We are grateful to the Research Corporation for Science Advancement (20092), the National Science Foundation (CHE-1362140), and Texas State University for their generous support.

1. Synthesis of Diamidocarbene (Compound 1)
<ol>
	<li>Connect an oven-dried 100 ml Schlenk flask to a high performance vacuum manifold, evacuate it and flush with nitrogen. Add a stir bar to the flask and cap with a rubber septum. Weigh out N,N&#8217;-dimesitylformamidine12 (1.5 g, 5.35 mmol) and add it to the flask while flushing with nitrogen.
	<ol>
		<li>Add (via dry, deoxygenated syringes) 30 ml of dry, degassed dichloromethane (DCM) followed by triethylamine (1.1 ml, 8.0 mmol, 1.5 equiv.). Cool the resulting solution to 0 &#176;C in an ice bath.</li>
		<li>Add dropwise (via a dry, deoxygenated syringe) dimethylmalonyl dichloride (0.75 ml, 5.60 mmol, 1.05 equiv.) to the cooled solution. Upon addition, allow the solution to stir at 0 &#176;C for 1 hr under an atmosphere of nitrogen. Once the solution has stirred for 1 hr, remove all the volatile materials under vacuum.</li>
		<li>Add a solvent mixture of dry, degassed hexanes:DCM (2:1 by volume, 24 ml total volume) to the residue in the reaction flask. Let the mixture triturate for 10 min.</li>
		<li>Meanwhile, attach a filter tube equipped with a medium porosity glass frit to the top of an oven-dried 100 ml Schlenk flask (see Figure 1). To the filter tube, add enough oven-dried Celite to create a filter plug approximately 2 inches&#160;tall. Seal the filter tube with a rubber septum, and pull vacuum, on the system by attaching the Schlenk flask to the vacuum manifold. 
		<img alt="Figure 1" fo:content-width="2.5in" src="/files/ftp_upload/52149/52149fig1highres.jpg" width="250" /> 
		Figure 1. Filtration apparatus assembly for removal of [HNEt3][Cl] from carbene precursors 1-HCl and 2-HCl. This apparatus is also used to remove NaCl formed during the synthesis of carbenes 1 and 2.</li>
		<li>Filtration apparatus assembly for removal of [HNEt3][Cl] from carbene precursors 1-HCl and 2-HCl. This apparatus is also used to remove NaCl formed during the synthesis of carbenes 1 and 2.</li>
		<li>Once the filtration apparatus has been assembled and is under vacuum, transfer the white suspension in the reaction flask via cannula into the filter tube. Be sure to periodically pull vacuum on the collection Schlenk flask to ensure that all of the solution filters through the Celite.</li>
		<li>Wash the Celite by adding a solvent mixture of dry, degassed hexanes:DCM (2:1, volume, 18 ml total volume) using a syringe through the rubber septum. Again, periodically pull vacuum on the collection Schlenk flask to ensure that all of the solution filters through the Celite.</li>
		<li>Disconnect the collection Schlenk flask from the filter tube under a flush of nitrogen, then seal the collection Schlenk flask with a glass stopper. Remove all of the solvent from the collection Schlenk flask under vacuum to afford the precursor for diamidocarbene 1 (1-HCl) as an air/moisture sensitive white powder in approximately 92% yield (2.04 g). The product can be verified by 1H and 13C NMR spectroscopy (CDCl3)13,14. Transfer compound 1-HCl into a glove box for storage prior to the next step.</li>
	</ol>
	</li>
	<li>To prepare the diamidocarbene 1, first transfer an oven-dried 100 ml Schlenk flask equipped with a stir bar and a glass stopper into a nitrogen-filled glove box.
	<ol>
		<li>Weigh out carbene precursor 1-HCl (0.600 g, 1.45 mmol) and sodium hexamethyldisilazide (NaHMDS, 0.267 g, 1.46 mmol) and place both solids into the Schlenk flask.</li>
	</ol>
	</li>
	<li>Add dry, degassed benzene (25 ml) to the two solids in the Schlenk flask and then stopper the flask. At this point, the Schlenk flask can be removed from the glove box.</li>
	<li>Stir the solution of the carbene at RT for 30 min. Throughout the reaction, the solution will become a turbid yellow-orange color. During this time, set up a filtering apparatus similar to the one described above (using a 1 inch plug of Celite) for the synthesis of 1-HCl.</li>
	<li>Filter the carbene solution (to remove precipitated NaCl) as described for the precursor 1-HCl. Once the solution has been filtered, remove all of the volatiles using vacuum to afford the crude carbene 1 as a yellow-orange powder. Further purify carbene 1 by washing the solid with cold hexanes (~10 ml) to afford analytically pure compound as an air/moisture sensitive white powder in approximately 85% yield (0.462 g). Verify the product by 1H and 13C NMR spectroscopy (C6D6)13.</li>
</ol>
2. Synthesis of Monoamidocarbene (Compound 2)
<ol>
	<li>Connect an oven-dried 250 ml Schlenk flask to a high performance vacuum manifold, evacuate it and flush with nitrogen. Add a stir bar to the flask and cap with a rubber septum. Weigh out N,N&#8217;-dimesitylformamidine (3.00 g, 10.70 mmol) and add it to the flask while flushing with nitrogen.
	<ol>
		<li>Add (via dry, deoxygenated syringes) 125 ml of dry, degassed DCM followed by triethylamine (2.25 ml, 16.05 mmol, 1.5 equiv.). Cool the resulting solution to 0 &#176;C in an ice bath.</li>
		<li>Add dropwise (via a dry, deoxygenated syringe) 3-chloropivaloyl chloride (1.54 ml, 11.77 mmol, 1.1 equiv.) to the cooled solution. Upon addition, allow the solution to stir at 0 &#176;C for 30 min&#160;under an atmosphere of nitrogen. Then gradually warm the solution to RT, and then remove all the volatile materials under vacuum. After the solvent is removed, a white solid residue will remain.</li>
		<li>Add toluene (200 ml) to the white solid and allow the suspension to triturate for 1 hr. Then filter the mixture over a 1 inch plug of Celite using a medium porosity glass fritted B&#252;chner funnel.</li>
		<li>Transfer the toluene solution to a 500 ml round bottom flask equipped with a stir bar. Connect a reflux condenser to the flask and heat the solution to reflux (110 &#176;C) for 16 hr. Over the course of the reaction, a white precipitate will form.</li>
		<li>After 16 hr&#160;allow the suspension to cool to RT. During this time, more solid will precipitate from the solution. Collect the solid via vacuum filtration and wash the solid with cold toluene (3 x 20 ml).</li>
		<li>Dry the obtained white solid using vacuum to afford the precursor for monoamidocarbene 2 (2-HCl) as an air stable white powder in approximately 91% yield (3.32 g). The product can be verified by 1H and 13C NMR spectroscopy (CDCl3)15. Transfer compound 2-HCl into a glove box for storage prior to the next step.</li>
	</ol>
	</li>
	<li>To prepare the monoamidocarbene 2, first transfer an oven-dried 100 ml Schlenk flask equipped with a stir bar and a glass stopper into a nitrogen-filled glove box.
	<ol>
		<li>Weigh out carbene precursor 2-HCl (0.500 g, 1.25 mmol) and NaHMDS (0.241 g, 1.32 mmol) and place both solids into the Schlenk flask.</li>
		<li>Add dry, degassed benzene (45 ml) to the two solids in the Schlenk flask and then stopper the flask. At this point, the Schlenk flask can be removed from the glove box.</li>
		<li>Stir the solution of the carbene at RT for 30 min. During the reaction, the solution will become a turbid yellow color. Meanwhile, set up a filtering apparatus similar to the one described above for the synthesis of 1-HCl using a 1 inch plug of Celite.</li>
		<li>Filter the carbene solution (to remove precipitated NaCl) as described for carbene 1. Once the solution has been filtered, remove all of the volatiles using vacuum to afford the crude carbene 2 as a tan powder. Further purify carbene 2 by washing the solid repeatedly with pentane to afford analytically pure compound as an air/moisture sensitive white powder in approximately 62% yield (0.309 g). Verify the product by 1H and 13C NMR spectroscopy (C6D6)15.</li>
	</ol>
	</li>
</ol>
3. Synthesis of a Tris(phosphaalkenyl)phosphane (Compound 3)
Caution Statement: White phosphorus is extremely pyrophoric as well as toxic and should be handled cautiously in a glovebox whenever possible.
<ol>
	<li>To make a tris(phosphaalkenyl)phosphane (compound 3), weigh out diamidocarbene 1 (0.100 g, 0.266 mmol, 3 equiv.) and white phosphorus (P4, 0.011 g, 0.089 mmol, 1 equiv.) inside of a nitrogen-filled glove box with the lights turned off. Turn off as many lights in the lab during these first few steps as P4&#160;is light sensitive.</li>
	<li>Add the two solids to a 20 ml glass vial that is wrapped in aluminum foil. Add dry, degassed diethyl ether (Et2O, 10 ml) to the solids and then cap the vial. Stir the slurry in the dark for 2 hr. During the course of the reaction, a bright red-orange precipitate will form.</li>
	<li>Isolate the red solid via filtration using a 10 ml medium porosity glass fritted B&#252;chner funnel. Wash the red solid with Et2O (4 x 5 ml) and then dry it under vacuum to afford compound 3 as an analytically pure air stable compound in approximately 82% yield, 0.092 grams (based on P4). Verify the product by 1H and 31P NMR spectroscopy (C6D6)11.</li>
</ol>
4. Synthesis of a Carbene-stabilized P8&#160;Allotrope (Compound 4)
<ol>
	<li>Method A
	<ol>
		<li>To make a carbene-stabilized P8 allotrope (compound 4), weigh out monoamidocarbene 2 (0.100 g, 0.276 mmol, 3 equiv.) and P4 (11.4 mg, 0.092 mmol, 1 equiv.) inside of a nitrogen-filled glove box with the lights turned off. Turn off as many lights in the lab during these first few steps as P4&#160;is light sensitive.</li>
		<li>Add the two solids to a 20 ml glass vial that is wrapped in aluminum foil. Add dry, degassed diethyl ether (Et2O, 10 ml) to the solids and then cap the vial. Upon addition of the ether, the fleeting dark green color rapidly changes to bright orange. Stir the slurry in the dark for 2 hr. During the course of the reaction, a bright orange precipitate will form.</li>
		<li>Isolate the orange solid via filtration using a 10 ml medium porosity glass fritted B&#252;chner funnel. Wash the orange solid with Et2O (4 x 2 ml) and then dry it under vacuum to afford compound 4 as and analytically pure air stable compound in approximately 51% yield, 39.5 mg (based on P4). Verify the product by 1H and 31P NMR spectroscopy (THF-D8)11.</li>
	</ol>
	</li>
	<li>Method B
	<ol>
		<li>Weigh out monoamidocarbene 2 (0.100 g, 0.276 mmol, 2 equiv.) and P4 (17.1 mg, 0.138 mmol, 1 equiv.) inside of a nitrogen-filled glove box with the lights turned off. Turn off as many lights in the lab during these first few steps as P4&#160;is light sensitive.</li>
		<li>Add the two solids to a 20 ml glass vial that is wrapped in aluminum foil. Add dry, degassed hexanes (10 ml) to the solids and then cap the vial. Upon addition of the ether, the fleeting dark green color rapidly changes to bright orange. Stir the slurry in the dark for 2 hr. During the course of the reaction, a bright orange precipitate will form.</li>
		<li>Isolate the orange solid via filtration using a 10 ml medium porosity glass fritted B&#252;chner funnel, and then dry it under vacuum to afford compound 4 as and analytically pure air stable compound in approximately 75% yield, 87.7 mg (based on P4). Verify the product by 1H and 31P NMR spectroscopy (THF-D8).11</li>
	</ol>
	</li>
</ol>
5. Trapping a Transient E-1,2-bis(phosphaalkenyl)diphosphene via [4+2] Cycloaddition: Synthesis of Compound 5
<ol>
	<li>To prepare compound 5, weigh out monoamidocarbene 2 (0.300 g, 0.828 mmol, 2 equiv.) and P4 (51.3 mg, 0.414 mmol, 1 equiv.) separately inside of a nitrogen-filled glove box with the lights turned off. Turn off as many lights in the lab during these first few steps as P4&#160;is light sensitive.</li>
	<li>Add the P4 to a 20 ml glass vial, and then add dry, degassed hexanes (18 ml) to the vial. Next, add 2,3-dimethyl-1,3-butadiene (2 ml) to the P4 suspension in hexanes.</li>
	<li>While the hexane/P4 suspension is rapidly stirring, add the carbene 2 as a solid in one portion. The suspension will instantly become bright yellow. Over a period of about 10 min, all of the solids will dissolve followed by the precipitation of a bright yellow solid. At this point, let the suspension stir for 4 hr.</li>
	<li>After 4 hr, isolate the yellow solid via filtration using a 10 ml medium porosity glass fritted B&#252;chner funnel. This yellow solid is compound 5 (&#62; 90% purity by both 1H and 31P NMR). Concentrate the yellow supernatant solution to dryness and combine the yellow residue with the filtered yellow solid.</li>
	<li>To purify compound 5, recrystallize the combined yellow solids from a 1:3 (by volume) DCM:hexanes solution (12 ml total volume) in a -30 &#176;C freezer in the glovebox O/N. This procedure will afford 5 as analytically pure air stable yellow crystals in approximately 71% yield, 0.301 grams (based on P4). Verify the product by 1H and 31P NMR spectroscopy (C6D6)11.</li>
</ol>

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The authors have nothing to disclose.

A straightforward procedure for generating carbonyl-decorated carbenes and their application in the activation of white phosphorus is presented here. The critical steps in the protocol for synthesizing the carbenes are: (a) make sure all solvents are properly dried before use, (b) make sure the addition of acid chlorides to the formamidine is done very slowly, (c) if the Celite is not oven-dried for a minimum of 12 hr&#160;at 180 &#176;C, hydrolysis of the 1-HCl as well as carbenes 1&#160;</stron...

Stable carbenes have emerged as ubiquitous reagents in homogeneous catalysis1, organocatalysis2,&#160;materials science3,4, and more recently main group chemistry5-9.&#160;In the context of the latter, stable carbenes have recently been used in the activation and functionalization of white phosphorus (P4)5-9.&#160;The ability to directly convert P4&#160;into organophosphorus compounds has become a topical research objective in an effort to develop &#8220;greener&#8221; methods that circumvent the use of chlorinated or oxychlorinated phosphorus precursors. Despite their widespread use, the preparation and handling of carbenes and reactive compounds such as P4 can be a daunting task. For this reason, we have written this manuscript to provide a clear and concise protocol that will allow synthetic chemists of all skill levels to synthesize and manipulate two very unique stable carbenes. Additionally, the activation of P4&#160;using the described carbenes is detailed.
Herein we detail a protocol for the synthesis of two electron-deficient carbonyl decorated carbenes. We have chosen these carbenes because they differ only in their electrophilic properties, and not their steric parameters, making them ideal for studying the effects of carbene electronics on reactivity. The importance of carbene electronics with regard reactivity is exemplified by two similar compounds of the general formula carbene-P2-carbene that have been reported by Bertrand and Robinson5,8.&#160;Bertrand&#8217;s P2&#160;derivative is supported by two cyclic alkyl amino carbene (CAAC) ligands, and is structurally, photophysically, and electrochemically different than Robinson&#8217;s compound which is a P2 fragment supported by two N-heterocyclic carbenes (NHCs)5,8. Indeed, Bertrand&#8217;s P2&#160;complex is characterized as a yellow solid that features carbene-to-phosphorus double bonds in the solid state, whereas the derivative reported by Robinson is a dark red solid that contains NHC&#8594;P dative bonds. This structural difference also manifests itself electrochemically such that Robinson&#8217;s compound contains more electron-rich phosphorus centers that can undergo reversible 1- or 2-electron oxidations in contrast to Bertrand&#8217;s compound which can only undergo a single reversible oxidation10.
Based on the studies described above, we became interested in studying the activation of P4 using the highly electrophilic diamido- and monoamidoamino carbenes to determine if novel carbene-stabilized allotropes of phosphorus could be prepared. We focused on diamidocarbene (DAC) 1, and monoamidoamino carbene (MAAC) 2 which differ only in their respective electrophilicities to interrogate what role carbene electronics play in P4 activation. Interestingly when the more electrophilic DAC is used, a tris(phosphaalkenyl)phosphane (3) could be isolated as the exclusive product, whereas when a MAAC is used, a carbene-stabilized P8 allotrope (4) can be obtained11. We also interrogated the mechanism for the formation (4), and found that it is formed via a [2+2] cylcoaddition dimerization reaction of a transient diphosphene. The existence of this diphosphene was confirmed by trapping it with 2,3-dimethyl-1,3-butadiene to furnish the [4+2] cycloaddition adduct 5. The protocol for synthesizing these carbonyl-decorated carbenes and their corresponding P4 activated compounds is described herein.

<table border="0" cellpadding="0" cellspacing="0" width="580">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:171px;">2,4,6-trimethylaniline</td>
			<td style="width:92px;">Alfa Aesar</td>
			<td style="width:123px;">AAA13049-0E</td>
			<td style="width:195px;">98%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:171px;">Triethylorthoformate</td>
			<td style="width:92px;">Alfa Aesar</td>
			<td style="width:123px;">AAA13587</td>
			<td style="width:195px;">98%</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:171px;">Dimethylmalonyl dichloride</td>
			<td style="width:92px;">TCI</td>
			<td style="width:123px;">D2723</td>
			<td style="width:195px;">&#62;98%</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:171px;">3-chloro-pivaloyl chloride</td>
			<td style="width:92px;">Aldrich</td>
			<td style="width:123px;">225703-25G</td>
			<td style="width:195px;">98%</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:171px;">Triethylamine</td>
			<td style="width:92px;">Alfa Aesar</td>
			<td style="width:123px;">AAA12646</td>
			<td style="width:195px;">Stored over dried, activated 3 &#197; molecular sieves</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:171px;">Celite&#8482; 545</td>
			<td style="width:92px;">EMD</td>
			<td style="width:123px;">CX0574-3D</td>
			<td style="width:195px;">Oven-dried at 180 &#176;C for a minimum of 12 hrs</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:171px;">Sodium hexamethyldisilazide</td>
			<td style="width:92px;">Across</td>
			<td style="width:123px;">200014-462</td>
			<td style="width:195px;">95+%</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:171px;">2,3-dimethyl-1,3-butadiene</td>
			<td style="width:92px;">Alfa Aesar</td>
			<td style="width:123px;">AAAL04207-09</td>
			<td style="width:195px;">98%</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:171px;">dichloromethane</td>
			<td style="width:92px;">EMD</td>
			<td style="width:123px;">DX0835-5</td>
			<td style="width:195px;">Purified through solvent purification system, or standard methods</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:171px;">tetrahydrofuran</td>
			<td style="width:92px;">Mallinckrodt</td>
			<td style="width:123px;">8498-09</td>
			<td style="width:195px;">Purified through solvent purification system, or standard methods</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:171px;">Hexanes</td>
			<td style="width:92px;">EMD</td>
			<td style="width:123px;">HX0299-3</td>
			<td style="width:195px;">Purified through solvent purification system, or standard methods</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:171px;">Benzene</td>
			<td style="width:92px;">EMD</td>
			<td style="width:123px;">BX0220-5</td>
			<td style="width:195px;">Purified through solvent purification system, or standard methods</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:171px;">Toluene</td>
			<td style="width:92px;">BDH</td>
			<td style="width:123px;">1151-19L</td>
			<td style="width:195px;">Purified through solvent purification system, or standard methods</td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:171px;">white phosphorus</td>
			<td style="width:92px;">Generously donated from the Texas A&#38;M chemistry store room.</td>
			<td style="width:123px;">NA</td>
			<td style="width:195px;">Purified through sublimation and transferred directly into a glovebox while under vacuum in the sublimator</td>
		</tr>
	</tbody>
</table>

preparation and use of carbonyl-decorated carbenes in the activation of white phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

The ability to isolate a tris(phosphaalkenyl)phosphane such as 3&#160;or the P8-allotrope (4) from white phosphorus relies on the use of an electrophilic carbene to activate the P4&#160;tetrahedron11,16.&#160;Therefore, it is critical to prepare carbenes with enhanced &#960;-acidity, and by extension electrophilicity. Figure 2&#160;illustrates the synthesis of carbene precursor 1-HCl and its subsequent deprotonation to af...

Watch this Scientific Journal Video about Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus at JoVE.com

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

preparation-use-carbonyl-decorated-carbenes-activation-white

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JoVE Journal

Chemistry

13.5K Views.  Texas State University. Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

Video: Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Preparation and Use of Samarium Diiodide (SmI2) in Organic Synthesis: The Mechanistic Role of HMPA and Ni(II) Salts in the Samarium Barbier Reaction

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of molecular iodine to samarium metal in THF.1,2-3 It is a mild and selective single electron reductant and its versatility is a result of its ability to initiate a wide range of reductions including C-C bond-forming and cascade or sequential reactions. SmI2 can reduce a variety of functional groups including sulfoxides and sulfones, phosphine oxides, epoxides, alkyl and aryl halides, carbonyls, and conjugated double bonds.2-12 One of the fascinating features of SmI-2-mediated reactions is the ability to manipulate the outcome of reactions through the selective use of cosolvents or additives. In most instances, additives are essential in controlling the rate of reduction and the chemo- or stereoselectivity of reactions.13-14 Additives commonly utilized to fine tune the reactivity of SmI2 can be classified into three major groups: (1) Lewis bases (HMPA, other electron-donor ligands, chelating ethers, etc.), (2) proton sources (alcohols, water etc.), and (3) inorganic additives (Ni(acac)2, FeCl3, etc).3
Understanding the mechanism of SmI2 reactions and the role of the additives enables utilization of the full potential of the reagent in organic synthesis. The Sm-Barbier reaction is chosen to illustrate the synthetic importance and mechanistic role of two common additives: HMPA and Ni(II) in this reaction. The Sm-Barbier reaction is similar to the traditional Grignard reaction with the only difference being that the alkyl halide, carbonyl, and Sm reductant are mixed simultaneously in one pot.1,15 Examples of Sm-mediated Barbier reactions with a range of coupling partners have been reported,1,3,7,10,12 and have been utilized in key steps of the synthesis of large natural products.16,17 Previous studies on the effect of additives on SmI2 reactions have shown that HMPA enhances the reduction potential of SmI2 by coordinating to the samarium metal center, producing a more powerful,13-14,18 sterically encumbered reductant19-21 and in some cases playing an integral role in post electron-transfer steps facilitating subsequent bond-forming events.22 In the Sm-Barbier reaction, HMPA has been shown to additionally activate the alkyl halide by forming a complex in a pre-equilibrium step.23
Ni(II) salts are a catalytic additive used frequently in Sm-mediated transformations.24-27 Though critical for success, the mechanistic role of Ni(II) was not known in these reactions. Recently it has been shown that SmI2 reduces Ni(II) to Ni(0), and the reaction is then carried out through organometallic Ni(0) chemistry.28
These mechanistic studies highlight that although the same Barbier product is obtained, the use of different additives in the SmI2 reaction drastically alters the mechanistic pathway of the reaction. The protocol for running these SmI2-initiated reactions is described.

Preparation and Use of Samarium Diiodide SmI2 in Organic Synthesis: The Mechanistic Role of HMPA and NiII Salts in the Samarium Barbier Reaction

A straightforward procedure for the preparation of samarium diiodide (SmI2) in THF is described. The role of two main additives namely hexamethylphosphoramide (HMPA) and Ni(acac)2 in Sm mediated reactions is demonstrated in the Sm-Barbier reaction.

Although initially considered an esoteric reagent, SmI2 has become a common tool for synthetic organic chemists. SmI2 is generated through the addition of ...

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of biomacromolecules, including DNA. Therefore, efficient detection of DNA oxidation is important for a variety of research disciplines, including medicine and toxicology. A common biomarker of oxidatively damaged DNA is 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo; often erroneously referred to as 8-hydroxy-2&#39;-deoxyguanosine (8-OH-dGuo or 8-oxo-dG)). Several protocols for 8-oxo-dGuo measurement by high pressure liquid chromatography with electrochemical detection (HPLC-ED) have been described. However, these were mainly applied to purified DNA treated with pro-oxidants. In addition, due to methodological differences between laboratories, mainly due to differences in analytical equipment, the adoption of published methods for detection of 8-oxo-dGuo by HPLC-ED requires careful optimization by each laboratory. A comprehensive protocol, describing such an optimization process, is lacking. Here, a detailed protocol is described for the detection of 8-oxo-dGuo by HPLC-ED, in DNA from cultured cells or animal tissues. It illustrates how DNA sample preparation can be easily and rapidly optimized to minimize undesirable DNA oxidation that can occur during sample preparation. This protocol shows how to detect 8-oxo-dGuo in cultured human alveolar adenocarcinoma cells (i.e., A549 cells) treated with the oxidizing agent KBrO3, and from the spleen of mice exposed to the polycyclic aromatic hydrocarbon dibenzo(def,p)chrysene (DBC, formerly known as dibenzo(a,l)pyrene, DalP). Overall, this work illustrates how an HPLC-ED methodology can be readily optimized for the detection of 8-oxo-dGuo in biological samples.

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2&amp;#8217;-deoxyguanosine, in Cultured Cells and Animal Tissues

The goal of this protocol is the detection of the DNA oxidation marker, 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxo-dGuo) by HPLC-ED, in DNA from cultured cells or animal tissues.

Oxidative stress is associated with many physiological and pathological processes, as well as xenobiotic metabolism, leading to the oxidation of ...

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 ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of Phosphorus(I)

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable low-valent-phosphorus-containing compound acts as a useful P+ transfer agent. Unlike traditional methods employed to access low-coordinate phosphorus species which usually require pyrophoric phosphorus-containing precursors (white phosphorus, Tris(trimethylsilyl)phosphine, etc.), or harsh reducing agents (alkali metals, potassium graphite, etc.), the current approach does not involve pyrophoric or explosive reagents and can be done on large scales (&#62;20 g) in excellent yields by undergraduates with basic air-free synthetic training. The bromide counter ion is readily exchanged with other anions such as tetraphenyl borate (described herein) using typical salt metathesis reagents to obtain materials with desired properties and reactivities. The versatility of this P+ transfer approach is exemplified by the reactions of these triphosphenium precursors with an N-heterocyclic carbene and an anionic bisphosphine, each of which readily displace the neutral bisphosphine to give an NHC-stabilized phosphorus(I) cation and a phosphorus(I) containing zwitterion, respectively.

Preparation and Reactivity of a Triphosphenium Bromide Salt: A Convenient and Stable Source of PhosphorusI

The synthesis of a triphosphenium bromide salt is described and its use as a P+ transfer agent is outlined by reactions with an N-heterocyclic carbene and an anionic bisphosphine, yielding an NHC-stabilized P(I) cation and a P(I) containing zwitterion, respectively.

We present herein the optimized synthesis of a triphosphenium bromide salt. Apart from being a versatile metathesis reagent, this unusually stable ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

The MPLEx Protocol for Multi-omic Analyses of Soil Samples

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and characterize microbial communities in environmental and biological systems. These measurements are even enabling enhanced analyses of complex soil microbial communities, which are the most complex microbial systems known to date. Multi-omic analyses, however, do have sample preparation challenges, since separate extractions are typically needed for each omic study, thereby greatly amplifying the preparation time and amount of sample required. To address this limitation, a 3-in-1 method for the simultaneous extraction of metabolites, proteins, and lipids (MPLEx) from the same soil sample was created by adapting a solvent-based approach. This MPLEx protocol has proven to be both simple and robust for many sample types, even when utilized for limited quantities of complex soil samples. The MPLEx method also greatly enabled the rapid multi-omic measurements needed to gain a better understanding of the members of each microbial community, while evaluating the changes taking place upon biological and environmental perturbations.

A protocol is presented for simultaneously extracting metabolites, proteins, and lipids from a single soil sample, allowing reduced sample preparation times and enabling multi-omic mass spectrometry analyses of samples with limited quantities.

Mass spectrometry (MS)-based integrated metaproteomic, metabolomic, and lipidomic (multi-omic) studies are transforming our ability to understand and ...

Application of Elemental Lanthanides in the Selective C-F Activation of Trifluoromethylated Benzofulvenes Providing Access to Various Difluoroalkenes

The selective activation of one carbon-fluorine bond in polyfluorinated aromatic molecules or in trifluoromethyl-containing substrates offers the possibility of accessing unique fluorine-containing molecules, which are difficult to obtain by other synthetic pathways. Among various metals, which can undergo C-F activation, lanthanides (Ln) are good candidates as they form strong Ln-F bonds. Lanthanide metals are strong reducing agents with a redox potential Ln3+/Ln&#160;of approximately -2.3 V, which is comparable to the value of the Mg2+/Mg&#160;redox couple. In addition, lanthanide metals display a promising functional group tolerance and their reactivity can vary along the lanthanide series, making them suitable reagents for fine-tuning reaction conditions in organic and organometallic transformations. However, due to their oxophilicity, lanthanides react readily with oxygen and water and therefore require special conditions for storage, handling, preparation, and activation. These factors have limited a more widespread use in organic synthesis. We herein present how dysprosium metal - and by analogy all lanthanide metals - can be freshly prepared under anhydrous conditions using glovebox and Schlenk techniques. The freshly filed metal, in combination with aluminum chloride, initiates the selective C-F activation in trifluoromethylated benzofulvenes. The resulting reaction intermediates react with nitroalkenes to obtain a new family of difluoroalkenes.

This protocol describes the preparation of elemental lanthanides under inert atmosphere and their application in a selective C-F activation process involving trifluoromethylated benzofulvenes.