“…Thus, we investigated the use of an "explorative solid-phase extraction" (E-SPE) protocol using SAX, Oasis MAX, SCX, and LH-20 columns for targeted exploitation of chemical functionalities. E-SPE provides a minimum of fractions (15) for chemical and biological analyses and implicates development into a preparative scale methodology. Overall, this allows fast extract prioritization, easier dereplication, mapping of biological activities, and formulation of a purification strategy.…”
mentioning
confidence: 99%
“…This is especially a problem with low yielding extracts typical of those arising from marine microorganisms . For this type of extract, orthogonal purification strategies are required; classically, this can be achieved by combining RP with normal-phase chromatography on silica gel (low cost) or alternatively using bonded phases such as polyethyleneimine , or diol. , Ion-exchange has been used less frequently − even though many natural products contain ionizable groups.…”
Microbial natural products (NP) cover a high chemical diversity, and in consequence extracts from microorganisms are often complex to analyze and purify. A distribution analysis of calculated pK(a) values from the 34390 records in Antibase2008 revealed that within pH 2-11, 44% of all included compounds had an acidic functionality, 17% a basic functionality, and 9% both. This showed a great potential for using ion-exchange chromatography as an integral part of the separation procedure, orthogonal to the classic reversed-phase strategy. Thus, we investigated the use of an "explorative solid-phase extraction" (E-SPE) protocol using SAX, Oasis MAX, SCX, and LH-20 columns for targeted exploitation of chemical functionalities. E-SPE provides a minimum of fractions (15) for chemical and biological analyses and implicates development into a preparative scale methodology. Overall, this allows fast extract prioritization, easier dereplication, mapping of biological activities, and formulation of a purification strategy.
“…Thus, we investigated the use of an "explorative solid-phase extraction" (E-SPE) protocol using SAX, Oasis MAX, SCX, and LH-20 columns for targeted exploitation of chemical functionalities. E-SPE provides a minimum of fractions (15) for chemical and biological analyses and implicates development into a preparative scale methodology. Overall, this allows fast extract prioritization, easier dereplication, mapping of biological activities, and formulation of a purification strategy.…”
mentioning
confidence: 99%
“…This is especially a problem with low yielding extracts typical of those arising from marine microorganisms . For this type of extract, orthogonal purification strategies are required; classically, this can be achieved by combining RP with normal-phase chromatography on silica gel (low cost) or alternatively using bonded phases such as polyethyleneimine , or diol. , Ion-exchange has been used less frequently − even though many natural products contain ionizable groups.…”
Microbial natural products (NP) cover a high chemical diversity, and in consequence extracts from microorganisms are often complex to analyze and purify. A distribution analysis of calculated pK(a) values from the 34390 records in Antibase2008 revealed that within pH 2-11, 44% of all included compounds had an acidic functionality, 17% a basic functionality, and 9% both. This showed a great potential for using ion-exchange chromatography as an integral part of the separation procedure, orthogonal to the classic reversed-phase strategy. Thus, we investigated the use of an "explorative solid-phase extraction" (E-SPE) protocol using SAX, Oasis MAX, SCX, and LH-20 columns for targeted exploitation of chemical functionalities. E-SPE provides a minimum of fractions (15) for chemical and biological analyses and implicates development into a preparative scale methodology. Overall, this allows fast extract prioritization, easier dereplication, mapping of biological activities, and formulation of a purification strategy.
“…Furthermore, treatment of b with NaBD 4 increased the MS signal of b ′ to m / z 229.1050. Based on these results, b was assigned as pentopyranone (PPN, 19 ), a metabolite which has been previously isolated from the BLS-producing strain . Therefore, b ′ could be assigned as 20 (Figure D).…”
The
biosynthesis of blasticidin S has drawn attention due to the
participation of the radical S-adenosyl methionine
(SAM) enzyme BlsE. The original assignment of BlsE as a radical-mediated,
redox-neutral decarboxylase is unusual because this reaction appears
to serve no biosynthetic purpose and would need to be reversed by
a subsequent carboxylation step. Furthermore, with the exception of
BlsE, all other radical SAM decarboxylases reported to date are oxidative
in nature. Careful analysis of the BlsE reaction, however, demonstrates
that BlsE is not a decarboxylase but instead a lyase that catalyzes
the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4′-keto-3′-deoxy-d-glucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated
analogues, as well as computational models based on X-ray crystal
structures of the BlsE·SAM (2.09 Å) and BlsE·SAM·CGA
(2.62 Å) complexes suggests that BlsE catalysis likely proceeds
via direct elimination of water from the CGA C4′ α-hydroxyalkyl
radical as opposed to 1,2-migration of the C3′-hydroxyl prior
to dehydration. Biosynthetic and mechanistic implications of the revised
assignment of BlsE are discussed.
“…As part of our work to elucidate the intermediates between CGA ( 3 ) and cytosinine ( 4 ), and to explore the relationships between the various cytosinyl metabolites, the in vivo utilization of known inhibitors of enzymes expected to function in the biosynthesis of 1 was investigated. This proved to be an effective approach to study the biosynthesis of 1 , resulting in accumulated intermediates and the enhanced production of other cytosinyl metabolites. , Here, we report additional in vivo inhibitor studies leading to the production of a new cytosine nucleoside, 7 . …”
mentioning
confidence: 88%
“…This proved to be an effective approach to study the biosynthesis of 1, resulting in accumulated intermediates and the enhanced production of other cytosinyl metabolites. 19,20 Here, we report additional in vivo inhibitor studies leading to the production of a new cytosine nucleoside, 7.…”
The identification of new cytosine glycosides and intermediates in the biosynthetic pathway of the antifungal antibiotic blasticidin S (1) was investigated using in vivo enzyme inhibition. Fermentations of Streptomyces griseochromogenes, the organism that produces 1, supplemented with the arginine analogue argininic acid or the argininosuccinate synthase inhibitor 2-methylaspartic acid were found to produce a new metabolite (7).
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