Single‐Entity Heparan Sulfate Glycomimetic Clusters for Therapeutic Applications

Heparan sulfate (HS) is a highly sulfated glycosaminoglycan with a variety of critical functions in cell signaling and regulation. HS oligosaccharides can mimic or interfere with HS functions in biological systems; however, their exploitation has been hindered by the complexity of their synthesis. Polyvalent displays of small specific HS structures on dendritic cores offer more accessible constructs with potential advantages as therapeutics, but the synthesis of single‐entity HS polyvalent compounds has not previously been described. Herein we report the synthesis of a novel targeted library of single‐entity glycomimetic clusters capped with varied HS saccharides. They have the ability to mimic longer natural HS saccharides in their inhibition of the Alzheimer’s disease (AD) protease BACE‐1. We have identified several single‐entity HS clusters with IC₅₀ values in the low‐nanomolar range. These HS clusters are drug leads for AD and offer a novel framework for the manipulation of heparan sulfate–protein interactions in general.     

Expedient Synthesis of Core Disaccharide Building Blocks from Natural Polysaccharides for Heparan Sulfate Oligosaccharide Assembly

The complex sulfation motifs of heparan sulfate glycosaminoglycans (HS GAGs) play critical roles in many important biological processes. However, an understanding of their specific functions has been hampered by an inability to synthesize large numbers of diverse, yet defined, HS structures. Herein, we describe a new approach to access the four core disaccharides required for HS/heparin oligosaccharide assembly from natural polysaccharides. The use of disaccharides rather than monosaccharides as minimal precursors greatly accelerates the synthesis of HS GAGs, providing key disaccharide and tetrasaccharide intermediates in about half the number of steps compared to traditional strategies. Rapid access to such versatile intermediates will enable the generation of comprehensive libraries of sulfated oligosaccharides for unlocking the “sulfation code” and understanding the roles of specific GAG structures in physiology and disease.     

Total Synthesis of Camptothecin and Related Natural Products by a Flexible Strategy

A flexible strategy for constructing natural products containing indolizinone or quinolizinone scaffolds and their analogues was developed, which was based on a cascade exo hydroamination followed by spontaneous lactamization. This method was applied in the total synthesis of camptothecin in nine steps in a new ring‐forming approach. It was also used to efficiently prepare five biogenetically or structurally related natural alkaloids, including 22‐hydroxyacuminatine, oxypalmatine, norketoyobyrine, naucleficine, and nauclefine, as well as 35 natural‐product‐like molecules. We believe that this method and the small‐molecule library prepared with it can open new avenues for studying the bioactivity of camptothecin and Nauclea natural products.     

Conformation and size of humic substances: Effects of major cation concentration and type,pH, salinity,and residence time

The macromolecular structure of humic substances (HS), i.e. molecular size, shape, conformation, and weight is believed to control key physico-chemical reactions in aquatic systems. However, these properties of HS are still deeply a matter of debate. Furthermore, they are affected by solution physico-chemical parameters such as ionic strength, pH, and chemical composition, etc. To better understand aggregation processes of HS in natural waters, the effects of cation concentration and type, water composition, and pH on the size and conformation of the Suwannee River Humic Acid (SRHA) were investigated with synthetic solutions. The size and the conformation of the SRHA in NaCl and CaCl₂ solutions at different concentrations (0.001–0.5 M) and pH (4.5–9.3) were determined using the photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. In addition, dissolved organic matter (DOM) samples collected from the Adour estuary along a salinity gradient were imaged by TEM. The average size of the SRHA measured by PCS increased with pH and ionic strength. This behavior was confirmed by TEM micrographs. The increase in pH and CaCl₂ engendered aggregation of the SRHA. Two aggregation steps were identified, a big increase in HS size occurred for small changes in CaCl₂ concentrations in the range <0.02 M CaCl₂, and a slight increase occured for higher concentrations in the range >0.02 M CaCl₂. The parallel analysis of SRHA by PCS and TEM allowed overcoming the limits for both techniques.Comparable results were found for the DOM samples from the Adour estuary (South West of France). The conformation and size of the HS network of these samples varied with the salinity gradient. TEM images demonstrated that, HS have a branched open network in freshwater whereas they have a more compacted and close network with increasing salinity.     

Free volume hypothetical scanning molecular dynamics method for the absolute free energy of liquids

The hypothetical scanning (HS) method is a general approach for calculating the absolute entropy, S, and free energy, F, by analyzing Boltzmann samples obtained by Monte Carlo (MC) or molecular dynamics (MD) techniques. With HS applied to a fluid, each configuration i of the sample is reconstructed by gradually placing the molecules in their positions at i using transition probabilities (TPs). With our recent version of HS, called HSMC-EV, each TP is calculated from MC simulations, where the simulated particles are excluded from the volume reconstructed in previous steps. In this paper we remove the excluded volume (EV) restriction, replacing it by a "free volume" (FV) approach. For liquid argon, HSMC-FV leads to an improvement in efficiency over HSMC-EV by a factor of 2-3. Importantly, the FV treatment greatly simplifies the HS implementation for liquids, allowing a much more natural application of the method for MD simulations. Given the success and popularity of MD, the present development of the HSMD method for liquids is an important advancement for HS methodology. Results for the HSMD-FV approach presented here agree well with our HSMC and thermodynamic integration results. The efficiency of HSMD-FV is equivalent to HSMC-EV. The potential use of HSMC(MD)-FV in protein systems with explicit water is discussed.     

Synthesis of a Systematic 64-Membered Heparan Sulfate Tetrasaccharide Library

Heparan sulfate (HS) has multifaceted biological activities. To date, no libraries of HS oligosaccharides bearing systematically varied sulfation structures are available owing to the challenges in synthesizing a large number of HS oligosaccharides. To overcome the obstacles and expedite the synthesis, a divergent approach was designed, where 64 HS tetrasaccharides covering all possible structures of 2-O-, 6-O- and N-sulfation with the glucosamine-glucuronic acid-glucosamine-iduronic acid backbone were successfully produced from a single strategically protected tetrasaccharide intermediate. This extensive library helped identify the structural requirements for HS sequences to have strong fibroblast growth factor-2 binding but a weak affinity for platelet factor-4. Such a strategy to separate out these two interactions could lead to new HS-based potential therapeutics without the dangerous adverse effect of heparin-induced thrombocytopenia.     

Design and synthesis of a diverse morpholine template library

Efficient and general procedures have been developed for the solution-phase preparation of substituted morpholine derivatives, and a library has been produced around generic structure 1. This library was designed with proprietary modeling software for use as a general screening library. The 30 R1 reagents were phenols, and the 275 R2 reagents were taken from five different reagent classes, giving a variety of product classes in the final library of 8250 potential products. All of the library members were generated from a common intermediate, mesylate (5), which was synthesized efficiently, in bulk, in three steps from N-benzylethanolamine (2). High-throughput chemistry using robotics was carried out to produce the 7907 library members, which were individually characterized by reversed-phase LC/MS analysis.     

Chemoinformatics methods for systematic comparison of molecules from natural and synthetic sources and design of hybrid libraries

Until recently, the field of diversity and library design has more or less ignored natural products as a compound source. This is probably due to at least two reasons. First, combinatorial and reaction-based approaches have been major focal points in the early days of computational library design. In addition, a widespread view is that natural products are often highly complex and not amenable to medicinal chemistry efforts. This contribution introduces recent computational approaches to systematically analyze natural molecules and bridge the gap between natural products and synthetic chemistry programs. Large scale comparisons of natural and synthetic molecules are discussed as well as studies designed to identify `synthetic mimics' of natural products with specific activity. In addition, a concept for the design of natural/synthetic hybrid libraries is introduced. Although research in this area is still in its early stages, an important lesson to be learned from computational analyses is that there is no need to a priori `shy away' from natural products as a source for molecular design.     

Chemical Structure and Biological Activity of Humic Substances Define Their Role as Plant Growth Promoters

Humic substances (HS) are dominant components of soil organic matter and are recognized as natural, effective growth promoters to be used in sustainable agriculture. In recent years, many efforts have been made to get insights on the relationship between HS chemical structure and their biological activity in plants using combinatory approaches. Relevant results highlight the existence of key functional groups in HS that might trigger positive local and systemic physiological responses via a complex network of hormone-like signaling pathways. The biological activity of HS finely relies on their dosage, origin, molecular size, degree of hydrophobicity and aromaticity, and spatial distribution of hydrophilic and hydrophobic domains. The molecular size of HS also impacts their mode of action in plants, as low molecular size HS can enter the root cells and directly elicit intracellular signals, while high molecular size HS bind to external cell receptors to induce molecular responses. Main targets of HS in plants are nutrient transporters, plasma membrane H⁺-ATPases, hormone routes, genes/enzymes involved in nitrogen assimilation, cell division, and development. This review aims to give a detailed survey of the mechanisms associated to the growth regulatory functions of HS in view of their use in sustainable technologies.     

A solution- and solid-phase approach to tetrahydroquinoline-derived polycyclics having a 10-membered ring

With the goal of library generation using a polycyclic derivative 5 having an enamide functional group, a simple and practical, enantioselective synthesis of tetrahydroquinoline derivative 2 was achieved. The phenolic hydroxyl group in compound 2 was utilized as an anchoring site for solid-phase synthesis. The ring closing metathesis approach yielded the desired polycyclic product 5 on solid phase in five steps (overall 40% yield). Compound 5 is a novel scaffold for the library generation of natural product-like polycyclics having a functionalized medium ring for obtaining a new class of small molecules to be utilized as chemical probes.     

Photoreduction of Terrigenous Fe‐Humic Substances Leads to Bioavailable Iron in Oceans

Humic substances (HS) are important iron chelators responsible for the transport of iron from freshwater systems to the open sea, where iron is essential for marine organisms. Evidence suggests that iron complexed to HS comprises the bulk of the iron ligand pool in near‐coastal waters and shelf seas. River‐derived HS have been investigated to study their transport to, and dwell in oceanic waters. A library of iron model compounds and river‐derived Fe‐HS samples were probed in a combined X‐ray absorption spectroscopy (XAS) and valence‐to‐core X‐ray emission spectroscopy (VtC‐XES) study at the Fe K‐edge. The analyses performed revealed that iron complexation in HS samples is only dependent on oxygen‐containing HS functional groups, such as carboxyl and phenol. The photoreduction mechanism of Fe^III‐HS in oceanic conditions into bioavailable aquatic Fe^II forms, highlights the importance of river‐derived HS as an iron source for marine organisms. Consequently, such mechanisms are a vital component of the upper‐ocean iron biogeochemistry cycle.     

Synthesis of a Targeted Library of Heparan Sulfate Hexa‐ to Dodecasaccharides as Inhibitors of β‐Secretase: Potential Therapeutics for Alzheimer’s Disease

Heparan sulfates (HS) are a class of sulfated polysaccharides that function as dynamic biological regulators of the functions of diverse proteins. The structural basis of these interactions, however, remains elusive, and chemical synthesis of defined structures represents a challenging but powerful approach for unravelling the structure–activity relationships of their complex sulfation patterns. HS has been shown to function as an inhibitor of the β‐site cleaving enzyme β‐secretase (BACE1), a protease responsible for generating the toxic Aβ peptides that accumulate in Alzheimer’s disease (AD), with 6‐O‐sulfation identified as a key requirement. Here, we demonstrate a novel generic synthetic approach to HS oligosaccharides applied to production of a library of 16 hexa‐ to dodecasaccharides targeted at BACE1 inhibition. Screening of this library provided new insights into structure–activity relationships for optimal BACE1 inhibition, and yielded a number of potent non‐anticoagulant BACE1 inhibitors with potential for development as leads for treatment of AD through lowering of Aβ peptide levels.     

Structural Insights into Pixatimod (PG545) Inhibition of Heparanase,a Key Enzyme in Cancer and Viral Infections

Pixatimod (PG545), a heparan sulfate (HS) mimetic and anticancer agent currently in clinical trials, is a potent inhibitor of heparanase. Heparanase is an endo-β-glucuronidase that degrades HS in the extracellular matrix and basement membranes and is implicated in numerous pathological processes such as cancer and viral infections, including SARS−CoV-2. To understand how PG545 interacts with heparanase, we firstly carried out a conformational analysis through a combination of NMR experiments and molecular modelling which showed that the reducing end β-D-glucose residue of PG545 adopts a distorted conformation. This was followed by docking and molecular dynamics simulations to study the interactions of PG545 with heparanase, revealing that PG545 is able to block the active site by binding in different conformations, with the cholestanol side-chain making important hydrophobic interactions. While PG545 blocks its natural substrate HS from binding to the active site, small synthetic heparanase substrates are only partially excluded, and thus pentasaccharide or larger substrates are preferred for assaying this class of inhibitor. This study provides new insights for the design of next-generation heparanase inhibitors and substrates.     

Efficient synthetic method of Psammaplin A

A new concise and efficient synthetic method of Psammaplin A was developed. Psammaplin A was obtained with 50% overall yield in nine steps from p-hydroxybenzaldehyde and ethyl acetoacetate via Knoevenagel condensation and direct nitrosation as key steps. This method might be very efficient to construct a quite diverse library of Psammaplin A type analogs.     

Solution- and solid-phase synthesis of natural product-like tetrahydroquinoline-based polycyclics having a medium size ring

A solid-phase synthesis of tetrahydroquinoline-derived polycyclic 4, having a medium size ring with an enamide functionality, was achieved from tetrahydroquinoline derivative 3 in five steps with overall 40-45% yield. An enantiopure, tetrahydroquinoline-derived beta-amino ester, 1, was converted into compound 2 that has a free phenolic hydroxyl group as an anchoring site for solid-phase synthesis. The solid-phase worked well for this sequence, in which the synthesis of the unsaturated eight-membered enamide lactam was obtained by a ring-closing metathesis approach. Compound 4 is a novel, natural product-like polycyclic derivative that could further be utilized in library generation for developing small molecule chemical probes.     

A rapid quantitative method for humic substances determination in natural waters

A simple and rapid method was proposed for humic substances (HS) determination at microgram levels in natural waters. This assay method is based on the binding of a dye, Toluidine Blue (TB), to HS molecules to produce a dye-HS complex, which causes a decrease in absorbance at 630 nm. This method was calibrated with HS samples with up to a concentration of 40 mg L⁻¹, which covered the range of dissolved HS concentrations present in natural waters. The detection limit was 0.8 mg L⁻¹ of HS, and the relative standard deviation of 10 replicate measurements for a 20-mg L⁻¹ standard sample was 3.5%. From the Langmuir adsorption isotherm theory, the binding equilibrium constant and total number of binding sites at neutral pH were calculated to be (8.17 ± 0.42) × 10⁵ L mol⁻¹ and N of 1.45 ± 0.04 mmol g⁻¹ HS, respectively. The determination results with five water samples from lake, river and pond were consistent with those measured with the reference methods, demonstrating that this quantification method for HS determination was rapid, sensitive and feasible.     

Silver-promoted Friedel-Crafts reaction: concise total synthesis of (-)-ardeemin, (-)-acetylardeemin and (-)-formylardeemin

Total syntheses of multidrug resistant inhibitors (-)-acetylardeemin 2a, (-)-ardeemin 2b, and (-)-formylardeemin 3 have been achieved within 10 steps starting from bromopyrroloinoline 13. The key step involves direct alkylation of 13 with prenyl tributylstannane 11 to yield 12 via a silver-promoted asymmetric Friedel-Crafts reaction. Highly efficient installation of the isoprenyl group allowed excellent overall yield. Moreover, the substrate scope of the asymmetric Friedel-Crafts reaction of 13 was expanded to include a variety of arenes 14 to afford natural product-like library analogues 15.     

Using an enzymatic combinatorial approach to identify anticoagulant heparan sulfate structures

Heparan sulfate (HS) represents a major class of glycans that perform central physiological functions. Emerging HS and glycosaminoglycan microarray techniques are used to interrogate the structure and function relationship to develop novel therapeutic agents. Availability of HS with specific sulfation patterns has been a limiting factor and impedes the accuracy of HS glycomics studies. Although organic synthesis provides oligosaccharides, these may not fully represent the biological functions of polysaccharides. Here, we present a study for developing an enzyme-based approach to synthesize a polysaccharide library with different sulfation patterns. Using different combinations of biosynthetic enzymes, we synthesized eight unique polysaccharides. We discovered that polysaccharides without the iduronic acid residue displayed strong binding affinity to antithrombin and high anti-Xa and anti-IIa activities. The enzyme-based synthetic approach could become a general method for discovering new HS structures with unique biological functions.     

Toward the assembly of heparin and heparan sulfate oligosaccharide libraries: efficient synthesis of uronic acid and disaccharide building blocks

The monosaccharide moieties found in heparin (HP) and heparan sulfate (HS), glucosamine and two kinds of uronic acids, glucuronic and iduronic acids, were efficiently synthesized by use of glucosamine hydrochloride and glucurono-6,3-lactone as starting compounds. In the synthesis of the disaccharide building block, the key issues of preparation of uronic acids (glucuronic acid and iduronic acid moieties) were achieved in 12 steps and 15 steps, respectively, without cumbersome C-6 oxidation. The resulting monosaccharide moieties were utilized to the syntheses of HP/HS disaccharide building blocks possessing glucosamine-glucuronic acid (GlcN-GlcA) or iduronic acid (GlcN-IdoA) sequences. The disaccharide building blocks were also suitable for further modification such as glycosylation, selective deprotection, and sulfation.     

Microwave-assisted synthesis and antioxidant properties of hydrazinyl thiazolyl coumarin derivatives

ABSTRACT: Coumarin derivatives exhibit a wide range of biological properties including promising antioxidant activity. Furthermore, microwave-assisted organic synthesis has delivered rapid routes to N- and O-containing heterocycles, including coumarins and thiazoles. Combining these features, the use of microwave-assisted processes will provide rapid access to a targeted coumarin library bearing a hydrazino pharmacophore for evaluation of antioxidant properties. RESULTS: Microwave irradiation promoted 3 of the 4 steps in a rapid, convergent synthesis of a small library of hydrazinyl thiazolyl coumarin derivatives, all of which exhibited significant antioxidant activity comparable to that of the natural antioxidant quercetin, as established by DPPH and ABTS radical assays. CONCLUSIONS: Microwave dielectric heating provides a rapid and expedient route to a series of hydrazinyl thiazolyl coumarins to investigate their radical scavenging properties. Given their favourable properties, in comparison with known antioxidants, these coumarin derivatives are promising leads for further development and optimization.