Ene-nucleic acids: a different paradigm to DNA chemistry

Abstract

Acyclic prochiral nucleic acids such as FNA, UNA, GNA and cyclic chiral TNA are all considered as precursors of DNA and RNA in the chemical etiology of nucleic acids. The chemical reasoning would suggest that unsaturated precursors with constrained flexibility and selectivity based on cis/trans isomers could be the missing link between the prochiral-acyclic and chiral-cyclic structures mentioned above. We find that ene-nucleic acids derived from an isoprenoid skeleton possess requisite flexibility and rigidity while forming stable duplex structures with complementary DNA and RNA.

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Introduction

The path-breaking work of Eschenmoser indicated that Watson–Crick base pairing in nucleic acids could be achieved from the other alternatives of ribose-based natural nucleic acids.^1,2 It is suggested that simple acyclic nucleic acids might be preliminary nucleic acids³ which ultimately have evolved as present day carriers of genetic information.

Acyclic nucleic acids such as flexible nucleic acids (FNAs),⁴ unlocked RNA⁵ and glycerol-based nucleic acids⁵ destabilize duplexes with cDNA, probably due to the flexibility in the backbone and a large entropic loss during duplex formation. GNA would be less flexible than FNA or UNA due to a lower number of flexible bonds in the monomer unit. An attempt was made to counter the entropic loss by introducing a double bond into the acyclic structure. Incorporation of these thymidine nucleoside mimics (Fig. 1, t^α and t^β) in oligomers was also found to be detrimental to the duplex stability,⁶ similar to the other acyclic derivatives. We presume that the attachment of a nucleobase directly to the double bond in this case may have conferred considerable unnecessary rigidity, leading to a reduced ability of the nucleobase to take part in specific W–C hydrogen bonding. Later, a homooligomeric GNA was synthesized by Meggers.⁷ The optically pure (S)-GNA could also cross-pair with RNA, though with much reduced stability. This means that the reduced flexibility in GNA compared to FNA could lead to stable duplex structures when the nucleobase attachment is kept flexible through a methylene group. The isoGNA later studied by Krishnamurthy et al. also destabilized duplexes, probably as the nucleobase attachment was directly to the backbone.⁸ In an earlier study, the cis/trans olefinic peptide nucleic acids (Fig. 1, E/Z OPAs) were synthesized to delineate the ambiguity regarding rotameric conformations and to elucidate the structural and electronic role of the tertiary amide group in PNA.⁹ This design prompted us to visualize an acyclic ene-nucleic acid (Fig. 2, ene-NA) in which the nucleobase attachment is to a planar double bonded structure through a methylene group, having the same number of bonds as a natural ribose sugar and a constraint of double bond unsaturation instead of the sugar ring. The cis or trans geometry of the proposed ene-nucleotides would be interesting to study with respect to the thermal stability of the nucleic acid complexes as well as the stability of the modified oligomers against enzymatic degradation. In this paper we describe the synthesis of the cis and trans thymine containing monomers and the synthesis of the oligomers comprising these monomers, and show that the mixed Pu/Py duplexes with cDNA and cRNA are quite stable. The pyrimidine sequences with multiple units were destabilized, and the replacement of thymidines in the loop region of quadruplex DNA was found to be less acceptable. Interestingly, the cis isomer was found to impart stability towards enzymatic digestion compared to the trans isomer.

Fig. 1 Chemical structures of DNA/RNA and nucleotide mimics with an ethylene linker to the nucleobase.

Fig. 2 Proposed cis- and trans-ene-nucleic acids.

Results and discussion

Synthesis of monomers

The nucleoside derivative 1 was synthesized according to a known procedure in the literarture.¹⁰ Conversion of 1 to mono-DMTr derivatives could be accomplished but the trans 1a and cis 1b compounds could not be separated (Scheme 1a) using repeated flash column chromatography as there was no difference in the Rf values on silica gel.

Scheme 1 Synthesis of cis- and trans-ene-nucleoside phosphoramidites.

We therefore started the synthesis all over again from dihydroxy acetone 2. Compound 2 was monoprotected¹¹ with TBDMS to get 3 and subsequently with DMTr to get 4 (Scheme 1b). Wittig reaction¹² with ethyl bromoacetate and triphenyl phosphine yielded a mixture of trans 5a and cis 5b α,β-unsaturated esters in more than 90% yield in a 6 : 4 ratio. At this stage the two compounds trans 5a and cis 5b could be separated with very careful column chromatography and were identified using a nOe experiment (ESI†). The DMTrO-group is considered to be corresponding to the 5′-position and the compound with a nucleobase on the side of the 5′-position is considered as the cis isomer. DIBAL-H reduction of each ester gave allylic alcohols 6a and 6b. Compounds 6a and 6b were then converted to nucleoside derivatives using N3–Bz–thymine under Mitsunobu conditions.¹³ The trans compound 7 was isolated using column chromatography but the cis isomer was contaminated with triphenyl phosphine oxide. Further deprotection of the silyl group using TBAF in THF gave pure compounds 8a and 8b in 55–57% overall yield. The silyl-deprotection followed by ammonia treatment in dioxane : water gave DMTr-protected trans 1a and cis 1b ene-nucleosides. The compounds 1a and 1b were then subjected to phosphitylation¹⁴ to get corresponding amidites 9a and 9b, respectively. All new compounds in Scheme 1b were characterized using ¹H, ¹³C, HRMS analysis. The phosphoramidite derivatives 9a and 9b were characterized using ³¹P NMR spectroscopy (ESI†).

Synthesis of oligomers and UV-melting studies

These ene-thyminyl amidites were used to synthesize modified DNA sequences by substituting the thymidine residues at predetermined positions in the sequences using solid phase DNA synthesis.¹⁵ We used the deprotection and cleavage conditions for obtaining the modified oligomers as described earlier to avoid cleavage at the site of modification.⁶ All the oligomers were purified using HPLC and the purity was checked using gel-electrophoretic mobility studies (ESI†). The unmodified 18 mer DNA sequence (DNA1) used in this study and the modified sequences are listed in Table 1. It is seen that the sequences modified with T-cis as well as T-trans are able to form stable sequences with both DNA as well as RNA independent of the site of modification, i.e. towards the 3′-end or in the middle of the sequence. The destabilization observed is 2–4 °C in each case (ESI†). The results obtained are indeed in accordance with our design and as the base is separated by a methylene group away from the central C=C, the deviation caused is minimal for each individual case of cis/trans isomers as well. This is much more satisfactory than that observed earlier, i.e. about 10–15 °C in the case of FNA⁴ and UNA,⁵ about 8 °C in the case of GNA,⁵ and 5–6 °C in the case of t^α/t^β6 when a single modified unit was present in the center of the sequence. In the case of a 10 mer PNA, containing a cis- or trans-olefinic PNA modification in the center of the sequence, the complexes with cDNA were destabilized by 14 °C and 6.5 °C, respectively.⁹

Table 1 18 mer DNA sequence and DNA sequences in which the T^cis and T^trans units are present at the defined position, MALDI-TOF mass analysis and their UV-melting studies with complementary DNA and RNA sequences

Name	Sequence^a 5′ → 3′	MALDI TOF mass cal/obs	UV Tm^b °C
			DNA	RNA
a Lower case denotes the DNA backbone, upper case denotes the modified site in the sequence.b The Tm values correspond to the mean values of a minimum of three experiments where the strand concentration is 1 μM each. All values are an average of at least 3 experiments and accurate to within ±0.5 °C.
DNA1	caccattgtcacactcca	5363/5367	63.5	62.7
DNA1-15T^trans	caccattgtcacacT^transcca	5347/5342	60.2	59.7
DNA1-9T^trans	caccattgT^transcacactcca	5347/5347	59.6	61
DNA1-15T^cis	caccattgtcacacT^ciscca	5347/5343	59.3	59.1
DNA1-9T^cis	caccattgT^ciscacactcca	5347/5344	60.8	59.6

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We further studied multiple modifications in the sequence containing a continuous stretch of cis-thymine units so that the modified units could be inserted continuously or alternately in the sequence. The destabilization was about 8 °C per modification when the thymine units were continuously substituted and ∼5 °C per modification when they were alternately substituted. The results are documented in Table 2. In homothyminyl sequences, the acyclic units were seldom tolerated and the duplexes formed were destabilized. Similar results were also obtained earlier in the cases of GNA and UNA,⁵ isoGNA,⁸ and OPA.⁹

Table 2 12 mer DNA sequence and DNA sequences in which the T^cis units are present at the defined position, MALDI-TOF mass analysis and their UV-melting studies with complementary DNA sequences

Name	Sequence^a 5′ → 3′	MALDI TOF mass cal/obs	UV Tm^b °C cDNA
a Lower case letters denote the DNA backbone, upper case letters denote the modified site in the sequence.b The Tm values correspond to the mean values of a minimum of three experiments where the strand concentration is 1 μM each. All values are an average of at least 3 experiments and accurate to within ±0.5 °C.
DNA2	gcgttttttgct	3633/3635	51
DNA3	gcgttT^cisT^cisT^cistgct	3585/3585	26
DNA4	gcgT^cistT^cistT^cistgct	3585/3582	34

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Stability of oligonucleotide to SVPD

The phosphodiester linkages of DNA are cleaved by snake venom phosphodiesterase (SVPD) as a 3′-exonuclease. We introduced the cis- and trans-thymine monomers at the 3′-end of the thyminyl 10 mer sequence (t₁₀, t₈T^transt and t₈T^cist, Fig. 3). In our experiments with SVPD, we found that the unmodified t₁₀ oligomer was completely digested within 10 min as expected. Surprisingly, we found a differential tolerance of the cis/trans isomers to SVPD digestion. After the cleavage of 3′-terminal thymidine, the 9 mer t₈T^trans oligomer containing the trans isomer was completely digested by SVPD within 15 min, whereas the 9 mer t₈T^cis oligomer containing the cis isomer was stable with a half life of 1 h and was about 10–15% available after 5 h (Fig. 3). The 9 mer sequence was isolated using HPLC and was confirmed using MALDI-TOF mass spectrometry (ESI†).

Fig. 3 Stability assay of the ONs to degradation by SVPD; digestion conditions: enzymatic hydrolysis of the ONs (7.5 μM) was carried out at 37 °C in buffer containing 100 mM Tris–HCl (pH 8.5), 15 mM MgCl₂, 100 mM NaCl and SVPD (100 μg mL⁻¹).

Such kind of discrimination towards hydrolytic enzymes is observed only in the case of enantiomers¹⁶ and probably would be the first example in the literature when cis–trans isomers are differentiated by the SVPD enzyme digestion reaction.

Synthesis of G-quadruplex forming TBA sequences

The acyclic UNA analogues mentioned earlier⁵ were used by Wengel and co-workers to moderate the unrequired high stability of LNA–DNA duplexes.¹⁷ In addition to modulating the DNA–RNA duplex stability in LNA/UNA mixmers, the acyclic UNA analogue found excellent application in stabilizing the loop structure in aptamers due to its ability to alleviate strain in the quadruplex loop structure¹⁸ of thrombin binding aptamer (TBA).¹⁹ We studied the constrained flexibility parameter of our ene-NA modification by introducing it in the loop region of the TBA quadruplex in comparison with unmodified TBA and with the UNA modification of TBA. The replacement of T3 or T7 positions of thymidine by UNA units was found to stabilize the quadruplex structure of TBA. We chose these two positions for replacing the thyminyl units of TBA by T^trans and T^cis monomers to study their effect on the quadruplex stability. The synthesized sequences are listed in Table 3. The stability of the quadruplexes formed was studied using temperature dependent CD studies (Table 3, ESI†).^18,20 Substitution of thymidines by UNA stabilized the quadruplex structures by 1.6 °C and 4 °C at T3 and T7 positions, respectively, whereas in our studies the structures were destabilized at both T3 and T7 positions by cis as well as trans modified units. This may indicate that the ene-NA modification is indeed more constrained compared to UNA and is less suitable for quadruplex formation compared to the highly evolved DNA quadruplexes.

Table 3 15 mer TBA sequence^a and TBA sequences in which the T^cis and T^trans units are present at the defined position, MALDI-TOF mass analysis and their CD melting studies

Name	Sequence^a 5′ → 3′	MALDI TOF mass cal/obs	CD Tm^b °C
a Lower case letters denote the DNA backbone, upper case letters denote the modified site in the sequence.b All values are an average of at least 3 experiments and accurate to within ±0.5 °C.
TBA	ggttggtgtggttgg	4726/4730	49.5
TBA-3T^cis	ggT^cistggtgtggttgg	4710/4709	38
TBA-7T^cis	ggttggT^cisgtggttgg	4710/4709	41.4
TBA-3T^trans	ggT^transtggtgtggttgg	4710/4714	36.1
TBA-7T^trans	ggtggT^transgtggttgg	4710/4708	43.7

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The reduced stability of the G-quadruplex structures due to the introduction of modification by T^cis or T^trans units was also evident due to the CD signals at 295 nm (+ve band) and 265 nm (−ve band), known to be the signature signals for antiparallel G-quadruplexes,²¹ formed by TBA sequences (Fig. 4). The positive CD signal at 295 nm showed a reduced intensity in each case where the modified T^cis or T^trans units were present. The −ve CD band was absent when the modified units destabilized the structure to a larger extent (ΔT_m = 8–12 °C). Only the T^trans unit, when present at the T7 position, retained all the CD signals as in unmodified TBA when the destabilization was minimum (ΔT_m = 6 °C).

Fig. 4 CD spectra of TBA and modified TBA sequences.

Conclusions

In conclusion, we designed, synthesized and studied the compatibility of a novel prebiotically plausible ene-NA analogue in duplex and quadruplex DNA for the first time. The stability of the duplexes formed by ene-NA modified oligomers with cDNA/RNA was found to be better compared to other reported acyclic DNA analogues. The constrained structure however, destabilized the quadruplex TBA structure compared to UNA. The cis/trans ene-NA showed differential enzymatic stability towards the hydrolytic enzyme, the trans isomer being almost as prone to hydrolytic cleavage as natural DNA in comparison to the more stable cis isomer. This may suggest that this novel ene-DNA analogue could be a missing link between the other suggested acyclic prochiral nucleic acids and the evolved chiral DNA/RNA.

Experimental

General information

All the reagents were purchased from Sigma-Aldrich and used without further purification. SVPD was purchased from Sigma. DMF and pyridine were dried over KOH and 4 Å molecular sieves. TLCs were run on pre-coated silica gel GF254 sheets (Merck 5554). All reactions were monitored using TLC and usual workup implies sequential washing of the organic extract with water and brine, followed by drying over anhydrous sodium sulphate and evaporation under vacuum. Column chromatography was performed for the purification of the compounds on silica gel (60–120 mesh or 100–200 mesh, Merck). TLCs were carried out on precoated silica gel 60 F254 (Merck), and were performed using dichloromethane–methanol or petroleum ether–ethyl acetate solvent systems for most compounds. Compounds were visualized with UV light and/or by spraying with 30% perchloric acid/EtOH solution and heating. ¹H (200 MHz) and ¹³C (50 MHz) NMR spectra were recorded using a Bruker ACF 200 spectrometer fitted with an Aspect 3000 computer and ³¹P NMR spectra were recorded using a 400 MHz Bruker ACF instrument. All the chemical shifts (δ/ppm) are referenced to the internal TMS for ¹H and chloroform-d/DMSO-d6 for ¹³C NMR. ¹H NMR data are reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet and/or multiple resonance), number of protons. Mass spectra were recorded using an APQSTAR spectrometer, LC-MS using a Finnigan-Matt instrument. High resolution mass spectra were recorded using a Thermo Fisher Scientific Q Exactive mass spectrometer. DNA oligomers were synthesized on a CPG solid support using the Bioautomation Mer-Made 4 synthesizer. The RNA oligonucleotides were obtained commercially (Sigma-Aldrich). RP-HPLC was carried out on a C18 column using a Waters system (Waters Delta 600e quaternary solvent delivery system and 2998 photodiode array detector and Empower2 chromatography software). MALDI-TOF spectra were recorded using an AB Sciex TOF/TOF™ Series Explorer™ 72085 instrument and the matrix used for analysis was THAP (2′,4′,6′-trihydroxyacetophenone). UV experiments were performed using a Varian Cary 300 UV-VIS spectrophotometer fitted with a Peltier-controlled temperature programmer. CD spectra were recorded using a Jasco J-715 Spectropolarimeter, with a ThermoHaake K20 programmable water circulator for temperature control of the sample.

1-((tert-Butyldimethylsilyl)oxy)-3-hydroxypropan-2-one (3). Compound 2 (9.4 g, 104.4 mmol) was first dissolved in dry DMF (100 mL) and then TBS-Cl (5.0 g, 33.5 mmol) and imidazole (2.95 g, 43.4 mmol) were added under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 10 h and then quenched with water (100 mL). The compound was extracted with ethyl acetate from the crude reaction mixture and the organic layer was washed with brine solution, dried over Na₂SO₄ and concentrated using a rotavapor in vacuo. The crude compound was purified through column chromatography (pet ether : EtOAc, 90 : 10) to give 3 (6.2 g, 55%) as a colourless thick liquid. ¹H NMR (200 MHz, CDCl₃) δ 0.10 (s, 6H), 0.93 (s, 9H), 3.01 (t, J = 4.99 Hz, 1H), 4.32 (s, 2H), 4.51 (d, J = 4.93 Hz, 2H) ppm; ¹³C NMR (50 MHz, CDCl₃) δ −5.7, 18.1, 25.7, 66.6, 67.7, 211.1 ppm; HRMS (EI): mass calculated for C₉H₂₀O₃NaSi (M + Na), 227.1074, found 227.1069.

1-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-((tert-butyl dimethyl silyl)oxy)propan-2-one (4). To a solution of 3 (5 g, 24.5 mmol) in pyridine (15 mL), DMTr chloride (10 g, 29.5 mmol) and a catalytic amount of DMAP were added, and the mixture was stirred at rt for 6 h. Pyridine was removed in vacuo and the residue was diluted with EtOAc. A water wash and a brine wash were carried out for the organic layer, it was dried over Na₂SO₄ and concentrated in vacuo. The residue was subjected to silica gel column chromatography (pet ether : EtOAc, 95 : 5) to afford 4 (9.3 g) in 75% yield.

¹H NMR (200 MHz, CDCl₃) δ 0.02 (s, 6H), 0.85 (s, 9H), 3.80 (s, 7H), 3.96 (s, 2H), 4.38 (s, 2H), 6.79–6.89 (m, 5H), 7.26–7.39 (m, 9H), 7.41–7.49 (m, 2H) ppm; ¹³C NMR (50 MHz, CDCl₃) δ −5.6, 18.2, 25.7, 55.2, 68.2, 68.4, 86.9, 113.3, 127.0, 128.0, 130.0, 135.4, 144.3, 158.7, 206.8 ppm; HRMS (EI): mass calculated for C₃₀H₃₈O₅NaSi (M + Na) 529.2381, found 529.2369.

Ethyl (Z)-4-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-(((tertbutyldimethylsilyl)oxy)methyl)but-2-enoate (5a & 5b). A solution of 4 (10 g, 19.7 mmol) and a two-carbon Wittig ylide (9.5 g, 29.6 mmol) in 100 mL toluene was refluxed for 4 h. The solvent was removed in vacuo, the residue was diluted with EtOAc and a water wash, a saturated aqueous NaHCO₃ wash, and finally a brine wash were given. The organic layer was dried over Na₂SO₄, and concentrated in vacuo followed by column chromatography (pet ether : EtOAc, 98 : 2) to give 5a and 5b (90%) in a 60 : 40 ratio. ¹H NMR (5a) (200 MHz, CDCl₃) δ −0.08 (s, 6H), 0.72 (s, 9H), 1.34 (t, J = 7.14 Hz, 4H), 3.80 (s, 6H), 3.90 (s, 2H), 4.21 (q, J = 7.07 Hz, 2H), 4.81 (s, 2H), 6.38 (t, J = 1.77 Hz, 1H), 6.84 (d, J = 8.72 Hz, 4H), 7.23, 7.39 (m, 8H), 7.41–7.49 (m, 2H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ −5.7, 14.4, 18.0, 25.7, 55.2, 59.9, 61.9, 64.0, 86.6, 112.2, 113.2, 126.8, 127.9, 128.0, 129.9, 136.1, 144.8, 158.5, 160.1, 166.7 ppm; HRMS (EI): mass calculated for C₃₄H₄₄O₆NaSi (M + Na) 599.2799, found 599.2789. ¹H NMR (5b) (200 MHz, CDCl₃) δ 0.10 (s, 6H), 0.94 (s, 9H), 1.19 (t, J = 7.14 Hz, 4H), 3.77–3.81 (m, 8H), 4.05 (q, J = 7.07 Hz, 2H), 4.39 (s, 2H), 4.52 (s, 2H), 5.94–5.99 (m, 1H), 6.82 (d, J = 8.84 Hz, 5H), 7.25–7.43 (m, 13H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ −5.4, 14.3, 18.4, 26.0, 55.2, 59.8, 62.3, 63.5, 86.5, 113.0, 113.1, 126.8, 127.8, 128.1, 129.1, 129.9, 130.0, 136.0, 144.8, 158.5, 158.8, 166.4 ppm; HRMS (EI): mass calculated for C₃₄H₄₄O₆NaSi (M + Na) 599.2799, found 599.2790.

(Z)-4-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-(((tertbutyldimethylsilyl)oxy)methyl)but-2-en-1-ol (6a). DIBAL-H was added to a solution of ester 5a (1 g, 2.6 mmol) in DCM at −78 °C. After 45 min at the same temperature, aq. sodium potassium tartarate and diethyl ether were added. The resultant cloudy reaction mixture was then vigorously stirred for 1 h, after which the organic layer appeared like a clear solution. The organic layer was separated, washed with brine solution, extracted using DCM and dried over Na₂SO₄. The compound was purified through column chromatography (pet ether : EtOAc, 70 : 30) to obtain 6a (0.71 g) in 79% yield. ¹H NMR (200 MHz, CDCl₃) δ −0.01 (s, 6H), 0.82 (s, 9H), 3.62 (s, 2H), 3.80 (s, 8H), 4.19 (s, 2H), 4.22–4.31 (m, 2H), 6.02 (t, J = 6.57 Hz, 1H), 6.83 (d, J = 8.84 Hz, 6H), 7.25 (d, J = 2.65 Hz, 2H), 7.28–7.52 (m, 11H) ppm; ¹³C NMR (50 MHz, CDCl₃) δ −5.5, 18.2, 25.8, 55.2, 58.7, 59.9, 65.3, 86.2, 113.1, 126.2, 126.7, 127.8, 128.1, 130.0, 136.3, 139.5, 145.0, 158.4 ppm; HRMS (EI): mass calculated for C₃₂H₄₂O₅NaSi (M + Na) 557.2694, found 557.2677.

(Z)-3-Benzoyl-1-(4-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-(((tertbutyldimethylsilyl)oxy)methyl)but-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)dione (7). To a solution of 6a (0.5 g, 0.93 mmol) in dry dioxane (4 mL), triphenyl phosphine (0.37 g, 1.4 mmol) and N³-benzoyl protected thymine (0.32 g, 1.4 mmol) were added and the mixture was stirred for 15 min. DIAD (0.36 mL, 1.86 mmol) was dissolved in 1 mL of dry dioxane and added to the reaction mixture; the stirring was continued overnight at room temperature. Dioxane was removed in vacuo and the residue was diluted with EtOAc. A water wash and a brine wash were carried out on the organic layer, it was dried over Na₂SO₄ and concentrated in vacuo. The crude residue was subjected to silica gel column chromatography (pet ether : EtOAc, 70 : 30) to obtain 7 (0.38 g) in 55% yield. ¹H NMR (200 MHz, CDCl₃) δ 0.01 (s, 6H), 0.83 (s, 9H), 1.97 (s, 3H), 3.67 (s, 2H), 3.80 (s, 6H), 4.23 (s, 2H), 4.57 (d, J = 7.58 Hz, 2H), 5.79 (t, J = 7.71 Hz, 1H), 6.84 (d, J = 8.84 Hz, 5H), 7.23 (br. s., 2H), 7.29–7.54 (m, 13H), 7.59–7.69 (m, 1H), 7.91–7.98 (m, 2H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ −5.3, 12.6, 18.3, 25.9, 25.9, 44.3, 55.3, 59.7, 65.4, 86.6, 110.9, 113.2, 120.0, 126.9, 128.0, 128.1, 129.2, 130.0, 130.6, 131.7, 135.0, 136.1, 139.6, 142.9, 144.9, 150.1, 158.6, 163.3, 169.3 ppm.

(Z)-3-Benzoyl-1-(4-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-(hydroxymethyl)but-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8a). Compound 7 (1 g, 1.28 mmol) was dissolved in 15 mL of THF and TBAF (0.394 g, 1.5 mmol) was added. The reaction mixture was stirred for 2 h at room temperature. The solvent was removed in vacuo. The residue was dissolved into 50 mL of ethyl acetate, washed with water (3 × 25 mL) and then with brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The resulting residue was purified using silica gel column chromatography (pet ether : EtOAc, 60 : 40) to yield 8a (0.7 g) in 83%. ¹H NMR (200 MHz, CDCl₃) δ 1.89 (s, 3H), 3.68 (s, 3H), 3.71 (s, 8H), 4.10 (s, 2H), 4.41 (d, J = 7.58 Hz, 2H), 5.62 (t, J = 7.64 Hz, 1H), 6.76 (d, J = 8.84 Hz, 5H), 7.12–7.24 (m, 9H), 7.29–7.43 (m, 6H), 7.48–7.61 (m, 2H), 7.79–7.88 (m, 3H) ppm; ¹³C NMR (100 MHz, CDCl₃) δ 12.4, 25.8, 44.2, 55.2, 59.6, 65.3, 86.5, 110.8, 113.1, 119.9, 126.8, 127.9, 128.0, 129.1, 129.9, 130, 131.6, 134.9, 136.0, 139.5, 140.0, 142.8, 144.7, 150.0, 158.5, 163, 169.2 ppm; HRMS (EI): mass calculated for C₃₈H₃₆O₇N₂Na (M + Na) 655.2415, found 655.2398.

(E)-4-(Bis(4-methoxyphenyl)(phenyl)methoxy)-3-(((tertbutyldimethylsilyl)oxy)methyl)but-2-en-1-ol (6b). DIBAL-H was added to a solution of ester 5b (1 g, 2.6 mmol) in DCM at −78 °C. After 45 min at the same temperature, aq. sodium potassium tartarate and diethyl ether were added. The resultant cloudy reaction mixture was then vigorously stirred for 1 h after which the organic layer appeared like a clear solution. The organic layer was separated, washed with brine solution, extracted with DCM and dried over Na₂SO₄. The compound was purified through column chromatography (pet ether : EtOAc, 70 : 30) to obtain 6b (0.67 g) in 75% yield. ¹H NMR (200 MHz, CDCl₃) δ 0.08 (s, 6H), 0.92 (s, 9H), 3.66 (s, 2H), 3.80 (s, 7H), 4.07 (d, J = 6.82 Hz, 2H), 4.22 (s, 2H), 5.88 (t, J = 6.82 Hz, 1H), 6.85 (d, J = 8.84 Hz, 5H), 7.24–7.50 (m, 12H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ −5.3, 18.4, 26.0, 55.2, 58.8, 59.6, 65.1, 86.6, 113.2, 113.3, 113.3, 126.4, 126.8, 127.9, 128.0, 128.1, 129.9, 130.0, 130.0, 136.1, 139.3, 144.9, 158.5 ppm; HRMS (EI): mass calculated for C₃₂H₄₂O₅NaSi (M + Na) 557.2694, found 557.2684.

(E)-3-Benzoyl-1-(4-(bis (4-methoxyphenyl)(phenyl)methoxy)-3-(hydroxymethyl)but-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8b). Compound 6b was subjected to the Mitsunobu reaction and without purification, the crude mixture was used for TBDMS deprotection to obtain 8b in 57% yield over two steps. ¹H NMR (500 MHz, CDCl₃) δ 1.86 (s, 3H), 3.78 (s, 7H), 3.80 (s, 2H), 4.19–4.23 (m, 4H), 5.65 (t, J = 7.02 Hz, 1H), 6.85 (d, J = 8.85 Hz, 5H), 7.27–7.37 (m, 8H), 7.42–7.50 (m, 5H), 7.90 (d, J = 7.32 Hz, 2H) ppm; ¹³C NMR (125 MHz, CDCl₃) δ 12.3, 44.9, 55.3, 59.6, 65.5, 87.0, 110.9, 113.4, 121.7, 127.1, 128.0, 128.1, 129.1, 130.0, 130.5, 131.7, 135.0, 135.5, 139.5, 142.2, 144.4, 149.8, 158, 163.1, 169.1 ppm; HRMS (EI): mass calculated for C₃₈H₃₆O₇N₂Na (M + Na) 655.2415, found 655.2396.

(Z)-1-(4-(Bis (4-methoxyphenyl)(phenyl)methoxy)-3-(hydroxymethyl)but-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1a). 30% aq. ammonia solution (0.5 mL) was added to a solution of 8a (0.5 g, 0.76 mmol) in 10 mL of dioxane and the mixture was stirred for 7 h at room temperature. The solvent was removed under reduced pressure. The residue was dissolved into 50 mL of ethyl acetate, washed with water (3 × 25 mL) and then with brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The resulting residue was purified using a silica gel column. The product was eluted with 50% ethyl acetate in petroleum ether to afford 1a (0.4 g, 85%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 1.83 (s, 3H), 3.80 (s, 7H), 3.81 (br s, 2H), 4.17 (d, J = 7.02 Hz, 2H), 4.23 (br s, 2H), 5.62 (t, J = 7.02 Hz, 1H), 6.85 (d, J = 9.16 Hz, 5H), 7.21–7.26 (m, 1H), 7.28–7.36 (m, 7H), 7.44 (d, J = 7.02 Hz, 2H), 8.82 (br. s., 1H) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 12.2, 44.7, 55.3, 59.7, 65.5, 87.0, 110.9, 113.3, 122.1, 127.1, 128.0, 128.0, 130.0, 135.5, 139.8, 141.8, 144.4, 150.8, 158.7, 164.1 ppm; HRMS (EI): mass calculated for C₃₁H₃₂O₆N₂Na (M + Na) 551.2153, found 551.2147.

(E)-1-(4-(Bis (4-methoxyphenyl)(phenyl)methoxy)-3-(hydroxymethyl)but-2-en-1-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (1b). 30% aq. ammonia solution (0.5 mL) was added to a solution of 8 (0.5 g, 0.76 mmol) in 10 mL of dioxane and the mixture was stirred for 7 h at room temperature. The solvent was removed under reduced pressure. The residue was dissolved into 50 mL of ethyl acetate, washed with water (3 × 25 mL) and then with brine. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The resulting residue was purified using a silica gel column. The product was eluted with 50% ethyl acetate in petroleum ether to afford 9 (0.33 g, 80%) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 1.92 (s, 3H), 3.76 (s, 2H), 3.77 (s, 6H), 4.19 (s, 2H), 4.43 (d, J = 7.63 Hz, 2H), 5.62 (t, J = 7.63 Hz, 1H), 6.81 (d, J = 8.85 Hz, 4H), 7.21 (d, J = 7.32 Hz, 1H), 7.24–7.32 (m, 7H), 7.40 (d, J = 7.32 Hz, 2H), 9.41 (br. s., 1H) ppm; ¹³C NMR (126 MHz, CDCl₃) δ 12.3, 45.4, 55.2, 58.7, 66.3, 86.8, 111.3, 113.2, 120.6, 126.9, 127.9, 128.1, 130.0, 135.9, 140.2, 142.6, 144.7, 151.2, 158.6, 164 ppm; HRMS (EI): mass calculated for C₃₁H₃₂O₆N₂Na (M + Na) 551.2153, found 551.2145.

General procedure followed for the synthesis of phosphoramidite derivatives 9a and 9b. To the compounds 1a and 1b (100 mg, 0.17 mmol) dissolved in dry DCM (3 mL), DIPEA (0.64 mmol, 0.12 mL) was added. 2-Cyanoethyl-N,N-diisopropyl-chloro phosphine (0.35 mmol, 0.08 mL) was added to the reaction mixture at 0 °C and stirring continued at room temperature for 1 h. The contents were diluted with DCM and washed with 5% NaHCO₃ solution. The organic phase was dried over anhydrous sodium sulphate and concentrated to a white foam. The residue was re-dissolved in DCM and the compound was precipitated with n-hexane to obtain the corresponding phosphoramidite derivatives in 70–75% yield. ³¹P NMR 9a (500 MHz, CDCl₃) δ 148.82 HRMS (EI): mass calculated for C₄₀H₄₉O₇N₄NaP (M + Na) 751.3231, found 751.3212. ³¹P NMR 9b (500 MHz, CDCl₃) δ 148.27 HRMS (EI): mass calculated for C₄₀H₄₉O₇N₄NaP (M + Na) 751.3231, found 751.3212.

Synthesis of oligonucleotides

The 18 mer DNA sequence chosen for the current study is of biological relevance, DNA1 is used for miRNA down-regulation.¹⁴ Unmodified oligomers were synthesized using commercially available phosphoramidite building blocks. The modified oligonucleotides were synthesized using phenoxyacetyl (Pac) protected cyanoethyl phosphoramidites and modified amidite building blocks, 9a and 9b. The modified phosphoramidites (0.1 M in CH₃CN) were manually coupled for 6 min, followed by a washing step with 10% H₂O, 0.2% Ac₂O and 0.2% lutidine v/v/v in THF. This was done to avoid the unwanted phosphitylation at the bases of the highly reactive acyclic olefinic monomers. After washing, capping followed by oxidation with 0.5 M tert-butyl hydroperoxide in CH₂Cl₂–acetone (1 : 1) used instead of iodine/water because it is known that iodine/water cleaves the allylic C–O bond. This is known to occur for other phosphites with allylic or tertiary substituents.¹² For the modified units, double coupling (300 s × 2) was performed. Deprotection and cleavage were performed by shaking the support-bound oligonucleotide with neat dry diisopropylamine and by washing with diethyl ether followed by shaking with conc aq. ammonia for 2 h at rt.¹² The crude oligomer was purified using RP-HPLC. The purity of the oligomers was confirmed by gel-electrophoretic mobility studies and characterization using MALDI-TOF mass spectrometry.

UV-T_m measurements

The concentration was calculated on the basis of the absorbance from the molar extinction coefficients of the corresponding nucleobases of DNA/RNA. The experiments were performed at 1 μM concentrations. The complexes were prepared in 10 mM sodium phosphate buffer, pH 7.2, containing NaCl (150 mM) and were annealed by keeping the samples at 90 °C for 2 min. This was followed by slow cooling to room temperature and refrigeration for at least two hours prior to running the experiments. Absorbance versus temperature profiles were obtained by monitoring the absorbance at 260 nm from 10–85 °C at a ramp rate of 0.5 °C per minute. The data were processed using Microcal Origin 6.1 and the T_m (°C) values were derived from the maxima of the first derivative plots.

CD experiments

CD experiments were done for the TBA sequences. A 5 μM concentration of each strand was used for the sample preparation. The complexes were prepared in 10 mM potassium phosphate buffer, pH 7.2, containing KCl (100 mM) and were annealed by keeping the samples at 90 °C for 2 min. This was followed by slow cooling to room temperature and refrigeration for at least four hours prior to running the experiments. CD spectra were recorded with a 2 mm pathlength cuvette, using a resolution of 1 nm, a bandwidth of 1 nm, a sensitivity of 20 m deg, a response of 1 s and a scan speed of 100 nm min⁻¹. Spectral scans were collected at 4 °C over a wavelength range of 200–320 nm at a scanning rate of 100 nm min⁻¹. CD melting was performed for the entire sample by monitoring the CD intensity at 295 nm against the temperature over a range of 5–90 °C. Three scans were averaged for each sample.

Nuclease resistance study

Enzymatic hydrolysis of the ONs (7.5 μM) was carried out at 37 °C in buffer (100 μL) containing 100 mM Tris–HCl (pH 8.5), 15 mM MgCl₂, 100 mM NaCl and SVPD (100 μg mL⁻¹). Aliquots were removed at several time-points; a portion of each reaction mixture was removed and heated to 90 °C for 2 min to inactivate the nuclease. The amount of intact ONs was analyzed at several time points using RP-HPLC. The percentage of intact ON was then plotted against the exposure time to obtain the ON degradation curve with time.

Gel experiments

The purity of the synthesized oligomers was assessed using non-denaturing 30% polyacrylamide gel electrophoresis. A pre-run was done by loading each well with 2 μL of bromophenol blue dye in 40% sucrose solution (1 : 1) and the run was carried out in 1× TBE buffer applying 200 V of voltage at 4 °C for 1 h until the marker dye had travelled down and washed out along with any unpolymerised gel. The DNA oligomer control and the samples in 2 μL of solution (350 μM concentration) mixed with an equal volume of the 40% sucrose solution were loaded into the appropriately numbered wells. The gel was run with the voltage set at 150 V for 120 min until the marker was visible at 3/4 of the gel height. After the run, the gels were washed with DI water and then were visualized using UV-shadowing. For denaturing gel experiments, 7 M urea was used for gel casting, while 2 μL of the 350 μM sample in DI-water was mixed with 2 μL of formamide for loading, and the gels were run in 1× TBE buffer at 25 °C by applying 150 V of voltage.

Acknowledgements

VAK thanks CSIR, New Delhi for financial support (Genecode BSC0123). MV thanks CSIR, New Delhi for the research fellowship. ND thanks UGC, New Delhi for the research fellowship.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra15673d

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