Recent advances in the synthesis of extensive libraries of heparan sulfate oligosaccharides for structure-activity relationship studies.

Heparan sulfate (HS) is a linear, sulfated and highly negatively-charged polysaccharide that plays important roles in many biological events. As a member of the glycosaminoglycan (GAG) family, HS is commonly found on mammalian cell surfaces and within the extracellular matrix. The structural complexities of natural HS polysaccharides have hampered the comprehension of their biological functions and structure-activity relationships (SARs). Although the sulfation patterns and backbone structures of HS can be major determinants of their biological activities, obtaining significant amounts of pure HS from natural sources for comprehensive SAR studies is challenging. Chemical and enzyme-based synthesis can aid in the production of structurally well-defined HS oligosaccharides. In this review, we discuss recent innovations enabling the syntheses of large libraries of HS and how these libraries can provide insights into the structural preferences of various HS binding proteins.

An identification method to distinguish monomeric sugar isomers on glycopeptides.

A one-step protocol for the automated flow synthesis of protected glycosylated amino acids is described using pumps with open-source controls in overall yields of 21-50%. The resulting glycosylated amino acids could be used directly in solid-phase peptide synthesis (SPPS) protocols to quickly produce glycopeptide standards. Access to a variety of stereoisomers of the sugar enabled the development of an LC-MS/MS protocol that can distinguish between peptides modified with carbohydrates having the same exact mass. This method could definitively identify fucose in an O-glycosylation site on the transmembrane protein, Notch1.

Efficient platform for synthesizing comprehensive heparan sulfate oligosaccharide libraries for decoding glycosaminoglycan-protein interactions.

Glycosaminoglycans (GAGs) are abundant, ubiquitous carbohydrates in biology, yet their structural complexity has limited an understanding of their biological roles and structure-function relationships. Synthetic access to large collections of well defined, structurally diverse GAG oligosaccharides would provide critical insights into this important class of biomolecules and represent a major advance in glycoscience. Here we report a new platform for synthesizing large heparan sulfate (HS) oligosaccharide libraries displaying comprehensive arrays of sulfation patterns. Library synthesis is made possible by improving the overall synthetic efficiency through universal building blocks derived from natural heparin and a traceless fluorous tagging method for rapid purification with minimal manual manipulation. Using this approach, we generated a complete library of 64 HS oligosaccharides displaying all possible 2-O-, 6-O- and N-sulfation sequences in the tetrasaccharide GlcN-IdoA-GlcN-IdoA. These diverse structures provide an unprecedented view into the sulfation code of GAGs and identify sequences for modulating the activities of important growth factors and chemokines.

Automated, Multistep Continuous-Flow Synthesis of 2,6-Dideoxy and 3-Amino-2,3,6-trideoxy Monosaccharide Building Blocks.

An automated continuous flow system capable of producing protected deoxy-sugar donors from commercial material is described. Four 2,6-dideoxy and two 3-amino-2,3,6-trideoxy sugars with orthogonal protecting groups were synthesized in 11-32 % overall yields in 74-131.5 minutes of total reaction time. Several of the reactions were able to be concatenated into a continuous process, avoiding the need for chromatographic purification of intermediates. The modular nature of the experimental setup allowed for reaction streams to be split into different lines for the parallel synthesis of multiple donors. Further, the continuous flow processes were fully automated and described through the design of an open-source Python-controlled automation platform.

Fully automated fast-flow synthesis of antisense phosphorodiamidate morpholino oligomers.

Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases.

Structural insight into the binding of human galectins to corneal keratan sulfate, its desulfated form and related saccharides.

Glycosaminoglycan chains of keratan sulfate proteoglycans appear to be physiologically significant by pairing with tissue lectins. Here, we used NMR spectroscopy and molecular dynamics (MD) simulations to characterize interactions of corneal keratan sulfate (KS), its desulfated form, as well as di-, tetra- (N-acetyllactosamine and lacto-N-tetraose) and octasaccharides with adhesion/growth-regulatory galectins, in particular galectin-3 (Gal-3). The KS contact region involves the lectin canonical binding site, with estimated KD values in the low µM range and stoichiometry of ~ 8 to ~ 20 galectin molecules binding per polysaccharide chain. Compared to Gal-3, the affinity to Gal-7 is relatively low, signaling preferences among galectins. The importance of the sulfate groups was delineated by using desulfated analogs that exhibit relatively reduced affinity. Binding studies with two related di- and tetrasaccharides revealed a similar decrease that underscores affinity enhancement by repetitive arrangement of disaccharide units. MD-based binding energies of KS oligosaccharide-loaded galectins support experimental data on Gal-3 and -7, and extend the scope of KS binding to Gal-1 and -9N. Overall, our results provide strong incentive to further probe the relevance of molecular recognition of KS by galectins in terms of physiological processes in situ, e.g. maintaining integrity of mucosal barriers, intermolecular (lattice-like) gluing within the extracellular meshwork or synaptogenesis.

Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries.

Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.

Automated Solution-Phase Synthesis of S-Glycosides for the Production of Oligomannopyranoside Derivatives.

Thioglycosides are more resistant to enzymatic hydrolysis than their O-linked counterparts, thereby becoming attractive targets for carbohydrate-based therapeutic development. We report the first development of methods for the site-selective incorporation of S-linkages into automated solution-phase oligosaccharide protocols. The protocols were shown to be compatible with the formation of S- or O-glycosides for the synthesis of mannopyranoside trimmers that incorporate both S- and O-linkages to allow the selective incorporation of an S-glycoside in various stages in an automated program.

Addition of Sialic Acid to Insulin Confers Superior Physical Properties and Bioequivalence.

Native insulin is susceptible to biophysical aggregation and fibril formation, promoted by manual agitation and elevated temperatures. The safety of the drug and its application to alternative forms of administration could be enhanced through the identification of chemical modifications that strengthen its physical stability without compromising its biological properties. Complex polysialic acids (PSAs) exist naturally and provide a means to enhance the physical properties of peptide therapeutics. A set of insulin analogues site-specifically derivatized with sialic acid were prepared in an overall yield of 50-60%. Addition of a single or multiple sialic acids conferred remarkable enhancement to the biophysical stability of human insulin while maintaining its potency. The time to the onset of fibrillation was extended by more than 10-fold relative to that of the native hormone. These results demonstrate that simplified sialic acid conjugates represent a viable alternative to complex natural PSAs in increasing the stability of therapeutic peptides.

Modular continuous flow synthesis of orthogonally protected 6-deoxy glucose glycals.

An efficient, modular continuous flow process towards accessing two orthogonally protected glycals is described with the development of reaction conditions for several common protecting group additions in flow, including the addition of benzyl, naphthylmethyl and tert-butyldimethylsilyl ethers. The process affords the desired target compounds in 57-74% overall yield in just 21-37 minutes of flow time. Furthermore, unlike batch conditions, the flow processes avoided the need for active cooling to prevent unwanted exotherms and required shorter reaction times.

Automated solution-phase syntheses of alpha 1 → 2, 1 → 3 type rhamnans and rhamnan sulfate fragments.

Rhamnan and rhamnan sulfate are naturally occurring carbohydrates that have important biological functions and possible therapeutic applications, but studies are limited to the microheterogeneous mixtures from natural sources. This work reports the first synthesis of any sulfated rhamnan fragments and successful automation of the process with a recently developed automated solution-phase approach using N-iodosuccinimide/trimethylsilyl triflate (NIS/TMSOTf) promotor and levulinoyl ester deprotection conditions. The automated solution-phase activation/deprotection approach was initially able to create alpha 1 → 2, 1 → 3 type rhamnan di- and trisaccharide in moderate yields. Once these targets were achieved, a process to use SO3•pyridine complex in DMF for sulfation compatible with an automated solution-phase liquid handling system was developed and successfully applied to carbohydrate sulfation to create two rhamnan sulfate fragments with differing monosulfation patterns.

Advancing Solutions to the Carbohydrate Sequencing Challenge.

Carbohydrates possess a variety of distinct features with stereochemistry playing a particularly important role in distinguishing their structure and function. Monosaccharide building blocks are defined by a high density of chiral centers. Additionally, the anomericity and regiochemistry of the glycosidic linkages carry important biological information. Any carbohydrate-sequencing method needs to be precise in determining all aspects of this stereodiversity. Recently, several advances have been made in developing fast and precise analytical techniques that have the potential to address the stereochemical complexity of carbohydrates. This perspective seeks to provide an overview of some of these emerging techniques, focusing on those that are based on NMR and MS-hybridized technologies including ion mobility spectrometry and IR spectroscopy.

Probing deoxysugar conformational preference: A comprehensive computational study investigating the effects of deoxygenation.

Deoxysugars are intrinsic components in a number of antibiotics, antimicrobials, and therapeutic agents that often dictate receptor binding, improve efficacy, and provide a diverse toolbox in modifying glycoconjugate function due to an extensive number of unique isomers and inherent conformational flexibility. Hence, this work provides a comprehensive examination of the conformational effects associated with deoxygenation of the pyranose ring. Both the location and degree of deoxygenation were evaluated by interrogating the energetic landscape for a number of mono- and dideoxyhexopyranose derivatives using DFT methods (M05-2X/cc-pVTZ(-f)). Both anomeric forms and in some cases, the alternate chair form, have been investigated in the gas phase. As was documented in a preceding study, variation of the C-6 oxidation state has been shown to affect the anomeric preference of select glucose stereoisomers. Similar results were also observed for several deoxysugar isomers in this work, wherein the alternate anomer was favored upon reduction to the 6-deoxyhexose derivative or oxidation to the hexonic acid. Additionally, comparison of relative Gibbs free energies revealed C-3 deoxygenation imparts greater instability compared to C-2 or C-4 deoxygenation, as indicated by an increase in free energy for 3-deoxysugars. A polarizable continuum solvation model was also applied to empirically validate theoretical results for several deoxysugars, wherein good agreement with both carbon (σ = 1.6 ppm) and proton (σ = 0.20 ppm) NMR shifts was observed for the majority of isomers. Solvated and gas phase anomeric ratios were also calculated and compared favorably to reported literature values, although some discrepancies are noted.

Development of a Post-Column Liquid Chromatographic Chiral Addition Method for the Separation and Resolution of Common Mammalian Monosaccharides.

The first solely MS-based methodology for the identification and resolution of the ten common mammalian monosaccharides is presented. Based on Cooks' fixed ligand kinetic method, this technique is effective on multiple classes of monosaccharides and includes the first example of two fixed ligand combinations used in a single multiplexed experiment. Subsequently, a post-HPLC chiral addition method is used in conjunction with this newly developed MS methodology for the separation and identification of mixtures of common neutral mammalian monosaccharides. This proposed technique is able to overcome a limitation of present carbohydrate analysis methods, namely the simultaneous isomeric resolution of multiple monosaccharides in a mixture. Graphical Abstract.

Synthesis of protected glucose derivatives from levoglucosan by development of common carbohydrate protecting group reactions under continuous flow conditions.

Common carbohydrate protecting group reactions under continuous flow processes are reported in the context of producing partially-protected glucose building blocks from levoglucosan. Benzyl ether protection was demonstrated without the use of NaH using barium oxide, which, however, pointed to the need for forms of this catalyst not as susceptible to close packing under flow. Acylation conditions were developed under continuous flow in acetonitrile and avoiding pyridine. Ring-opening the derivatized levoglucosan with propanethiol was also demonstrated producing S-alkyl 2,4-di-O-benzyl-glucopyranoside building block in 2 rather than 12 steps in increased overall yield.

Scope and limitations of carbohydrate hydrolysis for de novo glycan sequencing using a hydrogen peroxide/metallopeptide-based glycosidase mimetic.

Acidic hydrolysis is commonly used as a first step to break down oligo- and polysaccharides into monosaccharide units for structural analysis. While easy to set up and amenable to mass spectrometry detection, acid hydrolysis is not without its drawbacks. For example, ring-destruction side reactions and degradation products, along with difficulties in optimizing conditions from analyte to analyte, greatly limits its broad utility. Herein we report studies on a hydrogen peroxide/CuGGH metallopeptide-based glycosidase mimetic design for a more efficient and controllable carbohydrate hydrolysis. A library of methyl glycosides consisting of ten common monosaccharide substrates, along with oligosaccharide substrates, was screened with the artificial glycosidase for hydrolytic activity in a high-throughput format with a robotic liquid handling system. The artificial glycosidase was found to be active towards most screened linkages, including alpha- and beta-anomers, thus serving as a potential alternative method for traditional acidic hydrolysis approaches of oligosaccharides.

Challenges in the Conversion of Manual Processes to Machine-Assisted Syntheses: Activation of Thioglycoside Donors with Aryl(trifluoroethyl)iodonium Triflimide.

The steps needed to adapt a stable iodonium promoter for use in automated fluorous-assisted solution-phase oligosaccharide synthesis are described. Direct adaptation of the originally reported batch procedure resulted in the formation of an orthoester or protecting group transfer to the glycosyl acceptor. Fortunately, the addition of inexpensive ß-pinene as an acid scavenger avoided both of these side reactions. The utility of this newly developed protocol was applied to the automated solution-phase synthesis of a ß-glucan fragment.

Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions.

Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated with Leishmania major and trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated with L. major alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected with L. major alone, coinoculated with carrier beads and L. major, or coinoculated with trimannose-coated beads and L. major Trimannose treatment of L. major-infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR-/-) mice lack the ability to detect trimannose. When MR-/- mice were infected with L. major and treated with trimannose beads, they did not have decreased lesion size. Leishmania-derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.