Biocompatible and Water-Soluble Shortwave-Infrared (SWIR)-Emitting Cyanine-Based Fluorescent Probes for In Vivo Multiplexed Molecular Imaging.

Extending molecular imaging into the shortwave-infrared (SWIR, 900-1400 nm) region provides deep tissue visualization of biomolecules in the living system resulting from the low tissue autofluorescence and scattering. Looking at the Food and Drug Administration-approved and clinical trial near-infrared (NIR) probes, only indocyanine green (ICG) and its analogues have been approved for biomedical applications. Excitation wavelength less than 800 nm limits these probes from deep tissue penetration and noninvasive fluorescence imaging. Herein, we present the synthesis of ICG-based π-conjugation-extended cyanine dyes, ICG-C9 and ICG-C11 as biocompatible, and water-soluble SWIR-emitting probes with emission wavelengths of 922 and 1010 nm in water, respectively. Also, ICG-, ICG-C9-, and ICG-C11-based fluorescent labeling agents have been synthesized for the development of SWIR molecular imaging probes. Using the fluorescence of ICG, ICG-C9, and ICG-C11, we demonstrate three-color SWIR fluorescence imaging of breast tumors by visualizing surface receptors (EGFR and HER2) and tumor vasculature in living mice. Furthermore, we demonstrate two-color SWIR fluorescence imaging of breast tumor apoptosis using an ICG-conjugated anticancer drug, Kadcyla and ICG-C9 or ICG-C11-conjugated annexin V. Finally, we show long-term (38 days) SWIR fluorescence imaging of breast tumor shrinkage induced by Kadcyla. This study provides a general strategy for multiplexed fluorescence molecular imaging with biocompatible and water-soluble SWIR-emitting cyanine probes.

Stereochemistry of Sphingolipids in Ganglioside GM3 Enhances Recovery of Nervous Functionality.

GM3 is a simple monosialylated ganglioside (NeuAcα(2-3)Galß(1-4)Glcß1-1'-ceramide). Its aberrant expression in adipocytes is involved in a variety of physiological and pathological processes in diabetes mellitus and obesity. GM3 is exposed on the outer surface of cell membranes and is strongly associated with type 2 diabetes and insulin resistance. Exogenously added GM3 promotes neurite outgrowth in a variety of different neuroblastoma cell lines. Neurite outgrowth is a key process in the development of functional neuronal circuits and neuro-regeneration following nerve injury. Therefore, regulating GM3 levels in nerve tissues might be a potential treatment method for these disorders. Here, we demonstrate the comprehensive synthesis of stereoisomeric GM3s and compare their physicochemical properties with those of natural GM3 and diastereomers of sphingolipids in GM3 to examine the enhancement of biological activity. l-erythro-GM3 was confirmed to increase neurite outgrowth, providing valuable insights for potential neuro-regenerative treatments.

Stereochemistry-activity relationship of ceramide-induced exosome production to clear amyloid-ß in Alzheimer's disease.

Stereochemistry has a substantial impact on the biological activity of various drugs. We investigated the role of stereochemistry of ceramides in inducing the production of exosomes, a type of extracellular vesicle, from neuronal cells, with a potential benefit in improving the clearance of amyloid-ß (Aß), a causal agent of Alzheimer's disease. A stereochemical library of diverse ceramides with different tail lengths was synthesized with the purpose of varying stereochemistry (D-erythro: DE, D-threo: DT, L-erythro: LE, L-threo: LT) and hydrophobic tail length (C6, C16, C18, C24). The exosome levels were quantified using TIM4-based exosome enzyme-linked immunosorbent assay after concentrating the conditioned medium using centrifugal filter devices. The results revealed a pivotal role of stereochemistry in determining the biological activity of ceramide stereoisomers, with the superiority of those based on DE and DT stereochemistry with C16 and C18 tails, which demonstrated significantly higher exosome production, without a significant change in the particle size of the released exosomes. In transwell experiments with Aß-expressed neuronal and microglial cells, DE- and DT-ceramides with C16 and C18 tails significantly decreased extracellular Aß levels. The results reported here are promising in the design of non-classic therapies for the treatment of Alzheimer's disease.

A near-infrared fluorescent long-chain fatty acid toward optical imaging of cardiac metabolism in living mice.

Non-invasive fatty acid (FA) metabolic imaging is crucial for the evaluation of cardiac function in the heart. Currently, single-photon emission computed tomography (SPECT) and positron emission tomography (PET) are widely employed for cardiac metabolic imaging both in pre-clinical and clinical studies. Although SPECT and PET enable highly sensitive cardiac metabolic imaging, there are several disadvantages such as the high cost of instruments and radioactive tracer synthesis. In contrast, near-infrared (NIR) optical imaging using fluorescent FAs provides a simple and useful platform for in vivo imaging of cardiac metabolism. In this work, we synthesized a NIR fluorescence labelled long-chain fatty acid (LCFA) for real-time imaging of cardiac metabolism in vivo. A NIR fluorescence labelled LCFA was designed as an analogue of ß-methyl [123I] iodophenyl-pentanedecanoic acid (123I-BMIPP), which is widely used for the diagnosis of heart diseases in clinical practice. As a NIR fluorescent label, we used an Alexa 680 fluorophore that emits over 700 nm. By conjugation of Alexa 680 to Amino-BMPP (15-(4-(3-aminopropyl)phenyl)-3-methylpentadecanoic acid), we prepared a NIR fluorescent BMIPP analogue, Alexa680-BMPP. NIR fluorescence imaging showed that Alexa680-BMPP is taken up by the mouse heart tissue after intravenous injection, showing that Alexa680-BMPP can act as a fluorescent LCFA analogue. Among Alexa680 conjugated FA analogues including short and middle chain NIR fluorescent FAs, Alexa680-BMPP was most efficiently taken up by heart tissues. For fasted and fed mice, the difference in the degree of the uptake of Alexa680-BMPP in their heart tissues was clearly observed by in vivo and ex vivo NIR fluorescence imaging. Herein, we present the synthesis of a NIR fluorescent LCFA, Alexa680-BMPP, and its capability for real-time optical imaging of cardiac metabolism in living mice.

Shortwave-infrared (SWIR) emitting annexin V for high-contrast fluorescence molecular imaging of tumor apoptosis in living mice.

Recently, shortwave infrared (SWIR) fluorescence imaging over 1000 nm has attracted much attention for in vivo optical imaging because of the higher signal to background ratios in the SWIR region. For the application of SWIR fluorescence imaging to biomedical fields, the development of SWIR fluorescent molecular probes with high biocompatibility is crucial. Although many researchers have designed a variety of SWIR emitting probes based on organic dyes, the synthesis of biocompatible SWIR fluorescent molecular imaging probes is still challenging. In this work we synthesized indocyanine green (ICG) and π-conjugation extended ICG (ICG-C11) labelled annexin V as SWIR fluorescent probes for tumor apoptosis. Annexin V is an endogenous protein with binding ability to phosphatidylserine (PS) which appears on the outer monolayer of apoptotic cell membranes. Although there are many types of visible and NIR fluorescent annexin V, there are no SWIR emitting fluorescent probes that can be used for high contrast fluorescence imaging of apoptosis in vivo. Herein, we report the synthesis and application of ICG and ICG-C11 conjugated annexin V for SWIR fluorescence imaging of tumor apoptosis. The presented fluorescent annexin V is the first SWIR emitting probe for in vivo optical imaging of tumor apoptosis. We demonstrate that SWIR emitting ICG- and ICG-C11 conjugated annexin V enable high-contrast fluorescence imaging of tumor apoptosis in living mice. We further demonstrate that ICG-C11-annexin V can be used for long-term (ca. two weeks) SWIR fluorescence imaging of tumor apoptosis. The SWIR fluorescent annexin V will greatly contribute not only to the study of tumor-apoptosis induced by anti-cancer drugs, but also to the study of apoptosis-related diseases in a living system.

Shortwave-Infrared Fluorescent Molecular Imaging Probes Based on π-Conjugation Extended Indocyanine Green.

Recently, shortwave-infrared (SWIR) fluorescence imaging for the optical diagnostics of diseases has attracted much attention as a new noninvasive imaging modality. For this application, the development of SWIR molecular imaging probes with high biocompatibility is crucial. Although many types of biocompatible SWIR fluorescent probes based on organic dyes have been reported, there are no SWIR-emitting molecular imaging probes that can be used for the detection of specific biomolecules in vivo. To apply SWIR-emitting molecular imaging probes to biomedical fields, we developed a biocompatible SWIR fluorescent dye based on π-conjugation extended indocyanine green (ICG), where ICG is the only approved near-infrared dye by the US Food and Drug Administration (FDA) for use in the clinic. Using the π-conjugation extended ICG, we prepared SWIR molecular imaging probes that can be used for in vivo tumor imaging. Herein, we demonstrate noninvasive SWIR fluorescence imaging of human epidermal growth factor receptor 2 (HER2)-positive and epidermal growth factor receptor (EGFR)-positive breast tumors using π-conjugation extended ICG and monoclonal antibody conjugates. The presented π-conjugation extended ICG analog probes will be a breakthrough to apply SWIR fluorescence imaging in biomedical fields.

Daurichromenic Acid from the Chinese Traditional Medicinal Plant Rhododendron dauricum Inhibits Sphingomyelin Synthase and Aß Aggregation.

Species of the genus Rhododendron have been used in traditional Chinese medicine, with the medicinal herb "Manshanfong" used as an expectorant and for the treatment of acute bronchitis. Daurichromenic acid (DCA), a constituent of Rhododendron dauricum, is a meroterpenoid with antibacterial, anti-HIV, and anti-inflammatory activities. However, the mechanisms underlying these pharmacologic activities are poorly understood. To develop new drugs based on DCA, more information is required regarding its interactions with biomolecules. The present study showed that DCA inhibits the activity of the enzyme sphingomyelin synthase, with an IC50 of 4 µM. The structure-activity relationships between DCA and sphingomyelin synthase were evaluated using derivatives and cyclized hongoquercin A. In addition, DCA was found to inhibit amyloid ß aggregation. These results may help in the design of effective drugs based on DCA.

Determination of the Absolute Configurations and Sensory Properties of the Enantiomers of a Homologous Series (C6-C10) of 2-Mercapto-4-alkanones.

The enantiomers of a homologous series (C6-C10) of 2-mercapto-4-alkanones were obtained by lipase-catalyzed kinetic resolution of the corresponding racemic 2-acetylthio-4-alkanones. Their configurations were assigned via vibrational circular dichroism and 1H NMR anisotropy based methods. Odor thresholds and odor qualities were determined by capillary gas chromatography-olfactometry using chiral stationary phases. There were minima of the odor thresholds for the chain lengths C7 and C8. Except for chain length C8, the enantiomers of the other homologues showed similar odor thresholds. The odor qualities ranged from pungent (C5) to mushroom (C9 and C10) and were similar to those known for the corresponding 1-alken-3-ones with one less C atom. In contrast to their positional isomers (4-mercapto-2-alkanones), the investigated 2-mercapto-4-alkanones do not meet the requirements of a "tropical olfactophore" (i.e., compounds possessing a 1,3-oxygen-sulfur functionality and specific arrangements of the substituents).

Structure-inspired design of a sphingolipid mimic sphingosine-1-phosphate receptor agonist from a naturally occurring sphingomyelin synthase inhibitor.

Ginkgolic acid obtained as a sphingomyelin synthase inhibitor from a plant extract library inspired the concept of sphingolipid mimics. Ginkgolic acid-derived N-acyl anilines and ginkgolic acid 2-phosphate (GA2P) respectively mimic ceramide and sphingosine 1-phosphate (S1P) in structure and function. The GA2P-induced phosphorylation of ERK and internalization of S1P receptor 1 (S1P1) indicated potent agonist activity. Docking studies revealed that GA2P adopts a similar binding conformation to the bound ligand ML5, which is a strong antagonist of S1P1.

Reducing Molecular Flexibility by Cyclization for Elucidation of Absolute Configuration by CD Calculations: Daurichromenic Acid.

Circular dichroism (CD) calculations of flexible natural products have been difficult because of the large number of low-energy conformers and ambiguous Boltzmann distributions. In this article, through electronic (ECD) and vibrational (VCD) studies on a natural product, (+)-daurichromenic acid, we demonstrate that derivatization of a flexible molecule can dramatically reduce its flexibility. This work also shows the usefulness of derivatization for diminishing computational expenses required for optimization and CD calculations, and for increasing the reliability of the assignment of absolute configuration. Chirality 28:453-459, 2016. © 2016 Wiley Periodicals, Inc.

Stereochemistry of a Rhododaurichromanic Acid Derivative.

A rhododaurichromanic acid A derivative was synthesized from the naturally occurring daurichromenic acid in enantiomeric pure form. Its absolute configuration was elucidated by applying VCD, ECD and DFT calculations.

Garcinol sensitizes human head and neck carcinoma to cisplatin in a xenograft mouse model despite downregulation of proliferative biomarkers.

Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated whether garcinol, a polyisoprenylated benzophenone can chemosensitize HNSCC to cisplatin. We found that garcinol inhibited the viability of a panel of diverse HNSCC cell lines, enhanced the apoptotic effect of cisplatin, suppressed constitutive as well as cisplatin-induced NF-κB activation, and downregulated the expression of various oncogenic gene products (cyclin D1, Bcl-2, survivin and VEGF). In vivo study showed that administration of garcinol alone (0.5 mg/kg body weight, i.p. five times/week) significantly suppressed the growth of the tumor, and this effect was further increased by cisplatin. Both the markers of proliferation index (Ki-67) and microvessel density (CD31) were downregulated in tumor tissues by the combination of cisplatin and garcinol. The pharmacokinetic results of garcinol indicated that good systemic exposure was achievable after i.p. administration of garcinol at 0.5 mg/kg and 2 mg/kg with mean peak concentration (Cmax) of 1825.4 and 6635.7 nM in the mouse serum, respectively. Overall, our results suggest that garcinol can indeed potentiate the effects of cisplatin by negative regulation of various inflammatory and proliferative biomarkers.

Naphthoquinone-mediated inhibition of lysine acetyltransferase KAT3B/p300, basis for non-toxic inhibitor synthesis.

Hydroxynaphthoquinone-based inhibitors of the lysine acetyltransferase KAT3B (p300), such as plumbagin, are relatively toxic. Here, we report that free thiol reactivity and redox cycling properties greatly contribute to the toxicity of plumbagin. A reactive 3rd position in the naphthoquinone derivatives is essential for thiol reactivity and enhances redox cycling. Using this clue, we synthesized PTK1, harboring a methyl substitution at the 3rd position of plumbagin. This molecule loses its thiol reactivity completely and its redox cycling ability to a lesser extent. Mechanistically, non-competitive, reversible binding of the inhibitor to the lysine acetyltransferase (KAT) domain of p300 is largely responsible for the acetyltransferase inhibition. Remarkably, the modified inhibitor PTK1 was a nearly non-toxic inhibitor of p300. The present report elucidates the mechanism of acetyltransferase activity inhibition by 1,4-naphthoquinones, which involves redox cycling and nucleophilic adduct formation, and it suggests possible routes of synthesis of the non-toxic inhibitor.