This research was financed by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Nos. 15K00408, 24659011, 24640156, 245900425 and 22390005 for Shin Aoki), by a grant from the Tokyo Biochemical Research Foundation, Tokyo, Japan, and by the TUS (Tokyo University of Science) fund for strategic research areas. We would like to thank Noriko Sawabe (Faculty of Pharmaceutical Sciences, Tokyo University of Science) for the measurements of the NMR spectra, Fukiko Hasegawa (Faculty of Pharmaceutical Sciences, Tokyo University of Science) for the measurements of the mass spectra, and Tomoko Matsuo (Research Institute for Science and Technology, Tokyo University of Science) for the measurements of the elemental analyses.

NOTE: All experimental data [NMR, infrared spectroscopies (IR), mass spectroscopies (MS), optical rotations, and elemental analyses data] of the synthesized compounds were reported in a previous paper51.
1. Procedure for O-Glycosylation Reactions
<ol>
	<li>Synthesis of compound &#945;/&#946;-12 (Entry 12 in Table 1) 
	NOTE: Entries 1 - 13 in Table 1 were carried out using a similar procedure.
	<ol>
		<li>Temporary protection of 2&#39;,3&#39;-diol of ribonucleoside40
		<ol>
			<li>In a 10 mL pear-shaped flask (flask 1), dissolve mannosyl donor &#945;-9 (28.4 mg, 0.0486 mmol)52, uridine 10 (7.9 mg, 0.0324 mmol) and 4-(trifluoromethyl)phenylboronic acid 11c (9.3 mg, 0.0490 mmol) in anhydrous pyridine (0.40 mL). 
			NOTE: The use of a 10 mL pear-shaped flask is recommended because, in step 1.1.3.1, the reaction mixture will be transferred to flask 2 (a 10 mL two-neck round-bottom flask with a septum attached to it) containing molecular sieves powder.</li>
			<li>Co-evaporate the reaction mixture (obtained in step 1.1.1.1.) with anhydrous pyridine (0.40 mL, 3x) and anhydrous 1,4-dioxane (0.40 mL, 3x) at room temperature to ca. 40 &#176;C to remove any water.</li>
			<li>Dissolve the residue (obtained in step 1.1.1.2.) in anhydrous 1,4-dioxane (0.32 mL) and stir the reaction mixture at its reflux temperature for 1 h to form a boronic ester (the temporary protection).</li>
			<li>Remove the solvent using a rotary evaporator followed by a vacuum pump.</li>
		</ol>
		</li>
		<li>Activation of molecular sieves
		<ol>
			<li>In a 10 mL two-neck round-bottom flask with a septum attached to it (flask 2), add 4 &#197; molecular sieves powder (64 mg). 
			NOTE: Appropriate molecular sieves should be selected according to the solvent used for the glycosylation (3 &#197; for acetonitrile and 4 &#197; for 1,4-dioxane, dichloromethane, and propionitrile).</li>
			<li>Heat the molecular sieves in a microwave under atmospheric pressure and cool them under reduced pressure evacuated by a vacuum pump (3x), and then dry them with a heat gun under reduced pressure while replacing the air with argon gas several times.</li>
		</ol>
		</li>
		<li>Glycosylation
		<ol>
			<li>Dissolve the residue of step 1.1.1.4. in flask 1 in propionitrile (0.64 mL) or other solvents and transfer this solution to flask 2. 
			NOTE: Acetonitrile, 1,4-dioxane, dichloromethane, and propionitrile were used for Entries 1 - 7 and 9, Entry 10, Entry 11, and Entries 8, 12, and 13, respectively.</li>
			<li>Stir the reaction mixture in flask 2 at room temperature for 0.5 h followed by cooling it to -40 &#176;C. 
			NOTE: The temperature was changed according to the solvent used for the glycosylation (-40 &#176;C for dichloromethane and propionitrile, room temperature for 1,4-dioxane, and -20 &#176;C for acetonitrile).</li>
			<li>Add silver triflate (49.9 mg, 0.194 mmol) and p-toluenesulfenyl chloride (12.8 &#181;L, 0.0968 mmol) to the reaction mixture at the same temperature as used in step 1.1.3.2.</li>
			<li>Stir the reaction mixture&#160;at the same temperature&#160;for 1.5 h.</li>
			<li>Check the reaction by thin-layer chromatography (TLC) with hexane/ethyl acetate [3/1 (v/v)] to check the glycosyl donors [the retention factor (Rf) (donor &#945;-9) = 0.63] and with chloroform/methanol [10/1 (v/v))] to check the glycosyl acceptors and products [Rf (acceptor 10) = 0.03, Rf (desired product) = 0.50].</li>
			<li>Quench the reaction mixture with saturated aqueous sodium bicarbonate (1.0 mL), dilute it with chloroform (2.0 mL), remove the insoluble materials with Celite, and carefully wash the Celite with chloroform (20 mL).</li>
			<li>Wash the filtrate (organic layer) with saturated aqueous sodium bicarbonate (20 mL, 3x) and brine (20 mL) using a 100 mL separatory funnel.</li>
			<li>Dry the resulting organic layer with sodium sulfate, filter the insoluble materials, and concentrate the filtrate using a rotary evaporator.</li>
			<li>Roughly purify the remaining residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 50/1 (v/v)] to afford crude 5&#39;-O-(6&#34;-O-acetyl-2&#34;,3&#34;,4&#34;-tri-O-benzyl-&#945;/&#946;-&#7429;-mannopyranosyl)uridine containing a small quantity of byproducts (15.2 mg, colorless syrup).</li>
		</ol>
		</li>
		<li>Acetylation
		<ol>
			<li>In a 5 mL vial, dissolve the resulting crude compound prepared in step 1.1.3.9 in anhydrous pyridine (0.20 mL).</li>
			<li>Add N,N-dimethyl-4-aminopyridine (a catalytic amount) and acetic anhydride (20.4 &#181;L, 0.0216 mmol: 10 equivalents based on the crude compound) to the solution at 0 &#176;C.</li>
			<li>Stir the reaction mixture at the same temperature for 0.5 h followed by a warming to room temperature.</li>
			<li>After stirring overnight, check the reaction by TLC with chloroform/methanol [30/1 (v/v)] [Rf (&#945;/&#946;-12) = 0.45].</li>
			<li>Dilute the reaction mixture with chloroform (20 mL).</li>
			<li>Wash the organic layer with 1 M hydrochloric acid (20 mL, 3x), saturated aqueous sodium bicarbonate (20 mL, 3x), and brine (20 mL) using a 100 mL separatory funnel.</li>
			<li>Dry the resulting organic layer with sodium sulfate, filter the insoluble materials, and concentrate the filtrate using a rotary evaporator.</li>
			<li>Purify the remaining residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 90/1 (v/v)] to give &#945;/&#946;-12 (15.8 mg, 61%, &#945;/&#946; = 1.6/1, colorless amorphous solid).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Synthesis of compounds &#946;-22 to &#946;-30 (Table 2) and &#946;-33 (Table 3) 
	NOTE: The synthesis of &#946;-22 - &#946;-30 and &#946;-33 was carried out using a similar procedure.
	<ol>
		<li>Synthesis of compound &#946;-22 (Entry 1 in Table 2)
		<ol>
			<li>Temporary protection of 2&#39;, 3&#39;-diol of ribonucleoside
			<ol>
				<li>In a 10 mL pear-shaped flask (flask 3), dissolve adenosine 13 (20.4 mg, 0.0763 mmol), galactosyl donor &#946;-21 (80.4 mg, 0.114 mmol)53, and 4-(trifluoromethyl)phenylboronic acid 11c (21.7 mg, 0.114 mmol) in anhydrous pyridine (0.76 mL). 
				NOTE: The use of a 10 mL pear-shaped flask is recommended because the reaction mixture will be transferred to flask 4 (a 10 mL two-neck round-bottom flask with a septum attached to it) containing molecular sieves powder in the step 1.2.1.3.1.</li>
				<li>Co-evaporate the reaction mixture (obtained in step 1.2.1.1.1.) with anhydrous pyridine (0.76 mL, 3x) and anhydrous 1,4-dioxane (0.76 mL, 3x) at room temperature to ca. 40 &#176;C to remove any water.</li>
				<li>Dissolve the residue (obtained in step 1.2.1.1.2.) in anhydrous 1,4-dioxane (0.76 mL) and stir the reaction mixture at its reflux temperature for 1 h to form a boronic ester (a temporary protection).</li>
				<li>Remove the solvent using a rotary evaporator followed by a vacuum pump.</li>
			</ol>
			</li>
			<li>Activation of molecular sieves
			<ol>
				<li>In a 10 mL two-neck round-bottom flask with a septum attached to it (flask 4), add 4 &#197; molecular sieves powder (150 mg).</li>
				<li>Heat the molecular sieves in a microwave under atmospheric pressure and cool them under reduced pressure evacuated by a vacuum pump (3x), and then dry them with a heat gun under reduced pressure while replacing the air with argon gas several times.</li>
			</ol>
			</li>
			<li>Glycosylation
			<ol>
				<li>Dissolve the residue of step 1.2.1.1.4. in flask 3 in propionitrile (1.50 mL) and transfer this solution to flask 4.</li>
				<li>Stir the reaction mixture at room temperature for 0.5 h, followed by cooling it to -40 &#176;C.</li>
				<li>Add silver triflate (117.6 mg, 0.458 mmol) and p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol) to the reaction mixture at the same temperature as mentioned in step 1.2.1.3.2.</li>
				<li>Stir the reaction mixture,&#160;at the same temperature for 1.5 h.</li>
				<li>Check the reaction by TLC with hexane/ethyl acetate [2/1 (v/v)] to check the glycosyl donors [Rf (donor &#946;-21) = 0.62] and with chloroform/methanol [10/1 (v/v)] to check the glycosyl acceptors and products [Rf (acceptor 13) = 0.05, Rf (desired product) = 0.30].</li>
				<li>Quench the reaction mixture with saturated aqueous sodium bicarbonate (2.0 mL), dilute it with chloroform (3.0 mL), remove the insoluble materials through Celite, and carefully wash the Celite with chloroform (30 mL).</li>
				<li>Wash the filtrate (organic layer) with saturated aqueous sodium bicarbonate (30 mL, 3x) and brine (30 mL) using a 100 mL separatory funnel.</li>
				<li>Dry the resulting organic layer with sodium sulfate, filter the insoluble materials, and concentrate the filtrate using a rotary evaporator.</li>
				<li>Purify the remaining residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 30/1 (v/v)] to afford &#946;-22 (27.4 mg, 42%, colorless solid).</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-23 (Entry 2 in Table 2)
		<ol>
			<li>Conduct the reaction using 14 (28.4 mg, 0.0765 mmol)54, &#946;-21 (80.5 mg, 0.115 mmol), 11c (21.8 mg, 0.115 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.8 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 50/1 (v/v)] to give &#946;-23 (21.9 mg, 30%, colorless solid). TLC: Rf (&#946;-23) = 0.37 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-24 (Entry 3 in Table 2)
		<ol>
			<li>Conduct the reaction using 15 (21.6 mg, 0.0763 mmol), &#946;-21 (80.5 mg, 0.115 mmol), 11c (21.8 mg, 0.115 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.6 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 8/1 (v/v)] to give &#946;-24 (8.1 mg, 12%, colorless solid). TLC: Rf (&#946;-24) = 0.20 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-25 (Entry 4 in Table 2)
		<ol>
			<li>Conduct the reaction using 16 (27.0 mg, 0.0764 mmol)55, &#946;-21 (80.5 mg, 0.115 mmol), 11c (21.8 mg, 0.115 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.8 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 20/1 (v/v)] to give &#946;-25 (31.4 mg, 44%, colorless solid). TLC: Rf (&#946;-25) = 0.27 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-26 (Entry 5 in Table 2)
		<ol>
			<li>Conduct the reaction using 10 (18.6 mg, 0.0762 mmol), &#946;-21 (80.4 mg, 0.114 mmol), 11c (21.7 mg, 0.114 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.6 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 40/1 (v/v)] to give &#946;-26 (26.1 mg, 42%, colorless solid). TLC: Rf (&#946;-26) = 0.45 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-27 (Entry 6 in Table 2)
		<ol>
			<li>Conduct the reaction using 17 (19.7 mg, 0.0763 mmol), &#946;-21 (80.5 mg, 0.115 mmol), 11c (21.8 mg, 0.115 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.6 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 40/1 (v/v)] to give &#946;-27 (33.8 mg, 53%, colorless solid). TLC: Rf (&#946;-27) = 0.50 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-28 (Entry 7 in Table 2)
		<ol>
			<li>Conduct the reaction using 18 (20.0 mg, 0.0763 mmol), &#946;-21 (80.4 mg, 0.114 mmol), 11c (21.7 mg, 0.114 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.6 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform then ethyl acetate/chloroform = 1/1 (v/v)] to give &#946;-28 (38.8 mg, 61%, colorless solid). TLC: Rf (&#946;-28) = 0.33 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-29 (Entry 8 in Table 2)
		<ol>
			<li>Conduct the reaction using 19 (18.5 mg, 0.0761 mmol), &#946;-21 (80.4 mg, 0.114 mmol), 11c (21.7 mg, 0.114 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.6 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 10/1 (v/v)] to give &#946;-29 (34.1 mg, 55%, colorless solid). TLC: Rf (&#946;-29) = 0.25 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-30 (Entry 9 in Table 2)
		<ol>
			<li>Conduct the reaction using 20 (26.6 mg, 0.0766 mmol)56, &#946;-21 (80.6 mg, 0.115 mmol), 11c (21.8 mg, 0.115 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.8 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 50/1 (v/v)] to give &#946;-30 (28.0 mg, 40%, colorless solid). TLC: Rf (&#946;-30) = 0.48 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
		<li>Synthesis of compound &#946;-33 (Entry 1 in Table 3)
		<ol>
			<li>Conduct the reaction using 18 (20.0 mg, 0.0762 mmol), &#946;-31&#160;(80.4 mg, 0.114 mmol)57, 11c (21.7 mg, 0.114 mmol), p-toluenesulfenyl chloride (30.3 &#181;L, 0.229 mmol), silver triflate (117.6 mg, 0.458 mmol), anhydrous 1,4-dioxane (0.76 mL), anhydrous propionitrile (1.50 mL), and 4 &#197; molecular sieves (150 mg). Purify the resulting residue by column chromatography [silica gel, chloroform/methanol = 1/0 - 30/1 (v/v)] to give &#946;-33 (34.5 mg, 54%, colorless solid). TLC: Rf (&#946;-33) = 0.33 [chloroform/methanol = 10/1 (v/v)].</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Deprotection of &#946;-28 (Figure 2)
<ol>
	<li>In a 5 mL vial, add &#946;-28 (25.2 mg, 0.0300 mmol) and 10 M methylamine in methanol (2.0 mL)58.</li>
	<li>Stir the reaction mixture at 0 &#176;C for 2 h followed by warming it to room temperature.</li>
	<li>After stirring the mixture for 13 h, check the reaction by TLC with chloroform/methanol [10/1 (v/v)] [Rf (&#946;-35) = 0.20].</li>
	<li>Concentrate the reaction mixture using a rotary evaporator.</li>
	<li>Dissolve the resulting residue in water (15 mL) and wash the aqueous layer with dichloromethane (15 mL, 3x) using a 50 mL separatory funnel.</li>
	<li>Concentrate the aqueous layer using a rotary evaporator.</li>
	<li>Purify the remaining residue by preparative high-performance liquid chromatography (HPLC) [column: ODS (octadecylsilane) column (20&#934; x 250 mm), eluent: water (contains 0.1% [v/v] trifluoroacetic acid), flow rate: 8.0 mL/min, detection: 266 nm, temperature: 25 &#176;C, retention time: 20 min] to give &#946;-35 (7.9 mg, 62%, colorless amorphous solid)59.</li>
</ol>
3. NMR Studies of Cyclic Boronic Ester (Figure 3 and 4)
<ol>
	<li>Preparation and measurement of 36
	<ol>
		<li>In 10 mL pear-shaped flask, dissolve uridine 10 (34.3 mg, 0.140 mmol) and 4-(trifluoromethyl)phenylboronic acid 11c (40.0 mg, 0.211 mmol) in anhydrous pyridine (1.00 mL).</li>
		<li>Co-evaporate the reaction mixture with anhydrous pyridine (1.00 mL, 3x) and anhydrous 1,4-dioxane (1.00 mL, 3x) at room temperature to ca. 40 &#176;C to remove any water.</li>
		<li>Dissolve the residue in anhydrous 1,4-dioxane (1.40 mL) and stir the reaction mixture at its reflux temperature for 1 h to form a boronic ester (a temporary protection).</li>
		<li>Dispense the reaction mixture (0.14 mL) to a 5 mL vial.</li>
		<li>Remove the solvent from the 5 mL vial using a rotary evaporator followed by a vacuum pump.</li>
		<li>Dissolve the resulting residue 36 in acetonitrile-d3 (0.64 mL).</li>
		<li>Measure 1H, 11B and 19F NMR spectroscopies using a quartz NMR tube at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation and measurement of 38
	<ol>
		<li>Prepare the reaction mixture 38 from 11c (40.0 mg, 0.211 mmol) using the similar procedure as that of the step 3.1.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The purpose of this manuscript is to show a convenient synthetic method to prepare disaccharide nucleosides using unprotected ribonucleosides without tedious protecting group manipulations. We report herein on the regioselective O-glycosylations of nucleosides via the temporary 2&#39;,3&#39;-diol protection by a cyclic boronic ester (Figure 1B)51.
The preparation of the cyclic boronic ester intermediate is...

Disaccharide nucleosides, which are conjugates of a nucleoside and a carbohydrate moiety linked via an O-glycosidic bond, constitute a valuable class of naturally-occurring carbohydrate derivatives1,2,3,4,5,6,7. For instance, they are incorporated in biological macromolecules such as tRNA (transfer ribonucleic acid) and poly(ADP-ribose) (ADP = adenosine diphosphate), as well as in some antibacterial agents and other biologically-active substances (e.g., adenophostins, amicetins, ezomycin)5,6,8,9,10,11,12,13,14,15,16,17,18,19. Hence, disaccharide nucleosides and their derivatives are expected to be lead compounds for drug discovery research. The methodologies for the synthesis of disaccharide nucleosides are classified into three categories; enzymatic O-glycosylation20,21, chemical N-glycosylation5,9,16,22,23,24, and chemical O-glycosylation7,9,14,16,18,19,24,25,26,27,28,29,30,31,32,33,34,35,36,37. In particular, chemical O-glycosylation would be an efficient method for the stereoselective synthesis and large-scale synthesis of disaccharide nucleosides. Previous research has shown that the O-glycosylation of 2&#39;-deoxyribonucleoside 2 with the thioglycosyl donor 1, using the combination of p-toluenesulfenyl chloride and silver triflate, affords the desired disaccharide nucleoside 3 (Figure 1A; Ar = aryl and PG = protecting group)38.
Following these results, we decided to develop the O-glycosylation of ribonucleosides applying the p-toluenesulfenyl chloride/silver triflate promoter system. While several examples of the O-glycosylation of partially protected ribonucleosides have been demonstrated7,9,14,16,18,19,24,32,33,34,35,36,37, the use of unprotected or temporarily-protected ribonucleosides as a glycosyl acceptor for O-glycosylation has been negligibly reported. Therefore, the development of regioselective O-glycosylation of unprotected or temporarily-protected ribonucleosides would provide a more beneficial synthetic method without protecting group manipulations of ribonucleosides. In order to achieve the regioselective O-glycosylation of ribonucleosides, we focused on the boron compounds, because several examples of regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation of carbohydrate derivatives assisted by boronic or borinic acid have been reported39,40,41,42,43,44,45,46,47,48,49,50. In this article, we demonstrate the procedure for the synthesis of disaccharide nucleosides utilizing regioselective O-glycosylation at the 5&#39;-hydroxyl group of ribonucleosides via a boronic ester intermediate. In the strategy presented here, boronic ester intermediate 6 would be afforded by the esterification of the ribonucleoside 4 with the boronic acid 5, which allows the regioselective O-glycosylation at the 5&#39;-hydroxyl group with thioglycosyl donor 7 to give the disaccharide nucleoside 8 (Figure 1B)51. We also studied the interaction of a ribonucleoside and boronic acid by nuclear magnetic resonance (NMR) spectroscopy, to observe the formation of a boronic ester. Esterification to make a boronic ester and a glycosylation reaction require anhydrous conditions to prevent the hydrolysis of the boronic ester and the glycosyl donor. In this article, we demonstrate the typical procedures to obtain the anhydrous conditions for successful glycosylation reactions for researchers and students not only in chemistry but also in other research fields.

<table><tbody><tr><td>Silver trifluoromethanesulfonate</td><td>Nacalai Tesque</td><td>34945-61</td><td></td></tr><tr><td>Phenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>B0857</td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Methoxyphenylboronic acid</td><td>Wako Pure Chemical Industries</td><td>321-69201</td><td></td></tr><tr><td>4-(Trifluoromethyl)phenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>T1788</td><td></td></tr><tr><td>2,4-Difluorophenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>D3391</td><td></td></tr><tr><td>Cyclopentylboronic acid (contains varying amounts of Anhydride)</td><td>Tokyo Chemical Industry</td><td>C2442</td><td></td></tr><tr><td>4-Nitrophenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>N0812</td><td></td></tr><tr><td>4-Hexylphenylboronic acid (contains varying amounts of anhydride)</td><td>Tokyo Chemical Industry</td><td>H1489</td><td></td></tr><tr><td>Adenosine</td><td>Merck KGaA</td><td>862.</td><td></td></tr><tr><td>Guanosine</td><td>Acros Organics</td><td>411130050</td><td></td></tr><tr><td>Cytidine</td><td>Tokyo Chemical Industry</td><td>C0522</td><td></td></tr><tr><td>Uridine</td><td>Tokyo Chemical Industry</td><td>U0020</td><td></td></tr><tr><td>5-Fluorouridine</td><td>Tokyo Chemical Industry</td><td>F0636</td><td></td></tr><tr><td>5-Methyluridine</td><td>Sigma</td><td>M-9885</td><td></td></tr><tr><td>Methylamine (40% in Methanol,&amp;nbsp;ca. 9.8mol/L)</td><td>Tokyo Chemical Industry</td><td>M1016</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-dimethyl-4-aminopyridine</td><td>Wako Pure Chemical Industries</td><td>044-19211</td><td></td></tr><tr><td>Acetic anhydride</td><td>Nacalai Tesque</td><td>00226-15</td><td></td></tr><tr><td>Pyridine, Dehydrated</td><td>Wako Pure Chemical Industries</td><td>161-18453</td><td></td></tr><tr><td>Acetonitrile</td><td>Kanto Chemical</td><td>01031-96</td><td></td></tr><tr><td>1,4-Dioxane</td><td>Nacalai Tesque</td><td>13622-73</td><td></td></tr><tr><td>Dichloromethane</td><td>Wako Pure Chemical Industries</td><td>130-02457</td><td></td></tr><tr><td>Propionitrile</td><td>Wako Pure Chemical Industries</td><td>164-04756</td><td></td></tr><tr><td>Molecular sieves 4A powder</td><td>Nacalai Tesque</td><td>04168-65</td><td></td></tr><tr><td>Molecular sieves 3A powder</td><td>Nacalai Tesque</td><td>04176-55</td><td></td></tr><tr><td>Celite 545RVS</td><td>Nacalai Tesque</td><td>08034-85</td><td></td></tr><tr><td>Acetonitrile-D3 (D,99.8%)</td><td>Cambridge Isotope Laboratories</td><td>DLM-21-10</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Nacalai Tesque</td><td>34831-25</td><td></td></tr><tr><td>TLC Silica gel 60 F&lt;sub&gt;254&lt;/sub&gt;</td><td>Merck KGaA</td><td>1.05715.0001</td><td></td></tr><tr><td>Chromatorex</td><td>Fuji Silysia Chemical</td><td>FL100D</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Wako Pure Chemical Industries</td><td>191-01305</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Wako Pure Chemical Industries</td><td>080-01061</td><td></td></tr><tr><td>Sodium sulfate</td><td>Nacalai Tesque</td><td>31915-96</td><td></td></tr><tr><td>Chloroform</td><td>Kanto Chemical</td><td>07278-81</td><td></td></tr><tr><td>Sodium chloride</td><td>Wako Pure Chemical Industries</td><td>194-01677</td><td></td></tr><tr><td>Methanol</td><td>Nacalai Tesque</td><td>21914-74</td><td></td></tr><tr><td>JEOL Always 300</td><td>JEOL</td><td></td><td>Measurement of NMR</td></tr><tr><td>Lamda 400</td><td>JEOL</td><td></td><td>Measurement of NMR</td></tr><tr><td>PerkinElmer Spectrum 100 FT-IR Spectrometer</td><td>Perkin Elmer</td><td></td><td>Measurement of IR</td></tr><tr><td>JEOL JMS-700</td><td>JEOL</td><td></td><td>Measurement of MS</td></tr><tr><td>PerkinElmer CHN 2400 analyzer</td><td>Perkin Elmer</td><td></td><td>Measurement of elemental analysis</td></tr><tr><td>JASCO P-1030 digital polarimeter</td><td>JASCO</td><td></td><td>Measurement of optical rotation</td></tr><tr><td>JASCO PU-2089 Plus intelligent HPLC pump</td><td>JASCO</td><td></td><td>For HPLC</td></tr><tr><td>Jasco UV-2075 Plus Intelligent UV/Vis Detector</td><td>JASCO</td><td></td><td>For HPLC</td></tr><tr><td>Rheodyne Model 7125 Injector</td><td>Sigma-Aldrich</td><td>58826</td><td>For HPLC</td></tr><tr><td>Chromatopac C-R8A</td><td>Shimadzu</td><td></td><td>For HPLC</td></tr><tr><td>Senshu Pak Pegasil ODS</td><td>Senshu Scientific</td><td></td><td>For HPLC</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-Toluenesulfenyl&amp;nbsp;chloride</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 38</td></tr><tr><td>Phenyl 6-O-acetyl-2,3,4-tri-&lt;em&gt;O&lt;/em&gt;-benzyl-1-thio-a-D-mannopyranoside (a-&lt;strong&gt;9&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 52</td></tr><tr><td>4-Metylphenyl 2,3,4,6-tetra-&lt;em&gt;O&lt;/em&gt;-benzoyl-1-thio-b-D-galactopyranoside (b-&lt;strong&gt;21&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 53</td></tr><tr><td>4-Metylphenyl 2,3,4,6-tetra-&lt;em&gt;O&lt;/em&gt;-benzoyl-1-thio-b-D-glucopyranoside (b-&lt;strong&gt;31&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 57</td></tr><tr><td>4-Metylphenyl 2,3,4,6-tetra-&lt;em&gt;O&lt;/em&gt;-benzoyl-1-thio-a-D-Mannopyranoside (a-&lt;strong&gt;32&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 67</td></tr><tr><td>6-&lt;em&gt;N&lt;/em&gt;-Benzoyladenosine (&lt;strong&gt;14&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 54</td></tr><tr><td>2-&lt;em&gt;N&lt;/em&gt;-Isobutyrylguanosine (&lt;strong&gt;16&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 55</td></tr><tr><td>4-&lt;em&gt;N&lt;/em&gt;-Benzoylcytidine (&lt;strong&gt;20&lt;/strong&gt;)</td><td></td><td></td><td>Prepared&amp;nbsp; Ref. 56</td></tr></tbody></table>

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.

The results of the O-glycosylation of uridine 10 with thiomannoside &#945;-9 are summarized in Table 160,61. In Entry 1, the O-glycosylation of 10 with &#945;-9 in the absence of boronic acid derivatives resulted in the formation of a complicated mixture. In Entry 2, 10 and phenylboronic acid 11a...

Watch this Scientific Journal Video about Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides at JoVE

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.

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 ...

regioselective-o-glycosylation-nucleosides-via-temporary-2-3-diol

Research

JoVE Journal

Chemistry

8.6K Views.  Tokyo University of Science. Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2',3'-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.

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

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

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

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Immunology and Infection

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 ...

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.

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 ...

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 ...

Chemical Triphosphorylation of Oligonucleotides

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of oligonucleotides in biotechnology and synthetic biology. Oligonucleotide 5&#8242;-triphosphates have historically been prepared in vitro by enzymatic methods. However, these methods are limited to natural RNA oligonucleotides, have strong sequence preferences, and tend to produce heterogeneous products. New methods of chemical triphosphorylation complement both the reduced cost of automated oligonucleotide synthesis by phosphoramidite chemistry and the diverse range of nucleotide modifications now available. Thus, the synthesis of oligonucleotide triphosphates of arbitrary sequence and length, and optionally containing various nonnatural modifications, is now accessible.
This paper presents the appropriate methods and techniques for chemical triphosphorylation of oligonucleotides using salicyl phosphorochloridite and pyrophosphate. This method uses commercially available reagents, is compatible with most oligonucleotides prepared by standard solid-phase synthesis methods, and can be completed in 2 h following oligonucleotide synthesis, before deprotection and purification. Two uses of chemically triphosphorylated oligonucleotides as substrates for catalytic RNA enzymes are demonstrated, including the synthesis of a mirror-image version of the hammerhead ribozyme from nonbiological L-RNA triphosphates.

Oligonucleotide 5&#8242;-triphosphates are ubiquitous components in essential biological pathways and have seen increasing use in biotechnology applications. Here, we describe techniques for the routine synthesis and purification of oligonucleotide 5&#8242;-triphosphates, starting from oligonucleotides prepared by standard automated synthesis techniques.

The 5&#8242;-triphosphate is an essential nucleic acid modification found throughout all life and increasingly used as a functional modification of ...

Expansion Microscopy for Visualizing Intracellular Sialylated Glycans via MOE

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections.
Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques.
In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.

Here, we propose a simple protocol combining metabolic oligosaccharide engineering, click chemistry, and expansion microscopy that allows bioimaging of intracellular sialylated N-glycoproteins with improved resolution using routine microscopy equipment.

Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes ...

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

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Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

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

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

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

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

Chemical Triphosphorylation of Oligonucleotides

Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy