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Synthesis and pairing properties of oligoribonucleotide analogues containing a metal-binding site attached to [beta]-d-allofuranosyl cytosine
Introduction
Results
Synthesis of monomers
Functionalization
Synthesis of oligonucleotides
Pairing properties
Discussion
Conclusion And Outlook
Materials And Methods
General
Oligonucleotide synthesis
Thermal denaturation studies
3-O-Benzoyl-6-O-(5-bromopentyl)-1,2-O-isopropyliden-[alpha]-d-allofuranose (2)
6-O-(5-bromopentyl)-1,2,3-O-tribenzoyl-5-O-[(triisopropyl)-silyl]-[alpha],[beta]-d-allofuranose [3([alpha]/[beta])]
N4-Benzoyl-1-[6[prime]-O-(5-bromopentyl)-2[prime],3[prime]-di-O-benzoyl-[beta]-d-allofuranosyl]cytosine (5)
N4-Benzoyl-1-[6[prime]-O-(5-bromopentyl)-5[prime]-O-(4,4[prime]-dimethoxy-trityl)-[beta]-d-allofuranosyl]cytosine (7)
N4-Benzoyl-1-[6[prime]-O-(5-bromopentyl)-5[prime]-O-(4,4[prime]-dimethoxy-trityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofurano-syl]cytosine (9)
N4-Benzoyl-1-[6[prime]-O-(5-bromopentyl)-5[prime]-O-(4,4[prime]-dimethoxy-trityl)-3[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl]-cytosine (10)
N4-Acetyl-1-{6[prime]-O-[5-(1,4,7,10,13-pentaoxa-16-aza-cyclo-octadec-16-yl)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine (14)
N4-Acetyl-1-{6[prime]-O-[5-(1,4,7,10,13-pentaoxa-16-aza-cyclo-octadec-16-yl)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine 3[prime]-[(2-cyanoethyl) N,N-diisopropyl-phosphoramidite] (15)
6-O-[5-(N-Allyloxycarbonyl-methylamino)-pentyl]-1,2,3-tri-O-benzoyl-5-O-[(triisopropyl)silyl]-[beta]-d-allofuranose [4([alpha]/[beta])]
N4-Benzoyl-1-{6[prime]-O-[5-(N-allyloxycarbonyl-methylamino)-pentyl]-2[prime],3[prime]-di-O-benzoyl-[beta]-d-allofuranosyl}cytosine (6)
N4-Benzoyl-1-{6[prime]-O-[5-(N-allyloxycarbonyl-methylamino)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-[beta]-d-allofuranosyl}cytosine (8)
N4-Benzoyl-1-{6[prime]-O-[5-(N-allyloxycarbonyl-methylamino)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyl-oxy]methyl}-[beta]-d-allofuranosyl}cytosine (11)
N4-Benzoyl-1-{6[prime]-O-[5-(N-allyloxycarbonyl-N-methyl)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-3[prime]-O-{[(triisopropyl)silyl-oxy]methyl}-[beta]-d-allofuranosyl}cytosine (12)
N4-Benzoyl-1-{6[prime]-O-(N-methyl-5-aminopentyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine (13)
N4-Benzoyl-1-{6[prime]-O-(N-methyl-5-[(2-[2-methoxy-ethoxy]-ethoxy)-ethylamino]-pentyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine (16)
N4-Benzoyl-1-{6[prime]-O-(N-methyl-5-[(2-[2-methoxy-ethoxy]-ethoxy)-ethylamino]-pentyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine 3[prime]-[(2-cyanoethyl) N,N-diisopropyl-phosphoramidite] (17)
Supplementary Material
Acknowledgements
References
Synthesis and pairing properties of oligoribonucleotide analogues containing a metal-binding site attached to [beta]-d-allofuranosyl cytosine
ABSTRACT
INTRODUCTION
Modified and functionalized oligonucleotides play an important role as molecular tools and potential antisense drugs (1). Furthermore, there exists an as yet unexploited potential for catalysis of chemical reactions with tailored ribozymes containing additional side chains (2). So far, oligonucleotides have been functionalized at the nucleobases, at the 5[prime]- or 3[prime]-termini, at the phosphodiester linkages or at the 2[prime]-O position (for recent examples see 3-6).
During our ongoing investigations of the properties of hexofuranosyl oligonucleotides we developed a method for the synthesis of oligoribonucleotides containing 6[prime]-O-substituted [beta]-d-allofuranosyl and [beta]-l-talofuranosyl nucleosides, which can be regarded as C(5[prime])-substituted ribonucleosides. Preliminary melting curve studies revealed that single incorporations of d-allofuranosyl nucleosides in an A-type RNA duplex did not significantly change the pairing properties (relative to those of the parent duplex), whereas incorporation of [the C(5[prime])-epimeric] l-talofuranosyl nucleosides resulted in substantial weakening of the duplex (7; X.Wu, unpublished results).
Here we present a synthesis of the two [beta]-d-allofuranosyl cytosine building blocks 9 and 13, containing all protecting groups required for automated synthesis and a 6[prime]-O-bromopentyl (electrophilic) or a 6[prime]-O-methylaminopentyl (nucleophilic) substituent, respectively. The 2[prime]-O positions were protected with the Pri3SiOCH2 (TOM) group, which we recently introduced for the chemical synthesis of oligoribonucleotides under standard DNA coupling conditions (8; S.Pitsch, X.Wu, P.A.Weiss, S.Vonhoff and L.Jenny, in preparation).
The two reactive building blocks 9 and 13 potentially serve as starting materials for a variety of functionalized oligoribonucleotide analogues, allowing the straightforward introduction of different side chains at a very late stage of monomer synthesis. As first examples, we functionalized them with two metal chelating moieties and transformed them into phosphoramidites 15 and 17. Preliminary molecular model studies indicated the possibility of an interaction between a tethered metal complex (covalently bound to the 6[prime]-O position of an allofuranosyl nucleoside) and the phosphodiester backbone of an unmodified partner strand across the major groove. When an appropriate alignment is realized, such an interaction can potentially stabilize the duplex electrostatically or catalyse a specific strand scission reaction by providing a correctly positioned Lewis acid. Triethyleneglycol and 1-aza-18-crown-6 were chosen as ligands for their known ability to form complexes with the biologically abundant metal ions Na+, K+, Mg2+ and Ca2+ (9,10).
RESULTS
Synthesis of monomers
For synthesis of the key intermediates 9 and 13 we first prepared the appropriately pre-functionalized sugar building blocks 3 and 4 which allowed an efficient, stepwise introduction of the nucleobase, the dimethoxytrityl group and the 2[prime]-O protecting group (Scheme 1).
Scheme 1. Reagents and conditions. (a) (i) Bu2SnO, toluene, reflux, (ii) Br(CH2)5Br, CsF, Bu4NI, DMF, room temperature. (b) For 3: (i) Pri3Si-OTf, Et(Pri)2N, CH2Cl2, room temperature, (ii) CF3COOH, H2O, room temperature, (iii) BzCl, DMAP, py, CH2Cl2, room temperature; for 4: (i) Pri3Si-OTf, Et(Pri)2N, CH2Cl2, room temperature, (ii) MeNH2, EtOH, room temperature, (iii) AllOC(O)Cl, Et(Pri)2N, CH2Cl2, room temperature, (iv) CF3COOH, H2O, room temperature, (v) BzCl, DMAP, py, CH2Cl2, room temperature. (c) (i) Bis(trimethylsilyl)acetamide, N4-benzoylcytosine, MeCN, 705C, then Me3Si-OTf, (ii) HF, HCl, MeCN, room temperature; (d) (i) DMT-Cl, AgNO3, sym-collidine, CH2Cl2, room temperature, (ii) NaOH, THF/MeOH/H2O, 45C. (e) Bu2SnCl2, Pri3Si-OCH2Cl, Et(Pri)2N, (CH2Cl)2, 705C. (f) Pd(PPh3)4, Et2NH, PPh3, CH2Cl2, room temperature. Selective alkylation of the primary hydroxy group in diol 1 (11) with 1,5-dibromopentane gave bromide 2 in good yields. This reaction was accomplished by first forming the cyclic dibutyl tin-derivative, followed by alkylation in the presence of tetrabutylammonium iodide and caesium fluoride according to Nagashima and Ohno (12). The common precursor 2 could be elaborated by a series of reactions into sugar building blocks 3 and 4 without intermediate purification. The bromopentyl sugar 3 was obtained by silylation of 2 with triisopropylsilyl triflate, cleavage of the ketal group with 50% trifluoroacetic acid and dibenzoylation with benzoyl chloride. The N-allyloxycarbonyl-protected methylaminopentyl sugar 4 was obtained by silylation of 2 with triisopropylsilyl triflate, substitution of bromide (and cleavage of the 3-O-benzoyl group) with methylamine, selective N-acylation with allyl chloroformate, cleavage of the ketal group and perbenzoylation. Nucleosidation of 3 and 4 was achieved under Vorbrüggen conditions (13) with in situ trimethylsilylated N4-benzoylcytosine using trimethylsilyl triflate (with 3 and 4) or SnCl4 (with 3) as Lewis acid. Without isolation of the nucleosidation products the Pri3Si groups were removed with a mixture of aqueous HF and HCl in MeCN and nucleosides 5 and 6 were isolated in good yields. From these the dimethoxytritylated diols 7 and 8 were obtained in excellent yields by treatment with dimethoxytrityl chloride in the presence of collidine and AgNO3 according to Hakimelahi et al. (14), directly followed by O-debenzoylation. Scheme 2. Reagents and conditions. (a) 1-aza-18-crown-6, Bu4NI, Et(Pri)2N, EtOH, 755C. (b) (2-cyanoethyl)(N,N-diisopropylamino)-chlorophosphite, Et(Pri)2N, CH2Cl2, room temperature. (c) Me(OCH2CH2)3Cl, Bu4NI, Et(Pri)2N, toluene, 955C. Regioselective introduction of the Pri3SiOCH2 (TOM) group at the 2[prime]-O positions of diols 7 and 8 was carried out under conditions developed in our laboratory (11; S.Pitsch, X.Wu, P.A.Weiss, S.Vonhoff and L.Jenny, in preparation) and gave compounds 9 and 11 in satisfactory yields. The 3[prime]-O-alkylated regioisomers 10 and 12 were isolated as minor products. Both pairs of regioisomers were unambigously identified by their 1H NMR spectra according to Pitsch (11). From 11 the free methyl aminopentyl nucleoside 13 was obtained according to Hayakawa et al. (15).
Functionalization
Reaction of the bromopentyl-substituted nucleoside 9 and 1-aza-6-crown-18 led to the corresponding crown ether-substituted nucleoside. During this reaction partial loss of the benzoyl base-protecting group was observed. Therefore, it was completely removed with ammonia, protected again with Ac2O and isolated as the N4-acetylcytosine derivative 14 in fair yield. Under standard conditions, it was finally transformed into phosphoramidite 15 (Scheme 2).
Figure 1. Structure of TOM-protected phosphoramidites (TOM, triisopropylsilyl-oxymethyl). Figure 2. Pictures of modified A-type RNA duplexes, illustrating the position of the additional substituents present in functionalized allofuranosyl nucleosides. (a) The internally functionalized duplexes C.A and E.A; (b) the externally substituted duplexes D.A and F.A. Potentially, the functional group attached to oligonucleotides C and E can reach over the major groove and interact with the other strand (as indicated by the arrow). The RNA duplex was constructed with MacroModel and the substituents were added without further minimization. Reaction of the methyl aminopentyl nucleoside 13 and diethyleneglycol monomethyl monochloroethyl diether CH3(OCH2CH2)3Cl led to the corresponding triethyleneglycol-substituted nucleoside 16, which was finally transformed into phosphoramidite 17 (Scheme 2).
Synthesis of oligonucleotides
For our initial hybridization studies we designed a non-self-complementary tetradecamer RNA sequence in which we incorporated phosphoramidites 15 and 17 at two different positions, one near the 3[prime]- and one near the 5[prime]-end (Table
Table 1. The oligonucleotides were prepared under the following conditions
Pairing properties
Figure
We expected that an eventual positive interaction between the two pairing strands would lead to stabilization of the internally functionalized duplexes, but not of the externally functionalized ones. Therefore, the latter were prepared and investigated as reference compounds to detect intrinsic contributions which are not the result of a specific interaction across the major groove.
Initially, two sets of exploratory experiments were carried out. In the first set, the transition temperatures of all duplexes were determined at pH 7.4, varying the concentrations of NaCl, KCl and NaCl + MgCl2. In the second set, transition temperatures were determined in 150 mM NaCl + 2 mM MgCl2, varying the pH values from 5 to 9. These measurements revealed no significant differences among the relative duplex stabilities within the covered range of conditions. Further investigations were therefore performed under physiological conditions.
The thermodynamic stability of each duplex was determined at pH 7.4 in the presence of 150 mM NaCl, 150 mM KCl and 150 mM NaCl + 2 mM MgCl2. The results in Table
Table 2. Data obtained from measurements in 10 mM Tris-HCl (pH 7.4) and 150 mM NaCl, 150 mM KCl or 150 mM NaCl + 2 mM MgCl2
The transition temperatures (`melting points') of the duplexes formed from the functionalized oligoribonucleotides C, D, E and F and the complementary partner strand A were always lower than those of the corresponding unmodified duplex B.A. Usually, lower transition temperatures are taken as an indication of weaker pairing. However, determination of the [Delta]G° values of duplex formation revealed that at a physiologically relevant temperature of 37°C some of the functionalized oligonucleotides were in fact more strongly paired than the parent one (Table
The thermodynamic data for the crown ether-containing RNA strands in both NaCl and KCl solution at 37°C revealed a less favourable [Delta]G° for the internally functionalized duplex C.A than for the externally functionalized duplex D.A. In KCl solution the duplex C.A showed a weaker pairing than the unmodified duplex B.A, whereas in NaCl both sequences displayed about the same [Delta]G° value. In the presence of 2 mM MgCl2, however, the duplex C.A displayed a stronger pairing than the duplex D.A and than the parent duplex B.A. The more negative [Delta]G° value of duplex formation for the functionalized duplex C.A (relative to the parent duplex B.A) is the consequence of a more favourable [Delta]H° term, which below 50°C compensates for the less favourable [Delta]S° term (Fig.
The [Delta]G° values of duplex formation at 37°C obtained from pairing of the triethyleneglycol-substituted oligoribonucleotides E.A and F.A indicate a stronger association of sequence E compared with sequence F in all three environments investigated. In KCl and NaCl solution the difference in [Delta]G°37_C values was about -3 kcal/mol in favour of the internally functionalized duplex E.A. In the presence of 2 mM MgCl2, however, a very large energy difference of -8 kcal/mol (35% of total at 37°C), again in favour of sequence E, was observed. The externally functionalized duplex F.A uniformly displayed a weaker pairing than the unmodified duplex B.A, whereas the internally functionalized duplex E.A showed an equal pairing in KCl (-0.3 kcal/mol), a slightly stronger pairing in NaCl (-2.3 kcal/mol) and a much stronger pairing in NaCl + MgCl2 (-6.5 kcal/mol) than the unmodified duplex B.A. Again, the strong stabilization of the functionalized duplex E.A (relative to the unfunctionalized duplex B.A) is a consequence of a more favourable [Delta]H° term which below 65°C compensates for the less favourable [Delta]S° term (Fig.
DISCUSSION
These thermodynamic data for duplex stability indicate that in the presence of Mg2+ ions pairing is slightly stabilized by a 1-aza-18-crown-6 group and strongly stabilized by a triethyleneglycol group tethered to the 6[prime]-O position of an allofuranosyl nucleoside. In the absence of Mg2+ ions only the triethyleneglycol-substituted duplex is slightly stabilized.
Structurally, these observations indicate a specific interaction of the Mg2+-complexed ligand of the modified nucleosides with the negatively charged backbone of the other strand. The open chain ligand present in oligonucleotide E forms a relatively weak complex with the Mg2+ ion, still offering additional coordination sites. It was concluded that the strong duplex stabilization observed results from formation of a complex between the ethyleneglycol moiety, a Mg2+ ion and a phosphodiester group of the partner strand A. Thereby, one hydrated Mg2+ ion within the major groove is replaced by a chelated Mg2+ ion (Fig.
Figure 3. A comparison of the temperature dependance of [Delta]G° values among the parent duplex B.A, the crown ether-substituted duplexes C.A and D.A (a) and the triethylenglycol-substituted duplexes E.A and F.A (b). Data were obtained from concentration-dependant transition curves measured in 150 mM NaCl + 2 mM MgCl2, 10 mM Tris-HCl (pH 7.4). The additional non-covalent intramolecular interactions were reflected in the enthalpic stabilization of duplexes C.A and E.A. On the other hand, the conformational changes within the tethered group and/or the backbone required for such an interaction led to an entropic destabilization, which compensated largely, but at low temperature not entirely, for the enthalpic stabilization. In all cases where no stabilization of the duplex could be observed no significant differences in enthalpy and entropy terms were measured, indicating that the additional functional group was pointing into the solution. We were unable to detect any structural changes upon introduction of the functionalized allofuranosyl cytidines into RNA strands by CD spectroscopy. All duplexes essentially had the same CD spectrum, typical for an A-type RNA duplex.
CONCLUSION AND OUTLOOK
The strategy presented here allows a straightforward preparation of a variety of functionalized oligonucleotides and revealed the 5[prime]-position of nucleosides as a new and promising site for modification, labelling and conjugate formation. The metal-binding derivatives which have been prepared as first examples constitute a new principle for stabilizing oligonucleotide duplexes. We now are preparing reactive allofuranosyl and 2[prime]-deoxyallofuranosyl nucleosides with the other three nucleobases and with different tethers. We will also determine whether 5[prime]-triphosphates derived from those can be incorporated enzymatically into DNA or RNA. Employing nucleotide analogues related to those presented in this paper, we are trying to find oligoribo- and oligodeoxyribonucleotide analogues eventually capable of stabilizing RNA and/or DNA structure, catalysing specific strand scission reactions and enhancing cellular uptake.
Figure 4. Model representation (MacroModel) of the metal ion-mediated interaction between the two strands of the functionalized duplexes C.A and E.A as deduced from the thermodynamic data of duplex formation. For the sake of clarity, only the relevant part of the duplex is shown; the atoms of the phosphodiester backbones and the bonds of the functional group are in black.
MATERIALS AND METHODS
General
Work-up implies distribution of the reaction mixture between CH2Cl2 and saturated aqueous NaHCO3 solution, drying of the organic layer with MgSO4, filtration and evaporation of the filtrate. TLC: unless otherwise mentioned, precoated silica gel plates from Macherey & Nagel (exceptionally pre-coated Al2O3 plates from Merck), stained by dipping into a solution of 10 ml anisaldehyde, 10 ml concentrated H2SO4, 2 ml AcOH in 180 ml EtOH and subsequent heating with a heat gun. Column chromatography (CC): unless otherwise mentioned, silica gel 60 (230-400 mesh) from Fluka (exceptionally Al2O3, activity III, from ICN Adsorbentien). Optical rotation ([[alpha]]lpile {{2 5} above D}): Jasco-DIP-370, all measurements in CHCl3 (1 g/100 ml). UV spectra: Uvikon 931, [lambda]max in nm, [epsis] (dm3/mol/cm) indicated in parentheses, all measurements in MeOH. NMR: Varian-Gemini 300 (1H, 300 MHz; 31P, 121 MHz), chemical shift [delta]H in p.p.m. (Me4Si as internal standard), [delta]P in p.p.m. (85% H3PO4 as external standard), all measurements in CDCl3, coupling constants J in Hz. MS: VG-ZAB2-SEQ, all samples measured in FAB+ mode, 3-nitrobenzyl alcohol as matrix, relative intensity in % as indicated in parentheses.
Oligonucleotide synthesis
The oligoribonucleotides were assembled on CPG supports (1.5 µmol scale) on a Pharmacia Gene Assembler using the methods in Table
Thermal denaturation studies
Absorbance versus temperature profiles were recorded in fused quartz cuvettes at 260 nm on a Cary Bio-1 spectrophotometer equipped with a Peltier temperature control device. The samples were prepared under sterile conditions from stock solutions of the oligonucleotide, 1 M Tris-HCl buffer (pH 7.4) and 5 M NaCl solution and subsequently degassed. A layer of silicon oil was placed on the surface of the solution. The studies were carried out at 0.5, 1, 2, 4 and 8 µM concentrations of both strands. Prior to the measurements, each sample was briefly heated to 80°C. The curves were obtained with both a cooling and heating ramp of 0.3°C/min.
The transition temperatures were obtained after differentiation of the melting curves and analysed according to Marky and Breslauer (17).
3-O-Benzoyl-6-O-(5-bromopentyl)-1,2-O-isopropyliden-[alpha]-d-allofuranose (2)
In a Dean-Stark apparatus, a solution of 3-O-benzoyl-1,2-O-isopropylidene-[alpha]-d-allofuranose (1) (11) (9.72 g, 30 mmol) and Bu2SnO (11.2 g, 33 mmol) in toluene (75 ml) was refluxed for 1 h. The toluene was evaporated, the residue diluted with DMF (75 ml) and treated with CsF (6.84 g, 45 mmol), 1,5-dibromopentane (16.3 ml, 120 mmol) and Bu4NI (16.62 g, 45 mmol). The suspension was kept at room temperature for 8 h. Work-up and CC (hexane/EtOAc 9:1-6:4) gave 2 (9.4 g, 74%) as a colourless, viscous liquid. TLC (hexane/EtOAc 1:1) 0.64; [[alpha]]lpile {{2 5} above D} +28.0; [delta]H 1.33 (s, Me), 1.41-1.49 (m, CH2), 1.54 (s, Me), 1.58-1.61 (m, CH2), 1.81-1.85 (m, CH2), 2.46 (d, J 3.7, 5-OH), 3.37 (t, J 6.8, CH2), 3.40-3.50 (3 H, m, CH2O, H,H[prime]-6), 4.12 (m, H-5), 4.36 (dd, J 3.7, 8.4, H-4), 4.97 (dd, J 3.7, 5.0, H-2), 5.15 (dd, J 5.0, 8.4, H-3), 5.89 (d, J 3.7, H-1), 7.46 (m, 2 ArH), 7.59 (m, 1 ArH); 8.06 (m, 2 ArH); m/z 474 (1, MH+), 104 (100).
6-O-(5-bromopentyl)-1,2,3-O-tribenzoyl-5-O-[(triisopropyl)-silyl]-[alpha],[beta]-d-allofuranose [3([alpha]/[beta])]
At room temperature, a solution of 2 (3.52 g, 7.4 mmol) in CH2Cl2 (25 ml) was treated with Pri2NEt (3.8 ml, 22.2 mmol) and Pri3SiOSO2CF3 (2.6 ml, 9.7 mmol) for 1 h. After work-up, the crude product was treated with CF3COOH (50 ml) and H2O (50 ml) for 2 h. Work-up gave a colourless oil, which was dissolved in pyridine (3 ml) and CH2Cl2 (6 ml) and treated with benzoyl chloride (1.5 ml, 13 mmol) and dimethylamino pyridine (82 mg, 0.67 mmol) for 12 h. Work-up and CC (hexane/EtOAc 9:1-7:3) gave 3a([alpha]/[beta]) (4.7 g, 54%, [alpha]/[beta] 1:2 by NMR) as a colourless foam. TLC (hexane/EtOAc 4:1) 0.66; [delta]H 0.66-1.15 (21 H, m, Pri3Si), 1.33-1.53 (m, 2 CH2), 1.65-1.82 (m, CH2), 3.21-3.24 (m, H-6), 3.34-3.46 (3 H, m, H[prime]-6, CH2), 3.50-3.62 (m, CH2), 4.30 [m, H-C(5)], 4.67 [dd, J 4.4, 5.6, H-4([alpha])], 4.97 [m, H-4([beta])], 5.60 [dd, J 4.4, 6.5, H-2([beta])], 5.90 [dd, J 2.2, 5.3, H-2([alpha])], 6.03 [dd, J 5.3, 5.5, H-3([alpha])], 6.09 [dd, J 1.9, 6.5, H-3([beta])], 6.61 [d, J 2.2, H-1([alpha])], 6.87 [d, J 4.4, H-1([beta])], 7.17-7.61 (m, 9 ArH), 7.76-8.10 (m, 6 ArH); m/z 801 (6, MH+).
N4-Benzoyl-1-[6[prime]-O-(5-bromopentyl)-2[prime],3[prime]-di-O-benzoyl-[beta]-d-allofuranosyl]cytosine (5)
A suspension of 3([alpha]/[beta]) (5.4 g, 6.75 mmol), N4-benzoylcytosine (18) (1.6 g, 7.4 mmol) and bis(trimethylsilyl)acetamide (4.2 ml, 16.9 mmol) in CH3CN (27 ml) was stirred at 70°C for 1 h, treated with SnCl4 (3.2 ml, 27 mmol) and stirred at 70°C for 20 min. After work-up, the residue was dissolved in CH3CN (200 ml), treated with HCl (conc.) (2 ml) and HF (40% in H2O) (4 ml) and stirred at room temperature for 8 h. Work-up and CC (hexane/EtOAc 8:2-4:6) gave 5 (3.6 g, 71%) as a white foam. TLC (hexane/EtOAc 2:8) 0.55; [[alpha]]lpile {{2 5} above D} -97.2; [lambda]max 261 (21 000), 229 (28 900); [delta]H 1.44-1.64 (4 H, m, 2 CH2), 1.75-1.90 (m, CH2), 3.37 (t, J 6.8, CH2), 3.48-3.53 (m, 2 H-6[prime]), 3.65-3.67 (3 H, m, OCH2, OH-5[prime]), 4.30 (m, H-5[prime]), 4.45-4.49 [m, H-C(4[prime])], 5.88 (dd, J 5.6, 6.6, H-2[prime]), 5.99 (dd, J 2.5, 5.6, H-3[prime]), 6.58 (d, J 6.8, H-1[prime]), 7.26-8.03 (m, 15 ArH, H-5), 8.35 (d, J 7.8, H-6), 8.77 (s, NH); m/z 736 (11, M+).
N4-Benzoyl-1-[6[prime]-O-(5-bromopentyl)-5[prime]-O-(4,4[prime]-dimethoxy-trityl)-[beta]-d-allofuranosyl]cytosine (7)
A suspension of 5 (2.7 g, 3.6 mmol), AgNO3 (612 mg, 3.6 mmol) and sym-collidine (1.2 ml, 9 mmol) in CH2Cl2 (12 ml) was treated with 4,4[prime]-dimethoxytrityl chloride (1.84 g, 5.4 mmol) for 1 h at room temperature. After filtration and evaporation, the residue was dissolved in an ice-cold solution of THF/MeOH/H2O 5:4:1 (150 ml), treated with 10 N aqueous NaOH (3 ml) at 4°C for 15 min, then neutralized with AcOH (1.9 ml) and concentrated to 40 ml. Work-up and CC [CH2Cl2 to CH2Cl2/MeOH 97:3 (+2% NEt3)] gave 7 (2.46 g, 84%) as a white foam. TLC (MeOH/CH2Cl2 8:92) 0.50; [[alpha]]lpile {{2 5} above D} +28.4; [lambda]max 262 (14 700), 238 (22 300); [delta]H 1.44-1.63 (m, 2 CH2), 1.80-1.92 (m, CH2), 3.19-3.27 (m, OCH2, H-6[prime]), 3.42 (t, J 6.5, CH2), 3.40-3.45 (m, H[prime]-6[prime]), 3.55-3.64 (m, H-5[prime], OH), 3.80 (s, 2 OMe), 4.21-4.27 (m, H-2[prime], H-4[prime]), 4.44 (s, OH), 4.67 (dd, J 5.6, 5.8, H-3[prime]), 5.86(d, J 3.1, H-1[prime]), 6.82-6.86 (m, 4 ArH), 7.22-7.63 (m, 12 ArH, H-5), 7.88-7.89 (m, 2 ArH), 7.92 (d, J 7.5, H-6), 8.88 (s, NH); m/z 829 (18, MH+), 303 (100).
N4-Benzoyl-1-[6[prime]-O-(5-bromopentyl)-5[prime]-O-(4,4[prime]-dimethoxy-trityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofurano-syl]cytosine (9)
A solution of 7 (2.46 g, 3.0 mmol) and Pri2NEt (2.1 ml, 12 mmol) in ClCH2CH2Cl (10 ml) was treated with Bu2SnCl2 (1.1 g, 3.6 mmol) at room temperature for 1.5 h, then treated with Pri3SiOCH2Cl (1.0 g, 4.5 mmol) at 80°C for 15 min. Work-up and CC [hexane/EtOAc 5:5-2:8 (+ 2% NEt3)] gave 9 (1.28 g, 42%) and 10 (570 mg, 20%) as pale yellow foams. TLC (hexane/EtOAc 3:7) 0.63; [[alpha]]lpile {{2 5} above D} +36.8; [lambda]max 260 (17 800), 238 (23 800); [delta]H 1.08-1.22 (21 H, m, Pri3Si), 1.41-1.49 (m, 2 CH2), 1.81-1.86 (m, CH2), 3.15-3.22 (m, H-6[prime], CH2O), 3.39 (t, J 6.9, CH2), 3.50-3.55 (m, OH, H[prime]-6[prime]), 3.67 (s, H-5[prime]), 3.81 (s, 2 OMe), 4.15-4.20 (m, H-2[prime], H-4[prime]), 4.63 (dd, J 5.4, 6.4, H-3[prime]), 5.11 and 5.23 (2d, J 4.7, OCH2O), 6.01 (d, J 2.8, H-1[prime]), 6.84-6.87(m, 4 ArH), 7.25-7.61 (m, 12 ArH, H-5), 7.79-7.88 (m, 2 ArH), 7.89 (d, J 7.8, H-6), 8.61 (s, NH); m/z 1016 (100, MH+).
N4-Benzoyl-1-[6[prime]-O-(5-bromopentyl)-5[prime]-O-(4,4[prime]-dimethoxy-trityl)-3[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl]-cytosine (10)
From the reaction described above. TLC (hexane/EtOAc 3:7) 0.35; [[alpha]]lpile {{2 5} above D} -11.4; [delta]H 1.08-1.11 (21 H, m, Pri3Si), 1.39-1.48(m, 2 CH2), 1.81-1.86 (m, CH2), 3.13-3.21 (m, H-6[prime], OCH2), 3.40 (t, J 6.8, CH2), 3.37-3.42 (m, H[prime]-6[prime]), 3.50 (s, H-5[prime]), 3.80(s, 2 OMe), 3.77-3.84 (m, OH-2[prime]), 4.12 (m, H-2[prime]), 4.32 (m, H-4[prime]), 4.65 (dd, J 3.4, 5.6, H-3[prime]), 4.98 and 5.17 (2d, J 4.8, OCH2O), 5.96 (d, J 6.2, H-1[prime]), 6.83-6.85 (m, 4 ArH), 7.23-7.62 (m, 14 ArH, H-5), 7.90 (d, J 7.2, H-6), 8.61 (s, NH); m/z 1016 (54, MH+), 303 (100).
N4-Acetyl-1-{6[prime]-O-[5-(1,4,7,10,13-pentaoxa-16-aza-cyclo-octadec-16-yl)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine (14)
A solution of 9 (408 mg, 0.4 mmol), 1-aza-18-crown-6 (263 mg, 1.0 mmol), Pri2NEt (0.34 ml, 2 mmol) and Bu4NI (222 mg, 0.6 mmol) in EtOH (1 ml) was stirred at 75°C for 30 h. After work-up, the residue was treated with NH3 in EtOH/H2O 9:1 (2 ml) at room temperature for 10 h. Evaporation and filtration through a short Al2O3 column (CH2Cl2/MeOH 9:1) gave a product (277 mg, 0.25 mmol) which was dissolved in DMF (1 ml) and treated with Ac2O (31 µl, 0.32 mmol). After stirring for 4 h at room temperature, work-up and CC (Al2O3, CH2Cl2 to MeOH/CH2Cl2 3:97) 14 (192 mg, 43%) was obtained as a yellow foam. TLC (Al2O3, MeOH-CH2Cl2 4:96) 0.42; [[alpha]]lpile {{2 5} above D} +19.9; [lambda]max 261 (19 900), 238 (23 600); [delta]H 1.06-1.09 (21 H, m, Pri3Si), 1.20-1.28 (m, CH2), 1.37-1.49 (m, 2 CH2), 2.21 (s, MeCO), 2.46 (t, J 7.5, CH2), 2.74 (t, J 5.9, 4 CH2), 3.12-3.19 (m, H-6[prime], CH2O), 3.45-3.50 (dd, J 6.5, 10.5, H[prime]-6[prime]), 3.58-3.67 (22 H, m), 3.80(s, OMe), 4.12-4.15 (m, H-2[prime], H-4[prime]), 4.60 (m, H-3[prime]), 5.07 and 5.21 (2d, J 4.7, OCH2O), 5.98 (d, J 3.8, H-1[prime]), 6.81-6.84 (m, 4 ArH), 7.23-7.51 (m, ArH, H-5), 7.74-7.67 (d, J 7.5, H-6), 9.18 (s, NH); m/z 1136 (100, M+).
N4-Acetyl-1-{6[prime]-O-[5-(1,4,7,10,13-pentaoxa-16-aza-cyclo-octadec-16-yl)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine 3[prime]-[(2-cyanoethyl) N,N-diisopropyl-phosphoramidite] (15)
A solution of 14 (192 mg, 0.17 mmol) in CH2Cl2 (0.75 ml) was treated consecutively with Pri2NEt (73 µl, 0.43 mmol) and (2-cyanoethyl)(N,N-diisopropylamino)chlorophosphite (48 mg, 0.20 mmol). After stirring for 1 h at room temperature, the mixture was subjected to CC (Alox, hexane/EtOAc 6:4-3:7) and 15 (210 mg, 92%) was obtained as a pale yellow foam (1:1 mixture of diastereoisomers). TLC (Al2O3, MeOH/CH2Cl2 4:96) 0.52; [lambda]max 239 (23 600); [delta]H 1.00-1.07 (21 H, m, Pri3Si), 1.13-1.21 (m, 4 Me), 1.32-1.36 (m, 3 CH2), 2.21 (s, MeCO), 2.42-2.44 (m, CH2), 2.54 and 2.61 (2t, J 6.5, CH2), 2.73 (t, J 5.8, 4 CH2), 2.97-3.08 (m, OCH2, H-6[prime]), 3.39-3.67 (25 H, m), 3.79 (s, 2 OMe), 4.27 and 4.32 (2dd, J 5.0, 5.3, H-2[prime]), 4.40 (m, H-4[prime]), 4.64-4.77 (m, H-3[prime]), 4.96-5.06 (m, OCH2O), 6.05 (0.5 H, d, J 3.7, H-1[prime]), 6.06 (0.5 H, d, J 4.7, H-1[prime]), 6.81-6.84 (m, 4 ArH), 7.23-7.50 (m, 10 ArH, H-5), 7.61-7.66 (m, H-6), 9.18 (s, NH); [delta]P 150.2, 149.8; m/z 1336 (100, M+).
6-O-[5-(N-Allyloxycarbonyl-methylamino)-pentyl]-1,2,3-tri-O-benzoyl-5-O-[(triisopropyl)silyl]-[beta]-d-allofuranose [4([alpha]/[beta])]
At room temperature, a solution of 2 (9.78 g, 21 mmol) in CH2Cl2 (70 ml) was treated with Pri2NEt (10.8 ml, 63 mmol) and Pri3SiOSO2CF3 (7.34 ml, 27.3 mmol) for 1 h. Work-up gave a crude product, which was treated with MeNH2 in EtOH (8 M, 70 ml) for 1 h at room temperature. After evaporation and work-up, the residue was dissolved in CH2Cl2 (70 ml) and treated with Pri2NEt (7.2 ml, 42 mmol) and allyl chloroformate (2.2 ml, 21 mmol) at room temperature for 0.5 h. Work-up gave a yellow oil. The crude product obtained by treatment of the resulting oil with CF3COOH (50 ml) and H2O (50 ml) for 1 h at room temperature was dissolved in pyridine (15 ml) and CH2Cl2 (30 ml) and treated with benzoyl chloride (5.6 ml, 48 mmol) and dimethylaminopyridine (293 mg, 2.4 mmol) for 12 h. Work-up and CC (hexane/EtOAc 9:1-7:3) gave 4([alpha]/[beta]) (2.5 g, 42%, [alpha]/[beta] 1:2 by 1H NMR). TLC (hexane/EtOAc 7:3) 0.47; [delta]H 1.05-1.15 (7 H, m, Pri3Si), 1.21 (m, CH2), 1.44 (m, 2 CH2), 2.83 (s, CH3), 3.09-3.11 (m, CH2), 3.31-3.41 (m, 2 H-6), 3.59 (d, J 5.9, CH2), 4.26 [m, H-5([alpha])], 4.31 [m, H-5([beta])], 4.55 (m, CH2), 4.66 [dd, J 4.3, 5.1, H-4([alpha])], 4.78 [dd, J 1.8, 1.9, H-4([beta])], 5.23 (m, CH2), 5.60 [dd, J 4.3, 6.5, H-2([beta])], 5.90 [dd, J 2.5, 5.3, H-2([alpha])], 5.92-5.94 (m, CH), 6.02 [dd, J 2.2, 5.3, H-3([alpha])], 6.08 [dd, J 1.9, 6.5, H-3([beta])], 6.61 [d, J 2.1, H-1([alpha])], 6.87 [d, J 4.4, H-1([beta])], 7.17-7.61 (m, 9 ArH), 7.75-8.12 (m, 6 ArH); m/z 833 (2, MH+), 710 (100).
N4-Benzoyl-1-{6[prime]-O-[5-(N-allyloxycarbonyl-methylamino)-pentyl]-2[prime],3[prime]-di-O-benzoyl-[beta]-d-allofuranosyl}cytosine (6)
As described for 5, with 4([alpha]/[beta]) (4.7 g, 5.7 mmol), N4-benzoylcytosine (18) (1.36 g, 6.3 mmol), bis(trimethylsilyl)acetamide (3.5 ml, 14.4 mmol), CH3CN (20 ml), Me3Si-OTf (2.7 ml, 22.8 mmol), then CH3CN (100 ml), HCl (conc.) (1 ml), HF (40% in H2O) (2 ml). Work-up and CC (hexane/EtOAc 8:2-4:6) gave 6 (3.4 g, 74%) as a white foam. TLC (hexane/EtOAc 4:6) 0.53; [[alpha]]lpile {{2 5} above D} -54.2; [lambda]max 261 (26 600), 237 (30 100); [delta]H 1.34 (m, CH2), 1.57 (m, 2 CH2), 2.89 (s, CH3), 3.26 (br. s, CH2), 3.51-3.53(m, 2 H-6[prime]), 3.64-3.66 (m, CH2O, OH), 4.29 (s, H-5[prime]), 4.45(s, H-4[prime]), 4.57-4.59 (m, CH2), 5.25 (m, CH2), 5.83-5.97 (m, H-2[prime], H-3[prime], CH), 6.60 (s, H-1[prime]), 7.27-8.03 (m, 15 ArH, H-5), 8.36(m, H-6), 8.72 (s, NH); m/z 770 (100, MH+).
N4-Benzoyl-1-{6[prime]-O-[5-(N-allyloxycarbonyl-methylamino)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-[beta]-d-allofuranosyl}cytosine (8)
As described for 7, with 6 (2 g, 2.66 mmol), AgNO3 (687 mg, 4.0 mmol), sym-collidine (0.7 ml, 2 mmol), CH2Cl2 (12 ml), 4,4[prime]-dimethoxytrityl chloride (1.36 g, 4.0 mmol), then THF/MeOH/H2O 5:4:1 (120 ml), 10 N aqueous NaOH (2.4 ml), AcOH (1.5 ml). Work-up and CC [CH2Cl2 to CH2Cl2/MeOH 98:2 (+2% NEt3)] gave 8 (2.1 g, 91%) as a white foam. TLC (MeOH/CH2Cl2 8:92) 0.54; [[alpha]]lpile {{2 5} above D} +28.3; [lambda]max 258 (13 400), 237 (27 000); [delta]H 1.27 (m, CH2), 1.32 (m, 2 CH2), 2.90 (s, CH3), 3.21-3.30(m, CH2, CH2O, H-6[prime]), 3.41 (dd, J 4.2, 10.5, H[prime]-6[prime]), 3.62 (s, H-5[prime]), 3.80 (s, 2 OMe), 4.22 (m, H-2[prime], H-4[prime]), 4.56 (d, J 5.3, CH2), 4.66 (m, H-3[prime]), 5.16-5.30 (m, CH2), 5.88 (d, J 2.2, H-1[prime]), 5.89-5.97 (m, CH), 6.82-6.85 (m, 4 ArH), 7.21-7.62 (m, 12 ArH, H-5), 7.88-7.93 (m, 2 ArH, H-6), 8.65 (s, NH); m/z 864 (28, MH+), 303 (100).
N4-Benzoyl-1-{6[prime]-O-[5-(N-allyloxycarbonyl-methylamino)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyl-oxy]methyl}-[beta]-d-allofuranosyl}cytosine (11)
As described for 9, with 8 (862 mg, 1.0 mmol), Pri2NEt (0.85 ml, 5 mmol), ClCH2CH2Cl (4 ml), Bu2SnCl2 (340 mg, 1.1 mmol), Pri3SiOCH2Cl (230 mg, 1.2 mmol). Work-up and CC [hexane/EtOAc 5:5-2:8 (+2% NEt3)] gave 11 (470 mg, 45%) and 12 (209 mg, 20%) as pale yellow foams. TLC (hexane/EtOAc 15:85) 0.60; [[alpha]]lpile {{2 5} above D} +29.1; [lambda]max 260 (23 900), 238 (28 700); [delta]H 1.07-1.14 (21 H, m, Pri3Si), 1.23 (m, CH2), 1.46-1.56(m, 2 CH2), 2.89 (s, Me), 3.13-3.26 (m, 2 CH2, H-6[prime]), 3.48-3.56 (m, OH, H[prime]-6[prime]), 3.64-3.66 (m, H-5[prime]), 3.82 (s, 2 OMe), 4.11 (m, H-2[prime]), 4.20 (m, H-4[prime]), 4.56-4.67 (m, H-3[prime], CH2), 5.15 and 5.23 (2d, J 4.6, OCH2O), 5.17-5.31 (m, CH2), 5.88-5.97 (m, CH), 6.01 (d, J 2.3, H-1[prime]), 6.83-6.86 (m, 4 ArH), 7.23-7.63 (m, 12 ArH, H-5), 7.87-7.89 (m, 2 ArH, H-6), 8.57 (s, NH); m/z 1050 (4, MH+), 303 (100).
N4-Benzoyl-1-{6[prime]-O-[5-(N-allyloxycarbonyl-N-methyl)-pentyl]-5[prime]-O-(4,4[prime]-dimethoxytrityl)-3[prime]-O-{[(triisopropyl)silyl-oxy]methyl}-[beta]-d-allofuranosyl}cytosine (12)
Obtained from the reaction described above. TLC (hexane/AcOEt 15:85) 0.36; [[alpha]]lpile {{2 5} above D} -7.2; [lambda]max 260 (24 600), 238 (29 700); [delta]H 1.07-1.11 (21 H, m, Pri3Si), 1.20-1.26 (m, CH2), 1.42-1.64 (m, 2 CH2), 2.90 (s, CH3), 3.22-3.27 (m, NCH2, CH2O, H-6[prime]), 3.36-3.41 (m, H[prime]-6[prime]), 3.48-3.50 (m, H-5[prime]), 3.75-3.81 (m, OH-2[prime]), 3.80 (s, 2 OMe), 4.10-4.14 (m, H-2[prime]), 4.32 (m, H-4[prime]), 4.56-4.58 (m, CH2), 4.65 (m, H-3[prime]), 4.97 and 5.17 (2d, J 4.7, OCH2O), 5.20-5.31 (m, CH2), 5.89-5.98 (m, CH), 5.96 (d, J 6.2, H-1[prime]), 6.79-6.86 (m, 4 ArH), 7.21-7.64 (m, 14 ArH, H-5), 7.90 (d, J 6.2, H-6), 8.64 (s, NH); m/z 1059 (8, MH+), 303 (100).
N4-Benzoyl-1-{6[prime]-O-(N-methyl-5-aminopentyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine (13)
A suspension of Pd[P(Ph)3]4 (126 mg, 0.11 mmol) and PPh3 (60 mg, 0.23 mmol) in CH2Cl2 (3 ml) was added dropwise to a solution of 11 (604 mg, 0.58 mmol) and Et2NH (0.3 ml, 2.9 mmol) in CH2Cl2 (3 ml). The solution was stirred for 1 h at room temperature. After evaporation, the residue was subjected to CC [CH2Cl2 to MeOH/CH2Cl2 5:95 (+ 2% Et3N)] and 13 (488 mg, 88%) was obtained as a yellow foam. TLC (Al2O3, MeOH/CH2Cl2 4:96) 0.45; [[alpha]]lpile {{2 5} above D} +23.4; [lambda]max 261 (19 800), 238 (25 500); [delta]H 1.08 (21 H, m, Pri3Si), 1.29-1.37 (m, CH2), 1.44-1.51 (m, 2 CH2), 2.43 (s, NMe), 2.55 (t, J 1.2, NCH2), 3.15-3.20 (m, OCH2, H-6[prime]), 3.48-3.56 (m, H[prime]-6[prime], NH), 3.65-3.68 (m, H-5[prime]), 3.77-3.79 (m, OH-3[prime]), 3.81 (s, 2 OMe), 4.13-4.17(m, H-4[prime]), 4.21 (dd, J 3.1, 5.3, H-2[prime]), 4.63-4.65 (m, H-3[prime]), 5.10 and 5.22 (2 d, J 4.7, OCH2O), 5.99 (d, J 3.1, H-1[prime]), 6.83-6.86 m, 4 ArH), 7.25-7.63 (m, 12 ArH, H-5), 7.80-7.83 (m, 2 ArH), 7.91 (d, J 7.2, H-6); m/z 965 (100, M+).
N4-Benzoyl-1-{6[prime]-O-(N-methyl-5-[(2-[2-methoxy-ethoxy]-ethoxy)-ethylamino]-pentyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine (16)
A solution of 13 (244 mg, 0.253 mmol), Bu4NI (187 mg, 0.506 mmol), Pri2NEt (0.17 ml, 1.0 mmol) in 0.8 ml toluene was treated with Me(OCH2CH2)3Cl (18) (110 mg, 0.61 mmol) and stirred for 8 h at 95°C. Work-up and CC [CH2Cl2 to MeOH/CH2Cl2 3:97 (+ 2% Et3N)] gave 16 (120 mg, 44%) as a yellow foam. TLC (Al2O3, MeOH/CH2Cl2 3:97) 0.41; [lambda]max 259 (19 700), 234 (28 700); [delta]H 1.05-1.09 (21 H, m, Pri3Si), 1.26-1.29 (m, CH2), 1.32-1.39 (m, 2 CH2), 2.25 (s, NMe), 2.33-2.38(m, NCH2), 2.55-2.59 (m, CH2), 3.14-3.21 (m, OCH2, H-6[prime]), 3.36 (s, OMe), 3.50-3.66 (m, OH-3[prime], 2 CH2, H[prime]-6[prime], H-5[prime]), 3.80(s, 2 OMe), 4.14-4.21 (m, H-2[prime], H-4[prime]), 4.62-4.66 (m, H-3[prime]), 5.13 and 5.23 (2 d, J 4.7, OCH2O), 6.01 (d, J 2.8, H-1[prime]), 6.84-6.86(m, 4 ArH), 7.25-7.63 (m, 12 ArH, H-5), 7.82-7.85 (m, 2 ArH), 7.88 (d, J 7.2, H-6); m/z 1113 (3, MH+), 303 (100).
N4-Benzoyl-1-{6[prime]-O-(N-methyl-5-[(2-[2-methoxy-ethoxy]-ethoxy)-ethylamino]-pentyl)-5[prime]-O-(4,4[prime]-dimethoxytrityl)-2[prime]-O-{[(triisopropyl)silyloxy]methyl}-[beta]-d-allofuranosyl}cytosine 3[prime]-[(2-cyanoethyl) N,N-diisopropyl-phosphoramidite] (17)
As described for 15, with 16 (120 mg, 0.11 mmol), CH2Cl2 (0.3 ml), Pri2NEt (47 µl, 0.28 mmol) and (2-cyanoethyl)(N,N-diisopropylamino)chlorophosphite (31 mg, 0.12 mmol). CC (Al2O3, hexane/AcOEt 6:4-2:8) gave 17 (130 mg, 90%) as a pale yellow foam (1:1 mixture of diastereoisomers). TLC (Al2O3, MeOH/CH2Cl2 4:96) 0.52; [lambda]max 261 (18 700), 239 (27 700); [delta]H 1.02-1.08 (21 H, m, Pri3Si), 1.16-1.25 (m, 4 Me), 1.26-1.28(m, CH2), 1.34-1.39 (m, 2 CH2), 2.24 (s, NMe), 2.31-2.34 (m, NCH2), 2.54-2.63 (m, 2 CH2), 2.92-3.08 (m, OCH2, H-6[prime]), 3.36 and 3.37 (2s, CH3), 3.43-3.62 (m, 12 H, 2 CH2, H[prime]-6[prime], H-5[prime]), 3.80 (s, 2 OMe), 4.20-4.41 (m, H-2[prime], H-4[prime]), 4.66-4.69 (m, 0.5 H, H-3[prime]), 4.75-4.79 (m, 0.5 H, H-3[prime]), 5.00-5.05 (m, OCH2O), 6.07 and 6.09 (2 s, H-1[prime]), 6.82-6.85 (m, 4 ArH), 7.23-7.71 (m, 12 ArH, H-5), 7.89-7.91 (m, 2 ArH), 8.02 (d, J 7.2, H-6), 8.61 (s, NH); [delta]P 150.2, 149.8; m/z 1313 (26, MH+), 1312 (36, M+), 303 (100).
SUPPLEMENTARY MATERIAL
The 13C NMR and IR data of compounds 2-17, the CD spectra of all duplexes and a table of all concentration-dependant transition temperatures are available upon request.
ACKNOWLEDGEMENTS
We thank Prof. A. Vasella for continously supporting this work. We also thank Patrick A. Weiss (Xeragon AG, Switzerland) for providing us with numerous reagents and TOM-phosphoramidites and T. Vivlemore and A. Ernst for helpful suggestions. The ETH Zürich Research Council and the Alfred Werner Foundation are gratefully acknowledged for financial support.
REFERENCES
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