Oligonucleotides phosphate

Modification of the Phosphodiester Backbone. Oligonucleotides having modified phosphate backbones have been extensively studied (46). Because altering the backbone makes derivatives generally more resistant to degradation by cellular nucleases, these materials have the potential to be more resilient antisense dmgs. 

Both phosphoramidate and phosphate triester derivatives have been used as linkers to attach reporter groups to oligonucleotides. These derivatives are not entirely resistant to nucleases and they possess a chiral center. They have not been widely iavestigated as antisense dmgs. 

A number of standard synthetic reference books are available. A review article by Kossell and Seliger discusses protective groups used in oligonucleotide syntheses, including protection for the phosphate group, which is not included in this book, and a series of articles describe various aspects of protective group chemistry. 

In the case of oligonucleotides, the phosphate has been shown to increase the rate of formic-acid-induced TBDMS hydrolysis by internal phosphate participation. ... 

Phosphate esters and anhydrides dominate the living world. Major areas of synthetic interest include oligonucleotides (polymeric phosphate diesters), phospho-rylated peptides, phospholipids, glycosyl phosphates, and inositol phosphates. ... 

The steps involved in automated oligonucleotide synthesis illustrate the current use of protective groups in phosphate chemistry (Scheme 1). Oligonucleotide synthesis involves the protection and deprotection of the 5 -OH, the amino groups on adenine, guanine, and cytosine, and -OH groups on phosphorus. 

The disadvantage of this method is that the dichloridites and monochloridites are sensitive to water and thus could not be used readily in automated oligonucleotide synthesis. This problem was overcome by Beaucage and Caruthers, who developed the phosphoramidite approach. In this method, derivatives of the form R 0P(NR2)2 react with one equivalent of an alcohol (catalyzed by species such as l//-tetrazole) to form diesters, R OP(OR")NR2, which usually are stable, easily handled solids. These phosphoroamidites are easily converted to phosphite triesters by reaction with a second alcohol (catalyzed by l//-tetrazole). Here, again, oxidation of the phosphite triester with aqueous iodine affords the phosphate triester. Over the years, numerous protective groups and amines have been examined for use in this approach. Much of the work has been reviewed. ... 

This group, used for 5 -phosphate protection, has hydrophobicity similar to that of the dimethoxytrityl group and thus was expected to assist in reverse-phase HPLC purification of product from failure sequences in oligonucleotide synthesis. The group is cleaved with Bu4N F in DMSO at 70°. ... 

Bu4N F , THF, Pyr, H2O, rt, 30 min. These conditions result in the formation of a mixture of fluorophosphate and phosphate. In the case of oligonucleotides, some intemucleotide bond cleavage is observed with this reagent. 

The lipophilicity of this phosphate protective group helps in the chromatographic purification of oligonucleotides. It is removed by the oximate method. ... 

This highly lipophilic group is cleaved with isoamyl nitrite in Pyr/AcOH. The use of a lipophilic 5 -phosphate protective group aids in reverse-phase HPLC purification of oligonucleotides. 

This lipophilic group, developed for 5 -phosphate protection in oligonucleotide synthesis, is removed with 80% AcOH in 1 h. The related trityloxyethylamino group has been used in a similar capacity for phosphate protection and is also cleaved with 80% AcOH. ... 

Morpholine has been used for 5 -phosphate protection in oligonucleotide synthesis and can be cleaved with 0.01 N HCl without significant depurination of bases having free exocyclic amino functions. 

Step 4 With the coupling accomplished, the phosphite product is oxidized to a phosphate by treatment with iodine in aqueous tetrahydrofuran in the presence of 2,6-dimethylpyridine. The cycle (1) deprotection, (2) coupling, and (3) oxidation is then repeated until an oligonucleotide chain of the desired sequence has been built. 

CpG stands for cytosine phosphate guanine dinucleotide in a particular sequence context. CpG motifs are responsible for proliferative effects of antisense oligonucleotides, particularly with respect to B-lymphocytes. Die optimal immune-stimulatory consensus sequence surrounding CpG is R1R2CGY1Y2, where R1 is a purine (mild preference for G), R2 is a purine or T (preference for A), and Y1 and Y2 are pyrimidines (preference for T). 

Three classes of nucleic acid triple helices have been described for oligonucleotides containing only natural units. They differ according to the base sequences and the relative orientation of the phosphate-deoxyribose backbone of the third strand. All the three classes involve Hoogsteen or reverse Hoogsteen-like hydrogen bonding interaction between the triple helix form-... 

Carbodi-imides are used to mediate the formation of amide linkage betwen a carboxylate and an amine or phosphoramidate linkages between a phosphate and an amine [12]. The following is essentially the method of Rockwood [13] and is modified to give a phospho-diester link between the terminal monophosphate of the oligonucleotide and the hydroxyl group of 2-hydroxyethyl disulfide (HEDS) [14]. 

In the early solution phase syntheses of oligonucleotides, coupling of phosphate diesters was used. A mixed 3 -ester with one aryl substituent, usually o-chlorophenyl, was coupled with a deprotected 5 -OH nucleotide. The coupling reagents were sulfonyl halides, particularly 2,4,6-tri-i-propylbenzenesulfonyl chloride,53 and the reactions proceeded by formation of reactive sulfonate esters. Coupling conditions... 

Fig. 45. (a) Introduction of M(phen)31 complex into DNA oligomers, (b). Steady-state emission spectra of modified oligonucleotides in 0.01 m sodium phosphate buffer, pH 7.0, 0.1 NaCl. Top Ru(phen)3+-modified 20-mer duplex (solid line), a 1 1 mixture of non-comple-mentary Ru(phen)3+- and Os(phen)g+-modified 20-mers (- -) and a 20-mer duplex containing Ru(phen)g+ and Ps(phen)3+groups on different strands separated by one base pair (---). Bottom 20-mer duplex with 5 -terminal Rufphen) (solid line), and Ru(phen)jf/Os(phen)3 -containing analog (---). Reproduced with permission from Ref. (157). Copyright 1998, American Chemical Society. 

Nucleotides can be linked together into oligonucleotides through a phosphate bridge at the 5 position of one ribose unit and the 3 position of another. The purine bases, adenine and guanine, have two heterocyclic rings, while the pyrimidines cytosine, thymine, and uracil have one. The structure of adenosine monophosphate is shown in Figure 11. 

Analogues of Dinucleoside Di(Tri)phosphates and Oligonucleotides with a Di(Tri)phosphate Bridge... 

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