Shinya Hanashima

Synthesis of sulfoquinovosylacylglycerols, inhibitors of Eukaryotic DNA Polymerase α and β

Abstract Sulfoquinovosyldiacylglycerols (SQDGs) and sulfoquinovosylmonoacylglycerols (SQMGs), bearing diverse fatty acids, were synthesized from d -glucose, and were examined for enzymatic inhibitions of DNA polymerase α and β. These results indicated that the carbon numbers of the fatty acids were highly related to the activities, at least in vitro, of eukaryotic DNA polymerase inhibition.

Structure‐Activity Relationship of a Novel Group of Mammalian DNA Polymerase Inhibitors, Synthetic Sulfoquinovosylacylglycerols

We reported previously that sulfolipids in the sulfoquinovosylacylglycerol class from a fern and an alga are potent inhibitors of DNA polymerase α and β and potent anti‐neoplastic agents. In developing a procedure for chemical synthesis of sulfolipids, we synthesized many derivatives and stereoisomers of sulfoquinovosylmonoacylglycerol (SQMG)/sulfoquinovosyldiacylglycerol (SQDG). Some of these molecules were stronger inhibitors than the SQMG/SQDG originally reported as natural compounds. In this study, we examined the structure‐inhibitory function relationship of synthetic SQMG/SQDG and its relationship to cytotoxic activity. The inhibitory effect is probably mainly dependent on the fatty acid effect, which we reported previously, although each of the SQMG/SQDG was a much stronger inhibitor than the fatty acid alone that was present in the SQMG/SQDG. The inhibitory effect could be influenced by the chain size of fatty acids in the SQMG/SQDG. The sulfate moiety in the quinovose was also important for the inhibition. Lineweaver‐Burk plots of SQMG/SQDG indicated that DNA polymerase α was non‐competitively inhibited, but the SQMG/SQDG were effective as antagonists of both template‐primer DNA‐binding and nucleotide substrate‐binding of DNA polymerase β. The SQMG had an cytotoxic effect, but the SQDG tested did not. The SQDG might not be able to penetrate into cells. Based on these results, we discuss the molecular action of SQMG/SQDG and propose drug design strategies for developing new anti‐neoplastic agents.

Structural determination of sulfoquinovosyldiacylglycerol by chiral syntheses

Abstract Chiral sulfoquinovosyldiacylglycerols (SQDGs) have been synthesized to determine the absolute stereochemistry and the biological activities. The 1 H NMR spectrum of a natural SQDG is comparable to that of synthetic (2 S )-SQDG rather than that of the (2 R ) analogue. The biological activity of the respective isomers for DNA polymerase α and β inhibition was not distinguishable in the enzymatic assay.

Studies on a novel DNA polymerase inhibitor group, synthetic sulfoquinovosylacylglycerols: inhibitory action on cell proliferation

Some chemically synthesized sulfoquinovosylmonoacylglycerols (SQMG)-sulfoquinovosyldiacylglycerols (SQDG) have been reported to selectively and strongly inhibit the activities of mammalian DNA polymerases alpha and beta in vitro. In this study, using human cancer cell lines, we investigated the effects of SQMG-SQDG on the DNA polymerase in the cells. In the presence of n-decane, the IC(50) values on cell growth were approximately 1-5 microM for SQMG and about 0.3-1 microM for SQDG. The values were almost the same as the in vitro enzyme inhibitory levels. The cell lines were arrested in early S-phase by SQMG-SQDG at the concentrations of 0.1-4.7 microM in a manner dependent on incubation time, suggesting that SQMG-SQDG blocked the primary step of DNA replication by inhibiting DNA polymerase, possibly alpha-type. We also demonstrated the localization of SQMG in the cell using the fluorescent SQMG analog, SQMGalpha-NBDD, which was synthesized in our laboratory. SQMGalpha-NBDD was localized in the nucleus and on the nuclear surface, but the binding site seemed not to be the DNA/chromatin, suggesting that the SQMG-SQDG might interact with molecules located close to the DNA/chromatin and on the nuclear surface. These results suggested a correlation between the in vitro biochemical action of the SQMG-SQDGs and their intracellular mode of action.

Anti‐tumor Effect of Chemically Synthesized Sulfolipids Based on Sea Urchin's Natural Sulfonoquinovosylmonoacylglycerols

We recently reported that 3′‐sulfonoquinovosyl‐1′‐monoacylglycerol (designatedA‐5) extracted from sea urchin intestine was effective in suppressing the growth of solid tumors. Although the major fatty acid component of A‐5 was a saturated C16 acid, there were five other fatty acids, 14:0, 18:0, 14:1, 16:1, and 18:1, which constitute minor components of A‐5. Therefore, it remains unclear as to which of these six fatty acid components of A‐5 has the anti‐tumor effect. In this study, we synthesized sulfolipids each containing only one of these six fatty acids and tested their cytotoxicity against tumor cells and in vivo anti‐tumor effects on nude‐mice bearing solid tumors of human lung adenocarcinoma cell line A‐549. The IC50 values of all products against tumor cells were more than 10‐5M, suggesting weak cytotoxic activity compared with other chemotherapeutic compounds for cancer. On the other hand, in vivo anti‐tumor assay showed that sulfoquinovosyl‐monoacylglycerols (SQMG) composed of 14:1 and 18:1 (designated SQMG(14:1) and SQMG(18:1), respectively) were significantly effective in suppressing the growth of solid tumors. Our data suggested that these two SQMGs had a substantial anti‐tumor effect in vivo, and they are of interest as candidate drugs for anti‐cancer treatment.

An immunosuppressive effect by synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin.

Background. It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. Methods. We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-&bgr;-D-glucopyranosyl)-1,2-di-O-acylglycerol (&bgr;-SQDG) that contains a saturated C18 fatty acid, which is designated as &bgr;-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of &bgr;-SQDG(18:0). Results. &bgr;-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. &bgr;-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, &bgr;-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by &bgr;-SQDG(18:0) treatment. Conclusions. &bgr;-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by &bgr;-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(α-d-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.

Biotinylated lithocholic acids for affinity chromatography of mammalian DNA polymerases α and β

Biotinylated lithocholic acids have been synthesized. The compounds inhibited mammalian DNA polymerases α and β with dose-dependent manner. The streptavidine columns conjugated with the synthetic biotinylated compounds chromatographed both two enzymes eluted by KCl solution at the different concentrations.