Synthesis and hybridization property of novel 2′,5′-isoDNA mimic chiral peptide nucleic acids

Article abstract of DOI:10.1016/S0960-894X(03)00075-1

2′,5′-isoDNA mimic chiral peptide nucleic acid (isoPNA) monomers derived from D- and L-aspartic acids were synthesized. These novel monomers were incorporated in aminoethylglycine peptide nucleic acid (aegPNA) thymine dodecamers, and the hybridization pro

Full text of DOI:10.1016/S0960-894X(03)00075-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1041–1043
Synthesis and Hybridization Property of Novel 20,50-isoDNA
Mimic Chiral Peptide Nucleic Acids
Mohamed Abdel-Aziz, Tetsuo Yamasaki* and Masami Otsuka
Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan
Received 12 December 2002;accepted 16 January 2003
Abstract—20,50-isoDNA mimic chiral peptide nucleic acid (isoPNA) monomers derived from d- and l-aspartic acids were synthe-
sized. These novel monomers were incorporated in aminoethylglycine peptide nucleic acid (aegPNA) thymine dodecamers, and the
hybridization properties to RNA and DNA were demonstrated by UV thermal denaturation.
# 2003 Elsevier Science Ltd. All rights reserved.
A large variety of structural modifications of oligonu-
cleotides have been investigated as an exciting new con-
cept for drug design to modulate the expression of
genetic information by antisense and antigene oligonu-
cleotides. Aminoethylglycine peptide nucleic acids
(aegPNAs) developed by Nielsen and co-workers are
oligonucleotide analogues in which the sugar phosphate
backbone is replaced by a peptide chain.1 PNA is a very
potent DNA mimic capable of hybridizing to com-
plementary DNA, RNA or PNA. Therefore many kinds
of nucleotides with peptide backbone have been syn-
thesized in order to improve the limited solubility and to
investigate the structural flexibility and requirements for
nucleic acid recognition (by, e.g., introduction of a cer-
tain side chain2 and double bond3 to peptide backbones,
conversion of carbonyl group to sulfonyl group,4 and
construction of ethereal and/or cyclic structural peptide
backbones5). However, there is no report on the peptide
nucleic acids corresponding to the ‘non-genetic’ 20,50-
linked oligonucleotide (20,50-isoDNA),6 which is one of
the important candidates to search the effective anti-
sense oligonucleotides due to the intrinsic selective
RNA-binding activity of 20,50-isoDNA (although the
hybridization affinity of 2’,5’-DNA or RNA/30,50-RNA
is somewhat inferior to that of the 30,50-DNA/30,50-
RNA duplex, 20,50-DNA or RNA shows binding selec-
tivity for 30,50-RNA over 30,50-ssDNA). The favorable
RNA- and DNA-binding properties of PNA and the
selective binding activity of 20,50-isoDNA to RNA have
led us to design 20,50-isoDNA mimic chiral peptide
nucleic acids (isoPNAs) (Fig. 1). In this letter, we report
on the synthesis and hybridization properties of PNAs
containing novel isogaPNA monomers 4a and 4b with
glycyl-b-alanine backbone derived from d- and
l-aspartic acid.
The synthesis of glycyl-b-alanine isoPNA monomers
proceeds through hydroxymethyl-b-alanine 1a,b,7 which
were prepared from d- or l-aspartic acids protected
with Boc and benzyl groups by reaction with ethyl
chloroformate followed by NaBH4 reduction in 71 and
69% yields, respectively. As shown in Scheme 1,
hydroxymethylalanine 1a,b was treated with 3-ben-
zoylthymine under Mitsunobu conditions to give thy-
minylmethyl-b-alanine 2a,b.8 After removal of Boc
group of 2a,b by the addition of 3 N HCl, coupling with
FmocGly in the presence of TBTU followed by depro-
tection of benzyl group of 3a,b with 10% Pd/C and
1,4-cyclohexadiene provided thymine isogaPNA mono-
mer TR and TS (4a,b).9
In order to assess the hybridization properties of these
monomers to complementary DNA and RNA, the two
monomeres 4a and 4b were individually introduced into
aegPNA sequences in different positions to form differ-
ent thymine dodecamers. The preparation of these oli-
gomers was carried out using automated synthesis on an
Expedite 8909 (Applied Biosystems, Foster City, CA,
USA) and standard Fmoc-chemistry on Fmoc-XAL
PEG-PS resin. l-Lysine was introduced at the C-termi-
nus of the PNAs to reduce their self-aggregation and to
ensure adequate solubility in water. The monomers were
*Corresponding author. Fax: +81-96-371-4624;e-mail: tyamasak@
gpo.kumamoto-u.ac.jp
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00075-1

1042
M. Abdel-Aziz et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1041–1043
rechecked by HPLC and characterized by MALDI-
TOF mass spectrometry.
PNA sequences
5
6
H-T12-Lys-NH2
DNA T12
7
8
9
H-T11-TR-Lys-NH2
H-TR-T11-Lys-NH2
H-T9-TRTRTR-Lys-NH2
H-TRTRTR-T9-Lys-NH2
H-T6-TR-T5-Lys-NH2
H-TR12-Lys-NH2
H-T11-TS-Lys-NH2
H-TS-T11-Lys-NH2
H-T9-TSTSTS-Lys-NH2
H-TSTSTS-T9-Lys-NH2
H-T6-TS-T5-Lys-NH2
H-TS12-Lys-NH2
Figure 1. Design of 20,50-isoDNA mimic peptide nucleic acids (iso-
PNA).
10
11
12
13
14
15
16
17
18
T=thymine aegPNA monomer; TR=thymine isoga-
PNA monomer (4a); TS=thymine isogaPNA monomer
(4b).
The hybridization properties of PNA oligomers 7–18
with complementary DNA and RNA were investigated
in UV-melting experiments (260 nm) and the results of
these studies are shown in Table 1. The aegPNA T12Lys
control strand (5) gave Tm values of 65 and 73 ꢀC when
hybridized to dA12 and rA12, respectively. Destabiliza-
tion was observed upon incorporation of one and three
isoagPNA TR into the decamers (7–11), resulting in sig-
nificant close Tm values (ꢀTm 2–3 ꢀC) observed between
the PNA 7–11 DNA and the corresponding PNA 7–11
RNA, contrary to the characters that aegPNA and iso-
DNA hybridize with RNA preferentially. The complexes
Table 1. Melting temperature (Tm values) of complexes between
PNAs 5–16 and complementary DNA and RNA
Compd
Tm (ꢀC)a
Complex with dA12
Complex with rA12
5
6
7
8
65
32
65
65
73
27
67
67
67
62
56
nd
67
68
63
64
53
nd
Scheme 1. Synthesis of thymine isogaPNA monomers TR and TS (4a
and 4b): (a) DEAD (1.1 equiv), Ph3P (1.1 equiv), 3-benzoylthymine
(1 equiv), THF, À10 ꢀC, 45% (2a), 44% (2b);(b) (i) 3 N HCl, EtOAc;
(ii) FmocGly (1 equiv), TBTU (1 equiv), HoBt (1 equiv), DIEA
(2 equiv), DMF, 49% (3a), 50% (3b);(c) 1,4-cyclohexadiene
(20 equiv), 10% Pd/C, MeOH, 65% (4a), 62% (4b).
9
60
10
11
12
13
14
15
16
17
18
59 (50)b
53 (44)c
ndd
61
64
56
58 (56)b
46 (39)c
nd
coupled by activation with HATU in DMF/NMP (1/1).
After the final coupling, Fmoc-group was removed by
20% piperidine in DMF and cleavage of the PNAs from
the resin was performed using TFA/m-cresol (4/1). The
crude products were purified by C18-reverse-phase HPLC
using gradients of 0.1% TFA in water and 0.1% TFA in
acetonitrile. Also, complete isogaPNA dodecamers, which
consist of only 4a and 4b, were prepared in the same
manner, respectively. The purity of the oligomers was
aThe Tm was measured at a ratio of PNA/DNA or RNA=1:1, con-
centration of PNA strand=2.5 mM, 10 mM phosphate buffer, pH 7.4,
heating rate 0.5 ꢀC/min. All of these oligomers can be dissolved easily
in this buffer.
bThe values with triple mismatching DNA A9T3.
cThe values with single mismatching DNA A5TA6.
dnd, Not detected.

M. Abdel-Aziz et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1041–1043
1043
References and Notes
1. (a) Nielsen, P. E.;Egholm, M.;Berg, R. M.;Buchardt, O.
Science 1991, 254, 1497. (b) Egholm, M.;Buchardt, O.;Niel-
sen, P. E.;Berg, R. H. J. Am. Chem. Soc. 1992, 114, 1895. (c)
Egholm, M.;Nielsen, P. E.;Buchardt, O.;Berg, R. H. J. Am.
Chem. Soc. 1992, 114, 9677. (d) Egholm, M.;Buchardt, O.;
Christensen, L.;Behrens, C.;Freier, S. M.;Driver, D. A.;
Berg, R. H.;Kim, S. K.;Norden, B.;Nielsen, P. E.
1993, 365, 566. (e) Hyrup, B.;Egholm, M.;Nielsen, P. E.;
Witlung, P.;Norden, B.;Buchardt, O. J. Am. Chem. Soc.
Nature
1994, 116, 7964. (f) Hyrup, B.;Nielsen, P. E. Bioorg. Med.
Chem. 1996, 4, 5.
2. (a) Stammers, T. A.;Burk, M. J. Tetrahedron Lett. 1999,
40, 3325. (b) Falkiewicz, B.;Kowalska, K.;Kolodziejczyk,
A. S.;Winsniewski, K.;Lankiewicz, L. Nucleosides Nucleo-
tides 1999, 18, 353. (c) Sforza, S.;Corradini, R.;Ghirardi, S.;
Dossena, A.;Marchelli, R. Eur. J. Org. Chem. 2000, 2905.
3. Roberts, C. D.;Schutz, R.;Leumann, C. J. Synlett 1999, 819.
4. Liu, Y.;Hudson, R. H. E. Synlett 2001, 1626.
Figure 2. Job plots of absorbance at 25 ꢀC for 11 and dA12 at 260 nm.
The following extinction coefficients were used: T=8.6 mL/mmol cm
for PNA, A=15.3 mL/mmol cm, [11] and [dA12]=3.0 mM, 10 mM
phosphate buffer, pH 7.4.
5. (a) Kuwahara, M.;Arimitsu, M.;Sisido, M. J. Am. Chem.
Soc. 1999, 121, 256. (b) D’Costa, M.;Kumar, V.;Ganesh,
K. N. Org. Lett. 1999, 1, 1513. (c) Altmann, K.;Husken, D.;
Cuenoud, B.;Garcia-Echeverria, C. Bioorg. Med. Chem. Lett.
2000, 10, 929. (d) Shigeyuki, M.;Kuwahara, M.;Sisido, M.;
Ishikawa, T. Chem. Lett. 2001, 634.
6. Sheppard, T. L.;Breslow, R. C. J. Am. Chem. Soc. 1996,
118, 9810.
7. Hydroxymethyl-b-alanine 1a,b were obtained by reduction
of carboxyl groups of d- and l-BocAsp-(Bn)OH in analogy
to: Kokotos, G. Synthesis 1990, 299.
between 13–17 incorporated of isogaPNA TS and dA12
showed decreased Tm values (ꢀTm 1–7 ꢀC) compared to
the corresponding Tm values between PNA 7–11 incor-
porated TR and DNA dA12. Unfortunately, no hybridi-
zation was found for the complexes of complete
isogaPNA 12 and 18 with DNA and RNA, respectively.
Introduction of mismatch base T to dA12 at the position
to hybridize with isogaPNA TR and TS resulted in
decrease of the Tm (ꢀTm 9 ꢀC for 10 and 11, ꢀTm 2 ꢀC
and 7 ꢀC for 16 and 17 respectively). These results show
the conformation of backbone of TR is more effective
sterically than that of TS. Furthermore, the binding
stoichiometry was found by UV titration (Job-plot)10 to
be 2:1 (entry 11 vs dA12) indicating a PNA2-DNA tri-
plex structure (Fig. 2).
8. Diederichsen et al. reported adenine-adenine self-pairing of
b-homoalanyl PNAs, not binding activities with nucleic acids
Diederichsen, U.;Schmitt, H. W. Angew. Chem., Int. Ed. 1998,
37, 302.
9. 4a: mp 144 ꢀC; ꢀmax=cmÀ1 1714, 1638 (C¼O); dH (300 MHz,
DMSO-d6) 1.75 (3H, s, CH3), 2.35–2.46 (2H, m, CH2), 3.30
(1H, br, NH), 3.39–3.62 (2H, m, CH2), 3.83–4.01 (2H, m,
CH2), 4.21–4.27 (3H, m, CH and CH2), 4.40–4.56 (1H, m,
CH), 7.23 (1H, s, CH-6 of T), 7.32 (2H, dd, J 7.5 and 7.5, Ar–
H), 7.40 (2H, dd, J 7.5 and 7.5, Ar–H), 7.71 (2H, d, J 7.5, Ar–
H), 7.87 (2H, d, J 7.5, Ar–H), 10.81 (1H, brs, NH), 12.14 (1H,
br, COOH); dC (75 MHz, DMSO-d6) 12.48 (CH3), 37.03
(CH2), 42.72 (CH2), 43.39 (CH2), 44.55 (CH), 46.63 (CH),
65.74 (CH2), 107.13 (C ), 120.06 (CH), 125.27 (CH), 127.06
(CH), 127.60 (CH), 136.28 (CH), 140.69 (C), 143.85 (C),
151.54 (C), 156.30 (C), 164.14 (C), 168.48 (C), 171.94 (C);
FAB-MAS: m/z 507 (M+1).
The decreased Tm values compared to unmodified
aegPNA oligomer indicate that the glycylalanine back-
bone of isogaPNA may not be optimal length to hybri-
dize with DNA and RNA, and work is in progress to
examine isoPNA with different backbone.
Acknowledgements
10. (a) Nielsen, P. E.;Egholm, M., Eds. PNA-protocol and
Applications;Horizon Scientific: 1999. (b) Puglisi, J. D.;
Tinoco, I., Jr. Methods in Enzymol. 1989, 180, 304,
respectively.
The authors would like to thank Professor Shogo Mis-
umi of Kumamoto university for the measurement of
MALDI-TOF mass spectra.