This work was supported by Academia Sinica including the Summit Program, Ministry of Science and Technology [MOST 104-0210-01-09-02, MOST 105-0210-01-13-01, MOST 106-0210-01-15-02], and NSF (1664283).

1. Auto-CHO software manipulation
<ol>
	<li>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, &#8220;software initialization&#8221;; otherwise, download and install JRE according to the user&#8217;s operating system found at: &#60;https://www.oracle.com/technetwork/java/javase/downloads/index.html&#62;.</li>
	<li>Software initialization: go to the Auto-CHO website at &#60;https://sites.google.com/view/auto-cho/home&#62; and download the software according to the operating system. Currently, Auto-CHO supports Windows, macOS, and Ubuntu. The latest PDF user guide is provided on the Auto-CHO website.
	<ol>
		<li>For Windows users, unzip the Auto-CHO_Windows.zip and double-click on Auto-CHO.jar in the Auto-CHO_Windows folder to start the program. 
		NOTE: The user needs to install unzip software, such as 7-Zip, found at &#60;https://www.7-zip.org&#62;, for unpacking the zip file. The user may also use the java -jar Auto-CHO.jar command to start the program by the Windows Command Prompt.</li>
		<li>For macOS users, right-click on Auto-CHO.jar and choose Open to start the program.</li>
		<li>For Ubuntu users:
		<ol>
			<li>Install libcanberra-gtk using the following command: 
			$ sudo apt-get install libcanberra-gtk*</li>
			<li>Change the access permission of Auto-CHO_Ubuntu.sh: 
			$ chmod 755 Auto-CHO_Ubuntu.sh</li>
			<li>Run the Auto-CHO program: 
			$ ./Auto-CHO_Ubuntu.sh</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Input the desired glycan structure. Choose to draw a glycan structure or read an existing structure file.
	<ol>
		<li>Input by drawing:
		<ol>
			<li>Click on Edit Glycan by GlycanBuilder9,10 (Figure 1, btn1; Figure 2A) or the area of Click Here to Edit the Synthetic Target to draw and edit the query structure by GlycanBuilder. Linkage and chirality information should not be ignored. Click on the Globo-H, SSEA-4, or OligoLacNAc buttons (Figure 1, examples) to display the examples.</li>
			<li>Select File | Export to sequence formats | Export to GlycoCT condensed to save the edited structure (optional).</li>
			<li>Close the GlycanBuilder dialog to complete editing.</li>
		</ol>
		</li>
		<li>Input by reading a file:
		<ol>
			<li>Click on Edit Glycan by GlycanBuilder (Figure 1, btn1; Figure 2A) or the area of Click Here to Edit the Synthetic Target to edit the query structure.</li>
			<li>Select File | Import from sequence formats to choose the query structure file with the corresponding format.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Search parameter settings (optional).
	<ol>
		<li>Define the search parameters in the &#8220;Parameter Settings&#8221; tab (Figure 1, tab2) to get reasonable search results. 
		NOTE: 
		Threshold of High-Class RRV must be a real number and &#8805;0. 
		Threshold of Medium Class RRV must be a real number and &#8805;0. 
		Threshold of High-Class RRV should be &#62;Threshold of Medium Class RRV. 
		Max Fragment Number should be an integer and &#8805;1. 
		Min BBL Number in a Fragment must be an integer and between 1 and 3. 
		Max BBL Number in a Fragment must be an integer and between 1 and 3. 
		Max BBL Number in a Fragment must be &#8805;Min BBL Number in a Fragment. 
		Min Donor/Acceptor RRV Difference must be a positive real number. 
		Min Donor/Acceptor RRV Ratio must be a positive real number. 
		Max Donor/Acceptor RRV Ratio must be a positive real number. 
		Max Donor/Acceptor RRV Ratio must be &#62;Min Donor/Acceptor RRV Ratio.</li>
		<li>Click on the OK button to enable the new settings.</li>
	</ol>
	</li>
	<li>Select the building block library (Figure 1, tab5). The default setting is to search the experimental library only. If it is desired to search both the experimental and virtual libraries, check the following steps.
	<ol>
		<li>Select the Virtual Building Block Library tab (Figure 2C, tab5). Experimental and virtual building blocks can work together to enhance the searching ability of Auto-CHO. Currently, Auto-CHO provides more than 50,000 virtual building blocks with predicted RRVs in the library.</li>
		<li>Select Use Experimental and Virtual Libraries and apply Filtering to display virtual building blocks with certain criteria. Click on the Show Selected Virtual BBL(s) button (Figure 2C, btn5) to show only the selected virtual building block(s).</li>
		<li>Click on the Show Filtered Virtual BBL(s) button (Figure 2C, btn6) to show only virtual building blocks with certain criteria defined by the user.</li>
		<li>Click on the Show All Virtual BBL(s) button (Figure 2C, btn7) to show all available virtual building blocks and reset the filter.</li>
		<li>Check one or multiple desired virtual building blocks that the user would like to use for searching.</li>
	</ol>
	</li>
	<li>Select the Query Structure tab (Figure 1, tab1) and click on the Search Building Block Library button (Figure 1, btn2) to find the one-pot synthetic solutions for the query structure. Then, confirm the parameter settings.</li>
	<li>Search the result viewer. 
	NOTE: The search result is shown in the Result Visualization tab (Figure 1, tab6). The reducing end acceptors of different residue numbers are displayed in the Reducing End Acceptor column (Figure 1, viewer1).
	<ol>
		<li>Select a reducing-end acceptor, and solutions are displayed on the Synthetic Solution List (Figure 1, viewer2). Fragments are shown in the Fragment List (Figure 1, viewer3) to suggest how many fragments should be used in the synthesis. 
		NOTE: The system provides detailed information of each fragment, including the RRV of the fragment, computational yield as well as which protecting group should be deprotected for the subsequent use of the fragment in the one-pot reaction. The building blocks used to assemble the selected fragment are shown in the viewer4 of Figure 1. The viewer5 of Figure 1 also displays the fragment connection information.</li>
		<li>View and check chemical structures and detailed information of the selected building blocks in the regions of Chemical Structure of Building Block and Building Block Browser, respectively, for experimental building blocks (Figure 1, tab4).</li>
	</ol>
	</li>
	<li>Output the search result to text (optional).
	<ol>
		<li>Select the Result Text tab (Figure 1, tab7).</li>
		<li>Click on Save Result Text (Figure 2B, btn4) and choose the text file destination.</li>
	</ol>
	</li>
	<li>Feedback for virtual building blocks (optional). 
	NOTE: Feedback can be given on virtual building blocks through the online questionnaire. Feedback can help the community to keep useful virtual building blocks and remove ineffectual ones.
	<ol>
		<li>Select the Virtual Building Block tab (Figure 1, tab5).</li>
		<li>Click on the To rate link of the virtual building block of which it is desired to rate or comment in the Feedback column.</li>
		<li>Fill in the feedback form after the system opens up a webpage and submit it. 
		NOTE: Do not change the virtual BBL ID.</li>
	</ol>
	</li>
</ol>
2. RRV determination experiments
<ol>
	<li>In a 10 mL round-bottom flask, combine the two thioglycoside donors (0.02 mmol of each: Dr4 is the reference donor with known RRV; Dx1 is the donor molecule of unknown RRV), absolute methanol (0.10 mmol), and Drierite in dichloromethane (DCM, 1.0 mL), then stir at room temperature (RT) for 1 h.</li>
	<li>Take an aliquot of this mixture (30 &#181;L) and inject the mixture into high performance liquid chromatography (HPLC) in three separate injections (10 &#181;L for each injection). Measure the coefficient (a) between the absorption (A) and concentration of the donor molecule [D] under the baseline separation conditions (ether acetate/n-Hexane = 20/80).</li>
	<li>Add a solution of 0.5 M N-Iodosuccinimide (NIS) in acetonitrile (40 &#181;L, 0.02 mmol) into the reaction mixture, followed by addition of a 0.1 M trifluoromethanesulfonic acid (TfOH) solution (20 &#181;L, 0.002 mmol), and stir the mixture at RT for 2 h.</li>
	<li>Dilute the reaction mixture with DCM (4.0 mL), filter, and wash with saturated aqueous sodium thiosulfate containing 10% sodium hydrogen carbonate (2x with 5 mL volume each). Extract the aqueous layer with DCM (3x with 5 mL). Combine all organic layer, wash it with 5 mL of brine, and dry it with approximately 200 mg of anhydrous magnesium sulfate.</li>
	<li>Shake the mixture mildly for 30 s, filter it through a funnel with a fluted filter paper in order to remove the magnesium sulfate, then collect the filtrate in a 25 mL round-bottom flask. Remove the solvent using a rotary evaporator.</li>
	<li>Dissolve the residue in DCM (1.0 mL). Take an aliquot of this mixture (30 &#181;L) and inject it into HPLC in three separate injections (10 &#181;L for each injection). Measure the concentrations of the remaining donors ([Dx] and [Dref]) by HPLC under the same separation conditions (ether acetate/n-Hexane = 20/80) (Aref)t = 24417.0, (Ax)t = 23546.3.</li>
	<li>Measure the relative reactivity between Dx1 vs. Dr4, kx1/kr4 = 0.0932. Based on the relative reactivity value of Dr4, the relative reactivity value of Dx1 is 3. 
	NOTE: a = A/[D], (Aref)0 = 74530.1, (Ax)0 = 26143.0. kx/kref = (ln[Dx]t - ln[Dx]0)/(ln[Dref]t - ln[Dref]0) = (ln[Ax]t - ln[Ax]0)/(ln[Aref]t - ln[Aref]0) = 0.0932.</li>
</ol>
3. One-pot glycosylation of SSEA-4
<ol>
	<li>Place a 10 mL round-bottom flask under vacuum, flame-dry it, and allow the flask to cool to RT while still under vacuum. Remove the rubber septum to add a mixture of disaccharide 1 donor (38 mg, 1.1 eq., 0.057 mmol), the first acceptor 2 (40 mg, 1.0 eq., 0.053 mmol) and a Teflon-coated magnetic stir bar into the flask.</li>
	<li>Transfer 100 mg of powdered molecular sieves 4 &#197; into a 5 mL round-bottom flask. Keep this flask under vacuum, flame-dry it, and allow the flask to cool to RT while still under vacuum. Transfer the freshly dried 4 &#197; molecular sieves into the first flask that contains the starting material.</li>
	<li>Transfer 1 mL of freshly dried DCM into the flask. Stir the reaction mixture for 1 h at RT and then place it under a temperature of -40 &#176;C. Transfer the NIS (13 mg, 1.1 eq., 0.057 mmol) into the flask.</li>
	<li>Inject TfOH (34 &#956;L, 0.3 eq., 0.017 mmol, 0.5 M in ether) into the flask through the septum using a micro-volume syringe at -40 &#176;C. Keep stirring at -40 &#176;C for 3 h.</li>
	<li>After the first acceptor 2 is almost consumed, inject the solution of acceptor 3 in DCM into the flask through the septum.</li>
	<li>Warm the reaction mixture up to -20 &#176;C and transfer NIS (19 mg, 1.6 eq., 0.083 mmol) into the flask. Inject TfOH (34 &#956;L, 0.3 eq., 0.017 mmol, 0.5 M in ether) into the flask through the septum at -20 &#176;C. Keep stirring at -20 &#176;C for 3 h.</li>
	<li>After the product of the first step reaction is consumed, quench the reaction by injecting two equivalents of triethyl amine. Remove the molecular sieves through a filter funnel packed with Celite, collect the filtrate into a 25 mL round-bottom flask and further wash the filter with 10 mL of DCM.</li>
	<li>Transfer the filtrate into a separatory funnel and wash it with saturated aqueous sodium thiosulfate containing 10% NaHCO3 (2x with 10 mL each). Extract the aqueous layer with DCM (3x with 10 mL). Combine the organic layers and wash the mixture with brine (10 mL) and dry it by adding anhydrous MgSO4. Filter it and collect the filtrate in a 100 mL round-bottom flask.</li>
	<li>Remove the solvent using a rotary evaporator. Dissolve the crude mixture with approximately 1 mL of DCM and load it on top of the silica bed. Elute the product with a mixture of ethyl acetate and toluene (EtOAc/toluene, 1/4 to 1/2) and collect the fractions.</li>
	<li>Remove the solvent using a rotary evaporator. Dry the residue under reduced pressure to give fully protected SSEA-4 derivative 4 (74 mg, 50% based on acceptor 2) as white foam.</li>
</ol>

<ol><li>Apweiler, R., Hermjakob, H., Sharon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+frequency+of+protein+glycosylation%2C+as+deduced+from+analysis+of+the+SWISS-PROT+database%5BTitle%5D)+AND+%22Biochimica+Et+Biophysica+Acta%22%5BJournal%5D)">On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.</a> Biochimica Et Biophysica Acta. 1473 (1), 4-8 (1999).</li>
<li>Sears, P., Wong, C. -H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toward+Automated+Synthesis+of+Oligosaccharides+and+Glycoproteins%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Toward Automated Synthesis of Oligosaccharides and Glycoproteins.</a> Science. 291 (5512), 2344-2350 (2001).</li>
<li>Kulkarni, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%E2%80%9COne-Pot%E2%80%9D+Protection%2C+Glycosylation%2C+and+Protection-Glycosylation+Strategies+of+Carbohydrates%5BTitle%5D)+AND+%22Chemical+Reviews%22%5BJournal%5D)">“One-Pot” Protection, Glycosylation, and Protection-Glycosylation Strategies of Carbohydrates.</a> Chemical Reviews. 118 (17), 8025-8104 (2018).</li>
<li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Programmable+One-Pot+Oligosaccharide+Synthesis%5BTitle%5D)+AND+%22Journal+of+the+American+Chemical+Society%22%5BJournal%5D)">Programmable One-Pot Oligosaccharide Synthesis.</a> Journal of the American Chemical Society. 121 (4), 734-753 (1999).</li>
<li>Cheng, C. -W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hierarchical+and+programmable+one-pot+synthesis+of+oligosaccharides%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Hierarchical and programmable one-pot synthesis of oligosaccharides.</a> Nature Communications. 9 (1), 5202(2018).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22ChemDraw%22">ChemDraw</a>. , PerkinElmer Informatics. (2019).</li>
<li>Cheeseman, J. R., Frisch, Æ <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCheeseman%2C+JR+%22Predicting+magnetic+properties+with+chemdraw+and+gaussian%22">Predicting magnetic properties with chemdraw and gaussian</a>. , (2000).</li>
<li>Yap, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PaDEL-descriptor%3A+An+open+source+software+to+calculate+molecular+descriptors+and+fingerprints%5BTitle%5D)+AND+%22Journal+of+Computational+Chemistry%22%5BJournal%5D)">PaDEL-descriptor: An open source software to calculate molecular descriptors and fingerprints.</a> Journal of Computational Chemistry. 32 (7), 1466-1474 (2011).</li>
<li>Ceroni, A., Dell, A., Haslam, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+GlycanBuilder%3A+a+fast%2C+intuitive+and+flexible+software+tool+for+building+and+displaying+glycan+structures%5BTitle%5D)+AND+%22Source+Code+for+Biology+and+Medicine%22%5BJournal%5D)">The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures.</a> Source Code for Biology and Medicine. 2, 3(2007).</li>
<li>Damerell, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+GlycanBuilder+and+GlycoWorkbench+glycoinformatics+tools%3A+updates+and+new+developments%5BTitle%5D)+AND+%22Biological+Chemistry%22%5BJournal%5D)">The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments.</a> Biological Chemistry. 393 (11), 1357-1362 (2012).</li>
</ol>

The authors have nothing to disclose.

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 &#60;https://sites.google.com/view/auto-cho/home&#62;, and its source code with MIT license can be accessed from the GitHub at &#60;https://githu...

Carbohydrates are ubiquitous in nature1,2, 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 the selection of BBLs from a library of ~50 BBLs for sequential one-pot reactions. Each BBL was designed and synthesized with well-defined reactivity tuned by various protecting groups. Using this approach, the complexities of protecting manipulation and intermediate purification can be minimized during synthesis, which have been considered the most difficult issues to overcome in the development of automated synthesis. Despite this advance, the method is still quite restricted, as the number of BBLs is too small and the Optimer program can only handle certain small oligosaccharides. For more complex oligosaccharides that require more BBLs and multiple passes of one-pot reactions and fragment condensation, an upgraded version of the software program, Auto-CHO5, has been developed.
In Auto-CHO, more than 50,000 BBLs with defined reactivity to the BBL library have been added, including 154 synthetic and 50,000 virtual ones. These BBLs were designed by machine learning based on basic properties, calculated NMR chemical shifts6, 7, and molecular descriptors8, which affect the structure and reactivity of the BBLs. With this upgraded program and new set of BBLs available, the synthesis capacity is expanded, and as demonstrated, several oligosaccharides of interest can rapidly be prepared. It is believed that this new development will facilitate the synthesis of oligosaccharides for the study of their roles in various biological processes and their impacts on the structures and functions of glycoproteins and glycolipids. It is also thought that this work will benefit the glycoscience community significantly, given that this method is available to the research community free of charge. Synthesis of the essential human embryonic stem cell marker, SSEA-45, is demonstrated in this work.

<table><tbody><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>75-05-8</td><td></td></tr><tr><td>Anhydrous magnesium sulfate</td><td>Sigma-Aldrich</td><td>7487-88-9</td><td></td></tr><tr><td>Cerium ammonium molybdate</td><td>TCI</td><td>C1794</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma-Aldrich</td><td>75-09-2</td><td></td></tr><tr><td>Drierite</td><td>Sigma-Aldrich</td><td>7778-18-9</td><td></td></tr><tr><td>Ethyl acetate</td><td>Sigma-Aldrich</td><td>141-78-6</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>67-56-1</td><td></td></tr><tr><td>Molecular sieves 4 &amp;Aring;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>&lt;em&gt;n&lt;/em&gt;-Hexane</td><td>Sigma-Aldrich</td><td>110-54-3</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-Iodosuccinimide</td><td>Sigma-Aldrich</td><td>516-12-1</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>144-55-8</td><td></td></tr><tr><td>Sodium thiosulfate</td><td>Sigma-Aldrich</td><td>10102-17-7</td><td></td></tr><tr><td>Toluene</td><td>Sigma-Aldrich</td><td>108-88-3</td><td></td></tr><tr><td>Trifluoromethanesulfonic acid</td><td>Sigma-Aldrich</td><td>1493-13-6</td><td></td></tr></tbody></table>

hierarchical and programmable one-pot oligosaccharide synthesis

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 &#62;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.

The Auto-CHO search result based on default parameter settings indicates SSEA-4 can be synthesized by a [2 + 1 + 3] one-pot reaction. Figure 3 shows the software screenshot of the SSEA-4 search result. When a trisaccharide reducing end acceptor is selected (Figure 3, label 1), the program shows four potential solutions for the query. The first solution has one fragment (Figure 3, label 2), and its ca...

Watch this Scientific Journal Video about Hierarchical and Programmable One-Pot Oligosaccharide Synthesis at JoVE.com

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

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&#160;experiments and one-pot glycosylation of SSEA-4.

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for ...

hierarchical-and-programmable-one-pot-oligosaccharide-synthesis

Research

JoVE Journal

Chemistry

6.8K Views.  Academia Sinica. 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.

Video: Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other fields. For instance, trehalose analogues bearing detectable tags have been used to detect Mycobacterium tuberculosis and may have applications as tuberculosis diagnostic imaging agents. Hydrolytically stable versions of trehalose are also being pursued due to their potential for use as non-caloric sweeteners and bioprotective agents. Despite the appeal of this class of compounds for various applications, their potential remains unfulfilled due to the lack of a robust route for their production. Here, we report a detailed protocol for the rapid and efficient one-step biocatalytic synthesis of trehalose analogues that bypasses the problems associated with chemical synthesis. By utilizing the thermostable trehalose synthase (TreT) enzyme from Thermoproteus tenax, trehalose analogues can be generated in a single step from glucose analogues and uridine diphosphate glucose in high yield (up to quantitative conversion) in 15-60 min. A simple and rapid non-chromatographic purification protocol, which consists of spin dialysis and ion exchange, can deliver many trehalose analogues of known concentration in aqueous solution in as little as 45 min. In cases where unreacted glucose analogue still remains, chromatographic purification of the trehalose analogue product can be performed. Overall, this method provides a &#34;green&#34; biocatalytic platform for the expedited synthesis and purification of trehalose analogues that is efficient and accessible to non-chemists. To exemplify the applicability of this method, we describe a protocol for the synthesis, all-aqueous purification, and administration of a trehalose-based click chemistry probe to mycobacteria, all of which took less than 1 hour and enabled fluorescence detection of mycobacteria. In the future, we envision that, among other applications, this protocol may be applied to the rapid synthesis of trehalose-based probes for tuberculosis diagnostics. For instance, short-lived radionuclide-modified trehalose analogues (e.g., 18F-modified trehalose) could be used for advanced clinical imaging modalities such as positron emission tomography-computed tomography (PET-CT).

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

Chemically modified versions of trehalose, or trehalose analogues, have applications in biology, biotechnology, and pharmaceutical science, among other ...

Biochemistry

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Bioengineering

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5&#39;-[CUA CGG AAU CAU]-3&#39; containing zero, one, or two modifications located at the C2&#39;-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2&#39;-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism

This article provides a detailed procedure on the solid-phase synthesis, purification, and characterization of dodecamers of RNA modified at the C2&#39;-O-position. UV-vis and circular dichroism photometric analyses are used to quantify and characterize structural aspects, i.e., single-strands or double-strands.

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular ...

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate glycosyl donor. Following azido-nitration of a glycal, the product 2-azido-1-nitrate ester can be hydrolyzed under microwave-assisted irradiation. This transformation is usually achieved using strongly nucleophilic reagents and extended reaction times. Microwave irradiation induces hydrolysis, in the absence of reagents, with short reaction times. Following denitration, the intermediate anomeric alcohol is converted, in the same pot, to the corresponding 2-azido-1-trichloroacetimidate.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

Biology

Oligosaccharide Assembly

Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or glycosylation of proteins. Usually, these enzymes add sugars to the hydroxyl groups of selected serine or threonine residues to form O-linked glycans or the amino groups of asparagine residues to form N-linked glycans. Different positions on the same polypeptide chain can contain differently linked glycans.
Multiple sugar molecules that may or may not include mannose can be added to the precursor in the Golgi to generate complex oligosaccharides. N-linked sugars could be classified as high-mannose oligosaccharides based on whether sugars other than mannose and N-acetyl glucosamine are present. As soon as an N-linked oligosaccharide precursor generated in the ER enters the Golgi, it undergoes sugar trimming. Misfolded protein intermediates created during trimming are detected by a conformation sensing ER glucosyltransferase that adds a glucose residue, tagging the protein for another chance at folding. Proteins that fail to fold are transported to proteasomes for degradation.
In contrast, trimmed high-mannose oligosaccharides do not undergo sugar addition once they enter the Golgi apparatus. While the glycan core of the complex oligosaccharide is more accessible to adding sugars and tagging, the high mannose sugar cores remain tightly bound and inaccessible to the enzymes. Thus, resistance to specific glycosidases like Endo H is characteristic of maturing glycans. Because sugar addition is unidirectional, Endo H sensitivity is widely used to trace the forward transport of newly synthesized glycoproteins in the secretory pathway.

Protein glycosylation starts in the ER lumen and continues in the Golgi apparatus. Glycosyltransferases catalyze the addition of sugar molecules or ...

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

Summary

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Oligosaccharide Assembly