Name: solid-phase synthesis of [4.4] spirocyclic oximes
Uploaded: 2019-02-06T13:00:00
Description: Watch this Scientific Journal Video about Solid-phase Synthesis of [4.4] Spirocyclic Oximes at JoVE.com

This work was funded by a grant from the Faculty Research Council to K.S. Huang (Azusa Pacific University - United States). C.R. Drisko is a recipient of the John Stauffer Scholarship and the Gencarella Undergraduate Research Grant. S.A. Griffin received an S2S Undergraduate Research Fellowship from the Department of Biology and Chemistry.
<img alt="Image 1" src="/files/ftp_upload/58508/58508img1.jpg" />
Authors (left to right) Cody Drisko, Dr. Kevin Huang and Silas Griffin conducted the experiments and prepared the manuscript. Cody Drisko is a John Stauffer Fellow&#160;and a recipient of the Gencarela Research Grant. Silas is a S2S Azusa Pacific University Research Fellow. Dr. Kevin Huang provided the research mentoring and is a recipient of the Azusa Pacific University Faculty Research Council Grant.

CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in these syntheses are acutely toxic and carcinogenic. Please use all appropriate safety practices when performing the following reactions, including the use of engineering controls (fume hood and IR and NMR spectrometers) and personal protective equipment (safety goggles, gloves, lab coat, full-length pants, and closed-toe shoes).
1. Michael Addition of Furfurylamine to the REM Link...

In a typical REM linker/solid-phase synthetic strategy, prior to the release of an amine from the solid support, it is critical to form a quaternary ammonium salt, as described in section 4 of the protocol39. Due to the steric hindrance of the tricyclic system and bulky R2 groups (benzyl and octyl halides), only small alkylating reagents (methyl and allyl halides) could be utilized in this reaction46. With a simple modification, allowing for the addition and use ...

Despite recent discoveries using highly-functionalized spirocyclic heterocycles in a number of biological systems1, a convenient pathway is still necessary for their easy manufacture. Such systems and uses for these heterocycles include: MDM2 inhibition and other anticancer activities2,3,4,5, enzyme inhibition6,7,8, antibiotic activity9,10, fluorescent tagging10,11,12, enantioselective binding for DNA probes13,14,15 and RNA targeting16, along with numerous potential applications to therapeutics17,18,19. With an increasing demand for these heterocycles, current literature remains divided about which synthetic pathway is best. Modern synthetic approaches to this problem use isatin and isatin derivatives as starting materials for a variety of heterocycles20,21, complicated intramolecular rearrangements22,23,24,25, Lewis acid1,26,27 or transition metal catalysis17,28,29,30, or asymmetric processes31. While these procedures have had success in producing specific spirocyclic oximes with limited functionality, a synthetic strategy for producing a library of molecules with high diastereoselectivity has been explored relatively less32.
The technique presented here shows that these molecules of interest can be generated using a number of well-understood synthetic techniques in tandem. Starting with the synthesis of the molecule on a solid support using a REM linker and intramolecular silyl nitronate-olefin cycloaddition (ISOC), the proposed pathway deploys a nonlinear route, characterized by bond severing in a tricyclic system, leaving a highly functionalized heterocycle. REM linkers, known for their convenience and recyclability, utilize a solid support to synthesize tertiary amines33. Due to the ease of purification accredited to the REM linker via simple filtration, this solid-phase synthesis technique provides scientists with a recyclable and traceless linker, which has been used here. Once the reaction is complete, the REM linker is regenerated and can be reused multiple times. The REM linker is also traceless because, unlike many solid-phase linkers, the point of attachment between the product and the polymer is indistinguishable34,35. Also well-studied and understood is the ISOC reaction, useful in the synthesis of pyrrolidine oximes36,37. Perhaps better known as a 1,3-dipolar cycloaddition, these reactions form a number of heterocycles with high diastereoselectivity38,39,40,41,42,43,44,45. Using the modified REM-coupled-ISOC technique for the synthesis of spirocyclic molecules yields a highly diastereoselective product.Herein, we report on the efficient production of spirocyclic oximes using a new synthetic approach, combining two well-understood pathways and readily available starting materials.

<table>
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>REM Resin</td>
			<td>Nova Biochem</td>
			<td>8551010005</td>
			<td>Solid Polymer Support; 1.1 mmol/g loading</td>
		</tr>
		<tr>
			<td>Furfurylamine</td>
			<td>Acros Organics</td>
			<td>119800050</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Dimethylformamide (DMF)</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>trans-4-bromo-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>400017</td>
			<td>Nitro-olefin solid</td>
		</tr>
		<tr>
			<td>trans-3,4-dimethoxy-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>S752215</td>
			<td>Nitro-olefin solid</td>
		</tr>
		<tr>
			<td>trans-2,4-dichloro-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>642169</td>
			<td>Nitro-olefin solid</td>
		</tr>
		<tr>
			<td>trans-&#946;-nitrostyrene</td>
			<td>Sigma-Aldrich</td>
			<td>N26806</td>
			<td>Nitro-olefin solid</td>
		</tr>
		<tr>
			<td>Triethylamine (TEA)</td>
			<td>Sigma-Aldrich</td>
			<td>T0886</td>
			<td>Solvent</td>
		</tr>
		<tr>
			<td>Trimethylsilyl chloride (TMSCl)</td>
			<td>Sigma-Aldrich</td>
			<td>386529</td>
			<td>Reagent; CAUTION - highly volatile; creates HCl gas</td>
		</tr>
		<tr>
			<td>Tetra-n-butylammonium fluoride (TBAF) in Tetrahydrofuran (THF)</td>
			<td>Sigma-Aldrich</td>
			<td>216143</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Tetrahydrofuran (THF)</td>
			<td>Sigma-Aldrich</td>
			<td>401757</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>1-Bromooctane</td>
			<td>Sigma-Aldrich</td>
			<td>152951</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Iodomethane</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Allylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>337528</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Benzylbromide</td>
			<td>Sigma-Aldrich</td>
			<td>B17905</td>
			<td>Alkyl-halide</td>
		</tr>
		<tr>
			<td>Glassware/Instrumentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL solid-phase reaction vessel</td>
			<td>Chemglass</td>
			<td>CG-1861-02</td>
			<td>Glassware with filter</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nicole iS5</td>
			<td>Thermo Scientific</td>
			<td>IQLAADGAAGFAHDMAZA</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>AVANCE III NMR Spectrometer</td>
			<td>Bruker</td>
			<td>N/A</td>
			<td>Instrument; 300 MHz; Solvents: CDCl3 and CD3OH</td>
		</tr>
		<tr>
			<td>Wrist-Action Shaker Model 75</td>
			<td>Burrell Scientific</td>
			<td>757950819</td>
			<td>Instrument</td>
		</tr>
	</tbody>
</table>

solid-phase synthesis of [4.4] spirocyclic oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

As outlined in the procedure above, the synthetic route to spirocyclic oximes (see Figure 1) begins with the Michael addition of furfurylamine to compound 1, the REM linker, to afford 2. A subsequent Michael addition and 1,3-dipolar cycloaddition of the support 2 using various &#946;-nitrostyrene derivatives yield the tricyclic compound 3, an N-silyloxy isoxazolidine with four unique...

Watch this Scientific Journal Video about Solid-phase Synthesis of [4.4] Spirocyclic Oximes at JoVE.com

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

This solid-phase organic synthetic technique may find applications in the construction of a combinatorial library, while synthesis of het spirocyclic heterocycles, which can be tested for anti-cancer activities using bioassays. Though this method can be used for the synthesis of spirocyclic compounds, it can also be applied to other small molecules, such as isoxazolines and isoxazoles. To begin the synthesis, add to a 25 millileter solid-phase reaction vessel one gram of REM resin, 20 millileters of dimethylformamide, and 2.4 milliliters of furfurylamine.Cap the vessel and agitate it for 24 hours on a shaker at room temperature to load the resin with furfurylamine. Then drain the furfurylamine bearing resin and agitate it for about three minutes in five milliliters of DMF to wash it. After that, wash the resin four times, alternating between five milliliters of dichloromethane to swell the resin, and five milliliters of methanol to contract the resin.Then thoroughly dry the resin with compressed air, which usually takes about 30 minutes. Next, in a well ventilated fume hood, add 1.48 milliliters of triethylamine, 10 milliliters of anhydrous toluene, and 637 grams of beta nitrostyrene to the dry furfurylamine bearing resin. Add one milliliter of trimethylsilyl chloride to the reaction mixture.And leave the vessel open until the mixture has finished releasing hydrochloride gas. Then cap the vessel and agitate the mixture for 48 hours at room temperature to form the isoxazole intermediate. After that, add five milliliters of methanol to quench the reaction.Shake the mixture for three minutes, and then let it soak for two minutes. Then drain the resin and wash it four times, alternating between five milliliters of DCM, and five milliliters of methanol. Thoroughly dry the washed resin with compressed air.Next, add one milliliter of anhydrous tetrahydrofuran to the dry resin. Then add 1.24 milliliters of a one molar solution of tetra-n-butylammonium flouride in the THF and agitate the mixture at room temperature for 12 hours to open the isoxazole ring. Afterward, strain the resin and wash it with five milliliters of THF, followed by four washes alternating between five milliliters of DCM and five milliliters of methanol.Dry the resin thoroughly with compressed air. Next, add five milliliters of DMF to the resin, followed by one milliliter of one-bromooctane. Agitate the mixture for 24 hours at room temperature to form the quaternary amine.Then drain the resin and wash it with five milliliters of DMF. Wash the resin four more times, alternating between DCM and methanol, and dry with compressed air. Next, add three milliliters of DCM to the dry resin, followed by 1.5 milliliters of TEA.Agitate the mixture for 24 hours to cleave the spirocyclic oxime from the polymer support. Then, collect the crude product in a vial or flask. Wash the resin four times, alternating between DCM and methanol, and collect the washes in the product vial.Remove volatiles from the product mixture on a rotary evaporator. Then, triturate the crude product with 5 milliliters of boiling methanol, and carefully remove the liquid. Wash the resin twice with five milliliter portions of DCM and dry it with compressed air for reuse in subsequent experiments.Spirocyclic oximes with various substituents were synthesized with excellent stereoselectivity. The overall yield indicates an average of 80 to 88%yield for each synthetic step. The reaction progress up through the isoxazole ring opening can be monitored by IR spectroscopy.Generally, individuals new to this method struggle with selecting the appropriate polymer support linker. It is essential for the linker to be robust to all reaction conditions during the synthesis. While attempting this procedure, remember to wash and dry the resin before the next reaction step.Leftover reagents from the previous step will complicate the overall synthesis and will affect the yield and purity of the spirocyclic product.

solid-phase-synthesis-of-44-spirocyclic-oximes

Research

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Chemistry

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Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

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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.

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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 ...

Synthesis of Hierarchical ZnO/CdSSe Heterostructure Nanotrees

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

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

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Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

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

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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 ...

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Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

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Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...

Gas Chromatography-Mass Spectrometry Paired with Total Vaporization Solid-Phase Microextraction as a Forensic Tool

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including controlled substances, ignitable liquids, and explosives. GC-MS can be coupled with Solid-Phase Microextraction (SPME), in which a fiber with a sorptive coating is placed into the headspace above a sample or immersed in a liquid sample. Analytes are sorbed onto the fiber which is then placed inside the heated GC inlet for desorption. Total Vaporization Solid-Phase Microextraction (TV-SPME) utilizes the same technique as immersion SPME but immerses the fiber into a completely vaporized sample extract. This complete vaporization results in a partition between only the vapor phase and the SPME fiber without interference from a liquid phase or any insoluble materials. Depending upon the boiling point of the solvent used, TV-SPME allows for large sample volumes (e.g., up to hundreds of microliters). On-fiber derivatization may also be performed using TV-SPME. TV-SPME has been used to analyze drugs and their metabolites in hair, urine, and saliva. This simple technique has also been applied to street drugs, lipids, fuel samples, post-blast explosive residues, and pollutants in water. This paper highlights the use of TV-SPME to identify illegal adulterants in very small samples (microliter quantities) of alcoholic beverages. Both gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) were identified at levels that would be found in spiked drinks. Derivatization by a trimethylsilyl agent allowed for conversion of the aqueous matrix and GHB into their TMS derivatives. Overall, TV-SPME is quick, easy, and requires no sample preparation aside from placing the sample into a headspace vial.

Total Vaporization Solid Phase Microextraction (TV-SPME) completely vaporizes a liquid sample whilst analytes are sorbed onto a SPME fiber. This allows for partitioning of the analyte between only the solvent vapor and the SPME fiber coating.

Gas Chromatography &#8211; Mass Spectrometry (GC-MS) is a frequently used technique for the analysis of numerous analytes of forensic interest, including ...