Practical Remdesivir Synthesis through One-Pot Organocatalyzed Asymmetric (S)-P-Phosphoramidation.

Remdesivir, an inhibitor of RNA-dependent RNA polymerase developed by Gilead Sciences, has been used for the treatment of COVID-19. The synthesis of remdesivir is, however, challenging, and the overall cost is relatively high. Particularly, the stereoselective assembly of the P-chirogenic center requires recrystallization of a 1:1 isomeric p-nitrophenylphosphoramidate mixture several times to obtain the desired diastereoisomer (39%) for further coupling with the d-ribose-derived 5-alcohol. To address this problem, a variety of chiral bicyclic imidazoles were synthesized as organocatalysts for stereoselective (S)-P-phosphoramidation employing a 1:1 diastereomeric mixture of phosphoramidoyl chloridates as the coupling reagent to avoid a waste of the other diastereomer. Through a systematic study of different catalysts at different temperatures and concentrations, a mixture of the (S)- and (R)-P-phosphoramidates was obtained in 97% yield with a 96.1/3.9 ratio when 20 mol % of the chiral imidazole-cinnamaldehyde-derived carbamate was utilized in the reaction at -20 °C. A 10-g scale one-pot synthesis via a combination of (S)-P-phosphoramidation and protecting group removal followed by one-step recrystallization gave remdesivir in 70% yield and 99.3/0.7 d.r. The organocatalyst was recovered in 83% yield for reuse, and similar results were obtained. This one-pot process offers an excellent opportunity for industrial production of remdesivir.

Single-Step Per-O-Sulfonation of Sugar Oligomers with Concomitant 1,6-Anhydro Bridge Formation for Binding Fibroblast Growth Factors.

Many circulating cancer-related proteins, such as fibroblast growth factors (FGFs), associate with glycosaminoglycans-particularly heparan sulfate-at the cell surface. Disaccharide analogues of heparan sulfate had previously been identified as the shortest components out of the sugars that bind to FGF-1 and FGF-2. Taking note of the typical pose of l-iduronic acid, we conceived of per-O-sulfonated analogues of such disaccharides, and devised a single-step procedure for per-O-sulfonation of unprotected sugars with concomitant 1,6-anhydro bridge formation to achieve such compounds through direct use of SO3 ⋅Et3 N as sulfonation reagent and dimethylformamide as solvent. The synthesized sugars based on the oligomaltose backbone bound FGF-1 and FGF-2 mostly at the sub-micromolar level, although the tetrasaccharide analogue achieved low-nanomolar binding with FGF-2.

Structure of the Complex between a Heparan Sulfate Octasaccharide and Mycobacterial Heparin-Binding Hemagglutinin.

Heparin-binding hemagglutinin (HBHA) is a 199 amino acid virulence factor at the envelope of Mycobacterium tuberculosis that contributes to latent tuberculosis. The binding of HBHA to respiratory epithelial cells, which leads to extrapulmonary dissemination of the pathogen, is mediated by cell-surface heparan sulfate (HS). We report the structural characterization of the HBHA/HS complex by NMR spectroscopy. To develop a model for the molecular recognition, the first chemically synthesized uniformly 13 C- and 15 N-labeled HS octasaccharide and a uniformly 13 C- and 15 N-labeled form of HBHA were prepared. Residues 180-195 at the C-terminal region of HBHA show large chemical shift perturbation upon association with the octasaccharide. Molecular dynamics simulations conforming to the multidimensional NMR data revealed key electrostatic and even hydrophobic interactions between the binding partners that may aid in the development of agents targeting the binding event.

Chemoselective per-O-trimethylsilylation and homogeneous N-functionalisation of amino sugars.

A highly efficient CH3CN-promoted hexamethyldisilazane per-O-trimethylsilylation of amino sugars was developed. Its applications in homogenous N-functionalisation and a concise synthesis of glucosamine 6-phosphate are described.

Interactions that influence the binding of synthetic heparan sulfate based disaccharides to fibroblast growth factor-2.

Heparan sulfate (HS) is a linear sulfated polysaccharide that mediates protein activities at the cell-extracellular interface. Its interactions with proteins depend on the complex patterns of sulfonations and sugar residues. Previously, we synthesized all 48 potential disaccharides found in HS and used them for affinity screening and X-ray structural analysis with fibroblast growth factor-1 (FGF1). Herein, we evaluated the affinities of the same sugars against FGF2 and determined the crystal structures of FGF2 in complex with three disaccharides carrying N-sulfonated glucosamine and 2-O-sulfonated iduronic acid as basic backbones. The crystal structures show that water molecules mediate different interactions between the 3-O-sulfonate group and Lys125. Moreover, the 6-O-sulfonate group forms intermolecular interactions with another FGF2 unit apart from the main binding site. These findings suggest that the water-mediated interactions and the intermolecular interactions influence the binding affinity of different disaccharides with FGF2, correlating with their respective dissociation constants in solution.

Divergent synthesis of 48 heparan sulfate-based disaccharides and probing the specific sugar-fibroblast growth factor-1 interaction.

Several biological processes involve glycans, yet understanding their ligand specificities is impeded by their inherent diversity and difficult acquisition. Generating broad synthetic sugar libraries for bioevaluations is a powerful tool in unraveling glycan structural information. In the case of the widely distributed heparan sulfate (HS), however, the 48 theoretical possibilities for its repeating disaccharide call for synthetic approaches that should minimize the effort in an undoubtedly huge undertaking. Here we employed a divergent strategy to afford all 48 HS-based disaccharides from just two orthogonally protected disaccharide precursors. Different combinations and sequence of transformation steps were applied with many downstream intermediates leading up to multiple target products. With the full disaccharide library in hand, affinity screening with fibroblast growth factor-1 (FGF-1) revealed that four of the synthetic sugars bind to FGF-1. The molecular details of the interaction were further clarified through X-ray analysis of the sugar-protein cocrystals. The capability of comprehensive sugar libraries in providing key insights in glycan-ligand interaction is, thus, highlighted.

α-Glycosylation by D-glucosamine-derived donors: synthesis of heparosan and heparin analogues that interact with mycobacterial heparin-binding hemagglutinin.

Numerous biomolecules possess α-D-glucosamine as structural component. However, chemical glycosylations aimed at this backbone are usually not easily attained without generating the unwanted ß-isomer. We report herein a versatile approach in affording full α-stereoselectivity built upon a carefully selected set of orthogonal protecting groups on a D-glucosaminyl donor. The excellent stereoselectivity provided by the protecting group combination was found independent of leaving groups and activators. With the trichloroacetimidate as the optimum donor leaving group, core skeletons of glycosylphosphatidyl inositol anchors, heparosan, heparan sulfate, and heparin were efficiently assembled. The orthogonal protecting groups were successfully manipulated to further carry out the total syntheses of heparosan tri- and pentasaccharides and heparin di-, tetra-, hexa-, and octasaccharide analogues. Using the heparin analogues, heparin-binding hemagglutinin, a virulence factor of Mycobacterium tuberculosis, was found to bind at least six sugar units with the interaction notably being entropically driven.

Structural analysis of the extracellular domain of vaccinia virus envelope protein, A27L, by NMR and CD spectroscopy.

This study presents the molecular structure of the extracellular domain of vaccinia virus envelope protein, A27L, determined by NMR and CD spectroscopy. A recombinant protein, eA27L-aa, containing this domain in which cysteines 71 and 72 were replaced with alanine, was constructed to prevent self-assembly due to intermolecular disulfide bonds between these two cysteines. The soluble eA27L-aa protein forms an oligomer resembling that of A27L on vaccinia virions. Heteronuclear correlation NMR spectroscopy was carried out on eA27L-aa in the presence or absence of urea to determine backbone resonance assignments. Chemical shift index (CSI) propensity analysis showed that eA27L-aa has two distinct structural domains, a relatively flexible 22-amino acid random coil in the N-terminal region and a fairly rigid alpha-helix structure in the remainder of the structure. Binding interaction studies using isothermal titration calorimetry suggest that a 12-amino acid lysine/arginine-rich segment in the N-terminal region is responsible for glycosaminoglycan binding. The rigid alpha-helix portion of eA27L-aa is probably involved in the intrinsic self-assembly, and CSI propensity analysis suggests that region N37-E49, with a residual alpha-helix tendency, is probably the self-assembly core. Self-assembly was ascribed to three hydrophobic leucine residues (Leu(41), Leu(45), and Leu(48)) in this segment. The folding mechanism of eA27L-aa was analyzed by CD spectroscopy, which revealed a two-step transition with a Gibbs free energy of 2.5 kcal/mol in the absence of urea. Based on these NMR and CD studies, a residue-specific molecular model of the extracellular domain of A27L is proposed. These studies on the molecular structure of eA27L-aa will help in understanding how vaccinia virus enters cells.