Amines oligonucleotides

Manoharan et have demonstrated site specific cross-linking to an abasic site in a duplex incorporating a nucleoside with a 2 -0-pentylamino linker after reductive amination. Oligonucleotides containing the modifications 2 -0- 2-... 

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

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. ... 

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]. 

Smith and co-workers (194) have used this chemistry to prepare carboxyl-modified Si(lll) surfaces at which polylysine-tethered DNA is electrostatically adsorbed (Fig. 60). An alternative approach involved covalent attachment of a pre-synthesized oligonucleotide bearing a terminal carboxyl group to an amine-modified Si(001) surface (195). 

The syntheses, photophysical and electrochemical properties of [Ir(ppy)2(phen-NS-5)]PF6, (181), [Ir(ppy)2(phen-NHCOCH2I-5)]PF6, (182), and [Ir(ppy)2(phen-NH2-5)]PF6 are reported.342 Complexes (181) and (182) have been used to label amine- and sulfhydryl-modified oligonucleotides and human serum albumin to give luminescent bioconjugates. 

Hemiacetal hydroxyl groups of carbohydrate molecules also may be coupled to amine-containing molecules to form N-glycosidic linkages, such as those in nucleic acids and oligonucleotides. 

Chemical attachment of a detectable component to an oligonucleotide forms the basis for constructing a sensitive hybridization reagent. Unfortunately, the methods developed to crosslink or label other biological molecules such as proteins do not always apply to nucleic acids. The major reactive sites on proteins involve primary amines, sulfhydryls, carboxylates, or phenolates— groups that are relatively easy to derivatize. RNA and DNA contain none of these functionalities. 

Other non-protein molecules, such as nucleic acids and oligonucleotides, may not normally possess primary amines of sufficient nucleophilicity to react with common modification reagents. The ability to add amine functionalities to these molecules is sometimes the only route to successful conjugation. Creating amines at specific sites within these molecules allows for site-directed modification at known positions, thus better assuring active conjugates once formed. 

Molecules containing phosphate groups, such as the 5 phosphate of oligonucleotides, also may be conjugated to amine-containing molecules by using a carbodiimide-mediated reaction (Chapter 27, Section 2.1). The carbodiimide activates the phosphate to an intermediate phosphate ester similar to its reaction with carboxylates (Chapter 3, Section 1). In the presence of an amine, the ester reacts to form a stable phosphoramidate bond (Reaction 13). 

SIAB and sulfo-SIAB have been used to make a high-capacity RNA affinity column for the purification of human IRP1 and IRP2 (Allerson et al., 2003), to couple antibodies or Fab fragments to amine-modified microparticles (Harma et al., 2000), and in the attachment of oligonucleotides to surfaces for detection arrays (Adessi et al., 2000). 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

Many of the chemical derivatization methods employed in these strategies involve the use of an activation step that produces a reactive intermediary. The activated species then can be used to couple a molecule containing a nucleophile, such as a primary amine or a thiol group. The following sections describe the chemical modification methods suitable for derivatizing individual nucleic acids as well as oligonucleotide polymers. 

Figure 27.3 The reaction of cytosine with bisulfite in the presence of an excess of an amine nucleophile (such as a diamine compound) leads to transamination at the N-4 position. This process is a route to adding an amine functional group to cytosine residues in oligonucleotides.

Figure 27.5 Oligonucleotides containing a 5 -phosphate group can be reacted with EDC in the presence of imidazole to form an active phosphorimidazolide intermediate. This derivative is highly reactive with amine nucleophiles, forming a phosphoramidate linkage. Diamines reacted with the phosphorimidazolide result in amine-terminal spacers that can be modified with detectable components.

Figure 27.6 The 5 -phosphate group of oligonucleotides may be labeled with cystamine using the EDC/imid-azole reaction. This results in the formation of an amine-terminal spacer containing an internal disulfide group. Reduction of the disulfide provides a route to creating a free thiol for further derivatization.

Dissolve the amine-modified oligonucleotide to be thiolated in 250pi of 50mM sodium phosphate, pH 7.5. 

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