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.

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.

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.

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.

Clarifying the Use of Benzylidene Protecting Group for D-(+)-Ribono-1,4-Lactone, an Essential Building Block in the Synthesis of C-Nucleosides.

In the last two years, nucleosides analogues, a class of well-established bioactive compounds, have been the subject of renewed interest from the scientific community thanks to their antiviral activity. The COVID-19 global pandemic, indeed, spread light on the antiviral drug Remdesivir, an adenine C-nucleoside analogue. This new attention of the medical community on Remdesivir prompts the medicinal chemists to investigate once again C-nucleosides. One of the essential building blocks to synthetize these compounds is the D-(+)-ribono-1,4-lactone, but some mechanistic aspects linked to the use of different carbohydrate protecting groups remain unclear. Here, we present our investigations on the use of benzylidene as a ribonolactone protecting group useful in the synthesis of C-purine nucleosides analogues. A detailed 1D and 2D NMR structural study of the obtained compounds under different reaction conditions is presented. In addition, a molecular modeling study at the B3LYP/6-31G* level of theory with the SM8 solvation model for CHCl3 and DMSO to support the obtained results is used. This study allows for clarifying mechanistic aspects as the side reactions and structural rearrangements liked to the use of the benzylidene protecting group.

A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase.

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.

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.

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.

Analysis of hydroxy-cocaine metabolites as evidence of cocaine consumption: Identification by parent ion search and quantitation by UHPLC-MS/MS in hair.

Over the last decade, hair analysis has become a routine procedure in most forensic laboratories and, complementary to blood and urine, hair is a unique biological matrix which gives the opportunity to establish a temporal consumption profile. Despite hair is widely used to identify drug use, environmental contamination continues to represent a challenging factor of this procedure, especially for cocaine (COC). In the last few years several strategies have been proposed in order to distinguish between actual use and external contamination, however the commonly detected COC metabolites probably are insufficient for demonstrating cocaine use through hair testing. Thus, the aim of this study is to develop an ultra high performance liquid cromatography - tandem mass spectrometry (UHPLC-MS/MS) method able to detect and quantify hydroxy-COC metabolites, as specific markers of COC abuse, in hair samples from COC consumers, thus enabling unambiguous evidence of COC consumption. At the beginning, since no commercial reference materials were available, COC-positive hair samples were tested using parent ion scan-based analysis to extract hydroxy COC metabolites target ions. Once identified, the reference materials were synthesized by our analytical laboratory allowing the development of the first UHPLC-MS/MS validated method to quantify p- and m-isomers of hydroxy COC, as well as hydroxy benzoylecgonine (BE) and hydroxy norcocaine (NCOC). The method was successfully applied to a large number of COC-positive hair samples and introduced into a routine procedure for testing drug ingestion in order to evaluate for the first-time hydroxy metabolites of COC ranges in hair and their correlation with COC and BE.

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.

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.

Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade.

Elucidation of several neglected reactions in the GC-MS identification of sialic acids as heptafluorobutyrates calls for an urgent reassessment of previous claims.

The current analytical protocol used for the GC-MS determination of free or 1,7-lactonized natural sialic acids (Sias), as heptafluorobutyrates, overlooks several transformations. Using authentic reference standards and by combining GC-MS and NMR analyses, flaws in the analytical protocol were pinpointed and elucidated, thus establishing the scope and limitations of the method. It was demonstrated that (a) Sias 1,7-lactones, even if present in biological samples, decompose under the acidic hydrolysis conditions used for their release; (b) Sias 1,7-lactones are unpredicted artifacts, accidentally generated from their parent acids; (c) the N-acetyl group is quantitatively exchanged with that of the derivatizing perfluorinated anhydride; (d) the partial or complete failure of the Sias esterification-step with diazomethane leads to the incorrect quantification and structure attribution of all free Sias. While these findings prompt an urgent correction and improvement of the current analytical protocol, they could be instrumental for a critical revision of many incorrect claims reported in the literature.

Chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines formed by cross-link of bone and synovium collagen.

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

A simple synthesis of N-perfluoroacylated and N-acylated glycals of neuraminic acid with a cyclic aminic substituent at the 4α position as possible inhibitors of sialidases.

A simple protocol for the synthesis of N-perfluoroacylated and N-acylated glycals of neuraminic acid, with a secondary cyclic amine (morpholine or piperidine) at the 4α position, has been set-up, starting from peracetylated N-acetylneuraminic acid methyl ester that undergoes, sequentially to its direct N-transacylation followed by a C-4 amination, a ß-elimination, and a selective hydrolysis of the ester functions, without affecting the sensitive perfluorinated amide.

Simultaneous free and glycosylated pyridinium crosslink determination in urine: validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard.

Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free+bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (n=20, aged 27-41) and girls (n=20, aged 5-10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (p<0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (

Chemoselective synthesis of sialic acid 1,7-lactones.

The chemoselective synthesis of the 1,7-lactones of N-acetylneuraminic acid, N-glycolylneuraminic acid, and 3-deoxy-d-glycero-d-galacto-nononic acid is accomplished in two steps: a simple treatment of the corresponding free sialic acid with benzyloxycarbonyl chloride and a successive hydrogenolysis of the formed 2-benzyloxycarbonyl 1,7-lactone. The instability of the 1,7-lactones to protic solvents has been also evidenced together with the rationalization of the mechanism of their formation under acylation conditions. The results permit to dispose of authentic 1,7-sialolactones to be used as reference standards and of a procedure useful for the preparation of their isotopologues to be used as inner standards in improved analytical procedures for the gas liquid chromatography-mass spectrometry (GLC-MS) analysis of 1,7-sialolactones in biological media.