Presentation Information
[P01-027]Development of a cell-specific probe using DNJ-Conjugated Polyrotaxanes
Atsushi Uchida,Akihiko Hatano
Graduate School of Applied Chemistry, Shibaura Institute of Technology
○Atsushi Uchida1 (1. Shibaura Institute of Technology (Japan))
Keywords:
alpha-Glucosidaze,rotaxane,DNJ,beta-cyclodextrin
In recent years, research has been conducted to develop more efficient inhibitors by clustering inhibitor molecules onto a core structure. In particular, multivalent 1-deoxynojirimycin <DNJ> inhibitors—created by incorporating multiple DNJ units <an inhibitor of alpha-glucosidase, a membrane enzyme found in small intestinal epithelial cells> onto a core molecule—have been reported to exhibit significantly enhanced affinity for α-glucosidase due to increased local concentrations of DNJ.
However, a comparison of molecular sizes reveals that these multivalent DNJ inhibitors <45 Å> are only about half the size of α-glucosidase <92 Å>. Consequently, it has been found that previously reported multivalent DNJ inhibitors struggle to inhibit multiple α-glucosidase molecules simultaneously. Furthermore, when α-glucosidase and a multivalent DNJ inhibitor bind in a 1:1 ratio, the remaining DNJ molecules attached to the core do not contribute effectively to inhibitory activity.
In this study, we designed a mobile inhibitor by incorporating DNJ units onto a polyrotaxane core. Due to the unique characteristics of polyrotaxanes, where the axle and the cyclic molecules are not covalently bonded, the system was expected to autonomously search for and inhibit α-glucosidase an enzyme located on the fluid membrane of small intestinal epithelial cells, which constantly changes position due to membrane fluidity. Furthermore, this design was anticipated to enable the simultaneous inhibition of multiple membrane enzymes.
The design of the DNJ-clustered polyrotaxane was implemented using poly<propylene glycol> <PPG, Mw = 2000> as the axle and β-cyclodextrin <β-CD> covalently functionalized with DNJ, used as the cyclic components. The DNJ-conjugated β-CD was synthesized via organic synthesis and then mixed with the axle molecule, resulting in the spontaneous formation of a polyrotaxane with multiple cyclic units threaded onto the axle. By incorporating Rhodamine B as a fluorescent capping agent at both ends, we successfully synthesized a fluorescent, DNJ-clustered polyrotaxane.
Furthermore, 1H NMR analysis revealed that approximately 20 to 30 β-CD units were threaded per polyrotaxane molecule. When the DNJ-conjugated β-CD was evaluated for inhibitory activity against α-glucosidase and was found to exhibit high inhibitory potency.
We are currently culturing Caco-2 cells, which express α-glucosidase on their membrane surface, and are investigating whether the synthesized DNJ-clustered polyrotaxane can be used for specific cell staining.
References1> C. Deroocq, Chem. Eur. J., 2011, 17,13825.
However, a comparison of molecular sizes reveals that these multivalent DNJ inhibitors <45 Å> are only about half the size of α-glucosidase <92 Å>. Consequently, it has been found that previously reported multivalent DNJ inhibitors struggle to inhibit multiple α-glucosidase molecules simultaneously. Furthermore, when α-glucosidase and a multivalent DNJ inhibitor bind in a 1:1 ratio, the remaining DNJ molecules attached to the core do not contribute effectively to inhibitory activity.
In this study, we designed a mobile inhibitor by incorporating DNJ units onto a polyrotaxane core. Due to the unique characteristics of polyrotaxanes, where the axle and the cyclic molecules are not covalently bonded, the system was expected to autonomously search for and inhibit α-glucosidase an enzyme located on the fluid membrane of small intestinal epithelial cells, which constantly changes position due to membrane fluidity. Furthermore, this design was anticipated to enable the simultaneous inhibition of multiple membrane enzymes.
The design of the DNJ-clustered polyrotaxane was implemented using poly<propylene glycol> <PPG, Mw = 2000> as the axle and β-cyclodextrin <β-CD> covalently functionalized with DNJ, used as the cyclic components. The DNJ-conjugated β-CD was synthesized via organic synthesis and then mixed with the axle molecule, resulting in the spontaneous formation of a polyrotaxane with multiple cyclic units threaded onto the axle. By incorporating Rhodamine B as a fluorescent capping agent at both ends, we successfully synthesized a fluorescent, DNJ-clustered polyrotaxane.
Furthermore, 1H NMR analysis revealed that approximately 20 to 30 β-CD units were threaded per polyrotaxane molecule. When the DNJ-conjugated β-CD was evaluated for inhibitory activity against α-glucosidase and was found to exhibit high inhibitory potency.
We are currently culturing Caco-2 cells, which express α-glucosidase on their membrane surface, and are investigating whether the synthesized DNJ-clustered polyrotaxane can be used for specific cell staining.
References1> C. Deroocq, Chem. Eur. J., 2011, 17,13825.
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