Phosphate esters phosphation reagents

Modify a purified bait protein by adding an aliquot of crosslinker to achieve a molar excess of 2-10 moles per mole of protein (protect from light). For NHS ester-containing reagents, react in 0.1 M sodium phosphate buffer at pH 7.2-7A. Avoid using any other amine-containing buffer components, such as Tris or imidazole, which will interfere with the reaction. 

Keefe AD, Miller SL. Are polyphosphates or phosphate esters prebiotic reagents J. Mol. Evol. 1995 41 693-702. 

The principal phosphation reagents used to manufacture phosphate esters are phosphoric anhydride (P4O10), phosphorus oxychloride [P(0)Cl3], and polyphosphoric acid. It is important to understand the physical and chemical characteristics of each raw material. The structure of the phosphation agent affects its reactivity and selectivity, resulting in variation in the monoester, diester, and triester product distribution. 

Certain applications require products possessing a higher DAP content than attainable from the sesquiphosphate mixtures derived from phosphoric anhydride. Multiple reaction sites and a lack of selectivity characteristic of the phosphation reagents described earlier make than unsuitable for the preparation of high dialkyl content esters. 

Some esters of inorganic acids such as dimethyl sulfate are used as reagents m syn thetic organic chemistry Certain naturally occurring alkyl phosphates play an important role m biological processes... 

The formation of g-alkyl-a,g-unsaturated esters by reaction of lithium dialkylcuprates or Grignard reagents in the presence of copper(I) iodide, with g-phenylthio-, > g-acetoxy-g-chloro-, and g-phosphoryloxy-a,g-unsaturated esters has been reported. The principal advantage of the enol phosphate method is the ease and efficiency with which these compounds may be prepared from g-keto esters. A wide variety of cyclic and acyclic g-alkyl-a,g-unsaturated esters has been synthesized from the corresponding g-keto esters. However, the method is limited to primary dialkylcuprates. Acyclic g-keto esters afford (Zl-enol phosphates which undergo stereoselective substitution with lithium dialkylcuprates with predominant retention of stereochemistry (usually > 85-98i )). It is essential that the cuprate coupling reaction of the acyclic enol phosphates be carried out at lower temperatures (-47 to -9a°C) to achieve high stereoselectivity. When combined with they-... 

Fluormated organosilanes are used as reagents for the construction of carbon-carbon bonds and for the selective synthesis of phosphate esters... 

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... 

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. 

Dissolve an antibody or protein to be modified at a concentration of l-10mg/ml in 0.1 M sodium phosphate, 0.15M NaCl, pH 1.2-1.5. Lower concentrations of protein may result in decreased reaction yields and require increased quantities of reagent to obtain acceptable levels of biotinylation. Avoid amine-containing buffers or components, such as Tris or imidazole, which will react with the NHS ester and interfere with the biotinylation process. 

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