Name: split-and-pool synthesis and characterization of peptide tertiary amide library
Uploaded: 2014-06-20T13:00:00
Description: Watch this Scientific Journal Video about Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library at JoVE.com

The authors would like to thank Dr. Jumpei Morimoto and Dr. Todd Doran for valuable assistance. This work was supported by a contract from the NHLBI (NO1-HV-00242).

1. Basics of Split-and-pool Synthesis
In order to efficiently generate a large number of compounds on solid phase, split-and-pool synthesis is often employed as a general strategy. As shown in Figure 4, tentagel beads are first&#160;split into three portions. Each portion is reacted with a different reagent, generating the first residue on beads. After the first reaction, all three portions are pooled together, mixed, and then split again into three portions. Each portion will a...

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Peptide tertiary amides (PTAs) are a superfamily of peptidomimetic oligomers. Besides the well-studied peptides, peptoids and N-methylated peptides, a large portion of compounds within this family remains understudied, majorly due to lack of synthetic method to access general N-alkylated peptides. Here we describe an efficient method to synthesize PTAs with chiral building blocks derived from amino acids. Previously, we have reported to use a new sub-monomer route to synthesis libraries of PTA molecules23. We ...

Peptidomimetics are compounds that mimic the structure of natural peptides. They are designed to retain the bioactivity while overcoming some of the problems associated with natural peptides, including cell permeability and stability against proteolysis1-3. Due to the oligomeric nature of these compounds, large synthetic libraries can be readily accessed through monomeric or sub-monomeric synthetic routes4-7. One of the most studied classes of peptidomimetics is&#160;peptoids. Peptoids are oligomers of N-alkylated glycines that can be synthesized easily using a sub-monomer strategy8,9. Many useful protein ligands have been successfully identified from screening large synthetic peptoid libraries against protein targets1,10-14. Nonetheless, &#8220;hits&#8221; identified from peptoid libraries rarely archive very high affinity towards protein targets1,10-14,22. One major difference between peptoids and natural peptides is that most of the peptoids generally lack the ability to form secondary structure due to the lack of chiral centers and conformational constraints. In order to solve this problem, multiple strategies were developed over the past decade, largely focusing on the modification of side chains contained on the main chain nitrogen atoms15-22. Recently, we have developed a new synthetic route to introduce natural amino acid side chains onto a peptoid backbone to create peptide tertiary amides23.
Peptide tertiary amides (PTAs) are a super family of peptidomimetics that include but are not limited to peptides (R2&#160;= H), peptoids (R1&#160;= H) and N-methylated peptides (R1&#160;&#8800; H, R2&#160;= Me). (See Figure 1) Our synthetic route employs naturally occurring amino acids as the source of chirality and side chains on the &#945;-carbon, and commercially available primary amines to provide N-substitutions. Therefore, a larger chemical space than that of simple peptides, peptoids or N-methylated peptides can be explored. Circular dichroism spectra have shown that PTA molecules are highly structured in solution. Characterization of one of the PTA-protein complexes clearly shows that the conformational constraints of PTA are required for binding. Recently, we have also discovered that some of the PTA molecules possess improved cell permeability than their peptoid and peptide counterparts. We believe that these PTA libraries can be a good source of high-affinity ligands for protein targets. In this paper, we will discuss the synthesis of a sample one-bead one-compound (OBOC) PTA library in details along with some improved conditions for the coupling and cleavage of these compounds.

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		<tr height="20">
			<td height="20" style="height:21px;width:249px;">N,N&#39;-Diisopropylcarbodiimide (DIC)</td>
			<td style="width:169px;">ACROS</td>
			<td style="width:160px;">115211000</td>
			<td>CAS:693-13-0</td>
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		<tr height="20">
			<td height="20" style="height:21px;width:249px;">N,N-Diisopropylethylamine (DIPEA)</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:160px;">D125806</td>
			<td>CAS:7087-68-5</td>
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		<tr height="20">
			<td height="20" style="height:21px;">NaNO2</td>
			<td style="width:169px;">ACROS</td>
			<td style="width:160px;">424340010</td>
			<td>CAS:7631-99-4</td>
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		<tr height="20">
			<td height="20" style="height:21px;width:249px;">NAOD 40% solution in water</td>
			<td style="width:169px;">ACROS</td>
			<td style="width:160px;">200058-506</td>
			<td>CAS:7732-18-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Piperidine</td>
			<td style="width:169px;">ALFA AESAR</td>
			<td style="width:160px;">A12442-AE</td>
			<td>CAS:110-89-4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Piperonylamine</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:160px;">P49503&#160;</td>
			<td>CAS:2620-50-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Propylamine</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:160px;">240958</td>
			<td>CAS:107-10-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Trifluoroacetic acid</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:160px;">299537</td>
			<td>CAS:76-05-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">&#945;-Cyano-4-hydroxycinnamic acid&#160;</td>
			<td style="width:169px;">Sigma-Aldrich</td>
			<td style="width:160px;">39468</td>
			<td>CAS:28166-41-8&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">&#945;-ketoglutarate</td>
			<td style="width:169px;">ALFA AESAR</td>
			<td style="width:160px;">AAA10256-22</td>
			<td style="width:311px;">CAS:328-50-7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Tentagel Resin with RINK linker</td>
			<td style="width:169px;">Rapp-Polymere</td>
			<td style="width:160px;">S30023</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Alanine transaminase</td>
			<td style="width:169px;">Roche</td>
			<td style="width:160px;">10105589001</td>
			<td>AKA: Glutamate-Pyruvate Transaminase (GPT)</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Incubator</td>
			<td style="width:169px;">New Brunswick Scientific</td>
			<td style="width:160px;">Innova44</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:249px;">NMR</td>
			<td style="width:169px;">Bruker</td>
			<td style="width:160px;">400MHz</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:249px;">MALDI mass spectrometer</td>
			<td style="width:169px;">Applied Biosystems&#160;</td>
			<td style="width:160px;">4800 MALDI-TOF/TOF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Lyophilizer</td>
			<td style="width:169px;">SP Scientific</td>
			<td style="width:160px;">VirTis benchtop K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Syringe reactor</td>
			<td style="width:169px;">INTAVIS&#160;</td>
			<td style="width:160px;">Reaction Column</td>
			<td>3ml, 5ml, 10ml, 20ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:249px;">Vacuum manifold&#160;</td>
			<td style="width:169px;">Promega</td>
			<td style="width:160px;">A7231</td>
			<td>Vac-Man</td>
		</tr>
	</tbody>
</table>

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.

Here we show three representative MALDI spectrums from a PTA trimer with linker. As shown in Figure 6A, when cleaved under room temperature using 50% TFA/DCM solution, significant degradation is observed. In Figure 6A, peak 593 and 484 correspond to the linker and the PTA trimer respectively, show that the whole molecule was successfully synthesized on bead but degraded during cleavage. When cleaved under low temperature condition as described above, the amount of TFA-induced degradation...

Watch this Scientific Journal Video about Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library at JoVE.com

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

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 ...

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