Towards the Synthesis of Inosine Building Blocks for the Preparation of Oligonucleotides with Hydrophobic Alkyl Chains Between the Nucleotide Units †

The scientific objective of the research reported in this manuscript was the synthesis of novel phosphoramidite building blocks for the preparation of lipophilic oligonucleotides. Reaction of inosine (4) with 4-oxopentyl-4-methylbenzoate (2c) in the presence of triethyl orthoformate and 4M HCl in 1,4-dioxane gave a diastereoisomeric mixture of the ketals 5. Subsequent 4,4’-dimethoxytritylation at the 5’-hydroxyl afforded (R)-6 + (S)-6 which could be separated chromatographically. Detoluoylation gave compounds (R)-7 and (S)-7. Phosphitylation of a diastereoisomeric mixture of 7 led to a mixture of four diastereoisomers of the corresponding 2-cyanoethylphosphoramidites 8.


Introduction
The low cell membrane permeability of oligonucleotides is one major drawback of antisense gene therapy. Therefore, as early as the eighties antisense and antigene oligomers were prepared which carried lipophilic groups, mostly at the termini. Other lipophilic modifications comprise derivatization of: (i) the nucleobases, (ii) of the glyconic residues or (iii) of the phosphodiester moiety of the oligonucleotides with hydrophobic side groups [1]. Of particular importance for the stability and activity of antisense oligonucleotides proved to be their modification with terminal cholesterol residues OPEN ACCESS [2] which culminated in the development of the so-called "antagomirs" which are constructed from oligo(2'-OMe-ribonucleotides) carrying several phosphorothioate linkages at both termini and one cholesterol residue at the 5'-end. Such therapeutics have been successfully used for the in-vivo silencing of microRNAs [3]. The various applications of "nucleolipids" (nucleoside, nucleotide and oligonucleotide-based amphiphiles) were reviewed recently [4]. Appending a cholesterol moiety to an oligonucleotide, however, renders it so hydrophobic that its solubility in aqueous solvents is significantly reduced and its handling becomes problematic [5]. For example, a trimer consisting of three wobble nucleotides 2'-deoxyinosine [5'-d(IpIpI)] exhibits a calculated logP value of -6.67 ± 1.06 while its 5'-cholesterol derivative shown in Figure 1 exhibits a logP of +1.79 ± 1.27. We, therefore, designed an oligonucleotide building block on the basis of an O-2',3'-cyclic ketalfirst with hypoxanthine as wobble base -which allows a stepwise hydrophobization of an oligonucleotide and which can be incorporated into all positions of a growing nucleic acid chain by conventional solid-phase synthesis yielding a general structure shown in Figure 2. In this manuscript we report first steps toward the preparation of this new kind of oligonucleotide building block.

Results and Discussion
In order to get an impression about the hydrophobization effect of the incorporation of one or more O-2',3'-cyclic ketal derivative of inosine , I*, (with three CH 2 groups in the ketal side chain) into an oligonucleotide, we first calculated the logP values [6] of a series of inosine trimers with varying substitutions of inosine by inosine ketals (I*) with a chain length of three methylene groups. Figure 3 and Table 1 display the increase of lipophilicity with the increasing number of modifications as well as the influence of the position of incorporation.
It can be seen that in case of incorporation of either one or two modified units into an inosine trimer the resulting logP value depends on the position of incorporation: Substitution of one inosine by an inosine ketal at the 3'-end results in an oligomer (entry 2, Table 1) with a free primary hydroxyl group at the 5'-terminus; this oligomer shows a significantly lower lipophilicity than the corresponding trimers carrying the modified inosine derivative either in the middle position (entry 3) or at the 5'-end (entry 4). An analogous result can be seen for trimers carrying two modifications (entries 5-7, Table  1). The synthesis of 2'-cyanoethyl phosphoramidite building blocks of inosine O-2',3'-cyclic ketals is shown in Scheme 1. First, we converted the (ω-1) ketoalcohols 1a-c to the tolyl-protected compounds 2a-c. Exemplary, Figure 4 displays the three-dimensional structure of 2b obtained from an X-ray analysis [7].   Table 1. Calculated logP values of inosine oligonucleotide trimers as a function of the number and position of inosine O-2',3'-cyclic ketal derivatives (see Figure 2). Earlier, it had been shown that reaction of inosine (4) with (ω-1) ketoesters such as ethyl levulinate or unsymmetrical ketones such as pentan-2-one leads to O-2',3'-ketals with predominant or even exclusive formation of the (R) configuration at the newly formed stereogenic center [8][9][10][11][12] [the (R)and (S)-notation within this manuscript refers always to the configuration at the stereogenic center of the ketal moiety]. We now prepared the keto esters 2a-c, of which only compound 2c had been described earlier [13]. All attempts to react inosine with compound 2a in the presence of triethyl orthoformate and 4M HCl in 1,4-dioxane in various solvents failed. Next, we, therefore, converted compound 2a into the open acetal 3. A subsequent transacetalisation with inosine (4) which usually occurs under mild reaction conditions also failed. Also ketalisation of inosine with the elongated (ω-1) ketoester 2b did not show the desired result. The reason for this result might be an electrostatic repulsion of the ester oxygen atom and the ribose oxygen. At least, reaction of 4 with 4-oxopentyl-4-methylbenzoate, (2c) under the reaction conditions mentioned above gave the desired product 5, however, 1 H-and 13 C-NMR spectroscopy proved the formation of a diastereoisomeric mixture [(R)-5 + (S)-5]. Integration of the 1 H-NMR resonances of the clearly separated ketal Me groups indicated a ratio of 47% of the (R)-and 53% of the (S)diastereoisomer. The assignment of the NMR resonances of the different methyl groups as well as of other signals was made on the basis of a comparison with the 1 H-and 13 C-NMR spectra of (R)-2',3'-O-(3-carboxy-1-methylpropyliden)adenosine from which an X-ray analysis had been performed earlier [14] as well as by gradient-selected homo-and heteronuclear correlation spectroscopy.

Entry
A chromatographic separation of the mixture (R)-5 + (S)-5 proved extremely difficult; only a TLC with a 20-fold development in EtOAc/toluene (97:3, v/v) showed the presence of two products. Because the positioning of the ketal side chain is of decisive influence on the topology of oligonucleotides carrying such modified building blocks, a separation of the diastereoisomers is at least inevitable. Next, we converted the mixture (R)-5 + (S)-5 into the 4,4'-dimethoxytriphenylmethyl derivatives (R)-6 + (S)-6. On this stage the diastereoisomeric mixture could be separated silica gel chromatographically by elution with EtOAc/toluene (97:3, v/v). Both diastereoisomers were characterized by 1 H-NMR spectra.
In order to prove if a chromatographic separation is also possible on the stage of the de-toluoylated compounds a mixture of (R)-6 + (S)-6 was deprotected by treatment with conc. aq. ammonia/MeOH (1:1, v/v). After 72 h a mixture of (R)-7 + (S)-7 was isolated in moderate 38% yield. As it was found that a chromatographic separation of the diastereoisomers was very difficult, the separation was performed on the stage of compounds 6. De-toluoylation was performed on either the stage of a diastereoisomeric mixture or on the stage of the separated isomers and afforded (R)-7 and (S)-7. A first subsequent phosphitylation [15] of a mixture of (R)-7 + (S)-7 with chloro-(2-cyanoethoxy)-N,Ndiisopropylethylaminophosphane (CH 2 Cl 2 , Hünig's base, 3 h) gave, after chromatography, a mixture of four diastereoisomers (additional R P and S P diastereoisomers) in 67% total yield. The phosphitylation of the separated (R)-7 and (S)-7 isomers, their incorporation into oligonucleotides as well as the base pairing properties of such oligomers will be published in following manuscripts. We anticipate that appending of the novel building blocks to one or both termini of an unmodified oligonucleotide will lead to gap-mers with unaltered binding to their complementary strands but that their incorporation into the innermost part of a nucleic acid will give new and autonomous nucleic acid pairing systems.

2-Oxopropyl-4-methylbenzoate (2a)
3-Hydroxopropan-2-one (1a, 1.13 g, 15 mmol) was dissolved in anhydr. pyridine (3 mL) and cooled in an ice bath. After drop-wise addition of tolyl chloride (1.55 g, 10 mmol) the mixture was stirred for 30 min, warmed up to ambient temperature and stirred overnight. Then, the reaction mixture was poured into ice-water (100 mL), acidified by addition of conc. hydrochloric acid and warmed up to ambient temperature. After extraction with CHCl 3 the organic layer was separated and washed with 5% aqueous NaHCO 3 . The organic phase was dried (Na 2 SO 4 ) and evaporated. After drying under high vacuum 1.