T.G.K. acknowledges the International Graduate School for Science and Engineering (IGGSE) of the Technische Universit&#228;t M&#252;nchen for their financial support. H.K. acknowledges the Center for Integrated Protein Science Munich (CIPSM) for their support.

Note: All reagents and solvents were obtained from commercial suppliers and were used without further purification.
Caution: Please consult all relevant material safety data sheets (MSDS) before use. Please use all appropriate safety equipment when performing chemical syntheses (e.g., fume hood, safety glasses, gloves, lab coat, full-length pants, and closed-toe shoes).
1. Synthesis of the Guanidinylation Precursors
<ol>
	<li>Alkylated species
	<...

<ol>
	<li>Saczewski, F., Balewski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+guanidine+compounds.">Biological activities of guanidine compounds.</a> Exp. Opin. Ther. Patents. 19 (10), 1417-1448 (2009).</li><li>Saczewski, F., Balewski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+activities+of+guanidine+compounds,+2008-2012+update.">Biological activities of guanidine compounds, 2008-2012 update.</a> Exp. Opin. Ther. Patents. 23 (8), 965-995 (2013).</li><li>Wirth, T. H., Davidson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mercury+(II)+Comlexes+of+Guanidine+and+Ammonia,+and+a+general+discussion+of+the+Complexing+of+Mercury+(II)+by+Nitrogen+Bases.">Mercury (II) Comlexes of Guanidine and Ammonia, and a general discussion of the Complexing of Mercury (II) by Nitrogen Bases.</a> J. Am. Chem. Soc. 86 (20), 4325-4329 (1964).</li><li>Berlinck, R. G., Burtoloso, A. C., Kossuga, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemistry+and+biology+of+organic+guanidine+derivatives.">The chemistry and biology of organic guanidine derivatives.</a> Nat. Prod. Rep. 25, 919-954 (2008).</li><li>Peterlin-Ma&#353;i&#269;, L., Kikelj, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+mimetics.">Arginine mimetics.</a> Tetrahedron. 57 (33), 7073-7105 (2001).</li><li>Peterlin-Ma&#353;i&#269;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+mimetic+structures+in+biologically+active+antagonists+and+inhibitors.">Arginine mimetic structures in biologically active antagonists and inhibitors.</a> Curr. Med. Chem. 13 (30), 3627-3648 (2006).</li><li>Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins:+bidirectional,+allosteric+signaling+machines.">Integrins: bidirectional, allosteric signaling machines.</a> Cell. 110 (6), 673-687 (2002).</li><li>Liddington, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+aspects+of+integrins.">Structural aspects of integrins.</a> Adv. Exp. Med. Biol. 819, 111-126 (2014).</li><li>Plow, E. F., Haas, T. A., Zhang, L., Loftusi, J., Smith, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+binding+to+integrins.">Ligand binding to integrins.</a> J. Biol. Chem. 275 (29), 21785-21788 (2000).</li><li>Kapp, T. G., Fottner, M., Maltsev, O. V., Kessler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+cause,+great+impact+-+modification+of+guanidine+group+in+RGD+controls+subtype+selectivity.">Small cause, great impact - modification of guanidine group in RGD controls subtype selectivity.</a> Angew. Chem. Int. Ed. 55, 1540-1543 (2016).</li><li>Xiong, J. P., 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=Crystal+structure+of+the+extracellular+segment+of+integrin+alpha+Vbeta3.">Crystal structure of the extracellular segment of integrin alpha Vbeta3.</a> Science. 294, 339-345 (2001).</li><li>Xiong, J. P., 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=Crystal+structure+of+the+extracellular+segment+of+integrin+alpha+Vbeta3+in+complex+with+an+Arg-Gly-Asp+ligand.">Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand.</a> Science. 296 (5565), 151-155 (2002).</li><li>Nagae, M., 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=Crystal+structure+of+&#945;5&#946;1+integrin+ectodomain:+atomic+details+of+the+fibronectin+receptor.">Crystal structure of &#945;5&#946;1 integrin ectodomain: atomic details of the fibronectin receptor.</a> J. Cell Biol. 197 (1), 131-140 (2012).</li><li>Hersel, U., Dahmen, C., Kessler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGD+modified+polymers:+biomaterials+for+stimulated+cell+adhesion+and+beyond.">RGD modified polymers: biomaterials for stimulated cell adhesion and beyond.</a> Biomaterials. 24 (24), 4385-4415 (2003).</li><li>Auernheimer, J., Dahmen, C., Hersel, U., Bausch, A., Kessler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoswitched+Cell+Adhesion+on+Surfaces+with+RGD+Peptides.">Photoswitched Cell Adhesion on Surfaces with RGD Peptides.</a> J. Am. Chem. Soc. 127 (46), 16107-16110 (2005).</li><li>Corradini, D., Eksteen, E., Eksteen, R., Schoenmakers, P., Miller, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of HPLC. , (2011).</li><li>de Hoffmann, E., Stroobant, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mass Spectrometry: Principles and Applications. , (2007).</li><li>Frank, A. O., 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=Conformational+Control+of+Integrin-Subtype+Selectivity+in+isoDGR+Peptide+Motifs:+A+Biological+Switch.">Conformational Control of Integrin-Subtype Selectivity in isoDGR Peptide Motifs: A Biological Switch.</a> Angew. Chem. Int. Ed. 49 (48), 9278-9281 (2010).</li><li>Rossiter, S., 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=Selective+substrate-based+inhibitors+of+mammalian+dimethylarginine+dimethylaminohydrolase.">Selective substrate-based inhibitors of mammalian dimethylarginine dimethylaminohydrolase.</a> J. Med. Chem. 48 (14), 4670-4678 (2005).</li><li>Weiss, S., Keller, M., Bernhardt, G., Buschauer, A., K&#246;nig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N(G)-Acyl-argininamides+as+NPY+Y(1)+receptor+antagonists:+Influence+of+structurally+diverse+acyl+substituents+on+stability+and+affinity.">N(G)-Acyl-argininamides as NPY Y(1) receptor antagonists: Influence of structurally diverse acyl substituents on stability and affinity.</a> Bioorg. Med. Chem. 18 (17), 6292-6304 (2010).</li><li>Hammerschmidt, F., Kvaternik, H., Schweifer, A., Mereiter, K., Aigner, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Synthesis+of+No-Carrier-Added+[*I]MIBG+and+Its+Precursor.">Improved Synthesis of No-Carrier-Added [*I]MIBG and Its Precursor.</a> Synthesis. 44 (21), 3387-3391 (2012).</li></ol>

The authors have nothing to disclose.

The precursor for guanidinylation is an orthogonally deprotected cyclic peptide derivative, (c(OrnD(OtBu)Gf(NMe)V)), which is synthesized by a standard Fmoc protocol of solid-phase peptide synthesis (SPPS). Ornithin was used as the orthogonally protected derivative, (Fmoc-Orn(Dde)-OH), which can be deprotected with hydrazine in DMF after the cyclization of the peptide scaffold. The peptide precursor is purified by the precipitation of the compound and by the subsequent lyophilization.
<p cl...

Among the most abundant pharmacophoric groups in natural ligands is the guanidine group, which is involved in multiple interactions1,2. For example, it serves as a potential four-fold hydrogen donor in hydrogen bond interactions and is involved in electrostatic interactions, such as salt bridges or cation-&#960; interactions. In medicinal chemistry, this group is often found in drugs and drug candidates4, although very often as guanidine mimetics5,6. The reason for the development of guanidine mimetics is the removal of the ubiquitous, positively charged guanidine group, as well as the adjustment of the lipophilicity of the ligand. In peptidic ligands, the only guanidine group-containing amino acid is arginine, which is therefore often found in the bioactive region of peptidic ligands.
A very prominent example for an arginine-containing ligand family is the subfamily of the RGD-binding integrins. In general, integrins are a class of cell adhesion receptors, which take over important functions in all higher organisms. Some of these functions involve cell adhesion, migration, and cell survival. Thus, they are also involved in pathological indications, such as cancer and fibrosis. Integrins are transmembrane heterodimeric proteins consisting of an &#945;- and a &#946;-subunit that form 24 currently known integrin subtypes; 8 of them recognize the tripeptide sequence Arg-Gly-Asp (=RGD) in their ligands7. The binding region is located at the interface between these two subtypes in the extracellular part, the so-called integrin head group8. RGD is recognized by two common interactions: the metal-ion-dependent adhesion site (MIDAS) region, which is located in the beta subunit and which binds the carboxylic acid in the ligands (side chain of Asp); and the guanidine group in the ligands, which is located in the alpha subunit. Most of the integrin subtypes are promiscuous and share at least a part of their natural extracellular matrix (ECM) ligands9. Thus, for the development of artificial integrin ligands, the major focus is, besides a high binding affinity, the subtype selectivity. Recently, we were able to unveil a key element for the generation of subtype-selective ligands: the guanidine group. Through distinct modifications, biselective ligands for the &#945;v- and &#945;5-containing integrin subtypes can be turned into selective compounds by simple modifications on the guanidine group, which can then discriminate the different &#945;-subunits10.
In the pocket of &#945;v, the guanidine group interacts side-on via a bidentate salt bridge with Asp21811,12. This interaction can also be observed in &#945;5&#946;1 (here, with Asp227 in &#945;5), but additionally, an end-on interaction of the guanidine group with a Gln residue (Gln221) is observed there13. Thus, we modified the guanidine group in two opposite ways: in one case, by blocking the side-on interaction with the methylation of the N&#948; of the guanidine group, and in the other case, with the methylation of the guanidine N&#969;, blocking the end-on interaction. Surprisingly, this small modification led to a complete selectivity shift in the ligands. In addition to the alkylation, a new functionalization method was introduced in this publication. The classical functionalization method for this type of pentapeptidic ligand is through the side-chain conjugation of an amino acid not involved in binding (e.g., K in c(RGDfK))14,15. Here, we show that functionalization is also possible by modifying the guanidine &#8212; which is crucial for binding &#8212; with an acyl or alkylated linker. The positive charge that is essential for binding is retained, and models suggest that the long chain points out of the binding pocket, thus providing an ideal possibility for the attachment of further linkers and labeling units (e.g., a fluorescent label or a chelator for molecular imaging).
In this work, we concentrate on the preparative steps for the modification of the guanidine group in arginine-containing ligands. This involves the synthesis of N&#969;-methylated species, as well as guanidines with longer linker units. The different modifications comprise acyl and alkyl groups.

<table border="0" cellpadding="0" cellspacing="0" width="758">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">N,N&#8242;-Di-Boc-1H-pyrazole-1-carboxamidine, 98%&#160;</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:176px;">434167&#160;ALDRICH</td>
			<td style="width:111px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Triphenylphosphine, 99%</td>
			<td>Sigma Aldrich</td>
			<td>T84409&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofuran, &#62;99.5%</td>
			<td>Carl Roth</td>
			<td>4745</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofuran anhydrous, 99.8%</td>
			<td>Carl Roth</td>
			<td>5182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol anhydrous, 99.8%</td>
			<td>Sigma Aldrich</td>
			<td>322415&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diisopropyl azodicarboxylate, 98%</td>
			<td>Sigma Aldrich</td>
			<td>225541&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichlormethan, for synthesis, 99.5%</td>
			<td>Carl Roth</td>
			<td>8424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silica gel for flash chromtaography</td>
			<td>Sigma Aldrich</td>
			<td>60738&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Pentane, 99%</td>
			<td>Carl Roth</td>
			<td>8720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-Hexane, 99%</td>
			<td>Carl Roth</td>
			<td>CP47</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylacetate, 99.5%</td>
			<td>Carl Roth</td>
			<td>7338</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aminohexanol, 95%</td>
			<td>Sigma Aldrich</td>
			<td>A56353&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S-Methylisothiourea hemisulfate, 98%</td>
			<td>Sigma Aldrich</td>
			<td>M84445 ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Di-tert-butyl dicarbonate, 99%</td>
			<td>Sigma Aldrich</td>
			<td>205249&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamid, 99.8%</td>
			<td>Carl Roth</td>
			<td>A529</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Diisopropylethylamin, 99.5%</td>
			<td>Carl Roth</td>
			<td>2474</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic anhydrid, 99%</td>
			<td>Carl Roth</td>
			<td>4483</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chlortrityl resin</td>
			<td>Carbolution</td>
			<td>CC11006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Gly-OH, 98%</td>
			<td>Carbolution</td>
			<td>CC05014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Piperidin, 99%</td>
			<td>Sigma Aldrich</td>
			<td>104094&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Orn(Dde)-OH</td>
			<td>Iris-Biotech</td>
			<td>FAA1502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HATU, 99%</td>
			<td>Carbolution</td>
			<td>CC01011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HOAt, 99%</td>
			<td>Carbolution</td>
			<td>CC01004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Val-OH</td>
			<td>Carbolution</td>
			<td>CC05028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Nitrobenzenesulfonyl chloride, 97%</td>
			<td>Sigma Aldrich</td>
			<td>N11507&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,4,6-Collidine, 99%</td>
			<td>Sigma Aldrich</td>
			<td>27690&#160;SIGMA-ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mercaptoethanol, 99%&#160;</td>
			<td>Sigma Aldrich</td>
			<td>M6250&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diazabicycloundecen, 98%</td>
			<td>Sigma Aldrich</td>
			<td>139009&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-D-Phe-OH, 98%</td>
			<td>Sigma Aldrich</td>
			<td>47378&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fmoc-Asp(OtBu)-OH, 98%</td>
			<td>Carbolution</td>
			<td>CC05008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexafluoroisopropanol</td>
			<td>Carbolution</td>
			<td>CC03056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diphenylphosphoryl azide, 97%</td>
			<td>Sigma Aldrich</td>
			<td>178756&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TFA, 99.9%</td>
			<td>Carl Roth</td>
			<td>P088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triisopropylsilan, 98%</td>
			<td>Sigma Aldrich</td>
			<td>233781&#160;ALDRICH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile, HPLC grade</td>
			<td>Carl Roth</td>
			<td>HN44</td>
			<td></td>
		</tr>
	</tbody>
</table>

modification and functionalization of the guanidine group by tailor-made precursors

The guanidine group is one of the most important pharmacophoric groups in medicinal chemistry. The only amino acid carrying a guanidine group is arginine. In this article, an easy method for the modification of the guanidine group in peptidic ligands is provided, with an example of RGD-binding integrin ligands. It was recently demonstrated that the distinct modification of the guanidine group in these ligands allows for the selective modulation of the subtype (e.g., between the subtypes &#945;v and &#945;5). Moreover, a formerly unknown strategy for the functionalization via the guanidine group was demonstrated, and the synthetic approach is reviewed in this document. The modifications described here involve terminally (N&#969;) alkylated and acetylated guanidine groups. For the synthesis, tailor-made precursor molecules are synthesized, which are then subjected to a reaction with an orthogonally deprotected amine to transfer the pre-modified guanidine group. For the synthesis of alkylated guanidines, precursors based on N,N&#8242;-Di-Boc-1H-pyrazole-1-carboxamidine are used to synthesize acylated compounds, the precursor of choice being a correspondingly acylated derivative of N-Boc-S-methylisothiourea, which can be obtained in one- and two-step reactions.

The cyclic peptide precursor was synthesized as a linear peptide, cyclized, and orthogonally Dde-deprotected. After the precipitation, the purity of the compound was analyzed with HPLC-MS (Figure 1). To monitor the progress of the reaction, an HPLC analysis was performed after the 2-h reaction time (Figure 2).
For larger residues on the guanidine group, the reaction time of 2 h is often not enou...

Watch this Scientific Journal Video about Modification and Functionalization of the Guanidine Group by Tailor-made Precursors at JoVE.com

Modification and Functionalization of the Guanidine Group by Tailor-made Precursors

A protocol for the synthesis of alkyl-modified guanidines based on the use of the corresponding precursors is presented.

The guanidine group is one of the most important pharmacophoric groups in medicinal chemistry. The only amino acid carrying a guanidine group is arginine. ...

The overall goal of this procedure is to provide a convenient synthetic access to guanidine functionalized peptides. This method can help answer key questions in the field of muscle peptide chemistry such as the development of integrin ligands or GPCR ligands. The main advantage of this technique is that it allows an easy, synthetic access to functionalized guanidines, completely compatible with SPPS.The technology will be especially valuable for tactile molecules because the functional groups, which are modified, are typically deteriorating biomolecules. In our case, we aim for molecules which can be used fpr molecular imaging of distinct properties of individual cancers and for their specific treatment;an important issue for personalized medicine. To begin this specific procedure, add one gram of boc-pyrazol carboxamaidine and one gram of PPh3 to a 50 milliliter round-bottomed flask.Subsequently, add 10 milliliters of dried THF to a 50 milliliter round-bottomed flask in an inert atmosphere via a rubber septum. Using a syringe, add 194 microliters of dry methanol. Stir the solution at room temperature.Using a syringe, add a prepared solution of diisopropyl azodicarboxylate in THF dropwise over 15 minutes. Then, let the solution stir for two hours at room temperature. Using a rotary evaporator, remove the solvent under reduced pressure.After this, dissolve the crude product in one milliliter of DCM. Next, let a let a flash column with 100 grams of silica, in 10%ethyl acetate in a pentane solution. Load the dissolved crude product on the column and add the the solvent under pressure.Collect, identify and combine the fractions as outlined in the text protocol. Then, using a rotary evaporator, evaporate the solvent under reduced pressure to obtain the desired compound as a yellow, viscous oil. First, add one gram of boc-pyrazole carboxamidine, one gram of PPH3 and 0.9 grams of Dde-6-Aminohexanol to a 50 milliliter round-bottomed flask.Then, add 10 milliliters of dry THF in an inert atmosphere using a rubber septum. Afterwards, dissolve the starting materials at room temperature. Using a syringe, add a prepared solution of diisopropyl azodicarboxylate in THF dropwise over 15 minutes.Stir the solution for two hours at room temperature. After this, use a rotary evaporator to remove the solvent under reduced pressure. Dissolve the crude product in 1 milliliter of DCM.Next, load a flash column with 100 grams of silica in a two to one solution of pentane and ethyl acetate. Load the dissolved crude product on the column and add the solvent under pressure. Collect, identify and combine the fractions as outlined in the text protocol.Using a rotary evaporator, evaporate the solvent under reduced pressure to obtain the desired product. To begin, dissolve 1.4 grams of S-methylisothiourea and 2.2 grams of boc-anhydride in a vigorously-stirring biphasic solution of DCM and saturated aqueous sodium bicarbonate. Let the mixture stir for 24 hours at room temperature.After this, pour the mixture into a separatory funnel. Separate the layers and extract the aqueous phase three times with DCM. Then, combine the organic phases.Dry the combined organic phases with sodium sulfate. After removing the solvent using a rotary evaporator, dissolve the crude product in 2 milliliters of hexane. Load a flash column with 100 grams of silica in a four to one solution of hexane and ethyl acetate.After this, load the dissolved crude product on the column and add the solvent under pressure. After collecting and identifying the fractions, combine the desired fractions. Using a rotary evaporator, evaporate the solvent under reduced pressure to obtain 1.1 grams of Boc-S-methylisothiourea.Dissolve 250 milligrams of the collected Boc-S-methylisothiourea in 10 milliliters of dried DMF in a 50 milliliter round-bottomed flask in an inert atmosphere at room temperature. Use a syringe to add 440 microliters of DIPEA. Using a syringe, add 245 microliters of acetic anhydride dropwise while stirring.Let the mixture stir for one hour at room temperature. Then, use a syringe to add two milliliters of methanol and stir for 15 minutes. After removing the solvent under reduced pressure, using a rotary evaporator, dissolve the crude product in one milliliter of DCM.Load a flash column with 60 grams of silica in a three to one solution of hexane and ethyl acetate. Next, load the dissolved crude product and add the solvent under pressure. Collect, identify and combine the fractions as outlined in the text protocol.Using a rotary evaporator, evaporate the solvent under reduced pressure to obtain 210 milligrams of the desired product as a white solid. To begin, dissolve a small amount of the orthogonally deprotected cyclic peptide in a small volume of DMF. Add two equivalents of DIPEA.And two equivalents of the guanidinylation precursor. Then, let the solution stir for two hours at room temperature. Once the reaction is complete, remove the solvent.Re-dissolve the guanidinylated cyclic peptide in acetyl nitrile and perform semi-preparative HPLC purification as outlined in the text protocol. After this, in a small glass vile, add a prepared solution of trifluoroacetic acid, water and triisopropylsilane to the purified product. Stir this mixture for one hour at room temperature, and then observe the complete deprotection with ESIMS.Add 10 milliliters of ice-cold ether to a 50 milliliters centrifuge tube. Using a Pasteur pipette, add the deprotected peptide dropwise to the ether. Centrifuge the suspension at 5, 000 times g for five minutes.After this, wash the pellet with ice-cold ether. In centrifuge at 5, 000 times g for five minutes. Repeat this washing and centrifugation once more.Then, dissolve the product in 2 milliliters of HBLC-grade water and purify the product using semi-preparative HPLC as outlined in the text protocol. In this study, an easy method in the modification of the guanidine group in peptidic glygins is presented. The reactions'progress is monitored using high-pressure liquid chromatography.For larger residues on the guanidine group, two hours is often not enough time for the reaction to reach completion. Thus, the reaction is continued and the compounds are purified by semi-preparative HPLC. A small amount is then diluted with DMSO to obtain a stem solution for the biological evaluation in an al-ee-so-like solid phase binding assay.All the compounds analyzed show a relatively high affinity of the integrant subtype alpha v beta three, And high selectivity against the integrant subtype alpha five beta one. We first had the idea for this method when we were looking for a standard method for guanidinylation during SPPS. After watching this video, you should have a good understanding of how to prepare tailor made guanidinylation precursors, in order to modify or functionalize the guanidine group of your target peptide.

modification-functionalization-guanidine-group-tailor-made

Research

JoVE Journal

Biochemistry

10.4K vues.  Technische Universität München. The overall goal of this procedure is to provide a convenient synthetic access to guanidine functionalized peptides. This method can help answer key questions in the field of muscle peptide chemistry such as the development of integrin ligands or GPCR ligands. The main advantage of this technique is that it allows an easy, synthetic access to functionalized guanidines, completely compatible with SPPS.The technology will be especially valuable for tactile molecules because the functiona...