Synthesis of anionic peptide nucleic acid oligomers including γ-carboxyethyl thymine monomers

Article abstract of DOI:10.1016/j.mencom.2015.01.017

Synthesis of a modified thymine monomer based on l-Glu, solid phase synthesis of anionic peptide nucleic acid (PNA) oligomers containing the modified monomer and optimization of the deblocking procedure and cleavage conditions of the PNA oligomers from resin are accomplished.

Full text of DOI:10.1016/j.mencom.2015.01.017

Mendeleev
Communications
Mendeleev Commun., 2015, 25, 47–48
Synthesis of anionic peptide nucleic acid oligomers
including g-carboxyethyl thymine monomers
Andrey V. Dezhenkov,a Maria V. Tankevich,a Elena D. Nikolskaya,a Igor P. Smirnov,b
Galina E. Pozmogova,b Vitaly I. Shvetsa and Yulia G. Kirillova*a
a M. V. Lomonosov Moscow State University of Fine Chemical Technologies, 119571 Moscow,
Russian Federation. Fax: +7 495 434 8233; e-mail: pna-mitht@yandex.ru
b Research Institute of Physical-Chemical Medicine, 119435 Moscow, Russian Federation
DOI: 10.1016/j.mencom.2015.01.017
Synthesis of a modified thymine monomer based on l-Glu, solid phase synthesis of anionic peptide nucleic acid (PNA) oligomers
containing the modified monomer and optimization of the deblocking procedure and cleavage conditions of the PNA oligomers from
resin are accomplished.
Peptide nucleic acids (PNAs)1 are used in wide range of bio-
medical technologies.2 The original aminoethylglycine (aeg) PNAs
(Figure 1, B) are achiral non-charged molecules, which are not
difficult to produce.3 The development of this trend produced the
numerous aeg-PNA modifications including chiral acyclic a- and
g-modifications (Figure 1, C).4 Such g-PNA oligomers have a
number of advantages, in particular, l-amino acid derivatives
are preorganizated into a right-handed helix, and more effective
and specific to complementary nucleic acids targets.5 However,
nowadays broad use of PNAs and their modifications has the
number of limitations, namely, relatively low solubility of the
aeg-PNAs and another non-charged chiral PNAs,6 obstacles with
The synthesis of aeg-PNA monomers was performed by known
methods.17 The preparation of g-modified thymine monomer 1
using condensation of thymin-1-ylacetic acid with secondary
amine 2 was described earlier.18 However, high tendency of the
latter to form side cyclic b-lactam19 makes us to use the alternative
strategy20–23 of alkylation of thymine with bromoacetyl derivative
3 (Scheme 1).† The latter was obtained from secondary amine
2
19 by the reaction with bromoacetyl bromide in the presence of
TEA. The analysis of 13C NMR spectrum of fully protected mono-
mer 4 showed that alkylation of thymine occurs regioselectively
at N1-position: the chemical shift of C5 atom of alkylated thymine
was 109.5 ppm and equal to that obtained by another synthetic
approach18 (in case of N3-alkylation, chemical shift of C5 atom is
107.9 ppm24). The target monomer 1 was produced by cleavage
of allyl protecting group under catalysis with [Pd(PPh3)4] in
morpholine as scavenger.25 The overall yield was 85% that is
higher than that in case of conventional synthetic route (65%).18
Tetramer ACAgꢀT (Figure S1, Online Supplementary Mate-
rials) was synthesized according to standard protocol26 with some
distinctions: capping of amino groups was carried out with Ac2O
instead of Rappoport’s reagent,27 3 monomer equiv. instead of
5 equiv. were used at one condensation stage, and the resin was
washed with DMF–DCM system instead of pyridine. Earlier we
showed28,29 for a-PNA synthesis that when standard deblocking
delivery of potential therapeutics PNA oligomers to cellular targets 7
,
as well efficiency and selectivity of molecular recognition of com-
plementary targets.4 Introducing g-monomers based on dicarboxyl
acids into PNA may solve the problems of PNA solubility, since
the presence of a carboxyl function makes modified PNA some-
what analogues to nucleic acids (Figure 1, A). Negative charge
enables to form stable complex with cationic transfectants such
as cell-penetrating peptides8 and lipid-like substances.9 S-Chiral
center at g-position effects the required preorganization of PNA
oligomer and, hence, more directed interaction with targets.10
Recently, the attention to negatively charged PNAs appeared
again11–14 10 years later publications devoted to phosphono-
PNA.15,16 The reported11 Fmoc-protocol for the synthesis of
oligomers including the thymine monomers based on l-Asp
provided good complexing of negatively charged PNAs at
medium and high concentrations of salts with nucleic acids
targets, especially RNA. Therefore, here we describe the synthesis
of PNA oligomers containing g-carboxyethyl thymine monomers
based on l-Glu following Boc-protocol, which is more convenient
in terms of monomer synthesis.
BnO
O
BnO
O
Br
BrCH2COBr
Thy
O
TEA, 0°C
74%
K2CO3
86%
O
O
H
N
N
BocHN
OAll
BocHN
OAll
2
3
O
N
O
N
Me
OH
Me
HN
HN
O
P
O
BnO
O
BnO
O
HO
O
O
[Pd(PPh3)4]
morpholine
O
B
O
B
O
B
O
O
O
O
O
O
94%
O
O
N
N
N
N
H
BocHN
O
BocHN
OAll
N
N
(OH)
n
n
4
1
H
H
Boc = ButOC(O)
A
B
C
B = Thy, Cyt, Ade, Gua
Scheme 1
Figure 1 Structures of DNA (RNA) A, aeg-PNA B and g-carboxyethyl-
PNA C.
†
For synthetic procedures, see Online Supplementary Materials.
© 2015 Mendeleev Communications. Published by ELSEVIER B.V.
on behalf of the N. D. Zelinsky Institute of Organic Chemistry of the
Russian Academy of Sciences.
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Mendeleev Commun., 2015, 25, 47–48
protocol is employed for cleavage of aegPNA (‘low-high
This work was supported by the Ministry of Education and
Science of the Russian Federation (grant no. 4.128.2014/K).
TfOH’30), the isomeric a-PNAs oligomers underwent degradation
to amide bonds, so it is preferably to use the deblocking cocktail
including scavengers based on trialkylsilanes for their cleavage
from the polymer support.
Online Supplementary Materials
Supplementary data associated with this article can be found
in the online version at doi:10.1016/j.mencom.2015.01.017.
The cleavage of tetramer ACAgꢀT was carried out using two
deblocking protocols: (I) the standard ‘low-high TfOH’ treat-
ment of resin with solution of ‘low TfOH’–TFA/m-cresol/DMS/
TfOH (11:2:6:1, by volume) for 15 min at 0°C and solution
‘high TfOH’–TFA/m-cresol/TfOH (8:1:1, by volume) for 20 min
at 0°C with preliminary cooling of resin to –30°C and (II)
mixture TFA/TfOH/Pri3SiH (3:1:0.1, by volume) for 45 min at
0°C with preliminary cooling of resin to –30°C.29 RP HPLC of
produced reaction mixture are presented in Figure 2. In case of
employing protocol II with Pri3SiH, the formation of side pro-
ducts was not observed. The yield of oligomer was 31% after
purification by preparative RP HPLC. The initial loading of MBHA
resin was 0.14 mmol g–1. It was 0.114 mmol g–1 after four con-
densation stages.31 Therefore, the average yield of condensation
cycle was 95% before the cleavage of oligomer from resin, which
is similar to that for aeg-PNAs26 (97%). Thus, the major losses
occur on the final deblocking step and cleavage from the resin,
though the average coupling yield is 75% in this case. It is
important to note that earlier synthesis of a- and g-carboxyethyl
thymine decamers gave very low overall yields (0.2% and 0.7%,
respectively; the results will be published elsewhere).
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Figure 2 Profiles of RP HPLC of reaction mixture produced when cleaved
tetramer ACAgꢀT from polymer support using deblocking protocols (a) I and
(b) II, (c) UV and (d) MALDI-TOF mass spectra of oligomer ACAgT.
Received: 21st May 2014; Com. 14/4380
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