The authors thank Dr. Sai Kiran Sharma for helpful conversations.

1. The synthesis of 4-[5-(methylthio)-1,3,4-oxadiazol-2-yl]-aniline (1)
NOTE: Due to the light-sensitivity of the compound, keep all reactions in foil-covered vessels.
<ol>
	<li>In a 10 mL round bottom flask, dissolve 100 mg (0.517 mmol, 1 equivalent) of 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol in 3 mL of methanol.</li>
	<li>To this solution, add 360 &#956;L of diisopropylethylamine (DIPEA; 2.07 mmol; 4 equivalents; anhydrous) and a small magnetic stir bar. Cover the flask with a rubber stopper and stir the solution for 10 minutes at room temperature.</li>
	<li>Using a 1 mL glass syringe, poke a hole through the rubber stopper and quickly add 32 &#956;L (0.517 mmol, 1 equivalent) of iodomethane to this mixture. Allow the mixture to react for 45 minutes at room temperature. 
	NOTE: Due to the potential harmful effects of iodomethane, this reaction should be done in a chemical fume hood.</li>
	<li>Set the water bath of a rotary evaporator to 40 &#176;C and slowly reduce the pressure to remove the solvent to afford a white solid.</li>
	<li>Dissolve the solid in 3 mL of ethyl acetate and wash at least three times with a 5 mL solution of 0.1 M sodium carbonate using a separatory funnel. 
	NOTE: Periodically take spot-tests of the aqueous phase under a UV lamp; once nothing is seen under the lamp, you can stop the washes.</li>
	<li>Collect the organic phase in a separatory funnel and wash it with water until the pH of the aqueous phase reaches 6.8-7.0 (using pH paper).</li>
	<li>Collect the organic phase and add magnesium sulfate to remove any traces of water. 
	NOTE: The magnesium sulfate should be added with a small spatula, after which the solution should be swirled. If fine particles of the drying agent are still seen, the solution is dry. If not, add small amounts of magnesium sulfate until fine particles can be seen.</li>
	<li>Filter the mixture using a medium glass frit or filter paper.</li>
	<li>Evaporate the volatiles using a rotary evaporator, a process which should produce the desired product as white needles.</li>
</ol>
2. The synthesis of tert-butyl[18-({4-[5-(methylthio)-1,3,4-oxadiazol-2-yl]phenyl}amino)-15,18-dioxo-4,7,10-trioxa-14-azaoctadecyl] carbamate (2)
NOTE: Due to the light-sensitivity of the compound, keep all reactions in foil-covered vessels.
<ol>
	<li>In a 25 mL round bottom flask, dissolve 387 mg (0.92 mmol, 1.0 equivalent) of NBoc-N&#8242;-succinyl- 4,7,10-trioxa-1,13-tridecanediamine in 10 mL of dichloromethane.</li>
	<li>To this solution, add 480 &#956;L (2.76 mmol, 3 equivalents) of DIPEA, 264 mg (1.38 mmol; 1.5 equivalents) of N-ethyl-N&#8242;-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDCI), and 200 mg (0.97 mmol, 1.1 equivalents) of 1. Seal the vessel with a glass stopper and let the reaction stir for 5 days at room temperature. 
	NOTE: Be mindful of the evaporation of dichloromethane. If needed, add more throughout the week.</li>
	<li>Wash the mixture in a separatory funnel with a solution of 1 M hydrochloric acid (3 x 5 mL).</li>
	<li>Collect the organic phase and continue to wash it in a separatory funnel, first with a solution of 1 M Na2CO3 (2 x 5 mL) and then with water (3 x 5 mL).</li>
	<li>Collect the organic phase and add magnesium sulfate to remove any traces of water (see Step 1.7). Filter the mixture using a medium glass frit or filter paper.</li>
	<li>Using a rotary evaporator, remove the volatile solvents under reduced pressure to afford an off-white solid.</li>
	<li>Re-dissolve this solid in 10 mL of ethyl acetate and precipitate the product via the gradual (e.g., 2 mL at a time) addition of 30 mL of cyclohexane.</li>
	<li>Filter the solution with filter paper or a medium glass frit to obtain the product as a white powder.</li>
</ol>
3. The synthesis of tert-butyl[18-({4-[5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl]phenyl}amino)-15,18-dioxo-4,7,10-trioxa-14-azaoctadecyl] carbamate (3)
NOTE: Due to the light-sensitivity of the compound, keep all reactions in foil-covered vessels.
<ol>
	<li>In a 10 mL round-bottom flask, dissolve 30 mg (0.05 mmol; 1 equivalent) of 2 in 4 mL of dichloromethane.</li>
	<li>Slowly add in 49 mg (0.2 mmol; 4 equivalents) of 70% m-chloroperbenzoic acid to this mixture and cover the reaction vessel with a glass stopper. Stir the solution overnight at room temperature, ultimately yielding a yellow mixture.</li>
	<li>Wash the yellow mixture in a separatory funnel, first with a 0.1 M solution of NaOH (3 x 5 mL) and then with water (3 x 5 mL).</li>
	<li>Dry the organic phase with magnesium sulfate and filter the mixture using a medium glass frit or filter paper.</li>
	<li>Using a rotary evaporator, remove the solvents under reduced pressure to obtain the product as a pale solid.</li>
</ol>
4. The synthesis of N1-(3-{2-[2-(3-aminopropoxy)ethoxy]-ethoxy}propyl)-N4-{4-[5-(methylsulfonyl)-1,3,4-oxadiazol-2-yl] phenyl} succinamide (PODS)
<ol>
	<li>In a 25 mL round bottom flask, dissolve 30 mg of 3 in 2.0 mL of dichloromethane.</li>
	<li>Add 400 &#956;L of trifluoroacetic acid and seal the flask with a glass stopper.</li>
	<li>Stir the reaction mixture at room temperature for 3 hours.</li>
	<li>Using a rotary evaporator, remove the volatiles under reduced pressure at room temperature, leaving an oily residue.</li>
	<li>Dissolve the oily residue in 7 mL of water and, using a separatory funnel, wash with ethyl acetate (3 x 4 mL). Keep the aqueous layer.</li>
	<li>Lyophilize the aqueous layer to afford PODS as a white powder. 
	NOTE: The molar absorption coefficients for PODS at 280 and 298 nm are 9,900 and 12,400 cm-1M-1, respectively.</li>
</ol>
5. The synthesis of PODS-DOTA
<ol>
	<li>In a 1.5 mL microcentrifuge tube, dissolve 10 mg of PODS in 300 &#956;L of dimethyl sulfoxide (0.018 mmol; 1 equivalent) and add 26 &#956;L of N,N-diisopropylethylamine (0.15 mmol; 8 equivalents).</li>
	<li>Dissolve 15.2 mg of DOTA-Bn-NCS (0.02 mmol; 1.2 equivalents) in 100 &#956;L of dimethylsulfoxide and combine this solution with the solution from Step 5.1. Seal the microcentrifuge tube.</li>
	<li>Allow the reaction to incubate overnight at room temperature.</li>
	<li>Purify the product using reverse-phase C18 HPLC chromatography to remove any unreacted DOTA-Bn-NCS. 
	NOTE: Retention times are obviously highly dependent on the HPLC equipment of each laboratory (pumps, columns, tubing, etc.), and appropriate controls should be run prior to purification. However, to present an example, if a gradient of 5:95 MeCN/H2O (both with 0.1% TFA) to 70:30 MeCN/H2O (both with 0.1% TFA) over 30 min, a semi-preparative 19 x 250 mm C18&#160;column, and a flow rate of 6 mL/min are used, PODS, p-SCN-Bn-DOTA, and PODS-DOTA will have retention times of around 14.4, 18.8, and 19.6 min, respectively. All three compounds can be monitored at 254 nm.</li>
</ol>
6. The bioconjugation of PODS-DOTA to trastuzumab
NOTE: For this step, we started with a 16.4 mg/mL stock solution of trastuzumab.
<ol>
	<li>In a low protein binding 1.5 mL microcentrifuge tube, dilute 61 &#956;L of the trastuzumab stock solution (1 mg; 6.67 nmol, 1 equivalent) with 859 &#956;L of phosphate buffered saline (pH 7.4).</li>
	<li>To this mixture, add 6.7 &#956;L of a freshly made 10 mM solution of TCEP in H2O (66.7 nmol, 10 equivalents).</li>
	<li>Prepare a 1 mg/mL solution of PODS-DOTA in DMSO and add 73 &#956;L of this PODS-DOTA solution to the reaction mixture (66.67 nmol, 10 equivalents).</li>
	<li>Seal the microcentrifuge tube and incubate the solution for 2 hours at room temperature.</li>
	<li>After 2 hours, purify the immunoconjugate using a pre-packed disposable size exclusion desalting column.
	<ol>
		<li>First, equilibrate the size exclusion column as described by the supplier to remove any preservatives present in the column during storage. A typical procedure involves washing the column 5 times with a volume of PBS that corresponds to the volume of the column: 5 x 2.5 mL of PBS.</li>
		<li>Next, add the reaction mixture to the size exclusion column noting the volume of the reaction mixture.</li>
		<li>After the reaction mixture has entered the column, add an appropriate amount of PBS to bring the total volume of solution added to the column up to 2.5 mL. For example, if the conjugation reaction resulted in a total volume of 1.3 mL, 1.2 mL of additional PBS would need to be added to the column.</li>
		<li>Finally, collect the product using 2 mL of PBS as the eluent.</li>
	</ol>
	</li>
	<li>Concentrate the final immunoconjugate with centrifugal filtration units with a 50 kDa molecular weight cut-off.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+procedure+for+labeling+immunoglobulins+by+conjugation+to+oligosaccharide+moieties.">A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties.</a> Immunology Letters. 8 (5), 273-277 (1984).</li><li>Panowski, S., Bhakta, S., Raab, H., Polakis, P., Junutula, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+antibody+drug+conjugates+for+cancer+therapy.">Site-specific antibody drug conjugates for cancer therapy.</a> Mabs. 6 (1), 34-45 (2014).</li><li>Hu, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+conjugation+of+HIV-1+tat+peptides+to+IgG:+a+potential+route+to+construct+radioimmunoconjugates+for+targeting+intracellular+and+nuclear+epitopes+in+cancer.">Site-specific conjugation of HIV-1 tat peptides to IgG: a potential route to construct radioimmunoconjugates for targeting intracellular and nuclear epitopes in cancer.</a> European Journal of Nuclear Medicine and Molecular Imaging. 33 (3), 301-310 (2006).</li></ol>

The authors have nothing to disclose.

In this report, we have chosen not to include any protocols for radiolabeling or in vivo experimentation. Our reasons are straightforward. With respect to the former, the radiolabeling of a PODS-based immunoconjugate does not differ at all from that of an immunoconjugate synthesized using other bioconjugation strategies, and these procedures have been comprehensively reviewed elsewhere2. With regard to the latter, the specifics of preclinical in vivo experiments (i.e., mouse models, doses, etc.) c...

Radiopharmaceutical chemists have long exploited the selectivity and specificity of antibodies for biomarkers of disease for both nuclear imaging and targeted radiotherapy1. Far and away the most common approach to the radiolabeling of antibodies is predicated on the indiscriminate attachment of radiolabeled prosthetic groups or radiometal chelators to amino acids &#8212; most often lysines &#8212; within the structure of the immunoglobulin (Figure 1A)2. While this strategy is certainly effective, its random, non-site-specific nature can create problems. Specifically, traditional bioconjugation approaches produce poorly-defined and heterogeneous immunoconjugates composed of mixtures of thousands of different regioisomers, each with its own set of biological and pharmacological properties3. Furthermore, random bioconjugation can impede the immunoreactivity of antibodies if the cargo is appended to the immunoglobulin&#8217;s antigen-binding domains.
Over the years, a variety of site-specific and site-selective bioconjugation strategies have been developed in order to address these problems4,5. The most common of these approaches relies on the ligation of maleimide-bearing probes to the sulfhydryl groups of cysteines (Figure 1B). IgG1 antibodies naturally contain 4 inter-chain disulfide bridges, linkages that can be selectively reduced to yield free thiols capable of undergoing Michael addition reactions with maleimides to form succinimidyl thioether bonds. The use of thiols and maleimides is certainly an improvement over traditional methods, and a wide variety of maleimide-bearing synthons and bifunctional chelators are currently available. However, it is important to note that this methodology has serious limitations as well. Maleimide-based immunoconjugates exhibit limited stability in vivo because the thioether linkage can undergo a retro-Michael reaction (Figure 2)6,7,8,9,10. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation (e.g., glutathione or serum albumin). Both of these processes can increase activity concentrations in healthy organs as well as decrease activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. Several alternative thiol-reactive reagents have been developed in an effort to circumvent these issues, including tosylates, bromo- and iodo-acetyls, and vinyl sulfones11,12,13,14,15,16,17. However, all of these approaches have limitations that have hampered their widespread application.
About five years ago, the laboratory of the late Carlos Barbas III at Scripps Research Institute pioneered the use of phenyloxadiazolyl methyl sulfones as reagents for the selective formation of highly stable linkages with thiols (Figure 1C and Figure 3)18,19. The authors employed a phenyloxadiazolyl methyl sulfone-bearing variant of fluorescein to modify several antibodies engineered to contain free cysteine residues, ultimately producing immunoconjugates with higher stability than analogous constructs created using maleimide-based probes. Upon seeing this promising work, we were somewhat surprised that this technology had only been used scarcely in radiochemistry and had not yet been used at all in the synthesis of bifunctional chelators or radioimmunoconjugates20,21. This dearth of applications, however, soon began to make more sense: several attempts at procuring the reagent from Sigma-Aldrich resulted in the receipt of complex mixtures of degradation products with &#60;15% of the desired compound. In addition, synthesizing the reported reagent ourselves was not a realistic option either, as the published synthetic route is somewhat cumbersome and requires sophisticated organic chemistry equipment that most radiochemistry and molecular imaging laboratories &#8212; including ours &#8212; simply do not possess.
In response to these obstacles, we set out to create an easily accessible and highly stable phenyloxadiazolyl methyl sulfone reagent that can be obtained via a robust and reasonably facile synthetic route. Earlier this year, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations (Figure 1C and Figure 3)22. The key difference between PODS and the reagent reported by Barbas, et al. is that the former employs an aniline ring attached to the phenyloxadiazolyl methyl sulfone moiety, while the latter features a phenol in the same position (Figure 4). This change facilitates a more straightforward and accessible synthetic route as well as &#8212; if our experience with the commercially available compound is emblematic &#8212; a more stable final reagent. In this work, we also synthesized a pair of PODS-bearing bifunctional chelators &#8212; PODS-DFO and PODS-CHX-A&#39;&#39;-DTPA &#8212; to facilitate the creation of 89Zr- and 177Lu-labeled radioimmunoconjugates, respectively. As we will discuss, we have demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations.
The over-arching goal of this work is to facilitate the creation of well-defined, homogeneous, highly stable, and highly immunoreactive immunoconjugates for in vitro and in vivo applications. The synthetic approach is simple enough to be performed in almost any laboratory, and the parent PODS reagent can be modified with a plethora of different chelators, fluorophores, or cargoes. In this protocol and the accompanying video, we will describe the simple, four-step synthesis of PODS (Figure 5); the creation of a PODS-bearing variant of DOTA, a widely used chelator for the coordination of 64Cu, 68Ga, 111In, 177Lu, and 225Ac (Figure 6); and the bioconjugation of PODS-DOTA to a model antibody, the HER2-targeting IgG1 trastuzumab (Figure 7).

<table>
	<tbody>
		<tr>
			<td>5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol</td>
			<td>Sigma-Aldrich</td>
			<td>675024</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL LoBind Microcentrifugal Tube</td>
			<td>Eppendorf</td>
			<td>925000090</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifugal Tube</td>
			<td>Fisherbrand</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A998-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit</td>
			<td>EMD Millipore</td>
			<td>EN300000141G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclohexane</td>
			<td>Fisher Scientific</td>
			<td>C556-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Scientific</td>
			<td>AC383780010</td>
			<td></td>
		</tr>
		<tr>
			<td>Diisopropylethylamine</td>
			<td>MP Biomedicals, LLC</td>
			<td>150915</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylsulfoxide</td>
			<td>Fisher Scientific</td>
			<td>31-727-5100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Fisher Scientific</td>
			<td>E145 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodomethane</td>
			<td>Sigma-Aldrich</td>
			<td>289566-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Acros Organics</td>
			<td>413485000</td>
			<td></td>
		</tr>
		<tr>
			<td>m-chloroperbenzoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>273031</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A412 1</td>
			<td></td>
		</tr>
		<tr>
			<td>NBoc-N&#8242;-succinyl-4,7,10-trioxa-1,13-tridecanediamine</td>
			<td>Sigma-Aldrich</td>
			<td>671401</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>N-ethyl-N&#8242;- [3- (dimethylamino)propyl] carbodiimide hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>3450</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10&#215; Concentration</td>
		</tr>
		<tr>
			<td>p-SCN-Bn-DOTA</td>
			<td>Macrocyclics</td>
			<td>B-205</td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Sephadex G-25 in PD-10 Desalting Columns</td>
			<td>GE Healthcare</td>
			<td>17085101</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S7795</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>ThermoFischer Scientific</td>
			<td>20490</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylamine</td>
			<td>Fisher Scientific</td>
			<td>AC157911000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid</td>
			<td>Fisher Scientific</td>
			<td>A116-50</td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis and bioconjugation of thiol-reactive reagents for the creation of site-selectively modified immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

The first four steps of this protocol&#160;&#8212; the synthesis&#160;of PODS&#160;&#8212; have been designed to be robust and reliable. The deprotonation and substitution of 5-(4-aminophenyl)-1,3,4-oxadiazole-2-thiol to form the desired thioether product affords the thioether in &#62;99% yield after just 45 minutes. Next, the ligation between 1 and N-Boc-N&#39;-succinyl-4,7,10-trioxa-1,13-tridecanediamine was achieved via a standard peptide coupling procedure, resulting ...

Watch this Scientific Journal Video about Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates at JoVE.com

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

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9.3K Views.  Hunter College of the City University of New York. In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Video: Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Synthesis of Hypervalent Iodonium Alkynyl Triflates for the Application of Generating Cyanocarbenes

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent reactions with azides to form cyanocarbene intermediates. The synthesis of hypervalent iodonium alkynyl triflates can be facile, but difficulties stem from their isolation and reactivity. In particular, the necessity to use filtration under inert atmosphere at -45 &deg;C for some HIATs requires special care and equipment. Once isolated, the compounds can be stored and used in reactions with azides to form cyanocarbene intermediates. 
The evidence for cyanocarbene generation is shown by visible extrusion of dinitrogen as well as the characterization of products that occur from O-H insertion, sulfoxide complexation, and cyclopropanation. A side reaction of the cyanocarbene formation is the generation of a vinylidene-carbene and the conditions to control this process are discussed. There is also potential to form a hypervalent iodonium alkenyl triflate and the means of isolation and control of its generation are provided. The O-H insertion reaction involves using a HIAT, sodium azide or tetrabutylammonium azide, and methanol as solvent/substrate. The sulfoxide complexation reaction uses a HIAT, sodium azide or tetrabutylammonium azide, and dimethyl sulfoxide as solvent. The cyclopropanations can be performed with or without the use of solvent. The azide source must be tetrabutylammonium azide and the substrate shown is styrene.

Herein is described the synthesis, isolation, and reactions of hypervalent iodonium alkynyl triflates (HIATs) with azides to generate cyanocarbenes. The procedures will involve air-sensitive and cryogen techniques, including cold filtration under an inert atmosphere. Handling and safety of the synthesized compounds will also be discussed.

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

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

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

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

Synthesis of 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 ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

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

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

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

Chemical Precipitation Method for the Synthesis of Nb2O5 Modified Bulk Nickel Catalysts with High Specific Surface Area

We demonstrate a method for the synthesis of NixNb1-xO catalysts with sponge-like and fold-like nanostructures. By varying the Nb:Ni ratio, a series of NixNb1-xO nanoparticles with different atomic compositions (x = 0.03, 0.08, 0.15, and 0.20) have been prepared by chemical precipitation. These NixNb1-xO catalysts are characterized by X-ray diffraction, X-ray photoelectron spectroscopy, and scanning electron microscopy. The study revealed the sponge-like and fold-like appearance of Ni0.97Nb0.03O and Ni0.92Nb0.08O on the NiO surface, and the larger surface area of these NixNb1-xO catalysts, compared with the bulk NiO. Maximum surface area of 173 m2/g can be obtained for Ni0.92Nb0.08O catalysts. In addition, the catalytic hydroconversion of lignin-derived compounds using the synthesized Ni0.92Nb0.08O catalysts have been investigated.

Chemical Precipitation Method for the Synthesis of Nb2O5 Modified Bulk Nickel Catalysts with High Specific Surface Area

A protocol for the synthesis of sponge-like and fold-like Ni1-xNbxO nanoparticles by chemical precipitation is presented.

We demonstrate a method for the synthesis of NixNb1-xO catalysts with sponge-like and fold-like nanostructures. By varying the Nb:Ni ratio, a series of ...

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.