The authors would like to thank Dr. Zachary Z. Brown and Jennifer Alleva for the initial development of this solid phase synthesis technique and Matthew F.L. Parker for helpful discussions. This work is supported by the Defense Threat Reduction Agency (DOD-DTRA) (HDTRA1-09-1-0009) and the Horst Witzel Fellowship Award supported by Cephalon, Inc.

1. Setup
<ol>
 <li>The reaction set-up for the solid-phase synthesis is a polypropylene filter cartridge or glass reactor that is connected via polypropylene tubing to an enclosed filtering flask under vacuum as shown in Figure 1. The reaction can be mixed by a magnetic stir bar or by bubbling nitrogen through the reactor.</li>
 <li>A gas manifold connected to an Argon cylinder equipped with a drying tube and oil bubbler is also recommended as it allows the reaction vessel to be contained under an inert atmosphere and allows the removal of reagents from sealed containers.</li>
 <li>All operations are performed in a fume hood and proper personal protective equipment (safety glasses, lab coat and nitrile gloves) is required.</li>
</ol>
2. Loading First Bis-Peptide Onto Resin
<ol>
 <li>Weigh 114 mg of HMBA-AM Resin (0.88 mmol / g loading, 100 μmol) into 8 mL reaction vessel and add magnetic stir bar. Cap the top of the vessel with a rubber septum and purge the tube with argon for at least 5 minutes.</li>
 <li>In the meantime, weigh 117.3 mg of compound 1 of Figure 3 (586.63 g / mol, 2eq) and 59.2 mg of 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT, 296.0 g / mol, 2eq) into a 15 mL disposable centrifuge tube and dissolve in 2 mL anhydrous dichloromethane (DCM). Add 24 μL of 1-methylimidazole (NMI, 80.81 mL / mol, 3eq) to solution and mix until completely dissolved.</li>
 <li>Transfer the activated solution to the reaction vessel via syringe and allow to stir under argon overnight (~10 hours).</li>
 <li>Remove the septum and drain the reaction mixture. Wash the resin with DCM (5x) and dimethylformamide (DMF) (5x). Perform the "methyl red test" described in section 10.1 to assess the degree of resin loading. If the resin remains red during the methyl red test then steps 2.2 and 2.3 should be repeated. A yellow color, indicative of a negative methyl red test, is preferred; however, since any remaining hydroxyl groups will be capped in the next step, a slightly positive result (light orange resin color) may be acceptable.</li>
</ol>

3. Deprotection of First Bis-Peptide and Simultaneous Resin Capping
<ol>
 <li>Add 1 mL of DCM to the reaction vessel then add 1 mL 33% hydrogen bromide in acetic acid dropwise over 30 seconds (bubbling occurs) and allow to stir for 15 minutes. Drain and wash the resin with DCM (5x) then repeat process once more.</li>
 <li>Wash the resin with DCM (5x) then DMF (5x). Neutralize the resin by washing twice with a 5% v/v solution of N,N-diisopropylethylamine (DIPEA) in DMF then wash with DCM (5x) and DMF (5x) again. Perform the "methyl red test" and "chloranil test" discussed in section 10.1 and 10.2. Results should be negative for the methyl red test and positive for the chloranil test.</li>
</ol>
4. Coupling Boc/tBu-Protected Functionalized Bis-Amino Acid
<ol>
 <li>Reintroduce an inert atmosphere to the resin-containing reaction vessel by washing three times with anhydrous DCM then attach a septum and argon line. Purge and wash the vessel by adding 1-2 mL of anhydrous DCM and letting stir for 30 seconds then draining the vessel until the argon line bubbler begins to rise. Do this at least once more.</li>
 <li>Prepare a solution of 0.15 M functionalized bis-amino acid (3eq) and 245 mg of 1-hydroxy-7-azabenzotriazole (HOAt, 136.11 g / mol, 18eq) in 2 mL of 2:1 DCM:DMF in a flame dried test tube under argon atmosphere. Add 47 μL of diisopropylcarbodiimide (DIC, 156.6 mL / mol, 3eq) and stir for 90 minutes.</li>
 <li>Add 35 μL DIPEA (174.19 mL / mol, 2eq) in 666 μL anhydrous DMF to resin and allow to stir for 5 minutes.</li>
 <li>Transfer the pre-activated bis-amino acid solution to reaction vessel via syringe and allow to stir overnight.</li>
 <li>Drain the reaction mixture and wash twice with anhydrous DCM while under argon.</li>
 <li>To promote the closure of the diketopiperazine, add a 0.25 M solution of HOAt (136.11 g / mol, 10eq) and DIC (156.6 mL / mol, 10eq) in a 4 mL of 1:1 DCM:DMF and allow to stir under argon for 1 hour.</li>
 <li>Remove the septum and drain the reaction mixture. Wash the resin with DCM (5x) and DMF (5x). If desired, perform the "chloranil test" discussed in section 10.2.</li>
</ol>
5. Deprotection of Boc/tBu-Protected Functionalized Bis-Amino Acid
<ol>
 <li>Add 2 mL of a solution of 95:5 trifluororacetic acid (TFA):triisopropylsilane (TIPS) to the reaction vessel and allow it to stir for 1 hour. Drain and wash the resin for about 30 seconds with DCM (5x) then repeat process once more.</li>
 <li>Wash the resin with DCM (5x) then DMF (5x). Neutralize the resin by washing twice with a 5%v/v solution of DIPEA in DMF then wash DCM (5x) and DMF (5x) again. If desired, perform the "chloranil test" discussed in section 10.2.</li>
</ol>
6. Repeat steps 4 and 5 as desired to synthesize targeted bis-peptide.
7. Functionalizing the Bis-Peptide Prolidine End
<ol>
 <li>The prolidine end of the growing bis-peptide may be acylated independently or together through a diketopiperazine. Also, this end can be left protected, which will be cleaved latter, affording the free amino acid. If desired, perform the "chloranil test" discussed in section 10.2 to assess coupling efficiency.</li>
</ol>
8. Deprotection of Fmoc and Acylation of the Quaternary End of the Bis-Peptide
<ol>
 <li>A 2mL solution of 20% piperidine in DMF is added and the reaction is mixed for 20 minutes. Drain and wash the resin with DMF (5x) then repeat process once more.</li>
 <li>Wash the resin with DCM (5x) then DMF (5x).</li>
 <li>Prepare a 0.15 M solution of amino acid (3eq) in 2 mL of N-methylpyrrolidone (NMP) with 114 mg 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU, 380.2 g / mol, 3eq) and 104.5 μL DIPEA (174.19 mL / mol, 6eq) and mix well. Add to reaction vessel and allow to stir for 6 hours.</li>
 <li>Wash the resin with DCM (5x) then DMF (5x).</li>
</ol>
9. Remove the Boc Group from the Resin Bound Amino Acid and Cleave from Resin
<ol>
 <li>Add 2 mL of a 1:1 TFA:DCM solution to the reaction vessel and allow to stir for 30 minutes. Drain and wash the resin with DCM (5x) then repeat process once more.</li>
 <li>Wash and drain the resin for 30 seconds with DCM (5x) then DMF (5x).</li>
 <li>Add 2 mL of a solution of 10% DIPEA in anhydrous DMF and allow to stir 24-48 hours.</li>
 <li>Collect reaction mixture into pre-weighed round bottom flask. Transfer 30 μL of this solution to 450 μL of THF in an LC-MS vial and submit for analysis. Wash the resin with additional aliquots of DMF and collect into the round bottom flask then remove solvent in vacuo.</li>
</ol>

10. Purification of Bis-Peptide
<ol>
 <li>Dissolve crude bis-peptide in a minimal amount of dimethyl sulfoxide (100-250 μL) and transfer into HPLC vial insert. Place insert into autosampler of semi-prepitive HPLC system (Hewlett Packard 1100 Series) equipped with an XTerra Prep MS C18 5 μm 7.8x150 mm column and a 100 μL injection loop.</li>
 <li>Perform multiple 50 μL injections of the sample using a gradient program of 5-95% acetonitrile in water with 0.1% formic acid over 30 minutes while monitoring at 274 nm. Collect the product peak in a pre-weighed disposable centrifuge tube and freeze dry using a lyophilizer. Caution should be taken with the first run as a slight shift in peak retention time compared to analytical LCMS is typically observed.</li>
</ol>

11. Assessment Methods
<ol>
 <li>METHYL RED TEST7: Remove ~1 mg of dry resin via disposable pipette and rinse into 4 mL reaction vessel. Add a solution of 20 mg methyl red, 50 μL N,N'-diisopropylcarbodiimide (DIC), and 5 mg 4-dimethylaminopyridine (DMAP) in 500 μL anhydrous DCM and allow to stir for 5-10 min. Drain and wash resin with DCM until the filtrate becomes colorless. Positive indication is the resin beads remaining orange or red.</li>
 <li>CHLORANIL TEST12: Transfer ~1 mg of dry resin into a small vial via disposable pipette. Add 3 drops of both a 0.8 mM chloranil in DMF solution and 2% acetaldehyde in DMF solution and let sit at room temperature for 5-10 minutes. Positive indication is the resin beads turning blue/purple.</li>
 <li>ACTIVATION TRAP TEST: Activated compounds during the synthesis can be assessed by transferring a small amount (5-10 μL) of the activated solution to a liquid chromatography-mass spectrometry (LC-MS) vial containing 50 μL of pyrrolidine. Mix by hand for a few seconds (solution should become yellow) then dilute with 450 μL of tetrahydrofuran (THF) and submit for LC-MS analysis.</li>
 <li>ANALYTICAL LC-MS: The final product and activated intermediates can be assessed using an HP 1200 series LC-MS system equipped with a Waters Xterra MS C18 3.5 μm 4.6 mm x 150 mm column and a gradient system of 5-95% acetonitrile in water with 0.1% formic acid over 30 minutes.</li>
</ol>
12. Representative Results
An example of both crude (Figure 4) and purified (Figure 5) LCMS traces are provided. Purified yields of approximately 10% are expected using the methods outlined above.
<img alt="Vacuum filtration setup diagram; reaction vessel on magnetic stir plate connected to collection flask." src="/files/ftp_upload/4112/4112fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Diagram of Experimental Set-Up for Solid Phase Synthesis.
<img alt="Chemical structure diagram of peptide featuring quaternary and proline end groups." src="/files/ftp_upload/4112/4112fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Relevant Nomenclature of Bis-Amino Acids/Bis-Peptides.
<img alt="Peptide synthesis diagram; chemical reactions, reagent steps, solid-phase method, compound structures." src="/files/ftp_upload/4112/4112fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Overall Synthetic Scheme. <a href="https://www.jove.com/files/ftp_upload/4112/4112fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="LC-MS UV chromatogram at 274 nm, showing crude trimer retention time distribution, data analysis." src="/files/ftp_upload/4112/4112fig4a.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4a"> 
Figure 4a. HPLC Trace of Crude Product at 274 nm.
<img alt="Mass spectrometry diagram of crude product peak at 21.3min, displaying M/Z and abundance." src="/files/ftp_upload/4112/4112fig4b.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4b"> 
Figure 4b. MS Spectrum of Crude Product Peak.
<img alt="Purified trimer LC-MS UV trace at 274nm; chromatography result showing retention time vs mAu." src="/files/ftp_upload/4112/4112fig5a.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5a"> 
Figure 5a. HPLC Trace of Purified Product at 274 nm.
<img alt="Mass spectrometry graph showing peptide fragmentation; molecular weight 930.01; m/z peaks labeled." src="/files/ftp_upload/4112/4112fig5b.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5b"> 
Figure 5b. MS Spectrum of Purified Product Peak.

<ol>
	<li>Atherton, E., Sheppard, 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=.">.</a> Solid Phase Peptide Synthesis: A Practical Approach. , (1989).</li><li>Brown, Z. Z., Alleva, J., Schafmeister, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-Phase+Synthesis+of+Functionalized+Bis-Peptides.">Solid-Phase Synthesis of Functionalized Bis-Peptides.</a> Biopolymers. 96, 578-585 (2010).</li><li>Schafmeister, C. E., Brown, Z. Z., Gupta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape-Programmable+Macromolecules.">Shape-Programmable Macromolecules.</a> Acc. Chem. Res. 41, 1387-1398 (2008).</li><li>Brown, Z. Z., Schafmeister, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+Hexa-+and+Pentasubstituted+Diketopiperazines+from+Sterically+Hindered+Amino+Acids.">Synthesis of Hexa- and Pentasubstituted Diketopiperazines from Sterically Hindered Amino Acids.</a> Org. Let. 12, 1436-1439 (2010).</li><li>Nielson, J., Lyngso, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+Solid-Phase+Synthesis+of+Balanol+Analogues.">Combinatorial Solid-Phase Synthesis of Balanol Analogues.</a> Tet. Lett. 37, 8439-8442 (1996).</li><li>Blankemeyer-Menge, B., Nimtz, M., Frank, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Efficient+Method+for+Anchoring+Fmoc-Amino+Acids+to+Hydroxyl-Functionalized+Solid+Supports.">An Efficient Method for Anchoring Fmoc-Amino Acids to Hydroxyl-Functionalized Solid Supports.</a> Tet. Lett. 31, 1701-1704 (1990).</li><li>Komba, S., Sasaki, S., Machida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+New+Colorimetric+Test+for+Detection+of+Hydroxyl+Groups+in+Solid-Phase+Synthesis.">A New Colorimetric Test for Detection of Hydroxyl Groups in Solid-Phase Synthesis.</a> Tet. Lett. 48, 2075-2078 (2007).</li><li>Demner, O., Dijkgraaf, I., Schottelius, M., Wester, H. J., 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=Introduction+of+Functional+Groups+into+Peptides+via+N-Alkylation.">Introduction of Functional Groups into Peptides via N-Alkylation.</a> Org. Lett. 10, 2015-2018 (2008).</li><li>Plas, S. E. V. a. n. d. e. r., Van Hoeck, E., Lynen, F., Sandra, P., Madder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+New+SPE+Material+for+EDCs:+Fully+Automated+Synthesis+of+a+Library+of+Tripodal+Receptors+Followed+by+Fast+Screening+by+Affinity+LC.">Toward a New SPE Material for EDCs: Fully Automated Synthesis of a Library of Tripodal Receptors Followed by Fast Screening by Affinity LC.</a> Eur. J. Org. Chem. 11, 1796-1805 (2009).</li><li>Kaiser, E., Colescot, R. L., Bossinger, C. D., Cook, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Color+Test+for+Detection+of+Free+Terminal+Amino+Groups+in+Solid-Phase+Synthesis+of+Peptides.">Color Test for Detection of Free Terminal Amino Groups in Solid-Phase Synthesis of Peptides.</a> Anal. Biochem. 34, 595-598 (1970).</li><li>Chan, W. C., White, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fmoc Solid Phase Peptide Synthesis: A Practical Approach. , (2000).</li><li>Vojkovsky, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Secondary+Amines+on+Solid-Phase.">Detection of Secondary Amines on Solid-Phase.</a> Peptide Research. 71, 236-237 (1995).</li></ol>

Production and Free Access to this article is sponsored by the United States Defense Threat Reduction Agency.

The synthetic approach presented herein provides a method for the synthesis of functionalized bis-peptides from bis-amino acid building blocks using common solid-phase peptide synthesis techniques. The monomer synthesis of these "Pro4" building blocks from trans-4-hydroxyproline3 is highly scalable and has been successfully completed to the hydantoin stage at a 600 mmol (234 g) scale (unpublished). Once the monomers are in hand, the use of solid-phase techniques provides a more rapid method of bis-peptide synthesis than our current solution-phase methodology4 by eliminating the need for reaction work-ups and intermediate purifications. 
The primary challenge in solid-phase synthesis is diagnosing synthetic progress and problem solving since no intermediates are isolated. This has lead to the development of many colorimetric tests including those to identify if free amines (Kaiser Test10) or free hydroxyls (Methyl Red Test7) are exposed on resin. Unfortunately, the commonly used Kaiser Test10 is not generally applicable in our solid-phase synthesis due to the almost exclusive use of secondary amines or amines attached to a quaternary carbon. Other options for assessment on HMBA resin include test cleavages using a nucleophile such as hydrazine11, quantitative Fmoc cleavage monitored by UV/Vis1,11, and trapping and analyzing incoming activated compounds. 
Another overlooked issue in solid-phase synthesis is the repetitive nature of synthetic steps required by the operator. With this is mind, the authors strongly recommend the use of a spreadsheet or checklist when performing any manual solid-phase peptide synthesis.
The difficultly in using bis-peptides for solid-phase synthesis compared to common &alpha;-amino acids includes the potential for more difficult couplings due to steric hindrance, the need for on-resin diketopiperazine closures, and simultaneous deprotections (Boc/tBu; Cbz/tBu). Another difficultly lies in achieving quantitative release from the resin using this "safety catch" method when compared to more conventional means. With these factors in mind, it is very possible that further optimization of this method can be achieved and current efforts are underway in our group to improve the method presented here.

<table><tbody><tr><td>HMBA-Am Resin</td><td>Novabiochem, EMD Millipore</td><td>855018</td><td></td></tr><tr><td>MSNT</td><td>Novabiochem, EMD Millipore</td><td>851011</td><td></td></tr><tr><td>NMI</td><td>Sigma-Aldrich</td><td>336092</td><td>Toxic, Corrosive</td></tr><tr><td>DCM</td><td>Sigma-Aldrich</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&amp;amp;N4=D65100%7CSIAL&amp;amp;N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&amp;amp;F=SPEC&quot; target=&quot;_blank&quot;&gt;D65100&lt;/a&gt;</td><td>Carcinogenic</td></tr><tr><td>Anhydrous DCM</td><td>Acros Organics</td><td>34846</td><td>Carcinogenic</td></tr><tr><td>33% Hydrogen Bromide in Acetic Acid</td><td>Sigma-Aldrich</td><td>248630</td><td>Toxic, Corrosive, Fumes when open</td></tr><tr><td>DIPEA</td><td>Sigma-Aldrich</td><td>387649</td><td>Flammable, Toxic, Corrosive</td></tr><tr><td>DMF</td><td>Fisher Scientific</td><td>AC27960</td><td>Flammable, Toxic</td></tr><tr><td>Anhydrous DMF</td><td>Acros Organics</td><td>34843</td><td>Flammable, Toxic</td></tr><tr><td>HOAt</td><td>GenScript</td><td>C01568</td><td></td></tr><tr><td>DIC</td><td>Acros Organics</td><td>BP590</td><td>Flammable, Toxic, Corrosive</td></tr><tr><td>TFA</td><td>Sigma-Aldrich</td><td>T6508</td><td>Toxic, Corrosive</td></tr><tr><td>TIPS</td><td>Acros Organics</td><td>21492</td><td>Flammable, Toxic</td></tr><tr><td>Piperidine</td><td>Sigma-Aldrich</td><td>104094</td><td>Flammable, Toxic, Corrosive</td></tr><tr><td>HATU</td><td>GenScript</td><td>C01566</td><td>Toxic</td></tr><tr><td>NMP</td><td>Acros Organics</td><td>36438</td><td>Toxic</td></tr><tr><td>DMAP</td><td>Novabiochem, EMD Millipore</td><td>851055</td><td>Toxic</td></tr><tr><td>Methyl Red</td><td>Sigma-Aldrich</td><td>250198</td><td></td></tr><tr><td>THF</td><td>Sigma-Aldrich</td><td>401757</td><td>Flammable, Toxic, Peroxide Forming</td></tr><tr><td>Pyrrolidine</td><td>Sigma-Aldrich</td><td>P73803</td><td>Flammable, Toxic, Corrosive</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Fisher Scientific</td><td>D1281</td><td></td></tr><tr><td>SPPS Reaction Vessels</td><td>Grace</td><td>211108</td><td></td></tr><tr><td>LCMS</td><td>Agilent Technologies</td><td>1200 Series</td><td></td></tr><tr><td>Semi-Prep LC</td><td>Hewlett-Packard</td><td>1100 Series</td><td></td></tr><tr><td>Lyophilizer</td><td>Labconco Corp.</td><td>7934027</td><td></td></tr><tr><td>Rotovapor</td><td>Buchi</td><td>R-210 Series</td><td></td></tr><tr><td>Argon</td><td>Airgas</td><td>AR PP300CT</td><td></td></tr></tbody></table>

solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

Watch this Scientific Journal Video about Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology at JoVE.com

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

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

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

solid-phase-synthesis-functionalized-bis-peptide-using-safety-catch

Research

JoVE Journal

Biology

24.6K Views.  Temple University. De efficiënte solid-phase peptide synthese van een gefunctionaliseerde bis-peptide trimeer gebruik te maken van een "veiligheidspal" decollete procedure vanaf HMBA hars wordt beschreven.

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

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Biochemistry

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Bioengineering

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

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

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

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

Chemistry

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

Versatile Home-Built Microwave-Assisted System for Peptide Synthesis

An Inexpensive Adaptation of a Commercial Microwave Reactor for Solid Phase Peptide Synthesis

A home-built apparatus to perform solid phase peptide synthesis (SPPS), assisted by microwave irradiation and heating, is presented. In contrast to conventional SPPS reaction vessels, which drain solvent and byproducts via a frit located at the bottom of the vessel, the presented apparatus employs a gas dispersion tube under vacuum to remove solvent, byproducts, and excess reagents. The same gas dispersion tube supplies nitrogen gas agitation of the SPPS beads during the reaction steps of coupling and deprotection. Microwave heating is beneficial for SPPS couplings of sterically hindered residues, such as alpha-aminoisobutyric acid (Aib), an alpha,alpha-dialkylated amino acid residue. This home-built apparatus has been used to prepare, via manual Fmoc SPPS methods, heptameric and octameric peptides dominated by the Aib residue, which is notoriously difficult to couple under standard room temperature conditions and reagents. Further, typical commercial microwave SPPS reactors are dedicated exclusively to SPPS synthesis rendering them inaccessible to non-SPPS users. In contrast, the presented apparatus preserves the versatility of the microwave reactor for conventional microwave acceleration of chemical reactions, as the apparatus is trivially removed from the commercial microwave reactor.

A simple, inexpensive, and novel apparatus for performing solid phase peptide syntheses in a commercial microwave reactor is presented.

A home-built apparatus to perform solid phase peptide synthesis (SPPS), assisted by microwave irradiation and heating, is presented. In contrast to ...

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

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