Synthesis of nucleotides with specific radiolabels in ribose. Primary 14C and secondary 3H kinetic isotope effects on acid-catalyzed glycosidic bond hydrolysis of AMP, dAMP, and inosine.

We present procedures for the enzymatic synthesis of adenine nucleotides of high purity and with specific labels of 3Hand "C in the ribose ring

D W Parkin

2021

Scholarcy highlights

  • Adenosine S'-phosphate was synthesized with 'H or 14Clabel located asAMP, AMP,AMP, andAMP
  • The results of secondary isotope effects indicate that the transition statesfor the acid-catalyzed hydrolysis of the N-glycosidic bonds of inosine, AMP, and dAMP have similar bonding to l'-'H in the transition state and have considerable carboxonium character
  • We present procedures for the enzymatic synthesis of adenine nucleotides of high purity and with specific labels of 3Hand "C in the ribose ring
  • Synthesis and Analysis of Labeled AMP-The enzymatic synthesis ofAMP,AMP,AMP, andAMPwas accomplished using the coupled enzymatic steps shown in Scheme 3
  • Labeled AMP samples were analyzed for the presence of ribose 5-Po4 or other material which would coelute with ribose &Po4 and thereby interfere with subsequent measurement of isotope effects
  • The product AMP was obtained in high purity without contaminants which would interfere with kinetic isotope effects measurements following hydrolysis of the N-glycosidic bond
  • The measured primary kinetic isotope effect for 12C/14Cat C-1’ of AMP of 1.049 f 0.009 is well within the range of 1.00 to 1.08 expected for a dissociative mechanism and establishes that bonding to C-1’ of AMP in the transition state is considerably looser than in the substrate or product

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