Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

Summary

This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination experiments and one-pot glycosylation of SSEA-4.

Abstract

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.

Introduction

Carbohydrates are ubiquitous in nature^1,2, but their presence and mode of action remain an uncharted territory, mainly due to difficult access to this class of molecules³. Unlike automated synthesis of oligopeptides and oligonucleotides, the development of automated synthesis of oligosaccharides remains a formidable task, and progress has been relatively slow.

To tackle this problem, Wong et al. developed the first automated method for the synthesis of oligosaccharides using a programmable software program called Optimer⁴, which guides....

Protocol

1. Auto-CHO software manipulation

1. Java Runtime Environment installation: make sure the Java Runtime Environment (JRE) has been installed in the device. If JRE has been installed, go to the next step, “software initialization”; otherwise, download and install JRE according to the user’s operating system found at: <https://www.oracle.com/technetwork/java/javase/downloads/index.html>.
2. Software initialization: go to the Auto-CHO website at <https://sites.google.com/view/aut.......

Discussion

The Auto-CHO software was developed for assisting chemists to proceed hierarchical and programmable one-pot synthesis of oligosaccharides⁵. Auto-CHO was built by Java programming language. It is a GUI software and cross-platform, which currently supports Windows, macOS, and Ubuntu. The software can be downloaded free of charge for the Auto-CHO website at <https://sites.google.com/view/auto-cho/home>, and its source code with MIT license can be accessed from the GitHub at <https://githu.......

Materials

Name	Company	Catalog Number	Comments
Acetonitrile	Sigma-Aldrich	75-05-8
Anhydrous magnesium sulfate	Sigma-Aldrich	7487-88-9
Cerium ammonium molybdate	TCI	C1794
Dichloromethane	Sigma-Aldrich	75-09-2
Drierite	Sigma-Aldrich	7778-18-9
Ethyl acetate	Sigma-Aldrich	141-78-6
Methanol	Sigma-Aldrich	67-56-1
Molecular sieves 4 Å	Sigma-Aldrich
n-Hexane	Sigma-Aldrich	110-54-3
N-Iodosuccinimide	Sigma-Aldrich	516-12-1
Sodium bicarbonate	Sigma-Aldrich	144-55-8
Sodium thiosulfate	Sigma-Aldrich	10102-17-7
Toluene	Sigma-Aldrich	108-88-3
Trifluoromethanesulfonic acid	Sigma-Aldrich	1493-13-6