Synthesis and hybridization properties of β- and α-oligodeoxynucleotides containing β- and α-1-(3-C-allyl-2-deoxy-D-erythro-pentofuranosyl)thymine and α-1-3-C-(3-aminopropyl)-2-deoxy-D-erythro-pentofuranosylthymine

摘要

J. Chem. Soc. Perkin Trans. 1 1997 3275 Synthesis and hybridization properties of ‚- and ·- oligodeoxynucleotides containing ‚- and ·-1-(3-C-allyl-2-deoxy-Derythro- pentofuranosyl)thymine and ·-1-3-C-(3-aminopropyl)-2- deoxy-D-erythro-pentofuranosylthymine Pia N. Jørgensen,a Ulrik S. Sørensen,†,a Henrik M. Pfundheller,a Carl E. Olsen b and Jesper Wengel *,c a Department of Chemistry Odense University DK-5230 Odense M Denmark b Department of Chemistry The Royal Veterinary and Agricultural University DK-1871 Frederiksberg C Denmark c Department of Chemistry Chemical Laboratory II University of Copenhagen Universitetsparken 5 DK-2100 Copenhagen Denmark Convergent synthesis of ‚- and ·-1-(3-C-allyl-2-deoxy-D-erythro-pentofuranosyl)thymine and their incorporation into ‚- and ·-oligodeoxynucleotides (ODNs) is described.The thermal stabilities of duplexes formed between modified ODNs and complementary single-stranded DNA and RNA have been evaluated. In all cases stable duplexes are formed but whereas ‚-ODNs containing ‚-39-C-allylthymidine show moderately lowered thermal stability towards both DNA and RNA ·-ODNs containing ·-39-Callylthymidine show significantly increased thermal stabilities compared with the corresponding ‚-ODN reference duplexes. Even more stable duplexes towards both DNA and RNA have been obtained using an ·-ODN containing one ·-1-3-C-(3-aminopropyl)-2-deoxy-D-erythro-pentofuranosylthymine monomer. Introduction Synthesis of modified oligodeoxynucleotides (ODNs) for use in control of gene expression has been the subject of active research during the last few years.1 In order to improve the binding affinity enzyme stability cell-uptake and other pharmacokinetic properties a variety of oligonucleotide analogues have been synthesized and evaluated.2,3 In connection with a project directed towards the synthesis of new ODN analogues containing 39-C-alkyl functionalities as attachment sites for e.g.intercalating agents or lipophilic carriers we decided to synthesize 1-(3-C-allyl-2-deoxy-b-D-erythro-pentofuranosyl)- thymine 13 (b) (Scheme 1). The allyl group is suitable for diverse structural manipulations and allows e.g. easy access to both the 39-C-(2-hydroxyethyl)- and the 39-C-(3-hydroxypropyl)- modified nucleosides by either oxidative cleavage followed by reduction or hydroboration followed by in situ oxidation. Additionally it would be interesting to evaluate 1-(3-C-allyl-2- deoxy-b-D-erythro-pentofuranosyl)thymine as a novel monomeric substitute in modified ODNs.Until recently only a few examples of 39-C-allyl-substituted nucleosides were known. 39- C-Allyl-29,39-deoxynucleosides were obtained by addition of a 39-centred radical to allyltributyltin which proceeded in a stereoselective way to afford only the erythro-isomer corresponding to addition from the less hindered a-face of the free-radical intermediate.4 Recently we have reported the synthesis of 39-Callyluridines by cerium-assisted Grignard additions of allylmagnesium bromide to 39-ketouridines.5 However as in the majority of Grignard reactions on ketonucleosides,6 the nucleophilic addition occurred preferentially from the a-face due to steric hindrance from the base moiety.Based on these results we decided to use a convergent synthetic strategy hoping to achieve a favourable ratio between the desired erythro- and the threo-configurated products. In addition the possibility of † Present address Department of Medicinal Chemistry The Royal Danish School of Pharmacy Universitetsparken 2 DK-2100 Copenhagen Denmark. straightforward introduction of various nucleobases appealed to us. Recently ODNs containing nucleoside monomers carrying aminoalkyl linkers tethered at the 19-C 29-O 39-O or 49-C positions of the carbohydrate moiety have been reported.7 Here we introduce an ODN containing one 39-C-aminopropylderivatized monomer synthesized from the parent 39-C-allyl nucleoside. Results and discussion 1-(3-C-Allyl-3,5-di-O-benzyl-b-D-ribofuranosyl)thymine8 1 was prepared in six steps from 59-O-silyl-protected 1,2-Oisopropylidene- a-D-ribofuranosid-3-ulose in an overall yield of 47.The reaction included a stereoselective Grignard addition of allylmagnesium bromide and a 29-O-acyl-assisted nitrogen glycosylation to afford exclusively the b-nucleoside. Unfortunately free-radical deoxygenation 9 of the 29-hydroxy group of compound 1 via the corresponding pentafluorophenyl thionocarbonate by Bu3SnH in the presence of azoisobutyronitrile (AIBN) in hot benzene was unsuccessful in our hands probably due to uncontrolled intramolecular free-radical cyclizations. To overcome this problem we decided to use 2-deoxy-D-ribose as the starting compound for the synthesis of amidite 16a (and 16b). Conversion of 2-deoxy-D-ribose into an anomeric mixture of methyl 2-deoxy-D-erythro-pentofuranoside was done under kinetic control as previously described.10 Reaction of methyl 2- deoxy-D-erythro-pentofuranoside with 1.03 mol equiv.of tertbutyldimethylsilyl chloride (TBDMSCl) and imidazole in dimethylformamide (DMF) 11 afforded after column chromatography pure 5-O-silylated a-anomer 2 (28 yield) the 1 T = thymin-1-yl O BnO OBn T OH 3276 J. Chem. Soc. Perkin Trans. 1 1997 corresponding b-anomer 3 (19 yield) and a fraction containing an anomeric mixture of the disilylated glycosides (9 yield). Assignment of the anomeric configuration was done by 1H–1H 2-D chemical-shift-correlation spectroscopy (COSY) and nuclear Overhauser effect (NOE) difference experiments. In an attempt to control the stereoselectivity of the nucleophilic addition of allylmagnesium bromide to give the erythroconfigurated product the a-anomer 2 was selected.Oxidation of compound 2 with CrO3–pyridine–acetic anhydride reagent 12 gave in 84 yield the corresponding 3-ulose 4 which was used in the next step without further purification. Grignard addition of 1.0 mol equiv. of allylmagnesium bromide afforded the 3-Callyl- a-D-erythro glycoside 5 in 16 yield whereas the 3-C-allyla- D-threo glycoside 6 was obtained in 50 yield. The stereochemistry of the Grignard products was confirmed by 1H–1H COSY and NOE difference experiments. Thus the a-erythro configuration of product 5 was confirmed by observing NOEs between H-1 and H-2b (~3) 3-OH and H-4 (2) and 3-OH and H-2a (3). Mutual NOEs between 3-OH and H-2b and lack of an NOE between 3-OH and H-4 confirmed the a-threo configuration of product 6.These results clearly show that the stereochemical outcome of the Grignard addition was determined by the bulky group at the 5-position and that no stereocontrolling effect was induced by the a-OMe substituent. In addition our experiments indicated that no more than 1.0 mol equiv. of allylmagnesium bromide should be used in the Grignard reaction. Thus reaction of 3-ulose 4 with an excess of allylmagnesium bromide afforded a mixture of acyclic derivatives as a result of additional nucleophilic attack at the anomeric carbon atom. Deprotection of the furanosides 5 and 6 using tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) 13 afforded the diols 7 and 8 in 91 and 82 yield respectively.Subsequent acetylation of both the primary and the tertiary hydroxy groups using acetic anhydride in anhydrous pyridine in the presence of 4-(dimethylamino)pyridine (DMAP) afforded compounds 9 and 10 in 77 and 45 yield respectively. Direct nitrogen glycosylation 14 of 9 and 10 with thymine using N,O-bis(trimethylsilyl)acetamide (BSA) as silylating agent and trimethylsilyl trifluoromethanesulfonate (TMS triflate) as a Lewis acid in anhydrous 1,2-dichloroethane afforded inseparable anomeric mixtures of nucleosides 11 (b:a ~ 1 2.3) and 12 (b:a ~ 1 1.1) in 52 and 64 yield respectively. Exchange of 1,2-dichloroethane with the more polar solvent CH3CN in the direct glycosylation reaction of compound 9 with thymine improved the yield to 74 and reversed the ratio between the b- and a-anomer (b:a ~ 1.5 1).Contrary to the alternative convergent strategy starting from 59-O-silylprotected 1,2-O-isopropylidene-a-D-ribofuranosid-3-ulose,8 this strategy gives access to both anomers which enables evaluation of b- as well as a-ODN analogues. In this context it is notable that a-DNA is interesting as a potential antisense agent as it forms a more stable duplex with complementary RNA than does the corresponding b-DNA strand,15 and is not cleaved by many nucleases.16 Deacetylation of the threo-anomers 12 using methanolic ammonia afforded the b-anomer 14a in 39 yield and the aanomer 14b in 44 yield after column chromatographic separation. These two novel nucleosides are currently being evaluated for biological activity. Treatment of the erythro anomers 11 with CH3ONa–CH3OH afforded an inseparable anomeric mixture 13 in 81 yield.Dimethoxytritylation of mixture 13 in anhydrous THF–anhydrous pyridine with AgNO3 as catalyst,17 afforded 1-3-C-allyl-2-deoxy-5-O-(4,49-dimethoxytrityl)-b-Derythro- pentofuranosylthymine 15a in 47 yield and 1-3-Callyl- 2-deoxy-5-O-(4,49-dimethoxytrityl)-a-D-erythro-pentofuranosyl thymine 15b in 26 yield. The structural assignment of the b and a nucleosides was done by NOE difference experiments and one-dimensional 1H NMR spectroscopy. The key mutual NOE between H-19 and H-49 observed for the b-anomer 15a irradiation of H-19 gave an NOE-effect in H-49 (3) while irradiation of H-49 gave an NOE-effect in H-19 (7) was not seen for the a-anomer 15b for which an NOE between H-49 and H-6 (6) was observed. These results were further supported by the relative chemical shifts of H-59a,b and H-49.The H-49 signal of the a-anomer is shifted downfield relative to the signal of the b-anomer causing the difference in the chemical shifts between the H-59a,b and the H-49 to be greater in the a-anomer than in the b-anomer.18–19 In the 1H NMR spectrum of the banomer 15a the H-59a,b and the H-49 signals appeared with a chemical-shift difference of 1.04 ppm which was extended to 1.29 ppm for the a-anomer 15b caused by a downfield shift of the H-49. Phosphitylation 20 of compound 15a and 15b by reaction with 2-cyanoethyl N,N-diisopropylphosphoramido- Scheme 1 (a) CrO3 Pyridine Ac2O CH2Cl2; (b) 1.0 mol equiv. allylMgBr Et2O; (c) TBAF THF; (d) Ac2O DMAP pyridine; (e) A thymine BSA TMS triflate CH2ClCH2Cl; B thymine BSA TMS triflate CH3CN; (f) NH3 in CH3OH; (g) CH3ONa in CH3OH; (h) DMTCl AgNO3 pyridine THF; (i) NC(CH2)2OP(Cl)NPri 2 DIPEA CH2Cl2.T = thymin-1-yl O R1O OH OCH3 O R1O OCH3 O O R1O OR2 OCH3 O R1O OCH3 OR2 2 R1 = TBDMS 3 R1 = TBDMS b-anomer 4 R1 = TBDMS 5 R1 = TBDMS R2 = H 7 R1 = R2 = H 9 R1 = R2 = Ac 6 R1 = TBDMS R2 = H 8 R1 = R2 = H 10 R1 = R2 = Ac e e O R1 O OR2 O R1O OR2 11 R1 = R2 = Ac 13 R1 = R2 = H 12 R1 = R2 = Ac T T c d g c d O HO OH O HO OH + 12 f 14a 14b T T O DMTO OR1 O DMTO OR1 + 13 h T T 15a R1 = H 16a R1 = NCCH22OPNPri 2 i 15b R1 = H 16b R1 = NCCH22OPNPri 2 i HN N CH3 O O T = + + a b J. Chem. Soc. Perkin Trans. 1 1997 3277 chloridite in the presence of N,N-diisopropylethylamine (DIPEA) in anhydrous CH2Cl2 afforded the nucleoside phosphoramidites 16a in 75 yield and 16b in 77 yield respectively after column chromatographic purification and precipitation from light petroleum.With the aim of evaluating the properties of 39-C-allyl b-Dribo ODN-analogues the phosphoramidite derivative 20 was synthesized (Scheme 2) from 1-(3-C-allyl-3,5-di-O-benzyl-b-Dribofuranosyl) thymine 1.8 Debenzylation of compound 1 using BCl3 in CH2Cl2 at 278 8C afforded nucleoside 17 in 73 yield. As expected removal of the benzyl groups using H2 and Pd(OH)2–C as catalyst in absolute EtOH at room temperature caused concomitant reduction or the allylic double bond. Dimethoxytritylation of triol 17 using 1.2 mol equiv. of 4,49- dimethoxytrityl chloride (DMTCl) in anhydrous pyridine afforded nucleoside 18 in 82 yield. This was followed by silylation of the free 29-hydroxy group using TBDMSCl in anhydrous DMF and imidazole as catalyst to afford compound 19 in 82 yield.Subsequent phosphitylation as described above for the preparation of compound 16a afforded the phosphoramidite 20 in 78 yield after column chromatographic purification and precipitation from light petroleum. The chemistry used to synthesize the phosphoramidite monomer 26 which was used on an automated DNA synthesizer to introduce a 39-C-(3-aminopropyl) linker into an oligonucleotide is shown in Scheme 3. Direct nitrogen glycosylation 14 of furanoside 5 with thymine using BSA as silylating agent and TMS triflate as Lewis acid in CH3CN afforded an inseparable anomeric mixture 21 of the a- and b-nucleoside (a:b ~ 3 1) in 83 yield. Hydroboration of this mixture with BH3?1,4-oxathiane in THF followed by in situ oxidation with alkaline hydrogen peroxide gave 39-C-(3-hydroxypropyl) nucleosides 22 in 63 yield.Mitsunobu21 reaction of alcohol 22 with phthalimide afforded the phthaloyl-protected primary amines 23 in 86 yield. Treatment of the protected bis-ether 23 with TBAF in THF removed the silyl groups in 33 yield and the deprotected nucleosides 24 were allowed to react with DMTCl in anhydrous THF containing anhydrous pyridine with AgNO3 as catalyst to protect the 59-hydroxy groups. At this stage the a- and b-nucleosides were easily separated by silica gel chromatography to give 1-2-deoxy-5-O-(4,49-dimethoxytrityl)- 3-C-(phthalimidopropyl)-a-D-erythro-pentofuranosyl- thymine 25 in 43 yield and the corresponding b-anomer in 15 yield. The structural assignment of the a-and b-nucleoside was based on one-dimensional 1H NMR analysis.Thus for the b-anomer the H-59a,b and the H-49 signals appeared with a chemical-shift difference of 0.91 ppm which was extended to 1.36 ppm for the a-anomer 25. The configuration of the aanomer was further confirmed by a significant NOE of H-49 (5) by irradiation of H-6. The a-anomer 25 was treated with 2-cyanoethyl N,N-diisopropylphosphoramidochloridite in the presence of DIPEA in anhydrous CH2Cl2 to give the phosphoramidite 26 in 41 yield. Owing to the low yield obtained for the b-anomer corresponding to compound 25 incorporation of this anomer awaits completion of an alternative synthetic strategy. Scheme 2 (a) BCl3 CH2Cl2 hexane; (b) DMTCl pyridine; (c) TBDMSCl imidazole DMF; (d) NC(CH2)2OP(Cl)NPri 2 DIPEA CH2Cl2.T = thymin-1-yl O BnO OBn O DMTO O O R1O OH T T T OH OR2 OTBDMS a d 1 17 R1 = R2 = H 18 R1 = DMT R2 = H 19 R1 = DMT R2 = TBDMS 20 NCCH2CH2OPNPri 2 b c Synthesis of DMT-on ODNs A–L (Table 1) was performed by use of standard phosphoramidite methodology on an automated DNA-synthesizer using the appropriate building blocks 16a 16b 26 a-thymidine 39-O-2-(cyanoethyl)phosphoramidite and commercial thymidine 39-O-2-(cyanoethyl)phosphoramidite. The coupling efficiency of the modified phosphoramidites 16a 16b and 26 were approximately 40 (two times 24 min coupling) compared with >99 for a-thymidine and standard phosphoramidites (2 min coupling). The coupling efficiency of the building block 20 was in all experiments below 5. The low coupling yield obtained with compound 20 can be explained by the tertiary nature of the phosphitylated hydroxy group in connection with steric hindrance from the 29-O-(tertbutyldimethylsilyl) group (compare with structures 16a 16b and 26).No ODNs containing compound 20 could therefore be obtained. The 59-O-dimethoxytrityl-protected oligomers were removed from the solid support by treatment with conc. ammonia at room temperature for 72 h which also removed the phosphate and nucleobase-protecting groups and the phthaloyl group in species L. Purification using disposable reversed-phase chromatography cartridges (which includes detritylation) afforded the unprotected oligomers A–L. The purity of the modified oligomers was confirmed by anion-exchange highperformance liquid chromatography (HPLC) analysis. The composition of the oligomers was verified by matrix-assisted laser desorption mass spectrometry (MALDI-MS) a powerful method for mass analysis of ligomers.22,23 The observed relative molecular masses correspond within experimental error to those calculated (Table 2).The hybridization properties of the modified oligomers towards their complementary DNA and RNA strands were checked by UV melting point (Tm) measurement as described.24 The results are given in Table 1. Incorporation of 16a (monomer X) once or twice in the middle of a 14-mer (ODNs A–D) induces a minor destabilization of the duplex formed with complementary DNA (DTm ~21 8C modification). However when RNA is used as the complementary strand the resulting duplexes are destabilized by 22.8 to 24.0 8C per modification. These results support the assumption that the monomer X adopts a 29-endo conformation as reported earlier for a similar nucleoside,25 which is favourable for DNA–DNA duplex form- Scheme 3 (a) Thymine BSA TMS triflate CH3CN; (b) BH3?1,4- oxathiane THF; NaOH 30 H2O2; (c) pthalimide Ph3P DEAD THF; (d) TBAF THF; (e) DMTCl AgNO3 pyridine THF; (f) NC(CH2)2OP(Cl)NPri 2 DIPEA CH2Cl2.T = thymin-1-yl. PhthN = phthalimido. O TBDMSO OH O TBDMSO OTMS O TBDMSO OTMS a b 23 R1 = TBDMS R2 = TMS 24 R1 = R2 = H d OCH3 T T HO 5 21 22 c O R1O OR2 T PhthN e 25 R1 = DMT R2 = H 26 R1 = DMT R2 = NCCH22OPNPri 2 f + b-anomer O R1O OR2 PhthN T 3278 J. Chem. Soc. Perkin Trans. 1 1997 Table 1 Sequences and melting experiments of synthesized b- and a-ODNs. Sequence 59-TTTTTTTTTTTTTT-39 59-TTTTTTTXTTTTTT-39 59-TTTTTTXXTTTTTT-39 59-TTTTTXTTTXTTTT-39 59-TTTTTTTYTTTTTT-39 59-TTTTTTYYTTTTTT-39 59-TTTTTYTTTYTTTT-39 59-a-(TTTTTTTTTTTTTT)C-39 59-a-(TTTTTTTYTTTTTT)C-39 59-a-(TTTTTTYYTTTTTT)C-39 59-a-(TTTTYTTTYTTTTT)C-39 59-a-(TTTTTTZTTTTTT)C-39 AB C DEF G HI J KL Tm ( 8C)a 35.5 34.5 34.0 33.0 18.5 15.0 14.5 38.0 37.0 37.0 37.0 38.0 DTm ( 8C)a 21.0 20.8 21.3 217.0 210.3 210.5 21.0 20.5 20.5 0.0 Tm ( 8C)b 29.0 25.0 23.5 23.0 43.5 40.0 37.0 36.0 41.0 DTm ( 8C)b 24.0 22.8 23.0 23.5 23.3 23.8 22.5 T = thymidine monomer; C = 29-deoxycytidine monomer; X = b-monomer derived from amidite 16a; Y = a-monomer derived from amidite 16b; Z = a-3-C-3-aminopropyl monomer derived from amidite 26.Tm = melting temperature; DTm = change in Tm per modification compared with the corresponding reference duplex. a Complexed with dA14.b Complexed with rA14. ation whereas the conformation of the carbohydrate moiety in a DNA–RNA duplex is known to be 39-endo.26 As expected incorporation of the a-monomer Y derived from compound 16b into a b-ODN (E–G) results in a large destabilization of the duplex formed with complementary DNA. This further con- firms the assigned configuration of the nucleosides. As mentioned a-ODNs form thermally more stable duplexes with RNA than do b-ODNs. The melting experiments depicted in Table 1 show the same tendency as a-ODN values are on average 14.5 8C higher towards complementary RNA than are those for the corresponding b-ODN–RNA duplexes. However incorporation of a-monomer Y once or twice in the middle of a a-T14 (ODNs H–K) induces a similar destabilizing effect (DTm ~ 23.5 8C/modification) on a duplex formed with complementary RNA as seen above for X when incorporated in a b-strand.When DNA is used as the complementary strand incorporation of Y has only a minor effect on the hybridization properties of the modified a-ODNs. The hybridization obtained by modified b-ODNs with complementary DNA is similar to that obtained earlier for the corresponding 39-C-(hydroxymethyl) thymidine.27 As depicted in Table 1 the hybridization properties of the ODN L containing one 39-C-aminopropyl a-monomer Z in the middle were the most promising obtained towards both complementary DNA and RNA as no (towards DNA) or only minor (towards RNA) destabilizing effect was observed. These results could originate from the cationic nature of the amino group possibly leading to favourable partial neutralization of the phosphate backbone.In summary synthesis of the novel b- and a-anomers of 39-Callylthymidine has been accomplished and these nucleosides have been incorporated into b- and a-ODNs. Incorporation of the b-anomer into b-ODNs and the a-anomer into a-ODNs have a similar impact on duplex formation with complementary DNA and RNA. The increased thermal stabilities observed for all the modified a-ODNs compared with the unmodified T14 Table 2 Mass analysis of synthesized ODNs. ODN AB C DEF G HI J KL Calc. M 1 H1 (m/z) 4198 4238 4278 4278 4238 4278 4278 4487 4527 4567 4567 4544 Found M 1 H1 (m/z) 4200 4241 4280 4280 4240 4280 4280 4487 4529 4565 4566 4545 suggest them to be promising as bioactive molecules. The most likely application of 39-C-allylnucleosides however will be as a precursor for the synthesis of 39-C-(2-hydroxyethyl)- or 39-C-(3- hydroxypropyl)nucleosides and their amine analogues.Interesting properties of the latter were demonstrated (ODN L) and further research in this direction seems justified. Experimental NMR spectra were recorded at 250 MHz for 1H NMR and 62.9 MHz for 13C NMR on a Bruker AC-250 spectrometer or at 500 MHz for 1H NMR 125 MHz for 13C NMR and 202.3 MHz for 31P NMR on a Varian Unity 500 spectrometer. Chemical shifts are in ppm relative to tetramethylsilane as internal standard (1H NMR and 13C NMR) and relative to 85 H3PO4 as external standard (31P NMR). J Values are given in Hz. 1H NMR peak assignments for compounds 2 3 5 6 14a 14b 15a 15b 17–19 and 21–25 were derived from 1H–1H COSY and/or NOE difference experiments.13C NMR peak assignments for compounds 3 5 6 and 17 were derived from intensive nuclei enhancement by polarization transfer (INEPT) and 1H–13C COSY experiments. Fast-atom bombardment (FAB) mass spectra were recorded on a Kratos MS 50 RF spectrometer. Microanalyses were performed at the Department of Chemistry University of Copenhagen. The silica gel used for column chromatography (0.040–0.063 mm) was purchased from Merck. ODNs were synthesized on an Assembler Gene Special“ DNA-Synthesizer (Pharmacia Biotech). Purification of 59-O-DMT-on ODNs was accomplished using disposable Oligopurification Cartridges (COP Cruachem). MALDI-MS was performed in positive mode on a Micromass TofSpec E mass spectrometer using a matrix of diammonium citrate and 2,6-dihydroxyacetophenone.Analytical anion-exchange HPLC (RESOURCETM Q 1 cm3 Pharmacia Biotech) was performed on a Waters Delta Prep 3000 Preparative Chromatography System. Melting profiles of duplexes were obtained on a Perkin-Elmer UV/VIS spectrometer fitted with a PTP-6 Peltiér temperature-programming element. The reference oligonucleotide (rA14) was purchased from DNA Technology ApS Aarhus Denmark. Light petroleum refers to the fraction with distillation range 60–80 8C. Methyl 5-O-(tert-butyldimethylsilyl)-2-deoxy-·-D-erythropentofuranoside 2 and methyl 5-O-(tert-butyldimethylsilyl)-2- deoxy-‚-D-erythro-pentofuranoside 3 An anomeric mixture of methyl 2-deoxy-D-erythro-pentofuranoside 10 (51.32 g 0.35 mmol) and imidazole (57.06 g 0.84 mol) was dissolved in anhydrous DMF (400 cm3) under argon. TBDMSCl (58.02 g 0.36 mol) as a solution in anhydrous DMF (100 cm3) was added dropwise over a period of 1 h.After 5 h the reaction mixture was concentrated under reduced pressure. Purification by silica gel column chromatography (0–25 J. Chem. Soc. Perkin Trans. 1 1997 3279 EtOAc in light petroleum) afforded a-anomer 2 (25.83 g 28) as the less polar compound stereoisomer 3 (17.84 g 19) and a fraction of the diprotected glycoside (12.29 g 9). Compound 2 dC(CDCl3) 25.64 and 25.42 Si(CH3)2 18.13 C(CH3)3 25.72 C(CH3)3 40.96 (C-2) 54.60 (OCH3) 63.63 (C-5) 73.03 (C-3) 87.69 (C-4) and 105.50 (C-1); dH(CDCl3) 0.02 s 6 H Si(CH3)2 0.85 s 9 H C(CH3)3 1.91– 2.15 (m 2 H H2-2) 2.84 (d J 10.4 1 H OH) 3.34 (s 3 H OCH3) 3.53 (dd J 4.8 and 10.9 1 H Ha-5) 3.70 (dd J 3.6 and 10.9 1 H Hb-5) 4.05–4.09 (m 1 H H-4) 4.11–4.18 (m 1 H H- 3) and 5.04 (m 1 H H-1).Compound 3 dC(CDCl3) 25.46 and 25.41 Si(CH3)2 18.27 C(CH3)3 25.88 C(CH3)3 40.93 (C-2) 54.98 (OCH3) 65.01 (C-5) 73.63 (C-3) 85.66 (C-4) and 105.02 (C-1); dH(CDCl3) 0.08 s 6 H Si(CH3)2 0.91 s 9 H C(CH3)3 1.99 (s 1 H OH) 2.06 (ddd J 5.3 6.6 and 13.3 1 H Ha-2) 2.22 (ddd J 2.1 6.8 and 13.3 1 H Hb-2) 3.32 (s 3 H OCH3) 3.54 (dd J 8.0 and 9.7 1 H Ha-5) 3.75–3.90 (m 2 H Hb-5 and H-4) 4.42–4.44 (m 1 H H-3) and 5.06 (dd J 2.1 and 5.3 1 H H-1). Anomeric mixture of compounds 2 and 3 FAB-MS m/z 261 M 2 H2 (Found C 54.74; H 9.64. Calc. for C12H26O4Si C 54.92; H 9.99). Methyl 5-O-(tert-butyldimethylsilyl)-2-deoxy-·-D-glycero-pentofuranoside- 3-ulose 4 To a suspension of CrO3 (5.00 g 50.0 mmol) in anhydrous CH2Cl2 (150 cm3) were added anhydrous pyridine (8.1 cm3 0.10 mol) and methyl glycoside 2 (5.86 g 22.3 mmol) followed by Ac2O (4.7 cm3 49.7 mmol).The reaction mixture was stirred under argon for 4 h at room temp. EtOAc (500 cm3) was added and the mixture was filtered through a silica gel column. Concentration of the organic phase under reduced pressure afforded title keto glycoside 4 as a pale yellow oil (4.77 g 82) which was used without further purification in the next step; dC(CDCl3) 25.70 and 5.51 Si(CH3)2 18.17 C(CH3)3 25.70 C(CH3)3 44.18 (C-2) 54.82 (OCH3) 62.25 (C-5) 78.81 (C-4) 104.80 (C-1) and 212.28 (C-3); dH(CDCl3) 0.04 and 0.05 2 × s 6 H Si(CH3)2 0.86 s 9 H C(CH3)3 2.33–2.60 (m 2 H H2-2) 3.44 (s 3 H OCH3) 3.83–3.98 (m 3 H H2-5 and H-4) and 5.36 (m 1 H H-1). Methyl 3-C-allyl-5-O-(tert-butyldimethylsilyl)-2-deoxy-·-Derythro- pentofuranoside 5 and methyl 3-C-allyl-5-O-(tert-butyldimethylsilyl)- 2-deoxy-·-D-threo-pentofuranoside 6 Compound 4 (4.29 g 16.46 mmol) was coevaporated with anhydrous toluene (2 × 25 cm3) and dissolved under argon in anhydrous Et2O (150 cm3).The mixture was cooled to 0 8C a 1 M solution of allylmagnesium bromide in anhydrous Et2O (16.5 cm3 16.5 mmol) was added dropwise and the mixture was stirred for 16 h at room temp. The reaction was quenched by addition of saturated aq. NH4Cl (400 cm3) and the aqueous phase was extracted with Et2O (3 × 250 cm3). The organic phase was dried (Na2SO4) and evaporated. Purification by silica gel column chromatography (0–3 EtOAc in light petroleum v/v) afforded erythro product 5 (789 mg 16) as the less polar compound and its threo stereoisomer 6 (2.51 g 50).Compound 5 dC(CDCl3) 25.65 and 25.49 Si(CH3)2 18.10 C(CH3)3 25.81 C(CH3)3 38.67 (C-2) 44.71 (CH2) 54.90 (OCH3) 62.57 (C-5) 79.88 (C-3) 89.20 (C-4) 104.93 (C-1) 117.30 ( CH2) and 134.49 ( CH); dH(CDCl3) 0.06 and 0.07 2 × s 6 H Si(CH3)2 0.89 s 9 H C(CH3)3 1.88–1.93 (m 1 H Ha-2) 2.14 (dd J 5.2 and 13.2 1 H Hb-2) 2.36–2.55 (m 2 H CH2) 3.39 (s 3 H OCH3) 3.59 (s 1 H OH) 3.66–3.69 (m 2 H H2-5) 4.05–4.08 (m 1 H H-4) 5.05–5.08 (m 2 H H-1 and CH2 a) 5.12–5.15 (m 1 H CH2 b) and 5.93–6.10 (m 1 H CH) (Found C 59.71; H 9.88. Calc. for C15H30O4Si C 59.56; H 10.00). Compound 6 dC(CDCl3) 25.61 and 5.49 Si(CH3)2 18.12 C(CH3)3 25.75 C(CH3)3 44.27 (CH2) 47.10 (C-2) 55.09 (OCH3) 62.42 (C-5) 80.64 (C-3) 81.69 (C-4) 103.93 (C-1) 118.24 ( CH2) and 133.80 ( CH); dH(CDCl3) 0.11 s 6 H Si(CH3)2 0.91 s 9 H C(CH3)3 2.01 (dd J 2.9 and 13.8 1 H Ha-2) 2.17 (dd J 5.6 and 13.8 1 H Hb-2) 2.37–2.50 (m 2 H CH2) 3.35 (s 3 H OCH3) 3.66 (s 1 H OH) 3.78–3.81 (m 1 H H-4) 3.93–3.95 (m 2 H H2-5) 5.07–5.11 (m 2 H H-1 and CH2 a) 5.15–5.16 (m 1 H CH2 b) and 5.85–5.99 (m 1 H CH); FAB-MS m/z 303 M 1 H1 (Found C 59.37; H 9.60).Methyl 3-C-allyl-2-deoxy-·-D-erythro-pentofuranoside 7 Compound 5 (1.08 g 3.57 mmol) was dissolved in anhydrous THF (15 cm3)under argon. 1.1 M TBAF in THF (3.2 cm3 3.57 mmol) was added and the mixture was stirred for 15 min. EtOAc (70 cm3) was added and the organic phase was washed with saturated aq. NaHCO3 (3 × 30 cm3). The water phase was extracted with EtOAc (60 cm3) and the organic phases were combined dried (Na2SO4) and concentrated under reduced pressure.Purification using silica gel column chromatography (20 EtOAc in light petroleum) afforded title diol 7 as an oil (612 mg 91); dC(CDCl3) 38.76 (C-2) 44.95 (CH2) 54.92 (OCH3) 61.99 (C-5) 79.41 (C-3) 89.20 (C-4) 104.53 (C-1) 117.79 ( CH2) and 133.72 ( CH); dH(CDCl3) 2.00–2.03 (m 2 H H2-2) 2.27–2.47 (m 2 H CH2) 2.65 (br s 1 H OH) 3.40 (s 3 H OCH3) 3.52–3.73 (m 2 H H2-5) 4.14 (dd J 3.7 and 4.9 1 H H-4) 5.09–5.17 (m 3 H H-1 and CH2) and 5.89–6.06 (m 1 H CH) (Found C 57.60; H 8.47. Calc. for C9H16O4 C 57.43; H 8.57). Methyl 3-C-allyl-2-deoxy-·-D-threo-pentofuranoside 8 Compound 6 (739 mg 2.44 mmol) was dissolved in anhydrous THF (10 cm3) under argon. 1.1 M TBAF in THF (2.2 cm3 2.4 mmol) was added and the mixture was stirred for 15 min.EtOAc (40 cm3) was added and the organic phase was washed with saturated aq. NaHCO3 (3 × 20 cm3). The water phase was extracted with EtOAc (40 cm3) and the organic phases were combined dried (Na2SO4) and concentrated under reduced pressure. Purification using silica gel column chromatography (20 EtOAc in light petroleum) afforded title diol 8 as an oil (377 mg 82); dC(CDCl3) 43.77 (CH2) 47.49 (C-2) 55.23 (OCH3) 61.23 (C-5) 80.82 (C-3) 81.87 (C-4) 103.81 (C-1) 119.18 ( CH2) and 133.11 ( CH); dH(CDCl3) 2.04 (dd J 2.9 and 14.0 1 H Ha-2) 2.20 (dd J 5.64 and 14.0 1 H Hb-2) 2.43–2.47 (m 2 H CH2) 2.70 (br s 1 H OH) 3.37 (s 3 H OCH3) 3.80– 3.82 (m 1 H H-4) 3.93–3.94 (m 2 H H2-5) 5.13–5.17 (m 2 H H-1 and CH2 a) 5.20–5.21 (m 1 H CH2 b) and 5.83–6.00 (m 1 H CH); FAB-MS m/z 187 M 2 H2.Methyl 3,5-di-O-acetyl-3-C-allyl-2-deoxy-·-D-erythropentofuranoside 9 To a solution of compound 7 (1.71 g 9.09 mmol) in anhydrous pyridine (30 cm3) under argon were added DMAP (1.11 g 9.10 mmol) and Ac2O (6.01 cm3 54.54 mmol). After stirring of the mixture for 48 h at room temperature the solvent was evaporated off and saturated aq. NaHCO3 (80 cm3) was added. The aqueous phase was extracted with CH2Cl2 (3 × 125 cm3) and the organic phases were combined dried (Na2SO4) and concentrated under reduced pressure. Silica gel column chromatography (10–30 EtOAc in light petroleum) gave title diacetate 9 (1.90 g 77); dC(CDCl3) 20.75 and 21.53 (2 × CH3) 37.23 (CH2) 44.66 (C-2) 54.98 (OCH3) 63.22 (C-5) 81.69 (C- 4) 87.25 (C-3) 103.35 (C-1) 118.87 ( CH2) 131.90 ( CH) and 170.24 and 170.49 (2 × COCH3); dH(CDCl3) 2.02 and 2.09 (2 s 6 H 2 × CH3) 2.25–2.45 (m 3 H H2-2 and CH2 a) 2.99 (dd J 7.4 and 14.5 1 H CH2 b) 3.37 (s 3 H OCH3) 4.20 (dd J 7.0 and 11.6 1 H Ha-5) 4.44 (dd J 2.9 and 7.0 1 H H-4) 4.57 (dd J 2.9 and 11.6 1 H Hb-5) 5.03 (dd J 2.3 and 4.9 1 H H-1) 5.07–5.10 (m 1 H CH2 a) 5.14–5.15 (m 1 H CH2 b) and 5.64– 5.80 (m 1 H CH); FAB-MS m/z 272 M 1 H1.3280 J. Chem. Soc. Perkin Trans. 1 1997 Methyl 3,5-di-O-acetyl-3-C-allyl-2-deoxy-·-D-threopentofuranoside 10 To a solution of compound 8 (2.92 g 1.55 mmol) in anhydrous pyridine (5 cm3) under argon were added DMAP (195 mg 1.6 mmol) and Ac2O (0.68 cm3 6.20 mmol). After stirring of the mixture for 48 h at room temperature additional Ac2O (0.68 cm3 6.20 mmol) was added.The reaction mixture was stirred for another 72 h the solvent was evaporated off and saturated aq. NaHCO3 (15 cm3) was added. The aqueous phase was extracted with CH2Cl2 (3 × 25 cm3) and the organic phases were combined dried (Na2SO4) and concentrated under reduced pressure. Silica gel column chromatography (30 EtOAc in light petroleum v/v) gave title diacetate 10 (191 mg 45); dC(CDCl3) 20.81 and 21.58 (2 × CH3) 40.05 and 44.74 (CH2 and C-2) 54.93 (OCH3) 63.61 (C-5) 81.06 (C-4) 87.82 (C-3) 103.59 (C-1) 119.34 ( CH2) 131.75 ( CH) and 169.65 and 170.72 (2 × COCH3); dH(CDCl3) 2.00 and 2.10 (2 s 6 H 2 × CH3) 2.33 (dd J 1.7 and 14.9 1 H Ha-2) 2.65 (dd J 5.7 and 15.0 1 H Hb-2) 2.65–2.73 (m 1 H CH2 a) 3.05–3.13 (m 1 H CH2 b) 3.36 (s 3 H OCH3) 4.12–4.21 (m 2 H H2-5) 4.38– 4.47 (m 1 H H-4) 5.05–5.17 (m 3 H H-1 and CH2) and 5.67– 5.81 (m 1 H CH).1-(3,5-Di-O-acetyl-3-C-allyl-2-deoxy-·,‚-D-erythropentofuranosyl) thymine 11 Method A. Methyl glycoside 9 (1.79 g 6.57 mmol) and thymine (1.66 g 13.14 mmol) were dissolved in anhydrous CH2ClCH2Cl (100 cm3) under argon and BSA (9.75 cm3 39.45 mmol) was added. The mixture was refluxed for 15 min at 78 8C. The resulting clear mixture was allowed to cool to room temperature and TMS triflate (1.83 cm3 9.20 mmol) was added dropwise over a period of 10 min. After being stirred for 48 h at room temperature the mixture was diluted with CH2Cl2 (100 cm3) and poured into ice–water (50 cm3) saturated with NaHCO3. The aqueous phase was extracted with CH2Cl2 (3 × 150 cm3) and the organic phase was combined dried (Na2SO4) and concentrated under reduced pressure.Purification using silica gel column chromatography (10–30 EtOAc in light petroleum) afforded an anomeric mixture of the b- and a-nucleoside 11 (b:a; 1 2.3) (1.25 g 52). Method B. To a stirred suspension of the methyl glycoside 9 (901 mg 3.32 mmol) and thymine (843 mg 6.68 mmol) in anhydrous CH3CN (30 cm3) under argon at room temperature was dropwise added BSA (5 cm3 20.23 mmol). The mixture was stirred for 1 h until clearness was attained. The reaction mixture was cooled to 230 8C and TMS triflate (0.95 cm3 5.25 mmol) was added dropwise. The reaction mixture was stirred for 72 h at room temperature then was diluted with CH2Cl2 (50 cm3) and the reaction was quenched with saturated aq. NaHCO3 (2 × 50 cm3). After being washed with water (2 × 50 cm3) the organic phase was dried (Na2SO4) and concentrated under reduced pressure.Purification using silica gel column chromatography (0–1 MeOH in CH2Cl2) afforded an anomeric mixture of the b- and a-nucleoside 11 (b:a; 1.5 1) (894 mg 74); dC(CDCl3) 12.41 and 12.45 (2 × CH3) 20.70 21.52 and 21.59 (4 × COCH3) 35.75 and 36.05 (2 × C-29) 42.85 and 43.62 (2 × CH29) 62.79 and 63.05 (2 × C-59) 82.24 83.92 84.04 85.76 88.49 and 88.68 (2 × C-19 -39 and -49) 109.55 and 111.04 (2 × C-5) 119.41 and 119.53 (2 × CH29) 131.03 and 131.16 (2 × CH9) 134.48 and 135.17 (2 × C-6) 150.39 and 150.48 (2 × C-2) 163.81 and 164.12 (2 × C-4) and 169.71 169.92 and 170.10 (4 × COCH3); dH(CDCl3) 1.94 1.96 2.07 and 2.13 (4 s 2 × CH3 4 × COCH3) 2.36–2.85 (m 2 × H2-2 and 2 × CH29a) 3.08–3.29 (m 2 × CH29b) 4.11–4.19 (m 2 × H-49) 4.33–4.41 and 4.69–4.72 (2 m 2 × H2-59) 5.00–5.17 (m 2 × CH29) 5.63– 5.75 (m 2 × CH9) 6.18–6.27 (m 2 × H-19) 7.36 and 7.39 (2 s 2 × H-6) and 9.99 and 10.03 (2 s 2 × NH); EI-MS m/z 366 M1 25 (Found C 55.54; H 6.17; N 7.31.Calc. for C17H22N2O7 C 55.73; H 6.05; N 7.65). 1-(3,5-Di-O-acetyl-3-C-allyl-2-deoxy-·,‚-D-threopentofuranosyl) thymine 12 Method A. Used amounts. Methyl glycoside 10 (129 mg 0.47 mmol) thymine (120 mg 0.95 mmol) anhydrous CH2ClCH2Cl (7 cm3) BSA (0.70 cm3 2.84 mmol) and TMS triflate (0.13 cm3 0.65 mmol). Purification using preparative TLC (PLC) (2 CH3OH in CH2Cl2 double run) afforded an anomeric mixture of the b- and a-nucleoside 12 (112 mg 64); dC(CDCl3) 12.33 and 12.44 (2 × CH3) 20.66 20.68 21.46 and 21.66 (4 × COCH3) 37.99 38.03 40.52 and 41.65 (2 × C-29 and CH29) 62.64 and 63.37 (2 × C-59) 83.13 83.59 84.08 86.38 86.81 and 88.39 (2 × C-19 -39 and -49) 110.46 and 110.95 (2 × C-5) 120.31 and 120.67 (2 × CH29) 130.55 and 131.01 (2 × CH9) 134.63 and 136.00 (2 × C-6) 150.31 and 150.34 (2 × C-2) 163.84 and 164.01 (2 × C-4) and 169.23 169.74 170.43 and 170.60 (4 × COCH3); dH(CDCl3) 1.86 1.87 1.93 2.00 2.04 and 2.05 (6 s 2 × CH3 4 × COCH3) 2.35 (dd J 7.6 and 15.0 CH29a) 2.62–2.95 (m 2 × H2-29 and CH29b) 3.16 (dd J 6.6 and 15.0 CH29b) 4.07–4.15 (m H2-49) 4.38–4.43 (m H2-59) 5.10– 5.25 (m 2 × CH29) 5.61–5.77 (m 2 × CH9) 5.99–6.06 (m 2 × H-19) 7.04 and 7.35 (2 s 2 × H-6) and 9.89 and 9.93 (2 s 2 × NH); FAB-MS m/z 367 M 1 H1 (Found C 54.99; H 6.00; N 7.53.Calc. for C17H22N2O7?0.25H2O C 55.06; H 6.12; N 7.55).1-(3-C-Allyl-2-deoxy-·,‚-D-erythro-pentofuranosyl)thymine 13 The anomeric mixture of nucleosides 11 (894 mg 2.41 mmol) was dissolved in anhydrous CH3OH (25 cm3) under argon and CH3ONa (660 mg 12.2 mmol) was added. After stirring of the mixture for 12 h at room temp. water (25 cm3) was added and the mixture was neutralized with 4 M HCl. The aqueous phase was extracted with 3-methylbutan-1-ol (3 × 100 cm3) and the organic phase was concentrated under reduced pressure. Purifi- cation using silica gel column chromatography (0–5 CH3OH in CH2Cl2) afforded an anomeric mixture of b- and anucleoside 13 (b:a; 1.5 1) (569 mg 81); dC(CD3OD) 12.50 and 12.57 (2 × CH3) 40.94 41.13 44.32 and 45.76 (2 × C-29 2 × CH29) 62.20 and 62.37 (2 × C-59) 80.48 81.51 85.97 87.64 90.00 and 91.87 (2 × C-19 -39 -49) 110.02 and 111.34 (2 × C-5) 118.67 and 118.85 (2 × CH29) 134.64 and 134.72 (2 × CH9) 138.61 and 139.26 (2 × C-6) 152.32 and 152.44 (2 × C-2) and 166.36 and 166.61 (2 × C-4); dH(CD3OD) 2.02 and 2.08 (2 s 2 × CH3) 2.14–2.38 (m 2 × Ha-29 and Hb-29) 2.57–2.73 (m 2 × CH29 and Hb-29) 3.73–3.98 (m 2 × H-49 and -59) 4.05 (m OH) 4.34 (m OH) 5.25–5.33 (m 2 × CH29) 6.00–6.16 (m 2 × CH9) 6.33 (dd J 2.1 and 7.7 H-19) 6.46 (dd J 5.4 and 9.3 H-19) 8.21 and 8.22 (2 s 2 × H-6) and 9.97 and 10.03 (2 s 2 × NH); EI-MS m/z 282 (M1 9); HR-MS (Found M1 282.1210.Calc. for C13H18N2O5 M 282.1216) (Found C 53.20; H 6.31; N 9.94. Calc. for C13H18N2O5? 0.25H2O C 53.60; H 6.57; N 9.62). 1-(3-C-Allyl-2-deoxy-‚-D-threo-pentofuranosyl)thymine 14a and 1-(3-C-allyl-2-deoxy-·-D-threo-pentofuranosyl)thymine 14b An anomeric mixture of nucleosides 12 (79 mg 0.22 mmol) was dissolved in a saturated solution of NH3 in methanol (10 cm3).After stirring of the mixture for 15 h at room temp. the solvent was removed under reduced pressure and the crude product was purified by silica gel column chromatography (3 CH3OH in CH2Cl2) to give anomers 14a (23 mg 39) and 14b (26 mg 44). b-Isomer 14a dC(CDCl3) 12.36 (CH3) 42.56 and 44.63 (C-29 CH29) 60.87 (C-59) 79.61 (C-49) 84.48 85.11 (C-19 and -39) 109.47 (C-5) 119.43 ( CH29) 132.57 ( CH9) 138.08 (C-6) 150.83 (C-2) and 164.50 (C-4); dH(CDCl3) 1.81 (s 3 H CH3) 2.32–2.58 (m 4 H H2-29 CH29) 3.74 (m 1 H H-49) 3.84 (br s 1 H OH) 4.08–4.16 (m 2 H H2-59) 4.78 (s 1 H OH) 5.15– 5.22 (m 2 H CH29) 5.84–6.00 (m 1 H CH9) 6.08 (dd J 2.9 and 7.5 1 H H-19) 7.99 (s 1 H H-6) and 9.93 (s 1 H NH).a-Isomer 14b dC(CDCl3) 12.46 (CH3) 42.05 and 45.01 (C-29, J. Chem. Soc. Perkin Trans. 1 1997 3281 CH29) 60.87 (C-59) 81.01 (C-49) 85.28 and 86.09 (C-19 and -39) 111.14 (C-5) 119.28 ( CH29) 132.69 ( CH9) 135.74 (C-6) 150.74 (C-2) and 164.39 (C-4); dH(CDCl3) 2.00 (s 3 H CH3) 2.36–2.45 (m 4 H H-29 CH29) 3.46 (s 1 H OH) 3.96–4.02 (m 2 H H2-59) 4.10 (m 1 H H-49) 4.60 (br s 1 H OH) 5.13–5.18 (m 2 H CH29) 5.82–5.98 (m 1 H CH9) 6.28 (dd J 6.1 and 8.1 1 H H-19) 7.19 (s 1 H H-6) and 10.05 (br s 1 H NH). 1-3-C-Allyl-2-deoxy-5-O-(4,49-dimethoxytrityl)-‚-D-erythropentofuranosyl thymine 15a and 1-3-C-allyl-2-deoxy-5-O-(4,49- dimethoxytrityl)-·-D-erythro-pentofuranosylthymine 15b An anomeric mixture of nucleoside 13 (569 mg 2.11 mmol) AgNO3 (358 mg 2.11 mmol) and DMTCl (1.785 g 5.27 mmol) were dissolved in anhydrous THF (50 cm3) and anhydrous pyridine (0.85 cm3 10.5 mmol) was added.The mixture was stirred at room temp. for 72 h. The mixture was filtered into 5 aq. sodium hydrogen carbonate (100 cm3). The product was extracted into CH2Cl2 (4 × 100 cm3) and the combined extract was dried (Na2SO4) and concentrated under reduced pressure. Purification by PLC (1 pyridine 3 CH3OH in CH2Cl2 v/v/v 3 runs) gave the a-anomer 15b (304 mg 26) as the less polar isomer and the b-anomer 15a (545 mg 47). b-Isomer 15a. dC(CDCl3) 11.22 (CH3) 39.89 (C-29) 44.16 (CH29) 55.22 (OCH3) 62.46 (C-59) 80.13 (C-39) 83.97 (C-19) 87.34 (CPh3 C-49) 111.21 (C-5) 113.18 127.92 128.01 128.44 129.92 130.25 130.28 134.71 134.91 143.64 and 158.82 (Carom) 119.84 ( CH29) 132.50 ( CH9) 136.12 (C-6) 150.59 (C-2) and 163.87 (C-4); dH(CDCl3) 1.22 (s 3 H CH3) 2.12–2.41 (m 4 H CH29 H2-29) 3.02 (dd J 2.4 and 10.8 1 H Ha-59) 3.67 (dd J 3.5 and 10.8 1 H Hb-59) 3.79 (s 6 H 2 × OCH3) 4.06 (m 1 H H-49) 4.90 (dd J 1.4 and 17.1 1 H CH29a) 5.09 (dd J 1.4 and 10.2 1 H CH29b) 5.68–5.82 (m 1 H CH9) 6.51 (dd J 5.1 and 9.4 1 H H-19) 6.84 (m 4 H ArH) 7.21–7.45 (m 9 H ArH) 7.86 (s 1 H H-6) and 9.20 (s 1 H NH).a-Isomer 15b dC(CDCl3) 12.50 (CH3) 39.93 (C-29) 45.43 (CH29) 55.19 (OCH3) 63.17 (C-59) 79.57 (C-39) 86.96 (C-49) 87.20 (CPh3) 89.32 (C-19) 109.15 (C-5) 113.27 126.91 127.96 128.04 129.91 129.93 135.43 135.72 144.37 and 158.57 (Carom) 120.02 ( CH29) 132.63 ( CH9) 137.58 (C-6) 150.52 (C-2) and 164.21 (C-4); dH(CDCl3) 1.90 (s 3 H CH3) 2.17–2.34 (m 3 H CH29 Ha-29) 2.79 (dd J 7.7 and 14.3 1 H Hb-29) 3.02 (dd J 2.6 and 10.8 1 H Ha-59) 3.44 (dd J 3.7 and 10.8 1 H Hb-59) 3.78 (s 6 H 2 × OCH3) 4.31 (m 1 H H-49) 4.98 (dd J 1.3 and 17.0 1 H CH29a) 5.10 (dd J 1.3 and 10.1 1 H CH29b) 5.69–5.80 (m 1 H CH9) 6.41 (m 1 H H-19) 6.85 (m 4 H ArH) 7.16–7.45 (m 9 H ArH) 7.71 (s 1 H H-6) and 9.13 (s 1 H NH); FAB-MS m/z 584 M1 (Found C 69.28; H 6.20; N 5.19.Calc. for C34H36N2O7?0.1H2O C 69.63; H 6.22; N 4.78). 1-{3-C-Allyl-3-O-cyanoethoxy(diisopropylamino)phosphino-2- deoxy-5-O-(4,49-dimethoxytrityl)-‚-D-erythro-pentofuranosyl}- thymine 16a Method C. General method for phosphitylation. Nucleoside 15a (545 mg 0.93 mmol) was coevaporated with anhydrous CH3CN (3 × 2 cm3) and was then dissolved under argon in anhydrous CH2Cl2 (3.7 cm3).DIPEA (1.02 cm3 5.97 mmol) was added followed by dropwise addition of 2-cyanoethyl N,Ndiisopropylphosphoramidochloridite (0.44 cm3 1.86 mmol). After 5 h CH3OH (1 cm3) was added and the reaction mixture was diluted with EtOAc (20 cm3) containing triethylamine (0.2 cm3) washed successively with saturated aq. NaHCO3 (3 × 30 cm3) and saturated aq. NaCl (2 × 30 cm3) dried (Na2SO4) and evaporated under reduced pressure. Purification using silica gel column chromatography (EtOAc–CH2Cl2–Et3N–light petroleum 15 30:5:50 v/v/v/v) followed by precipitation in light petroleum (200 cm3) at 220 8C after re-dissolution in anhydrous toluene (2 cm3) afforded compound 16a as a solid (534 mg 75); dP(CDCl3) 138.88 and 138.28.1-{3-C-Allyl-3-O-cyanoethoxy(diisopropylamino)phosphino-2- deoxy-5-O-(4,49-dimethoxytrityl)-·-D-erythro-pentofuranosyl}- thymine 16b Method C. Used amounts. Nucleoside 15b (304 mg 0.52 mmol) anhydrous CH2Cl2 (2.1 cm3) DIPEA (0.57 cm3 3.33 mmol) and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.25 cm3 1.06 mmol) as above. Purification using silica gel column chromatography (EtOAc–CH2Cl2–Et3N–light petroleum 15 30:5:50 v/v/v/v) followed by precipitation in light petroleum (150 cm3) at 220 8C after re-dissolution in anhydrous toluene (1.5 cm3) afforded compound 16b as a solid (306 mg 77); dP(CDCl3) 143.94. 1-(3-C-Allyl-‚-D-ribofuranosyl)thymine 17 To a solution of compound 18 (2.16 g 4.51 mmol) in anhydrous CH2Cl2 (70 cm3) under argon at 278 8C was added dropwise BCl3 (1 M solution in hexane; 18.1 cm3 18.1 mmol).The mixture was stirred for 3.5 h at 278 8C additional BCl3 was added (1 M hexane solution; 4.0 cm3 4.0 mmol) and the mixture was stirred for a further 2 h. MeOH (50 cm3) was added to the mixture which was then stirred overnight at room temp. After concentration under reduced pressure and coevaporation with MeOH (3 × 5 cm3) the residue was purified using silica gel column chromatography (3–6 MeOH in CH2Cl2) to give title compound 17 (977 mg 73) which was used in the next step without further purification. An analytical sample was obtained by recrystallization from MeOH; dC(CD3OD) 12.68 (CH3) 39.65 (CH29) 62.22 (C-59) 78.62 (C-29) 79.91 (C-39) 88.78 (C-49) 89.05 (C-19) 112.14 (C-5) 118.91 ( CH29) 135.11 ( CH9) 139.63 (C-6) 153.68 (C-2) and 166.90 (C-4); dH(CD3OD) 1.88 (s 3 H CH3) 2.49–2.56 (dd J 8.3 and 14.8 1 H CH29a) 2.56–2.63 (dd J 6.2 and 14.8 1 H CH29b) 3.72–3.77 (dd J 2.6 and 12.1 1 H Ha-59) 3.80–3.84 (dd J 2.3 and 12.2 1 H Ha-59) 3.94–3.96 (t J 2.6 1 H H-49) 4.16 (d J 7.9 1 H H- 29) 5.12–5.21 (m 2 H CH29) 5.99–6.05 (m 1 H CH9) 6.00 (d J 7.9 1 H H-19) and 8.05 (s 1 H H-6); FAB-MS m/z 299 M 1 H1 (Found C 52.39; H 5.84; N 9.26.Calc. for C13H18N2O6 C 52.34; H 6.08; N 9.39). 1-3-C-Allyl-5-O-(4,49-dimethoxytrityl)-‚-D-ribofuranosyl- thymine 18 Nucleoside 17 (977 mg 3.28 mmol) was coevaporated with anhydrous pyridine (3 × 10 cm3) and re-dissolved in anhydrous pyridine (8 cm3). DMTCl (1.33 g 3.93 mmol) was added and the mixture was stirred for 16 h under argon at room temp. The solution was evaporated under reduced pressure the residue was re-dissolved in CH2Cl2 (40 cm3) and the solution was washed with saturated aq.NaCl (3 × 30 cm3). The water phase was extracted with CH2Cl2 (2 × 10 cm3). The combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. Purification using silica gel column chromatography (20–60 EtOAc in light petroleum 0.5 pyridine v/v/v) gave title compound 18 (1.66 g 82); dC(CDCl3) 11.19 (CH3) 38.25 (CH29) 55.18 (OCH3) 62.04 (C-59) 77.83 and 78.31 (C-29 and -39) 86.00 87.39 and 87.58 (C-19 -49 and CPh3) 111.28 (C-5) 113.26 113.30 127.38 128.07 128.61 132.50 134.80 134.90 136.70 143.80 and 158.96 ( CH9 C-6 and Carom) 118.75 ( CH29) 152.01 (C-2) and 164.37 (C-4); dH(CDCl3) 1.13 (s 3 H CH3) 2.21–2.29 (dd J 8.3 and 14.5 1 H CH29a) 2.46–2.53 (dd J 5.6 and 14.5 1 H CH29b) 3.24–3.29 (dd J 2.2 and 10.7 1 H Ha-59) 3.64–3.68 (dd J 2.9 and 10.9 1 H Hb-59) 3.76 (s 6 H 2 × OCH3) 4.17 (m 1 H H-49) 4.28 (d 1 H J 7.2 H-29) 4.49 (d 1 H J 17.1 CH29a) 4.90 (d 1 H J 10.3 CH29b) 5.75–5.83 (m 1 H CH9) 6.15 (d 1 H J 7.2 H-19) 6.82–6.86 (m 4 H ArH) 7.23–7.38 (m 9 H ArH) and 7.79 (s 1 H H-6); FAB-MS m/z 600 M1 (Found C 67.88; H 5.86; N 5.13.Calc. for C34H36N2O8?0.2C5H5N C 68.19; H 6.05; N 5.00). 1-3-C-Allyl-2-O-(tert-butyldimethylsilyl)-5-O-(4,49-dimethoxytrityl)-‚- D-ribofuranosylthymine 19 Nucleoside 18 (1.61 g 2.68 mmol) was coevaporated with 3282 J. Chem. Soc. Perkin Trans. 1 1997 anhydrous CH3CN (3 × 10 cm3) and re-dissolved in anhydrous DMF (10 cm3). Imidazole (1.46 g 21.4 mmol) was added followed by the addition of TBDMSCl (1.61 g 10.7 mmol).The mixture was stirred at room temp. under argon for 72 h. MeOH (3 cm3) was added and the solution was concentrated under reduced pressure. The residue was re-dissolved in CH2Cl2 (30 cm3) and this solution was washed with water (3 × 20 cm3). The water phase was extracted with CH2Cl2 (20 cm3) and the combined organic phases were dried (Na2SO4) and concentrated under reduced pressure. Purification using silica gel column chromatography (20–30 EtOAc in light petroleum 0.5 pyridine v/v/v) gave title compound 19 (1.54 g 82); dC(CDCl3) 24.62 and 24.56 (2 × SiCH3) 10.43 (CH3) 17.70 C(CH3)3 25.52 C(CH3)3 38.40 (CH29) 55.19 and 55.28 (2 × OCH3) 61.68 (C-59) 78.00 and 78.46 (C-29 and -39) 84.57 85.93 and 87.66 (C-19 -49 and CPh3) 111.83 (C-5) 113.26 113.30 127.68 128.04 128.93 130.66 132.34 134.41 134.44 136.44 143.29 and 159.15 ( CH9 C-6 and Carom) 118.37 ( CH2) 150.94 (C-2) and 163.77 (C-4); dH(CDCl3) 0.00 and 0.19 (2 s 6 H 2 × SiCH3) 0.92 s 9 H C(CH3)3 0.95 (s 3 H CH3) 2.32 (d J 6.8 2 H CH29) 3.42 (d J 10.6 1 H Ha-59) 3.72–3.76 (dd J 3.1 and 10.1 1 H Hb-59) 3.79 (s 6 H 2 × OCH3) 4.03– 4.10 (m 2 H CH29a H-49) 4.38 (d J 7.5 1 H H-29) 4.78 (d J 10.7 1 H CH29b) 5.71–5.77 (m 1 H CH9) 6.22 (d J 7.4 1 H H-19) 6.84 (m 4 H ArH) 7.20–7.34 (m 9 H ArH) 7.83 (s 1 H H-6) and 8.76 (s 1 H NH); FAB-MS m/z 714 M1(Found C 66.78; H 6.90; N 3.99.Calc. for C40H50- N2O8Si?0.25 H2O C 66.78; H 7.08; N 3.89). 1-{3-C-Allyl-2-O-(tert-butyldimethylsilyl)-3-O-2-cyanoethoxy- (diisopropylamino)phosphino-5-O-(4,49-dimethoxytrityl)- ‚-D-ribofuranosylthymine 20 Method C.Used amounts. Nucleoside 19 (252 mg 0.35 mmol) anhydrous CH2Cl2 (3 cm3) DIPEA (2.63 mmol 0.45 cm3) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (1.4 mmol 0.33 cm3) as above. After 24 h the reaction was quenched by addition of CH3OH (1 cm3). Purification using silica gel column chromatography (0.5–1 Et3N in CH2Cl2 v/v) followed by precipitation in light petroleum (250 cm3) at 240 8C after re-dissolution in anhydrous toluene (2 cm3) afforded title compound 20 (249 mg 78); dP(CDCl3) 139.50 and 140.75. 1-3-C-Allyl-5-O-(tert-butyldimethylsilyl)-2-deoxy-3-Otrimethylsilyl- ·,‚-D-erythro-pentofuranosylthymine 21 To a stirred suspension of the methyl glycoside 5 (720 mg 2.38 mmol) and thymine (604 mg 4.79 mmol) in anhydrous CH3CN (25 cm3) under argon at room temp.was added dropwise BSA (4.7 cm3 19.01 mmol). The mixture was stirred for 1 h until clearness. The reaction mixture was then cooled to 230 8C and TMS triflate (0.86 cm3 4.75 mmol) was dropwise added. The reaction mixture was stirred for 7 days at room temp. before being diluted with CH2Cl2 (50 cm3) and the reaction quenched with saturated aq. NaHCO3 (2 × 50 cm3). After being washed with water (2 × 50 cm3) the organic phase was dried (Na2SO4) and concentrated under reduced pressure. Purification using silica gel column chromatography (0–1 MeOH in CH2Cl2 v/v) afforded a (1 2) anomeric mixture of nucleosides 21 (926 mg 83); dC(CDCl3) 25.92 25.81 25.76 and 25.61 2 × Si(CH3)2 1.92 2.03 and 2.17 2 × Si(CH3)3 12.39 and 12.45 ‡ (2 × CH3) 17.92 and 18.01 ‡ 2 × C(CH3)3 25.65 and 25.78 2 × C(CH3)3 39.41 ‡ and 39.84 (2 × C-29) 44.74 ‡ and 46.16 (2 × 62.56‡ and 62.93 (2 × C-59) 83.85 and 83.92‡ (2 × C-39) 85.44 ‡ and 85.92 (2 × C-19) 88.62‡ and 89.99 (2 × C-49) 109.66 and 109.99 ‡ (2 × C-5) 117.83 ‡ and 118.18 (2 × CH20) 133.63 (2 × CH0) 135.76 ‡ and 137.12 (2 × C-6) 150.47 ‡ and 150.65 (2 × C-2) and 164.28 ‡ and 164.38 (2 × C- 4); dH(CDCl3) 0.01–0.20 2 × Si(CH3)2 and 2 × Si(CH3)3 0.92 ‡ Minor anomer.s 2 × C(CH3)3 1.93 (s 2 × CH3) 1.98–2.11 (m 2 × Ha-29) 2.40–2.65 (m 2 × Hb-29 2 × CH29) 3.73–3.98 (m 2 × H2-59) 4.04‡ (m H-49) 4.22 (m H-49) 4.94–5.17 (m 2 × CH29) 5.74–5.99 (m 2 × CH9) 6.15 ‡ (dd J 4.9 and 9.2 H-19) 6.27 (dd J 2.5 and 8.1 H-19) 7.61 ‡ and 7.64 (2 s 2 × H-6) and 9.22 and 9.34 ‡ (2 s 2 × NH); FAB-MS m/z 469 M 1 H1 (Found C 56.47; H 8.20; N 5.78.Calc. for C22H40N2O5Si2 C 56.37; H 8.60; N 5.98). 1-5-O-(tert-Butyldimethylsilyl)-2-deoxy-3-C-(3-hydroxypropyl)- 3-O-trimethylsilyl-·,‚-D-erythro-pentofuranosyl- thymine 22 To a stirred solution of allyl compound 21 (918 mg 1.96 mmol) in anhydrous THF (4 cm3) under argon at room temp. was added BH3?1,4-oxathiane (0.27 cm3 of a 7.8 M solution in 1,4- oxathiane 2.11 mmol). The mixture was cooled to 0 8C and 2 M aq. NaOH (1.1 cm3) was slowly added followed by the dropwise addition of 30 aq. H2O2 (0.28 cm3); stirring was continued for 60 min at room temp. The reaction mixture was poured into ice–water (50 cm3) and extracted with Et2O. The organic phase was washed successively with saturated aq. NaHCO3 (2 × 50 cm3) and water (50 cm3) dried (Na2SO4) and evaporated under reduced pressure.The crude product was purified by silica gel column chromatography (0–40 EtOAc in light petroleum v/v) to give title compound 22 as a solid (602 mg 63); dC(CDCl3) 25.75 25.61 and 25.44 2 × Si(CH3)2 2.00 and 2.27 2 × Si(CH3)3 12.50 and 12.56 ‡ (2 × CH3) 18.07 and 18.17 ‡ 2 × C(CH3)3 25.78 and 25.90‡ 2 × C(CH3)3 27.71‡ and 27.82 (2 × C-20) 31.49 ‡ and 31.81 (2 × C-10) 44.67‡ and 46.01 (2 × C-29) 62.79 62.94 and 63.19 (2 × C-59 and 2 × C- 30) 84.38 and 84.48 ‡ (2 × C-39) 85.79 (2 × C-19) 88.61 ‡ and 89.93 (2 × C-49) 109.69 and 109.97 ‡ (2 × C-5) 135.88‡ and 137.30 (2 × C-6) 150.44 ‡ and 150.65 (2 × C-2) and 164.15‡ and 164.26 (2 × C-4); dH(CDCl3) 0.01–0.24 2 × Si(CH3)2 and 2 × Si(CH3)3 0.91 s 2 × C(CH3)3 1.59–2.04 (m 2 × CH3 Ha- 29 ‡ H2-10) 2.10 (dd J 1.7 and 14.1 Ha-29) 2.48 ‡ (m Hb-29) 2.53 (dd J 8.3 and 14.1 Hb-29) 3.67–3.90 (m 2 × H2-59 and H2- 30) 4.07 ‡ (m H-49) 4.23 (m H-49) 6.18 ‡ (dd J 4.9 and 9.2 H- 19) 6.27 (dd J 2.5 and 8.2 H-19) 7.63 ‡ and 7.69 (2 s 2 × H-6) and 9.00 (br s 2 × NH); FAB-MS m/z 487 M 1 H1 (Found C 54.34; H 8.44; N 5.59.Calc. for C22H42N2O6Si2 C 54.29; H 8.70; N 5.76). 1-5-O-(tert-Butyldimethylsilyl)-2-deoxy-3-C-(3-phthalimidopropyl)- 3-O-trimethylsilyl-·,‚-D-erythro-pentofuranosyl- thymine 23 Compound 22 (602 mg 1.24 mmol) phthalimide (237 mg 1.61 mmol) and triphenylphosphine (444 mg 1.69 mmol) were dissolved in anhydrous THF (1.3 cm3) under argon. The mixture was cooled to 0 8C and a solution of diethyl azodicarboxylate (DEAD) in anhydrous THF (0.62 cm3 THF; 1.59 mmol) was added dropwise.After 24 h at room temperature the solvent was evaporated off under reduced pressure. The crude product was dissolved in CH2Cl2 (50 cm3) and washed successively with saturated aq. NaHCO3 (2 × 40 cm3) and water (2 × 40 cm3). The organic phase was dried (Na2SO4) and concentrated under reduced pressure. Silica gel column chromatography (CH2Cl2) afforded title compound 23 as a solid (656 mg 86); dC(CDCl3) 26.07 and 25.86 2 × Si(CH3)2 1.81 and 2.05 2 × Si(CH3)3 12.42 and 14.28 ‡ (2 × CH3) 17.83 and 17.95 ‡ 2 × C(CH3)3 25.57 and 25.72‡ 2 × C(CH3)3 23.63‡ and 23.90 (2 × C-20) 32.40 ‡ and 32.81 (2 × C-10) 37.88 and 37.97 ‡ (2 × C-30) 45.94 (2 × C-29) 61.98 and 63.05‡ (2 × C-59) 84.18 ‡ and 84.20 (2 × C-39) 85.61 ‡ and 85.77 (2 × C-19) 88.45 ‡ and 89.76 (2 × C-49) 109.71 and 110.00 ‡ (2 × C-5) 123.30 ‡ 123.33 132.10 132.15 ‡ 134.06‡ and 134.09 (2 × Carom) 135.82 ‡ and 137.30 (2 × C-6) 150.28 ‡ and 150.50 (2 × C-2) 164.05 (2 × C-4) and 168.40 ‡ and 168.43 (2 × CON); dH(CDCl3) 0.01–0.18 2 × Si(CH3)2 and 2 × Si(CH3)3 0.81 J.Chem. Soc. Perkin Trans. 1 1997 3283 s 2 × C(CH3)3 1.71–1.96 (m 2 × CH3 Ha-29‡ H2-10 and H-20) 2.05 (dd J 14.3 Ha-29) 2.40 ‡ (dd J 4.8 and 12.5 Hb-29) 2.45 (dd J 8.2 and 14.2 Hb-29) 3.60–3.71 (m Ha-59 ‡ H-59a and H2-30) 3.81‡ (m Hb-59) 4.01 ‡ (m H-49) 4.16–4.23 (m H-49 and Hb-59) 6.15 ‡ (dd J 4.8 and 9.2 H-19) 6.24 (dd J 2.0 and 8.2 H-19) 7.58 ‡ and 7.65 (2 s H2-6) 7.66–7.81 (2 × ArH) and 8.58 and 8.69 ‡ (2 s 2 × NH); FAB-MS m/z 616 M 1 H1. 1-2-Deoxy-3-C-(3-phthalimidopropyl)-·,‚-D-erythro-pentofuranosyl thymine 24 To a solution of nucleoside 23 (668 mg 1.09 mmol) in anhydrous THF (27 cm3) was added 1.1 M TBAF in THF (2.2 cm3 2.42 mmol).The reaction mixture was stirred under argon for 20 min and then was concentrated to dryness. Purification using silica gel column chromatography (20 EtOAc in light petroleum v/v) afforded title compound 24 as solid (154 mg 33); dC(CDCl3) 12.12 and 12.22 ‡ (2 × CH3) 23.37 ‡ and 23.57 (2 × C-20) 32.21 ‡ and 32.80 (2 × C-10). 38.09‡ and 38.24 (2 × C-30) 42.60 ‡ and 44.56 (2 × C-29) 58.17 and 62.02 ‡ (2 × C-59) 79.60 80.78 ‡ 85.78 ‡ 87.32 88.93 ‡ and 91.51 (C- 19 -39 and -49) 108.43 and 110.74 ‡ (2 × C-5) 123.29 131.96 and 134.06 (2 × Carom) 137.29 ‡ and 137.93 (2 × C-6) 151.06‡ and 151.32 (2 × C-2) 164.34‡ and 164.72 (2 × C-4) and 168.72 ‡ and 168.83 (CON); dH(CDCl3) 1.75–2.62 (m 2 × CH3 H2-29 H2-10 and H2-20) 3.67–4.55 (m 2 × H-49 H2-59 and H2- 30) 6.17 (d J 5.0 H-19) 6.27 ‡ (dd J 5.2 and 9.2 H-19) 7.66– 7.81 (m 2 × H-6 and ArH) and 10.90 (br s 2 × NH); FAB-MS m/z 430 M 1 H1.1-2-Deoxy-5-O-(4,49-dimethoxytrityl)-3-C-(3-phthalimidopropyl)- ·-D-erythro-pentofuranosylthymine 25 An anomeric mixture of nucleoside 24 (368 mg 0.86 mmol) AgNO3 (174 mg 1.02 mmol) and DMTCl (1.45 g 4.28 mmol) were dissolved in anhydrous THF (35 cm3) and pyridine (0.35 cm3 4.33 mmol) was added. The mixture was stirred at room temp. for 72 h with exclusion of light. The mixture was filtered into 5 aq. NaHCO3 (50 cm3). The product was extracted into CH2Cl2 (4 × 100 cm3) and the organic phase was dried (Na2SO4) and concentrated under reduced pressure. Purification by PLC (2 pyridine 5 CH3OH in CH2Cl2 v/v/v two runs) gave the a-anomer 25 (267 mg 43) as the less polar isomer and the corresponding b-anomer (94 mg 15).b-Anomer 25 dC(CDCl3) 11.18 (CH3) 23.36 (C-20) 31.81 (C-10) 37.92 (C-30) 43.99 (C-29) 55.12 (OCH3) 62.69 (C-59) 80.98 (C-39) 84.15 (C-19) 87.32 (C-49) 87.84 (CPh3) 111.33 (C-5) 113.24 127.35 128.01 130.28 130.34 134.78 134.99 143.75 and 158.88 (DMT) 123.31 131.99 and 134.06 (Phth) 136.58 (C-6) 150.76 (C-2) 163.96 (C-4) and 168.62 (CON); dH(CDCl3) 1.22 (s 3 H CH3) 1.45–1.76 (m 4 H H2-10 and -20) 2.09 (dd J 9.5 and 12.4 2 H Ha-29) 2.40 (dd J 4.8 and 12.4 1 H Hb-29) 3.15 (dd J 2.2 and 10.8 1 H Ha-59) 3.53 (m 2 H H2-30) 3.62 (dd J 3.3 10.8 1 H Hb-59) 3.79 (s 6 H 2 × OCH3) 4.06 (m 1 H H-49) 6.49 (dd J 4.8 and 9.5 1 H H-19) 6.84 (m 4 H DMT) 7.23–7.38 (m 9 H DMT) 7.65–7.85 (m 5 H Phth and H-6) and 9.18 (s 1 H NH).a-Anomer 25 dC(CDCl3) 12.26 (CH3) 23.56 (C-20) 32.05 (C-10) 37.82 (C-30) 44.94 (C-29) 54.88 (OCH3) 63.28 (C-59) 80.08 (C-39) 86.74 (C-49) 87.36 (CPh3) 89.86 (C-19) 108.64 (C-5) 113.19 126.79 127.91 129.81 129.88 135.46 135.79 144.46 and 158.47 (DMT) 123.75 131.85 and 133.87 (Phth) 137.92 (C-6) 150.66 (C-2) 164.71 (C-4) and 168.37 (CON); dH(CDCl3) 1.24–1.79 (m 4 H H2-10 and -20) 1.84 (s 3 H CH3) 2.32 (d J 14.0 1 H Ha-29) 2.72 (dd J 7.6 and 14.4 1 H Hb-29) 2.99 (dd J 2.0 and 10.7 1 H Ha-59) 3.41 (dd J 3.6 and 10.7 1 H Hb-59) 3.59 (m 2 H H2-30) 3.78 (s 6 H 2 × OCH3) 4.35 (m 1 H H-49) 6.36 (d J 7.0 1 H H-19) 6.84 (m 4 H DMT) 7.17–7.43 (m 9 H DMT) 7.64–7.77 (m 5 H Phth H-6) and 10.03 (s 1 H NH); FAB-MS m/z 731 M1.1-{3-O-2-Cyanoethoxy(diisopropylamino)phosphino-2-deoxy- 5-O-(4,49-dimethoxytrityl)-3-C-phthalimidopropyl-·-D-erythropentofuranosyl} thymine 26 Nucleoside 25 (267 mg 0.37 mmol) was coevaporated with anhydrous CH3CN (3 × 2 cm3) and dissolved under argon in anhydrous CH2Cl2 (2 cm3). DIPEA (0.4 cm3 2.34 mmol) was added followed by dropwise addition of 2-cyanoethyl N,Ndiisopropylphosphoramidochloridite (0.17 cm3 0.72 mmol). After stirring of the mixture for 5 h CH3OH (0.5 cm3) was added and the reaction mixture was diluted with EtOAc (20 cm3) containing triethylamine (0.2 cm3) washed successively with saturated aq. NaHCO3 (3 × 30 cm3) and saturated aq. NaCl (2 × 30 cm3) dried (Na2SO4) and evaporated under reduced pressure. Purification using silica gel column chromatography (EtOAc–CH2Cl2–Et3N–petroleum ether 15 30:5 50 v/v/v/v) followed by precipitation in light petroleum (30 cm3) at 220 8C after re-dissolution in anhydrous toluene (1 cm3) afforded compound 26 as a solid (141 mg 41); dP(CDCl3) 140.78 and 140.90.Synthesis of oligonucleotides The synthesis of ODNs was carried out on a 0.2 mmol scale (5 mmol amidite per cycle Pharmacia Primer SupportTM) using compounds 16a 16b 26 aT-cyanoethylphosphoramidite and commercial bT-cyanoethylphosphoramidite. The synthesis followed the regular protocol for the DNA synthesizer. For the modified phosphoramidites the coupling time was increased from 2 to 24 min and the cycle was repeated twice. The ODNs were removed from the support and deblocked by treatment with conc. ammonia at room temp. for 72 h.Purification and detritylation was achieved on Cruachem oligonucleotide purifi- cation cartridges using the standard procedure. The purity of the ODNs was confirmed by analytical anion-exchange HPLC. The solvent systems consisting of 10 mM NaOH (A) and 10 mM NaOH 1 1.8 M NaCl (B) were used in the following order 10 min linear gradient of 25–30 B in A 40 min linear gradient of 30–45 B in A 1 min 45–100 B in A 1 min 100 B 1 min linear gradient of 100–25 B in A. Flow rate 1.67 cm3 min21. The purified ODNs eluted as one peak after approximately 14 min for products H–L and after approximately 20 min for products A–G. Melting experiments The melting experiments were carried out in medium salt buffer 1 mM ethylenediaminetetra-acetic acid (EDTA) 10 mM Na2HPO4 140 mM NaCl pH 7.2 at a concentration of 2 nmol for each strand.The increase in absorbance at 260 nm as a function of time was recorded while the temperature was raised from 10 to 60 8C at a rate of 1 8C per min. The melting temperature was determined as the maximum of the first-derivative plot of the 260 nm transition. Acknowledgements The Danish Natural Science Research Council is thanked for financial support. References 1 R. W. Wagner Nature 1994 372 333. 2 E. Uhlmann and A. Peyman Chem. Rev. 1990 90 543. 3 S. L. Beaucage and R. P. Iyer Tetrahedron 1993 49 6123. 4 C. K. Chu B. Doboszewski W. Schmidt and G. V. Ullas J. Org. Chem. 1989 54 2767; J. Fiandor and S. Y. Tam Tetrahedron Lett. 1990 31 597. 5 P. Nielsen K. Larsen and J. Wengel Acta Chem. Scand. 1996 50 1030. 6 P. N. Jørgensen and J. 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