Synthesis of two new thymine-containing negatively charged PNA monomers

Full text of DOI:10.1134/S0012500806050016

ISSN 0012-5008, Doklady Chemistry, 2006, Vol. 408, Part 1, pp. 57–60. © Pleiades Publishing, Inc., 2006.
Original Russian Text © N.P. Boyarskaya, Yu.G. Kirillova, E.A. Stotland, D.I. Prokhorov, E.N. Zvonkova, V.I. Shvets, 2006, published in Doklady Akademii Nauk, 2006, Vol. 408,
No. 1, pp. 55–58.
CHEMISTRY
Synthesis of Two New Thymine-Containing
Negatively Charged PNA Monomers
N. P. Boyarskaya, Yu. G. Kirillova, E. A. Stotland, D. I. Prokhorov,
E. N. Zvonkova, and V. I. Shvets
Presented by Academician N.F. Myasoedov December 6, 2005
Received December 6, 2005
DOI: 10.1134/S0012500806050016
Two new thymine-containing monomers of negatively to control the PNA hybridization with complementary
charged peptide nucleic acids (PNAs) based on L-glutamic sections of nucleic acids by changing the solution ionic
and L-aspartic acids and glycine were synthesized.
strength, which is of interest for their use in diagnostics,
molecular biology, and medicine [2–4]. In addition, it has
been found that the presence of the carboxyl group of
glutamic acid in position 5 (Fig. 1c) of the pseudopeptide
backbone of the PNA oligomer [5] has a substantial influ-
ence on the properties of the PNA/DNA duplex.
The purpose of this study is to prepare thymine PNA
monomers bearing an anionic group in position 8
(Fig. 1d) according to Scheme 1. It is noteworthy that
the pseudopeptides containing a hydrocarbon radical
(Fig. 1c) were described previously [6], whereas the
structure of PNA monomers with a radical in position 8
is synthesized for the first time. The extension of the
range of negatively charged PNA monomers increases
the possibility of variation of the strength of duplexes
with another nucleic acid.
The design and study of modified oligonucleotides
have become a central line of research in biotechnol-
ogy, which is due to the possibility of using them as
therapeutic agents and tools of molecular biology.
Among all the synthetic analogues of nucleic acids, a
separate class is represented by peptide nucleic acids,
which are structural homologues of DNA where the
sugar phosphate backbone of natural nucleic acids
(Fig. 1a) is replaced by a pseudopeptide one con-
structed from N-(2-aminoethyl)glycine repeating units
(Fig. 1b) [1]; this replacement provides stability against
intracell degradation with retention of the capacity for
hybridization with complementary sections of nucleic
acids. The introduction of groups bearing a negative
charge into the structure of these polyamide DNA-
mimetics would decrease the tendency for self-aggre-
The key intermediates, namely, o-NBS-protected
gation and increase the solubility and would enable one pseudopeptides 9 and 10, were obtained in 75 and 74%
HN
HN
HN
COO–
O
n
*
1
B
1
B
2
B
B
B
2
3
4
N
5
N
*
N
N
3
4
O
O
O
O
5 O
n
O
–OOC
–OOC
O
O
O
–O P O
6
COO–
6
7
HN
HN
HN
*
7
O
8
8
n
13
B
12
13
B
12
O
9
B
9
10
11
N
N
*
11
O 10
O
O
n
O
O
–O P O
O
HN
HN
HN
n = 1
n = 2
(a)
(b)
(c)
(d)
Fig. 1. Structure of (a) DNA and (b) classical (Aeg) and (c, d) negatively charged PNAs based on dicarboxylic amino acids.
Lomonosov State Academy of Fine Chemical Technology, pr. Vernadskogo 86, Moscow, 117571 Russia
57

58
BOYARSKAYA et al.
yields, respectively, by the Mitsunobu condensation [7] of BH3 in THF [9]. The glycine derivative 8 was synthe-
of amino alcohols 6 and 7 with the o-NBS glycine
derivative under conditions described previously [6]
and were fully characterized [8]. The alcohol compo-
nents 6 and 7 were prepared in three steps from
L-aspartic and L-glutamic acids in 18 and 33% yields,
respectively. The free α-carboxyl group in dicarboxylic
sized in 59% yield in four steps from glycine 5.
Free pseudopeptides 11 and 12 produced in 53
and 65% yields, respectively, upon the removal of
the o-NBS group in derivatives 9 and 10 by mild thi-
olysis were introduced into acylation with thymin-1-
amino acid derivatives was recovered by a 1 M solution ylacetic acid 15.
H2N
COOH
H2N
COOH
I–III
n(H2C)
IV–VII
COOH
NO2
O S O O
HN
5
3 n = 1
4 n = 2
BocHN
n(H2C)
OH
O
COOBzl
8
6 n = 1
7 n = 2
VIII
R
N
BocHN
O
(CH2)n
O
9 n = 1 R = o-NBS
10 n = 2 R = o-NBS
BzlOOC
IX
11 n = 1 R = H
12 n = 2 R = H
X
O
N
HN
O
O
BocHN
n(H2C)
OR'
N
13 n = 1 R' = CH2–CH=CH2
14 n = 1 R' = CH2–CH=CH2
O
COOBzl
XI
1 n = 1 R'=H
2 n = 2 R'=H
(I) BzlOH, H2SO4, Et2O;
(II) Boc2O, for n = 1: TEA, THF,
for n = 2: NaHCO3, dioxane/H2O;
(III) 1 M BH3THF, THF;
(IV) Boc2O, NaHCO3, dioxane/H2O;
(V) DEAD, PPh3, CH2=CH–CH2OH, THF;
(VI) CF3COOH, DCM;
(VII) o-NBS–Cl, TEA, DCM;
(VIII) DEAD, PPh3, THF;
(IX) PhSH, K2CO3, CH3CN;
(X) ThyCH2COOH (15), iBuOCOCl, NMM, TEA, DMF;
(XI) [Pd(PPh3)4], morpholine, THF.
Scheme 1.
DOKLADY CHEMISTRY Vol. 408 Part 1 2006

SYNTHESIS OF TWO NEW THYMINE-CONTAINING NEGATIVELY CHARGED PNA MONOMERS
59
It was shown by 1H NMR spectroscopy that
pseudopeptides 9 and 10 as the bases tend to form
cyclization products 15 and 16 (Scheme 2) whose chro-
matographic mobility is similar to that of the protected
monomers 13 and 14. The cyclization at room temper-
ature proceeds up to 100% within 12 h. For chromatog-
raphy on silica gel, a 95 : 5 ethyl acetate–methanol sys-
tem was selected. This system is capable of separating
the target products (13 and 14) from the by-products
(15 and 16).
As a result of investigations, we chose the method of
mixed anhydrides with the use of threefold excesses of
thymin-1-ylacetic acid and N-isobutyl chloroformate in
the presence of N-methylmorpholine (DMF, –20°C) as
the optimal approach to the formation of an amide bond
between the pseudopeptide fragments and the thymine
derivative. Using this approach, we attained acceptable
yields of compounds 13 and 14 (67 and 55%, respec-
tively).
BzlOOC
(CH2)n
H
O
Basic
n(H2C)
BocHN
conditions
COOAll
–BzlOH
N
N
COOAll
BocHN
11 n = 1
12 n = 2
15 n = 1
16 n = 2
Scheme 2.
3
t-Bu); 13C NMR (DMSO-d6): 172.42, 170.25, 167.31,
164.37, 155.48, 150.97, 141.90, 136.20, 128.41,
127.97, 108.17, 78.07, 65.47, 51.04, 48.26, 47.66,
40.76, 30.43, 28.16, 26.84, 11.93.
The selective removal of the allylic protection from
the α-carboxyl group was carried out under mild neu-
tral conditions (tetrakis(triphenylphosphine)palla-
dium(0) [Pd(PPh3)4], 10 mol % in morpholine) [10].
After pretreatment with an aqueous solution of NaCl
brought to pH 3.0 by adding 1 N HCl, monomers 1 and
2 were formed as white (1) or light yellow (2) crystals
in 70 and 74% yields, respectively. The structures and
the purities of these products and the structures of the
For C25H32N4O9 anal. calcd. (wt %): C, 56.39;
H, 6.02; N, 10.53.
Found (wt %): C, 55.68; H, 5.85; N, 9.72.
MS, m/z: 433.5 [M-Boc + H]+.
4
[Boc-Glu(g-OBzl)y[ëH2N(ThyAc)]Gly] (2). Mp
1
182–186°ë; [α]2D0 –1.3° (c = 1, methanol). H NMR
1
protected monomers 13 and 14 were confirmed by H
and 13C NMR spectroscopy, mass spectrometry, and
elemental analysis.
(DMSO-d6, δ, J, ppm): 11.25 (s, 1H, NH-Thy), 7.35 (s,
2H, –ë6H5), 7.25 (s, 1H, HThy), 6.75 (dd, 1H, J1 =
9.54 Hz, J2 = 65.29 Hz, BocNH), 5.12 (s, 2H, –ëç2-Ph),
4.65 (s, 2H, CH2-Thy), 4.20 (m, 1H, α-CH), 3.95 (m,
2H, CH2Gly), 3.60 (m, 2H, CH2Glu), 2.39 (m, 2H,
γ-CH2), 1.75 (s, 3H, CH3-Thy), 1.55 (m, 2H, β-CH2),
Thus, we developed and optimized a strategy for the
synthesis of negatively charged PNA monomers con-
taining a substituent in position 8 of the pseudopeptide
skeleton; two new thymine-containing monomers of
negatively charged PNAs were synthesized and charac-
terized.
1.35 (s, 9H, t-Bu); 13C NMR (DMSO-d6): 171.02,
170.74, 167.19, 164.43, 155.75, 150.96, 142.05,
136.10, 128.43, 127.83, 108.20, 78.12, 65.64, 57.44,
47.82, 47.66, 40.77, 34.40, 28.16, 11.93.
[Boc-Asp(b-OBzl)y[ëH2N(ThyAc)]Gly] (1). Mp
For C26H34N4O9 anal. calcd. (wt %): C, 57.14; H,
6.23; N, 10.26.
20 1
2
233–235°C; [α]D +12.3° (c = 1, methanol). 1H NMR
(DMSO-d6, δ, J, ppm): 11.22 (s, 1H, NH-Thy), 7.35
(2H, s, –ë6H5), 7.21 (1H, s, HThy), 6.98 (d, 1H, J =
8.80 Hz, BocNH), 5.10 (2ç, s, –CH2-Ph), 4.42 (2H, s,
CH2-(Thy)), 4.06 (m, 1H, α-CH), 3.75 (m, 2H, CH2Gly),
3.45 (m, 2H, CH2Asp), 2.65 (dd, 2H, J1 = 5.13 Hz, J2 =
15.65 Hz, β-ëç2), 1.75 (s, 3H, CH3-Thy), 1.35 (s, 9H,
Found (wt %): C, 55.91; H, 5.97; N, 9.96.
MS, m/z: 446.9 [M-Boc]+.
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13
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2
1
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