Metallic radionuclide-labeled tetrameric 2,6-diisopropylphenyl azides for cancer treatment.

This study proposes a new method for radionuclide therapy that involves the use of oligomeric 2,6-diisopropylphenyl azides and a chelator to form stable complexes with metallic radionuclides. The technique works by taking advantage of the endogenous acrolein produced by cancer cells. The azides react with the acrolein to give a diazo derivative that immediately attaches to the nearest organelle, effectively anchoring the radionuclide within the tumor. Preliminary in vivo experiments were conducted on a human lung carcinoma xenograft model, demonstrating the feasibility of this approach for cancer treatment.

Therapeutic efficacy of 211At-radiolabeled 2,6-diisopropylphenyl azide in mouse models of human lung cancer.

Targeted α-particle therapy (TAT) is an attractive alternative to conventional therapy for cancer treatment. Among the available radionuclides considered for TAT, astatine-211 (211At) attached to a cancer-targeting molecule appears very promising. Previously, we demonstrated that aryl azide derivatives could react selectively with the endogenous acrolein generated by cancer cells to give a diazo compound, which subsequently forms a covalent bond with the organelle of cancer cells in vivo. Herein, we synthesized 211At-radiolabeled 2,6-diisopropylphenyl azide (ADIPA), an α-emitting molecule that can selectively target the acrolein of cancer cells, and investigated its antitumor effect. Our results demonstrate that a single intratumor or intravenous administration of this simple α-emitting molecule to the A549 (human lung cancer) cell-bearing xenograft mouse model, at a low dose (70 kBq), could suppress tumor growth without inducing adverse effects. Furthermore, because acrolein is generally overproduced by most cancer cells, we believe ADIPA is a simple TAT compound that deserves further investigation for application in animal models and humans with various cancer types and stages.

Biofunctional chemistry and reactivity of biogenic acrolein for cancer diagnosis and therapy.

Acrolein holds excellent potential as a biomarker in various oxidative stress-related diseases, including cancer, Alzheimer's, Parkinson's, and inflammatory disorders. Consequently, a direct method to target and visualize acrolein in biological systems might be essential to provide tools for diagnosis and therapeutic purposes. Previously, we discovered 1,3-dipolar cycloaddition between aryl azides and acrolein, which proceeds without a catalyst to give α-diazocarbonyl derivatives. The reaction proceeds with high reactivity and selectivity even under physiological conditions. We have successfully utilized the reaction as a robust method for detecting acrolein generated by cancer cells. This review discusses the utilization of the endogenous acrolein reaction with aryl azide to (1) distinguish breast cancer from normal tissue during breast-conserving surgery and (2) treat cancer through selective prodrug activation in a mouse model without causing adverse effects. The methods have potential clinical application for breast-conserving surgery and are highly advantageous for cancer therapy.

Inhibition of amyloid formation of amyloid ß (1-42), amylin and insulin by 1,5-diazacyclooctanes, a spermine-acrolein conjugate.

Amyloid aggregates of proteins are known to be involved in various diseases such as Alzheimer's disease (AD). It is therefore speculated that the inhibition of amyloid formation can play an important role in the prevention of various diseases involving amyloids. Recently, we have found that acrolein reacts with polyamines, such as spermine, and produces 1,5-diazacyclooctane, such as cyclic spermine (cSPM). cSPM could suppress the aggregation of amyloid ß 1-40 (Aß40), one of the causative proteins of AD. This result suggests the potential inhibitory effect of cSPM against Aß 1-42 (Aß42) and other amyloid protein aggregation which are the main pathological features of AD and other diseases. However, the effect on the aggregation of such proteins remains unclear. In this study, the effect of cSPM on the amyloid formation of Aß42, amylin, and insulin was investigated. These three amyloidogenic proteins forming amyloids under physiological conditions (pH 7.4 and 37℃) served as model and are thought to be the causative proteins of AD, type 2 diabetes, and insulin-derived amyloidosis, respectively. Our results indicate that cSPM can suppress the amyloid aggregation of these proteins and reduce cytotoxicity. This study contributes to a better understanding of means to potentially counteract diseases by the means of polyamine and acrolein.

Application of Acrolein Imines to Organic Synthesis, Biofunctional Studies, and Clinical Practice.

N-alkyl unsaturated imines derived from acrolein, a toxin produced during oxidative stress, and biogenic alkyl amines occur naturally and are considered biologically relevant compounds. However, despite the recent conceptual and technological advances in organic synthesis, research on the new reactivity of these compounds is lacking. This personal account discusses research on the reactivity that has been overlooked in acrolein imines, including the discovery of new methods to synthesize biologically active compounds, the determination of new functions of relevant imines and their precursors, i. e., aldehydes and amines, and the application of these methods for clinical diagnosis.

Targeted 1,3-dipolar cycloaddition with acrolein for cancer prodrug activation.

Cytotoxic anticancer drugs used in chemotherapy are often antiproliferative agents that preferentially kill rapidly growing cancer cells. Their mechanism relies mainly on the enhanced proliferation rate of cancer cells and is not genuinely selective for cancer cells. Therefore, these drugs can also significantly affect healthy cells. Prodrug therapy provides an alternative approach using a less cytotoxic form of anticancer drug. It involves the synthesis of inactive drug derivatives which are converted to an active form inside the body and, preferably, only at the site of cancerous tissues, thereby reducing adverse drug reaction (ADR) events. Herein, we demonstrate a prodrug activation strategy by utilizing the reaction between aryl azide and endogenous acrolein. Since acrolein is generally overproduced by most cancer cells, we anticipate our strategy as a starting point for further applications in mouse models with various cancers. Furthermore, cancer drugs that have had therapeutic index challenges might be reconsidered for application by utilizing our strategy.

Disease-associated acrolein: A possible diagnostic and therapeutic substrate for in vivo synthetic chemistry.

Acrolein, a highly reactive α,ß-unsaturated aldehyde, is a compound to which humans are exposed in many different situations and often causes various human diseases. This paper summarizes the reports over the past twenty-five years regarding disease-associated acrolein detected in clinical patients and the role acrolein plays in various diseases. In several diseases, it was found that the increased acrolein acts as a pathogenetic factor. Thus, we propose the utility of over-produced acrolein as a substrate for a promising therapeutic or diagnostic method applicable to a wide range of diseases based on an in vivo synthetic chemistry strategy.

A Strategy for Tumor Targeting by Higher-Order Glycan Pattern Recognition: Synthesis and In Vitro and In Vivo Properties of Glycoalbumins Conjugated with Four Different N-Glycan Molecules.

Natural glycoconjugates that form glycocalyx play important roles in various biological processes based on cell surface recognition through pattern recognition mechanisms. This work represents a new synthesis-based screening strategy to efficiently target the cancer cells by higher-order glycan pattern recognition in both cells and intact animals (mice). The use of the very fast, selective, and effective RIKEN click reaction (6π-azaelectrocyclization of unsaturated imines) allows to synthesize and screen various structurally well-defined glycoalbumins containing two and eventually four different N-glycan structures in a very short time. The importance of glycan pattern recognition is exemplified in both cell- and mouse-based experiments. The use of pattern recognition mechanisms for cell targeting represents a novel and promising strategy for the development of diagnostic, prophylactic, and therapeutic agents for various diseases including cancers.

Enantioselective synthesis of cyclic and linear diamines by imine cycloadditions.

Imine is one of the most versatile functional groups in chemistry and biochemistry fields. Although many biochemical reactions involve imine formation, the inherently unstable property of N-alkyl-α,ß-unsaturated imines still hindered their utilization in organic synthesis. In this article, we described that the N-alkyl-α,ß-unsaturated imines, which prepared from alkylamines and acrolein, could smoothly react through [4 + 4] cycloaddition to give eight-membered diazacyclooctane derivatives in excellent yields. Under a similar condition, in the presence of formaldehyde, the [4 + 2] and [4 + 2 + 2] cycloadditions could lead to the formation of six-membered hexahydropyrimidine or eight-membered triazacyclooctanes, depending on the substituent of aldehydes. Moreover, an easy functional group manipulation of the cyclic products obtained from these cycloadditions can provide variously substituted chiral linear diamines. We can utilize these novel reactivities to reveal the unknown and essential properties of many biological processes that involve N-alkyl-unsaturated imines.

Emerging Technologies for Real-Time Intraoperative Margin Assessment in Future Breast-Conserving Surgery.

Clean surgical margins in breast-conserving surgery (BCS) are essential for preventing recurrence. Intraoperative pathologic diagnostic methods, such as frozen section analysis and imprint cytology, have been recognized as crucial tools in BCS. However, the complexity and time-consuming nature of these pathologic procedures still inhibit their broader applicability worldwide. To address this situation, two issues should be considered: 1) the development of nonpathologic intraoperative diagnosis methods that have better sensitivity, specificity, speed, and cost; and 2) the promotion of new imaging algorithms to standardize data for analyzing positive margins, as represented by artificial intelligence (AI), without the need for judgment by well-trained pathologists. Researchers have attempted to develop new methods or techniques; several have recently emerged for real-time intraoperative management of breast margins in live tissues. These methods include conventional imaging, spectroscopy, tomography, magnetic resonance imaging, microscopy, fluorescent probes, and multimodal imaging techniques. This work summarizes the traditional pathologic and newly developed techniques and discusses the advantages and disadvantages of each method. Taking into consideration the recent advances in analyzing pathologic data from breast cancer tissue with AI, the combined use of new technologies with AI algorithms is proposed, and future directions for real-time intraoperative margin assessment in BCS are discussed.

Facile Access to Optically Active 2,6-Dialkyl-1,5-Diazacyclooctanes.

The chiral substituted 1,5-diazacyclooctane (1,5-DACO) is of considerable importance and has attracted attention from a wide range of fields due to their unique chemical and biological properties. Despite the application potential, further study has not been optimized due to difficulties in their synthetic accessibility. Here, we report that the 1,5-DACO bearing a chiral auxiliary obtained from the formal [4+4] cycloaddition of N-alkyl-α,ß-unsaturated imines can be further derivatized by nucleophilic alkylation to give various chiral substituted 1,5-DACO derivatives. The removal of the chiral auxiliary was effectively carried out using hydrogenation over Pearlman's catalyst. This methodology allows the production of a broad range of unprecedented optically active 2,6-dialkyl-1,5-DACO, which could not be accessed by other methods.

In vitro and in vivo cancer cell apoptosis triggered by competitive binding of Cinchona alkaloids to the RING domain of TRAF6.

TRAF6 is highly expressed in many tumors and plays an important role in the immune system. The aim of this study is to confirm anti-tumor activities of all naturally occurring Cinchona alkaloids that have been screened using computational docking program, and to validate the accuracy and specificity of the RING domain of TRAF6 as a potential anti-tumor target, and to explore their effect on the immune system. Results reported herein would demonstrate that Cinchona alkaloids could induce apoptosis in HeLa cells, inhibit the ubiquitination and phosphorylation of both AKT and TAK1, and up-regulate the ratio of Bax/Bcl-2. In addition, these compounds could induce apoptosis in vivo, and increase the secretion of TNF-α, IFN-γ, and IgG, while not significantly impacting the ratio of CD4+T/CD8+T. These investigations suggest that the RING domain of TRAF6 could serve as a de novo biological target for therapeutic treatment in cancers.

Tetramethylrhodamine is an essential scaffold of azide probe in detecting cellular acrolein.

Tetramethylrhodamine (TAMRA)-phenyl azide is a chemical probe used to detect intracellular acrolein directly in live cells. Herein, we demonstrated that TAMRA is the optimum fluorophore for the probe. TAMRA-phenyl azide was used to reveal that high levels of acrolein are generated in a variety of breast cancer cells, regardless of the tumor subtype. These findings corroborate the analysis presented in our previous report, in which TAMRA-phenyl azide was used to label breast cancer tissues resected from breast cancer patients. Because high levels of acrolein were generated in all cancer cell types, we believe that acrolein detection may be useful as a general method for labeling cancerous tissues.

Cascade Reaction in Human Live Tissue Allows Clinically Applicable Diagnosis of Breast Cancer Morphology.

Clean operating margins in breast cancer surgery are important for preventing recurrence. However, the current methods for determining margins such as intraoperative frozen section analysis or imprint cytology are not satisfactory since they are time-consuming and cause a burden on the patient and on hospitals with a limited accuracy. A "click-to-sense" probe is developed based on the detection of acrolein, which is a substance released by oxidatively stressed cancer cells and can be visualized under fluorescence microscopy. Using live breast tissues resected from breast cancer patients, it is demonstrated that this method can quickly, selectively, and sensitively differentiate cancer lesion from normal breast gland or benign proliferative lesions. Since acrolein is accumulated in all types of cancers, this method could be used to quickly assess the surgical margins in other types of cancer.

Expanding the Applicability of the Metal Labeling of Biomolecules by the RIKEN Click Reaction: A Case Study with Gallium-68 Positron Emission Tomography.

Radiolabeled biomolecules with short half-life times are of increasing importance for positron emission tomography (PET) imaging studies. Herein, we demonstrate an improved and generalized method for synthesizing a [radiometal]-unsaturated aldehyde as a lysine-labeling probe that can be easily conjugated into various biomolecules through the RIKEN click reaction. As a case study, 68 Ga-PET imaging of U87MG xenografted mice is demonstrated by using the 68 Ga-DOTA-RGDyK peptide, which is selective to αV ß3 integrins.

Effect of spermine-derived AGEs on oxidative stress and polyamine metabolism.

Non-enzymatic glycation between proteins and carbohydrates, such as advanced glycation end products (AGEs), are naturally occurring compounds implicated in aging and numerous degenerative diseases. Methyl glyoxal (MG), which is an intermediate of the AGE biosynthetic pathway, is known to react with primary amines of proteins to create a wide range of AGE modifications, such as carboxyethyl lysine (CEL) and methylglyoxal-derived lysine dimer (MOLD). As a means to investigate and probe the ROS production pathways of AGEs, low molecular weight compounds carboxyethyl spermine (CES) and methylglyoxal-derived spermine dimer (MOSD) were synthesized, which replace lysine with another highly nucleophilic biological amine, spermine (SPM). Contrary to expectations, results show CES- and MOSD-induced oxidative stress proceeds through different pathways. As such, we have developed useful probes that can be used to better understand and investigate pathways related to acrolein-based oxidative stress and/or polyamine metabolic pathways.

Unexplored Chemical Reactions of Endogenous Acrolein: Detection, Toxicity, and Biological Roles.

Acrolein, a highly toxic α, ß-unsaturated aldehyde, occurs as pollutant in the environment (e.g., tobacco smoke and exhaust gas) and is ubiquitously generated in biosystems (e.g., the lipid peroxidation process and metabolism of polyamine or amino acids). High accumulation of acrolein in biosystems is often linked pathologically with several oxidative stress-related diseases, including cancer and Alzheimer's disease. Accordingly, acrolein holds great potential as a key biomarker in oxidative stress-related diseases, and direct measurement of acrolein in biological samples is important to provide information for diagnostic and therapeutic purposes. Recently, we have serendipitously discovered the unrecognized reactivity of phenyl azide to acrolein. Phenyl azide can rapidly and selectively react with acrolein in a "click" manner to provide 4-formyl-1,2,3-triazoline through 1,3-dipolar cycloaddition. We have successfully utilized the acrolein-azide click reaction as a simple but robust method for detecting and visualizing acrolein generated by live cells in the context of oxidative stress processes. In addition, we also serendipitously discovered novel cycloaddition reactions of N-alkyl-α,ß-unsaturated imines derived from acrolein and biogenic amines (e.g., polyamines, norepinephrine, and sphingosine), to yield 8-membered cyclic compounds. We then examined the biological functions of the cyclic products and revealed for the first time their roles in the oxidative stress mechanism and inhibition of amyloid ß(1-40) fibrillization.

Cinchonine induces apoptosis of HeLa and A549 cells through targeting TRAF6.

BACKGROUND: Cancer cells are known to over-express TRAF6 that is critical for both AKT and TAK1 activations. The Really Interesting New Gene (RING) domain of TRAF6 is believed to be responsible for the E3 ligase activity, ZINC fingers of TRAF6 provide critical support for the activity of the RING domain which is critical for both AKT and TAK1 activations. METHODS: We employed computational docking program to identify small molecules that could effectively and competitively bind with the RING domain of TRAF6, which is believed to be responsible for its E3 ligase activity. MTT assay and flow cytometry were employed to analyze apoptosis of cancer cells. Signaling pathways were detected using immunoprecipitation and western blotting, and immunofluorescence was pursued to assess the nature of binding of cinchonine to TRAF6. We also performed animal experiments to test effect of cinchonine in vivo. RESULTS: Cinchonine, a naturally occurring Cinchona alkaloid identified from the docking study, could bind to TRAF6 in HeLa and A549 cells and induce apoptosis of these cancer cells. We found that AKT ubiquitination and phosphorylation as well as phosphorylation of TAK1 were decreased. These activities would lead to subsequent suppression anti-apoptotic protein Bcl-2, while elevating pro-apoptotic protein Bax. Immunofluorescence staining unambiguously demonstrated the binding of cinchonine specifically at the RING domain of TRAF6 in cells, thereby validating the computational modeling. Animal experiments showed that cinchonine could suppress tumor growth in mice without showing significant acute toxicity. CONCLUSION: These investigations suggest that through competitive binding with the RING domain of TRAF6, cinchonine could induce apoptosis via inhibiting AKT and TAK1 signaling pathways.

In†Vivo Gold Complex Catalysis within Live Mice.

Metal complex catalysis within biological systems is largely limited to cell and bacterial systems. In this work, a glycoalbumin-AuIII complex was designed and developed that enables organ-specific, localized propargyl ester amidation with nearby proteins within live mice. The targeted reactivity can be imaged through the use of Cy7.5- and TAMRA-linked propargyl ester based fluorescent probes. This targeting system could enable the exploitation of other metal catalysis strategies for biomedical and clinical applications.

Synthesis of characteristic Mycobacterium peptidoglycan (PGN) fragments utilizing with chemoenzymatic preparation of meso-diaminopimelic acid (DAP), and their modulation of innate immune responses.

Peptidoglycan (PGN) is a major component of bacterial cell wall and is recognized as a potent immunostimulant. The PGN in the cell envelope of Mycobacterium Tuberculosis has been shown to possess several unique characteristics including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues, and amidation of the free carboxylic acid of d-Glu or of meso-DAP in the peptide chains. Using a newly developed, highly stereoselective, chemoenzymatic approach for the synthesis of meso-DAP in peptide stems, we successfully synthesized for the first time, a series of Mycobacterium PGN fragments that include both mono- and disaccharides of MurNGlyc or 1,6-anhydro-MurNGlyc, as well as peptide-amidated variants. The ability of these PGN fragments to stimulate the immune system through activation of human Nod1 and Nod2 was examined. The PGN fragments were found to modulate immune stimulation, specifically, amidation at the d-Glu and meso-DAP in the peptide stem strongly reduced hNod1 activation. This effect was dependent on modification position. Additionally, N-glycolyl (instead of acetyl) of muramic acid was associated with slightly reduced human Nod1 and Nod2 stimulatory capabilities.