566–575
Nucleic Acids Research, 1998, Vol. 26, No. 2
 1998 Oxford University Press
Synthesis and evaluation of some properties of
chimeric oligomers containing PNA and
phosphono-PNA residues
Vladimir A. Efimov*, Michael V. Choob, Alla A. Buryakova, Anna L. Kalinkina and
Oksana G. Chakhmakhcheva
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, ul.Miklukho-Maklaya 16/10, Moscow 117871, Russia
Received September 4, 1997; Revised and Accepted November 17, 1997
ABSTRACT
In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs),
particularly water solubility and cellular uptake, the
synthesis of chimeric oligomers consisted of PNA and
phosphono-PNA analogues (pPNAs) bearing the four
natural nucleobases has been accomplished. To
produce these chimeras, pPNA monomers of
two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers
representing derivatives of N-(2-aminoethyl)glycine, or
N-(2-hydroxyethyl)glycine. The oligomers obtained
were composed of either PNA and pPNA stretches or
alternating PNA and pPNA monomers. The examination
of hybridization properties of PNA–pPNA chimeras to
DNA and RNA complementary strands in comparison
with pure PNAs, and pPNAs as well as DNA–pPNA
hybrids and DNA fragments confirmed that these
chimeras form stable complexes with complementary
DNA and RNA fragments. They were found to be
resistant to degradation by nucleases. All these
properties together with good solubility in water make
PNA–pPNA hybrids promising for further evaluation as
potential therapeutic agents.
INTRODUCTION
Last years, peptide nucleic acids (PNAs), nuclease resistant DNA
analogues, were reported as antisense and antigene agents, and their
binding properties to nucleic acids as well as biological applications
were evaluated (1–3). PNAs consist of achiral monomer subunits
derived from N-(2-aminoethyl)glycine bearing nucleoside heterocyclic bases, which are coupled by standard methods of the amide
bond formation. It was shown that PNA oligomers can hybridize to
complementary DNA or RNA chains forming very stable duplexes
and triple helixes. Due to their excellent binding properties, these
oligonucleotide mimics are currently of much interest as potential
substances for therapeutic use. However, the biological applications
of PNAs are limited by their poor water solubility, tendency to
self-aggregation, inability to activate RNAase H and insufficient
cellular uptake (3,4). To improve the solubility characteristics of
PNAs, the synthesis of PNA–DNA chimeras has been recently
reported by different authors (4–8), and it was demonstrated that
PNA–DNA chimeras hybridize specifically to DNA and RNA
chains (4–7).
In a previous study on the search of alternative compounds, which
would open the way to solution of these problems, a novel class of
DNA mimics representing phosphono-PNA analogues (pPNAs)
containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone has been designed (Fig. 1)
(9–11). Procedures to obtain corresponding thymine and cytosine
monomers has been developed, and the synthesis of pyrimidine
containing oligomers by solid phase approach has been
accomplished (9,10). Similarly, a synthesis of the homo-thymine
phosphono-PNA analogue in solution has been reported by
E.Uhlmann and co-workers (11). A preliminary study into some
properties of homo-pyrimidine pPNAs demonstrated that they have
promising binding properties to complementary DNA and RNA
sequences, and the introduction of negative charges into the PNA
backbone lead to excellent solubility characteristics of pPNAs
(9–11). However, the stability of their complexes is lower than the
stability of corresponding complexes formed by PNAs (11).
To continue this investigation, we decided to obtain the hybrids of
pPNAs with PNAs composed of either PNA and pPNA stretches,
or alternating PNA and pPNA monomers. As both these DNA
analogues are isosteric, it is possible that PNA–pPNA chimeras
might combine hybridization characteristics of PNA with high water
solubility of pPNAs and improve the properties of both these
mimics. This paper reports the synthesis of PNA–pPNA hybrids
depicted in Figure 1 and evaluation of their hybridization properties
and nuclease stability in comparison with pure PNA and pPNA
oligomers as well as DNA fragments.
MATERIALS AND METHODS
Solvents and reagents were obtained from commercial suppliers and
were used without further purification. 1H and 31P NMR spectra
were recorded in CDCl3 on a Bruker WM500 spectrometer.
Chemical shifts are given in p.p.m. relative to tetramethylsilane (1H)
or H3PO4 (31P). Mass spectra were recorded using either
electrospray ionisation (ES) or matrix-assisted laser
desorption-ionization time of flight (MALDI-TOF). TLC was
carried out on Merck Silica Gel 60 F254 plates in CHCl3-CH3OH
(9:1, v/v) (solvent A) or CHCl3-CH3OH-triethylamine
*To whom correspondence should be addressed. Tel: +7 095 336 5911; Fax: +7 095 336 5911; Email: [email protected]
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Figure 1. General chemical structures of PNAs, pPNAs, PNA–pPNA hybrids and monomer building units used for their construction.
(8.4:1.5:0.1, v/v/v) (solvent B) Silica gel column chromatography
was performed using Merk silica gel 60. Synthesis of oligomers
was performed using Applied Biosystems Synthesizer 381A.
Anion-exchange separations were performed using Pharmacia
Mono-Q column/FPLC system and a linear gradient of NaCl
(0–1.2 M) in 0.02 M NaOH (pH 12). Reversed phase FPLC was
performed using Pharmacia ProRPC column and a linear gradient
of acetonitrile (0–30%) in 0.1 M triethylammonium acetate (pH
7). Preparative electrophoresis of oligomers was performed using
15% polyacrylamide gels/7 M urea with 0.1 M Tris–borate/
EDTA buffer (pH 8.3).
General methods for preparation of pPNA monomers
The synthesis of pPNA monomers (Scheme 1) was accomplished
via a common intermediate representing N-[2-(4,4’-dimethoxytrityl)oxyethyl]-aminomethyl diphenylphosphonate (3a), or
N-[2-(4-monomethoxytrityl)-aminoethyl]-aminomethyl diphenylphosphonate (3b), obtained as described previously (9,10).
Thymine-N1-acetic acid (12), N4-benzoylcytosine-N1-acetic acid
(5), N2-isobutyrylguanine-N9-acetic acid (13) and N4-benzoyladenine-N9-acetic acid (5) were obtained according to the
published procedures.
The synthesis of pyrimidine containing pPNA monomers 1 was
carried out essentially as described (9). Thymine-, or N4-benzoylcytosine-N1-acetic acid (13 mmol) was mixed with a solution of the
intermediate 3 (10 mmol) [δp = 20.2 (3a), or 20.5 (3b)] in CH3CN
(40 ml). N,N’-Dicyclohexylcarbodiimide (DCC) (13 mmol) was
added, and the reaction mixture was shaken for 1–2 h. The
reaction was terminated by the addition of water (5 ml) and
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (4 ml). After 1 h, the
precipitate formed was removed by filtration, and the filtrate
containing monophenyl ester 5 was evaporated to a gum. The
latter compound was isolated by silica gel column chromatography using a gradient of methanol (0–7%) in CH2Cl2/1%
triethylamine. [Yield 85–90%. For Thy derivatives: Rf (solvent
B) = 0.45 (5a), or 0.40 (5b), δp = 11.0 and 11.1 (5a); or 11.4 and
11.6 (5b); for Cyt derivatives: Rf (solvent B) = 0.52 (5a), or 0.49
(5b), δp = 11.4 and 11.6 (5a); or 11.6 and 11.75 (5b)]. To introduce
a catalytic protective group, compound 5 (5 mmol) was allowed
to react with 1-oxido-4-methoxy-2-pyridinemethanol (6 mmol)
in 50 ml of dry acetonitrile-pyridine (4:1, v/v) in the presence of
1-(2,4,6-triisopropylbenzenesulfonyl)-3-nitro-1,2,4-triazole
(TPSNT) (7.5 mmol) for 0.5 h. The reaction was terminated by
the addition of 5% NaHCO3 (50 ml). The solution obtained was
extracted with CH2Cl22 (2 × 50 ml), and the combined organic
fractions were evaporated to dryness. The residue was treated
with 20 ml of 0.5 M DBU solution in CH3CN-H2O (9:1, v/v) for
1 h. After evaporation, the desired product was isolated by silica
gel column chromatography using 0–10% gradient of methanol
in CH2Cl2/1% triethylamine. The fractions containing compound
1 were concentrated by evaporation, the residue was dissolved in
CH2Cl2 and precipitated with hexane-ether (2:1, v/v). The
precipitate was collected by centrifugation. Yield 80–85%.
Analytical data: Thy-1a - Rf = 0.35 (solvent B); m/z (ES) = 761.5
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Scheme 1.
(M + H)+ (C38H42N4O11P1 requires 761.7); 1H NMR (CDCl3)
δ = 1.20 (t, 9H, +NHEt3-CH3), 1.80 (s, 3H, Thy-CH3), 2.95
(q, 6H, +NHEt3-CH2), 3.30 (m, 2H, DMTrO-CH2), 3.40 (m, 2H,
DMTrO-CH2-CH2), 3.75 (s, 6H, DMTr-OCH3), 3.80 (s, 3H,
picolyl-OCH3), 3.82-4.0 (m, 2H, CH2-P), 4.75-4.92 (2xs,
rotamers, 2H, N-CO-CH2), 5.1-5.2 (2xd, rotamers, 2H, P-OCH2), 6.70 (m, 1H, picolyl-H-3), 6.75 (m, 1H, picolyl-H-5),
6.80-7.50 (m, 14H, DMTr and Thy-H-6), 8.07-8.17 (2xd, 1H,
picolyl-H-6); 31P NMR (CDCl3) δ = 15.0 and 15.7. Thy-1b Rf = 0.3 (solvent B); m/z (ES) = 730.4 (M+H)+ (C37H41N5O9P1
requires 730.7); 1H NMR δ = 1.18 (t, 9H, +NHEt3-CH3), 1.80
(s, 3H, Thy-CH3), 2.35 (m, 2H, MMTrNH-CH2), 2.90 (q, 6H,
+NHEt -CH ), 3.37 (m, 2H, MMTrNH-CH -CH ), 3.75 (s, 3H,
3
2
2
2
MMTr-OCH3), 3.80 (s, 3H, picolyl-OCH3), 3.85-3.95 (m, 2H,
CH2-P), 4.75-4.92 (2xs, rotamers, 2H, N-CO-CH2), 5.1-5.2 (2xd,
rotamers, 2H, P-O-CH2), 6.70 (m, 1H, picolyl-H-3), 6.75 (m, 1H,
picolyl-H-5), 6.80-7.50 (m, 15H, MMTr and Thy-H-6), 8.07-8.17
(2xd, 1H, picolyl-H-6), 8.65 (s, 1H, NH); 31P NMR δ = 15.25 and
15.75. Cyt-1a: Rf = 0.42 (solvent B); m/z (ES) = 850.4 (M + H)+
(C44H45N5O11P1 requires 850.8); 1 NMR δ = 1.18 (t, 9H,
+NHEt -CH ), 2.95 (q, 6H, +NHEt -CH ), 3.30 (m, 2H, DMTrO3
3
3
2
CH2), 3.38 (m, 2H, DMTrO-CH2-CH2), 3.75 (s, 6H, DMTrOCH3), 3.78 (s, 3H, picolyl-OCH3), 3.80-3.90 (m, 2H, CH2-P),
4.92-5.10 (2xs, rotamers, 2H, N-CO-CH2), 5.04-5.18 (2xd,
rotamers, 2H, P-O-CH2), 6.64 (m, 1H, picolyl-H-3), 6.72 (d, 1H,
picolyl-H-5), 6.75-7.60 (m, 15H, Ar-H and Cyt-H-5), 7.70 (d, 2H,
m-Ar-H), 7.95 (m, 1H, Cyt-H-6), 8.0 (m, 2H, o-Ar-H), 8.05-8.12
(2xd, 1H, picolyl-H-6); 31P NMR δ = 15.0 and 15.4. Cyt-1b: Rf
= 0.38 (solvent B); m/z (ES) = 819.7 (M + H)+ (C43H44N6O9P1
requires 819.8); 1H NMR (CDCl3) δ = 1.18 (t, 9H, +NHEt3-CH3),
2.30 (m, 2H, MMTrNH-CH2), 2.95 (q, 6H, +NHEt3-CH2), 3.40
(m, 2H, MMTrNHCH2-CH2), 3.75 (s, 3H, MMTr-OCH3), 3.78
(s, 3H, picolyl-OCH3), 3.80-3.90 (m, 2H, CH2-P), 4.92-5.10 (2xs,
rotamers, 2H, N-CO-CH2), 5.04-5.18 (2xd, rotamers, 2H,
P-O-CH2), 6.64 (m, 1H, picolyl-H-3), 6.72 (d, 1H, picolyl-H-5),
6.75-7.60 (m, 16H, Ar-H and Cyt-H-5), 7.70 (d, 2H, m-Ar-H),
7.95 (m, 1H, Cyt-H-6), 8.0 (m, 2H, o-Ar-H), 8.05-8.12 (2xd, 1H,
picolyl-H-6); 31P NMR δ = 15.15 and 15.7 .
The synthesis of purine containing pPNA monomers 1 was
accomplished according to the following general procedure.
N2-Isobutyrylguanine- or N4-benzoyladenine-N9-acetic acid
(15 mmol) was mixed with a solution of the intermediate 3
(10 mmol) in CH3CN-CCl4 (9:1, v/v; 100 ml) containing
diisopropylethylamine (50 mmol). Triphenyl phosphine
(30 mmol) was added, and the mixture was shaken for 30 min at
room temperature. The reaction was terminated by the addition
of methanol (5 ml). Then, 0.5 M TEAB (100 ml) was added, and
the mixture was extracted with CH2Cl2 (2 × 100 ml). The organic
fraction was evaporated to a gum containing the desired
diphenylphosphonate 4 (yield 65–70%). [For Gua derivatives: Rf
(solvent A) = 0.6 (4a), or 0.55 (4b), δp = 14.7 (4a), or 15.3 (4b);
for Ade derivatives: Rf (solvent A) = 0.72 (4a), or 0.65 (4b),
δp = 14.7 (4a), or 15.3 (4b)]. The latter was dissolved in 50 ml of
acetonitrile-water (9:1, v/v) and treated with 25 mmol of DBU for
1 h. The reaction was terminated by the addition of 0.5 M TEAB
(50 ml), and monophenyl ester 5 was isolated by extraction with
CH2Cl2 (2 × 50 ml) followed by silica gel column chromatography using a gradient of methanol (0–7%) in CH2Cl2/1%
triethylamine. [Yield 90–95%. For Gua derivatives: Rf (solvent
B) = 0.35 (5a), or 0.33 (5b), δp = 11.1 and 11.3 (5a); or 11.5 and
11.7 (5b); for Ade derivatives: Rf (solvent B) = 0.53 (5a), or 0.5
(5b), δp = 11.2 and 11.4 (5a); or 11.4 and 11.6 (5b)]. The
conversion of guanine or adenine, containing monophenyl ester
5 to pPNA monomer 1 was carried out as described for the
thymine derivative. Yield 85–90 %. Analytical data: Gua-1a:
Rf = 0.2 (solvent B); m/z (ES) = 856.8 (M + H)+ (C42H47N7O11P1
requires 856.9); 1H NMR (CDCl3) δ = 1.12 (d, 6H, iBu-CH3),
1.20 (t, 9H, +NHEt3-CH3), 2.95 (q, 6H, +NHEt3-CH2), 3.25
(m, 2H, DMTrO-CH2), 3.40 (m, 2H, DMTrO-CH2-CH2), 3.70
(s, 3H, picolyl-OCH3), 3.75 (s, 6H, DMTr-OCH3), 3.80-3.95
(m, 2H, CH2-P), 5.1-5.2 (2xd, rotamers, 2H, P-O-CH2),
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Scheme 2.
5.40-5.62 (2xs, rotamers, 2H, N-CO-CH2), 6.65 (m, 1H, picolylH-3), 6.72 (m, 1H, picolyl-H-5), 6.80-7.50 (m, 13H, DMTr),
7.75-7.80 (2xs, rotamers, 1H, Gua-H-8), 8.05-8.15 (2xd, 1H,
picolyl-H-6); 31P NMR (CDCl3) δ = 15.0 and 15.6. Gua-1b:
Rf = 0.17 (solvent B); m/z (ES) = 825.8 (M+H)+ (C41H46N8O9P1
requires 825.8); 1H NMR δ = 1.12 (d, 6H, iBu-CH3), 1.20 (t, 9H,
+NHEt -CH ), 2.20 (m, 3H, MMTrNH-CH and iBu-CH), 2.95
3
3
2
(q, 6H, +NHEt3-CH2), 3.45 (m, 2H, MMTrNH-CH2-CH223.70
(s, 3H, MMTr-OCH3), 3.80 (s, 3H, picolyl-OCH3), 3.85-3.95
(m, 2H, CH2-P), 5.1-5.2 (2xd, rotamers, 2H, P-O-CH2),
5.40-5.62 (2xs, rotamers, 2H, N-CO-CH2), 6.65 (m, 1H, picolylH-3), 6.72 (m, 1H, picolyl-H-5), 6.80-7.50 (m, 14H, MMTr),
7.75–7.80 (2xs, rotamers, 1H, Gua-H-8), 8.05-8.15 (2xd, 1H,
picolyl-H-6); 31P NMR δ = 15.2 and 15.7. Ade-1a: Rf = 0.4
(solvent B); m/z (ES) = 874.8 (M+H)+ (C45H45N7O10P1 requires
874.9);1H NMR δ = 1.20 (t, 9H, +NHEt3-CH3), 2.95 (q, 6H,
+NHEt -CH ), 3.30 (m, 2H, DMTrO-CH ), 3.45 (m, 2H,
3
2
2
DMTrO-CH2-CH2), 3.70 (s, 3H, picolyl-OCH3), 3.75 (s, 6H,
DMTr-OCH3), 3.80-3.95 (m, 2H, CH2-P), 5.10-5.22 (2xd,
rotamers, 2H, P-O-CH2), 5.40-5.50 (2xs, rotamers, 2H, N-COCH2), 6.65 (m, 1H, picolyl-H-3), 6.75 (d, 1H, picolyl-H-5),
6.80-7.63 (m, 16H, Ar-H), 8.05 (m, 3H, Ade-H-2 and o-Ar-H),
8.08-8.15 (2xd, 1H, picolyl-H-6), 8.70 (2xs, rotamers, 1H,
Ade-H-8); 31P NMR δ = 15.0 and 15.4. Ade-1b: Rf = 0.35
(solvent B); m/z (ES) = 843.8 (M + H)+ (C44H44N8O8P1 requires
843.9); 1H NMR (CDCl3) δ = 1.20 (t, 9H, +NHEt3-CH3), 2.30
(m, 2H, MMTrNH-CH2), 2.95 (q, 6H, +NHEt3-CH2), 3.40 (m,
2H, MMTrNHCH2-CH2), 3.70 (s, 3H, picolyl-OCH3), 3.75
(s, 3H, MMTr-OCH3), 3.80-3.95 (m, 2H, CH2-P), 5.10-5.22
(2xd, rotamers, 2H, P-O-CH2), 5.40-5.50 (2xs, rotamers, 2H,
N-CO-CH2), 6.65 (m, 1H, picolyl-H-3), 6.75 (d, 1H, picolylH-5), 6.80-7.63 (m, 17H, Ar-H), 8.05 (m, 3H, Ade-H-2 and
o-Ar-H), 8.08-8.15 (2xd, 1H, picolyl-H-6), 8.70 (2xs, rotamers,
1H, Ade-H-8); 31P NMR δ = 15.1 and 15.7.
Preparation of PNA–pPNA dimers
To obtain pPNA–PNA dimer of type 9a (Scheme 2),
N-[2-(4,4’-dimethoxytrityloxyethyl)]-N-(thymin-1-ylacetyl) glycine
methyl ester 6a (2 mmol) synthesized as described (6) was treated
with 80% aqueous acetic acid (20 ml) for 1 h. The reaction mixture
was evaporated to dryness in vacuo and co-evaporated with
acetonitrile (2 × 20 ml) and toluene (20 ml). The residue containing
intermediate 7a was dissolved in dry pyridine (4 ml), and a solution
of Thy-containing compound 1b (9) (2.1 mmol, δp = 15.0 and 15.7)
in 12 ml of CH3CN and TPSNT (3 mmol) were added. The mixture
was allowed to react for 5 min, then 5% NaHCO3 (50 ml) was
added, and the reaction mixture was extracted with CH2Cl2 (3 ×
50 ml). The combined organic phases were evaporated to a gum.
The latter was dissolved in 5% aqueous CH3CN (25 ml) containing
DBU (5 mmol). After 2 h, 0.5 M TEAB (50 ml) was added. The
desired dimer 9a was isolated as a foam (yield 75%) by extraction
with CH2Cl2 followed by silica gel column chromatography using
a gradient of methanol (0–10%) in CH2Cl2/1.5% triethylamine. Rf
= 0.2 (solvent B); m/z (ES) = 997.9 (M + H)+ (C48H54N8O14P1
requires 998); 31P NMR δ = 22.6 and 23.2.
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Table 1. Elongation cycles for the solid phase synthesis of oligomers*
*Reactions were performed using 30 mg of CPG support (1 µmol of the first nucleoside).
** Before coupling, carboxylic component was pre-activated by mixing with 1-(2,4,6-triisopropylbenzenesulfonyl)-3-nitro-1,2,4-triazole (TPSNT) and 1-methylimidazole.
To obtain pPNA–PNA dimer of type 9b (Scheme 2), 2 mmol of
N-[2-(4-methoxytrityl-aminoethyl)]-N-(thymin-1-ylacetyl)-glycine
methyl ester 6b obtained as described (13) was treated with picric
acid (2.1 mmol) in acetonitrile-water (9.5:0.5 v/v, 10 ml) for 15 min.
The reaction mixture was evaporated to dryness in vacuo and
co-evaporated with acetonitrile (3 × 15 ml). The residue
containing intermediate 7b was dissolved in dry pyridine (8 ml),
and a solution of the thymine containing compound 1b
(1.9 mmol) in 8 ml of CH3CN-CCl4 (7:1, v/v) and triphenylphosphine (4 mmol) were added. The mixture was allowed to
stand for 40 min. Then, methanol (3 ml) and diisopropylethylamine (4 mmol) were added. After 15 min, 0.5 M TEAB (40 ml)
was added, and the reaction mixture was extracted with CH2Cl2
(2 × 50 ml). The combined organic phases were evaporated to a
gum. The removal of methyl protecting group and isolation of the
desired dimer 9b (yield 65%) was performed as described for 9a.
Rf = 0.17 (solvent B); m/z (ES) = 996.9 (M + H)+
(C48H55N9O13P1 requires 997); 31P NMR δ = 26.2 and 26.9.
For the preparation of PNA–pPNA dimers of type 12 (Scheme 2),
the Thy-containing diphenyl phosphonate 4b (9) (2 mmol) was
treated with picric acid (2.1 mmol) in 5% aqueous acetonitrile
(10 ml) for 15 min. The reaction mixture was evaporated in vacuo
and then co-evaporated with acetonitrile (2 × 15 ml). The residue
was dissolved in pyridine (8 ml) and a solution of 2 mmol of 2a
(6), or 2b (13), in 6 ml of pyridine and DCC (3 mmol) were added.
The reaction was completed in 1 h. Then water (1 ml) and DBU
(7 mmol) were added to the reaction mixture containing diphenyl
phosphonate 11 to remove one of phenyl protecting groups. After
40 min, the precipitate formed was removed by filtration, and a
solution obtained was evaporated to dryness and co-evaporated
with toluene. Then, the monophenyl protected dimer obtained
was converted to the corresponding 4-methoxy-1-oxido-pyridine-2-methyl phosphonate 12 and isolated as described for
pPNA monomers as a colourless foam. Yield 75–80%. Analytical
data: 12a: Rf = 0.18 (solvent B); m/z (ES) = 1027.9 (M + H)+
(C49H56N8O15P1 requires 1028), 31P NMR δ = 15.0 and 15.7;
12b: Rf = 0.12 (solvent B); m/z (ES) = 996.8 (M + H)+
(C48H55N9O13P1 requires 997); 31P NMR δ = 15.2 and 15.7.
Solid phase synthesis of oligonucleotides and mimics
Synthesis of oligodeoxyribonucleotides was performed by the
standard phosphoramidite method, or by the phosphotriester method
using O-nucleophilic intramolecular catalysis (14). PNA monomers
were synthesized and purified as described (6,13). The protected
mononucleotides for the phosphotriester synthesis were prepared
according to the procedure published by us earlier (14). Chain
elongation in the solid phase synthesis of pPNA, PNA and
PNA–pPNA oligomers was carried out according to conditions
given in Table 1. Deblocking and isolation procedures for
oligonucleotides were carried out according to standard protocols.
Deblocking of mimics was started from the removal of a terminal
trityl protecting group by acidolysis as described in Table 1. In the
case of oligomers containing terminal amino function, the latter was
capped by the action of acetic anhydride-1-methylimidazole-pyidine
(1:1:8, v/v/v) for 5 min. The deprotection of pPNAs and their
hybrids included the removal of catalytic P-protective groups by the
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action of thiophenol-triethylamine-dioxane (1:2:2 v/v/v; 2 ml/30 mg
of the support) for 3–4 h at room temperature. The removal of
N-protecting groups from heterocycles and cleavage from the
support were achieved by the treatment with concentrated aqueous
ammonia. Oligomers were purified by anion-exchange FPLC and/or
gel-electrophoresis as described (14). The identity and purity of the
oligomers obtained, were confirmed by mass spectrometry
(MALDI-TOF) and reversed phase FPLC.
Thermal denaturation and enzyme degradation experiments
Absorbance (260 nm) versus temperature curves of complexes
formed by mimics or oligodeoxyribonucleotides with
corresponding DNA (RNA) templates were measured using a
Gilford 250 UV-VIS spectrophotometer equipped with a Gilford
2527 thermocontroller and a heating/cooling rate of 0.5C/min.
Solutions contained 3–5 µM of each oligomer in 150 mM
NaCl/10 mM Tris–HCl (pH 7–8)/5 mM EDTA in the absence or
in the presence of 10 mM MgCl2. Melting temperatures (Tm)
were taken to be the temperature of half-dissociation and were
obtained from a plot of the derivative of 1/T versus absorbance at
260 nm. Molar extinction coefficients used for nucleobase were
as follows: Ade, 15.4; Thy, 8.8; Gua, 11.7 and Cyt, 7.3 µmol.cm.
Snake venom phosphodiesterase (VPDE) and S1 nuclease assays
were performed as described (15). The digestion products were
analysed using 20% denaturing PAGE.
Gel mobility assays
The corresponding DNA, or RNA, target (50 µM) was mixed
with the complementary oligomer in various molar ratio in
200 mM NaCl/0.01 M Tris–HCl (pH 7)/1 mM EDTA in the
presence or in the absence of 10 mM MgCl2. The mixture was
heated at 90C for 2 min and slowly cooled to +10C. Aliquots
of the reaction mixtures (50 µl) were resolved at 10C in a 15%
native polyacrylamide gel containing either 100 mM Tris–borate
(pH 8.3) or 90 mM Tris–borate (pH 8.3)/10 mM MgCl2. The
bands were detected by UV-shadowing and visualised by staining
with ethidium bromide or ‘Stains all’.
RESULTS AND DISCUSSION
Synthesis of building blocks for construction of PNA–pPNA
chimeras
To construct PNA–pPNA hybrids depicted in Figure 1, the
monomer building units of four types were used. Two of them
represent pPNA monomers (1a and 1b) having a combination of
blocking groups compatible with the DNA phosphotriester
synthesis using the intramolecular O-nucleophylic catalysis
(14,16). They were designed for the solid phase automated
synthesis of corresponding oligomers containing either phosphonate ester or phosphonamide bonds between monomer units.
Similarly to the synthesis of PNA monomers (2), a general
synthetic route developed to obtain pPNA monomers employs a
common intermediate 3 representing either N-[2-(4,4′-dimethoxytrityl)oxyethyl]-aminomethyl (3a), or N-[2-(4-monomethoxytrityl)aminoethyl]-aminomethyl diphenylphosphonate (3b), to which
any nucleobase can be attached via a methylene carbonyl linker
(Scheme 1) (9,10). The attachment of thymine-N1-acetic acid and
N4-benzoylcytosine-N1-acetic acid was performed using
N,N′-dicyclohexylcarbodiimide (DCC) as a condensing agent with
Figure 2. FPLC analysis of oligomers. Anion-exchange chromatography of
crude PNA–pPNA hybrids s657 (A), s677 (B) and s686(C) (Table 2) and
reversed-phase chromatography of purified homo-thymine oligomers (D) and
oligomers with mixed purine-pyrimidine sequence (E).
the yields of 85–90% as it was described previously (9). At the
same time, for the introduction of the carboxymethylated N-protected adenine and guanine bases to the backbone we used a
mixture of triphenylphosphine and CCl4 (17), which in these cases
was more effective than DCC and allowed us to obtain the purine
containing intermediates 4a,b with 65–70% yields. The transformation of 4 into the pPNA building units 1, carrying the catalytic
4-methoxy- 1-oxido-2-picolyl phosphonate protecting group was
accomplished in three step procedure (9,10). The latter includes the
conversion of diphenylphosphonate 4 to corresponding monophenyl phosphonate 5 by the action of DBU/H2O, the condensation of
compounds 5 with 2-hydroxymethyl-4-methoxy-1-oxydo-pyridine in the presence of a condensing agent and the final treatment
the phosphonate triester obtained with DBU/H2O to remove the
second phenyl phosphonate protecting group to give the desired
monomer 1 in an overall yield varying from 70–75% for
pyrimidine to 50–60% for purine derivatives.
Other two types of monomer building units represented PNA
derivatives on the base of N-(2-hydroxyethyl)glycine (2a) and
N-(2-aminoethyl)glycine (2b) backbones (Fig. 1), which were
obtained according to the published procedures (6,13). The
synthesis of PNA monomers of type 2b, in which acid-labile
monomethoxytrityl group was used for protection of the amino
function of the aminoethylglycine residue, has been reported by
several groups (13,18,19). Monomers of type 2a were described
as linker units in the synthesis of DNA–PNA chimeras (6,7).
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In addition, the dimer building blocks with mixed PNA–pPNA
backbone were synthesized in solution starting from the adequately
protected PNA and pPNA monomers as it is shown in Scheme 2.
Dimers of type 9 containing terminal N-monomethoxytritylamino
and carboxyl functions and the internal phosphonate diester or
phosphonamide, linkage between monomers were designed for the
synthesis of hybrid oligomers using PNA methodology, whereas
dimers 12 bearing the internal amide bond and terminal phosphonate
function were obtained for the solid-supported synthesis of PNA–
pPNA hybrids with the use of the DNA phosphotriester chemistry.
Design and synthesis of chimeric oligomers
We have started our experiments on the synthesis of mimics
containing mixed PNA–pPNA backbone with molecules consisting
of alternating PNA and pPNA residues (Fig. 1). The first type of
hybrids obtained consisted of a PNA monomer 2a representing the
derivative of N-(2-hydroxyethyl)glycine linked through an amide
bond to the pPNA monomer of the type 1b, which in its turn was
connected to the next PNA monomer 2a by the phosphonate
monoester bond. Similarly, the synthesis of the second type of
hybrids (PNA–pPNA-b) containing alternating phosphonamide and
amide linkages between monomer units was completed started from
the standard PNA monomer 2b and pPNA monomer 1b. The
synthesis of hybrids containing two and four pPNA residues of type
1b embedded in various positions of a chain composed mainly from
PNA residues of type 2b, has been also accomplished.
To assemble the chains of homo-thymine PNA–pPNA-a,b
hybrids, two approaches have been used. The first one was to couple
monomers in a stepwise manner changing a type of monomer unit
and coupling conditions after the each elongation cycle. The second
approach was to use preliminary prepared PNA–pPNA dimers as
synthons for chain elongation in conjunction with only pPNA
(dimers of type 9), or PNA (dimers of type 12) synthetic route. The
dimers of type 9 were also applied for the introduction of individual
pPNA units into an oligomer composed mainly from PNA
residues.
To prepare chimeric molecules containing PNA and pPNA
segments connected by a linker unit (Fig. 1, PNA–pPNA-c,d), we
adopted an approach similar to that used for the synthesis of
PNA–DNA hybrids (4–8). The chimeras were assembled with
PNA sections both at the pseudo 3′-COOH terminus (PNA–
pPNA-c) or 5′-NH2 terminus (PNA–pPNA-d). For the synthesis
of 5′pPNA–3′PNA chimera, the PNA part was synthesized first
on a CPG support derivatized with 3′-O-succinate of
N-(monomethoxytrityl)-5′-aminonucleoside (20). Then, PNA
monomer 2a representing the derivative of N-(2-hydroxyethyl)glycine was coupled to the N-terminus as a linker. This
allowed the following synthesis of the 5′-pPNA part of the
molecule. For the construction of the 5′PNA–3′pPNA chimera,
the synthesis was started from a pPNA part with the use of CPG
support derivatized with a DMTr-nucleoside and the monomer
1a. The pPNA monomer 1b was then coupled to the terminal
OH-group on the support as a linker to the 5′-terminal PNA part
of the chimera, which was synthesized from the monomer 2b.
As reference compounds, the pure PNA and pPNA oligomers
as well as corresponding oligodeoxyribonucleotides have been
synthesized. The synthesis of a homo-thymine hybrid consisted
of DNA and pPNA stretches has been also accomplished by the
phosphotriester method using O-nucleophilic intramolecular
catalysis (14). The sequences of the oligomers obtained are
shown in Table 2.
The solid phase synthesis of all oligomers was carried out
‘online’ using an automated synthesizer and adequately
derivatized CPG supports (Table 1). At each step, the 5-fold
excess of a monomer component over the resin capacity was used.
The coupling reactions between the monoester phosphonate unit
1a or 1b, and the terminal HO- or NH2 -group on the support were
performed in the presence of 1-(2,4,6-triisopropylbenzenesulfonyl)-3-nitro-1,2,4-triazole (TPSNT) as a condensing agent
with coupling yields of 95–98%. Similarly to the procedure
described by Jorba et al. for peptide synthesis (21), we used the
same condensing agent in the presence of 1-methylimidazole as
a nucleophilic catalyst for the amide bond formation between the
support-bounded terminal NH2-group and the COOH-group of
PNA unit 2a or 2b. With the use of this procedure, an average
yield on the step of amide bond formation was ∼95%. The dimer
synthons 9 and 12 were applied for the synthesis of chimeras
using the same protocols as for corresponding monomer building
units with 90–95% coupling yields. The deprotection of the
dimethoxytrityl group from the OH-function on the support was
performed by the action of 3% dichloroacetic acid in dichloromethane, where as the monomethoxytrityl group from amino
functions of PNAs, or pPNAs was removed by the action of 3%
pentafluorophenol in dichloromethane. We have found that the
latter reagent is strong enough to provide fast removal of
monomethoxytrityl group from the amino function, and at the
same time it practically does not interfere with the phosphonamide bonds. After a completion of the chain elongation, the
oligomers were deprotected starting from the removal of a
terminal trityl protecting group. In the case of oligomers
containing the terminal amino function, prior to the next
deprotection steps their free amino group was acetylated to
suppress possible side reactions, such as cyclization and nucleobase migration (22), during the following treatment of oligomers
under the basic conditions. 1-Oxido-4-alkoxy-2-picolyl phosphonate protecting groups were removed from the support-bound
oligomers by the treatment with triethylammonium thiophenolate
(14). The final deprotection of oligomers and cleavage from the
support was effected by ammonolysis. The oligomers were
purified by anion-exchange FPLC (Fig. 2) or gel-electrophoresis
in denaturing conditions (Table 2), and their purity and identity
were confirmed by reversed phase FPLC and mass spectrometry.
Hybridization and nuclease stability studies
The binding affinity of the PNA–pPNA chimeric oligomers as well
as pure pPNAs to complementary nucleic acid sequences was
investigated using ultraviolet melting experiments. The melting
temperatures (Tm) values of complexes formed by mimics with
complementary DNA and RNA targets are shown in Table 2. The
investigation of hybridization properties of pure pPNAs indicate that
they are able to form stable complexes with complementary DNA
and RNA fragments as well as with complementary PNA and pPNA
sequences. Thus, homo-thymine and homo-adenine pPNAs form
with complementary single-stranded DNA or RNA, complexes with
melting temperatures very close to those formed by DNA fragments,
but lower than Tm values for corresponding complexes formed by
PNAs. However, the thermal stability of complexes between pPNAs
containing mixed purine/pyrimidine sequenses and the
complementary single-stranded fragments of nucleic acids was
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Table 2. Properties of oligomers obtained in this study
a(t, a, c, g), PNA residues of type 9b; (t*, a*, c*, g*), PNA residues of type 9a; (T*,A*,C*,G*), pPNA residues of type 4a; (T’’, A’’, C’’, G’’), pPNA
residues of type 4b; d(NHT) and d(NHA), residues of 5′-aminonucleosides.
in denaturing 15% PAGE relative to dT15.
cMelting temperatures of complexes formed by oligomers with complementary DNA/RNA targets measured in 0.15 M NaCl/0.02 M Tris–HCl/0.01
M MgCl2/0.1 mM EDTA. In the cases of two transitions in Tm curves, the values corresponding to both transitions are given.
d∆T is the difference between dissociation and re-association temperatures obtained in melting-cooling experiments.
eYields of oligomers are given relative to the first nucleoside attached to the support and were estimated by spectroscopic analysis of a trityl function liberated
from the support.
bMobility
Figure 3. UV-Melting curves of complexes formed by mimics and oligonucleotides (Table 2). (A) Tm curves of complexes formed by mimics (s677, s686, s687 or
s694) with the complementary target d(TCATGGTGTCCTTTGCAG) and of that formed by the oligodeoxyribonucleotide (s638) with the same target. (B) Tm curves
of complexes between homo-Thy PNA (s659) and homo-Ade pPNA (s674), homo-Thy PNA (s659) and dA15, homo-Thy pPNA (s631) and dA15, homo-Thy pPNA
(s631) and homo-Ade pPNA (s674).
significantly lower than that of duplexes formed by non-modifed
oligonucleotides.
Ultraviolet melting studies indicate some increase in stability
of complexes formed by PNA–pPNA hybrids with DNA ( RNA)
targets with respect to the similar complexes formed by pure
pPNAs. In the case of homo-thymine mimics, molecules with
alternated PNA and pPNA residues gave more stable complexes
than the hybrids constructed from PNA and pPNA stretches.
Complexes of PNA–pPNA hybrids with complementary DNA
and RNA sequences had a considerably higher stability than
(DNA–pPNA)–DNA and (DNA–pPNA)–RNA complexes. At
the same time, PNA–DNA and PNA–RNA complexes were the
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Figure 4. Titration of oligodeoxyribonucleotide targets with complementary oligomers at 20C in 0.15 M NaCl/0.02 M Tris–HCl/0.01 M MgCl2/0.1 mM EDTA pH
7.0. (A) Titration of dA15 target by homo-Thy oligomers. (B) Titration of a 18mer d(TCATGGTGTCCTTTGCAG) target with complementary oligomers: pPNA
(s686), PNA–pPNA hybrid (s677), PNA (s675) and oligodeoxyribonucleotide (s638).
most stable. As could be expected, Tm values of complexes
formed by PNA–pPNA hybrids containing between two and four
pPNA residues, were higher as compared to those formed by
hybrids containing seven pPNA residues. It should be noted that
already the presence of two pPNA residues in a 14-unit PNA–
pPNA chain ensured good water solubility of a hybrid (>10 mg/
ml). Similar results were obtained with mimics and their hybrids
containing mixed purine-pyrimidine sequences (Fig. 3A).
In most of the cases, we observed only one transition in the
dissociation curves, whereas in some cases of homo-thymine
oligomers dissociation curves exhibited two transitions. In contrast
to dissociation curves, re-association (cooling) curves usually
exhibited transitions at lower temperatures than in the corresponding dissociation curves that is similar to the results observed for
pure PNAs (23). Hysteresis between main peaks in the dissociation
and re-association curves ranged from 3 to 25C (Table 2).
The control experiments with oligomers having
non-complementary sequences, or mismatches, have shown that
the interaction between chimeras and the complementary DNA
strands occurs in a sequence specific manner. Thus, we did not
detect the formation of a complex between pPNA oligomer
(s686) or PNA–pPNA hybrid (s687) and a complementary DNA
target containing two mismatches in the sixth and tenth positions
of the sequence. The introduction of one mismatch (a cytosine)
in the centre of dA15 target gave a drop in Tm values of
corresponding complexes with homo-thymine mimics of
12–15C (data not shown).
Binding of mimics to complementary oligonucleotides was
indicated by ∼15–20% hypochromicity upon mixing, and from the
titration data it can be concluded that in the case of homo-thymine
sequences a (PNA–pPNA)2–DNA (RNA) triplex is formed (Fig.
4A). In contrast to the data reported by Dr E.Uhlmann and
co-workers (9), we have found that homo-thymine pPNAs also form
triplexes with single-stranded DNA (and RNA) targets binding them
in 2:1 ratio. At the same time, the experiments on the titration of
oligonucleotides with mixed purine-pyrimidine sequences with
complementary pPNAs or PNA–pPNA hybrids revealed that they
form duplexes (Fig. 4B).
We have also evaluated the binding ability of homo-thymine and
homo-adenine pPNAs and PNA–pPNA hybrids with comp-
lementary PNA and pPNA targets. From the melting data, it was
apparent that these mimics bind each other quite efficiently, and the
stability of pPNA–PNA and PNA–pPNA–PNA complexes was also
relatively higher as compared to the corresponding complexes
formed with DNA, or pPNA targets (Fig. 3B).
To demonstrate the ability of PNA–pPNA hybrids as well as pure
pPNAs to give stable complexes with complementary nucleic acid
sequences, we also exploited shift mobility assays, which are
successfully used now for evaluation of complex formation between
DNA, RNA and their analogues (11,23). Gel-shift experiments
were performed with the use of non-denaturing 15% polyacrylamide
gels at 10C. The results of these experiments suggest that
PNA–pPNA hybrids and pPNA oligomers bind to complementary
DNA and RNA sequences and form stable complexes (Fig. 5). The
mobility of these complexes depends on the presence or the absence
of MgCl2 as well as on the nature of the corresponding target (DNA
or RNA). In general, complexes of pPNAs and PNA–pPNA hybrids
with DNA and RNA move on a gel slower than natural duplexes and
triplexes but faster than complexes of PNAs with nucleic acids.
Similarly, single-stranded pPNAs and PNA–pPNA hybrids run on
the gel slower than DNA fragments of the same length (see also
Table 2). In the case of complexes formed by homo-thymine mimics
with homo-adenine targets, it was found that their 1:1 mixture gave
a sufficient amount of the unbound target together with a slower
running band corresponding to a complex formed (Fig. 5, lanes 9,
10, 13 and 14), whereas in the 2:1 mimic target mixture the only
band of a complex is seen (Fig. 5, lanes 3–6). These results proved
the data obtained on UV-titration experiments that triplexes are
formed in the case of homo-pyrimidine pPNAs and PNA–pPNA
hybrids interaction with complementary targets. The similar experiments with pPNAs, or PNA–pPNA hybrids, containing mixed
pyrimidine-purine sequences confirmed the formation of duplexes
between these oligomers and complementary nucleic acids fragments (data not shown).
The examination of the stability of PNA–pPNA hybrids to
hydrolysis by exo- and endonucleases revealed that they were
completely resistant to the degradation by snake venom
phosphodiesterase and single-strand-specific S1 nuclease under the
conditions, in which the corresponding unmodified oligonucleotides
were totally degraded (Table 2). With these enzymes, the
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stranded fragments. They form more stable complexes than do
the equivalent pPNA and DNA–pPNA oligomers. Some increase
in stability of complexes formed by PNA–pPNA hybrids with
DNA and RNA targets with respect to the similar complexes
formed by pure DNA fragments was also observed. The hybrids
containing a few pPNA residues embedded into a PNA chain
form complexes with nucleic acid fragments with melting
temperatures comparable with those of complexes formed by
pure PNAs that makes them more promising for further
evaluation as potential therapeutic agents. The results obtained
indicate that homo-pyrimidine PNA–pPNA chimeras as well as
pure pPNAs form with the single-stranded complementary DNA
or RNA template triple helixes, whereas molecules with mixed
pyrimidine-purine sequences form duplexes. The extending of
these studies is aimed at further characterisation and development
of these and related molecules for binding and inhibition of viral
and cellular nucleic acid sequences.
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Figure 5. Electrophoresis in 15% native polyacrylamide gel in the presence
(A and B) or in the absence (C) of MgCl2 of complexes formed by homo-Thy
pPNAs and PNA–pPNA hybrids with dA15 and rA15 targets and those formed
by dT15 with oligo-A targets. Staining with ‘Stains all’.
degradation was monitored with the help of 20% denaturing PAGE
by the loss of the full length oligomer.
CONCLUSION
We have demonstrated that PNA–pPNA chimeras can be
automatically synthesized on solid phase using PNA and pPNA
monomers and specially constructed PNA–pPNA dimers. It was
confirmed that these chimeras have good water solubility and
hybridize specifically to complementary DNA and RNA single-
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