Enzymatic Synthesis of Guanine Nucleotides Labeled with 15N at the 2-Amino Group of the Purine Ring

✍ Scribed by A. Bouhss; H. Sakamoto; N. Palibroda; M. Chiriac; R. Sarfati; J.M. Smith; C.T. Craescu; O. Barzu

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 521 KB
• Volume: 225
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1995.1101

✦ Synopsis

GMP and dGMP labeled with ({ }^{15} \mathrm{~N}) at the 2 -amino group of the purine ring was obtained enzymatically from (\mathrm{NH}_{4} \mathrm{Cl}\left(>99\right.) at. (%{ }^{15} \mathrm{~N}) ) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at. (%{ }^{15} \mathrm{~N}). The proton NMR spectrum of ({ }^{15} \mathrm{~N}) ]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 (\mathrm{Hz}). When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at (6.47 \mathrm{ppm}), indicating that the corresponding proton is bound to ({ }^{15} \mathrm{~N}). 1995 Academic Press, Inc.