期刊
ACS CHEMICAL BIOLOGY
卷 3, 期 8, 页码 499-511出版社
AMER CHEMICAL SOC
DOI: 10.1021/cb800066p
关键词
-
资金
- National Institutes of Health [GM-74330]
A general method for isotopic labeling of the purine base moiety of nucleotides and RNA has been developed through biochemical pathway engineering in vitro. A synthetic scheme was designed and implemented utilizing recombinant enzymes from the pentose phosphate and de novo purine synthesis pathways, with regeneration of folate, aspartate, glutamine, ATP, and NADPH cofactors, in a single-pot reaction. Syntheses proceeded quickly and efficiently in comparison to chemical methods with isolated yields up to 66% for C-13-, N-15-enriched ATP and GTP. The scheme is robust and flexible, requiring only serine, NH4+ glucose, and CO, as stoichiometric precursors in labeled form. Using this approach, U-C-13- GTP, U-C-13,N-15- TP, C-13(2,8)- TP, and U-N-15- GTP were synthesized on a millimole scale, and the utility of the isotope labeling is illustrated in NMR spectra of HIV-2 transactivation region RNA containing C-13(2,8)-adenosine and N-15(1,3,7,9,2)-guanosine. Pathway engineering in vitro permits complex synthetic cascades to be effected, expanding the applicability of enzymatic synthesis.
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