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Heparin and heparan sulfate represent key members of the glycosaminoglycan family of carbohydrates and underpin considerable repertoires of biological importance. As such, their efficiency of synthesis represents a key requirement, to further understand and exploit the H/HS structure-to-biological function axis. In this review we focus on chemical approaches to and methodology improvements for the synthesis of these essential sugars (from 2015 onwards). We first consider advances in accessing the heparin-derived pentasaccharide anticoagulant fondaparinux. This is followed by heparan sulfate targets, including key building block synthesis, oligosaccharide construction and chemical sulfation techniques. We end with a consideration of technological improvements to traditional, solution-phase synthesis approaches that are increasingly being utilised., (© 2021 The Authors. Published by The Chemical Society of Japan & Wiley-VCH GmbH.)

The dependence of development and homeostasis in animals on the interaction of hundreds of extracellular regulatory proteins with the peri- and extracellular glycosaminoglycan heparan sulfate (HS) is exploited by many microbial pathogens as a means of adherence and invasion. Heparin, a widely used anticoagulant drug, is structurally similar to HS and is a common experimental proxy. Exogenous heparin prevents infection by a range of viruses, including S-associated coronavirus isolate HSR1. Here, we show that heparin inhibits severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) invasion of Vero cells by up to 80% at doses achievable through prophylaxis and, particularly relevant, within the range deliverable by nebulisation. Surface plasmon resonance and circular dichroism spectroscopy demonstrate that heparin and enoxaparin, a low-molecular-weight heparin which is a clinical anticoagulant, bind and induce a conformational change in the spike (S1) protein receptor-binding domain (S1 RBD) of SARS-CoV-2. A library of heparin derivatives and size-defined fragments were used to probe the structural basis of this interaction. Binding to the RBD is more strongly dependent on the presence of 2- O or 6- O sulfate groups than on N -sulfation and a hexasaccharide is the minimum size required for secondary structural changes to be induced in the RBD. It is likely that inhibition of viral infection arises from an overlap between the binding sites of heparin/HS on S1 RBD and that of the angiotensin-converting enzyme 2. The results suggest a route for the rapid development of a first-line therapeutic by repurposing heparin and its derivatives as antiviral agents against SARS-CoV-2 and other members of the Coronaviridae ., Competing Interests: None declared., (Thieme. All rights reserved.)

d-Glucosamine derivatives bearing latent O4 functionality provide modified H/HS-type disaccharide donors for a final stage capping approach enabling introduction of conjugation-suitable, non-reducing terminal functionality to biologically important glycosaminoglycan oligosaccharides. Application to the synthesis of the first O4-terminus modified synthetic LMWH decasaccharide and an HS-like dodecasaccharide is reported.

L-Idofuranoside cyanohydrin 1 is converted on large scale into a mixture of L-IdoA methyl pyranosides and furanosides, which is converged to provide short 2-step routes to bicyclic [3.2.1] or [2.2.2] L-iduronate lactones. The former is obtained via a 100 g scale synthesis of 3-OBn L-IdoA. A two-step conversion of this mixture provides either pure anomer of the novel [2.2.2] l-iduronate thioglycoside lactones. Both [3.2.1] and [2.2.2] lactones are converted into GlcN-IdoA heparin precursor disaccharides. The [2.2.2] lactone enables a scalable 3-step route from 1 to a new type of highly disarmed O-4 iduronate thioglycoside, which is an effective acceptor with glucoazide thioglycoside donors. The resulting new iduronic [2.2.2] lactone disaccharides are readily rearmed by mild methanolysis to provide GlcN-IdoA thiophenyl disaccharide donors, intercepting their established utility for the assembly of both heparin- and heparan sulfate-like oligosaccharides. The [2.2.2] lactonization acts as a conformational switch to superdisarm iduronate components, reversible by lactone ring opening. In addition, the separated 2,4-diacetates also provide short access to all four anomeric and ring size isomers of l-iduronic acid methyl glycosides, including the first syntheses of the parent idofuranosides. X-ray structures are reported for a [2.2.2] iduronate lactone and examples of both methyl L-idopyranoside and novel methyl-L-idofuranoside systems.

Synthesis of an array of differentially sulfated GlcN-IdoA disaccharides, accessible on good scale, directly from l-iduronate components is described. These are specifically directed to provide the sulfation variability at the key most common biologically relevant sulfation-variable l-IdoA O-2 and d-GlcN O-6 and amino sites of this heparin disaccharide. This sulfation-varied matrix has allowed the first evaluation of using Raman/ROA spectroscopy to characterize changes in spectra as a function of both site and level of sulfation with pure, defined heparin-related disaccharide species. This provides analysis of both similarities and differences to digest native heparin and this shows evidence of different types of changes in conformations and conformational freedom as a function of some specific sulfation changes at the disaccharide level. It is anticipated that this data set will open the way for applications to further site-specific sulfated saccharides and demonstrates the capability offered by Raman-ROA towards fingerprinting sulfation in heparin fragments., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The first example of a gram-scale synthesis of a structurally defined, heparin-related dodecasaccharide is reported. An iterative 14-step process using an iduronate donor disaccharide delivers >1g quantities of the dodecasaccharide sequence [GlcNS-IdoA2S](6)-OMe in 15% overall yield from the reducing terminal disaccharide, a 2 orders of magnitude increase in scale for access to synthetic heparanoid dodecasaccharide mimetics. The synthesis also delivers multigram amounts of the protected oligosaccharides from tetra- through to dodecasaccharide.

A diastereomerically pure cyanohydrin, preparable on kilogram scale, is efficiently converted in one step into a novel L-iduronamide. A new regioselective acylation of this iduronamide and a new mild amide hydrolysis method mediated by amyl nitrite enables short, scalable syntheses of an L-iduronate diacetate C-4 acceptor, and also L-iduronate C-4 acceptor thioglycosides. Efficient conversions of these to a range of heparin-related gluco-ido disaccharide building blocks (various C-4 protection options) including efficient multigram access to key heparin-building block ido-thioglycoside donors are described. A 1-OAc disaccharide is converted into a heparin-related tetrasaccharide, via divergence to both acceptor and donor disaccharides. X-ray and NMR data of the 1,2-diacetyl iduronate methyl ester and the analogous iduronamide show that while both adopt (1)C(4) conformations in solution, the iduronate ester adopts the (4)C(1) conformation in solid state. An X-ray structure is also reported for the novel, (4)C(1)-conformationally locked bicyclic 1,6-anhydro iduronate lactone along with an X-ray structures of a novel distorted (4)C(1) iduronate 4,6-lactone. Deuterium labeling also provides mechanistic insight into the formation of lactone products during the novel amyl nitrite-mediated hydrolysis of iduronamide into the parent iduronic acid functionality.

Heparin and heparan sulphate (H/HS) are important members of the glycosaminoglycan family of sugars that regulate a substantial number of biological processes. Such biological promiscuity is underpinned by hetereogeneity in their molecular structure. The degree of O-sulfation, particularly at the 6-position of constituent D-GlcN units, is believed to play a role in modulating the effects of such sequences. Synthetic chemistry is essential to be able to extend the diversity of HS-like fragments with defined molecular structure, and particularly to deconvolute the biological significance of modifications at O6. Here we report a synthetic approach to a small matrix of protected heparin-type oligosaccharides, containing orthogonal D-GlcN O-6 protecting groups at programmed positions along the chain, facilitating access towards programmed modifications at specific sites, relevant to sulfation or future mimetics. [ABSTRACT FROM AUTHOR]

Only palliative therapeutic options exist for the treatment of Alzheimer's Disease; no new successful drug candidates have been developed in over 15 years. The widely used clinical anticoagulant heparin has been reported to exert beneficial effects through multiple pathophysiological pathways involved in the aetiology of Alzheimer's Disease, for example, amyloid peptide production and clearance, tau phosphorylation, inflammation and oxidative stress. Despite the therapeutic potential of heparin as a multi-target drug for Alzheimer's disease, the repurposing of pharmaceutical heparin is proscribed owing to the potent anticoagulant activity of this drug. Here, a heterogenous non-anticoagulant glycosaminoglycan extract, obtained from the shrimp Litopenaeus vannamei, was found to inhibit the key neuronal β-secretase, BACE1, displaying a more favorable therapeutic ratio compared to pharmaceutical heparin when anticoagulant activity is considered. [ABSTRACT FROM AUTHOR]

The clinically important anticoagulant heparin, a member of the glycosaminoglycan family of carbohydrates that is extracted predominantly from porcine and bovine tissue sources, has previously been shown to inhibit the β-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a key drug target in Alzheimer's Disease. In addition, heparin has been shown to exert favourable bioactivities through a number of pathophysiological pathways involved in the disease processes of Alzheimer's Disease including inflammation, oxidative stress, tau phosphorylation and amyloid peptide generation. Despite the multi-target potential of heparin as a therapeutic option for Alzheimer's disease, the repurposing of this medically important biomolecule has to-date been precluded by its high anticoagulant potential. An alternative source to mammalian-derived glycosaminoglycans are those extracted from marine environments and these have been shown to display an expanded repertoire of sequence-space and heterogeneity compared to their mammalian counterparts. Furthermore, many marine-derived glycosaminoglycans appear to retain favourable bioactivities, whilst lacking the high anticoagulant potential of their mammalian counterparts. Here we describe a sulphated, marine-derived glycosaminoglycan extract from the Atlantic Sea Scallop, Placopecten magellanicus that displays high inhibitory potential against BACE-1 (IC 50 = 4.8 μg.mL−1) combined with low anticoagulant activity; 25-fold less than that of heparin. This extract possesses a more favourable therapeutic profile compared to pharmaceutical heparin of mammalian provenance and is composed of a mixture of heparan sulphate (HS), with a high content of 6-sulphated N-acetyl glucosamine (64%), and chondroitin sulphate. [Display omitted] • A GAG extract from the Atlantic Sea Scallop, P. magellanicus , inhibits BACE-1, a key drug-target in Alzheimer's disease. • The GAG extract is predominantly HS, containing a high content of UA-GlcNAc(6S), uncommon in mammalian-derived HS. • The GAG extract possesses highly attenuated anticoagulant potential compared to mammalian-derived heparin. [ABSTRACT FROM AUTHOR]