Synthesis and characterization of phosphocitric acid, a potent inhibitor of hydroxylapatite crystal growth.
Human urine and extracts of rat liver mitochondria contain apparently identical agents capable of inhibiting the precipitation or crystallization of calcium phosphate. Its general properties, as well as 1H NMR and mass spectra, have suggested that the agent is phosphocitric acid. This paper reports the synthesis of phosphocitric acid via the phosphorylation of triethyl citrate with o-phenylene phosphochloridate, hydrogenolysis of the product to yield triethyl phosphocitrate, hydrolytic removal of the blocking ethyl groups  and also chromatographic purification. An enzymatic assay of phosphocitrate is described. Synthetic phosphocitrate was found to be an exceedingly potent inhibitor of the growth of hydroxylapatite seed crystals in a medium supersaturated with respect to Ca2+ and phosphate. Comparative assays showed phosphocitrate to be much more potent than the most active precipitation-crystallization inhibitors previously reported, which include pyrophosphate and ATP. 14C-Labeled phosphocitrate was bound very tightly to hydroxylapatite crystals. Such binding appeared to be essential for its inhibitory activity on crystal growth. Citrate added before  but not after, phosphocitrate greatly enhanced the inhibitory potency of the latter. This enhancement effect was not given by other tricarboxylic acids. The monoethyl ester of phosphocitrate had no inhibitory effect on hydroxylapatite crystal growth.
D002130:Calcium Phosphates; D002625:Chemistry, Organic; D002951:Citrates / Q000138:chemical synthesis*; D003460:Crystallization; D006882:Hydroxyapatites; D007700:Kinetics; D013058:Mass Spectrometry; D055601:Organic Chemistry Phenomena; D009943:Organophosphorus Compounds / Q000138:chemical synthesis
D002130:Calcium Phosphates; D002951:Citrates; D006882:Hydroxyapatites; D009943:Organophosphorus Compounds; C014148:phosphocitrate