Supriya Dey

The clinically approved Fondaparinux (Arixtra) has been used for the treatment of deep vein thrombosis and acute pulmonary embolism since 2002 and is considered to be better than the low-molecular weight heparin in terms of anticoagulation response, duration of action, and biosafety. However, the synthetic methods previously developed for its manufacture are relatively complicated, thus restricting its extensive use. We report here a potentially scalable and programmable one-pot synthesis of Fondaparinux using the [1,2,2] strategy and designed thioglycosides with well-defined reactivity as building blocks.

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and >50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.

Idraparinux is a fully O-sulfated α-methyl glycoside of heparin pentasaccharide motif known to inter-act with the antithrombin III domain and act as anti-coagulant. The current most effective synthesis of Idraparinux is complicated and non-stereoselective, requiring numerous step-wise procedures with low yields. We report here an efficient modular one-pot synthesis of Idraparinux, involving the use of a glycosyl phosphate with 6-O-tert-butyl diphenyl silyl group and a D-glucuronic acid-containing di-saccharide thioglycoside with 6-O-acetyl group as donor building blocks for the α-directing one-pot glycosylations with an L-iduronic acid-containing disaccharide acceptor building block. The uronic acid was incorporated in a disaccharide module used in the one-pot synthesis to avoid the complicated late-stage installation of these acidic sugars. The one-pot synthesis of Idraparinux demonstrated here is an effective strategy and should be applicable to the modular assembly of other heparan sulfates with regiodefined sulfation pattern for functional study.

Fluorinated glycosides are known to resist the glycosidase-catalyzed glycosidic bond cleavage; however the synthesis of such glycans, especially 3-fluoro-sialic acid (3F-Neu5Ac) containing sialosides, has been a major challenge. Though the enzymatic synthesis of α-2,3-linked 3F-sialosides was reported, until recently there has not been any effective method available for the synthesis of 3F-sialosides in the α-2,6-linkage. In order to understand the biological effect of such modification, we report here a chemical synthesis of 3F^ax-Neu5Ac-α2,6-Gal as building block for the assembly of 3F^ax-Neu5Ac-containing sialosides and a representative homogeneous antibody glycoform. Our results showed that the sialosides are stable under sialidase catalysis and the rituximab glycoform with a sialylated complex-type biantennary glycan terminated with 3F^ax-Neu5Ac in the α-2,6-linkage (α2,6-F-SCT) has a similar binding avidity as its parent glycoform. These findings open up new opportunities for the development of therapeutic glycoproteins with improved pharmacokinetic parameters.

The programmable one-pot oligosaccharide synthesis method was designed to enable the rapid synthesis of a large number of oligosaccharides, using the software Optimer to search Building BLocks (BBLs) with defined relative reactivity values (RRVs) to be used sequentially in the one-pot reaction. However, there were only about 50 BBLs with measured RRVs in the original library and the method could only synthesize small oligosaccharides due to the RRV ordering requirement. Here, we increase the library to include 154 validated BBLs and more than 50,000 virtual BBLs with predicted RRVs by machine learning. We also develop the software Auto-CHO to accommodate more data handling and support hierarchical one-pot synthesis using fragments as BBLs generated by the one-pot synthesis. This advanced programmable one-pot method provides potential synthetic solutions for complex glycans with four successful examples demonstrated in this work.

Heparin (H) and heparan sulfate (HS) belong to the glycosaminoglycan (GAG) family of oligosaccharides, and their sequences and sulfation patterns are known to regulate the functions of various proteins in biological processes. Among these, the 6-O-sulfation of HS/H contributes most significantly to the structural diversity and binding interactions. However, the synthesis of HS with defined sulfation patterns remains a major challenge. Herein, we report a highly efficient and programmable one-pot method for the synthesis of protected heparin pentasaccharides using thioglycoside building blocks with optimized relative reactivities to allow the selective deprotection and preparation of regiodefined sulfate derivatives.