Solid phase chemical technologies for combinatorial chemistry

Balasubramanian, Shankar

doi:10.1002/jcb.10063

Cited by 3 publications

(2 citation statements)

References 13 publications

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“…The diversity of compound libraries is one of the keys to increasing the number of hits in HTS. A variety of methods for synthesizing numerous compounds have been developed [38]–[40]; for example, methods using phage libraries have been used for HTS [41,42]. However, there has been little work on the modification of small molecules or the production of new compounds by the direct use of enzymes.…”

Section: Resultsmentioning

confidence: 99%

Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli

Uno

Okamoto

Masuda

et al. 2008

Biotech and App Biochem

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P450 (cytochrome P450) enzymes catalyse the mono-oxygenation of a wide range of compounds such as steroids, fatty acids, vitamins and drugs. In the present paper we demonstrate a system for bioconverting diverse compounds [flavanone, DHEA (dehydroepiandrosterone) and 7-ethoxycoumarin] using P450 species expressed in Escherichia coli. First, we expressed four P450 species: rabbit CYP2B (P450 family 2, subfamily B), fruitfly (Drosophila) CYP317A, rat CYP3A23 and mouse CYP2J5. Next, we added substrates directly to the incubation medium. The resulting metabolites were extracted and analysed by HPLC and spectrofluorimetry. The first substrate, 7-ethoxycoumarin, was de-ethylated by CYP2B; CYP2J5 and CYP3A23 showed weak activity, and CYP317A had no activity for 7-ethoxycoumarin. We next used flavanone, a flavonoid, as a substrate for these four P450 species and other P450 species expressed previously. As a result, CYP2B, CYP2C43 and CYP2C29 catalysed flavanone 2-hydroxylation. CYP2A5 catalysed 2- and 4-hydroxylations. Finally, to produce diverse modified compounds, variants of CYP2A5 with point mutations were incubated with a steroid (DHEA) and an antioxidant (flavanone) in vivo. HPLC analysis indicated that two P450 species produced a 7-beta-hydroxy-DHEA and two P450 species produced a 2-alpha-hydroxy-DHEA. Four P450 species catalysed flavanone 2- and 4-hydroxylations. These results indicate that bioconversion by P450 is a useful technique to modify small molecules (steroids, coumarin and flavanone) and produce new, diverse hydroxylated compounds, which could be used for high-throughput screening for drug discovery.

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Section: Resultsmentioning

confidence: 99%

Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli

Uno

Okamoto

Masuda

et al. 2008

Biotech and App Biochem

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“…The diversity of compound libraries is one of the keys to increasing the number of hits in HTS. A variety of methods of synthesizing numerous compounds have been developed [2,4,18]. For example, methods using phage libraries have been used for HTS [6,22].…”

Section: Introductionmentioning

confidence: 99%

Modification of small molecules by using cytochrome P450 expressed in Escherichia coli

Uno

Nakao

Masuda

et al. 2006

J Ind Microbiol Biotechnol

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We developed a system for bioconverting diverse compounds using P450s produced in Escherichia coli. Vectors for the expressing various P450 cDNAs quickly and easily in E. coli were developed by using several restriction enzyme sites. Three types of P450 (2C2, 2C29, and 2D22) were produced using these plasmids. Substrates were directly added to the incubation medium and metabolized. To obtain pure product from the medium, we first tried production of P450 in synthetic medium. The amount of another P450 2C43 produced in the synthetic medium was similar to the amount produced in Luria broth (LB) medium. Next, estradiol, a steroid, was added as a substrate, incubated, and the metabolite was extracted and analyzed by high-performance liquid chromatography. The metabolite extracted from synthetic medium was purer than that obtained from LB medium. Three P450s (2C29, 2C2, and 2A4) metabolized testosterone at different positions. P450 2C29 metabolized 7-ethoxycoumarin, androstendione, and dehydroepiandrosterone in this medium. P450s produced in the synthetic medium may be useful for producing various modified compounds for high-throughput screening.

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ChemInform Abstract: Solid‐Phase Chemical Technologies for Combinatorial Chemistry

Balasubramanian

2002

ChemInform

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Solid phase chemical technologies for combinatorial chemistry

Cited by 3 publications

References 13 publications

Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli

Bioconversion of small molecules by cytochrome P450 species expressed in Escherichia coli

Modification of small molecules by using cytochrome P450 expressed in Escherichia coli

ChemInform Abstract: Solid‐Phase Chemical Technologies for Combinatorial Chemistry

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