天然有機化合物討論会講演要旨集
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アクロレインのインビボクリック反応性に基づく酸化ストレス疾患イメージングと生体内合成化学治療
プラディプタ アンバラ田中 克典
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アクロレインのインビボクリック反応性に基づく酸化ストレス疾患イメージングと生体内合成化学治療

Acrolein, a highly toxic a,b-unsaturated aldehyde, exists both as ubiquitous pollutants in environment (e.g. in tobacco smoke or exhaust gas) and as endogenous metabolites generated by cells through enzymatic oxidation of polyamines or through reactive oxygen species (ROS)-mediated lipid peroxidation. Acrolein, which is sometimes generated on millimolar scale in cells under oxidative stress, has been reported to be more toxic than ROS molecules (e.g. H2O2, •OH). Considering that acrolein has been used as longstanding key biomarker in numerous oxidative stress-related diseases including cancer and Alzheimer’s, consequently detection of acrolein level in biosystems is becoming of significant importance for defining pathogenesis of the disease and to provide information for the therapeutic and diagnostic remedies.

The conventional analytical method for detection of acrolein, e.g. HPLC analysis after derivatization with 3-aminophenol under harsh reaction conditions, is not suitable for high-throughput assay and frequently provide poor selectivity when other aldehydes are present. Furthermore, while notable, the use of antibodies that can recognize 3-formyl- 3,4-dehydropiperidine (FDP), an acrolein-lysine adducts, is not only costly but also requires procedures that are time-consuming. More critically, the formation of FDP is quite slow, and thus, this method suffers from lack of detection sensitivity in a time-dependent manner. Consequently, developing new analytical tools for acrolein detection that are straightforward, cost-effective, selective, and preferably feasible in live cells remains a highly essential pursuit in the therapeutic treatment of oxidative stress related diseases. Herein we would like to demonstrate a simple but robust method for detecting and imaging acrolein generated by cells in the context of oxidative stress processes or introduced via environmental exposure.

1. Discovery of 1,3-dipolar cycloaddition between phenyl azide and acrolein

Recent advances in Huisgen 1,3-dipolar cyclo- addition between azide and terminal acetylene has led to extensive application in the fields of chemical biology and organic functional materials. The reaction may be accele- rated in the presence of Cu(I) catalyst or by placing the acetylene group within a strained ring. Aside from these “click reactions”, we serendipitously uncovered that phenyl azide can participate in similar 1,3-dipolar cycloaddition with acrolein to produce triazoline and triazole derivatives.

Reaction of phenyl azide with 10 equiv. of acrolein present in THF at millimolar level smoothly gave the heterocyclic products (Fig. 1-i). The reaction is generally complete within 30 minutes at room temperature in the absence of catalyst. After silica gel column chromatography, the products were identified to be 4-formyl-1,2,3-triazoline 2 and 4-formyl-1,2,3-triazole 3 (Fig. 1-i). Interestingly, in the case of 4-formyl-1,2,3-triazoline 2, the double bond isomerized at conjugated position of C4-aldehyde, and decomposition was not observed even over an incubation period of several days. Thus, the thermally labile triazoline cycloadduct was stabilized by this isomerization. Significantly, the 1,3-dipolar cycloaddition between phenyl azide and acrolein is highly chemoselective for acrolein. Under the same conditions, no discernable products were found when phenyl azide was reacted with a- or b- substituted acrolein (e.g. methacrolein, crotonaldehyde, trans-2- octenal) and activated olefins serving as model of lipid metabolites (Fig. 1-ii). Surprisingly, to the best of our knowledge, such inherent reactivity of acrolein towards phenyl

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Acrolein, a highly toxic a,b-unsaturated aldehyde, exists both as ubiquitous pollutants in environment (e.g. in tobacco smoke or exhaust gas) and as endogenous metabolites generated by cells through enzymatic oxidation of polyamines or through reactive oxygen species (ROS)-mediated lipid peroxidation. Acrolein, which is sometimes generated on millimolar scale in cells under oxidative stress, has been reported to be more toxic than ROS molecules (e.g. H2O2, •OH). Considering that acrolein has been used as longstanding key biomarker in numerous oxidative stress-related diseases including cancer and Alzheimer’s, consequently detection of acrolein level in biosystems is becoming of significant importance for defining pathogenesis of the disease and to provide information for the therapeutic and diagnostic remedies.

The conventional analytical method for detection of acrolein, e.g. HPLC analysis after derivatization with 3-aminophenol under harsh reaction conditions, is not suitable for high-throughput assay and frequently provide poor selectivity when other aldehydes are present. Furthermore, while notable, the use of antibodies that can recognize 3-formyl- 3,4-dehydropiperidine (FDP), an acrolein-lysine adducts, is not only costly but also requires procedures that are time-consuming. More critically, the formation of FDP is quite slow, and thus, this method suffers from lack of detection sensitivity in a time-dependent manner. Consequently, developing new analytical tools for acrolein detection that are straightforward, cost-effective, selective, and preferably feasible in live cells remains a highly essential pursuit in the therapeutic treatment of oxidative stress related diseases. Herein we would like to demonstrate a simple but robust method for detecting and imaging acrolein generated by cells in the context of oxidative stress processes or introduced via environmental exposure.

1. Discovery of 1,3-dipolar cycloaddition between phenyl azide and acrolein

Recent advances in Huisgen 1,3-dipolar cyclo- addition between azide and terminal acetylene has led to extensive application in the fields of chemical biology and organic functional materials. The reaction may be accele- rated in the presence of Cu(I) catalyst or by placing the acetylene group within a strained ring. Aside from these “click reactions”, we serendipitously uncovered that phenyl azide can participate in similar 1,3-dipolar cycloaddition with acrolein to produce triazoline and triazole derivatives.

Reaction of phenyl azide with 10 equiv. of acrolein present in THF at millimolar level smoothly gave the heterocyclic products (Fig. 1-i). The reaction is generally complete within 30 minutes at room temperature in the absence of catalyst. After silica gel column chromatography, the products were identified to be 4-formyl-1,2,3-triazoline 2 and 4-formyl-1,2,3-triazole 3 (Fig. 1-i). Interestingly, in the case of 4-formyl-1,2,3-triazoline 2, the double bond isomerized at conjugated position of C4-aldehyde, and decomposition was not observed even over an incubation period of several days. Thus, the thermally labile triazoline cycloadduct was stabilized by this isomerization. Significantly, the 1,3-dipolar cycloaddition between phenyl azide and acrolein is highly chemoselective for acrolein. Under the same conditions, no discernable products were found when phenyl azide was reacted with a- or b- substituted acrolein (e.g. methacrolein, crotonaldehyde, trans-2- octenal) and activated olefins serving as model of lipid metabolites (Fig. 1-ii). Surprisingly, to the best of our knowledge, such inherent reactivity of acrolein towards phenyl azide has not been reported before in the literature. Several example of this type of reaction between alkyl azide and unsaturated ketones, esters, or amides have been reported, but the reactions required elevated temperatures, and under such conditions the 1,2,3-triazoline cycloadduct readily decompose into highly reactive diazo intermediate, from which a variety of byproducts are produced.

In order to check the applicability of the azide–acrolein “click reaction” towards biologically relevant conditions, we performed the reaction in aqueous and physiological conditions. Gratifyingly, the azide–acrolein 1,3-dipolar cycloaddition proceed, even in the presence of various metals or interferences, to give the “clicked” products in the range of 20% to 50% yields (Fig. 2-i). Furthermore, we also found that the phenyl azide was inert to 0.1 M H2O2and 1.0 M glutathione (GSH) under the experimental conditions investigated in this research (Fig. 2-ii). When H2S gas is bubbled into the reaction mixture, most of the phenyl azide was recovered. On the other hand, 1.0 M NaHS rapidly reduced phenyl azide to aniline, but the concentration used in the experiment is much higher than the real concentration in biosystems, which usually contains only submicromolar level of hydrosulfide; hence it will not affect the reaction with acrolein when excess phenyl azide is applied (Fig. 2-ii).

2. Direct imaging of extracellular acrolein released from oxidatively stressed cells

Given such impressive reactivity and selectivity of the phenyl azide with acrolein at millimolar concentration under physiological conditions, we envisioned that phenyl azide with functional group, e.g. fluorescent group, could be used to detect the extracellular acrolein generated by cells under oxidative stress or introduced via environmental exposure. If the reaction is efficient and the triazoline or triazole products internalized into the cells, the oxidative stress cells could also be selectively imaged simply by treating the cells with functionalized azides.

The reaction-based cell imaging approach was put into practice using the tetramethylrhodamine (TAMRA)-labeled phenyl azide 4 (Fig. 3). Human umbilical vein endothelial cells (HUVECs) were treated with 10 mM solution of TAMRA-labeled phenyl azide 4 at room temperature for 30 minutes, accompanied by [1] pretreatment with excess acrolein (Fig. 3b); [2] exposure to tobacco smoke (Fig. 3c and 3d); and [3] the presence of hydrogen peroxide (Fig. 3e-3h), which induced cellular oxidative stress. Accordingly, fluorescently labeled phenyl azide 4 reacted rapidly and selectively with acrolein included in tobacco smoke or that generated from oxidatively stressed cells. Figure 3 shows microscopy images of the HUVECs. Gratifyingly, the fluorescence intensity across the whole cells increased significantly upon treatment with TAMRA-labeled phenyl azide 4, which pretreated with acrolein (Fig. 3b). In the case of fluorescence images of cells exposed to tobacco smoke, we found that the fluorescence uptake by the cells was notably enhanced in a dose-dependent manner (Fig. 3c and 3d). Furthermore, acrolein that generated by the cells after treatment with hydrogen peroxide was also detected using our TAMRA-labeled phenyl azide 4 (Fig. 3e-3h). Thus, detection of acrolein in situ generated from the live cells was still possible under exposure of 50 mM of hydrogen peroxide (Fig. 3e), and the fluorescence intensity increased with the increasing consentration of hydrogen peroxide.

To evaluate the sensitivity for acrolein detection, we then compared our protocol with the conventional HPLC- and fluorescence-based detection using 3-aminophenol, which produces fluorescence 7-hydroxyquinoline by the reaction with acrolein. Our method is superior in detection sensitivity; while the traditional method only detected 2 mM of acrolein in cell medium, our new protocol can detect even 1 nM of acrolein. We could then evaluate the acrolein produced during the treatment of the cells with a certain concentration of hydrogen peroxide. Based on calculation of the standard fluorescence graph, we estimated about 100 nM of acrolein could be generated by treatment of HUVECs with 50 mM of hydrogen peroxide (see Fig. 3e).

Furthermore, by utilizing both ROS probes and TAMRA- labeled phenyl azide 4, we demonstrated that our method could be used to independently detect and image the early formation of ROS generated by menadione and the late forma- tion of acrolein following lipid peroxidation by the produced ROS on the live cells (Fig. 4). Significant accumulation of ROS in the cells was detected as early as 10 minutes after treatment with menadione (Fig. 4-i). On the other hand, the late production of acrolein by cells was detected after 30 minutes of treatment with menadione (Fig. 4-ii). Accordingly, the stability of TAMRA-labeled phenyl azide 4 under physiologically relevant conditions makes it possible to detect the late formation of acrolein. Thus, it provides evidence that the reaction is highly selective towards extracellular acrolein released from the cells.

3. Mechanism for the cellular uptake of “clicked” products

The mechanism by which the cells took up the azide–acrolein conjugates (i.e. the fluorescently labeled 4-formyl-1,2,3-triazoline and/or triazole derivatives) were examined by assessing the intracellular localization of the azide–acrolein conjugates after accumulation in cells. Organelle staining with Hoechst 33342 (nucleic acid staining), ER- Tracker Green (ER staining), LysoTracker Green DND-26 (lysosome staining), and MitoTracker Green FM (mitochondrial staining) were applied to the detection of TAMRA- labeled azide–acrolein conjugate accumulation in the cellular organelles without cell fixation. Confocal microscopy images revealed a high colocalization of the azide–acrolein conjugate in ER (Fig. 5-i) and lysosome (Fig. 5-ii), but weak colocalization in mitochondria (Fig. 5-iii), and there is no flow of the azide–acrolein conjugate into the nucleus.

At low temperature (4 °C), which inhibited energy dependent cellular uptake pathways, including clathrin-mediated endocytosis, and reduced membrane fluidity, the cellular uptake efficacy of the TAMRA-labeled azide–acrolein conjugates decreased significantly. Treatment of dynasore, which is an inhibitor of dynamin for clathrin- or caveolae- mediated endocytosis, reduced cellular uptake of the azide–acrolein conjugates. These results suggested endocytosis was an important pathway for the cellular uptake of the azide–acrolein conju- gates. The conjugates penetrated the membrane and accumulated intra- cellularly at the ER and lysosome during the very early stages of endocytosis. Membrane fluidity may play crucial role in membrane penetration by the azide–acrolein conjugates. Henceforth, the acrolein introduced from tobacco smoke or produced from oxidatively stressed cells could be efficiently and conveniently visualized, simply by treating the live cells with a fluorescently labeled phenyl azide.

4. Conclusion

The detection method described here is convenient and simple. Conceptually, it based on an “overlooked” and serendipitously discovered reactivity of phenyl azide in the context of 1,3-dipolar cycloadditions. Because environmental exposures to acrolein and oxidative stressess in cells lead to detrimental diseases, this method provides an important foundation for developing new therapeutic diagnostic tools. It is also noteworthy that the enhanced organelle accumulation of azide–acrolein conjugates should be advantageous when considering future development of small molecule-based diagnostic and therapeutic, and their controlled intracellular delivery at the organelle level.

Based on the above results, we have also utilized the fluorescently labeled aryl azides for selective imaging of oxidative stress in cancer cells. Furthermore, the remarkable selectivity and stability of aryl azide under physiologically relevant conditions encouraged us to utilize the azide–acrolein 1,3-dipolar cycoladdition in “therapeutic in vivo synthetic chemistry”. These details will be discussed at the symposium.

Acknowledgement:

We are grateful to Dr. Ikuhiko Nakase at Osaka Prefecture University, Dr. Shinobu Kitazume and Dr. Naoyuki Taniguchi at Disease Glycomics Team – RIKEN for their support and advice in the cell-based experiments. This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 22651081, 23681047, 25560410); by a MEXT Grant-in-Aid for Scientific Research on Innovative Areas (Nos. 26102743, 15H05843); by Suntory Foundation for Life Sciences, Kyoto; and by a subsidy of the Russian Government “Program of Competitive Growth of Kazan Federal University among World’s Leading Academic Centers”.

References:

(1) A. R. Pradipta, K. Tanaka, et al., ACS Sens. 2016, 1, 623; (2) A. Tsutsui, K. Tanaka, et al., Med. Chem. Commun. 2015, 6, 431; (3) A. Tsutsui, K. Tanaka, et al., Adv. Sci. 2016, DOI: 10.1002/advs. 201600082; (4) A. R. Pradipta, K. Tanaka, et al., 有機合成化学協会誌, 2016, 7月号.

 
© 2016 天然有機化合物討論会電子化委員会
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