Name: development of a backbone cyclic peptide library as potential antiparasitic therapeutics using microwave irradiation
Uploaded: 2016-01-26T20:00:00
Description: Watch this Scientific Journal Video about Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation at JoVE.com

We thank Lauren Van Wassenhove, Sunhee Hwang, and Daria Mochly-Rosen for helpful discussions. The work was supported by the National Institutes of Health Grant NIH RC4 TW008781-01 C-IDEA (SPARK) to N.Q. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Equipment and Reagents Preparation
<ol>
	<li>Preparing equipment
	<ol>
		<li>Perform all steps inside a fume hood using proper personal protective equipment.</li>
		<li>Chemically synthesize peptides on solid support using a Microwave Peptide Synthesizer with an additional module of Discover equipped with a fiber-optic temperature probe for controlling the microwave power delivery in a Teflon reaction vessel (30 ml, with a glass frit) or in a disposable polypropylene cartridge (12 ml, with a coarse frit).</li>
		<li...

The synthesis of a focused library of backbone cyclic peptides derived from the LACK protein of the Leishmania parasite using a fully automated microwave synthesizer is described. A focused library of cyclic peptides was developed with conserved pharmacophores and various linkers. Addition of various linkers such as glutaric anhydride, succinic anhydride, adipic acid, pimelic acid, lysine, ornithine, and other building blocks can be used to increase the variety of the conformational space of the cyclic peptides....

Protein-protein interactions (PPIs) play a pivotal role in most biological processes, from intracellular signal transduction to cell death1. Hence, targeting PPIs is of fundamental importance to basic research and therapeutic applications. PPIs can be regulated by specific and stable antibodies, but antibodies are expensive and difficult to manufacture and have poor bioavailability. Alternatively, PPIs can be targeted by small molecules. Small molecules are easier to synthesize and inexpensive compared to antibodies; however, they are relatively less flexible and fit better to small cavities than to large protein-protein interfaces2,3. Diverse studies have demonstrated that peptides, which are simpler and cheaper than antibodies and more flexible than small molecules, can bind protein interfaces and regulate PPIs4,5. The global therapeutic peptide market was valued around fifteen billion dollars in 2013 and is growing 10.5% annually6. Furthermore, there are more than 50 marketed peptides, around 270 peptides in different phases of clinical testing, and about 400 peptides in advanced preclinical phases7. Although numerous peptides are being used as drugs, peptides still pose several challenges that limit their widespread application including poor bioavailability and stability, inefficiency in crossing cell membranes, and conformational flexibility8,9. One alternative to surmount these drawbacks is to apply different modifications such as local (D-amino acid and N-alkylation) and global (cyclization) constraints8,10-12. These modifications also occur naturally. For example, cyclosporin A, an immunosuppressant cyclic natural peptide, contains a single D-amino acid and undergoes N-alkylation modifications13,14.
Modification of natural amino acids to induce local constraints, such as D- and N-alkylation, often affects the peptide&#39;s biological activity. However, cyclization, in which the sequence of interest can remain the same, is more likely to preserve biological activity. Cyclization is a highly attractive way to restrict peptide conformational space by reducing the equilibrium between different conformations. It usually increases biological activity and selectivity by restricting the peptide to the active conformation that mediates only one function. Cyclization also improves peptide stability by keeping the peptide in a conformation that is less recognized by degrading enzymes. Indeed, cyclic peptides were shown to have improved metabolic stability, bioavailability, and selectivity compared to their linear counterparts15-17.
However, cyclization can be a double-edged sword since in some cases the restriction may prevent the peptides from achieving a bioactive conformation. To overcome this hurdle, a focused library in which all peptides have the same primary sequence and consequently constant pharmacophores can be synthesized. Peptides in the library differ in parameters that influence their structure, such as ring size and position, in order to subsequently screen for the most bioactive conformation9,18.
Peptides can be synthesized both in solution and by a solid-phase peptide synthesis (SPPS) approach, which is now the more prevalent peptide synthesis approach and will be discussed further. SPPS is a process by which chemical transformations are performed on a solid support via a linker to prepare a wide range of synthetic compounds19. SPPS enables assembling peptides by consecutive coupling of amino acids in a stepwise manner from the C-terminus, which is attached to a solid support, to the N-terminus. The N-&#945;-amino acid side-chains must be masked with protecting groups that are stable in the reaction conditions used during peptide elongation to ensure the addition of one amino acid per step. In the final step, the peptide is released from the resin and the side-chain protecting groups are concomitantly removed. While the peptide is being synthesized, all soluble reagents can be removed from the peptide-solid support matrix by filtration and washed away at the end of each coupling step. With such a system, a large excess of reagents at high concentration can drive coupling reactions to completion and all the synthesis steps can be performed in the same vessel without any transfer of material20 .
Although SPPS has some limitations such as the production of incomplete reactions, side reactions, impure reagents, as well as difficulties monitoring the reaction21, the advantages of SPPS have made it the &#34;gold standard&#34; for peptide synthesis. These advantages include the option to incorporate non-natural amino acids, automation, easy purification, minimized physical losses, and the use of excess reagents, resulting in high yields. SPPS has been shown to be extremely useful in the synthesis of difficult sequences21,22, fluorescent modifications23, and peptide libraries24,25. SPPS is also very useful for other poly-chain assemblies such as oligonucleotides26,27, oligosaccharides28,29, and peptide nucleic acids30,31. Interestingly, in some cases, SPPS was shown to be advantageous for synthesizing small molecules that are traditionally made in solution32,33. SPPS is used both in small scale for research and teaching34,35 as well as large scale in industry36-38.
Two synthesis strategies that are mainly used in SPPS methodology for the synthesis of peptides are butyloxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). The original strategy introduced for SPPS was Boc, which requires strong acid conditions to remove side-chain protecting groups and cleave the peptide from the resin. Fmoc-based peptide synthesis, however, utilizes moderate base conditions and is a milder alternative to the acid-labile Boc protocol39. The Fmoc strategy utilizes orthogonal t-butyl (tBu) side-chain protection that is removed in the last step of the synthesis while cleaving the peptide from the resin under acid conditions.
The general principle for peptide synthesis on solid support is presented in Figure 1. The initial amino acid, masked by a temporary protecting group on the N-&#945;-terminus, is loaded onto the resin from the C-terminus. A semi-permanent protecting group to mask the side chain is also used if necessary (Figure 1, Step 1). The synthesis of the target peptide is assembled from the C-terminus to the N-terminus by repetitive cycles of deprotection of the N-&#945;-temporary protecting group (Figure 1, Step 2) and coupling of the next protected amino acid (Figure 1, Step 3). After the last amino acid is loaded (Figure 1, Step 4), the peptide is cleaved from the resin support and the semi-permanent protecting groups are removed (Figure 1, Step 5).
<img alt="Figure 1" src="/files/ftp_upload/53589/53589fig1.jpg" /> 
Figure 1. General scheme of solid phase peptide synthesis. The N-&#945;-protected amino acid is anchored using the carboxyl group via a linker to the resin (Step 1). The desired peptide is assembled in a linear fashion from the C-terminus to the N-terminus by repetitive cycles of deprotection of the temporary protecting group (TPG) from the N-&#945; (Step 2) and amino acid coupling (Step 3). After accomplishing the synthesis (Step 4), the semi-permanent protecting groups (SPG) are deprotected during peptide cleavage (Step 5). <a href="https://www.jove.com/files/ftp_upload/53589/53589fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After assembly of the complete peptide chain, cyclization can be achieved by several alternatives: (A) head-to-tail cyclization &#8212; this is a convenient way but limited since it provides only one option for cyclization (Figure 2A), (B) cyclization using the amino acids from the sequence of interest that contain bioactive functional groups&#160;&#8212; however, the use of these amino acids may influence the biological activity (Figure 2B), and (C) cyclization by adding amino acids (or other building blocks) without disturbing the bioactive sequence. Introducing these molecules is widespread as it allows production of focused libraries without modifying the sequence of interest (Figure 2C).
<img alt="Figure 2" src="/files/ftp_upload/53589/53589fig2.jpg" /> 
Figure 2. Alternative peptide cyclization strategies. (A) head to tail cyclization, through a peptide bond between the C-terminus and N-terminus; (B) cyclization between functional groups such as a disulfide bond between cysteine residues (1), or an amide bond between the side chains of lysine to aspartic/glutamic acid (2), or side chain to N- or C-terminus (3-4); (C) cyclization by adding extra amino acids or amino acid derivatives or small molecules, for example before (R0) and after (R7) the bioactive sequence. <a href="https://www.jove.com/files/ftp_upload/53589/53589fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Microwave-assisted synthesis uses microwave irradiation to heat reactions, thus accelerating organic chemical transformations40,41. Microwave chemistry is based on the ability of the reagent/solvent to absorb the microwave energy and convert it to heat42. Before the technology became widespread, major drawbacks had to be overcome, including the controllability and reproducibility of synthesis protocols and lack of available systems for adequate temperature and pressure controls43,44. The first report of microwave-assisted peptide synthesis was done using a kitchen microwave to synthesize several short peptides (7-10 amino acids) with significant improvement of the coupling efficiency and purity 45. Moreover, microwave energy was shown to decrease chain aggregation, reduce side reactions, limit racemization, and improve coupling rates, which are all critical for difficult and long sequences46-53.
Currently the use of microwave irradiation for the synthesis of peptides or related compounds on a solid support is extensive, including (A) synthesis in water instead of organic solvent54; (B) synthesis of peptides with common post-translational modifications, such as glycopeptides55-58 or phosphopeptides59-61, whose synthesis is typically difficult due to the low coupling efficiency of sterically hindered amino acid derivatives; (C) synthesis of peptides with modification in the backbone, such as azapeptides, which can be formed by the replacement of the C(&#945;) of an amino acid residue with a nitrogen atom62, or peptoids, whose side chain is connected to the amide nitrogen rather than the C&#945; atom63,64; (D) synthesis of cyclic peptides65-71; and (E) synthesis of combinatorial libraries51,72. In numerous cases, the authors reported higher efficiency and reduced synthesis time using microwave irradiation as compared to the conventional protocol.
Using a rational design73-75, we developed anti-parasitic peptides that were derived from the scaffold Leishmania&#39;s receptor for activated C-kinase (LACK). LACK plays an important role in the early phase of Leishmania infection76. Parasites expressing lower levels of LACK fail to parasitize even immune-compromised mice77 as LACK is involved in essential parasite signaling processes and protein synthesis78. Therefore, LACK is a key scaffold protein79 and a valuable drug target. Focusing on sequences in LACK that are conserved in the parasites, but not in the host mammalian homolog RACK, we identified an 8 amino acid peptide (RNGQCQRK) that decreased Leishmania sp. viability in culture.
Here, we describe a protocol for the synthesis of backbone cyclic peptides derived from the LACK protein sequence described above. The peptides were synthesized on a solid support using microwave heating by SPPS methodology with Fmoc/tBu protocol. Peptides were conjugated to a TAT47-57 (YGRKKRRQRRR) carrier peptide through an amide bond as part of the SPPS. TAT-based transport of a variety of cargoes into cells has been used for over 15 years and delivery of the cargo into subcellular organelles has been confirmed80. Four different linkers, succinic and glutaric anhydride as well as adipic and pimelic acid, were used to perform the cyclization to generate carboxylic acid linkers of two to five carbons. Cyclization was done using microwave energy, and the final cleavage and side-chain deprotection steps were done manually without microwave energy. The use of an automated microwave synthesizer improved the product purity, increased the product yield, and reduced the duration of the synthesis. This general protocol can be applied to other studies that utilize peptides to understand important molecular mechanism in vitro and in vivo and further develop potential drugs for human diseases.

<table border="0" cellpadding="0" cellspacing="0" width="519">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">REAGENTS</td>
			<td style="width:123px;"></td>
			<td style="width:107px;"></td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Solid support, Rink Amide AM resin ML</td>
			<td style="width:123px;">CBL</td>
			<td style="width:107px;">BR-1330</td>
			<td style="width:151px;">loading: 0.49 mmol/g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Ala-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FA2100</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Arg(Pbf)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FR2136</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Asn(Trt)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FN2152</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Asp(OBut)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FD2192</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Cys(Trt)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FC2214</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Gln(Trt)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FQ2251</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Glu(OtBu)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FE2237</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Gly-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FG2275</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-His(Trt)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FH2316</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Ile-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FI2326</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Leu-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FL2350</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Lys(Boc)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FK2390</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Met-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FM2400</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Phe-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FF2425</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Pro-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FP2450</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Ser-(tBu)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FS2476</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Thr(tBu)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FT2518</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Trp(Boc)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FW2527</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Tyr(But)-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FY2563</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Fmoc-Val-OH</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">FV2575</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">1-Methyl-2-pyrrolidinone (NMP)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">328634</td>
			<td style="width:151px;">Caution Toxic/Highly flammable/Irritant.</td>
		</tr>
		<tr height="20">
			<td height="140" rowspan="2" style="height:140px;width:140px;">N,N-Dimethylformamide (DMF)</td>
			<td rowspan="2" style="width:123px;">Alfa Aesar</td>
			<td rowspan="2" style="width:107px;">43465</td>
			<td style="width:151px;">Caution Toxic</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:151px;">Use high quality DMF to eliminate side reactions such as Fmoc removal as a result of the dimethylamine traces from DMF decomposition.</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Dichloromethane (DCM)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">D65100</td>
			<td style="width:151px;">Caution Harmful</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Dibromomethane (DBM)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">D41868</td>
			<td style="width:151px;">Caution Harmful</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Trifluoroacetic acid (TFA)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">T62200</td>
			<td style="width:151px;">Caution Corrosive/Toxic</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Trifluoroacetic acid, HPLC grade (TFA)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">91707</td>
			<td style="width:151px;">Caution Corrosive/Toxic</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Diethylether</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">31690</td>
			<td style="width:151px;">Caution Highly flammable/Harmful</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Triisopropylsilane (TIS)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">233781</td>
			<td style="width:151px;">Caution Irritant/Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Water, HPLC grade</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">270733</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:140px;">Acetonitroile, HPLC grade (ACN)</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:107px;">A998-4</td>
			<td style="width:151px;">Caution Flammable/Irritant/Harmful</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:140px;">N,N-Diisopropylethylamine (DIEA)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">3440</td>
			<td style="width:151px;">Caution Corrosive/Highly flammable</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Piperidine</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">W290807</td>
			<td style="width:151px;">Caution Toxic/Highly flammable</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Pyridine</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">270970</td>
			<td style="width:151px;">Caution Highly flammable/Harmful</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Ethanol (EtOH)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">459844</td>
			<td style="width:151px;">Caution Highly flammable/Irritant</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:140px;">1-Hydroxybenzotriazole hydrate (HOBt)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">157260</td>
			<td style="width:151px;">Caution Highly flammable/Irritant/Harmful</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:140px;">O-(Benzotriazol-1-yl)-N,N,N&#8242;,N&#8242;-tetramethyluronium hexafluorophosphate (HBTU)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">12804</td>
			<td style="width:151px;">Caution Irritant/Harmful</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:140px;">Benzotriazole-1-ly-oxy-tris-pyrrolidinophosphonium hexafluorphosphate (PyBOP)</td>
			<td style="width:123px;">Advanced Chemtech</td>
			<td style="width:107px;">RC8602</td>
			<td style="width:151px;">Caution Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Ninhydrin</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">454044</td>
			<td style="width:151px;">Caution Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Phenol</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">P3653</td>
			<td style="width:151px;">Caution Corrosive/Toxic</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Potassium cyanide (KCN)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">11813</td>
			<td style="width:151px;">Caution Very Toxic</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Potassium hydroxide (KOH)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">221473</td>
			<td style="width:151px;">Caution Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:140px;">N,N&#8217;-</td>
			<td rowspan="2" style="width:123px;">Sigma</td>
			<td rowspan="2" style="width:107px;">38370</td>
			<td rowspan="2" style="width:151px;">Caution Flammable/ Toxic</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Diisopropylcarbodiimide (DIC)</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:140px;">4-Dimethylaminopyridine (DMAP)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">522805</td>
			<td style="width:151px;">Caution Toxic/Irritant</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:140px;">Glutaric anhydride</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">G3806</td>
			<td style="width:151px;">Caution Flammable/Irritant/Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Succinic anhydride</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">239690</td>
			<td style="width:151px;">Caution Irritant/Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Adipic acid</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">A26357</td>
			<td style="width:151px;">Caution Toxic/Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Pimelic acid</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">P45001</td>
			<td style="width:151px;">Caution Toxic/Irritant</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Chloranil</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">23290</td>
			<td style="width:151px;">Caution Toxic/Irritant</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Acetaldehyde</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:107px;">402788</td>
			<td style="width:151px;">Caution Flammable/ Toxic</td>
		</tr>
		<tr height="20">
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			<td></td>
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			<td height="21" style="height:21px;width:140px;">EQUIPMENT</td>
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			<td height="21" style="height:21px;width:140px;">Name&#160;</td>
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			<td style="width:151px;">Comments</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Centrifuge</td>
			<td style="width:123px;">Beckman Coulter</td>
			<td style="width:107px;">Allegra 6R centrifuge</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Lyophilizer</td>
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			<td style="width:151px;">Alcatel pump with Franklin Motor&#160;</td>
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		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Polypropylene cartridge 12 ml</td>
			<td style="width:123px;">Applied Separation</td>
			<td style="width:107px;">2419</td>
			<td style="width:151px;"></td>
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		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Cap plug for 12 ml polypropylene cartridge</td>
			<td style="width:123px;">Applied Separation</td>
			<td style="width:107px;">8157</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Polypropylene cartridge 3 ml</td>
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			<td style="width:107px;">2413</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Cap plug for 3 ml polypropylene cartridge</td>
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			<td style="width:107px;">8054</td>
			<td style="width:151px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Stop cocks PTFE</td>
			<td style="width:123px;">Applied Separation</td>
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			<td height="21" style="height:21px;width:140px;">Tubes flat, 50 ml</td>
			<td style="width:123px;">VWR</td>
			<td style="width:107px;">21008-240</td>
			<td style="width:151px;"></td>
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			<td height="35" style="height:35px;width:140px;">Extraction manifold, 20 pos, 16 x 100 mm tubes</td>
			<td style="width:123px;">Waters</td>
			<td style="width:107px;">WAT200609</td>
			<td style="width:151px;"></td>
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			<td height="35" style="height:35px;width:140px;">Shaker, BD adams&#8482; nutator mixer</td>
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			<td style="width:107px;">22363152</td>
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			<td height="52" style="height:52px;width:140px;">Nalgene HDPE narrow mouth IP2 bottles, 125 ml</td>
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			<td style="width:107px;">03-312-8</td>
			<td style="width:151px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Erlenmeyer flask</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:107px;">FB-501, 500 ml</td>
			<td style="width:151px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Heating block</td>
			<td style="width:123px;">Thermolyne</td>
			<td style="width:107px;">1760 dri bath</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:140px;">Disposable borosilicate glass tubes with plain end</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:107px;">14-961-25</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Micropipettes and tips Finnpipette</td>
			<td style="width:123px;">Thermo</td>
			<td style="width:107px;">20&#8211;200 and 100&#8211;1,000 &#956;l</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">HPLC vials - micro vl pp 400 &#181;l PK100&#160;&#160;</td>
			<td style="width:123px;">VWR</td>
			<td style="width:107px;">69400-124</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">HPLC vial- Blue Snap-It Cap</td>
			<td style="width:123px;">VWR</td>
			<td style="width:107px;">66030-600</td>
			<td style="width:151px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Analytical HPLC column</td>
			<td style="width:123px;">Peeke Scientific</td>
			<td style="width:107px;">U1-5C18Q-JJ</td>
			<td style="width:151px;">ultro 120 5 &#181;m C18Q, 4.6 mm ID 150 mm</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Prep HPLC column, XBridge&#160;</td>
			<td style="width:123px;">Waters</td>
			<td style="width:107px;">OBD C18 5 &#181;m column</td>
			<td style="width:151px;">19 mm &#215; 150 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Mass spectrometer</td>
			<td style="width:123px;">Applied Biosystems</td>
			<td style="width:107px;">Voyager DE-RP&#160;</td>
			<td style="width:151px;"></td>
		</tr>
	</tbody>
</table>

development of a backbone cyclic peptide library as potential antiparasitic therapeutics using microwave irradiation

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is a major effort to target PPIs in basic research and in the pharmaceutical industry. Protein-protein interfaces are usually large, flat, and often lack pockets, complicating the discovery of small molecules that target such sites. Alternative targeting approaches using antibodies have limitations due to poor oral bioavailability, low cell-permeability, and production inefficiency.
Using peptides to target PPI interfaces has several advantages. Peptides have higher conformational flexibility, increased selectivity, and are generally inexpensive. However, peptides have their own limitations including poor stability and inefficiency crossing cell membranes. To overcome such limitations, peptide cyclization can be performed. Cyclization has been demonstrated to improve peptide selectivity, metabolic stability, and bioavailability. However, predicting the bioactive conformation of a cyclic peptide is not trivial. To overcome this challenge, one attractive approach it to screen a focused library to screen in which all backbone cyclic peptides have the same primary sequence, but differ in parameters that influence their conformation, such as ring size and position.
We describe a detailed protocol for synthesizing a library of backbone cyclic peptides targeting specific parasite PPIs. Using a rational design approach, we developed peptides derived from the scaffold protein Leishmania receptor for activated C-kinase (LACK). We hypothesized that sequences in LACK that are conserved in parasites, but not in the mammalian host homolog, may represent interaction sites for proteins that are critical for the parasites&#39; viability. The cyclic peptides were synthesized using microwave irradiation to reduce reaction times and increase efficiency. Developing a library of backbone cyclic peptides with different ring sizes facilitates a systematic screen for the most biological active conformation. This method provides a general, fast, and facile way to synthesize cyclic peptides.

Here we describe the development of a focused small library of backbone cyclic peptides that specifically target vital PPIs of the Leishmania parasite and act as antiparasitic agents (for review about peptides that target PPIs as antiparasitic agents87). Through the synthesis of novel backbone cyclic peptides, pharmacophores are conserved in a scaffold of extendable size. The strength of the focused library proposed here is the ability to vary peptide scaffold sizes while allowing a restricted degree ...

Watch this Scientific Journal Video about Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation at JoVE.com

Development of a Backbone Cyclic Peptide Library as Potential Antiparasitic Therapeutics Using Microwave Irradiation

A simple and general method for the synthesis of cyclic peptides using microwave irradiation is outlined. This procedure enables the synthesis of backbone cyclic peptides with a collection of different conformations while retaining the side chains and the pharmacophoric moieties., and therefore, allows to screen for the bioactive conformation.

Protein-protein interactions (PPIs) are intimately involved in almost all biological processes and are linked to many human diseases. Therefore, there is ...

The overall goal of this procedure is to develop a focused backbone cyclic peptidomimetic library with conformational diversity using microwave irradiation for novel anti-parasitic therapeutics. This method can help develop novel tools to specifically target protein-protein interactions. To begin this procedure, add 2.5 milliliters of the desired amino acid, one milliliter of activator, and 0.5 milliliters of activator base to a polypropylene cartridge.Place the cartridge in an automated microwave synthesizer and allow the coupling reaction to proceed for 300 seconds at 25 watts and 75 degrees Celsius. Once the reaction is complete, wash the resin with seven milliliters of N, N-dimethylformamide, or DMF, for 120 seconds at room temperature. After repeating the previous step five times, add seven milliliters of 20%piperidine in DMF with 0.1 molar 1-hydroxybenzotriazole, or HOBt, to the polypropylene cartridge and incubate for 30 seconds at 45 watts and 75 degrees Celsius.When finished, drain the reaction mixture. Next, add seven milliliters of 20%piperidine in DMF with 0.1 molar HOBt to the polypropylene cartridge and incubate for 180 seconds at 45 watts and 75 degrees Celsius. After draining the reaction mixture, wash the resin with seven milliliters of DMF for 120 seconds at room temperature.Then, wash the resin with seven milliliters of dichloromethane, or DCM, for 120 seconds at room temperature. For anhydride coupling, wash the resin with seven milliliters of 1-methyl-2-pyrrolidinone, or NMP, for 120 seconds at room temperature. When finished, drain the reaction mixture.After repeating the previous step three times, dissolve 10 equivalents of the corresponding anhydride in five milliliters of NMP in a 50 milliliter polypropylene tube. Then, add one equivalent of 4-Dimethylaminopyridine, or DMAP, and 10 equivalents of N, N-diisopropylethylamine, or DIEA, to the solution. Add this mixture to the resin, and incubate for 300 seconds at 25 watts and 75 degrees Celsius.After washing the resin with NMP, wash it with seven milliliters of DMF for 120 seconds at room temperature. At this point, transfer the resin to a polypropylene cartridge equipped with a cap plug and stopcock. Once the resin has been washed with DCM, add 15 to 25 milliliters of a mixture of 1%trifluoroacetic acid, 5%triisopropylsilane, and 94%DCM to the polypropylene cartridge per 1 gram of resin.Place the polypropylene cartridge on a shaker, and shake for five minutes at room temperature. Following this, drain the solution from the polypropylene cartridge by applying a vacuum. After repeating the previous steps three times, wash the resin with seven milliliters of DCM for 120 seconds at room temperature.These steps are highly important, as the trityl protecting groups in several resins were found to be partially cleaved in the DCM-trifluoroacetic acid solutions. Removing methyltrityl groups is a slow process that takes multiple washes. In a 50 milliliter polypropylene tube, dissolve five equivalents of Benzotriazole-1-ly-oxy-tris-pyrrolidinophosphonium hexafluorphosphate in five milliliters of dibromomethane.Then, add 10 equivalents of DIEA to the solution. Add the mixture to the DCM-rinsed resin and incubate for 300 seconds at 25 watts and 75 degrees Celsius. Following this, wash the resin with seven milliliters of DCM for 120 seconds at room temperature.After two washes with DCM, transfer the resin to a polypropylene cartridge and wash twice with diethyl ether. Dry the resin in a vacuum desiccator at room temperature for at least three hours over potassium hydroxide. Next, add 10 milliliters of a pre-cooled trifluoroacetic acid cleavage cocktail to every one gram of resin.Place the polypropylene cartridge on a shaker, and shake for 3 hours at room temperature. Following this, collect the cleavage solution by filtering the resin into a 50 milliliter polypropylene tube. Add 35 milliliters of cold diethyl ether to the tube.Centrifuge for five minutes at 1, 207 times g at four degrees Celsius. Then, decant the ether layer. For the Kaiser test, prepare Solution A by dissolving 16.5 milligrams of potassium cyanide in 25 milliliters of distilled water.Dilute one milliliter of the solution with 49 milliliters of peridine. Next, prepare Solution B by dissolving one gram of ninhydrin in 20 milliliters of ethanol. Prepare Solution C by dissolving 40 grams of phenol in 20 milliliters of ethanol.After the solutions have been prepared, transfer a few beads from the resin to a test tube. Add three drops of each solution to the tube, and mix. Finally, heat the test tube on a heating block at 110 degrees Celsius for five minutes.Synthesis of the backbone cyclic peptides was performed using an automated microwave synthesizer on solid support following the FMOC, t-butyl protocol. The product was analyzed by mass spectrometry, and its degree of purity was determined using HPLC. Biological screening showed that peptide pL1 was active against Leishmania donovani, a parasite causing visceral leishmaniasis, the most severe leishmaniasis in humans.Peptide pL1 reduced parasite viability by 75%as compared with the control treatment. Once mastered, this technique can be done in three to five days if it is performed properly. After it's development, this technique paved the way for researchers in a wide array of fields to explore peptides as pharmacological regulators in basic research and therapeutics.After watching this video, you should have a good understanding of how to develop a focused library of peptidomimetics with conformational diversity to specifically target protein-protein interactions. Don't forget that working with trifluoroacetic acid can be extremely dangerous, and precautions, such as wearing eye protection, a lab coat, and gloves while working in a well ventilated hood should always be taken while performing this procedure.

development-backbone-cyclic-peptide-library-as-potential

Research

JoVE Journal

Chemistry

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

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 ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

Atom Transfer Radical Polymerization of Functionalized Vinyl Monomers Using Perylene as a Visible Light Photocatalyst

A standardized technique for atom transfer radical polymerization of vinyl monomers using perylene as a visible-light photocatalyst is presented. The procedure is performed under an inert atmosphere using air- and water-exclusion techniques. The outcome of the polymerization is affected by the ratios of monomer, initiator, and catalyst used as well as the reaction concentration, solvent, and nature of the light source. Temporal control over the polymerization can be exercised by turning the visible light source off and on. Low dispersities of the resultant polymers as well as the ability to chain-extend to form block copolymers suggest control over the polymerization, while chain end-group analysis provides evidence supporting an atom-transfer radical polymerization mechanism.

A method for the atom transfer radical polymerization of functionalized vinyl monomers using perylene as a visible-light photocatalyst is described.

A standardized technique for atom transfer radical polymerization of vinyl monomers using perylene as a visible-light photocatalyst is presented. The ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Failure of Cleaning Verification in Pharmaceutical Industry Due to Uncleanliness of Stainless Steel Surface

The aim of this work is to identify the parameters that affect the recovery of pharmaceutical residues from the surface of stainless steel coupons. A series of factors were assessed, including drug product spike levels, spiking procedure, drug-excipient ratios, analyst-to-analyst variability, intraday variability, and cleaning procedure of the coupons. The lack of a well-defined procedure that consistently cleaned the coupon surface was identified as the major contributor to low and variable recoveries. Assessment of cleaning the surface of the coupons with clean-in-place solutions (CIP) gave high recovery (&#62;90%) and reproducible results (Srel&#8804;4%) regardless of the conditions that were assessed previously. The approach was successfully applied for cleaning verification of small molecules (MW &#60;1,000 Da) as well as large biomolecules (MW up to 50,000 Da).

The lack of a well-defined procedure that consistently cleaned coupon surfaces was identified as the major contributor to low and variable recoveries in cleaning verification. This manuscript describes the correct protocol of cleaning stainless steel coupons.

The aim of this work is to identify the parameters that affect the recovery of pharmaceutical residues from the surface of stainless steel coupons. A ...

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate glycosyl donor. Following azido-nitration of a glycal, the product 2-azido-1-nitrate ester can be hydrolyzed under microwave-assisted irradiation. This transformation is usually achieved using strongly nucleophilic reagents and extended reaction times. Microwave irradiation induces hydrolysis, in the absence of reagents, with short reaction times. Following denitration, the intermediate anomeric alcohol is converted, in the same pot, to the corresponding 2-azido-1-trichloroacetimidate.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance (EPR) or ultraviolet-visible and near-infrared (UV-Vis-NIR) spectroscopies, we obtain useful information such as electron affinity, ionization potential, band-gap energies, the type of charge carriers, and degradation information that can be used to synthesize stable organic electronic devices. In this study, we present electrochemical and spectroelectrochemical methods to analyze the processes occurring in active layers of an organic device as well as the generated charge carriers.

In this article, we describe electrochemical, electron paramagnetic resonance, and ultraviolet-visible and near-infrared spectroelectrochemical methods to analyze organic compounds for application in organic electronics.

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

A Microwave-Assisted Direct Heteroarylation of Ketones Using Transition Metal Catalysis

Heteroarylation introduces heteroaryl fragments to organic molecules. Despite the numerous available reactions reported for arylation via transition metal catalysis, the literature on direct heteroarylation is scarce. The presence of heteroatoms such as nitrogen, sulfur and oxygen often make heteroarylation a challenging research field due to catalyst poisoning, product decomposition and the rest. This protocol details a highly efficient direct &#945;-C(sp3) heteroarylation of ketones under microwave irradiation. Key factors for successful heteroarylation include the use of XPhos Palladacycle Gen. 4 Catalyst, excess base to suppress side reactions and the high temperature and pressure achieved in a sealed reaction vial under microwave irradiation. The heteroarylation compounds prepared by this method were fully characterized by proton nuclear magnetic resonance spectroscopy (1H NMR), carbon nuclear magnetic resonance spectroscopy (13C NMR) and high-resolution mass spectrometry (HRMS). This methodology has several advantages over literature precedents including broad substrate scope, rapid reaction time, greener procedure and operational simplicity by eliminating the preparation of intermediates such as silyl enol ether. Possible applications for this protocol include, but are not limited to, diversity-oriented synthesis for the discovery of biologically active small molecules, domino synthesis for the preparation of natural products and ligand development for new transition metal catalytic systems.

Heteroaryl compounds are important molecules utilized in organic synthesis, medicinal and biological chemistry. A microwave-assisted heteroarylation using palladium catalysis provides a rapid and efficient method to attach heteroaryl moieties directly to ketone substrates.

Heteroarylation introduces heteroaryl fragments to organic molecules. Despite the numerous available reactions reported for arylation via transition metal ...

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.