Name: Modification and Functionalization of the Guanidine Group by Tailor-made Precursors (Video) | JoVE Journal
Uploaded: 2017-04-27T13:00:00.000+00:00
Duration: 9 min 45 s
Description: Learn a convenient synthetic method for guanidine functionalized peptides using tailor-made precursors compatible with solid-phase peptide synthesis. This technique advances muscle peptide chemistry and personalized medicine applications.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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>

<ol><li>Saczewski, F., Balewski, L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/19780701">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="https://www.ncbi.nlm.nih.gov/pubmed/23617396">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://pubs.acs.org/doi/abs/10.1021/ja01074a018">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/pubmed?term=((The+chemistry+and+biology+of+organic+guanidine+derivatives%5BTitle%5D)+AND+%22Nat.+Prod.+Rep%22%5BJournal%5D)">The chemistry and biology of organic guanidine derivatives.</a> Nat. Prod. Rep. 25, 919-954 (2008).</li>
<li>Peterlin-Mašič, L., Kikelj, D. <a target="_blank" href="http://www.sciencedirect.com/science/article/pii/S0040402001005075">Arginine mimetics.</a> Tetrahedron. 57 (33), 7073-7105 (2001).</li>
<li>Peterlin-Mašič, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arginine+mimetic+structures+in+biologically+active+antagonists+and+inhibitors%5BTitle%5D)+AND+%22Curr.+Med.+Chem%22%5BJournal%5D)">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/pubmed?term=((Integrins%3A+bidirectional%2C+allosteric+signaling+machines%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Structural+aspects+of+integrins%5BTitle%5D)+AND+%22Adv.+Exp.+Med.+Biol%22%5BJournal%5D)">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="https://www.ncbi.nlm.nih.gov/pubmed/10801897">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/pubmed?term=((Small+cause%2C+great+impact+-+modification+of+guanidine+group+in+RGD+controls+subtype+selectivity%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">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/pubmed?term=((Crystal+structure+of+the+extracellular+segment+of+integrin+alpha+Vbeta3%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Crystal+structure+of+the+extracellular+segment+of+integrin+alpha+Vbeta3+in+complex+with+an+Arg-Gly-Asp+ligand%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((Crystal+structure+of+%CE%B15%CE%B21+integrin+ectodomain%3A+atomic+details+of+the+fibronectin+receptor%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Crystal structure of α5β1 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/pubmed?term=((RGD+modified+polymers%3A+biomaterials+for+stimulated+cell+adhesion+and+beyond%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Photoswitched+Cell+Adhesion+on+Surfaces+with+RGD+Peptides%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCorradini%2C+D+%22Handbook+of+HPLC%22">Handbook of HPLC</a>. , CRC Press. (2011).</li>
<li>de Hoffmann, E., Stroobant, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3ade+Hoffmann%2C+E+%22Mass+Spectrometry%3A+Principles+and+Applications%22">Mass Spectrometry: Principles and Applications</a>. , John Wiley & Sons. (2007).</li>
<li>Frank, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conformational+Control+of+Integrin-Subtype+Selectivity+in+isoDGR+Peptide+Motifs%3A+A+Biological+Switch%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">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/pubmed?term=((Selective+substrate-based+inhibitors+of+mammalian+dimethylarginine+dimethylaminohydrolase%5BTitle%5D)+AND+%22J.+Med.+Chem%22%5BJournal%5D)">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., König, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N%28G%29-Acyl-argininamides+as+NPY+Y%281%29+receptor+antagonists%3A+Influence+of+structurally+diverse+acyl+substituents+on+stability+and+affinity%5BTitle%5D)+AND+%22Bioorg.+Med.+Chem%22%5BJournal%5D)">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="https://www.thieme-connect.com/products/ejournals/abstract/10.1055/s-0032-1316789">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><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

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...

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

Research

JoVE Journal

Biochemistry

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

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.

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

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.

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

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.

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

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.

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

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

Facile Preparation of 4-Substituted Quinazoline Derivatives

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.

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

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

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

Here, we present protocols for the synthesis of disaccharide nucleosides by the regioselective O-glycosylation of ribonucleosides via a temporary protection of their 2',3'-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.

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

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

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

This protocol describes a method for producing and evaluating the antimicrobial activity and cytotoxicity of the cationic peptide Mel4 and peptide mimic RK758. By integrating chemical synthesis, recombinant expression, and standardized functional assays, it provides a streamlined workflow for the production and testing of peptides and small-molecule mimics.

Production and Testing of Antimicrobial Peptides and Their Mimics

Learn a convenient synthetic method for guanidine functionalized peptides using tailor-made precursors compatible with solid-phase peptide synthesis. This technique advances muscle peptide chemistry and personalized medicine applications.

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

Explore More Videos