Synthesis of peptide nucleic acid dimer containing modified cytosine

Full text of DOI:10.1002/bkcs.11340

Communication
DOI: 10.1002/bkcs.11340
BULLETIN OF THE
Y. D. Lee et al.
KOREAN CHEMICAL SOCIETY
Synthesis of Peptide Nucleic Acid Dimer Containing Modified Cytosine
Yeong Deok Lee,† Kyung Sun Yoon,‡ and Keun Ho Chun†,
*
†Department of Chemistry, Soongsil University, Seoul 06978, Republic of Korea.
*E-mail: kchun@ssu.ac.kr
‡Department of Information Communication, Materials, and Chemistry Convergence Technology,
Soongsil University, Seoul 06978, Republic of Korea
Received August 28, 2017, Accepted November 4, 2017, Published online December 21, 2017
Keywords: Peptide nucleic acid, Peptide nucleic acid dimer, Modified cytosine, Peptide synthesis
Peptide nucleic acid (PNA) has been widely used in the devel-
opment of antisense drugs and gene diagnosis sensors since it
was released by Neilson et al. in 1991.1 Although PNA has
excellent binding ability with natural DNA or RNA, it has been
pointed out that it is difficult to be synthesized in a large scale,
and has low cell membrane permeability. To overcome these
problems, new synthetic methodologies and development of
various derivatives have been proposed. Most of these deriva-
tives were designed to increase complementary binding capac-
ity and cell permeability by introducing a new functional group
into the peptide backbone or modifying DNA bases.2 Recently
Israel researchers have found that a PNA dimer forms a specific
quaternary structure by self-replication of bases at a specific pH
and exhibits a unique fluorescence property, which can be used
as an organic light-emitting diode (OLED) device. This result
is significant in that PNA has a new application for an elec-
tronic material in addition to the biological field.3
To further investigate the applicability of PNA as an elec-
tronic material, we tried to make a PNA dimer with a modified
cytosine base (C*, Figure 1). The C* was designed to have
strong complementary bonds by G-clamp formation.4 There-
fore, we aimed to investigate the effect of modified cytosine on
the self-replication pattern and fluorescence properties. For this
purpose, we needed to make C*G and GC* PNA dimers.
Modified base C* could be synthesized through the method
described in the literature.5
fmoc protecting group is removed during peptide coupling or
final deprotection of fmoc group (Figure 2(a)).6 In addition,
unlike natural C, it was found the PNA monomer containing
C* showed significantly reduced reactivity during peptide cou-
pling step with G-monomer. Therefore, in our synthesis, mono-
methoxy trityl (MMTr) was chosen for the N-terminal amine
protection of the PNA monomer because MMTr could be
selectively removed at the weak acidity.
MMTr protection is not often used for PNA oligomer syn-
thesis, but acyl migration is rarely observed in the deprotec-
tion step, and also it can be selectively removed in the
presence of the Boc protecting groups, which were already
existing in C*, G bases. Starting from ethylene diamine,
compound 6 was synthesized in three steps. C* or G base
could be coupled with 6 by EDC coupling, and hydrolysis
of ester group gave C* or G monomer, respectively (9a, 9b)
in which N-terminal was protected with MMTr (Scheme 1).
However, the direct coupling between the PNA mono-
mer 9a or 9b and other N-deprotected PNA monomers also
showed severe acyl migration as a side reaction (Figure 2
(b)). To prevent such acyl migration, we first prepared a PNA
unit 11, which had no DNA base attached and tried to couple
Typical PNA oligomer synthesis is performed by solid phase
peptide synthesis using fluorenylmethyloxycarbonyl (fmoc)
resin. However, the synthesis of PNA dimer using PNA mono-
mers protected by fmoc group in the N-terminal amine is very
likely to occur acyl migration, a fatal side reaction, when the
Figure 1. Modified cytosine–guanine complex.
Figure 2. Acyl migration of PNA.
Bull. Korean Chem. Soc. 2018, Vol. 39, 10–11
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BULLETIN OF THE
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Scheme 3. Synthesis of C*G and GC* PNA dimers. Reagent and
conditions: (i) 9a–b, NMM, ClCO2iBu, CH2Cl2, 11, 60 min,
Scheme 1. Synthesis of C* and G PNA monomer. Reagent and
conditions: (i) chloroacetic acid, 22 h, 96%; (ii) HCl, MeOH,
reflux 3 h, 98%; (iii) TEA, MmtCl, CH2Cl2 65%; (iv) 7a or 7b,
EDC, HOBt, CH2Cl2, 6, 22 h, 91%; (v) 8a or 8b, 1 M LiOH, 1,4-
dioxane, 6 h, 95%.
91%; (ii) 20% piperidine, CH2Cl2, 60 min, 55%; (iii) C*(Boc) acid
diBoc)
or G(OBn,
acid, EDC, HOBt, CH2Cl2, 13a–b, 2 h, 94%;
(iv) 14a–b, 1 M LiOH, 1,4-dioxane, TFA/CH2Cl2, 92%.
improvement in the yield, and finally the desired C*G and
GC* PNA dimers could be synthesized effectively.
Scheme 2. Synthesis of PNA unit. Reagent and conditions:
(i) (Boc)2O, CH2Cl2, 22 h, 82%; (ii) ethyl bromoacetate, TEA,
CH3CN, 2 h, 55%; (iii) FmocCl, TEA, CH2Cl2, 30 min, 82%;
(iv) TFA/CH2Cl2, 1:1, 4 h, 99%.
Supporting Information. Additional supporting informa-
tion is available in the online version of this article.
11 with PNA monomer 9a or 9b (Scheme 2). Although cou-
pling of 11 with PNA monomer 9a or 9b can avoid acyl
migration, the protection group of sec-amine of 11 should be
carefully chosen. Without sec-amine protection, during the
coupling reaction with PNA monomer, side products are gen-
erated because sec- and pri-amine are competing.7
In the literature, as a protecting group for sec-amine, a rela-
tively rare and expensive protection group such as Alloc or
Troc group is generally used.8 However, in our synthesis, the
fmoc group could be used as the sec-amine protecting group
of 10. The fmoc protection of sec-amine in 10 was successful,
and coupling between 11 and PNA monomer 9a or 9b
showed the best results in the reaction with isobutyl chlorofor-
mate (Scheme 33).9 After the fmoc group of 12 was removed,
C* or G base was introduced by EDC coupling to yield 14.
The ethyl ester of the C-terminal and the MMTr of the N-
terminal were sequentially removed, and finally C*G and
GC* PNA dimers were effectively synthesized.
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In conclusion, the synthesis of PNA dimer containing
modified cytosine required a different synthetic strategy
than the conventional method in order to minimize the acyl
migration and improve the yield of coupling process
between PNA monomers. The N-terminal protection using
MMTr was successful. Adding the second base later after
attaching the PNA unit 11 first led to a significant
Bull. Korean Chem. Soc. 2018, Vol. 39, 10–11
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