Acyclic analogs of nucleosides based on tris(hydroxymethyl)phosphine oxide: synthesis and incorporation into short DNA oligomers

Synthesis of protected THPO-acyclic nucleoside phosphoramidites 11

Protection of two hydroxyl groups in THPO

For selective protection of only one hydroxyl group, tris(hydroxymethyl)phosphine oxide 5 was treated with one molar equivalent of 4,4′-dimethoxytrityl chloride (DMT-Cl) in pyridine at room temperature (Scheme 1). The O-DMT derivative 6 was obtained in 51% yield after silica gel chromatography. The second hydroxyl group in 6 was protected with tert-butyldimethylsilyl (TBDMS) moiety upon treatment with one molar equivalent of TBDMS-Cl (in the presence of a four-fold molar excess of imidazole) furnishing compound 7A (R = TBDMS). Compound 6 was also treated with benzoyl chloride (Bz-Cl) in pyridine to yield the O-DMT-O′-Bz derivative 7B(R = Bz). When 5 was allowed to react with a two-fold molar excess of DMT-Cl, O,O′-di-DMT derivative 7C (R = DMT) was obtained in good yield.

Scheme 1

Transformation of tris(hydroxymethyl)phosphine oxide into acyclic nucleoside phosphoramidites.

Conditions: (i) 1 eq. DMT-Cl/pyridine, 20 h, room temperature (RT); (ii) for 7A: TBDMS-Cl, imidazole, CH₃CN, 48 h, RT; for 7B: BzCl, pyridine, 24 h, RT; for 7C: 2 eq. DMT-Cl/pyridine, 20 h, RT; (iii) DMAP, p-TsCl/CH₂Cl₂, 2 h, 0–5°C; (iv) cytosine, NaH, DMF, 60°C, 3 h; (v) BzCl, pyridine, 24 h, RT; (vi): N3-benzoylthymine, N6-benzoyladenine or N2-isobutyryl-O6-diphenylcarbamoylguanine, PhP₃, DIAD, THF, 48 h; (vii): for 8a,c,d: 1 m (t-Bu)₄NF in THF, 24 h, RT; for 8a′: 28% aq. NH₄OH, 24 h; for 8b: 1 M p-TsOH/MeOH, TLC control, pyridine; (viii) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, CH₂Cl₂, RT.

Coupling of the protected THPO derivatives with nucleobases

Compound 7A was used as a starting material for the PPh₃/DIAD promoted coupling (the Mitsunobu reaction conditions) [26] with N3-benzoyl-thymidine [27], N6-benzoyl-adenine [28, 29], or N2-isobutyryl-O6-diphenylcarbamoyl-guanine [30, 31]. The first reaction gave the derivative 8a (B^PG= N3-Bz-Thy) in 95% yield (Table 1). Similarly effective was the condensation of 7B with the same nucleobase, which produced 8a′.

Table 1

Spectral characteristics and yields of THPO derivatives 8, 10 and 11.

Compound	R	31P NMR δ (ppm)	FAB MS (m/z)		Yield (%)
			[M+H]+	[M-H]-
8a	TBDMS	38.52	912.8d	758.5	92a
8a′	Bz	40.87	921.5d	767.5	95a
8b	DMT	41.15	942.2	940.3	44b
8c	TBDMS	42.02	778.5	776.4	51
8d	TBDMS	40.89	955.2	953.2	90
10a		38.40	–	653.5	38
10b		45.15	640.3	638.2	60
10c		42.39	664.3	662.0	84
10d		36.92	841.5	839.2	51
10e		45.46	551.6	549.0	60c, 41c
10f		43.64	646.4	644.3	40
11b		152.54; 152.34; 152.03f 39.76; 39.64; 39.46; 39.33g	NDe	ND	45
11c		151.88; 151.72; 151.57f 151.41; 38.10; 37.77g	ND	ND	77
11e		150.95; 150.71; 150.64;f 37.20; 37.02; 36.90; 36.72g	ND	ND	70
11f		151.91; 151.59; 151.27;f 39.54; 39.22; 38.99; 38.68g	ND	ND	41

^aThe product was in ca. 30% contaminated with Ph₃PO.^bThe combined yield of transformations 9→8g→8b. ^cThe yields of two transformations: 8a→10e – 60%, and 8a′→10e – 41%. ^dThe [M+153]⁺ ion was registered for 8a and 8a′ in a complex with m-nitrobenzyl alcohol (NBA) matrix molecule (molecular mass 153). ^eND – Not determined. ^fResonances at δ>150 ppm correspond to the phosphorus atom in the phosphoramidite part of the molecule. ^gResonances at δ<40 ppm correspond to the phosphorus atom in the THPO part of the molecule.

The coupling of 7A with N6-benzoyl-adenine was more challenging, since two products (most likely regioisomers) at ca. 1:1 ratio were obtained. Chromatographic separation delivered the required N9-adenine derivative 8c (fast-eluting, B^PG= N6-Bz-Ade) in 51% yield. Its structure was confirmed by a 2D NMR analysis in a heteronuclear multiple bonds coherence (HMBC) experiment, since the spin-spin coupling between the hydrogen atom at C1′ in the THPO moiety and C4 and C8 atoms of adenine confirmed the presence of the P-C-N9 linkage (Figure 2).

Figure 2

The detected spin-spin coupling (by a HMBC 2D NMR experiment) between hydrogen atoms at C1′ in the THPO moiety and selected neighboring carbon atoms in 8c, 8g, and 10e.

The reaction of 7A with N2-isobutyryl-O6-diphenylcarbamoylguanine furnished two products at a 95:5 ratio (as determined by ³¹P NMR), and the major product (efficiently isolated in 90% yield by silica gel chromatography) was the required N9-derivative 8d (B^PG= N2-iBu-O6-Dpc-Gua) the structure of which was confirmed as previously described [25].

Unfortunately, under the Mitsunobu reaction conditions, N4-benzoylcytosine did not react with any compound 7 and the required compound 8b was not obtained, probably because of insufficient acidity of the N1-H function. Therefore, cytosine was treated with NaH in DMF to generate anion, which was allowed to react with O,O′-bis-DMT-O″-tosyl-derivative 9 (obtained from 7C by routine tosylation with tosyl chloride) furnishing a 95:5 mixture of regioisomers. The required N1-substituted regioisomer 8g (B^PG= Cyt) was isolated by silica gel column chromatography and its structure was confirmed by 2D HMQC and HMBC analysis, as shown previously [25] (Figure 2). It was further benzoylated at the exoamine function by treatment with benzoyl chloride in pyridine to yield 8b (B^PG= N4-Bz-Cyt).

Synthesis of chimeric oligomers containing the THPO motif

Phosphoramidite derivatives of THPO-based acyclic nucleosides 11b, 11c, 11e and 11f, together with commercial phosphoramidites of suitably protected thymidine, 2′-deoxycytidine, 2′-deoxyadenosine and 2′-deoxyguanosine were used for the automated solid phase synthesis of chimeric DNA oligomers 12-21 (Table 2, the lower-case italicized letters a, c, g and t represent the modified acyclic units bearing Ade, Cyt, Gua or Thy nucleobases, respectively) [32]. The syntheses were performed on a succinyl-linked LCAA CPG solid support using an ABI 394 synthesizer (Applied Biosystems Inc., Foster City, CA, USA). The only modification made in the manufacturer’s protocol was a prolonged coupling time (up to 600 s) applied for the monomers 11. For monomers 11c and 11e the coupling efficiency (determined by the DMT-cation assay) was in the range of 98–99%, while for 11b and 11f the efficiency dropped below 90%.

After synthesis of 12-19, standard cleavage from the solid support and deprotection of phosphate groups and nucleobases was done (28% aqueous NH₄OH, 55°C, 16 h). Notably, in the case of fully modified decamers 20 and 21, containing only the THPO-based units a, c, g, or t), an LCAA-CPG support with a universal linker was used [33], for which the synthesis started with a phosphoramidite monomer 11e. The assembled oligomers were then cleaved from the support with a 20% ethanolic solution of gaseous ammonia (55°C, 4 h), followed by the treatment with 28% aq. NH₄OH/40% aq. MeNH₂ (room temperature, 16 h) [34, 35].

To purify the oligomers, a two-step RP-HPLC (DMT-on/DMT-off) was applied [36]. The DMT-tagged oligomers (isolated during the first step) were detritylated with 50% aq. acetic acid and purified in the DMT-off form. The respective retention times (R_t) are given in Table 2. Illustrative RP-HPLC profiles for 5′-T₉gT₉-3′ (16) in its DMT-on and DMT-off forms are shown in Figure 3. The structures of all oligomers listed in Table 2 were confirmed by MALDI-TOF mass spectrometry.

Figure 3

Reverse-phase HPLC profiles for purification of 16 in its DMT-on (A) and DMT-off form (B), and for DMT-21 oligomer (C).

Interestingly, the RP-HPLC profiles recorded for DMT-20 and DMT-21 showed two closely eluted main peaks of similar intensity (Figure 3C). These four products (two pairs: DMT-20-fast and DMT-20-slow; DMT-21-fast and DMT-21-slow) were collected separately and detritylated. Interestingly, within each pair of fully deprotected products the RP-HPLC retention times were almost identical and mass spectrometry analysis showed identical m/z values (Table 2). Moreover, all four detritylated samples exhibited identical electrophoretic mobility in 20% polyacrylamide/7 m urea gel (data not shown). Therefore, we concluded that each pair consists of molecules of the same sequence, which differ in absolute configuration of the P-stereogenic centers present in the THPO acyclic nucleotide first from the 5′-end. Similar observations were noted in earlier works on P-chiral, 5′-DMT protected oligo(nucleoside phosphorothioate)s [37]. Perhaps for the same reason, a relatively small difference in the chromatographic mobility was also seen after removal of the DMT group (Table 2). This assumption is supported by reports that short oligo(nucleoside phosphorothioate)s (trimers, tetramers and pentamers) were chromatographically separated into diastereomeric species but the efficiency of this process strongly depended upon the sequence of nucleobases and composition of the buffered eluent [38].

Physicochemical characterization of oligomers 12-21

Circular dichroism analysis of duplexes formed by oligomers 20 and 21

The helical structures of duplexes formed by 20 and 21 with complementary DNA templates, as well as the structure of the homo-THPO duplex 20/21, were examined by circular dichroism spectroscopy (CD) (Figure 4). The CD spectra for 20 and 21 alone indicate that these single-stranded oligomers do not adopt any ordered helical structure. To analyze the duplexes, the 1:1 equimolar mixtures of complementary oligomers (in 10 mm Tris-HCl, pH 7.4, 10 mm MgCl₂, 100 mm NaCl buffer) were annealed by slow cooling from 95°C, down to the room temperature. The CD spectra recorded for 20/5′-d(ATAATTAAAT)-3′ and 21/5′-d(ATTTAATTAT)-3′ duplexes as well as for the 20/21 duplex exhibit positive Cotton effects at λ = 276 nm, and negative Cotton effects at λ = 253 nm. All three spectra are characteristic for the B-type DNA duplex helical structures, with the most ordered structure of the 20/21 duplex. Interestingly, in CD spectra of 20/21 and 21/5′-d(ATTTAATTAT)-3′ the two isoelliptic points are present at similar wavelengths (at 235 and 265 nm), while the CD spectrum of 20/5′-d(ATAATTAAAT)-3′ very much differs from those previously mentioned, especially at the short-wave region (220–250 nm, no isoelliptic point below 260 nm). This difference is quite surprising because at first glance 20 and 21 differ by only two nucleobases (4×Ade, 6×Thy vs. 6×Ade, 4×Thy).

Figure 4

The CD spectra of the duplexes: 20/21(circles), 20/5′-d(ATAATTAAAT)-3′ (triangles) and 21/5′-d(ATTTAATTAT)-3′ (crosses), as well as single stranded THPO-oligomers 20 and 21(solid line) (2 μm duplex in a 10 mm Tris-HCl, pH 7.4, 10 mm MgCl₂, 100 mm NaCl buffer).

Stability of the oligomer 13 (5′-T₈tttT₈-3′) towards selected 5′- and 3′-exonucleases

Chimeric oligomer 13 (5′-T₈tttT₈-3′), possessing three THPO-thymine units (t) located in the central part of the homothymidine nonadecamer, was tested as a substrate for 3′- and 5′-exonucleases, i.e. for snake venom phosphodiesterase (svPDE or PDE I) and for calf spleen phosphodiesterase (csPDE, PDE II), respectively. The digestion products were identified by MALDI-TOF MS analysis (Figure 5). From the samples of 13 treated with PDE I (reaction A) or PDE II (reaction B) small aliquots were taken after 15, 45 and 60 min, or after 10, 30 and 90 min, respectively.

Figure 5

MALDI-TOF mass spectrometry analysis of the products of the enzymatic hydrolysis of oligomer 13 with snake venom phosphodiesterase (PDE I, panel A) and with calf spleen phosphodiesterase (PDE II, panel B).

The time of incubation is indicated above the consecutive plots.

Both sets of spectra show the ladders of products, which differ by m/z 304, corresponding to the products of consecutive removal of _PT (with PDE I) or T_P (with PDE II) from the parent oligonucleotide 13 (m/z 5732). After 45 min of the reaction A, accumulation of the oligonucleotide 5′-T₈ttt-3′ (a band at m/z 3298) was observed. No further degradation of this product was observed over next 6 h (data not shown). After 60 min of the reaction B, accumulation of the oligonucleotide 5′-TtttT₈-3′ (a band at m/z 3602) was observed, so the reaction stopped at the last natural nucleotide upstream of the modification site. Thus, the THPO modified unit inserted within the DNA chain is recognized by both 3′- and 5′-exonucleases tested. Consequently, the use of the THPO units at the 3′- and 5′-ends of therapeutic nucleic acids will make them resistant towards cellular exonucleases and will improve their pharmacokinetics. Similar abasic phosphinic acid ‘clamps’(BHPA, 3) were already successfully applied in in vitro experiments for protection of deoxyribozymes (directed towards the HIV-1 viral RNA sequence) in HIV-1 infected cells (unpublished results), and of deoxyribozymes designed to cleave bcr-abl mRNA fragments [44].

Synthesis of an O-DMT derivative of THPO (6)

To a solution of 10 g (0.07 mol) of tris(hydroxymethyl)phosphine oxide (5) in pyridine (100 mL) 4,4′-dimethoxytrityl chloride (DMT-Cl) (24 g, 0.07 mol) was added and the mixture was stirred for 20 h at room temperature. Pyridine was evaporated, the residue was (three times) dissolved in ethanol and evaporated to dryness. This step was repeated with toluene. The desired product 6 was obtained in 51% yield (16 g, 0.036 mol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→97:3, v/v, and 0.1% vol. of Et₃N). ¹H NMR (200 MHz, CDCl₃): δ 7.41–6.74 (m, 13H, aromatic protons), 4.19 (m, J_P-H= 3 Hz, 4H, CH₂OH), 3.74 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 3.64 (d, J_P-H= 6 Hz, 2H, CH₂ODMT); ³¹P NMR (CDCl₃): δ 46.51; MS FAB (m/z): 544.3 [M+102 (Et₃NH)]⁺.

Synthesis of an O-DMT-O′-TBDMS derivative of THPO (7A)

To a solution of 2 g (4.5 mmol) of 6 in acetonitrile (60 mL), imidazole (1.2 g, 18 mmol) was added, followed by TBDMS-Cl (0.68 g, 4.5 mmol), and the mixture was stirred for 20 h at room temperature. Then, triethylamine (5 mL) was added and the solvent was evaporated. The desired (DMT-oxymethyl)(tert-butyldimethylsilyloxymethyl)(hydroxymethyl)phosphine oxide 7A was obtained in 45% yield (1.1 g, 2.0 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→95:5, v/v). ¹H NMR (200 MHz, CDCl₃): δ 7.46–6.80 (m, 13H, aromatic protons), 4.14–4.07 (m, 4H, CH₂OTBDMS), 3.78 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.62–3.53 (m, 2H, CH₂ODMT), 0.81 (s, 9H, 3×CH₃), 0.06 (s, 3H, Si-CH₃), 0.02 (s, 3H, Si-CH₃); ³¹P NMR (CDCl₃): δ 44.55; MS FAB (m/z): 579.4 [M+Na]⁺, 709.3 [M+153]⁺.

Synthesis of an O-DMT-O′-Bz derivative of THPO (7B)

To a solution of 3.5 g (7.8 mmol) of 6 in pyridine (50 mL) benzoyl chloride (1.1 g, 7.8 mmol) was added and the mixture was stirred for 16 h at room temperature. Pyridine was evaporated, the residue was (three times) dissolved in ethanol and evaporated to dryness. This step was repeated with toluene. The desired (DMT-oxymethyl)(benzoyl-oxymethyl)(hydroxymethyl)phosphine oxide 7B was obtained in 49% yield (2.1 g, 3.9 mmol) by chromatographic separation on a silica gel column (CHCl₃:MeOH, 100:0→95:5, v/v). ¹H NMR (CDCl₃): δ 8.12–6.73 (m, 18H, aromatic protons), 4.90–4.77 (m, 2H), 4.24 (s, 2H), 3.79–3.73 (m, J = 7 Hz, J = 3 Hz, 2H), 3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃); ³¹P NMR (CDCl₃): δ 42.40; FAB MS (m/z): 699.2 [M+153]⁺.

Synthesis of a O,O′-di-DMT derivative of THPO (7C)

To a solution of 1.4 g (10 mmol) of tris(hydroxymethyl)phosphine oxide (5) in pyridine (150 mL) 4,4′-dimethoxytrityl chloride (DMT-Cl) (6.8 g, 20 mmol) was added and the mixture was stirred for 20 h at room temperature. Pyridine was evaporated, the residue was (three times) dissolved in ethanol and evaporated to dryness. This step was repeated with toluene. The desired (bis-(DMT-oxymethyl))(hydroxymethyl)phosphine oxide 7C was obtained in 50% yield (3.11 g, 5 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→98:2, v/v). ¹H NMR (200 MHz, CDCl₃): δ 7.72–6.51 (m, 26H, aromatic protons), 4.02 (d, J = 4.7 Hz, 2H), 3.90–3.80 (m, 2H), 3.77 (s, 6H, 2×OCH₃), 3.76 (s, 6H, 2×OCH₃), 3.54 (dd, J = 5.5 Hz, J = 12.4 Hz, 2H); ³¹P NMR (200 MHz, CDCl₃): δ 43.87; FAB MS (m/z): 767.3 [M+Na]⁺, 897.6 [M+153]⁺.

Synthesis of an (N2-isobutyryl-O4-diphenylocarbamoyl-guanine) derivative of 7A (8d)

A suspension of N2-isobutyryl-O6-diphenylocarbamoyl-guanine (163 mg, 0.39 mmol) in anhydrous THF was heated under reflux for 20 min and cooled to room temperature. Then 7A (166 mg, 0.3 mmol), triphenylphosphine (165 mg, 0.63 mmol) and DIAD (diisopropyl azodicarboxylate) (124 μL, 0.63 mmol) were added (argon atmosphere, daylight protected). After 48 h the solvent was evaporated. The desired ((N2-isobutyryl-O4-diphenylocarbamoyl-guanine)-9-methyl)(DMT-oxymethyl)(tert-butyldimethylsilyloxymethyl)phosphine oxide (8d) was obtained in 90% yield (257 mg, 0.26 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→95:5, v/v). ¹H NMR (200 MHz, CDCl₃): δ 8.21 (s, 1H, H-8), 7.81–6.78 (m, 23H, aromatic protons), 4.75 ppm (d, 2H, J_P-H= 6.7 Hz, CH₂N), 4.15 (dd, 2H, J = 2.5 Hz, J = 5.5 Hz, CH₂Si), 3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.57 (d, 2H, J_P-H= 6.2 Hz, CH₂ODMT), 2.95–2.81 (m, 1H, CH(CH₃)₂), 1.22 (d, 6H, J = 6.8 Hz, 2×CH₃), 0.76 (s, 9H, 3×CH₃), -0.03 (s, 3H, Si-CH₃), -0.05 (s, 3H, Si-CH₃); ³¹P NMR (CDCl₃): δ 40.89; FAB MS (m/z): [M+H]⁺ 955.2, [M-H]^- 953.2.

Synthesis of an N3-benzoyl-thymine derivative of 7A (8a)

To a solution of N3-benzoyl-thymine (110 mg, 0.48 mmol) in anhydrous THF, 7A (200 mg, 0.37 mmol), triphenylphosphine (204 mg, 0.77 mmol) and DIAD (152 μL, 0.77 mmol) were added (argon atmosphere, daylight protected). After 48 h the solvent was evaporated. The desired product [(N3-benzoyl-thymine)-1-methyl](DMT-oxymethyl)(tert-butyldimethylsilyl-oxymethyl)phosphine oxide (8a) was obtained in 92% yield (260 mg, 0.34 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→95:5, v/v).

¹H NMR (200 MHz, CDCl₃): δ 7.91–6.33 (m, 19H, aromatic protons, H6), 5.08–4.91 (m, 2H, CH₂N), 4.05 (d, 2H, J = 5.4 Hz, CH₂Si), 3.78 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.61 (d, 2H, J = 6.3 Hz, CH₂ODMT), 1.93 (d, J = 1 Hz, CH₃), 0.77 (s, 9H, 3×CH₃), 0.01 (s, 3H, CH₃), -0.02 (s, 3H, CH₃); ³¹P NMR (CDCl₃): δ 40.87; FAB MS (m/z): [M+154]⁺921.5, [M-H]^‑ 767.5.

Synthesis of N6-benzoyl-adenine derivative of 7A (8c)

To a solution of N6-benzoyl-adenine (115 mg, 0.48 mmol) in anhydrous THF, 7A (200 mg, 0.37 mmol), triphenylphosphine (204 mg, 0.77 mmol) and DIAD (152 μL, 0.77 mmol) were added (argon atmosphere, daylight protected). After 48 h the solvent was evaporated. The desired product [(N6-benzoyl-adenine)-9-methyl](DMT-oxymethyl)(tert-butyldimethylsilyl-oxymethyl)phosphine oxide (8c) was obtained in 51% yield (148 mg, 0.19 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→95:5, v/v).

¹H NMR (200 MHz, CDCl₃): δ 8.93 (s, 1H, NH), 8.76 (s, 1H, H-2), 8.32 (s, 1H, H-8), 7.96–6.68 (m, 18H, aromatic protons), 4.82 (d, 2H, J = 6.5 Hz, CH₂N), 4.08 (dd, J = 5 Hz, J = 14 Hz, 1H, CH₂Si), 4.02 (dd, J = 6.5 Hz, J = 14 Hz, 1H, CH₂Si), 3.78 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.60–3.59 (m, 2H, CH₂ODMT), 0.80 ( s, 9H, 3×CH₃), 0.01 (s, 3H, CH₃), -0.02 (s, 3H, CH₃); ³¹P NMR (CDCl₃): δ 42.02; FAB MS (m/z): [M+H]⁺ 778.5, [M-H]^- 776.4.

Synthesis of an N3-benzoylthymine derivative of 7B (8a′)

To a solution of N3-benzoyl-thymine (110 mg, 0.48 mmol) in anhydrous THF, 7B (202 mg, 0.37 mmol), triphenylphosphine (204 mg, 0.77 mmol) and DIAD (152 μL, 0.77 mmol) were added (argon atmosphere, daylight protected). After 48 h the solvent was evaporated. The desired product ((N3-benzoylthymine)-1-methyl)(DMT-oxymethyl)(tert-butyldimethylsilyl-oxymethyl)phosphine oxide (8a′) was obtained in 90% yield (253 mg, 0.33 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→95:5, v/v).

¹H NMR (200 MHz, CDCl₃): δ 7.93–6.73 (m, 24H, aromatic protons + H6); 4.88 (dd, J = 5 Hz, J = 14 Hz, 1H, CH′N), 4.75 (dd, J = 4 Hz, J = 14 Hz, 1H, CH″N), 4.58 (dd, J = 4 Hz, J = 16 Hz, 1H, CH″OBz), 4.13 (dd, J = 6 Hz, J = 16 Hz, 1H, CH′OBz), 3.82–3.71 (m, CH₂ODMT), 3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 1.90 (d, J = 1 Hz, CH₃); ³¹P NMR (CDCl₃): δ 38.52; FAB MS (m/z): [M-H]^- 758.5, [M+153]⁺912.8.

Synthesis of an O,O′-di-DMT-O″-tosyl derivative of THPO (9): Conversion of 7C to 9

To a solution of 7C (400 mg, 0.53 mmol) and DMAP (410 mg, 3.36 mmol), in anhydrous methylene chloride (50 mL, cooled to 0°C) p-toluenesulfonic chloride (303 mg, 1.59 mmol) was added. The mixture was kept at 0–5°C for 2 h and then was diluted with dichloromethane (100 mL) and washed with saturated aqueous NaHCO₃. The organic layer was dried with MgSO₄ and evaporated to dryness. The desired [bis (DMT-oxymethyl)](p-toluenesulfonyloxymethyl)phosphine oxide 9 was obtained in 40% yield (193 mg, 0.21 mmol) by chromatographic separation on a silica gel column using CHCl₃ as an eluent.

¹H NMR (200 MHz, CDCl₃): δ 7.69–6.66 (m, 20H, aromatic protons), 4.35 (d, J = 6 Hz, 2H, CH₂OTs), 3.78 (s, 6H, 2×OCH₃), 3.77 (s, 6H, 2×OCH₃), 3.70–3.52 (m, 4H, CH₂ODMT), 2.43 (s, 3H, CH₃); ³¹P NMR (CDCl₃): δ 39.87; FAB MS (m/z): [M-H]^-898.1, [M+Na]⁺ 922.5.

Synthesis of an O,O′-di-DMT-cytosine derivative of THPO (8g)

To a solution of cytosine (101 mg, 0.91 mmol) in anhydrous DMF (30 mL) sodium hydride (22 mg, 0.91 mmol, 60% suspension in mineral oil) was added. The mixture was kept at room temperature for 20 min and then a solution of 9 (672 mg, 0.75 mmol) in DMF (10 mL) was added dropwise. The mixture was kept at 60°C for 3 h and the solvent was evaporated under reduced pressure. The desired (cytosine-1-methyl)[bis(DMT-oxymethyl)]phosphine oxide (8g) was obtained in 60% yield (376 mg, 0.45 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→90:10, v/v). ¹H NMR (500 MHz, CDCl₃): δ 7.32–6.66 (m, 27H, aromatic protons, H6), 5.59 (d, J = 7.0 Hz, 1H, H-5), 4.12 (d, J = 5.6 Hz, 2H, CH₂N), 3.81 (dd, J = 8.5 Hz, J = 12.5 Hz, CH₂ODMT), 3.73 (s, 6H, 3×OCH₃), 3.72 (s, 6H, 3×OCH₃), 3.52 (dd, J = 5.0 Hz, J = 12.5 Hz, 2H, CH₂ODMT); ³¹P NMR (CDCl₃): δ 41.92; ¹³C NMR (125 MHz, CDCl₃): δ 165.8 (C4), 156.2 (C-2), 145.2 (C6), 95.1 (C5), 88.4 (d, J_CP= 12 Hz, C-Ph), 58.6 (d, J_CP= 83 Hz, CH₂O), 44.3 (d, J_CP= 67 Hz, CH₂N), 55.1 (CH₃O), 143.6–113.3 (36C, 6×Ph); FAB MS (m/z): [M+H]⁺ 838.2, [M-H]^- 836.5.

Synthesis of an O,O′-bis-DMT-N4-benzoylcytosine derivative of THPO (8b)

To a solution of (cytosine-1-methyl)[bis(DMT-oxymethyl)]phosphine oxide (8g) (350 mg, 0.42 mmol) in anhydrous pyridine (20 mL, cooled to 0°C) benzoyl chloride (240 μL, 2.09 mmol) was added dropwise. The mixture was kept at room temperature for 16 h and then water (12 μL) and 28% aqueous NH₄OH (12 μL) were added. After 0.5 h the solvent was evaporated under reduced pressure. The residue was diluted with dichloromethane (100 mL) and washed with saturated aqueous NaHCO₃. The organic layer was dried with MgSO₄ and evaporated to dryness. The desired [(N4-benzoylcytosine)-1-methyl][bis(DMT-oxymethyl)]phosphine oxide (8b) was obtained in 74% yield (289 mg, 0.31 mmol) by chromatographic separation on a silica gel column (CHCl₃:MeOH, 100:0→90:10, v/v).

¹H NMR (200 MHz, CDCl₃): δ 8.62–6.73 ppm (m, 33H, aromatic protons, H6, H5); 4.40 (d, J = 7 Hz, 2H, CH₂N), 3.8–3.7 (m, 2H, CH₂ODMT), 3.74 (s, 6H, 2×OCH₃), 3.73 (s, 6H, 2×OCH₃), 3.60 (dd, J = 6.4 Hz, J = 12.8 Hz, 2H, CH₂ODMT); ³¹P NMR (CDCl₃): δ 41.15; FAB MS (m/z): [M+H]⁺942.2, [M-H]^-940.3.

General procedure for removal of TBDMS protecting group in 8a,c,d,f: synthesis of 10a,c-f

To a solution of 8 (8a,c,d,f, 200 mg, ≈ 0.25 mmol) in anhydrous THF (3 mL), 1 m THF solution of tetra-n-butylammonium fluoride (311 μL, 0.31 mmol) was added. After 24 h the solvent was evaporated and the residue was dissolved in water and extracted with chloroform (3 times). The organic layer was dried with MgSO₄ and evaporated to dryness. The products 10a,c-f were isolated by chromatography on a silica gel column (CHCl₃/MeOH, 100:0→90:10, v/v).

Removal of the benzoyl protecting group in 8a′: synthesis of 10e

To a solution of 8a′ (200 mg, 0.26 mmol) in methanol (10 mL), 28% aqueous NH₄OH (10 mL) was added. After 24 h the solvent was evaporated and the residue was evaporated with ethanol and then with toluene (three times). The desired (thymine-1-methyl)(DMT-oxymethyl)(hydroxymethyl)phosphine oxide 10e was obtained in 41% yield (82 mg, 0.15 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→95:5, v/v).

Removal of the DMT protecting group in 8b: synthesis of 10b

To a solution of 8b (170 mg, 0.18 mmol) in dichloromethane (5 mL), 1 m methanolic solution of p-tolueneosulfonic acid (180 μL, 0.18 mmol) was added. The reaction progress was monitored by TLC. The mixture was quenched with pyridine (1 mL) and the volatile components were evaporated. The desired ((N4-benzoyl-cytosine)-1-methyl)(DMT-oxymethyl)(hydroxymethyl)phosphine oxide 10b was obtained in 60% yield (69 mg, 0.11 mmol) by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→95:5, v/v). ¹HNMR (200 MHz, CDCl₃): δ 8.09–6.69 (m, 20H, aromatic protons, H6, H5); 4.85 (d, J = 15.2, 2H); 4.29 (dd, J = 9.8 Hz, J = 16 Hz, 2H); 3.74 (s, 3H, OCH₃); 3.73 (s, 3H, OCH₃); 3.23–3.09 (m, 2H); ³¹P NMR (CDCl₃): δ 45.15; FAB MS (m/z): [M+H]⁺640.3, [M-H]^- 638.2.

General procedure for phosphitylation of compounds 10: synthesis of 11b,c,e,f

To a solution of 10 (0.15 mmol) in a mixture of CH₃CN (1 mL) and CH₂Cl₂ (1 mL), N,N-diisopropylamine (78 μL, 0.45 mmol) was added, followed by 2-cyanoethyl-N,N-diisopropyloaminochlorophosphite (35 μL, 0.18 mmol). The reaction progress was monitored by TLC. After ca. 1 h the volatile components were evaporated. The desired products were isolated by chromatographic separation on a silica gel column (CHCl₃/MeOH, 100:0→98:2, v/v).

Synthesis of oligomers 12-21

Chimeric THP-DNA oligomers 12-19 and homo-THPO oligomers 20 and 21 were prepared on an ABI 394 synthesizer (Applied Biosystems Inc., Foster City, CA, USA) at a 1 μmol scale. Acetonitrile solutions of phosphoramidites 11b, 11c, 11e and 11f, and of commercial thymidine, 2′-deoxycytidine, 2′-deoxyadenosine and 2′-deoxyguanosine phosphoramidites, at concentrations 0.08–0.12 m were used. For the monomers 11, a coupling time of 600 s was applied. The syntheses of oligomers 12-19 were performed on a thymidine succinyl-linked LCAA CPG solid support, and for oligomers 20 and 21, the LCAA-CPG support with a universal linker was used. The cleavage of oligomers 12-19 was done in 28% aqueous NH₄OH over 16 h at 55°C. Fully modified oligomers 20 and 21were cleaved from the support with a 20% ethanolic solution of ammonia (55°C, 4 h), followed by the treatment with 28% aq. NH₄OH/40% aq. MeNH₂ (room temperature, 16 h). All oligomers were purified by a two-step RP-HPLC (DMT-on/DMT-off). The DMT-tagged oligomers (isolated during the first step) were detritylated with 50% aq. acetic acid and purified in the DMT-off form. RP-HPLC purification was performed on a C18 column (4.6×250 mm, ThermoQuest) with a linear gradient of a buffer A (0.1 m triethylammonium bicarbonate, pH 7.5) and a buffer B (40% acetonitrile in 0.1 m triethylammonium bicarbonate, pH 7.5); a gradient for DMT-on analysis: 15–100% B, for DMT-off: 0–100% B over 30 min, flow rate 1 mL/min.

Assays for enzymatic digestion of THPO-DNA oligomers 13

Digestion of the chimeric oligomer 13with snake venom and calf spleen phosphodiesterases (PDE I and PDE II, respectively) was performed as described earlier [8, 24]. Briefly, to a 1 μL aliquot of a 5 μm solution of oligonucleotide 13 diluted with water (3 μL), a solution of PDE I from Crotalus durissus (1 μL, EC 3.1.15.1, Boehringer Mannheim GmbH, Germany, 0.1 mU/μL) or PDE II from calf spleen (1 μL, EC 3.1.16.1, Sigma, St. Louis, MO, USA, 0.1 μg/μL) was added. The mixtures were incubated at 37°C. The 1 μL samples were withdrawn from the reaction mixture after 0, 5, 15 and 45 min (for PDE I) or after 0, 10, 30 and 60 min (for PDE II), and analyzed by MALDI-TOF MS.

MALDI-TOF mass spectrometry measurements

Samples (1 μL) withdrawn from the digestion reaction mixtures were loaded on a sample plate, mixed with the matrix solution [1 μL of an 8/1 (v/v) mixture of 2,4,6-trihydroxyacetophenone (10 μg/mL in ethanol) and diammonium citrate (50 μg/mL in water)] and left for crystallization. MALDI-TOF spectra were recorded on a Voyager-Elite instrument (PerSeptive Biosystems, CT, USA) in a reflector mode, at a resolution of 2000. The m/z negative ion peaks are shown in the spectra.

Melting temperature (Tm) measurements

The Tm measurements were performed on a Cintra 40 instrument (GBC Australia). The samples (2 μm concentration of duplexes) were prepared by hybridization of modified oligomers (in a 10 mm Tris-HCl, pH 7.4, 10 mm MgCl₂, 100 mm NaCl buffer) with the complementary single stranded DNA or RNA as listed in Table 3. Before melting, the duplexes were annealed from 95°C to 5°C at a temperature gradient of 0.5°C/min. and kept at 5°C for 5 min. Melting was performed up to 86°C at a temperature gradient of 0.2°C/min. The melting temperatures Tm were calculated using the first order derivative method.

Circular dichroism measurements

Samples of the duplexes 20/21, 20/5′-d(ATAATTAAAT)-3′ and 21/5′-d(ATTTAATTAT)-3′, as well as single stranded THPO-oligomers 20 and 21were prepared at 2 μm concentration in a 10 mm Tris-HCl, pH 7.4, 10 mm MgCl₂, 100 mm NaCl buffer. The spectra were recorded on a Jobin Yvon CD6 dichrograf. Measurements were made at room temperature using 0.5 cm path-length quartz cuvettes of 1 mL capacity, 2 nm bandwidth and 1-2 s integration time. Each spectrum was smoothed with a 9- or 15-point algorithm (included in the manufacturer’s software, version 2.2.1) after averaging of at least three scans.