Phosphoramidate linkage

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]. 

Figure 1.72 Cystamine may be used to label phosphate groups, such as at the 5 -end of oligonucleotides, via a carbodiimide reaction using EDC. The resultant phosphoramidate linkage is a common way to modify oligonucleotides at the 5 -end.

Alkylphosphate Group Adipic Acid Dihydrazide Phosphoramidate Linkage with... 

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.

Phosphoramidate Linkage with Terminal Hydrazide Group... 

Carbodiimides are used to mediate the formation of amide or phosphoramidate linkages between a carboxylate and an amine or a phosphate and an amine, respectively (Hoare and Koshland, 1966 Chu etal., 1986 Ghosh etal., 1990). Regardless of the type of carbodiimide, the reaction proceeds by the formation of an intermediate o-acylisourea that is highly reactive and short-lived in aqueous environments. The II attack of an amine nucleophile on the carbonyl group of this ester results in the loss an... 

Chu et al. (1983, 1986) and Ghosh et al. (1990) describe modified carbodiimide protocols using the water-soluble reagent EDC instead of DCC. They also incorporate a second reactive intermediate, a phosphorimidazolide, created from the reaction of the phosphomonoester at the 5 -terminus of DNA with EDC in the presence of imidazole. A reactive phosphorimidazolide will rapidly couple to amine-containing molecules to form a phosphoramidate linkage (Fig. 398). The chemical reaction had been used previously to effect the formation of phosphodiester linkages between short DNA strands (Shabarova et al., 1983). 

Sekine has reported the synthesis and anticancer activity of Phosmidosine (18) and its demethylated parent (19). The Phosmidosines were obtained by reaction of an appropriately protected 8-oxoadenosine 5 -0-phosphoramidite and N-protected prolinamide in the presence of 5-(3,5-dinitrophenyl)-l//-tetrazole, followed by in situ oxidation with t-BuOOH to form the iV-acyl phosphoramidate linkage. These syntheses required extensive work with regard to the choice of protecting groups on the adenine moiety, as this was crucial for successful P-N bond formation. ... 

BNA (also known as LNA) has been used in TFOs in a pyrimidine motif at neutral pH. The binding constant of the BNA TFO was about 20 times larger than that of DNA as a result of a large decrease in the dissociation rate constant. A 3 -amino-2, 4 -BNA nucleotide has been prepared to introduce N3 -P5 phosphoramidate linkages. It is introduced as a dinucleotide unit (61), and in thermal stability studies was shown to exhibit superior duplex and triplex stability compared to either BNA or DNA, and shows enhanced resistance to digestion by SVPDE. These properties have previously been observed for N3 -P5 phosphoramidate linkages in DNA. ... 

The conformationally-locked 3 -amino thymidine derivative (47) was prepared and incorporated into DNA as a dimer unit, attaching the amino group via a carbamate or phosphoramidate linkage.Both modifications were found to be destabilising towards both RNA and DNA targets. The hybridisation properties of an amino-modified bicyclic thymine nucleotide (48) have been exam-ined. Using alternate T and (48) residues there is an increase in duplex stability with d(A)io, with no pH dependence. When the amino group is acetylated, the duplex stability is decreased. The locked C-nucleoside oxazole derivative (49) has been incorporated into TFOs where it was found to selectively enhance the stability with a C.G base pair. ... 

The phosphoramidate linkage in its aliphatic variant has been exploited by Gryaznov and Letsinger [252] for solid phase synthesis of oligonucleotide 3 -phosphates (Figure 19.9). After phosphate 2-cyanoethyl group removal by prior ammonolysis the phosphoramidate required milder conditions to break down (80% acetic acid, 4h at ambient temperature) that are tolerable for the purine... 

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