Analysis of the intramolecular 1,7-lactone of N-acetylneuraminic acid using HPLC-MS: relationship between detection and stability.

A subclass of the sialic acid family consists of intramolecular lactones that may function as key indicators of physiological and pathological states. However, the existence of these compounds in free form is highly improbable, since they are unlikely to exist in an aqueous solution due to their lability. Current analytical method used to detect them in biological fluids has not recognized their reactivity in solution and is prone to misidentification. However, recent advances in synthetic methods for 1,7-lactones have allowed the preparation of these sialic acid derivatives as authentic reference standards. We report here the development of a new HPLC-MS method for the simultaneous detection of the 1,7-lactone of N-acetylneuraminic acid, its γ-lactone derivative, and N-acetylneuraminic acid that overcomes the limitations of the previous analytical procedure for their identification.

Small-Molecules as Chemiluminescent Probes to Detect Lipase Activity.

The set-up of highly sensitive detection tools to evaluate lipase activity remains a central goal in different fields. In this context, we proposed new chemiluminescent 1,2-dioxetane luminophores, sharing an octanoyl triggerable group, to monitor lipase activity. We herein report the synthesis and both the evaluation of their luminescence emission profile and their enzyme-substrate specificity, generated by three different commercial lipases (Candida cylindracea, Pseudomonas fluorescens, and Mucor miehei) and one esterase (porcine liver esterase, PLE, as a literature control). Remarkably, the present study confirmed the applicability of these 1,2-dioxetane luminophores as (i) highly efficient, broad-range, chemiluminescent probes for the detection and the enzymatic activity evaluation of lipases and as (ii) promising candidates for the future development of both flash- and glow-type luminescence assays.

Human Sarcopenic Myoblasts Can Be Rescued by Pharmacological Reactivation of HIF-1α.

Sarcopenia, an age-related decline in muscle mass and strength, is associated with metabolic disease and increased risk of cardiovascular morbidity and mortality. It is associated with decreased tissue vascularization and muscle atrophy. In this work, we investigated the role of the hypoxia inducible factor HIF-1α in sarcopenia. To this end, we obtained skeletal muscle biopsies from elderly sarcopenic patients and compared them with those from young individuals. We found a decrease in the expression of HIF-1α and its target genes in sarcopenia, as well as of PAX7, the major stem cell marker of satellite cells, whereas the atrophy marker MURF1 was increased. We also isolated satellite cells from muscle biopsies and cultured them in vitro. We found that a pharmacological activation of HIF-1α and its target genes caused a reduction in skeletal muscle atrophy and activation of PAX7 gene expression. In conclusion, in this work we found that HIF-1α plays a role in sarcopenia and is involved in satellite cell homeostasis. These results support further studies to test whether pharmacological reactivation of HIF-1α could prevent and counteract sarcopenia.

Neu3 Sialidase Activates the RISK Cardioprotective Signaling Pathway during Ischemia and Reperfusion Injury (IRI).

Coronary reperfusion strategies are life-saving approaches to restore blood flow to cardiac tissue after acute myocardial infarction (AMI). However, the sudden restoration of normal blood flow leads to ischemia and reperfusion injury (IRI), which results in cardiomyoblast death, irreversible tissue degeneration, and heart failure. The molecular mechanism of IRI is not fully understood, and there are no effective cardioprotective strategies to prevent it. In this study, we show that activation of sialidase-3, a glycohydrolytic enzyme that cleaves sialic acid residues from glycoconjugates, is cardioprotective by triggering RISK pro-survival signaling pathways. We found that overexpression of Neu3 significantly increased cardiomyoblast resistance to IRI through activation of HIF-1α and Akt/Erk signaling pathways. This raises the possibility of using Sialidase-3 activation as a cardioprotective reperfusion strategy after myocardial infarction.

The antithetic role of ceramide and sphingosine-1-phosphate in cardiac dysfunction.

Cardiovascular diseases (CVDs) are the leading cause of death globally and the number of cardiovascular patients, which is estimated to be over 30 million in 2018, represent a challenging issue for the healthcare systems worldwide. Therefore, the identification of novel molecular targets to develop new treatments is an ongoing challenge for the scientific community. In this context, sphingolipids (SLs) have been progressively recognized as potent bioactive compounds that play crucial roles in the modulation of several key biological processes, such as proliferation, differentiation, and apoptosis. Furthermore, SLs involvement in cardiac physiology and pathophysiology attracted much attention, since these molecules could be crucial in the development of CVDs. Among SLs, ceramide and sphingosine-1-phosphate (S1P) represent the most studied bioactive lipid mediators, which are characterized by opposing activities in the regulation of the fate of cardiac cells. In particular, maintaining the balance of the so-called ceramide/S1P rheostat emerged as an important novel therapeutical target to counteract CVDs. Thus, this review aims at critically summarizing the current knowledge about the antithetic roles of ceramide and S1P in cardiomyocytes dysfunctions, highlighting how the modulation of their metabolism through specific molecules, such as myriocin and FTY720, could represent a novel and interesting therapeutic approach to improve the management of CVDs.

2ß-3,4-Unsaturated sialic acid derivatives: Synthesis optimization, and biological evaluation as Newcastle disease virus hemagglutinin-neuraminidase inhibitors.

The optimization of the synthetic protocol to obtain the 3,4-unsaturated sialic acid derivatives, through the fine-tuning of both the Ferrier glycosylation conditions and the subsequent hydrolysis work-up, is herein reported. The accomplishment of the desired ß-anomers and some selected α-ones, in pure form, led us to evaluate their specific inhibitory activity towards NDV-HN and human sialidase NEU3. Importantly, the resulting data allowed the identification, for the first time, of three active 3,4-unsaturated sialic acid analogs, showing IC50 values against NDV-HN in the micromolar range.

Intramolecular Lactones of Sialic Acids.

The so-called "sialo-chemical-biology" has become an attractive research area, as an increasing number of natural products containing a sialic acid moiety have been shown to play important roles in biological, pathological, and immunological processes. The intramolecular lactones of sialic acids are a subclass from this crucial family that could have central functions in the discrimination of physiological and pathological conditions. In this review, we report an in-depth analysis of the synthetic achievements in the preparation of the intramolecular lactones of sialic acids (1,4-, 1,7- and γ-lactones), in their free and/or protected form. In particular, recent advances in the synthesis of the 1,7-lactones have allowed the preparation of key sialic acid derivatives. These compounds could be used as authentic reference standards for their correct determination in biological samples, thus overcoming some of the limitations of the previous analytical procedures.

Lactonization Method To Assign the Anomeric Configuration of the 3,4-Unsaturated Congeners of N-Acetylneuraminic Acid.

Assigning the correct configuration at C2 in sialosides is a standing problem because of the absence of an anomeric hydrogen. All different empirical rules that have been proposed over the years lack general applicability. In particular, the correct configuration of several 3,4-unsaturated derivatives of N-acetylneuraminic acid (Neu5Ac), which have been recently shown to be novel sialidase/neuraminidase inhibitors, could only be tentatively assigned by similarity with the reported 3,4-unsaturated 2O-methyl sialosides. In this work, we overcome this problem as we devised a rapid synthetic method to unequivocally resolve the anomeric configuration of the 3,4-unsaturated Neu5Ac derivatives through the synthesis of the corresponding unreported unsaturated 1,7-lactones. Moreover, we discovered a diagnostic 13C nuclear magnetic resonance signal that allows the formulation of a new empirical rule for the direct assignment of the C2 stereochemistry of these molecules, even when only one of the two C2 epimers is available.

Interplay of Modified Sialic Acid Inhibitors and the Human Parainfluenza Virus 1 Hemagglutinin-Neuraminidase Active Site.

In the search for effective antivirals against Paramyxoviridae, the dynamics of human parainfluenza virus type 1 hemagglutinin-neuraminidase (hPIV1-HN) inhibition offers a promising perspective. This study focuses on the potential of C5- and C4-modified 2,3-unsaturated sialic acid (DANA) inhibitors and highlights their interaction with the hPIV1-HN enzyme. We show that a strategic substitution, replacing the C5 isopropyl group in BCX 2798 with a trifluoroacetyl function, increases inhibitory potency 3- to 4-fold. At the same time, we explore the special properties of the catalytic site of hPIV1-HN, which harbors only small substituents and favors a C4 sulfonylamido function over a carbonyl function, in contrast to the C4 pocket of Newcastle disease virus hemagglutinin-neuraminidase (NDV-HN). Based on these findings, we present a newly identified potent inhibitor that has the preferred C5 trifluoroacetamido and C4 trifluorosulfonylamide groups. The results of this study pave the way for a deeper understanding of the C4 and C5 binding pockets of hPIV1-HN and promote the development of new, more selective inhibitors.

Design, Synthesis, and Antiviral Evaluation of Sialic Acid Derivatives as Inhibitors of Newcastle Disease Virus Hemagglutinin-Neuraminidase: A Translational Study on Human Parainfluenza Viruses.

Global infections with viruses belonging to the Paramyxoviridae, such as Newcastle disease virus (NDV) or human parainfluenza viruses (hPIVs), pose a serious threat to animal and human health. NDV-HN and hPIVs-HN (HN hemagglutinin-neuraminidase) share a high degree of similarity in catalytic site structures; therefore, the development of an efficient experimental NDV host model (chicken) may be informative for evaluating the efficacy of hPIVs-HN inhibitors. As part of the broad research in pursuit of this goal and as an extension of our published work on antiviral drug development, we report here the biological results obtained with some newly synthesized C4- and C5-substituted 2,3-unsaturated sialic acid derivatives against NDV. All developed compounds showed high neuraminidase inhibitory activity (IC50 0.03-13 µM). Four molecules (9, 10, 23, 24) confirmed their high in vitro inhibitory activity, which caused a significant reduction of NDV infection in Vero cells, accompanied by very low toxicity.

Sphingolipids and Atherosclerosis: The Dual Role of Ceramide and Sphingosine-1-Phosphate.

Sphingolipids are bioactive molecules that play either pro- and anti-atherogenic roles in the formation and maturation of atherosclerotic plaques. Among SLs, ceramide and sphingosine-1-phosphate showed antithetic properties in regulating various molecular mechanisms and have emerged as novel potential targets for regulating the development of atherosclerosis. In particular, maintaining the balance of the so-called ceramide/S1P rheostat is important to prevent the occurrence of endothelial dysfunction, which is the trigger for the entire atherosclerotic process and is strongly associated with increased oxidative stress. In addition, these two sphingolipids, together with many other sphingolipid mediators, are directly involved in the progression of atherogenesis and the formation of atherosclerotic plaques by promoting the oxidation of low-density lipoproteins (LDL) and influencing the vascular smooth muscle cell phenotype. The modulation of ceramide and S1P levels may therefore allow the development of new antioxidant therapies that can prevent or at least impair the onset of atherogenesis, which would ultimately improve the quality of life of patients with coronary artery disease and significantly reduce their mortality.

Hexahydrocannabinol on the Light Cannabis Market: The Latest "New" Entry.

Introduction: Hexahydrocannabinols (HHCs), referred to as (9R)-HHC and (9S)-HHC diastereoisomers, are poorly studied cannabinoids naturally found in small concentrations in the pollen and the seeds of the hemp plants. Aim: In this study, for the first time, we describe the finding of (9R)-HHC and (9S)-HHC in two commercialized hemp derived products. Methods: The achievement of reference standards by semisynthetic or isolation approach allows us to develop and validate a gas chromatography mass spectrometry method for the identification and quantification of HHCs in hemp-derived resin. Results: The two analyzed samples showed percentage of 42.5 and 41.5 for (9R)-HHC and of 23.6 and 23.6 for (9S)-HHC. Conclusions: Despite the lack of in-depth studies about HHCs activity, potency, toxicity, and safety, these cannabinoids are emerging on the light-cannabis (hemp) market probably because legislations still do not clearly regulate them. Since analytical assay for hemp-derived products usually include only Δ9-THC, THC-A, CBD, and CBD-A, a thorough investigation could be carried out to reveal the possible addition of "new" compounds that might be a matter of safety.

The acidic hydrolysis of N-acetylneuraminic 4,5-oxazoline allows a direct functionalization of the C5 position of Neu5Ac2en (DANA).

The acidic hydrolysis of N-acetylneuraminic 4,5-oxazoline affords the corresponding 2,3-unsaturated amino ester, which was not previously detected (nor isolated) due to the unexpected rearrangement into its corresponding alcohol. In this work, we unveiled the mechanism of these reactions and optimized the conditions to obtain either synthetic intermediate.

Potent Inhibitors against Newcastle Disease Virus Hemagglutinin-Neuraminidase.

Neuraminidase activity is essential for the infection and propagation of paramyxoviruses, including human parainfluenza viruses (hPIVs) and the Newcastle disease virus (NDV). Thus, many inhibitors have been developed based on the 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid inhibitor (DANA) backbone. Along this line, herein we report a series of neuraminidase inhibitors, having C4 (p-toluenesulfonamido and azido substituents) and C5 (N-perfluorinated chains) modifications to the DANA backbone, resulting in compounds with 5- to 15-fold greater potency than the currently most active compound, the N-trifluoroacetyl derivative of DANA (FANA), toward the NDV hemagglutinin-neuraminidase (NDV-HN). Remarkably, these inhibitors were found to be essentially inactive against the human sialidase NEU3, which is present on the outer layer of the cell membrane and is highly affected by the current NDV inhibitor FANA.