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>Methylated species
		<ol>
			<li>Dissolve 1.0 g of N,N&#8242;-di-Boc-1H-pyrazole-1-carboxamidine (3.2 mmol, 1 eq.) and 1.0 g of triphenylphosphine (PPh3, 3.8 mmol, 1.2 eq.) in 10 mL of dry tetrahydrofuran (THF) in a 50-mL round-bottom flask in an inert atmosphere using a rubber septum at room temperature (RT). Add 194 &#181;L of dry methanol using a syringe (4.8 mmol, 1.5 eq.) and let it stir at RT.</li>
			<li>Separately, prepare a solution with 747 &#181;L of diisopropyl azodicarboxylate (DIAD, 3.8 mmol, 1.2 eq.) in 2 mL of dry THF in a small glass vial. While stirring, add the solution of DIAD in THF dropwise to the solution of N,N&#8242;-di-Boc-1H-pyrazole-1-carboxamidine, PPh3, and methanol using a syringe (over 15 min). Let the solution stir for 2 h at RT.</li>
			<li>Remove the solvent under reduced pressure using a rotary evaporator and dissolve the crude product in 1 mL of dichloromethane (DCM). Load a flash column (2.5 cm x 20 cm) with 100 g of silica in 10% ethyl acetate (EtOAc) in a pentane solution. Load the dissolved crude product on the column and add the solvent under pressure (1.5 bar).</li>
			<li>Collect the fractions (solvent system: isocratic 10% EtOAc in pentane), identify the desired compound spot by thin layer chromatography (TLC), and collect it (Rf = 0.4, EtOAc/pentane 1:9, UV). Combine the desired fractions and evaporate the solvent under reduced pressure using a rotary evaporator to obtain 0.85 g of the title compound as a yellow, viscous oil (2.6 mmol, 81% yield). Store the yielded compound at RT for further use.</li>
		</ol>
		</li>
		<li>Hexylamine-modified species
		<ol>
			<li>Dissolve 1.0 g of N,N&#8242;-Di-boc-1H-pyrazole-1-carboxamidine (3.2 mmol, 1.0 eq.), 0.9 g of Dde-6-aminohexanol (3.8 mmol, 1.2 eq.), and 1.0 g of PPh3 (3.8 mmol, 1.2 eq.) in 10 mL of dry THF in a 50-mL round-bottom flask in inert atmosphere using a rubber septum at RT.</li>
			<li>Separately, prepare a solution with 747 &#181;L of DIAD (3.8 mmol, 1.2 eq.) in 2 mL of dry THF in a small glass vial. While stirring, add the solution of DIAD in THF dropwise to the solution of N,N&#8242;-di-Boc-1H-pyrazole-1-carboxamidine, PPh3, and Dde-6-aminohexanol using a syringe (over 15 min). Let the solution stir for 2 h at RT.</li>
			<li>Remove the solvent under reduced pressure using a rotary evaporator and take the crude product up in 1 mL of DCM. Load a flash column (2.5 cm x 20 cm) with 100 g of silica in a solution of pentane/EtOAc (2:1). Apply the dissolved crude product on the column and add solvent under pressure (1.5 bar).</li>
			<li>Collect the fractions (solvent system: isocratic 2:1 pentane/EtOAc), identify the desired fractions with TLC, and collect them (Rf = 0.66, 2:1 pentane/EtOAc, UV). Combine the desired fractions and evaporate the solvent under reduced pressure using a rotary evaporator to obtain 1.6 g of the title compound as a yellow, viscous oil (2.8 mmol, 88% yield). Store the yielded compound at RT for further use.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Acylated species (2 reaction steps)
	<ol>
		<li>Dissolve 1.4 g of S-methylisothiuronium hemisulfate (10 mmol, 1.0 eq.) and 2.2 g of di(tert-butyl) dicarbonate (10 mmol, 1.0 eq.) in a vigorously stirring biphasic solution of DCM/ sat. aq. NaHCO3 and let it stir for 24 h at RT.</li>
		<li>Separate the layers in a separatory funnel and extract the aqueous phase three times with DCM. Combine the organic phases, dry them with Na2SO4, and remove the solvent under reduced pressure using a rotary evaporator. Take the crude product up in 1 mL of hexane.</li>
		<li>Load a flash column (2.5 cm x 20 cm) with 100 g of silica in a solution of 4:1 hexane/EtOAc. Load the dissolved crude product on the column. Add solvent under pressure. Identify the desired fractions with TLC (solvent system: 4:1 isocratic hexane/EtOAc (detection UV: 254 nm), and collect them (Rf = 0.30).</li>
		<li>Combine the desired fractions and evaporate the solvent under reduced pressure using a rotary evaporator to obtain 1.1 g of the title compound as a yellow, viscous oil (5.8 mmol, 58% yield). Store the reagent, N-(tert-butoxycarbonyl)-S-methylisothiourea, at RT for further use.</li>
		<li>Dissolve 250 mg of N-(tert-butoxycarbonyl)-S-methylisothiourea (1.3 mmol, 1.0 eq.) in 10 mL of dry N,N-dimethylformamide (DMF) in a 50-mL round-bottom flask in an inert atmosphere at RT. Add 440 &#181;L of N, N-diisopropylethylamine (DIPEA) (2.6 mmol, 2 eq.) using a syringe. While stirring, add 245 &#181;L of Ac2O (5.2 mmol, 4.0 eq.) dropwise using a syringe and let it stir for 1 h at RT.</li>
		<li>Add an excess of methanol (2 mL) to the mixture using a syringe and let it stir for 15 min at RT. Remove the solvent under reduced pressure using a rotary evaporator. Take the crude product up in 1 mL of DCM.</li>
		<li>Load a flash column (2.5 cm x 20 cm) with 60 g of silica in a solution of 3:1 hexane/EtOAc 3:1. Apply the dissolved crude product to the column. Add solvent (3:1 hexane/EtOAc) and pressure (1.5 bar). Identify the desired fractions with TLC (3:1 hexane/EtOAc, UV: 220 nm), and collect them (Rf = 0.62).</li>
		<li>Combine the desired fractions and evaporate the solvent under reduced pressure using a rotary evaporator to obtain 210 mg of the title compound as a white solid (0.9 mmol, 70% yield). Store the compound at RT for further use.</li>
	</ol>
	</li>
</ol>
2. Synthesis of Cyclic Peptide Precursors
<ol>
	<li>Loading and capping TCP resin
	<ol>
		<li>Add 1.00 g of 2-chlortriyl chloride (CTC) resin (0.9 mmol/g) and 320 mg of Fmoc-Gly-OH (1.2 eq.) into a plastic 20-mL syringe with a frit. Prepare a solution of 313 &#181;L of DIPEA (2 eq.) in 5 mL of dry DCM and add it to the syringe. Let it rotate for 1 h at RT using a rotary unit.</li>
		<li>Meanwhile, prepare a 2-mL solution of 5:1 methanol/DIPEA (v/v; capping solution). Add the capping solution to the syringe and let it rotate for another 15 min. Wash the resin with DCM (5x) and DMF (3x).</li>
	</ol>
	</li>
	<li>On-resin Fmoc deprotection
	<ol>
		<li>Treat the resin-bound Fmoc-Gly with 20% piperidine in DMF for 10 min and then for 5 min. Wash the resin with DMF (3x).</li>
	</ol>
	</li>
	<li>On-resin coupling of Fmoc-Orn(Dde)-OH
	<ol>
		<li>Add 934 mg of Fmoc-l-Orn(Dde)-OH (2 eq.), 684 mg of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) (2 eq.), and 245 mg of 1-hydroxy-7-azabenzotriazole (HOAt) (2 eq.) to a small glass vial and dissolve it in 5 mL of DMF. Add 814 &#181;L of DIPEA.</li>
		<li>Add this solution to the Fmoc-deprotected, washed, and resin-bound Gly and let it rotate for 1 h. Wash the resin with DMF (3x).</li>
	</ol>
	</li>
	<li>On-resin Fmoc deprotection
	<ol>
		<li>Treat the resin-bound Fmoc-Orn(Dde)-Gly with 20% piperidine in DMF for 10 min and then for 5 min. Wash the resin with DMF (3x).</li>
	</ol>
	</li>
	<li>On-resin coupling of Fmoc-Val-OH
	<ol>
		<li>Add 610 mg of Fmoc-Val-OH (2 eq.), 684 mg of HATU (2 eq.), and 245 mg of HOAt (2 eq.) to a small glass vial and dissolve it in 5 mL of DMF. Add 814 &#181;L of DIPEA.</li>
		<li>Add this solution to the Fmoc-deprotected, washed, and resin-bound Orn(Dde)-Gly and let it rotate for 1 h.</li>
	</ol>
	</li>
	<li>On-resin Fmoc deprotection
	<ol>
		<li>Treat the resin-bound Fmoc-Val-Orn(Dde)-Gly with 20% piperidine in DMF for 10 min and then for 5 min. Wash the resin with DMF (3x).</li>
	</ol>
	</li>
	<li>On-resin N- Methylation
	<ol>
		<li>Wash the resin with DCM (3x). Dissolve 887 mg of 2-nitrobenzenesulfonylchloride (o-NBS-Cl, 4 eq.) in DCM and add 1.2 mL of 2,4,6-collidine (10 eq.).</li>
		<li>Add the solution to the resin-bound peptide and let it incubate for 20 min at RT.</li>
		<li>Wash the resin with CH2Cl2 (3x) and with THF (5x). Prepare a solution with 1.18 g of PPh3 (5 eq.) and 365 &#181;L of methanol in THF. Add this solution to the syringe.</li>
		<li>Prepare a solution with 883 &#181;L of DIAD in 2 mL of THF and add it to the syringe. Let it incubate for 15 min. Wash the resin with THF (5x) and with DMF (5x).</li>
	</ol>
	</li>
	<li>o -Ns deprotection
	<ol>
		<li>Prepare a solution with 570 &#181;L of mercaptoethanol (10.0 eq.) and 672 &#181;L of diazabicycloundecen (DBU) in 2 mL of DMF. Add this solution to the syringe and let it incubate for 5 min.</li>
		<li>Repeat the deprotection step and then wash the resin with DMF (5x).</li>
	</ol>
	</li>
	<li>On-resin coupling of Fmoc- d-Phe-OH
	<ol>
		<li>Add 697 mg of Fmoc-D-Phe-OH (2 eq.), 684 mg of HATU (2 eq.), and 245 mg of HOAt (2 eq.) to a small glass vial and dissolve it in 5 mL of DMF. Add 814 &#181;L of DIPEA.</li>
		<li>Add this solution to the Fmoc-deprotected, washed, and resin-bound peptide and let it rotate for 1 h.</li>
	</ol>
	</li>
	<li>On-resin Fmoc deprotection
	<ol>
		<li>Treat the resin-bound peptide with 20% piperidine in DMF for 10 min and then for 5 min. Wash the resin with DMF (3x).</li>
	</ol>
	</li>
	<li>On-resin coupling of Fmoc-Asp(OtBu)-OH
	<ol>
		<li>Add 741 mg of Fmoc- Asp(OtBu)-OH (2 eq.), 684 mg of HATU (2 eq.), and 245 mg of HOAt (2 eq.) to a small glass vial and dissolve it in 5 mL of DMF. Add 814 &#181;L of DIPEA.</li>
		<li>Add this solution to the Fmoc-deprotected, washed, and resin-bound peptide and let it rotate for 1 h.</li>
	</ol>
	</li>
	<li>Cleavage of the linear peptide from the resin
	<ol>
		<li>Wash the resin with DCM (3x) and subsequently prepare 10 mL of a solution of hexafluoroisopropanol (HFIP) in DCM (1:4; v/v).</li>
		<li>Add the solution to the resin and let it rotate for 15 min. Collect the solution in a round-bottom flask. Repeat the cleavage and evaporate the solvent using a rotary evaporator.</li>
	</ol>
	</li>
	<li>Cyclization of the linear peptide
	<ol>
		<li>Dissolve 384 mg of NaHCO3 (5.0 eq.) and 582 &#181;L of diphenylphosphoryl azide (DPPA) (3.0 eq.) in 50 mL of DMF and add it to the crude product. Let it stir overnight or until no linear peptide is observed with ESI-MS (10 h)17.</li>
		<li>Under reduced pressure, reduce the solvent to a small volume using a rotary evaporator. Prepare a saturated aqueous solution of NaCl (brine). Using a Pasteur pipette, add the cyclized peptide dropwise to 40 mL of saturated aqueous solution of NaCl in a centrifuge tube.</li>
		<li>Centrifuge the suspension (5,000 x g, 5 min) and wash the precipitate with HPLC-grade water (2x). Lyophilize the product.</li>
	</ol>
	</li>
</ol>
3. Guanidinylation and Deprotection in Solution
<ol>
	<li>Guanidinylation with the tailor-made precursors
	<ol>
		<li>Dissolve a small amount (e.g., 25 mg) of the orthogonally deprotected cyclic peptide in a small volume of DMF (e.g., 2 mL). Add 2 eq. of the guanidinylation precursor and 2 eq. of DIPEA to the solution and let it stir for 2 h at RT.</li>
		<li>Monitor the progress of the reaction with HPLC-MS. After the reaction is complete, remove the solvent, re-dissolve the guanidinylated cyclic peptide in acetonitrile, and perform semipreparative HPLC purification16.</li>
	</ol>
	</li>
	<li>Removal of acid labile side chain protecting groups
	<ol>
		<li>Prepare 2 mL of a solution containing trifluoroacetic acid (TFA), water, and triisopropylsilane (TIPS) (95/2.5/2.5; v/v/v). Add this solution to the purified product in a small glass vial and let it stir at RT for at least 1 h. Observe the complete deprotection with ESI-MS17.</li>
		<li>Evaporate the solvent under reduced pressure to a small volume. Prepare ice-cold ether and add 10 mL in a 50-mL centrifuge tube. Precipitate the deprotected peptide in ice-cold ether using a Pasteur pipette by adding it dropwise. Centrifuge the suspension (5,000 x g, 5 min) to obtain a pellet.</li>
		<li>Wash the precipitate with ice-cold ether and centrifuge it (5,000 x g, 5 min, 2x). Dissolve the product in 2 mL of HPLC-grade water and purify it with semipreparative HPLC16.</li>
	</ol>
	</li>
</ol>
4. Analytical Data and Parameters for Purification
<ol>
	<li>Analytical HPLC-ESI-MS
	<ol>
		<li>Perform analytical HPLC-ESI-MS with an LCQ mass spectrometer using a C18 column (12-nm pore size, 3-&#181;m particle size, 125 mm &#215; 2.1 mm) or a C8 column (20-nm pore size, 5-&#181;m particle size, 250 mm &#215; 2.1 mm), with H2O (0.1% v/v formic acid)/acetonitrile (0.1% v/v formic acid) as eluents.</li>
	</ol>
	</li>
	<li>Semi-preparative HPLC
	<ol>
		<li>Perform semi-preparative HPLC using a preparative HPLC instrument with an ODS-A column (20 x 250 mm, 5 &#181;m), a flow rate of 8 mL/min, and linear gradients of H2O (0.1% v/v TFA) and acetonitrile (0.1% v/v TFA).</li>
	</ol>
	</li>
</ol>

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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.
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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><tbody><tr><td>&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-Di-Boc-1H-pyrazole-1-carboxamidine, 98%&amp;nbsp;</td><td>Sigma Aldrich</td><td>434167&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Triphenylphosphine, 99%</td><td>Sigma Aldrich</td><td>T84409&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>Tetrahydrofuran, &gt;99.5%</td><td>Carl Roth</td><td>4745</td><td></td></tr><tr><td>Tetrahydrofuran anhydrous, 99.8%</td><td>Carl Roth</td><td>5182</td><td></td></tr><tr><td>Methanol anhydrous, 99.8%</td><td>Sigma Aldrich</td><td>322415&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>Diisopropyl azodicarboxylate, 98%</td><td>Sigma Aldrich</td><td>225541&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Dichlormethan, for synthesis, 99.5%</td><td>Carl Roth</td><td>8424</td><td></td></tr><tr><td>Silica gel for flash chromtaography</td><td>Sigma Aldrich</td><td>60738&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>n-Pentane, 99%</td><td>Carl Roth</td><td>8720</td><td></td></tr><tr><td>n-Hexane, 99%</td><td>Carl Roth</td><td>CP47</td><td></td></tr><tr><td>Ethylacetate, 99.5%</td><td>Carl Roth</td><td>7338</td><td></td></tr><tr><td>Aminohexanol, 95%</td><td>Sigma Aldrich</td><td>A56353&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>&lt;em&gt;S&lt;/em&gt;-Methylisothiourea hemisulfate, 98%</td><td>Sigma Aldrich</td><td>M84445 ALDRICH</td><td></td></tr><tr><td>Di-&lt;em&gt;tert&lt;/em&gt;-butyl dicarbonate, 99%</td><td>Sigma Aldrich</td><td>205249&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamid, 99.8%</td><td>Carl Roth</td><td>A529</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Diisopropylethylamin, 99.5%</td><td>Carl Roth</td><td>2474</td><td></td></tr><tr><td>Acetic anhydrid, 99%</td><td>Carl Roth</td><td>4483</td><td></td></tr><tr><td>Chlortrityl resin</td><td>Carbolution</td><td>CC11006</td><td></td></tr><tr><td>Fmoc-Gly-OH, 98%</td><td>Carbolution</td><td>CC05014</td><td></td></tr><tr><td>Piperidin, 99%</td><td>Sigma Aldrich</td><td>104094&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>Fmoc-Orn(Dde)-OH</td><td>Iris-Biotech</td><td>FAA1502</td><td></td></tr><tr><td>HATU, 99%</td><td>Carbolution</td><td>CC01011</td><td></td></tr><tr><td>HOAt, 99%</td><td>Carbolution</td><td>CC01004</td><td></td></tr><tr><td>Fmoc-Val-OH</td><td>Carbolution</td><td>CC05028</td><td></td></tr><tr><td>2-Nitrobenzenesulfonyl chloride, 97%</td><td>Sigma Aldrich</td><td>N11507&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>2,4,6-Collidine, 99%</td><td>Sigma Aldrich</td><td>27690&amp;nbsp;SIGMA-ALDRICH</td><td></td></tr><tr><td>Mercaptoethanol, 99%&amp;nbsp;</td><td>Sigma Aldrich</td><td>M6250&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Diazabicycloundecen, 98%</td><td>Sigma Aldrich</td><td>139009&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Fmoc-D-Phe-OH, 98%</td><td>Sigma Aldrich</td><td>47378&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>Fmoc-Asp(OtBu)-OH, 98%</td><td>Carbolution</td><td>CC05008</td><td></td></tr><tr><td>Hexafluoroisopropanol</td><td>Carbolution</td><td>CC03056</td><td></td></tr><tr><td>Diphenylphosphoryl azide, 97%</td><td>Sigma Aldrich</td><td>178756&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>TFA, 99.9%</td><td>Carl Roth</td><td>P088</td><td></td></tr><tr><td>Triisopropylsilan, 98%</td><td>Sigma Aldrich</td><td>233781&amp;nbsp;ALDRICH</td><td></td></tr><tr><td>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. ...

modification-functionalization-guanidine-group-tailor-made

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

10.5K Views.  Technische Universität München. A protocol for the synthesis of alkyl-modified guanidines based on the use of the corresponding precursors is presented.

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

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Chemistry

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Bioengineering

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important structural element found in many complex natural products and pharmaceuticals. Owing to the continual discovery of new guanidine-containing natural products and designed small molecules, rapid and efficient guanidinylation methods are of keen interest to synthetic and medicinal organic chemists. Because the nucleophilicity and basicity of guanidines can affect subsequent chemical transformations, traditional, indirect guanidinylation is typically pursued. Indirect methods commonly employ multiple protection steps involving a latent amine precursor, such as an azide, phthalimide, or carbamate. By circumventing these circuitous methods and employing a direct guanidinylation reaction early in the synthetic sequence, it was possible to forge the linear terminal guanidine containing backbone of clavatadine A to realize a short and streamlined synthesis of this potent factor XIa inhibitor. In practice, guanidine hydrochloride is elaborated with a carefully constructed protecting array that is optimized to survive the synthetic steps to come. In the preparation of clavatadine A, direct guanidinylation of a commercially available diamine eliminated two unnecessary steps from its synthesis. Coupled with the wide variety of known guanidine protecting groups, direct guanidinylation evinces a succinct and efficient practicality inherent to methods that find a home in a synthetic chemist&#39;s toolbox.

Here, we present a protocol for direct, early stage guanidinylation that enables rapid total synthesis of aminoguanidine-containing small organic molecules. An advanced synthetic intermediate used in the synthesis of a blood coagulation factor XIa inhibitor was prepared using this protocol.

The guanidine functional group, displayed most prominently in the amino acid arginine, one of the fundamental building blocks of life, is an important ...

Facile Preparation of 4-Substituted Quinazoline Derivatives

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted 2-aminobenzophenones and thiourea in the presence of dimethyl sulfoxide (DMSO). This is a unique complementary reaction system in which thiourea undergoes thermal decomposition to form carbodiimide and hydrogen sulfide, where the former reacts with 2-aminobenzophenone to form 4-phenylquinazolin-2(1H)-imine intermediate, whilst hydrogen sulfide reacts with DMSO to give methanethiol or other sulfur-containing molecule which then functions as a complementary reducing agent to reduce 4-phenylquinazolin-2(1H)-imine intermediate into 4-phenyl-1,2-dihydroquinazolin-2-amine. Subsequently, the elimination of ammonia from 4-phenyl-1,2-dihydroquinazolin-2-amine affords substituted quinazoline derivative. This reaction usually gives quinazoline derivative as a single product arising from 2-aminobenzophenone as monitored by GC/MS analysis, along with small amount of sulfur-containing molecules such as dimethyl disulfide, dimethyl trisulfide, etc. The reaction usually completes in 4-6 hr at 160 &#186;C in small scale but may last over 24 hr when carried out in large scale. The reaction product can be easily purified by means of washing off DMSO with water followed by column chromatography or thin layer chromatography.

A protocol for facile preparation of 4-substituted quinazoline derivatives from 2-aminobenzophenones, thiourea and dimethyl sulfoxide is presented.

Reported in this paper is a very simple method for direct preparation of 4-substituted quinazoline derivatives from a reaction between substituted ...

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

Immunology and Infection

Preparation of N-(2-alkoxyvinyl)sulfonamides from N-tosyl-1,2,3-triazoles and Subsequent Conversion to Substituted Phthalans and Phenethylamines

Decomposition of N-tosyl-1,2,3-triazoles with rhodium(II) acetate dimer in the presence of alcohols forms synthetically versatile N-(2-alkoxyvinyl)sulfonamides, which react under a variety of conditions to afford useful N- and O-containing compounds. Acid-catalyzed addition of alcohols or thiols to N-(2-alkoxyvinyl)sulfonamide-containing phthalans provides access to ketals and thioketals, respectively. Selective reduction of the vinyl group in N-(2-alkoxyvinyl)sulfonamide-containing phthalans via hydrogenation yields the corresponding phthalan in good yield, whereas reduction with sodium bis(2-methoxyethoxy)aluminumhydride generates a ring-opened phenethylamine analogue. Because the N-(2-alkoxyvinyl)sulfonamide functional group is synthetically versatile, but often hydrolytically unstable, this protocol emphasizes key techniques in preparing, handling, and reacting these pivotal substrates in several useful transformations.

Preparation of N-2-alkoxyvinylsulfonamides from N-tosyl-1,2,3-triazoles and Subsequent Conversion to Substituted Phthalans and Phenethylamines

Representative experimental procedures for the synthesis of N-(2-alkoxyvinyl)sulfonamides and subsequent conversion to phthalan and phenethylamine derivatives are presented in detail.

Decomposition of N-tosyl-1,2,3-triazoles with rhodium(II) acetate dimer in the presence of alcohols forms synthetically versatile ...

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having multifarious bioactivities. Although chemical O-glycosylation is a commonly beneficial strategy to synthesize disaccharide nucleosides, the preparation of substrates such as glycosyl donors and acceptors requires&#160;tedious protecting&#160;group manipulations and a purification at each synthetic step. Meanwhile, several research groups have reported that boronic and borinic esters serve as a protecting or activating group of carbohydrate derivatives to achieve the regio- and/or stereoselective acylation, alkylation, silylation, and glycosylation. In this article, we demonstrate the procedure for the regioselective O-glycosylation of unprotected ribonucleosides utilizing boronic acid. The esterification of 2&#39;,3&#39;-diol of ribonucleosides with boronic acid makes the temporary protection of diol, and, following O-glycosylation with a glycosyl donor in the presence of p-toluenesulfenyl chloride and silver triflate, permits the regioselective reaction of the 5&#39;-hydroxyl group to afford the disaccharide nucleosides. This method could be applied to various nucleosides, such as guanosine, adenosine, cytidine, uridine, 5-metyluridine, and 5-fluorouridine. This article and the accompanying video represent&#160;useful (visual) information for the O-glycosylation of unprotected nucleosides and their analogs for the synthesis of not only disaccharide nucleosides, but also a variety of biologically relevant derivatives.

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2&#39;,3&#39;-diol moieties utilizing a cyclic boronic ester. This method applies to several unprotected nucleosides such as adenosine, guanosine, cytidine, uridine, 5-methyluridine, and 5-fluorouridine to give corresponding disaccharide nucleosides.

Disaccharide nucleosides, which consist of disaccharide and nucleobase moieties, have been known as a valuable group of natural products having ...

Preparation of 6-aminocyclohepta-2,4-dien-1-one Derivatives via Tricarbonyl(tropone)iron

aza-Michael adducts of tricarbonyl(tropone)iron are synthesized by two different methods. Primary aliphatic amines and cyclic secondary amines participate in a direct aza-Michael reaction with tricarbonyl(tropone)iron under solvent-free conditions. Less nucleophilic aniline derivatives and more hindered secondary amines add efficiently to the cationic tropone complex formed by protonation of tricarbonyl(tropone)iron. While the protocol utilizing the cationic complex is less efficient overall for accessing the aza-Michael adducts than the direct, solvent-free addition to the neutral complex, it allows the use of a broader range of amine nucleophiles. Following protection of the amine of the aza-Michael adduct as a tert-butyl carbamate, the diene is decomplexed from the iron tricarbonyl fragment upon treatment with cerium(IV) ammonium nitrate to provide derivatives of 6-aminocyclohepta-2,4-dien-1-one. These products can serve as precursors to diverse compounds containing a seven-membered carbocyclic ring. Because the demetallation requires protection of the amine as a carbamate, the aza-Michael adducts of secondary amines cannot be decomplexed using the protocol described here.

Preparation of 6-aminocyclohepta-2,4-dien-1-one Derivatives via Tricarbonyltroponeiron

Representative experimental procedures for the addition of amine nucleophiles to tricarbonyl(tropone)iron and subsequent demetallation of the resulting complexes are presented in detail.

aza-Michael adducts of tricarbonyl(tropone)iron are synthesized by two different methods. Primary aliphatic amines and cyclic secondary amines participate ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

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

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Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

Facile Preparation of 4-Substituted Quinazoline Derivatives

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Preparation of N-(2-alkoxyvinyl)sulfonamides from N-tosyl-1,2,3-triazoles and Subsequent Conversion to Substituted Phthalans and Phenethylamines

Regioselective O-Glycosylation of Nucleosides via the Temporary 2',3'-Diol Protection by a Boronic Ester for the Synthesis of Disaccharide Nucleosides

Preparation of 6-aminocyclohepta-2,4-dien-1-one Derivatives via Tricarbonyl(tropone)iron

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications