Phosphates reaction with carbodiimides

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). 

Conjugation via Carbodiimide Reaction with the 5 Phosphate of DNA (Phosphoramidate Formation)... 

Via Carbodiimide Reaction with 5 Phosphates of DNA (Phosphoran)idate Formation)... 

Compounds possessing the cyclic phosphate structure may be prepared by the action of phosphorus oxychloride or phenyl phosphorodichloridate on a glycol, or by treating phosphate monoesters with trifluoroacetic anhydride (173) or a carbodiimide reagent (f74). The latter is the preferred reagent since it may be used in the presence of water and usually gives a good yield of a pure product. The reaction is formulated as follows ... 

The carbodiimide of choice used to couple cystamine to carboxylate- or phosphate-containing molecules is most often the water-soluble carbodiimide, EDC hydrochloride Chapter 3, Section 1.1). This reagent rapidly reacts with carboxylates or phosphates to form an active ester intermediate, which is highly reactive toward primary amines. The reaction is efficient from pH 4.7 to 7.5, and a variety of buffers may be used, providing they don t contain competing groups. 

DNA or RNA also may be modified with cystamine at the 5 -phosphate group using a carbodiimide reaction. See Chapter 27, Section 2.2 for a complete discussion of the labeling protocol. 

Some procedures recommend the use of water as the solvent in an EDC reaction, while the pH is maintained constant by the addition of HC1. Buffered solutions are more convenient, because the pH does not have to be monitored during the course of the reaction. For acidic pH conjugations, MES [2-(N-morpholino)ethane sulfonic acid] buffer at 0.1 M works well. When doing neutral pH reactions, a phosphate buffer at 0.1 M is appropriate. Any buffers may be used that do not interfere with the reaction, but avoid amine- or carboxylate-containing buffer salts or other components in the medium that may react with the carbodiimide. 

Carbodiimide formation is markedly accelerated when phosphine oxides (R PO) or phosphates are used as catalysts. Intermediates with P—NR bonds have been postulated as intermediates in these reactions (59,60). 

Phosphate-containing carbohydrates that are stable, such as the 5 -phosphate of the ribose derivatives of oligonucleotides, may be targeted for modification using a carbodiimide-facilitated reaction (Section 4.3). The water-soluble carbodiimide EDC can react with the phosphate groups to form highly reactive phospho-ester intermediates. These intermediates can react with amine- or hydrazide-containing molecules to form stable phosphoramidate bonds. 

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