Towards the semi-synthesis of phosphorylated mimics of glycosaminoglycans: Screening of methods for the regioselective phosphorylation of chondroitin

Glycosaminoglycan (GAG) mimics carrying phosphate rather than sulfate anionic groups have been poorly investigated, in spite of their interesting perspectives. While some GAG-mimicking phosphorylated polymers have been reported, to the best of our knowledge no phosphorylated polysaccharides having the same backbone of natural sulfated GAGs have been accessed yet. To fill this gap, in this work two standard phosphorylation protocols and two recently reported procedures have been screened on a set of polysaccharide species composed by microbial sourced chondroitin and three partially protected, semi-synthetic derivatives thereof. A detailed structural characterization by 1H, 13C and 31P NMR spectroscopy revealed the higher versatility of the innovative, biomimetic reaction employing monopotassium salt of phosphoenolpyruvate (PEP–K) with respect to standard phosphorylating agents (phosphoric acid or phosphorus oxychloride). Indeed, PEP-K and H3PO4 gave similar results in the regioselective phosphorylation of the primary hydroxyls of unprotected chondroitin, while only the former reacted on partially protected chondroitin derivatives in a controlled, regioselective fashion, affording chondroitin phosphate (CP) polysaccharides with different derivatization patterns. The reported results represent the first, key steps towards the systematic semi-synthesis of phosphorylated GAGs as a new class of GAG mimics and to the evaluation of their biological activities in comparison with native sulfated GAGs

Solvent-Free Approaches in Carbohydrate Synthetic Chemistry: Role of Catalysis in Reactivity and Selectivity

Owing to their abundance in biomass and availability at a low cost, carbohydrates are very useful precursors for products of interest in a broad range of scientific applications. For example, they can be either converted into basic chemicals or used as chiral precursors for the synthesis of potentially bioactive molecules, even including nonsaccharide targets; in addition, there is also a broad interest toward the potential of synthetic sugar-containing structures in the field of functional materials. Synthetic elaboration of carbohydrates, in both the selective modification of functional groups and the assembly of oligomeric structures, is not trivial and often entails experimentally demanding approaches practiced by specialized groups. Over the last years, a large number of solvent-free synthetic methods have appeared in the literature, often being endowed with several advantages such as greenness, experimental simplicity, and a larger scope than analogous reactions in solution. Most of these methods are catalytically promoted, and the catalyst often plays a key role in the selectivity associated with the process. This review aims to describe the significant recent contributions in the solvent-free synthetic chemistry of carbohydrates, devoting a special critical focus on both the mechanistic role of the catalysts employed and the differences evidenced so far with corresponding methods in solution

Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise “glycocodes” that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation. The lack of dedicated bioinformatic tools for mining GAG data sets contributes to a partial characterization of the structural and functional landscape and interactions of GAGs. These pending issues will benefit from the development of new approaches reviewed here, namely (i) the synthesis of GAG oligosaccharides to build large and diverse GAG libraries, (ii) GAG analysis and sequencing by mass spectrometry (e.g., ion mobility-mass spectrometry), gas-phase infrared spectroscopy, recognition tunnelling nanopores, and molecular modeling to identify bioactive GAG sequences, biophysical methods to investigate binding interfaces, and to expand our knowledge and understanding of glycocodes governing GAG molecular recognition, and (iii) artificial intelligence for in-depth investigation of GAGomic data sets and their integration with proteomics

A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate

Abstract Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core

Synthetic and semi-synthetic chondroitin sulfate oligosaccharides,polysaccharides, and glycomimetics

Chondroitin sulfate (CS) is a sulfated polysaccharide involved in a myriad of biological processes. Due to the variable sulfation pattern of CS polymer chains, the need to study in detail structure–activity relationships regarding CS biomedical features has provoked much interest in obtaining synthetic CS species. This paper reviews two decades of synthetic and semi-synthetic CS oligosaccharides, polysaccharides, and glycomimetics obtained by chemical, chemoenzymatic, enzymatic, and microbiological-chemical strategies

A new, improved synthesis of the trisaccharide repeating unit of the O-antigen from Xanthomonas campestris pv. campestris 8004

Recent studies have revealed that lipid-A and core fragments of the lipopolysaccharide from Xanthomonas campestris pv. campestris 8004 (Xcc), a phytopathogenic Gram-negative bacterium, are able to elicit plant immunity with two independent mechanisms. To date, nothing is known about the effect of the O-antigen portion. Since its separation from the core region by selective chemical degradation is very difficult, the chemical synthesis of related oligosaccharides is strictly necessary. In this paper a new, improved synthesis of the O-antigen repeating unit is presented. The main improvements in the synthesis are: (1) a shorter, high-yielding preparation of an efficient glycosyl donor of the rare sugar 3-acetamido-3,6-dideoxy-d-galactopyranose (3-acetamido-d-fucose, d-Fucp3NAc); (2) a new protecting group pattern, which is demonstrated to open a path to the future synthesis of higher oligomers