Synthesis of [guanidino‐13C]creatine and measurement of the creatine phosphokinase reaction in vitro by 13C NMR spectroscopy

[guanidino-13C]Creatine has been synthesized by reaction involving synthesis of [13C]cyanamide followed by its condensation with sarcosine. The creatine guanidino carbon 13C NMR frequency shifts upon the formation of phosphocreatine by 0.83 ppm with a splitting (2JCP = 3.3 Hz) due to two-bond phosphorus-carbon coupling. The high 13C enrichment (99%) at the guanidino carbon of creatine gave rise to an adequate signal-to-noise ratio for both creatine and phosphocreatine so that a study of the time course of the creatine phosphokinase reaction could be performed and provided the means for a determination of the Km (15.5 mM) of the reaction.