Preparative solvent partition chromatography of butyrylated cyclic AMP analogues
✍ Scribed by Francis A. Neelon; Bruce M. Birch; Harold E. Lebovitz
- Publisher
- Elsevier Science
- Year
- 1973
- Tongue
- English
- Weight
- 527 KB
- Volume
- 56
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A simple and effective separation of cyclic adenosine-3',5'-monophosphate (CAMP) and its butyryl-substituted analogues using partition chromatography on columns of Sephadex gel in isopropanol/0.5M ammonium acetate (4:l) is described. The technique is suitable for preparative separations as demonstrated by revised uv spectral data obtained on butyrylated CAMP'S purified by this technique. In addition, it has analytical utility in that it allows complete separation of NE-monobutyryl CAMP from O"-monobutyryl CAMP, thereby permitting simultaneous and independent assessment of the rate of acyl substituent hydrolysis from the disubstituted derivative (NB.O"dibutyryl CAMP), and this is demonstrated under several conditions. Acylated derivatives of cyclic adenosine-3',5'-monophosphate (CAMP) are known to be more active in vivo than the parent compound (1,2) and have been widely used to mimic CAMP effects, since their synthesis was first reported by Posternak, Sutherland, and Henion (3). Despite such widespread use, however, significant questions remain regarding the chemistry and the mode of action of the acylated derivates, especially the N6,02'-dibutyryl derivative (Bu,cAMP) (4-6). In part, these questions derive from the fact that commercial Bu,cAMP is contaminated with a number of secondary products (7) ; from the fact tha,t Bu,cAMP is unstable in aqueous solut.ion ( 8)) and that use of paper chromatography for purification of Bu,cAMP requires elution and concentration procedures during which the Bu,cAMP decomposes (8), and from the fact that the N"-monobutyryl CAMP (Ng-BueAMP) and 02'-monobutyryl CAMP (02-BucAMP) derivatives do not separate on paper chromatography (3).
In this report we describe a column chromatographic procedure for analytic and preparative separation of Bu,cAMP, N6-BucAMP, O"-BucAMP, and CAMP, as well as other minor components of commercial Bu,cAMP. This technique has permitted purification of the various