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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+frequency+of+protein+glycosylation,+as+deduced+from+analysis+of+the+SWISS-PROT+database.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+Automated+Synthesis+of+Oligosaccharides+and+Glycoproteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#8220;One-Pot&#8221;+Protection,+Glycosylation,+and+Protection-Glycosylation+Strategies+of+Carbohydrates.">&#8220;One-Pot&#8221; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+One-Pot+Oligosaccharide+Synthesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+and+programmable+one-pot+synthesis+of+oligosaccharides.">Hierarchical and programmable one-pot synthesis of oligosaccharides.</a> Nature Communications. 9 (1), 5202 (2018).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ChemDraw. , (2019).</li><li>Cheeseman, J. R., Frisch, &. #. 1. 9. 8. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Predicting magnetic properties with chemdraw and gaussian. , (2000).</li><li>Yap, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PaDEL-descriptor:+An+open+source+software+to+calculate+molecular+descriptors+and+fingerprints.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GlycanBuilder:+a+fast,+intuitive+and+flexible+software+tool+for+building+and+displaying+glycan+structures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GlycanBuilder+and+GlycoWorkbench+glycoinformatics+tools:+updates+and+new+developments.">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 &#197;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>n-Hexane</td>
			<td>Sigma-Aldrich</td>
			<td>110-54-3</td>
			<td></td>
		</tr>
		<tr>
			<td>N-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

5.8K 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

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.

A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming.

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Hierarchical ZnO/CdSSe Heterostructure Nanotrees

A two-step chemical vapor deposition procedure is here employed to prepare tree-like hierarchical ZnO/CdSSe hetero-nanostructures. The structures are composed of CdSSe branches grown on ZnO nanowires that are vertically aligned on a transparent sapphire substrate. The morphology was measured via scanning electron microscopy. The crystal structure was determined by X-ray powder diffraction analysis. Both the ZnO stem and CdSSe branches have a predominantly wurtzite crystal structure. The mole ratio of S and Se in the CdSSe branches was measured by energy dispersive X-ray spectroscopy. The CdSSe branches result in strong visible light absorption. Photoluminescence (PL) spectroscopy showed that the stem and branches form a type-II heterojunction. PL lifetime measurements showed a decrease in the lifetime of emission from the trees when compared to emission from individual ZnO stems or CdSSe branches and indicate fast charge transfer between CdSSe and ZnO. The vertically aligned ZnO stems provide a direct electron transport pathway to the substrate and allow for efficient charge separation after photoexcitation by visible light. The combination of the abovementioned properties makes ZnO/CdSSe nanotrees promising candidates for applications in solar cells, photocatalysis, and opto-electronic devices.

Here, we prepare and characterize novel tree-like hierarchical ZnO/CdSSe nanostructures, where CdSSe branches are grown on vertically aligned ZnO nanowires. The resulting nanotrees are a potential material for solar energy conversion and other opto-electronic devices.

A two-step chemical vapor deposition procedure is here employed to prepare tree-like hierarchical ZnO/CdSSe hetero-nanostructures. The structures are ...

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 ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products and notably to generate chiral carbon centers. The crucial first step consists in the selective double hydroboration of CO2 catalyzed by an iron hydride complex. The product obtained with this 4 e- reduction is a rare bis(boryl)acetal, compound 1, which is subjected in situ to three different reactions in a second step. The first reaction concerns a condensation reaction with (diisopropyl)phenylamine affording the corresponding imine 2. In the second and third reaction, intermediate 1 reacts with triazol-5-ylidene (Enders&#39; carbene) to afford compounds 3 or 4, depending on the reaction conditions. In both compounds, C-C bonds are formed, and chiral centers are generated from CO2 as the only source of carbon. Compound 4 exhibits two chiral centers obtained in a diastereoselective manner in a formose-type mechanism. We proved that the remaining boryl fragment plays a key role in this unprecedented stereocontrol. The interest of the method stands on the reactive and versatile nature of 1, giving rise to various complex molecules from a single intermediate. The complexity of a two-step method is compensated by the overall short reaction time (2 h for the larger reaction time), and mild reaction conditions (25 &#176;C to 80 &#176;C and 1 to 3 atm of CO2).

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

CO2 transformations are conducted in a one-pot two-step procedure for the synthesis of complex molecules. The selective 4 e- reduction of CO2 with a hydroborane reductant affords a reactive and versatile bis(boryl)acetal intermediate which is subsequently involved in condensation reaction or carbene-mediated C-C coupling generation.

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

Radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor proliferation, epilepsy, neurodegenerative diseases, and psychiatric illnesses due to their immune-modulatory, neuro-modulatory, and neurotoxic effects. The most extensively used positron emission tomography (PET) agent for mapping tryptophan metabolism, &#945;-[11C]methyl-L-tryptophan ([11C]AMT), has a short half-life of 20 min with laborious radiosynthesis procedures. An onsite cyclotron is required to radiosynthesize [11C]AMT. Only a limited number of centers produce [11C]AMT for preclinical studies and clinical investigations. Hence, the development of an alternative imaging agent that has a longer half-life, favorable in vivo kinetics, and is easy to automate is urgently needed. The utility and value of 1-(2-[18F]fluoroethyl)-L-tryptophan, a fluorine-18-labeled tryptophan analog, has been reported in preclinical applications in cell line-derived xenografts, patient-derived xenografts, and transgenic tumor models.
This paper presents a protocol for the radiosynthesis of 1-(2-[18F]fluoroethyl)-L-tryptophan using a one-pot, two-step strategy. Using this protocol, the radiotracer can be produced in a 20 &#177; 5% (decay corrected at the end of synthesis, n &#62; 20) radiochemical yield, with both radiochemical purity and enantiomeric excess of over 95%. The protocol features a small precursor amount with no more than 0.5 mL of reaction solvent in each step, low loading of potentially toxic 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222), and an environmentally benign and injectable mobile phase for purification. The protocol can be easily configured to produce 1-(2-[18F]fluoroethyl)-L-tryptophan for clinical investigation in a commercially available module.

Radiosynthesis of 1-2-[18F]Fluoroethyl-L-Tryptophan using a One-pot, Two-step Protocol

Here, we describe the radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-tryptophan, a positron emission tomography imaging agent for studying tryptophan metabolism, using a one-pot, two-step strategy in a radiochemistry synthesis system with good radiochemical yields, high enantiomeric excess, and high reliability.

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor ...