2650–2658 Nucleic Acids Research, 1998, Vol. 26, No. 11
1998 Oxford University Press
Novel internucleotide 3′-NH-P(CH3)(O)-O-5′ linkage. Oligo(deoxyribonucleoside methanephosphonamidates); synthesis, structure and hybridization properties Barbara Nawrot*, Malgorzata Boczkowska, Marzena Wójcik, Marek Sochacki, Slawomir Kazmierski and Wojciech J. Stec Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lódz, Poland Received February 13, 1998; Revised and Accepted April 17, 1998
ABSTRACT Diastereomeric dithymidine methanephosphonamidates (TnpmT) were synthesized by reaction of 3′-amino3′-deoxythymidine with 3′-O-acetylthymidin-5-ylmethanephosphonochloridate. Separated dinucleotide TnpmT(fast) and TnpmT(slow) diastereomers were used as building blocks to prepare chimeric dodecathymidylates, possessing one to four modified linkages, by means of phosphoramidite automated solid phase synthesis. As expected, the methanephosphonamidate internucleotide linkage is resistant to nuclease P1, snake venom PDE and 3′-exonuclease from human plasma. Degradation of dodecathymidylates possessing modified internucleotide linkages in alternate positions proved the ‘hopping’ properties of 3′-exonuclease. Oligo(deoxyribonucleotide methanephosphonamidates) were tested for their binding affinity to complementary oligomers in thermal denaturation experiments. All the oligomers showed lower binding affinity to DNA and RNA targets, however, oligomers originating from the TnpmT(fast) dimeric unit exhibited better hybridization properties than their diastereomeric TnpmT(slow) counterparts. A lowering of Tm of ∼2.4C (1.0–1.8C) was observed for each introduced TnpmT(fast) modification and 6.0C (4.2–5.0C) for each TnpmT(slow) modification in duplexes of modified dodecathymidylates with dA12 (A12) oligomers. The oligo(deoxyribonucleoside methanephosphonamidate) designated F4, possessing four modified methanephosphonate linkages originating from the TnpmT(fast) diastereomeric unit, exhibits a tendency for triplex formation, as was demonstrated in thermal denaturation experiments with the d(A21C4T21) hairpin oligomer. INTRODUCTION It is generally accepted that the high avidity of oligonucleotide analogs for complementary DNA or RNA strands promotes them as
good candidates for the antisense strategy (1). Other required properties of potential antisense therapeutics include a requirement for stability against endo- and exonucleases, enhanced cellular uptake and low affinity for proteins (2). With respect to the first generation of antisense constructs such expectations were met by oligo(nucleoside methanephosphonates) of the [RP] configuration (3,4), albeit that their limited solubility in physiological media and pharmacokinetics (fast influx and fast efflux; 5) limited their practical application. However, mixed backbone oligonucleotides possessing phosphorothioate core oligonucleotides flanked by short fragments of [RP] methanephosphonates have recently been presented as promising second generation antisense constructs (6,7). In parallel, Gryaznov et al. (8–12) introduced another second generation antisense construct possessing an internucleotide phosphoramidate 3′-HN-P(O)O–-O5′ backbone. N3′-P5′ phosphoramidate oligonucleotides, besides stability to phosphodiesterases, possess excellent hybridization properties towards RNA and single- or double-stranded DNA (9). The mostly appreciated property of N3′-P5′ oligonucleotides is their low affinity for proteins, in contrast to oligo(nucleoside phosphorothioates) (12). Taking into account the advantageous properties of [Rp] methanephosphonates and N3′-P5′ phosphoramidates it was tempting to prepare new oligonucleotides with incorporated structural motifs related to both aforementioned classes of oligomers, namely methanephosphonamidates. Here we describe synthesis of nucleotide dimers linked by a new class of internucleotide linkage, 3′-NH-P(CH3)(O)-O-5′. Analogous to methanephosphonates, non-stereospecific synthesis of dinucleoside methanephosphonamidates leads to a mixture of two, [Sp] and [Rp], diastereomers. Dinucleoside methanephosphonamidates are nuclease P1- and svPDE-resistant and relatively stable in basic and acidic media. Therefore, dinucleoside methanephosphonamidates are attractive building blocks for the synthesis of chimeric oligodeoxyribonucleotides designed as potential antisense agents. Several thymidine dodecamers with one to four modified internucleotide bonds, originating from particular diastereomeric dithymidine methanephosphonamidates, have been prepared. Their stability to nuclease P1 and 3′-exonuclease from human plasma as well hybridization properties are discussed.
*To whom correspondence should be addressed. Tel: +48 42 81 97 44; Fax: +48 42 81 54 83; Email:
[email protected]
2651 Nucleic Acids Acids Research, Research,1994, 1998,Vol. Vol.22, 26,No. No.111 Nucleic MATERIALS AND METHODS The nuclear magnetic resonance spectra were recorded using a Bruker AC-200 instrument at 200.13 MHz for 1H and 81.33 MHz for 31P. Chemical shifts (δ) are given in p.p.m. If not indicated samples were dissolved in CDCl3. The solvent signal was used as internal standard for 1H (δ CDCl3 = 7.26 p.p.m., δ CD3OD = 3.31 p.p.m.) and 85% H3PO4 as the external standard for 31P. ROESY spectra were recorded using a Bruker DRX 500 at 500.13 MHz for 1H in a 2 × 0.5k (F2 × F1) data point matrix. Total deviation of the spin lock was set to 350 ms. Data were processed with the SINE function in both dimensions. Zero filling up to 1k data points was applied in F1. No zero filling was applied in F2. Spectra were collected for samples dissolved in CDCl3 (5 mM). The LSIMS spectra (Cs+, 13 keV) were recorded on a Finnigan MAT 95 spectrometer and MALDI-TOF MS spectra were done on a Voyager-Elite instrument (PerSeptive Biosystems, CT) in reflector mode, at a resolution of 2000. Ultraviolet (UV) spectra were recorded on a Kontron Uvikon 860 spectrophotometer. Circular dichroism (CD) spectra were recorded on a Jobin Yvon CD 6 Dichrograph in 10 mM Tris, pH 7.5, 100 mM NaCl, 10 mM MgCl2. HPLC analyses were performed on an LDC Analytical System (pumps CM 3500 and CM 3200) SpectroMonitor SM4100 equipped with an Econosphere C18 column (4.6 × 25 mm; Alltech). The following solvent systems were used: method A, an acetonitrile gradient in 0.1 M NH4OAc (0–40% acetonitrile) over 20 min; method B, 0.1 M triethylammonium bicarbonate/ acetonitrile 0–40% gradient over 30 min, flow rate, 1 ml/min. Preparative purifications were performed on an LDC Milton Roy instrument equipped with a PRP-1 column (305 × 7.0 mm; Hamilton) eluted with a 0.1 M triethylammonium bicarbonate/ acetonitrile 0–50% gradient, flow rate 3 ml/min. Evaporations were carried out at 40C using a water or oil pump vacuum. Freeze drying was done in a Lyovac instrument. TLC was carried out on silica gel 60 F254 plates (Merck, Germany) in chloroform/ methanol; (A) 9/1 v/v, (B) 8/2 v/v. Thymidine was purchased from Pharma Waldhof (Germany). Melting curves were recorded with a GBC 916 UV/VIS spectrophotometer in 10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2. Snake venom phosphodiesterase (svPDE, EC 3.1.15.1) and alkaline phosphatase (EC 3.1.3.1) were from Boehringer Mannheim (Mannheim, Germany). Nuclease P1 (EC 3.1.30.1) was from Sigma. T4 polynucleotide kinase (EC 2.7.1.78) was from Amersham USA. Human plasma was isolated from human blood as described (13).
N-[5′-O-(4,4′-Dimethoxytrityl)-3′-amino-3′-deoxythymidin3′-yl]-O-(3′-O-acetylthymidin-5′-yl)-methanephosphonamidates (4a and 4b) and bis-O,O-(3′-O-acetylthymidin5′-yl)-methanephosphonate (4c) 3′-O-Acetylthymidine (1, 1.7 mmol, 483 mg) was dissolved in anhydrous pyridine (5 ml) and added at 0C to the stirred solution of dichloromethanephosphonate (2.5 mmol, 340 mg) (14) in anhydrous pyridine (5 ml). After 15 min 5′-O-DMT3′-amino-3′-deoxythymidine (3, 1.7 mmol, 937 mg) (10) in anhydrous pyridine (5 ml) was added dropwise to the reaction mixture and stirred for 2 h at room temperature. Addition of chloroform (300 ml) followed by subsequent washings with saturated sodium bicarbonate (2 × 100 ml) and water (100 ml) and
2651
concentration in vacuo afforded a mixture of products which were separated on silica gel by flash column chromatography with a gradient (0–5%) of methanol in chloroform. 4a (110 mg, 7.3%), 4b (138 mg, 9.1%) and 4c (131 mg, 10.5%) were obtained. Rf, 31P NMR and MS data for dinucleotides 4a, 4b and 4c are given in Table 1. 1H NMR data are available in Supplementary Material. Removal of acetyl protecting group from 4a and 4b Protected dimer 4a (120 mg) was treated with concentrated ammonium hydroxide (7 ml)/ethanol (7 ml) mixture and kept for 17 h at 4C. Removal of solvents in vacuo and subsequent co-evaporation with anhydrous toluene (3 × 5 ml) followed by flash column chromatography purification on silica gel in a methanol/chloroform gradient (0–7 %) afforded 5a (103 mg, 90%). Analogous deprotection of the acetyl group of 4b gave 5b with a yield of ∼90%. Selected spectral and chromatographic data for 5a and 5b are given in Table 1. 1H NMR data are available in Supplementary Material. Removal of the DMT protecting group from 5a and 5b Deprotection was performed with 80% acetic acid at 50C for 20 min. After freeze drying the products were purified by flash column chromatography on silica gel in a methanol/chloroform (0–10%) gradient. Deprotected dimers 6a and 6b were obtained with yields >90%. Selected spectral and chromatographic data for 6a and 6b are given in Table 1. 1H NMR data are available in Supplementary Material. Table 1. Spectral and chromatographic characteristics of dithymidine methanephosphonamidates 4–7 Dimer TnpmT
TLC mobilitya
31P-NMRc
δ (p.p.m.)
JP–Me (Hz)
FAB MS Calculated
Found
4a
0.60
34.19
16.57
886.3064
886.3063
4b
0.51
34.78
16.48
886.3064
886.3068
4c
0.63
33.03
17.50
628.1782
628.1729
5a
0.29
35.13
16.70
844.2959
844.2935
5b
0.29
36.15
16.55
844.2959
844.2927
6a
0.11b
36.96d
16.68
542.1641
542.1652
6b
0.12b
37.30d
16.67
542.1641
542.1652
7a
0.57
34.53
1044
1043.5 [M-H]+
34.23 149.44 149.22e 7b
0.54
34.55 34.44
aIf
1044
1045.2 [M+H]+
149.64
1043.0
149.06e
[M-H]+
not indicated otherwise, TLC was determined in solvent system (A). system (B), as described in Materials and Methods. cIf not indicated otherwise, NMR spectra were recorded in CDCl . 3 dIn CD OD. 3 eIn methylene chloride. bSolvent
2652 Nucleic Acids Research, 1998, Vol. 26, No. 11 Table 2. Sequences and HPLC and MS data for oligo(deoxyribonucleoside methanephosphonamidates) F1–F4 and S1–S4 ODN T12 F1 F2 F3 F4 S1 S2 S3 S4 aAnalytical
Parent dimer TnpmT
4a, fast 4a, fast 4a, fast 4a, fast 4b, slow 4b, slow 4b, slow 4b, slow
HPLCa (Rt/purity)
Oligonucleotide sequence TpTpTpTpTpTpTpTpTpTpTpT TpTpTpTpTpTnpmTpTpTpTpTpT TpTpTpTpTnpmTpTnpmTpTpTpTpT TpTpTpTnpmTpTnpmTpTnpmTpTpTpT TpTpTnpmTpTnpmTpTnpmTpTnpmTpTpT TpTpTpTpTpTnpmTpTpTpTpTpT TpTpTpTpTnpmTpTnpmTpTpTpTpT TpTpTpTnpmTpTnpmTpTnpmTpTpTpT TpTpTnpmTpTnpmTpTnpmTpTnpmTpTpT
Min
%
16.55 17.05 17.25 17.20 16.80 17.08 17.02 17.30
98.0 >99.0 >99.0 >99.0 99.0 >99.0 >99.0 >99.0
MALDI-TOF MS Calculated Found 3587.6 3587.6 3584.6 3583.8 3581.6 3581.3 3578.6 3578.7 3575.6 3575.7 3584.6 3584.6 3581.6 3581.1 3578.6 3578.7 3575.6 3575.9
purity determination was performed chromatographically on a C18 column according to the conditions described in Materials and Methods.
Phosphitylation of 5a and 5b 2-Cyanoethyl tetraisopropylphosphorodiamidite (75 mg, 0.21 mmol) was added under argon to a solution of dimer 5a (175 mg, 0.2 mmol) and IH-tetrazole (84 mg, 0.2 mmol) in anhydrous methylene chloride (2 ml). The reaction mixture was stirred for 3 h at room temperature and then loaded under argon onto a silica gel column. The product was eluted with a gradient (0–5%) of methanol in methylene chloride, concentrated in vacuo and stored under argon at –20C. Yield of 7a 193 mg (92%). Phosphitylation of 5b was done analogously to afford 7b (160 mg, 71%). Selected spectral and chromatographic data for 7a and 7b are given in Table 1. Oligonucleotide synthesis Modified dodecathymidylates, F1–F4 and S1–S4, were prepared by the phosphoramidite method on an ABI 391 synthesizer at the 0.5–1.0 µmol scale. Dinucleotide phosphoramidities 7a and 7b were used as solutions in acetonitrile at concentrations of 0.08–0.12 M. Purification of all oligonucleotides was carried out by two-step RP-HPLC (DMT-on and DMT-off) (15). Oligonucleotide labeling Oligonucleotides listed in Table 2 were 5′-end-labeled with [γ-32P]ATP and T4 polynucleotide kinase according to Koziolkiewicz et al. (13).
incubated for 15 h at 37C. The products were analyzed by means of HPLC according to method A. Assay for enzymatic digestion of dodecathymidylates F1–F4 and S1–S4 Dodecathymidylates F1–F4 and S1–S4 (0.1 mM) were dissolved in 100 mM Tris–HCl, pH 7.2, and 1 mM Zn2+ buffer (20 µl) and incubated with nuclease P1 (2 µg) at 37C for 18 h. Then the reaction mixture was incubated with alkaline phosphatase (2 µg) at 37C for 3 h. The enzymes were denaturated by heating at 95C for 10 min and the reaction mixture was centrifuged for 10 min at 10 000 r.p.m. The reaction products were analyzed by means of HPLC according to method A. Assay for digestion of oligonucleotides with 3′-exonuclease from human plasma The samples of oligonucleotides (10 µM) in phosphate-buffered saline, pH 7.5, were mixed with an equal volume of human plasma and incubated at 37C. At various times (0, 20, 60, 120 and 240 min) 10 µl aliquots were withdrawn and the enzymatic reaction was quenched by heating for 2 min at 95C. Then, 100 µl water were added to each denatured sample. After vigorous shaking, the protein precipitates were spun down and the aqueous solutions were dissolved in formamide containing 0.03% bromophenol blue and 0.03% xylene cyanol (5–8 µl) and analyzed by 20% polyacrylamide/7 M urea gel electrophoresis. Melting experiments
Assay for enzymatic digestion of diastereomers 6a and 6b Diastereomers 6a or 6b (0.57 mM) was dissolved in 100 mM Tris–HCl, pH 7.2, and 1 mM Zn2+ buffer (100 µl) or in 100 mM Tris–HCl, pH 8.0, and 20 mM MgCl2 (100 µl) and incubated with nuclease P1 (2 µg) or with svPDE (2 µg) respectively at 37C for 15 h. The enzyme was denaturated by heating at 95C for 10 min and the reaction mixture was centrifuged for 10 min at 10 000 r.p.m. (Biofuge 13; Heraeus). The reaction products were analyzed by means of HPLC according to method A. Assay for stability of diastereomers 6a and 6b at pH 3 and pH 11 Diastereomers 6a or 6b (0.57 mM) were dissolved in 100 mM ammonium hydroxide/acetic acid pH 3 or pH 11 buffer and
Melting temperature measurements of duplexes obtained by hybridization of modified oligomers F1–F4 and S1–S4 with the dA12 target were performed in 10 mM Tris–HCl, pH 7.5, 100 mM NaCl and 10 mM MgCl2 with various oligonucleotide concentrations (1 × 10–6–6 × 10–6 M). Measurements of melting temperatures of oligomers F2–F4 and S2–S3 with A12 were done at a concentration of 3 × 10–6 M duplex. Triplex melting experiments for oligonucleotides T12, F4 or S4 with the d(A21C4T21) hairpin oligonucleotide were carried out in 10 mM Tris–HCl, pH 7.5, 0.5 M NaCl and 10 mM MgCl2 at a concentration of 2 × 10–6 M each oligomer. Oligonucleotides at the appropriate concentrations based on UV 260 nm absorbance were mixed and heated at 60C for 5 min, allowed to cool to room temperature and kept overnight at 4C. Melting profiles were recorded from 3 to 80C with a temperature gradient of
2653 Nucleic Acids Acids Research, Research,1994, 1998,Vol. Vol.22, 26,No. No.111 Nucleic
2653
Scheme 1.
0.2C/min. All measurements were carried out in a 1 cm path length cell with a GBC UV/VIS 916 spectrophotometer equipped with a Peltier Thermocell.
phosphoramidities 7a and 7b in 92 and 71% yield respectively (Scheme 1). Structure of dinucleoside methanephosphonamidates
RESULTS Synthesis of dithymidine methanephosphonamidates A one-pot strategy has been chosen for the synthesis of dimeric nucleotides with a modified internucleotide bond. As monomeric units we used 5′-O-DMT-protected 3′-amino-3′-deoxythymidine 3 (10) and 3′-O-acetyl-protected thymidine 1 (Scheme 1). The applied standard protecting groups allowed the use of basic deprotection for the removal of acetyl groups and further functionalization of the 3′-end of the dimer, while the acid-labile dimethoxytrityl group at the 5′-end was suitable for solid phase synthesis of oligomers. Reaction of 3′-O-acetylthymidin-5′-ylmethanephosphonochloridate 2 with 3′-amino-3′-deoxythymidine 3 resulted in a mixture of two diastereomeric dimers, 4a [TnpmT(fast), TLC-fast migrating diastereomer] and 4b [TnpmT(slow), TLC-slow migrating diastereomer] as well as symmetrical bis-(3′-O-acetylthymidyn-5′-yl)-methanephosphonate 4c in the ratio 1:1:2. Diastereomers 4a and 4b and by-product 4c were separated by column chromatography on silica gel in a methanol/chloroform gradient. The total yield of isolated products was 60C represents transition of the hairpin structure to single-stranded oligomer (Fig. 4). All the melting profiles for T12/d(A21C4T21), F4/d(A21C4T21) and S4/d(A21C4T21) triplexes are similar in this region. However, the profiles of those melting curves are significantly different at lower temperatures (3–50C, insert in Fig. 4). The curve observed for oligonucleotide F4 complexed with hairpin oligomer d(A21C4T21) clearly shows an upper part of triplex–duplex transition, in contrast to the lack of such clear transitions for the remaining complexes. Despite a low binding affinity of the dodecathymidylates, due to the length of the chain, for hairpin d(A21C4T21), F-type oligodeoxyribonucleotide modification enhances triplex formation (34). In contrast, S-type modification,
2658 Nucleic Acids Research, 1998, Vol. 26, No. 11 present in oligomer S4, does not improve its affinity for the hairpin d(A21C4T21) and decreases triplex stability, as compared with the unmodified triplex structure. Heating and cooling curves (0.2C/min) recorded for T12/d(A21C4T21) or F4/d(A21C4T21) triplexes do not exhibit any significant hysteresis for triplex–duplex transitions (data not shown). Further hybridization studies on longer chimeric oligo(deoxyribonucleoside methanephosphonamidates) concerning their tendency to form triplex structures are in progress. CONCLUSIONS Diastereomeric dithymidine methanephosphonamidates 4a and 4b were obtained by reaction of 3′-amino-3′-deoxythymidine with 3′-O-acetylthymidin-5-yl-methanephosphonchloridate. Successful separation of the diastereomers was achieved by silica gel column chromatography with a methanol/chloroform gradient, giving rise to fast migrating diastereomer 4a [TnpmT(fast)] and slow migrating diastereomer 4b [TnpmT(slow)]. By comparison with the stereochemistry of dinucleoside methanephosphonates tentative assignment of the absolute configuration at the phosphorus atom of [Rp] for diastereomer 4a and [Sp] for diastereomer 4b is proposed. Selective removal of the 3′-terminal protecting group from 4a and 4b and subsequent phosphitylation with 2-cyanoethyl tetraisopropylphosphordiamidite gave 7a and 7b respectively, which were used as building blocks for automated solid phase synthesis of oligodeoxyribonucleotides. Chimeric dodecathymidylates F1–F4 and S1–S4, possessing one to four modified linkages originating from diastereomers TnpmT(fast) or TnpmT(slow) respectively, were obtained. Stability of dimeric methanephosphonamidates 6a and 6b to nuclease P1 and svPDE as well stability of dodecathymidylates F1–F4 and S1–S4 to nuclease P1 and 3′-exonuclease from human plasma show a complete resistance of the methanephosphonamidate linkage to nucleolytic degradation. The binding affinities of chimeric dodecathymidylates for single-stranded DNA and RNA as well as double-stranded DNA strongly depend on the stereochemistry of the methanephosphonamidate units. In general, both chimeric dodecathymidylate series possess lower binding affinities for complementary single-stranded oligonucleotides in comparison with the binding properties of non-modified dodecathymidylate. However, chimeric dodecathymidylates F1–F4, originating from diastereomer TnpmT(fast), exhibit better hybridization properties in comparison with dodecathymidylates S1–S4, originating from diastereomer TnpmT(slow). Moreover, chimeric dodecathymidylate F4, possessing four methanephosphonamidate linkages in alternate positions, exhibits a higher binding affinity for hairpin d(A21C4T21) in comparison with the T12 and S4 oligomers. Modification of the oligodeoxyribonucleotide phosphate backbone with F-type methanephosphonamidate linkages enhances triplex stability. In summary, the stability against enzymatic degradation and the configurational differentiation with respect to binding affinity of [Rp] and [Sp] oligo(deoxyribonucleoside methanephosphonamidates) for single-stranded DNA and RNA as well doublestranded DNA could be of interest for further investigation. ACKNOWLEDGEMENTS This project was supported by The State Committee for Scientific Research (KBN) grant 4P05F 02310 (to W.J.S.). The authors are indebted to Mrs Wieslawa Goss for excellent technical assistance.
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