Synthesis of a novel pyrrolidine-based peptide nucleic acid that contains tertiary amines in the main chain and its internalization into cells

Article abstract of DOI:10.1246/cl.2006.300

A novel thymine monomer of pyrrolidine-based peptide nucleic acid that contains tertiary amines in the main chain (pyrrolidine-based amino peptide nucleic acid = PAPNA) was synthesized. A mixed oligomer of two PAPNA(T) units and seven units of pyrrolidine-based oxypeptide nucleic acid (POPNA), was synthesized by a solid-phase method. The mixed oligomer was internalized into CHO cells more readily than the previous version of POPNA oligomers. Copyright

Full text of DOI:10.1246/cl.2006.300

300
Chemistry Letters Vol.35, No.3 (2006)
Synthesis of a Novel Pyrrolidine-based Peptide Nucleic Acid that Contains Tertiary Amines
in the Main Chain and Its Internalization into Cells
Mizuki Kitamatsu,ꢀ Tomoko Kashiwagi, Rino Matsuzaki, and Masahiko Sisidoꢀ
Department of Bioscience and Biotechnology, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530
(Received December 9, 2005; CL-051515; E-mail: sisido@cc.okayama-u.ac.jp)
OH
N
OH
OH
N
A novel thymine monomer of pyrrolidine-based peptide
nucleic acid that contains tertiary amines in the main chain (pyr-
rolidine-based amino peptide nucleic acid = PAPNA) was syn-
thesized. A mixed oligomer of two PAPNA(T) units and seven
units of pyrrolidine-based oxypeptide nucleic acid (POPNA),
was synthesized by a solid-phase method. The mixed oligomer
was internalized into CHO cells more readily than the previous
version of POPNA oligomers.
ii
iii
iv
N
COOH
COOEt
COOEt
R
Boc
Boc
3
4
1
2
R = H
i
R = Boc
OTBDPS
N
OTBDPS
OTBDPS
v
vii
N
COOtBu
R
N
N
COOEt
Boc
Boc
Boc
5
R = OH
R = Br
6
7
8
vi
Peptide nucleic acids developed by Nielsen et al. (PNA)
form more stable duplexes with the complementary nucleic acids
than the corresponding hybrids between nucleic acids.1 Thus, the
PNAs are expected to work as effective antisense and antigene
drugs. However, water solubility of PNA is not high enough
for their practical applications. To improve water solubility,
we developed an oxypeptide nucleic acid (OPNA) that contains
ether linkages in the main chain.2 The ether linkages also afford-
ed high flexibility in the main chain and caused sharp melting
curves with DNAs. However, OPNA did not show high affinity
to RNAs. Recently, we reported a conformationally restricted
version of OPNA that possesses a pyrrolidine ring in a monomer
unit (pyrrolidine-based oxypeptide nucleic acids = POPNAs).3
The two chiral centers on the pyrrolidine ring gave an opportu-
nity to optimize the configuration for hybridization with DNAs
and RNAs. Of the four stereoisomeric POPNAs, the trans-L-con-
figurated POPNA (Scheme 1, left) formed the most stable duplex
with RNA. Here, we report an attempt to provide the POPNA
with an ability of internalization into cells by introducing tertiary
amino groups into the main chain.
TBz
OH
viii
ix
x
N
COOtBu
N
COOtBu
N
N
Boc
Boc
9
10
T
Solid-phase peptide
sysnthesis
a
a
N
COOH
H-T GG T GC GAA-Lys-NH
N
2
Fmoc
11 (Ta
)
Figure 1. Synthetic route of Fmoc-protected thymine monomer
of trans-L-PAPNA (Ta) and the sequence of PAPNA-containing
oligomer. i) Boc2O, NaHCO3 in water:dioxane (1:1), overnight
(95%); ii) a) DCHA in AcOEt, b) EtBr in DMF, overnight
(94%); iii) a) formic acid, DEAD, Ph3P in THF, overnight, b)
25% NH3 aq in MeOH, 6 h (98%); iv) TBDPS–Cl, imidazole,
DIEA in DMF, overnight (77%); v) NaBH4 in EtOH, overnight
(70%); vi) CBr4, Ph3P in THF, 3 h (85%); vii) H–Sar–OtBu,
Na2CO3 in DMF, 75 ꢁC, 2 days (56%); viii) TBAF in THF,
4 h (99%); ix) N3–Bz–Thymine, DEAD, Ph3P in THF, overnight
(crude); x) a) 30% HBr in AcOH, 30 min, b) Fmoc–OSu,
NaHCO3 in water:MeCN (1:1), overnight (60% at 2 steps).
The structure of a tertiary amine-containing pyrrolidine-
based peptide nucleic acid (PAPNA) is shown in Scheme 1
(right). At the physiological pH (7.4), the tertiary amino groups
will be protonated and the cationic charges will facilitate the
internalization into cells. Cell penetrating peptides (CPP)–PNA
conjugates have been commonly used for the internalization.4
However, the positively charged CPP will cause non-specific
binding to nucleic acids. In this work, POPNAs containing the
least number of cationic charges are designed to achieve cell
internalization without scarifying their hybridization property.
The synthetic route of trans-L-PAPNA carrying a thymine
base 11 is shown in Figure 1. First, a secondary amine of
trans-L-hydroxyproline (1) was protected with a Boc group.
The product 2 was reacted with ethyl bromide to give the corre-
sponding ethyl ester 3. Then, the configuration of the hydroxy
group of the 4th position on the pyrrolidine ring was reversed
by the reaction with formic acid with DEAD, followed by the
treatment with aqueous ammonia (4). The hydroxy group of
4 was then protected with a tert-butyldiphenylsilyl (TBDPS)
group (5). Reduction of cis-L-configurated compound 5 with
NaBH4 resulted in the corresponding alcohol 6. Then, the latter
was treated with CBr4 to give the bromide 7. Nucleophilic sub-
stitution of 7 with sarcosine tert-butyl ester gave the correspond-
ing tertiary amine 8. Removal of the TBDPS group with tetrabu-
tylammonium fluoride (TBAF) gave a cis-L-configurated inter-
midiate 9. The intermediate 9 was obtained in an overall yield
22% from 1.
The alcohol 9 was reacted with N3-benzoylthymine under
standard Mitsunobu conditions to give compound 10. Then,
compound 10 was treated with 30% HBr in acetic acid to remove
the Boc, tert-butyl ester, and the N3-benzoyl group. The amino
group of free compound was protected again with an Fmoc
group to give the final product 11 in an overall yield of 60% from
the key intermediate. The new compound 11 was characterized
Base
N
Base
N
O
O
trans-L-POPNA
trans-L-PAPNA
O
N
Scheme 1. Chemical structures of monomer units of trans-L-
POPNA (left) and trans-L-PAPNA (right).
Copyright ꢀ 2006 The Chemical Society of Japan

Chemistry Letters Vol.35, No.3 (2006)
301
Figure 3. Confocal microscopy images of CHO cells cultured
for 6 h in the presence of POPNA 9-mer (left) and PAPNA 9-
mer (right). [PAPNA] = [POPNA] = 10 mM.
Figure 2. Temperature dependence of the absorption at 260 nm
for equimolar mixtures of PAPNA 9-mer and the complementa-
ry DNA (dashed line), and that for POPNA 9-mer and the com-
plementary DNA (solid line). [POPNA] = [PAPNA] = [DNA]
= 5.0 mM in phosphate buffer (10 mM NaH2PO4, 0.1 M EDTA,
100 mM NaCl, pH 7.0).
In summary, we successfully synthesized a novel peptide
nucleic acid unit that carries a cationic charge, PAPNA. A 9-
mer POPNA oligomer containing two PAPNA thymine units
formed stable hybrid with DNA as well as a noncationic POPNA
9-mer. The cationic oligomer was effectively internalized into
CHO cells.
by NMR (single species) and high resolution mass spectrometry
(calcd ðM þ HÞþ ¼ 519:2165, obsd. ðM þ HÞþ ¼ 519:2255).
A mixed 9-mer POPNA oligomer containing two thymine
PAPNA units (PAPNA 9-mer) was prepared by a solid-phase
peptide synthesis (Fmoc/HATU). The base sequence was H–
TaGG TaGC GAA–Lys–NH2, where Ta indicates the PAPNA
thymine monomer and G, C, and A indicates the corresponding
POPNA monomers. As a noncharged control compound, a 9-mer
POPNA oligomer without the PAPNA unit (POPNA 9-mer, H–
TGG TGC GAA–Lys–NH2) was also prepared. The crude
oligomers were purified by preparative HPLC to single peaks
and the purified oligomers were identified by MALDI-TOF
mass spectroscopy (PAPNA 9-mer, calcd ðM þ HÞþ ¼ 2661:19,
References and Notes
1
P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science
1991, 254, 1497.
2
a) M. Kuwahara, M. Arimitsu, M. Sisido, J. Am. Chem. Soc.
1999, 121, 256. b) M. Kuwahara, M. Arimitsu, M. Sisido,
Tetrahedron 1999, 55, 10067. c) M. Kuwahara, M. Arimitsu,
M. Shigeyasu, N. Saeki, M. Sisido, J. Am. Chem. Soc. 2001,
123, 4653.
3
4
a) M. Shigeyasu, M. Kuwahara, M. Sisido, T. Ishikawa,
Chem. Lett. 2000, 634. b) M. Kitamatsu, M. Shigeyasu,
T. Okada, M. Sisido, Chem. Commun. 2004, 1208. c) M.
Kitamatsu, M. Shigeyasu, M. Saitoh, M. Sisido, Biopoly-
mers, Peptide Sci., in press.
a) U. Koppelfus, S. K. Awasthi, V. Zachar, H. U. Holst, P.
Ebbesen, P. E. Nielsen, Antisense Nucleic Acid Drug Dev.
2002, 12, 51. b) R. Villa, M. Folini, S. Lualdi, S. Veronese,
M. G. Daidone, N. Zaffaroni, FEBS Lett. 2000, 473, 241.
c) P. Sazani, S. H. Kang, M. A. Maier, C. Wei, J. Dillman,
J. Summerton, M. Manoharan, R. Kole, Nucleic Acids Res.
2001, 29, 3965.
obsd. ðM þ HÞþ ¼ 2659:39; POPNA 9-mer, calcd ðM þ HÞþ
2635:13, obsd. ðM þ HÞþ ¼ 2634:26).
¼
UV melting curve of an equimolar mixture of the PAPNA
9-mer (TaGG TaGC GAA) and the complementary DNA
showed the melting temperature (Tm), 25.8 ꢁC (Figure 2). The
Tm was a little lower than that of the POPNA 9-mer (TGG
TGC GAA) measured under the same conditions, 26.8 ꢁC. Thus,
the incorporation of PAPNA units into POPNA oligomer did not
largely affect the stability of POPNA–DNA hybrid.
5
CHO cells were cultured in Dulbecco’s modified Eagle’s
medium. The medium was supplemented with penicillin/
streptomycin (10 mg/mL) and 10% fetal bovine serum.
The cells were incubated at 37 ꢁC under 5% CO2 to give
ca. 70% confluence. Then, subculture was performed on 35
mm glass-based dishes that were coated with poly(L-lysine).
The cells were incubated at 37 ꢁC under 5% CO2 until 40–
60% confluence. Before the cellular uptake, the cells were
incubated at 37 ꢁC for 6 h on a fresh medium containing
FAM-labeled PAPNA 9-mer or FAM-labeled POPNA 9-
mer. The final concentration of each peptide in the medium
was 10 mM. The cells were then washed three times with
PBS and examined on a confocal laser-scanning microscope
without fixation.
The internalization of the PAPNA oligomer into CHO cells
was examined on a confocal laser-scanning microscopy.5 The
fluorescence image of the CHO cells after equilibration with
the FAM-labeled PAPNA oligomer6 is shown in Figure 3 (right).
The oligomer was successfully taken up by the CHO cells. The
intracellular location of the PAPNA was mostly confined in
small vesicular compartments, but some fraction seems to be
spread out from the vesicles. On the other hand, no internaliza-
tion was observed for the FAM-labeled POPNA 9-mer under the
same conditions (Figure 3, left). These results indicate that cat-
ionic PAPNA units in the oligomer facilitate the internalization
into CHO cells. Internalization has been reported for peptide nu-
cleic acids that carry cationic sequences like cell penetrating
peptides.4 However, since the long cationic peptides may cause
nonspecific binding to DNAs and RNAs, an alternative method
has to be searched for. This work indicates that the solubility-
improved version of peptide nucleic acids can be internalized
into cells by attaching the least number of cationic charges.
6
The PAPNA oligomer was labeled with FAM–OSu after it
was isolated from the resin. Thus, the FAM probe may be
covalently attached on either the N-terminal of the Lys unit
at the C-terminal (MALDI-TOF mass spectroscopy: calcd
ðM þ HÞþ ¼ 3019:50, obsd. ðM þ HÞþ ¼ 3019:58).
Published on the web (Advance View) February 11, 2006; DOI 10.1246/cl.2006.300