Name: regioselective o-glycosylation of nucleosides via the temporary 2',3'-diol protection by a boronic ester for the synthesis of disaccharide nucleosides
Uploaded: 2018-07-26T13:00:00
Description: 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

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>Temporar...

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 border="0" cellpadding="0" cellspacing="0" style="width:847px;" width="847">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Silver trifluoromethanesulfonate</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">34945-61</td>
			<td style="width:232px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Phenylboronic acid (contains varying amounts of anhydride)</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">B0857</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">p-Methoxyphenylboronic acid</td>
			<td style="width:255px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">321-69201</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:241px;">4-(Trifluoromethyl)phenylboronic acid (contains varying amounts of anhydride)</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">T1788</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:241px;">2,4-Difluorophenylboronic acid (contains varying amounts of anhydride)</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">D3391</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Cyclopentylboronic acid (contains varying amounts of Anhydride)</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">C2442</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:241px;">4-Nitrophenylboronic acid (contains varying amounts of anhydride)</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">N0812</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:241px;">4-Hexylphenylboronic acid (contains varying amounts of anhydride)</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">H1489</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Adenosine</td>
			<td style="width:255px;">Merck KGaA</td>
			<td style="width:119px;">862.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Guanosine</td>
			<td style="width:255px;">Acros Organics</td>
			<td align="right" style="width:119px;">411130050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Cytidine</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">C0522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Uridine</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">U0020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">5-Fluorouridine</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">F0636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">5-Methyluridine</td>
			<td style="width:255px;">Sigma</td>
			<td style="width:119px;">M-9885</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Methylamine (40% in Methanol,&#160;ca. 9.8mol/L)</td>
			<td style="width:255px;">Tokyo Chemical Industry</td>
			<td style="width:119px;">M1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">N,N-dimethyl-4-aminopyridine</td>
			<td style="width:255px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">044-19211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Acetic anhydride</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">00226-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Pyridine, Dehydrated</td>
			<td style="width:255px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">161-18453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Acetonitrile</td>
			<td style="width:255px;">Kanto Chemical</td>
			<td style="width:119px;">01031-96</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">1,4-Dioxane</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">13622-73</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Dichloromethane</td>
			<td style="width:255px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">130-02457</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Propionitrile</td>
			<td style="width:255px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">164-04756</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Molecular sieves 4A powder</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">04168-65</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Molecular sieves 3A powder</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">04176-55</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Celite 545RVS</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">08034-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Acetonitrile-D3 (D,99.8%)</td>
			<td style="width:255px;">Cambridge Isotope Laboratories</td>
			<td style="width:119px;">DLM-21-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Trifluoroacetic acid</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">34831-25</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:241px;">TLC Silica gel 60 F254</td>
			<td style="width:255px;">Merck KGaA</td>
			<td style="width:119px;">1.05715.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Chromatorex</td>
			<td style="width:255px;">Fuji Silysia Chemical</td>
			<td style="width:119px;">FL100D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Sodium hydrogen carbonate</td>
			<td style="width:255px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">191-01305</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Hydrochloric acid</td>
			<td style="width:255px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">080-01061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Sodium sulfate</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">31915-96</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Chloroform</td>
			<td style="width:255px;">Kanto Chemical</td>
			<td style="width:119px;">07278-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Sodium chloride</td>
			<td style="width:255px;">Wako Pure Chemical Industries</td>
			<td style="width:119px;">194-01677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Methanol</td>
			<td style="width:255px;">Nacalai Tesque</td>
			<td style="width:119px;">21914-74</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">JEOL Always 300</td>
			<td style="width:255px;">JEOL</td>
			<td style="width:119px;"></td>
			<td>Measurement of NMR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Lamda 400</td>
			<td style="width:255px;">JEOL</td>
			<td style="width:119px;"></td>
			<td>Measurement of NMR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">PerkinElmer Spectrum 100 FT-IR Spectrometer</td>
			<td style="width:255px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td>Measurement of IR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">JEOL JMS-700</td>
			<td style="width:255px;">JEOL</td>
			<td style="width:119px;"></td>
			<td>Measurement of MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">PerkinElmer CHN 2400 analyzer</td>
			<td style="width:255px;">Perkin Elmer</td>
			<td style="width:119px;"></td>
			<td>Measurement of elemental analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">JASCO P-1030 digital polarimeter</td>
			<td style="width:255px;">JASCO</td>
			<td style="width:119px;"></td>
			<td>Measurement of optical rotation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">JASCO PU-2089 Plus intelligent HPLC pump</td>
			<td style="width:255px;">JASCO</td>
			<td style="width:119px;"></td>
			<td>For HPLC</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Jasco UV-2075 Plus Intelligent UV/Vis Detector</td>
			<td style="width:255px;">JASCO</td>
			<td style="width:119px;"></td>
			<td>For HPLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Rheodyne Model 7125 Injector</td>
			<td style="width:255px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">58826</td>
			<td>For HPLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Chromatopac C-R8A</td>
			<td style="width:255px;">Shimadzu</td>
			<td style="width:119px;"></td>
			<td>For HPLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">Senshu Pak Pegasil ODS</td>
			<td style="width:255px;">Senshu Scientific</td>
			<td style="width:119px;"></td>
			<td>For HPLC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">p-Toluenesulfenyl&#160;chloride</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td>Prepared&#160; Ref. 38</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Phenyl 6-O-acetyl-2,3,4-tri-O-benzyl-1-thio-a-D-mannopyranoside (a-9)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td>Prepared&#160; Ref. 52</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:241px;">4-Metylphenyl 2,3,4,6-tetra-O-benzoyl-1-thio-b-D-galactopyranoside (b-21)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td>Prepared&#160; Ref. 53</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:241px;">4-Metylphenyl 2,3,4,6-tetra-O-benzoyl-1-thio-b-D-glucopyranoside (b-31)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td>Prepared&#160; Ref. 57</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:241px;">4-Metylphenyl 2,3,4,6-tetra-O-benzoyl-1-thio-a-D-Mannopyranoside (a-32)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td>Prepared&#160; Ref. 67</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">6-N-Benzoyladenosine (14)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td>Prepared&#160; Ref. 54</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">2-N-Isobutyrylguanosine (16)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td>Prepared&#160; Ref. 55</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">4-N-Benzoylcytidine (20)</td>
			<td style="width:255px;"></td>
			<td style="width:119px;"></td>
			<td>Prepared&#160; 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&#039;,3&#039;-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 ...

This method can be expected to help drug discovery research because of short process needed for the preparation of biologically-active disaccharide nucleosides as compared with traditional existing methods. The main advantage of this technique is that unprotected nucleosides can be applied for glycosylation without a tedious protecting group manipulation to synthesize disaccharide nucleosides. Visual demonstration of this method will present useful information for successful glycosylation of unprotected nucleosides for researchers and students, not only in chemistry but also in other research fields.To begin, optimize the reaction conditions for the synthesis of compound Alpha-and Beta-12 as described in the text protocol. Next, apply the optimized reaction conditions to the synthesis of Beta-22 to Beta-30, as well as for Beta-33, using a similar procedure. In this video, the synthesis of compound Beta-22 is demonstrated.Begin with the temporary protection of two prime, three prime-Diol of ribonucleoside by dissolving 20.4 milligrams of Adenosine-13 in 0.76 milliliters of anhydrous pyridine in a 10 milliliter pear-shaped flask. Then, add 80.4 milligrams of galactosyl donor Beta-21. Now add 21.7 milligrams of four-Trifluoromethyl phenylboronic acid 11C.This boronic acid alternative is used because it gave Alpha-and Beta-12 in the highest chemical yield during optimization. Evaporate the mixture, dissolved in 0.76 milliliters of anhydrous pyridine, using the rotary evaporator. Then, co-evaporate the resulting reaction mixture with 0.76 milliliters of anhydrous pyridine twice, followed by 0.76 milliliters of anhydrous one four-dioxane three times at room temperature to approximately 40 degrees Celsius to remove any water.Dissolve the residue in 0.76 milliliters of anhydrous one four-dioxane and stir the reaction mixture at its reflux temperature for one hour to form a boronic ester as a temporary protection. Remove the solvent using a rotary evaporator, followed by a vacuum pump. Meanwhile, in a 10 milliliter two-neck round-bottom flask with a septum attached to it, add 150 milligrams of four-angstrom molecular sieves powder.Heat the molecular sieves in a microwave under atmospheric pressure. Then, cool them under reduced pressure, evacuated by a vacuum pump three times. After cooling, dry the sieves with a heat gun under reduced pressure, while replacing the air with argon gas several times.To perform the glycosylation, dissolve the residue resulting from vacuum decompression in 1.50 milliliters of anhydrous propionitrile and transfer this solution to a flask containing the molecular sieves. Stir the reaction mixture at room temperature for 0.5 hours, followed by cooling it to minus 40 degrees Celsius. Now, add 117.6 milligrams of silver triflate followed by 30.3 microliters of para toluenesulfonyl chloride to the reaction mixture at the same temperature.Stir the reaction mixture at minus 40 degrees Celsius for 1.5 hours. Next, check the reaction by thin-layer chromatography with two to one hexane diethyl acetate to check the glycosyl donors and with 10 to one chloroform to methanol to check the glycosyl acceptors and products. Quench the reaction mixture with 2.0 milliliters of saturated aqueous sodium bicarbonate.Then, dilute the reaction with 3.0 milliliters of chloroform. Now, remove the insoluble materials through celite and carefully wash the celite with 30 milliliters chloroform. Using a 100 milliliter separatory funnel, wash the filtrate with 30 milliliters of saturated aqueous sodium bicarbonate three times.Then, wash the filtrate with 30 milliliters of brine. Dry the resulting organic layer with sodium sulfate. After filtering the insoluble materials, concentrate the filtrate using a rotary evaporator.Purify the remaining residue by silica gel column chromatography with a chloroform methanol gradient ranging from a one to zero ratio to a 30 to one ratio to afford the Beta-22. Continue synthesis of other compounds as detailed in the text protocol. In a 10 milliliter pear-shaped flask, dissolve 34.3 milligram of Uridine 10 in one milliliter of anhydrous pyridine.Add 40.0 milligrams of four trifluoromethyl phenylboronic acid 11C. Co-evaporate the reaction mixture with one milliliter of anhydrous pyridine three times and then one milliliter of anhydrous one four dioxane three times at room temperature to approximately 40 degrees Celsius to remove any water. Dissolve the residue in anhydrous 1.40 milliliters of one four dioxane.Then, stir the reaction mixture at its reflux temperature for one hour to form a boronic ester as a temporary protection. Now, dispense the 0.14 milliliters of the reaction mixture to a five milliliter vial. Remove the solvent from the five milliliter vial using a rotary evaporator, followed by a vacuum pump.Dissolve the resulting residue in 0.64 milliliters of tridutero acetonitrile. Finally, measure proton, boron 11 and fluorine 19 NMR spectrum using a quartz NMR tube at 25 degrees Celsius. Reaction conditions for the glycosylation of Uridine 10 with glycosyl donor Alpha-nine were optimized.The condition, as shown in entry 12, gave the disaccharide nucleoside Alpha-and Beta-12 in highest chemical yield. Shown here are the results of the glycosylations of various nucleosides 10 and 13 through 20 with glycosyl donor Beta-21, that when used with optimized reaction conditions, afford the corresponding disaccharide nucleosides Beta-22 through Beta-30 in the highest yields. While attempting this procedure, it's very important to obtain the anhydrous conditions using sufficient redried flask, molecular sieves and solvents to avoid the hydrolysis of boronic ester on the glycosyl donor.The application of the temporary protection of hydroxyl groups by boronic ester will be useful for the synthesis of a variety of natural and artificial compounds, as well as disaccharide nucleosides.

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

Research

JoVE Journal

Chemistry

Anticancer Metal Complexes: Synthesis and Cytotoxicity Evaluation by the MTT Assay

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; therefore their preparation should be conducted under an inert atmosphere. Evaluation of the anticancer activity of these complexes can be achieved by the MTT assay.
The MTT assay is a colorimetric viability assay based on enzymatic reduction of the MTT molecule to formazan when it is exposed to viable cells. The outcome of the reduction is a color change of the MTT molecule. Absorbance measurements relative to a control determine the percentage of remaining viable cancer cells following their treatment with varying concentrations of a tested compound, which is translated to the compound anticancer activity and its IC50 values. The MTT assay is widely common in cytotoxicity studies due to its accuracy, rapidity, and relative simplicity.
Herein we present a detailed protocol for the synthesis of air sensitive metal based drugs and cell viability measurements, including preparation of the cell plates, incubation of the compounds with the cells, viability measurements using the MTT assay, and determination of IC50 values.

A method for synthesis of air-sensitive titanium and vanadium anticancer agents is described, along with the evaluation of their cytotoxic activity towards human cancer cell line by the MTT Assay.

Titanium (IV) and vanadium (V) complexes are highly potent anticancer agents. A challenge in their synthesis refers to their hydrolytic instability; ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

HKUST-1 as a Heterogeneous Catalyst for the Synthesis of Vanillin

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately 200 different odorant compounds in addition to vanillin. The natural extraction of vanillin (from the orchid Vanilla planifolia, Vanilla tahitiensis and Vanilla pompon) represents only 1% of the worldwide production and since this process is expensive and very long, the rest of the production of vanillin is synthesized. Many biotechnological approaches can be used for the synthesis of vanillin from lignin, phenolic stilbenes, isoeugenol, eugenol, guaicol, etc., with the disadvantage of harming the environment since these processes use strong oxidizing agents and toxic solvents. Thus, eco-friendly alternatives on the production of vanillin are very desirable and thus, under current investigation. Porous coordination polymers (PCPs) are a new class of highly crystalline materials that recently have been used for catalysis. HKUST-1 (Cu3(BTC)2(H2O)3, BTC = 1,3,5-benzene-tricarboxylate) is a very well known PCP which has been extensively studied as a heterogeneous catalyst. Here, we report a synthetic strategy for the production of vanillin by the oxidation of trans-ferulic acid using HKUST-1 as a catalyst.

The conversion of trans-ferulic acid to vanillin was achieved by heterogeneous catalysis. HKUST-1 was employed in this synthesis and the essential step in the catalytic process was the generation of unsaturated metal sites. Thus, when the catalyst was activated under vacuum, full vanillin conversion (yield of 95%) was obtained.

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

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&#039;-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 ...

Synthesis of a Thiol Building Block for the Crystallization of a Semiconducting Gyroidal Metal-sulfur Framework

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and porous metal-dithiolene network in the highly ordered single crystalline state. Unlike the highly reactive free-standing thiols, which tend to decompose and complicate the crystallization of metal-thiolate open frameworks, the thioester reacts in situ to provide the thiol species, serving to mitigate the reaction between the mercaptan units and the metal centers, and to improve crystallization consequently. Specifically, the thioester was synthesized in a one-pot procedure: an aromatic bromide (hexabromotriphenylene) reacted with excess sodium thiomethoxide under vigorous conditions to first form the thioether intermediate product. The thioether was then demethylated by the excess thiomethoxide to provide the thiolate anion that was acylated to form the thioester product. The thioester was conveniently purified by standard column chromatography, and then used directly in the framework synthesis, wherein NaOH and ethylenediamine serve to revert in situ the thioester to the thiol linker for assembling the single-crystalline Pb(II)-dithiolene network. Compared with other methods for thiol synthesis (e.g., by cleaving alkyl thioether using sodium metal and liquid ammonia), the thioester synthesis here uses simple conditions and economical reagents. Moreover, the thioester product is stable and can be conveniently handled and stored. More importantly, in contrast to the generic difficulty in accessing crystalline metal-thiolate open frameworks, we demonstrate that using the thioester for in situ formation of the thiol linker greatly improves the crystallinity of the solid-state product. We intend to encourage broader research efforts on the technologically important metal-sulfur frameworks by disclosing the synthetic protocol for the thioester as well as the crystalline framework solid.

Here, we present a one-pot, transition-metal-free synthesis of thiols and thioesters from aromatic halides and sodium thiomethoxide, followed by the preparation of single crystals of a metal-dithiolene network using thiol species generated in situ from the more stable and tractable thioester.

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and ...

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.