Enzymatic synthesis of deoxyATP using DNA as starting material

The authors describe the synthesis of deoxyadenosine triphosphate (dATP) from DNA on a 200-mmol scale. Enzymatic digestion of DNA to a mixture of deoxynucleoside monophosphates was accomplished by a two-step process: initial conversion to a mixture of oligonucleotides using soluble DNase I, and subs...

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Bibliographic Details
Published in:Journal of organic chemistry 1985, Vol.50 (7), p.1076-1079
Main Authors: LADNER, W. E, WHITESIDES, G. M
Format: Article
Language:English
Subjects:
Bioconversions. Hemisynthesis
Biological and medical sciences
Biotechnology
Fundamental and applied biological sciences. Psychology
Methods. Procedures. Technologies
Online Access:Get full text
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Summary:The authors describe the synthesis of deoxyadenosine triphosphate (dATP) from DNA on a 200-mmol scale. Enzymatic digestion of DNA to a mixture of deoxynucleoside monophosphates was accomplished by a two-step process: initial conversion to a mixture of oligonucleotides using soluble DNase I, and subsequent hydrolysis of this mixture to mononucleoside monophosphates using nuclease P sub(1) immobilized in polyacrylamide gel. The overall conversion of the deoxynucleotide moieties in the original DNA to soluble deoxynucleoside monophosphates was 75-90%. Selective conversion of dAMP to dATP in the presence of a mixture of dAMP, dGMP, dCMP, and TMP was accomplished by enzymatic phosphorylation using PEP, a catalytic quantity of ATP, adenylate kinase, and pyruvate kinase. DeoxyATP was isolated from the reaction mixture as its barium salt in 67% yield and 60% purity. A subsequent simple purification yielded dATP with 95% purity, in 40% overall yield based on dAMP moieties present in the starting DNA.
ISSN:0022-3263
1520-6904
DOI:10.1021/jo00207a032