tethered PNA.
The thermal stability of the hybrids of flavin-tethered
PNA (FPNA) with the complementary DNA oligomer
was examined by measuring their melting temperatures
(Table 1). FPNA3 containing a flavin moiety at the C
terminal was slightly more unstable than PNA1, while
FPNA2 containing a flavin incorporated in the interior
of the hybrid was less stable than PNA1. In addition,
FPNA1 containing the flavin moiety near the amino
terminal region slightly stabilized the PNA–DNA
hybrid. These results suggested that the flavin moiety,
which was introduced near the amino terminal region
rather than the C-terminal or the middle of the PNA
oligomer, can contribute to the stabilization of the
PNA–DNA hybrid.
Flavin-containing PNA monomer unit 5 was prepared
from lumiflavin (Scheme 1).12 The facile alkylation of
the N-amino group of lumiflavin with ethyl bromo-
acatate (1) followed by acid hydrolysis of the resulting
ethyl ester gave 2. Flavin is known to be easily decom-
posed by alkaline hydrolysis.13 PNA monomer 5 was
prepared from the active ester 3 and N-(2-Boc-
aminoethyl)glycine 4.14 It is possible to know whether a
flavin moiety is incorporated into PNA oligomers by
simply measuring their UV spectra because UV absorp-
tion of 5 at 390 and 460 nm is not overlapped with
those of any PNA nucleobases, as shown in Fig. 1.15
Table 1. MALDI-TOF MS17 of FPNA1ꢀ3 and melting
temperatures of PNA–DNA hybridsa
PNA
MALDI-TOF (M+H)+ Tm (°C)
Several PNA oligomers were synthesized by solid-phase
tBoc chemistry as previously described.16 A Ninhydrin
test showed that each coupling of PNA monomers with
MBHA resin using HBTU proceeded quantitatively.
After completion of PNA oligomer elongation, the
resin was treated with TFA/TFMSA/thioanisole/p-
cresol (6:2:1:1) for release of the PNA oligomer from
the resin and for deprotection of the carbobenzyloxy
and Boc groups. After the acid solution was filtered off
to remove the MBHA resin, the supernatant was
poured into ether, and then the precipitate was col-
lected by centrifugation. The residue was redissolved in
TFA, and the solution was reprecipitated in ether and
centrifuged to give a crude PNA oligomer. It was
further purified by reversed-phase HPLC on a Wakosil
II 5-ODS-AR column (30×150 mm) using acetonitrile/
water/TFA solvent system. The eluted product, which
was detected by UV at 260 nm, was evaporated to
Calcd
Found
PNA1
H-(GC TCT
TCC
CGC)-NH2
H-(GC TCT
TCC
CFC)-NH2
H-(GC TCT
FCC
CGC)-NH2
H-(FC TCT
TCC
2907.79
2906.70
59.3
61.3
50.2
55.5
FPNA1
FPNA2
FPNA3
3012.92
3037.44
3012.92
3012.85
3038.11
3012.95
CGC)-NH2
a Each FPNA was annealed with 5%-d(CG AGA AGG GCG)-3% (50
mM, strand concentration) in sodium cacodylate buffer (50 mM, pH
7.0). The sample was graduately heated from 2 to 90°C in 1°C
increments with 1 min equilibration. Bold F means flavin PNA unit.

H. Ikeda et al. / Tetrahedron Letters 42 (2001) 2529–2531
2531
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Figure 2. CD spectra of the hybrids of PNA1 (—), FPNA1
(···), FPNA2 (– – –), and FPNA3 (—) with 5%-d(CG AGA
AGG GCG)-3%.
The CD spectra of the hybrids of FPNA1-3 with com-
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significant difference between PNA1 and FPNA1-3
(Fig. 2). These results suggested that the hybrids of
flavin-tethered PNA oligomers can serve as a function-
alized antisense oligonucleotide.
In summary, we have synthesized for the first time
flavin-tethered PNAs and examined their thermody-
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DNA. Flavin-tethered PNA oligomers might be utilized
as functionalized antisense molecules. We are also
investigating whether flavin-conjugated PNA oligomers
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