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.
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).
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 (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).
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.
Scheme 1.
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) [[delta]p = 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), [delta]p = 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), [delta]p = 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 (M + H)+ (C38H42N4O11P1 requires 761.7); 1H NMR (CDCl3) [delta] = 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-O-CH2), 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) [delta] = 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 [delta] = 1.18 (t, 9H, +NHEt3-CH3), 1.80 (s, 3H, Thy-CH3), 2.35 (m, 2H, MMTrNH-CH2), 2.90 (q, 6H, +NHEt3-CH2), 3.37 (m, 2H, MMTrNH-CH2-CH2), 3.75 (s, 3H, 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 [delta] = 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 [delta] = 1.18 (t, 9H, +NHEt3-CH3), 2.95 (q, 6H, +NHEt3-CH2), 3.30 (m, 2H, DMTrO-CH2), 3.38 (m, 2H, DMTrO-CH2-CH2), 3.75 (s, 6H, DMTr-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, 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 [delta] = 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) [delta] = 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 [delta] = 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), [delta]p = 14.7 (4a), or 15.3 (4b); for Ade derivatives: Rf (solvent A) = 0.72 (4a), or 0.65 (4b), [delta]p = 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), [delta]p = 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), [delta]p = 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) [delta] = 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), 5.40-5.62 (2xs, rotamers, 2H, N-CO-CH2), 6.65 (m, 1H, picolyl-H-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) [delta] = 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 [delta] = 1.12 (d, 6H, iBu-CH3), 1.20 (t, 9H, +NHEt3-CH3), 2.20 (m, 3H, MMTrNH-CH2 and iBu-CH), 2.95 (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, picolyl-H-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 [delta] = 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 [delta] = 1.20 (t, 9H, +NHEt3-CH3), 2.95 (q, 6H, +NHEt3-CH2), 3.30 (m, 2H, DMTrO-CH2), 3.45 (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), 5.10-5.22 (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, 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 [delta] = 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) [delta] = 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, picolyl-H-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 [delta] = 15.1 and 15.7.
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, [delta]p = 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 [delta] = 22.6 and 23.2.
To obtain pPNA-PNA dimer of type 9b (Scheme 2), 2 mmolofN-[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 [delta] = 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 [delta] = 15.0 and 15.7; 12b: Rf = 0.12 (solvent B); m/z (ES) = 996.8 (M + H)+ (C48H55N9O13P1 requires 997); 31P NMR [delta] = 15.2 and 15.7.
Table 1. 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 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.
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 0.5°C/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.
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 90°C for 2 min and slowly cooled to +10°C. Aliquots of the reaction mixtures (50 µl) were resolved at 10°C 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'.
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 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.
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.
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.
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 significantly lower than that of duplexes formed by non-modifed oligonucleotides.
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-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.
Nucleic Acids Research
Pages
Introduction
Materials And Methods
General methods for preparation of pPNA monomers
Preparation of PNA-pPNA dimers
Solid phase synthesis of oligonucleotides and mimics
Thermal denaturation and enzyme degradation experiments
Gel mobility assays
Results And Discussion
Synthesis of building blocks for construction of PNA-pPNA chimeras
Design and synthesis of chimeric oligomers
Hybridization and nuclease stability studies
Conclusion
References
bMobility 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[Delta]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.
REFERENCES
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