Las madres

In the design and synthesis of the vinyl bromide component, the initial aim was investigate the installation of the vinyl bromide moiety prior to addition of the nucleoside base. It was hoped that this strategy would overcome the problems experienced in the DNA series where the vinyl bromide could not be isolated as a single isomer when olefination was performed in the presence of the nucleoside base (see section 1.6.2.1).^129,154

At the start of the project, the commercially available 1,2-isopropylidene xylofuranose 86 was identified as a good starting point. Unfortunately, the analogous ribofuranose is not commercially available so the first steps involved inversion of the stereochemistry of the 3-OH.

The initial steps of this route followed work developed by R.

Bertram.¹²⁹ The selective protection of the primary alcohol, 5-OH as the TBS ether using standard conditions (imidazole, TBS-Cl, DMF), gave xylofuranose 87 with a pleasing 72% yield (Scheme 38).¹⁵⁹ The oxidation with Dess Martin periodinane (DMP) in dichloromethane^160,161 gave the ketone which was then reduced with NaBH4 (1.2 eq.) in the presence of methanol (20 eq.) in dry ether.

The reduction was entirely selective as the hydride addition occurs

in an exo fashion, at the more accessible top face, to access ribofuranose 81 in an excellent 89% yield.

a b-c

Scheme 38. Reagents & conditions: a. TBSCl, imidazole, DMF, 0^oC (72%); b.

DMP, CH₂Cl₂, 0 ^oC (91%); c. NaBH₄, MeOH, Et₂O, 0^oC (89%).

Previously, Bertram had used the PMB protecting group at the 3-position,¹²⁹ however, this proved to be too acid-labile at a later stage when hydrolysis of the 1,2-isopropylidene was carried out using acidic conditions. Therefore, at this stage the decision was made to use the 2-methylnaphthyl protecting group at the 3-OH position. A protecting group that is both acid and base stable is necessary for later steps in the synthesis. As first reported by Spencer et al.,¹⁶² Bertram has shown that the 2-methylnaphthyl could be utilised as an acid stable protecting group in the synthesis of the vinyl bromide nucleosides. This protecting group can be cleaved reductively using hydrogenation or oxidatively using DDQ or CAN.¹⁶³ Alcohol 81 was protected using the method of Bessodes et al.:¹⁶⁴ thus 2-bromomethyl naphthalene (1.2 eq.) in the presence of potassium hydroxide (1.8 eq.) and 18-crown-6 (0.04 eq.) in dry THF afforded 158 in a yield of 70% (Scheme 39). The TBS ether of 158 was then cleaved using TBAF (86%) and the resulting alcohol 88 was cleanly oxidised to aldehyde 89 using hypervalent iodine oxidising agent DMP in CH₂Cl₂(79%).¹²⁹

O

Scheme 39. Reagents & conditions: a. 2-NapCH₂Br, KOH, 18-c-6, THF, r.t.

(70%); b. TBAF, THF, r.t. (86%); c. DMP, CH2Cl2, r.t. (79%); d. Ph3P, CBr4, CH₂Cl₂, 0 ^oC (22%); e. (MeO)₂P(O)H, Et₃N, DMF, 110^oC (84%).

Our next task was to form the E-vinyl bromide, 160; this was achieved using our previously developed two-step procedure. Work in the Hayes group has developed a modified Hirao reduction¹⁶⁵ of a dibromo olefin to form the bromo olefin, favouring the trans geometry.¹⁵⁶ Dibromo olefin 159 was generated using the method of Ramirez et al.; by reaction of the aldehyde 89 with the Ph₃P-CBr₄ ylid, generated in situ by the reaction of Ph₃P (4.0 eq.) and CBr₄ (2.0 eq.) in CH₂Cl₂ at r.t. in a disappointing 22% yield (Scheme 39).153,166,167 Further attempts to optimise this reaction were unsuccessful.¹²⁹ The dibromo olefin 159 was then reduced using dimethyl phosphite and Et3N in DMF at 110 ^oC to obtain a 3:1 mixture of E- and Z-vinyl bromides isomers in 84% combined yield.

Due to the disappointing yields in the Wittig reaction to form the aldehyde 89, the decision was made to revert to the benzyl protecting group previously used by Solesbury in the development

of a similar route from the 1,2-isopropylidene xylofuranose 86 to an analogous terminal olefin (Scheme 56).¹⁴⁰ Similar to the 2-methylnaphthyl, the benzyl protecting group is stable in acid and base environments and can be removed under neutral conditions.

Benzylation of the 3-OH of ribofuranose 81 was achieved using BnBr (1.3 eq.), TBAI (0.05 eq.) and NaH (1.6 eq.) (Scheme 40).¹⁶⁸ This was followed by deprotection of the TBS ether using TBAF as a fluoride source to give alcohol 161 in yield of 83% over the two steps. The intermediate aldehyde was generated by oxidation of the alcohol 161 using Dess Martin periodinane and this was isolated in an excellent 96% yield. The dibromo olefin 162 was obtained from this aldehyde in 69% yield using the protocol of Ramirez et al.¹⁵³ This shows a marked improvement in yield compared to the 22%

yield when the 2-methylnaphthy protecting group was used presumably due to reduced steric bulk.

O

Scheme 40. Reagents & conditions: a. BnBr, NaH, TBAI, THF, 0^oC; b. TBAF, THF (83%, 2 steps); c. DMP, CH2Cl2, 0^oC (96%); d. Ph3P, CBr4, CH2Cl2, 0^oC (69%).

The dibromo olefin 162 was reduced using dimethyl phosphite (4.0 eq.) and Et3N (2.0 eq.) in dry DMF at 110 ^oC to produce E- and Z-vinyl bromides 163 and 164 in a 3:1 ratio respectively (Scheme

41).^165,156 The two isomers were separated by column chromatography and the desired E-vinyl bromide 163 was isolated in 51% yield. This is an improvement on the previous work in the DNA series of installing the vinyl bromide with the nucleotide base already present.

Scheme 41. Reagents & conditions: (MeO)2P(O)H, Et₃N, DMF, 110^oC.

Following the generation of E-vinyl bromide 163, the 1,2-acetonide was hydrolysed in refluxing 60% v/v aqueous acetic acid (Scheme 42). The diol was acetylated to form the bis-acetate 165 using Ac₂O and pyridine as a 10:1 mixture of anomers with a yield of 95%

over the two steps. Using the modified Vorbrüggen glycosylation conditions of Yang et al.; uracil was added with complete facial selectivity to form the uridine vinyl bromide 166 in 85% yield.¹⁶⁹

O

Scheme 42. Reagents & conditions: a. 60% AcOH, reflux; b. Ac₂O, pyridine, r.t. (95%, 2 steps); c. Uracil, BSA, MeCN, 65^oC then TMSOTf (85%).

Although the 2’-OAc serves as an adequate protecting group, it was decided to manipulate this to have the 2’-OTBS group as this would

be required later on, for the solid-phase synthesis of oligonucleotides. The 2’-OAc species 166 was deprotected using K₂CO₃ in MeOH to form the alcohol which was then protected to form TBS ether 167 in 66% yield using TBSCl, and imidazole in dry DMF (Scheme 43).¹⁵⁹

O

Scheme 43. Reagents & conditions: a. K2CO₃, MeOH, r.t. (95%); b. TBSCl, imid., NaH, DMF, 0^oC (66%).

2.1.1.1 Troubleshooting the First Generation Synthesis The initial synthesis of the vinyl bromide nucleoside from the isopropylidene starting material contains problems and needs improvement. In the ten steps it takes to get from the starting xylofuranose 86 to the 2’-OTBS uridine vinyl bromide 167, two oxidations are performed that require DMP. On its own, Dess Martin periodinane is a good, clean oxidising agent. However, it is required in a stoichiometric amount and use of the reagent at such early stages in the synthesis is not economical. The protecting group strategy must also be revised to use a protecting group at the 3’-position that can be removed at the desired stage. Parallel work in the Hayes’ group showed that the benzyl protecting group could not be removed without loss of the carbon-carbon double bond moiety of the vinyl bromide, so a second generation approach was required.