• Archives of Pharmacal Research
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• v.21 no.4
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• pp.458-464
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• 1998

Search for a new $\alpha$-methylene-$\gamma$-butyrolactone-bearing 6-substituted purine as a potental antitumor agent has led to synthesize seven, hitherto unreported, $5^1$-Methyl-$5^1$-[(6-substituted-9H-purin-9-yl)methyl]-$2^1$-oxo-$3^1$- methylenetetrahydrofurans (H, Cl, l, $CH_3$, $NH_2$, SH, >C=O) (6a-g). These include $5^1$-Methyl-$5^1$-[(9H-purin-9-yl)methyll-$2^1$-oxo-$3^1$ -methylenetetrahydrofurans (6a), $5^1$-Methyl-$5^1$-[(6-chloro-9H-purin-9-yl)methyl]-$2^1$-oxo-$3^1$-methylenetetrahydr ofurans (6b), $5^1$-Methyl-$5^1$-[(6-chloro-9H-purin-9-yl) methyl]-$2^1$-oxo-$3^1$-methylenetetrahydrofurans (6c), $5^1$-Methyl-$5^1$-[(6-methyl-9H-purin-9-yl) methyl]-$2^1$-oxo-$3^1$-methylenetetrahydrofurans (6d), $5^1$-Methyl-$5^1$-[(9H-adenin-9-yl)methyll-$2^1$-oxo-$3^1$-methylenetetrahydrofurans (6e), $5^1$-Methyl-$5^1$-[(6-mercapto-9H-purin-9-yl) methyl]-$2^1$-oxo-$3^1$-methylenetetrahydrofurans (6f) and $5^1$-Methyl-$5^1$-[(9H-hypoxanthin-9-yl)methyll-$2^1$-oxo-$3^1$-methylenetetrahydrof urans (6g) which were made by the Reformatsky-type reaction of ethyl $\alpha$-(bromomethyl) acrylate with the corresponding (6-substituted-9H-purin-9-yl)-2-propanone intermediates (5a-g). These ketone intermediates 5a-g, 1-(9H-purin-9-yl)-2-propanone (5a), 1-(6-chloro-9H-purin-9-yl)-2-propanone (5b), 1-(6-iodo-9H-purin-9-yi)-2-propanone (5c), 1-(6-methyl-9H-purin-9-yl)-2-propanone (5d), 1-(9H-adenin-9-yl)-2-propanone (Se), 1-(6-mercapto-9H-purin-9-yl)-2-propanone (5f), and 1-(9H-hypoxanthin-9-yl)-2-propanone (5g) were directly obtained by the alkylation of the 6-substituted purine bases with the chloroacetone in the presence of $K_2$$CO_3$ (or NaH) under DMF (or DMSO). The preliminary in vitro cytotoxcity assay for the synthetic .alpha.-methylene-y-butyro-lactone compounds (6a-g) were determined against three cell lines (PM-3A, P-388, and K-562) and showed the moderate antitumor activity ($IC_50$ ranged from 1.4 to 4.3 $\mu\textrm{g}$/ml) with the compound $5^1$-methyl-$5^1$ -[(9H-hypoxanthin-9-yl)methyl]-$2^1$-oxo-$3^1$-methylenetetrahydrofuran (6g) showing the least antitumor activity.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.117-123
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• 2003

For screening of autoregulator receptor gene from Saccharopolyspora erythraea, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHI site of pUC19 and transformed into the E. coli DH5$\alpha$. The isolated plasmid from transformant contained the fragment of 120 bp, which was detected on 2% gel after BamHI treatment. The insert, 120 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridization using Saccha. erythraea chromosomal DNA were performed with the insert as probe. The plasmid (pEsg) having 3.2 kbp SacI DNA fragment from Saccha. erythraea is obtained. The 3.2 kbp SacI DNA fragment was sequenced by the dye terminator sequencing. The nucleotide sequence data was analyzed with GENETYX-WIN (ver 3.2) computer program and DNA database. frame analyses of the nucleotide sequence revealed a gene encoding autoregulator receptor protein which is a region including KpnI and SalI sites on 3.2 kbp SacI DNA fragment. The autoregulator receptor protein consisting of 205 amino acid was named EsgR by author. In comparison with known autoregulator receptor proteins, homology of EsgR showed above 30%.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.96-105
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• 2005

For screening of autoregulator receptor gene from Streptomyces longwoodensis, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHIsite of pUC19 and transformed into the E. coli $DH5{\alpha}$. The isolated plasmid from transformant contained the fragment of 100 bp, which was detected on $2\%$ gel after BamHI treatment. The insert, 100 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridizations with the 100 bp fragment as a probe allowed to select a genomic clone of S. longwoodensis, pSLT harboring a 4.4 kb SphI fragment. Nucleotide sequencing analyses revealed a 651 bp open reading frame(ORF) were isolated protein showing moderate homology ($35{\sim}46\%$) with the ${\Gamma}$-butyrolactone autoregulator receptors from Streptomyces sp., and this ORF was named sltR The sltR/pET-17b plasmid was constructed to overexpress the recombinant SltR protein (rSltR) in E. coli BL21 (DE3)/pLysS, and the rSltR protein was purified to homogeneity by DEAE-Sephacel column chromatography, and DEAE-5PW chromatography (HPLC). The molecular mass of the purified rSltR protein was 55 kDa by HPLC gel-filtration chromatography and 28 kDa by SDS-PAGE, indicating that the rSltR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that the rSltR has clear binding activity with a A-factor type autoregulator as the most effective ligand.